Gene Literature Dashboard

Obesity is a leading contributor to colorectal cancer risk. We investigated whether the risk variants identified in genome-wide association studies of body mass index (BMI) and waist size are associated with colorectal cancer risk, independently of the effect of obesity phenotype due to a shared etiology. Twenty-four single nucleotide polymorphisms (SNPs) in 15 loci (BDNF, FAIM2, FTO, GNPDA2, KCTD15, LYPLAL1, MC4R, MSRA, MTCH2, NEGR1, NRXN3, SEC16B, SH2B1, TFAP2B and TMEM18) were genotyped in a case-control study of 2,033 colorectal cancer cases and 9,640 controls nested within the multiethnic cohort study, as part of the population architecture using genomics and epidemiology consortium. Risk alleles for two obesity SNPs were associated with colorectal cancer risk--KCTD15 rs29941 [odds ratio (OR) for C allele = 0.90, 95% confidence interval (CI) 0.83-0.98; p = 0.01] and MC4R rs17782313 (OR for C allele = 1.12, 95% CI 1.02-1.22; p = 0.02). These associations were independent of the effect of BMI. However, none of the results remained significant after adjustment for multiple comparisons. No heterogeneity was observed across race/ethnic groups. Our findings suggest that the obesity risk variants are not likely to affect the risk of colorectal cancer substantially.

<h4>Aim</h4>Huntingtin protein (Htt) was a neuropathological hallmark in human Huntington's Disease. The study aimed to investigate whether the macroautophagy regulator, Beclin1, was involved in the degradation of Htt.<h4>Methods</h4>PC12 cells and primary cultured brain neurons of rats were examined. pDC316 adenovirus shuttle plasmid was used to mediate the expression of wild-type Htt-18Q-552 or mutant Htt-100Q-552 in PC12 cells. The expression of the autophagy-related proteins LC3 II and Beclin1, as well as the lysosome-associated enzymes Cathepsin B and L was evaluated using Western blotting. The locations of Beclin1 and Htt were observed with immunofluorescence and confocal microscope.<h4>Results</h4>Htt552 expression increased the expression of LC3 II, Beclin1, cathepsin B and L in autophagy/lysosomal degradation pathway. Treatment with the autophagy inhibitor 3-MA or the proteasome inhibitors lactacystin and MG-132 increased Htt552 levels in PC12 cells infected with Ad-Htt-18Q-552 or Ad-Htt-100Q-552. The proteasome inhibitor caused a higher accumulation of Htt552-18Q than Htt552-100Q, and the autophagy inhibitor resulted in a higher accumulation of Htt552-100Q than Htt552-18Q. Similar results were observed in primary cultured neurons infected with adenovirus. In Htt552-expressing cells, Beclin1 was redistributed from the nucleus to the cytoplasm. Htt siRNA prevented Beclin1 redistribution in starvation conditions. Blockade of Beclin1 nuclear export by leptomycin B or Beclin1 deficiency caused by RNA interference induced the formation of mHtt552 aggregates.<h4>Conclusion</h4>Beclin1 regulates the accumulation of Htt via macroautophagy.

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.

As one of the most widely used drugs worldwide, heparin is an essential anticoagulant required for surgery, dialysis, treatment of thrombosis, cancer, and general circulatory management. Stabilin-2 is a scavenger clearance receptor with high expression in the sinusoidal endothelium of liver. It is believed that Stabilin-2 is the primary receptor for the clearance of unfractionated and low molecular weight heparins in the liver. Here, we identify the modifications and length of the heparin polymer that are required for binding and endocytosis by both human Stabilin receptors: Stabilin-2 and its homolog Stabilin-1 (also found in liver endothelium). Using enzymatically synthesized (35)S-labeled heparan sulfate oligomers, we identified that sulfation of the 3-OH position of N-sulfated glucosamine (GlcNS) is the most beneficial modification for binding and endocytosis via both Stabilin receptors. In addition, our data suggest that a decasaccharide is the minimal size for binding to the Stabilin receptors. These findings define the physical parameters of the heparin structure required for efficient clearance from blood circulation. These results will also aid in the design of synthetic heparins with desired clearance rates.

Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions of E. chaffeensis tandem repeat proteins 47 and 120 (TRP47 and -120) and the eukaryotic host cell have been described. In this investigation, yeast two-hybrid analysis demonstrated that an E. chaffeensis type 1 secretion system substrate, TRP32, interacts with a diverse group of human proteins associated with major biological processes of the host cell, including protein synthesis, trafficking, degradation, immune signaling, cell signaling, iron metabolism, and apoptosis. Eight target proteins, including translation elongation factor 1 alpha 1 (EF1A1), deleted in azoospermia (DAZ)-associated protein 2 (DAZAP2), ferritin light polypeptide (FTL), CD63, CD14, proteasome subunit beta type 1 (PSMB1), ring finger and CCCH-type domain 1 (RC3H1), and tumor protein p53-inducible protein 11 (TP53I11) interacted with TRP32 as determined by coimmunoprecipitation assays, colocalization with TRP32 in HeLa and THP-1 cells, and/or RNA interference. Interactions between TRP32 and host targets localized to the E. chaffeensis morulae or in the host cell cytoplasm adjacent to morulae. Common or closely related interacting partners of E. chaffeensis TRP32, TRP47, and TRP120 demonstrate a molecular convergence on common cellular processes and molecular cross talk between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that promote intracellular survival.

<h4>Purpose</h4>This 3-year, prospective, multicenter trial assessed the safety and efficacy of deferasirox in low- or intermediate-1-risk myelodysplastic syndrome (MDS).<h4>Patients and methods</h4>Eligible patients had serum ferritin ≥ 1,000 μg/L and had received ≥ 20 units of RBCs with ongoing transfusion requirements. The starting dose of deferasirox was 20 mg/kg/d, with dose escalation up to 40 mg/kg/d permitted.<h4>Results</h4>A total of 176 patients were enrolled, and 173 patients received therapy. Median serum ferritin decreased 23% in the 53% of patients who completed 12 months of treatment (n = 91), 36.7% in patients who completed 2 years (n = 49), and 36.5% in patients who completed 3 years (n = 33) despite continued transfusion requirement. Reduction in serum ferritin significantly correlated with ALT improvement (P < .001). Labile plasma iron (LPI) was measured quarterly during the first year of the study. Sixty-eight patients (39.3%) had elevated LPI at baseline. By week 13, LPI levels normalized in all patients with abnormal baseline level. Fifty-one (28%) of 173 patients experienced hematologic improvement by International Working Group 2006 criteria; of these, only seven patients received growth factors or MDS therapy. Over the 3-year study, 138 (79.8%) of 173 patients discontinued therapy, 43 patients (24.8%) because of adverse events or disease progression and 23 patients (13.2%) because of abnormal laboratory values. The most common drug-related adverse events were gastrointestinal disturbances and increased serum creatinine. There were 28 deaths, none of which were considered related to deferasirox.<h4>Conclusion</h4>Deferasirox reduces serum ferritin and LPI in transfusion-dependent patients with MDS. A subset of patients had an improvement in hematologic and hepatic parameters.

The microRNA miR-519 robustly inhibits cell proliferation, in turn triggering senescence and decreasing tumor growth. However, the molecular mediators of miR-519-elicited growth inhibition are unknown. Here, we systematically investigated the influence of miR-519 on gene expression profiles leading to growth cessation in HeLa human cervical carcinoma cells. By analyzing miR-519-triggered changes in protein and mRNA expression patterns and by identifying mRNAs associated with biotinylated miR-519, we uncovered two prominent subsets of miR-519-regulated mRNAs. One subset of miR-519 target mRNAs encoded DNA maintenance proteins (including DUT1, EXO1, RPA2, and POLE4); miR-519 repressed their expression and increased DNA damage, in turn raising the levels of the cyclin-dependent kinase (cdk) inhibitor p21. The other subset of miR-519 target mRNAs encoded proteins that control intracellular calcium levels (notably, ATP2C1 and ORAI1); their downregulation by miR-519 aberrantly elevated levels of cytosolic [Ca(2+)] storage in HeLa cells, similarly increasing p21 levels in a manner dependent on the Ca(2+)-activated kinases CaMKII and GSK3β. The rises in levels of DNA damage, the Ca(2+) concentration, and p21 levels stimulated an autophagic phenotype in HeLa and other human carcinoma cell lines. As a consequence, ATP levels increased, and the level of activity of the AMP-activated protein kinase (AMPK) declined, further contributing to the elevation in the abundance of p21. Our results indicate that miR-519 promotes DNA damage, alters Ca(2+) homeostasis, and enhances energy production; together, these processes elevate the expression level of p21, promoting growth inhibition and cell survival.

By using surface plasmon resonance (SPR) technology, the kinetics of the interaction of various fungal endopolygalacturonases (EPGs) (13 EPGs) with Phaseolus vulgaris (bean) PGIP2 was carried out to determine whether or not there is any interaction between polygalacturonases-inhibiting protein (PGIP) and EPG. The effect of polygalacturonic acid (PGA) on these interactions was also evaluated. The results show that all EPGs evaluated bind to PGIP2, except for AnPGb and the strength of the interaction depends on the EPG/PGIP2 pairing. Further, the presence of PGA has a moderate to strong effect on the EPG/PGIP2 interaction and the strength of the effect is dependent on the exact EPG/PGIP2 pairing. The differences in affinity in the absence and presence of PGA, suggest a certain level of cooperativity. These results indicate a three-component complex similar to that observed for the heparin-ATIII-thrombin, the FGF-FGFR-heparin, or the hedgehog-interference hedgehog-heparan complexes. This data points to an architecture in which the inhibitor binds at a location distant from the substrate binding site. Furthermore, we applied differential proteolysis mass spectrometry (DPMS) to study the location of the binding site between EPG and PGIP2. DPMS studies indicate that PGIP2 does not bind AnPGII, AnPGa, and AnPGc directly over the active site but instead binds on the face opposite to the active site, creating an allosteric interaction.

The extension of processes of oligodendrocyte (OLG) and their precursor cells are crucial for migration, axonal contact and myelination. Here we show that a non-lethal oxidative stress induced by 3-nitropropionic acid (3-NP) elicited a rapid shortening of processes (~24%) in primary OLGs and in oligodendroglial cell line (OLN-93) cells (~36%) as compared with vehicle-exposed cells. This was reversible and prevented by antioxidants. Proteomics of OLG lysates with and without 3-NP treatment yielded collapsin response mediator protein 2 (CRMP-2) as a candidate effector molecule. Inhibition of rho kinase was sufficient to prevent process retraction in both OLGs and OLN-93 cells. Oxidative stress increased phosphorylation of CRMP-2 at T555 that was completely prevented by Y27632. Moreover, transfection of OLN-93 cells with the mutant CRMP-2 T555A which cannot be phosphorylated by rho kinase, prevented process shortening induced by 3-NP as compared with wild-type CRMP-2. Our results suggest a role for endogenous reactive oxygen species in a pathway that regulates OLG process extension. The vulnerability of late myelinated neurons in the adult brain and the presence of white matter pathology in human dementias warrant the study of this oligodendroglial pathway in the early stages of neurodegenerative conditions characterized by oxidative stress.

The relation between cancer and coagulation is the subject of investigation since a relation between tumor and thrombosis has been determined. Antithrombin III is an important thrombin inhibitor, and increased thrombin-antithrombin (TAT) complex levels activate coagulation. Activated thrombin activatable fibrinolysis inhibitor (TAFI) inhibits the conversion of plasminogen to plasmin. In addition, it directly inactivates plasmin. Defective fibrinolysis increases the risk of thrombosis. In this study, we evaluated homeostatic parameters, TAFI, and TAT levels in patients with gastric cancer applying to the medical oncology outpatient clinic. Fifty-two patients and 35 healthy controls were included. ELISA was used to measure TAFI and TAT complex levels. These were statistically higher in the patient group (p < 0.05 and p = 0.001, respectively). D-dimer levels were higher in stage IV (p = 0.05). Correlations between lymph nodes and TAFI and TAT levels were examined. Weak but positive correlation between lymph nodes and TAFI was detected (R = 0.452, p = 0.027). TAFI and TAT levels were evaluated using relative operating characteristic analysis to differentiate the disease. TAT was more specific than TAFI according to this analysis (TAFI area under curve (AUC), 0.676; TAT AUC, 0.874). Thrombotic events and bleeding disorders need to be borne in mind in gastric cancer. This situation is due to the impairment of the balance between coagulation and fibrinolysis. Further studies are now needed to evaluate the effects of TAFI and TAT on survey and prognosis as well as the potential of these parameters as tumor markers for gastric cancer.

<h4>Background</h4>Hemoglobin A1c (HbA1c) levels diagnose diabetes, predict mortality and are associated with ten single nucleotide polymorphisms (SNPs) in white individuals. Genetic associations in other race groups are not known. We tested the hypotheses that there is race-ethnic variation in 1) HbA1c-associated risk allele frequencies (RAFs) for SNPs near SPTA1, HFE, ANK1, HK1, ATP11A, FN3K, TMPRSS6, G6PC2, GCK, MTNR1B; 2) association of SNPs with HbA1c and 3) association of SNPs with mortality.<h4>Methods</h4>We studied 3,041 non-diabetic individuals in the NHANES (National Health and Nutrition Examination Survey) III. We stratified the analysis by race/ethnicity (NHW: non-Hispanic white; NHB: non-Hispanic black; MA: Mexican American) to calculate RAF, calculated a genotype score by adding risk SNPs, and tested associations with SNPs and the genotype score using an additive genetic model, with type 1 error = 0.05.<h4>Results</h4>RAFs varied widely and at six loci race-ethnic differences in RAF were significant (p < 0.0002), with NHB usually the most divergent. For instance, at ATP11A, the SNP RAF was 54% in NHB, 18% in MA and 14% in NHW (p < .0001). The mean genotype score differed by race-ethnicity (NHW: 10.4, NHB: 11.0, MA: 10.7, p < .0001), and was associated with increase in HbA1c in NHW (β = 0.012 HbA1c increase per risk allele, p = 0.04) and MA (β = 0.021, p = 0.005) but not NHB (β = 0.007, p = 0.39). The genotype score was not associated with mortality in any group (NHW: OR (per risk allele increase in mortality) = 1.07, p = 0.09; NHB: OR = 1.04, p = 0.39; MA: OR = 1.03, p = 0.71).<h4>Conclusion</h4>At many HbA1c loci in NHANES III there is substantial RAF race-ethnic heterogeneity. The combined impact of common HbA1c-associated variants on HbA1c levels varied by race-ethnicity, but did not influence mortality.

<h4>Introduction</h4>We aimed to investigate whether the effect size of the systemic lupus erythematosus (SLE) risk alleles varies across European subpopulations.<h4>Methods</h4>European SLE patients (n = 1,742) and ethnically matched healthy controls (n = 2,101) were recruited at 17 centres from 10 different countries. Only individuals with self-reported ancestry from the country of origin were included. In addition, participants were genotyped for top ancestry informative markers and for 25 SLE associated SNPs. The results were used to compare effect sizes between the Central Eureopan and Southern European subgroups.<h4>Results</h4>Twenty of the 25 SNPs showed independent association with SLE, These SNPs showed a significant bias to larger effect sizes in the Southern subgroup, with 15/20 showing this trend (P = 0.019) and a larger mean odds ratio of the 20 SNPs (1.46 vs. 1.34, P = 0.02) as well as a larger difference in the number of risk alleles (2.06 vs. 1.63, P = 0.027) between SLE patients and controls than for Central Europeans. This bias was reflected in a very significant difference in the cumulative genetic risk score (4.31 vs. 3.48, P = 1.8 × 10-32). Effect size bias was accompanied by a lower number of SLE risk alleles in the Southern subjects, both patients and controls, the difference being more marked between the controls (P = 1.1 × 10-8) than between the Southern and Central European patients (P = 0.016). Seven of these SNPs showed significant allele frequency clines.<h4>Conclusion</h4>Our findings showed a bias to larger effect sizes of SLE loci in the Southern Europeans relative to the Central Europeans together with clines of SLE risk allele frequencies. These results indicate the need to study risk allele clines and the implications of the polygenic model of inheritance in SLE.

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.

Monocyte-derived cells display highly variable cytokine secretion upon pattern recognition receptor (PRR) stimulation across individuals; such variability likely affects interindividual inflammatory/autoimmune disease susceptibility. To define mechanisms for this heterogeneity, we examined PRR-induced monocyte-derived cell cytokine secretion from a large cohort of healthy individuals. Although cytokine secretion ranged widely among individuals, the magnitude of cytokine induction after individual nucleotide-binding oligomerization domain 2 (Nod2) and TLR2 stimulation (a cohort of 86 individuals) or stimulation of multiple TLRs (a cohort of 77 individuals), either alone or in combination with Nod2, was consistent intraindividually across these stimuli. Nod2 and TLRs signal through IFN regulatory factor 5 (IRF5), and common IRF5 polymorphisms confer risk for autoimmunity. We find that cells from rs2004640 IRF5 risk-associated allele carriers secrete increased cytokines upon individual or synergistic PRR stimulation in a gene dose- and ligand dose-dependent manner in both monocyte-derived dendritic cells and monocyte-derived macrophages. IRF5 expression knockdown in IRF5 risk allele carrier cells significantly decreases PRR-induced cytokines. Moreover, we find that IRF5 knockdown profoundly decreases Nod2-mediated MAPK and NF-κB pathway activation, whereas the PI3K and mammalian target of rapamycin pathways are not impaired. Finally, the IRF5 rs2004640 polymorphism is a major determinant of the variance (r(2) = 0.53) in Nod2-induced cytokine secretion by monocyte-derived cells from different individuals. We therefore show a profound contribution of a single gene to the variance in interindividual PRR-induced cytokines. The hyperresponsiveness of IRF5 disease-associated polymorphisms to a wide spectrum of microbial triggers has broad implications on global immunological responses, host defenses against pathogens, and inflammatory/autoimmune disease susceptibility.

<h4>Background</h4>Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown.<h4>Methods/findings</h4>In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K(D) for different ligands. Native mass spectrometry was used as an alternative method for measuring K(D) values.<h4>Conclusions/significance</h4>Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

Rotavirus is the leading cause of acute gastroenteritis in worldwide young children. Effective vaccines to prevent rotavirus infection are currently available, although their clinical use is still limited, and rotavirus still causes many episodes of infantile gastroenteritis, mainly during the winter season. The aim of this study was to evaluate the prevalence of rotavirus infection in children aged <5-years-old who were hospitalised for gastroenteritis. One hundred and sixty-three stool samples from hospitalised children (<5-years-old) complicated with severe diarrhoea, in two hospitals in Jahrom City, Iran were collected from 2009 to 2010. Antigenic prevalence of rotavirus group A was distinguished by enzyme immunoassay. The antigen of group A rotavirus was diagnosed by EIA in 75 of 163 collected samples. The genotype of EIA-positive samples was determined by nested RT-PCR. The frequency of rotavirus genotypes G1, G2, G3, G4 and G9 was 17.33, 13.34, 2.67, 30.66 and 2.67 %, respectively. Also, the frequency of mixed and non-typable genotypes was detected in 2.67 and 30.66 %, respectively. G1/G8 mixed infection was the first of these rotavirus genotypes to be reported in Iran. Detection of high prevalence of group A rotavirus infection in hospitalised children with diarrhoea, and determination of circulating rotavirus genotypes in this region of Iran, provide useful data for formulating effective vaccines; especially for infants less than 5-years-old.

<h4>Background</h4>Hereditary angioedema (HAE) is a rare but serious disease marked by swelling attacks in the extremities, face, trunk, airway, or abdominal areas that can be spontaneous or the result of trauma and other triggers. It can be life-threatening due to the risk of asphyxiation. While there have been major advancements in our understanding of the immunogenetics of HAE, there are significant gaps in the literature regarding understanding of the humanistic and economic impact of the disease, particularly in Europe. The purpose of the HAE Burden of Illness Study-Europe (HAE-BOIS-Europe), the development and methodology of which is described here, is to better understand the management and impact of HAE from the patient perspective in Europe.<h4>Methods/design</h4>This is a cross-sectional study in which retrospective data were also collected being conducted in Denmark, Germany and Spain. The study is open to patients ages 12 and older with a diagnosis of HAE-I or HAE-II. Data collection includes: (i) a survey on individuals' health care resource use, direct and indirect medical costs, impact on work and school, treatment satisfaction, and emotional functioning (via the Hospital Anxiety and Depression Scale); and (ii) one-on-one interviews to collect detailed descriptive data and patient testimonials on the impact of HAE on patients' health-related quality of life.<h4>Discussion</h4>The present manuscript describes the development and plans for implementing a multi-country European study with the aim of characterizing the humanistic and economic burden of HAE from the patient perspective. This study will help raise awareness of HAE as a rare but debilitating condition with wide-ranging impacts.

Ovarian cancer (OVCA) is the most lethal gynecological malignancy. The high mortality rate associated with this disease is due in large part to the development of resistance to chemotherapy; however, the biological basis of this remains unclear. Gemcitabine is frequently used for the treatment of patients with platinum-resistant OVCA. We report molecular signaling pathways associated with OVCA response to gemcitabine. Forty-one OVCA cell lines were subjected to gene expression analysis; in parallel, IC50 values for gemcitabine were quantified using CellTiter-Blue viability assays. Pearson's correlation coefficients were calculated for gene expression and gemcitabine IC50 values. The genes associated with gemcitabine sensitivity were subjected to pathway analysis. For the identified pathways, principal component analysis was used to derive pathway signatures and corresponding scores, which represent overall measures of pathway expression. Expression levels of the identified pathways were then evaluated in a series of clinico-genomic datasets from 142 patients with stage III/IV serous OVCA. We found that in vitro gemcitabine sensitivity was associated with expression of 131 genes (p<0.001). These genes include significant representation of three molecular signaling pathways (p<0.02): O-glycan biosynthesis, Role of Nek in cell cycle regulation and Antiviral actions of Interferons. In an external clinico-genomic OVCA dataset (n=142), expression of the O-glycan pathway was associated with overall survival, independent of surgical cytoreductive status, grade and age (p<0.001). Expression levels of Role of Nek in cell cycle regulation and Antiviral actions of Interferons were not associated with survival (p=0.31 and p=0.54, respectively). Collectively, expression of the O-glycan biosynthesis pathway, which modifies protein function via post-translational carbohydrate binding, is independently associated with overall survival from OVCA. Our findings shed light on the molecular basis of OVCA responsiveness to gemcitabine and also identify a signaling pathway that may influence patient survival.

Evidence for an interaction between alcohol consumption and the serotonin system has been observed repeatedly in both humans and animal models yet the specific relationship between the two remains unclear. Research has focused primarily on the serotonin transporter (SERT) due in part to its role in regulating extracellular levels of serotonin. The hippocampal formation is heavily innervated by ascending serotonin fibers and is a major component of the neurocircuitry involved in mediating the reinforcing effects of alcohol. The current study investigated the effects of chronic ethanol self-administration on hippocampal SERT in a layer and field specific manner using a monkey model of human alcohol consumption. [(3)H]Citalopram was used to measure hippocampal SERT density in male cynomolgus macaques that voluntarily self-administered ethanol for 18 months. Hippocampal [(3)H]citalopram binding was less dense in ethanol drinkers than in controls, with the greatest effect observed in the molecular layer of the dentate gyrus. SERT density was not correlated with measures of ethanol consumption or blood ethanol concentrations, suggesting the possibility that a threshold level of consumption had been met. The lower hippocampal SERT density observed suggests that chronic ethanol consumption is associated with altered serotonergic modulation of hippocampal neurotransmission.

There is growing evidence that genetic risk factors for common disease are caused by hereditary changes of gene regulation acting in complex pathways. Clearly understanding the molecular genetic relationships between genetic control of gene expression and its effect on complex diseases is essential. Here we describe the Brisbane Systems Genetics Study (BSGS), a family-based study that will be used to elucidate the genetic factors affecting gene expression and the role of gene regulation in mediating endophenotypes and complex diseases.BSGS comprises of a total of 962 individuals from 314 families, for which we have high-density genotype, gene expression and phenotypic data. Families consist of combinations of both monozygotic and dizygotic twin pairs, their siblings, and, for 72 families, both parents. A significant advantage of the inclusion of parents is improved power to disentangle environmental, additive genetic and non-additive genetic effects of gene expression and measured phenotypes. Furthermore, it allows for the estimation of parent-of-origin effects, something that has not previously been systematically investigated in human genetical genomics studies. Measured phenotypes available within the BSGS include blood phenotypes and biochemical traits measured from components of the tissue sample in which transcription levels are determined, providing an ideal test case for systems genetics approaches.We report results from an expression quantitative trait loci (eQTL) analysis using 862 individuals from BSGS to test for associations between expression levels of 17,926 probes and 528,509 SNP genotypes. At a study wide significance level approximately 15,000 associations were observed between expression levels and SNP genotypes. These associations corresponded to a total of 2,081 expression quantitative trait loci (eQTL) involving 1,503 probes. The majority of identified eQTL (87%) were located within cis-regions.

The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.

Eating Disorders are complex psychiatric problems that involve biologic and psychological factors. Brain imaging studies provide insights about how functionally connected brain networks may contribute to disturbed eating behavior, resulting in food refusal and altered body weight, but also body preoccupations and heightened anxiety. In this article, we review the current state of brain imaging in eating disorders, and how such techniques may help identify pathways that could be important in the treatment of those often detrimental disorders.

Renal injury induced by brain death is characterized by ischemia and inflammation, and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 h of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 h of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain-dead rats showed significant improvement in function over the first 2 to 4 days after transplantation compared with untreated brain-dead animals. Gene microarray analysis after 2 h of brain death without or with nitrite therapy showed that the latter significantly altered the expression of about 400 genes. Ingenuity Pathway Analysis indicated that multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription, and genes related to humoral immune responses. Thus, nitrite therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

Interaction of estrogen with iron at the systemic level is long suspected, but direct evidence linking the two is limited. In the present study, we examined the effects of 17β-estradiol (E2) on hepcidin, a key negative regulator of iron absorption from the liver. We found that transcription of hepcidin was suppressed by E2 treatment in human liver HuH7 and HepG2 cells, and this down-regulation was blocked by E2 antagonist ICI 182780. Chromatin immunoprecipitation, deletion, and EMSA detected a functional estrogen responsive element half-site that is located between -2474 and -2462 upstream from the start of transcription of the hepcidin gene. After cloning the human hepcidin promoter into the pGL3Luc-Reporter vector, luciferase activity was also down-regulated by E2 treatment in HepG2 cells. E2 reduced hepcidin mRNA in wild-type mice as well as in hemochromatosis Fe gene knockout mice. In summary, our data suggest that hepcidin inhibition by E2 is to increase iron uptake, a mechanism to compensate iron loss during menstruation. This mechanism may also contribute to increased iron stores in oral contraceptive users.

Recognition of RNA virus through TLR and RIG-I-like receptor results in rapid expression of type I IFNs, which play an essential role in host antiviral responses. However, the mechanisms to terminate the production of type I IFNs are not well defined. In the current study, we identified a member of the tripartite motif (TRIM) family, TRIM38, as a negative regulator in TLR3/4- and RIG-I-mediated IFN-β signaling. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of IFN regulatory factor 3 and enhanced expression of IFN-β, whereas overexpression of TRIM38 had opposite effects. Coimmunoprecipitation and colocalization experiments demonstrated that TRIM38 interacted with NF-κB-activating kinase-associated protein 1 (NAP1), which is required for TLR-induced IFN regulatory factor 3 activation and IFN-β production. As an E3 ligase, TRIM38 promoted K48-linked polyubiquitination and proteasomal degradation of NAP1. Thus, knockdown of TRIM38 expression resulted in higher protein level of NAP1 in primary macrophages. Consistent with the inhibitory roles in TLR3/4- and RIG-I-mediated IFN-β signaling, knockdown of TRIM38 significantly inhibited the replication of vesicular stomatitis virus. Overexpression of TRIM38 resulted in enhanced replication of vesicular stomatitis virus. Therefore, our results demonstrate that TRIM38 is a negative regulator for TLR and RIG-I-mediated IFN-β production by targeting NAP1 for ubiquitination and subsequent proteasome-mediated degradation.

We performed a genome-wide association study (GWAS) of antibody levels in a multi-ethnic group of 1071 healthy smallpox vaccine recipients. In Caucasians, the most prominent association was found with promoter SNP rs10489759 in the LOC647132 pseudogene on chromosome 1 (p=7.77×10(-8)). In African-Americans, we identified eight genetic loci at p<5×10(-7). The SNP association with the lowest p-value (rs10508727, p=1.05×10(-10)) was in the Mohawk homeobox (MKX) gene on chromosome 10. Other candidate genes included LOC388460, GPR158, ZHX2, SPIRE1, GREM2, CSMD1, and RUNX1. In Hispanics, the top six associations between genetic variants and antibody levels had p-values less than 5×10(-7), with p=1.78×10(-10) for the strongest statistical association (promoter SNP rs12256830 in the PCDH15 gene). In addition, SNP rs4748153 in the immune response gene PRKCQ (protein kinase C, theta) was significantly associated with neutralizing antibody levels (p=2.51×10(-8)). Additional SNP associations in Hispanics (p≤3.40×10(-7)) were mapped to the KIF6/LOC100131899, CYP2C9, and ANKLE2/GOLGA3 genes. This study has identified candidate SNPs that may be important in regulating humoral immunity to smallpox vaccination. Replication studies, as well as studies elucidating the functional consequences of contributing genes and polymorphisms, are underway.

Cilia are cell organelles that play important roles in cell motility, sensory and developmental functions and are involved in a range of human diseases, known as ciliopathies. Here, we search for novel human genes related to cilia using a strategy that exploits the previously reported tendency of cell type-specific genes to be coexpressed in the transcriptome of complex tissues. Gene coexpression networks were constructed using the noise-resistant WGCNA algorithm in 12 publicly available microarray datasets from human tissues rich in motile cilia: airways, fallopian tubes and brain. A cilia-related coexpression module was detected in 10 out of the 12 datasets. A consensus analysis of this module's gene composition recapitulated 297 known and predicted 74 novel cilia-related genes. 82% of the novel candidates were supported by tissue-specificity expression data from GEO and/or proteomic data from the Human Protein Atlas. The novel findings included a set of genes (DCDC2, DYX1C1, KIAA0319) related to a neurological disease dyslexia suggesting their potential involvement in ciliary functions. Furthermore, we searched for differences in gene composition of the ciliary module between the tissues. A multidrug-and-toxin extrusion transporter MATE2 (SLC47A2) was found as a brain-specific central gene in the ciliary module. We confirm the localization of MATE2 in cilia by immunofluorescence staining using MDCK cells as a model. While MATE2 has previously gained attention as a pharmacologically relevant transporter, its potential relation to cilia is suggested for the first time. Taken together, our large-scale analysis of gene coexpression networks identifies novel genes related to human cell cilia.

Apathy, characterized by generally reduced interest in and likelihood to perform goal-directed actions, is a recognized symptom of Huntington's disease (HD), a devastating neurological disorder caused by a CAG repeat expansion of the Htt gene located on chromosome 4. The present experiments used a modified progressive ratio task that incorporated a fixed-ratio schedule of reinforcement component to assess consummatory behavior, and a progressive-ratio schedule component that required increasing numbers of lever-presses for successive reinforcers (0.01 ml of evaporated milk). The studies revealed an apathetic phenotype in two mouse models of HD, with decreased response rates either overall or only at higher ratio requirements in the progressive-ratio component relative to wild-type controls. Based on the procedure used (within-session fixed- and progressive-ratio components), it is proposed that an observed phenotype can be ascribed either specifically to reduced motivation to work for food reinforcement or more generally to deficits in consummatory behavior. This procedure provides a simple means to assess this type of phenotype in rodents, with issues in consummatory vs. incentive motivation reflected in general alterations in fixed- versus progressive alterations on an escalating-ratio schedules respectively, providing translational measures of the amotivation/apathy construct of the human realm to the homologous construct of incentive motivation in preclinical models of human disease.

This work investigated the ability of electropolished Ti surface to induce Hydroxyapatite (HA) nucleation and growth in vitro via a biomimetic method in Simulated Body Fluid (SBF). The HA induction ability of Ti surface upon electropolishing was compared to that of Ti substrates modified with common chemical methods including alkali, acidic and hydrogen peroxide treatments. Our results revealed the excellent ability of electropolished Ti surfaces in inducing the formation of bone-like HA at the Ti/SBF interface. The chemical composition, crystallinity and thickness of the HA coating obtained on the electropolished Ti surface was found to be comparable to that achieved on the surface of alkali treated Ti substrate, one of the most effective and popular chemical treatments. The surface characteristics of electropolished Ti contributing to HA growth were discussed thoroughly.

<h4>Introduction</h4>CD226 genetic variants have been associated with a number of autoimmune diseases and recently with systemic sclerosis (SSc). The aim of this study was to test the influence of CD226 loci in SSc susceptibility, clinical phenotypes and autoantibody status in a large multicenter European population.<h4>Methods</h4>A total of seven European populations of Caucasian ancestry were included, comprising 2,131 patients with SSc and 3,966 healthy controls. Three CD226 single nucleotide polymorphisms (SNPs), rs763361, rs3479968 and rs727088, were genotyped using Taqman 5'allelic discrimination assays.<h4>Results</h4>Pooled analyses showed no evidence of association of the three SNPs, neither with the global disease nor with the analyzed subphenotypes. However, haplotype block analysis revealed a significant association for the TCG haplotype (SNP order: rs763361, rs34794968, rs727088) with lung fibrosis positive patients (PBonf = 3.18E-02 OR 1.27 (1.05 to 1.54)).<h4>Conclusion</h4>Our data suggest that the tested genetic variants do not individually influence SSc susceptibility but a CD226 three-variant haplotype is related with genetic predisposition to SSc-related pulmonary fibrosis.

The isolation of a single type of protein from a complex mixture is vital for the characterization of the function, structure, and interactions of the protein of interest and is typically the most laborious aspect of the protein purification process. In this work, a model system is utilized to show the efficacy of synthesizing a "baited" nanoparticle to capture and recycle enzymes (proteins that catalyze chemical reactions) from crude cell lysate. Enzyme trapping and recycling is illustrated with the carbazole 1,9a-dioxygenase (CARDO) system, an enzyme important in bioremediation and natural product synthesis. The enzymes are baited with azide-modified carbazolyl moieties attached to poly(propargyl acrylate) nanoparticles through a click transformation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicates the single-step procedure to immobilize the enzymes on the particles is capable of significantly concentrating the protein from raw lysate and sequestering all required components of the protein to maintain bioactivity. These results establish a universal model applicable to concentrating and extracting known substrate-protein pairs, but it can be an invaluable tool in recognizing unknown protein-ligand affinities.

Canonical Wnt signaling strongly inhibits chondrogenesis. Previously, we identified Twist1 as a critical downstream mediator of Wnt in repression of chondrocyte differentiation. However, the mechanistic basis for the antichondrogenic activity of Twist1 has not heretofore been established. Here, we show that Twist1 suppresses cartilage development by directly inhibiting the transcriptional activity of Sox9, the master regulator of chondrogenesis. Twist1, through its carboxyl-terminal Twist-box, binds to the Sox9 high mobility group DNA-binding domain, inhibiting Sox9 transactivation potential. In chondrocyte precursor cells, Twist1, in a Twist-box-dependent manner, inhibits Sox9-dependent activation of chondrocyte marker gene expression by blocking Sox9-enhancer DNA association. These findings identify Twist1 as an inhibitor of Sox9 and further suggest that the balance between Twist1 and Sox9 may determine the earliest steps of chondrogenesis.

While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here we compared the gene expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA-CA/04 was associated with the following: (i) an early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased NF-κB activation, as well as inhibition of the negative regulator TRIM24, (ii) early and persistent infiltration of immune cells, including inflammatory macrophages, and (iii) the absence of activation of lipid metabolism later in infection, which may be mediated by inhibition of nuclear receptors, including PPARG and HNF1A and -4A, with proinflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of various pathogenicities confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following infection with lethal H1N1 r1918 influenza virus. This study links differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.

Neuropeptide B/W receptor-1 (NPBWR1) is expressed in discrete brain regions in rodents and humans, with particularly strong expression in the limbic system, including the central nucleus of the amygdala. Recently, Nagata-Kuroiwa et al. reported that Npbwr1(-/-) mice showed changes in social behavior, suggesting that NPBWR1 plays important roles in the emotional responses of social interactions.The human NPBWR1 gene has a single nucleotide polymorphism at nucleotide 404 (404A>T; SNP rs33977775). This polymorphism results in an amino acid change, Y135F. The results of an in vitro experiment demonstrated that this change alters receptor function. We investigated the effect of this variation on emotional responses to stimuli of showing human faces with four categories of emotional expressions (anger, fear, happiness, and neutral). Subjects' emotional levels on seeing these faces were rated on scales of hedonic valence, emotional arousal, and dominance (V-A-D). A significant genotype difference was observed in valence evaluation; the 404AT group perceived facial expressions more pleasantly than did the 404AA group, regardless of the category of facial expression. Statistical analysis of each combination of [V-A-D and facial expression] also showed that the 404AT group tended to feel less submissive to an angry face than did the 404AA group. Thus, a single nucleotide polymorphism of NPBWR1 seems to affect human behavior in a social context.

<h4>Background</h4>The main goal of the present study was to analyse the genetic architecture of mRNA expression in muscle, a tissue with an outmost economic importance for pig breeders. Previous studies have used F(2) crosses to detect porcine expression QTL (eQTL), so they contributed with data that mostly represents the between-breed component of eQTL variation. Herewith, we have analysed eQTL segregation in an outbred Duroc population using two groups of animals with divergent fatness profiles. This approach is particularly suitable to analyse the within-breed component of eQTL variation, with a special emphasis on loci involved in lipid metabolism.<h4>Methodology/principal findings</h4>GeneChip Porcine Genome arrays (Affymetrix) were used to determine the mRNA expression levels of gluteus medius samples from 105 Duroc barrows. A whole-genome eQTL scan was carried out with a panel of 116 microsatellites. Results allowed us to detect 613 genome-wide significant eQTL unevenly distributed across the pig genome. A clear predominance of trans- over cis-eQTL, was observed. Moreover, 11 trans-regulatory hotspots affecting the expression levels of four to 16 genes were identified. A Gene Ontology study showed that regulatory polymorphisms affected the expression of muscle development and lipid metabolism genes. A number of positional concordances between eQTL and lipid trait QTL were also found, whereas limited evidence of a linear relationship between muscle fat deposition and mRNA levels of eQTL regulated genes was obtained.<h4>Conclusions/significance</h4>Our data provide substantial evidence that there is a remarkable amount of within-breed genetic variation affecting muscle mRNA expression. Most of this variation acts in trans and influences biological processes related with muscle development, lipid deposition and energy balance. The identification of the underlying causal mutations and the ascertainment of their effects on phenotypes would allow gaining a fundamental perspective about how complex traits are built at the molecular level.

Mechanistically unrelated developmental neurotoxicants often produce neural cell loss culminating in similar functional and behavioral outcomes. We compared an organophosphate pesticide (diazinon), an organochlorine pesticide (dieldrin) and a metal (Ni(2+)) for effects on the genes regulating cell cycle and apoptosis in differentiating PC12 cells, an in vitro model of neuronal development. Each agent was introduced at 30μM for 24 or 72h, treatments devoid of cytotoxicity. Using microarrays, we examined the mRNAs encoding nearly 400 genes involved in each of the biological processes. All three agents targeted both the cell cycle and apoptosis pathways, evidenced by significant transcriptional changes in 40-45% of the cell cycle-related genes and 30-40% of the apoptosis-related genes. There was also a high degree of overlap as to which specific genes were affected by the diverse agents, with 80 cell cycle genes and 56 apoptosis genes common to all three. Concordance analysis, which assesses stringent matching of the direction, magnitude and timing of the transcriptional changes, showed highly significant correlations for pairwise comparisons of all the agents, for both cell cycle and apoptosis. Our results show that otherwise disparate developmental neurotoxicants converge on common cellular pathways governing the acquisition and programmed death of neural cells, providing a specific link to cell deficits. Our studies suggest that identifying the initial mechanism of action of a developmental neurotoxicant may be strategically less important than focusing on the pathways that converge on common final outcomes such as cell loss.

In this paper, hydroxyapatite-carbon nanotube/titania (HA-CNT/TiO(2)) double layer coatings were successfully developed on titanium (Ti) substrates intended for biomedical applications. A TiO(2) coating was firstly developed by anodization to improve bonding between HA and Ti, and then the layer of HA and CNTs was coated on the surface by the sol-gel process to improve the biocompatibility and mechanical properties of Ti. The surfaces of double layer coatings were uniform and crack-free with a thickness of about 7 μm. The bonding strength of the HA-CNT/TiO(2) coating was higher than that of the pure HA and HA-CNT coatings. Additionally, in vitro cell experiments showed that CNTs promoted the adhesion of preosteoblasts on the HA-CNT/TiO(2) double layer coatings. These unique surfaces combined with the osteoconductive properties of HA exhibited the excellent mechanical properties of CNTs. Therefore, the developed HA-CNT/TiO(2) coatings on Ti substrates might be a promising material for bone replacement.

<h4>Background</h4>Many shortcomings exist in the traditional methods of treating bone defects, such as donor tissue shortages for autografts and disease transmission for allografts. The objective of this study was to design a novel three-dimensional nanostructured bone substitute based on magnetically synthesized single-walled carbon nanotubes (SWCNT), biomimetic hydrothermally treated nanocrystalline hydroxyapatite, and a biocompatible hydrogel (chitosan). Both nanocrystalline hydroxyapatite and SWCNT have a biomimetic nanostructure, excellent osteoconductivity, and high potential to improve the load-bearing capacity of hydrogels.<h4>Methods</h4>Specifically, three-dimensional porous chitosan scaffolds with different concentrations of nanocrystalline hydroxyapatite and SWCNT were created to support the growth of human osteoblasts (bone-forming cells) using a lyophilization procedure. Two types of SWCNT were synthesized in an arc discharge with a magnetic field (B-SWCNT) and without a magnetic field (N-SWCNT) for improving bone regeneration.<h4>Results</h4>Nanocomposites containing magnetically synthesized B-SWCNT had superior cytocompatibility properties when compared with nonmagnetically synthesized N-SWCNT. B-SWCNT have much smaller diameters and are twice as long as their nonmagnetically prepared counterparts, indicating that the dimensions of carbon nanotubes can have a substantial effect on osteoblast attachment.<h4>Conclusion</h4>This study demonstrated that a chitosan nanocomposite with both B-SWCNT and 20% nanocrystalline hydroxyapatite could achieve a higher osteoblast density when compared with the other experimental groups, thus making this nanocomposite promising for further exploration for bone regeneration.

Epididymal tumour incidence is at most 0.03% of all male cancers. It is an enigma why the human epididymis does not often succumb to cancer, when it expresses markers of stem and cancer cells, and constitutively expresses oncogenes, pro-proliferative and pro-angiogenic factors that allow tumour cells to escape immunosurveillance in cancer-prone tissues. The privileged position of the human epididymis in evading tumourigenicity is reflected in transgenic mouse models in which induction of tumours in other organs is not accompanied by epididymal neoplasia. The epididymis appears to: (i) prevent tumour initiation (it probably lacks stem cells and has strong anti-oxidative mechanisms, active tumour suppressors and inactive oncogene products); (ii) foster tumour monitoring and destruction (by strong immuno-surveillance and -eradication, and cellular senescence); (iii) avert proliferation and angiogenesis (with persistent tight junctions, the presence of anti-angiogenic factors and misplaced pro-angiogenic factors), which together (iv) promote dormancy and restrict dividing cells to hyperplasia. Epididymal cells may be rendered non-responsive to oncogenic stimuli by the constitutive expression of factors generally inducible in tumours, and resistant to the normal epididymal environment, which mimics that of a tumour niche promoting tumour growth. The threshold for tumour initiation may thus be higher in the epididymis than in other organs. Several anti-tumour mechanisms are those that maintain spermatozoa quiescent and immunologically silent, so the low incidence of cancer in the epididymis may be a consequence of its role in sperm maturation and storage. Understanding these mechanisms may throw light on cancer prevention and therapy in general.

The demographic and social changes of the past decades have determined improvements in public health and longevity. So, the number of centenarians is increasing as a worldwide phenomenon. Scientists have focused their attention on centenarians as optimal model to address the biological mechanisms of "successful and unsuccessful ageing". They are equipped to reach the extreme limits of human life span and, most importantly, to show relatively good health, being able to perform their routine daily life and to escape fatal age-related diseases, such as cardiovascular diseases and cancer. Thus, particular attention has been centered on their genetic background and immune system. In this review, we report our data gathered for over 10 years in Sicilian centenarians. Based on results obtained, we suggest longevity as the result of an optimal performance of immune system and an over-expression of anti-inflammatory sequence variants of immune/inflammatory genes. However, as well known, genetic, epigenetic, stochastic and environmental factors seem to have a crucial role in ageing and longevity. Epigenetics is associated with ageing, as demonstrated in many studies. In particular, ageing is associated with a global loss of methylation state. Thus, the aim of future studies will be to analyze the weight of epigenetic changes in ageing and longevity.

The ability to quantitatively evaluate the impact of a potential therapeutic intervention for Huntington disease (HD) in animal models for the disease is a critical step in the pathway to development of an effective therapy for this devastating neurodegenerative disorder. We report here an approach that combines a cell-based assay's quantitative accuracy and direct relationship to molecular processes with the ability to directly monitor effects in HD model mouse neurons. To accomplish this goal, we have developed an accurate quantitative reporter assay for a transcript known to be down-regulated as an early consequence of mutant huntingtin expression. This system uses mouse strains carrying a GFP reporter for the expression of the dopamine receptor D2, expressed in the medium spiny neurons of the basal ganglion. This receptor consistently demonstrates reduced expression in patients and murine models, and the FACS-based assay gives a highly accurate and quantitative readout of this pathology in mouse neurons expressing mutant huntingtin. For four genetic models and one viral model, a highly reproducible time course of loss of reporter expression is observed. This quantitative measure of HD pathology can be used to measure the effects of HD therapeutics in small cohorts with high confidence. We further demonstrate that the introduction of an shRNA against the huntingtin transgene by virus can improve this pathological status in medium spiny neurons transduced with the construct. We believe this system can be of great utility in the validation of effective therapeutic interventions for HD.

<h4>Background</h4>Pneumococcal infections cause major morbidity and mortality in developing countries. We report the epidemiology of S. pneumoniae carriage in a developing region, the Gaza strip, and evaluate the theoretical coverage of carriage strains by pneumococcal conjugate vaccines (PCVs).<h4>Methodology</h4>In 2009 we conducted a cross-sectional survey of S. pneumoniae carriage in healthy children and their parents, living throughout the Gaza strip. Data were collected and nasopharyngeal swabs were obtained. Antibiotic susceptibilities were determined by Vitek-2 and serotypes by the Quellung reaction.<h4>Principal findings</h4>S. pneumoniae carriage was detected in 189/379 (50%) of children and 30/376 (8%) of parents. Carriage prevalence was highest in children <6 months of age (63%). Significant predictors for child carriage were number of household members and DCC attendance. The proportion of pediatric and adults isolates with serotypes included in PCV7 were 32% and 20% respectively, and 46% and 33% in PCV13 respectively. The most prominent non-vaccine serotypes (NVT) were 35B, 15B/C and 23B. Penicillin-nonsusceptible strains were carried by 70% of carriers, penicillin-resistant strains (PRSP) by 13% and Multi-drug-resistant (MDR) by 30%. Of all PRSP isolates 54% belonged to serotypes included in PCV7 and 71% in the PCV13. Similarly, 59% and 73% of MDR-SP isolates, would theoretically be covered by PCV7 and PCV13, respectively.<h4>Conclusions</h4>This study demonstrates that, PCV13-included strains were carried by 46% and 33% of pediatric and adult subjects respectively. In the absence of definitive data regarding the virulence of the NVT strains, it is difficult to predict the effect of PCVs on IPD in this region.

The Metabochip is a custom genotyping array designed for replication and fine mapping of metabolic, cardiovascular, and anthropometric trait loci and includes low frequency variation content identified from the 1000 Genomes Project. It has 196,725 SNPs concentrated in 257 genomic regions. We evaluated the Metabochip in 5,863 African Americans; 89% of all SNPs passed rigorous quality control with a call rate of 99.9%. Two examples illustrate the value of fine mapping with the Metabochip in African-ancestry populations. At CELSR2/PSRC1/SORT1, we found the strongest associated SNP for LDL-C to be rs12740374 (p = 3.5 × 10(-11)), a SNP indistinguishable from multiple SNPs in European ancestry samples due to high correlation. Its distinct signal supports functional studies elsewhere suggesting a causal role in LDL-C. At CETP we found rs17231520, with risk allele frequency 0.07 in African Americans, to be associated with HDL-C (p = 7.2 × 10(-36)). This variant is very rare in Europeans and not tagged in common GWAS arrays, but was identified as associated with HDL-C in African Americans in a single-gene study. Our results, one narrowing the risk interval and the other revealing an associated variant not found in Europeans, demonstrate the advantages of high-density genotyping of common and rare variation for fine mapping of trait loci in African American samples.

The association between endometriosis and malignant transformation has often been described in the medical literature. A search was conducted between 1966 and 2010 through the English language literature (online Medline PubMed database) using the keywords endometriosis combined with malignant transformation. The search revealed an increase in reports describing endometriosis and malignancy. Approximately 1.0% of women with endometriosis have lesions that undergo malignant transformation. The malignant processes that are associated with endometriosis may be classified into three groups: i) epithelial ovarian cancers (endometrioid adenocarcinoma and clear cell carcinoma), ii) other Müllerian-type tumors, including Müllerian-type mucinous borderline tumor and serous borderline tumor and iii) sarcomas such as adenosarcoma and endometrial stromal sarcoma in the female pelvic cavity. Persistent oxidative stress induced by endometriosis-dependent hemorrhage may be associated with carcinogenesis. In conclusion, the malignant transformation of endometriosis has multiple pathways of development and may share a common pathogenic mechanism; iron-induced oxidative stress derived from repeated hemorrhage.

<h4>Objective</h4>To define altered gene expression networks in endometriosis.<h4>Design</h4>Experiments using endometriotic tissues and primary cells.<h4>Setting</h4>Division of Reproductive Biology Research, Northwestern University.<h4>Patient(s)</h4>Premenopausal women.<h4>Intervention(s)</h4>Matched samples of eutopic endometrium and ovarian endometriosis (n = 8 patients) were analyzed by microarray and verified in a separate set of tissues (n = 6 patients). Experiments to define signaling pathways were performed in primary endometriotic stromal cells (n = 12 patients).<h4>Main outcomes measure(s)</h4>Using a genome-wide in vivo approach, we identified 1,366 differentially expressed genes and a new gene network favoring increased glucocorticoid levels and action in endometriosis.<h4>Result(s)</h4>Transcript and protein levels of 11β-hydroxysteroid dehydrogenase (HSD11B1), which produces cortisol, the biologically active glucocorticoid, were strikingly higher, whereas messenger RNA (mRNA) levels of the cortisol-degrading HSD11B2 enzyme were significantly lower in endometriotic tissue. Glucocorticoid receptor mRNA and protein levels were significantly higher in endometriosis. The inflammatory cytokine tumor necrosis factor robustly induced mRNA and protein levels of HSD11B1 and glucocorticoid receptor but suppressed HSD11B2 mRNA in primary endometriotic stromal cells, suggesting that tumor necrosis factor stimulates cortisol production and action. We also uncovered a subset of genes critical for prostaglandin synthesis and degradation, which favor high eicosanoid levels and activity in endometriosis.<h4>Conclusion(s)</h4>The proinflammatory milieu of the endometriotic lesion stimulates cortisol synthesis and action in endometriotic lesions.

Cells precisely regulate mitochondrial movement in order to balance energy needs and avoid cell death. Neurons are particularly susceptible to disturbance of mitochondrial motility and distribution due to their highly extended structures and specialized function. Regulation of mitochondrial motility plays a vital role in neuronal health and death. Here we review the current understanding of regulatory mechanisms that govern neuronal mitochondrial transport and probe their implication in health and disease. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.

Alzheimer's disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.

Huntington's disease (HD) is a neurodegenerative disease characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and caused by a polyglutamine expansion in the huntingtin protein. There is so far neither cure nor approved disease-slowing therapy for HD, though recent clinical studies have shown a beneficial long-term effect of pridopidine in patients with HD. The nature of this effect, purely symptomatic or, in addition, neuroprotective, is difficult to elucidate in clinical trials. Pridopidine and (-)-OSU6162 are members of a new family of compounds referred to as dopaminergic stabilizers, which normalize abnormal dopamine neurotransmission. We investigated the effects of (-)-OSU6162 on huntingtin knocked-in striatal neurons in culture. Control neurons had normal full-length huntingtin with 7 glutamines while "mutant" neurons had large expansions (Q=111). We studied the dose-effect curves of (-)-OSU6162 on mitochondrial activity, LDH levels, necrosis and apoptosis in untreated Q7 and Q111 cells. In addition, we investigated the effects of (-)-OSU6162 on Q7 and Q111 neurons challenged with different neurotoxins such as sodium glutamate, H(2)O(2), rotenone and 3-nitropropionic acid (3NP). As we found prevention of toxicity of some of these neurotoxins, we investigated the putative neuroprotective mechanisms of action of (-)-OSU6162 measuring the effects of this dopaminergic stabilizer on expression and release of BDNF, the ratios of Bcl2/Bax proteins and of p-ERK/ERK, the levels of chaperones and GSH, and the effects of (-)-OSU6162 on dopamine uptake and release. We found that (-)-OSU6162, 3-150 μM, produces a dose dependent increase of mitochondrial activity and a reduction of cell death. (-)-OSU6162 does not change glutamate toxicity, but it partially prevents that of H(2)O(2), rotenone and 3-nitropropionic acid. (-)-OSU6162 increases the intracellular levels of BDNF and Bcl2/Bax and decreases those of p-ERK/ERK and CHIP in Q111 cells. (-)-OSU6162 increased (3)H-dopamine uptake and amphetamine-induced (3)H-dopamine release in E13 mouse mid brain neurons. Our studies demonstrate that (-)-OSU6162 improves survival and mitochondrial function in striatal Q111 neurons and the resistance of these cells to several striatal neurotoxins, suggesting that (-)-OSU6162 and related compounds should be tested for neuroprotection in animal models and, eventually, in patients with HD.

Cancer is a collection of very complex diseases that share many traits while differing in many ways as well. This makes a universal cure difficult to attain, and it highlights the importance of understanding each type of cancer at a molecular level. Although many strides have been made in identifying the genetic causes for some cancers, we now understand that simple changes in the primary DNA sequence cannot explain the many steps that are necessary to turn a normal cell into a rouge cancer cell. In recent years, some research has shifted to focusing on detailing epigenetic contributions to the development and progression of cancer. These changes occur apart from primary genomic sequences and include DNA methylation, histone modifications, and miRNA expression. Since these epigenetic modifications are reversible, drugs targeting epigenetic changes are becoming more common in clinical settings. Daily discoveries elucidating these complex epigenetic processes are leading to advances in the field of cancer research. These advances, however, come at a rapid and often overwhelming pace. This review specifically summarizes the main epigenetic mechanisms currently documented in solid tumors common in the United States and Europe.

Muscles are composed of multinucleated muscle fibers with different contractile and physiological properties, which result from specific slow or fast gene expression programs in the differentiated muscle cells. In the zebra fish embryo, the slow program is under the control of Hedgehog signaling from the notochord and floor plate. This pathway activates the expression of the conserved transcriptional repressor, Prdm1 (Blimp1), which in turn represses the fast program and promotes the slow program in adaxial cells of the somite and their descendants. In the mouse embryo, myogenesis is also initiated in the myotomal compartment of the somite, but the slow muscle program is not confined to a specific subset of cells. We now show that Prdm1 is expressed in the first differentiated myocytes of the early myotome from embryonic day (E)9.5-E11.5. During this period, muscle formation depends on the myogenic regulatory factors, Myf5 and Mrf4. In their absence, Prdm1 is not activated, in apparent contrast to zebra fish where Prdm1 is expressed in the absence of Myf5 and MyoD that drive myogenesis in adaxial cells. However, as in zebra fish, Prdm1 expression in the mouse myotome does not occur in the absence of Hedgehog signaling. Analysis of the muscle phenotype of Prdm1 mutant embryos shows that myogenesis appears to proceed normally. Notably, there is no requirement for Prdm1 activation of the slow muscle program in the mouse myotome. Furthermore, the gene for the transcriptional repressor, Sox6, which is repressed by Prdm1 to permit slow muscle differentiation in zebra fish, is not expressed in the mouse myotome. We propose that the lack of functional conservation for mouse Prdm1, that can nevertheless partially rescue the adaxial cells of zebra fish Prdm1 mutants, reflects differences in the evolution of the role of key regulators such as Prdm1 or Sox6, in initiating the onset of the slow muscle program, between teleosts and mammals.

DNA cleavage by the Type III Restriction-Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼ 200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can 'turnover', albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. 'DNA sliding').

In Huntington's disease (HD), the mutant huntingtin (mhtt) protein is associated with striatal dysfunction and degeneration. Excitotoxicity and early synaptic defects are attributed, in part, to altered NMDA receptor (NMDAR) trafficking and function. Deleterious extrasynaptic NMDAR localization and signalling are increased early in yeast artificial chromosome mice expressing full-length mhtt with 128 polyglutamine repeats (YAC128 mice). NMDAR trafficking at the plasma membrane is regulated by dephosphorylation of the NMDAR subunit GluN2B tyrosine 1472 (Y1472) residue by STriatal-Enriched protein tyrosine Phosphatase (STEP). NMDAR function is also regulated by calpain cleavage of the GluN2B C-terminus. Activation of both STEP and calpain is calcium-dependent, and disruption of calcium homeostasis occurs early in the HD striatum. Here, we show increased calpain cleavage of GluN2B at both synaptic and extrasynaptic sites, and elevated extrasynaptic total GluN2B expression in the YAC128 striatum. Calpain inhibition significantly reduced extrasynaptic GluN2B expression in the YAC128 but not wild-type striatum. Furthermore, calpain inhibition reduced whole-cell NMDAR current and the surface/internal GluN2B ratio in co-cultured striatal neurons, without affecting synaptic GluN2B localization. Synaptic STEP activity was also significantly higher in the YAC128 striatum, correlating with decreased GluN2B Y1472 phosphorylation. A substrate-trapping STEP protein (TAT-STEP C-S) significantly increased VGLUT1-GluN2B colocalization, as well as increasing synaptic GluN2B expression and Y1472 phosphorylation. Moreover, combined calpain inhibition and STEP inactivation reduced extrasynaptic, while increasing synaptic GluN2B expression in the YAC128 striatum. These results indicate that increased STEP and calpain activation contribute to altered NMDAR localization in an HD mouse model, suggesting new therapeutic targets for HD.

Mutations in PARK7/DJ-1 gene are associated to autosomal recessive early onset forms of Parkinson's disease (PD). Although large gene deletions have been linked to a loss-of-function phenotype, the pathogenic mechanism of missense mutations is less clear. The L166P mutation causes misfolding of DJ-1 protein and its degradation. L166P protein may also accumulate into insoluble cytoplasmic aggregates with a mechanism facilitated by the E3 ligase TNF receptor associated factor 6 (TRAF6). Upon proteasome impairment L166P activates the JNK/p38 MAPK apoptotic pathway by its interaction with TRAF and TNF Receptor Associated Protein (TTRAP). When proteasome activity is blocked in the presence of wild-type DJ-1, TTRAP forms aggregates that are localized to the cytoplasm or associated to nucleolar cavities, where it is required for a correct rRNA biogenesis. In this study we show that in post-mortem brains of sporadic PD patients TTRAP is associated to the nucleolus and to Lewy Bodies, cytoplasmic aggregates considered the hallmark of the disease. In SH-SY5Y neuroblastoma cells, misfolded mutant DJ-1 L166P alters rRNA biogenesis inhibiting TTRAP localization to the nucleolus and enhancing its recruitment into cytoplasmic aggregates with a mechanism that depends in part on TRAF6 activity. This work suggests that TTRAP plays a role in the molecular mechanisms of both sporadic and familial PD. Furthermore, it unveils the existence of an interplay between cytoplasmic and nucleolar aggregates that impacts rRNA biogenesis and involves TRAF6.

<h4>Background</h4>Our understanding of the molecular pathways that underlie melanoma remains incomplete. Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies. Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways.<h4>Methodology/principal findings</h4>Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules. Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients. The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis. An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study. Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active. Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro.<h4>Conclusions/significance</h4>This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis. Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response). The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets.

We previously showed that the -1131T→C polymorphism (rs662799) of the apolipoprotein A-V gene (APOA5) and the C→T polymorphism (rs6929846) of the butyrophilin, subfamily 2, member A1 gene (BTN2A1) were significantly associated with an increased serum concentration of triglycerides, a decreased serum concentration of high density lipoprotein (HDL)-cholesterol, and the prevalence of metabolic syndrome (MetS) in Japanese individuals. The purpose of the present study was to examine whether these polymorphisms synergistically affect the prevalence of dyslipidemia and MetS in East Asian populations. The study populations comprised 7471 Japanese and 3529 Korean individuals in the dyslipidemia study, and 3474 Japanese and 1671 Korean individuals in the MetS study. Multivariable logistic regression analysis of combined genotypes with adjustment for age, gender and diabetes mellitus revealed that rs662799 and rs6929846 significantly and synergistically affected dyslipidemia. Japanese or Korean individuals with the C allele of APOA5 and the T allele of BTN2A1 had a 2.05- or 1.92-fold increased risk for hypertriglyceridemia and a 1.82- or 1.56-fold increased risk for hypo-HDL-cholesterolemia, respectively, compared to those with the TT genotype of APOA5 and the CC genotype of BTN2A1. Similar analysis with adjustment for age and gender revealed that Japanese individuals, but not Korean individuals, with the C allele of APOA5 and the T allele of BTN2A1 had a 2.87-fold increased risk for MetS compared to those with the TT genotype of APOA5 and the CC genotype of BTN2A1. Genetic variants of APOA5 and BTN2A1 may synergistically affect the prevalence of dyslipidemia in East Asian populations and of MetS in Japanese individuals.

It has been suggested that xenoestrogens, a group of agents termed endocrine disruptors, may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. We previously demonstrated that the xenoestrogen, bisphenol A (BPA), was able to induce the transformation in vitro of human breast epithelial cells. The normal-like human breast epithelial cell line, MCF-10F, formed tubules in collagen (3-D cultures), although after treatment with BPA (10-5 M and 10-6 M BPA) the cells produced less tubules (73% and 80%, respectively) and some spherical masses (27% and 20%, respectively). In the present study, expression and DNA methylation analyses were performed in these cells after exposure to BPA. These cells showed an increased expression of BRCA1, BRCA2, BARD1, CtIP, RAD51 and BRCC3, all of which are genes involved in DNA repair, as well as the downregulation of PDCD5 and BCL2L11 (BIM), both of which are involved in apoptosis. Furthermore, DNA methylation analysis showed that the BPA exposure induced the hypermethylation of BCL2L11, PARD6G, FOXP1 and SFRS11, as well as the hypomethylation of NUP98 and CtIP (RBBP8). Our results indicate that normal human breast epithelial cells exposed to BPA have increased expressions of genes involved in DNA repair in order to overcome the DNA damage induced by this chemical. These results suggest that the breast tissue of women with BRCA1 or BRCA2 mutations could be more susceptible to the effects of BPA.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by the presence of telangiectasias and arteriovenous malformations in various organs and organ systems, including the liver. The most frequent clinical manifestation of HHT is epistaxis.In 2010 (January-December inclusive) 51 patients with HHT were admitted to the ENT ward of our hospital for epistaxis, and in accordance with routine protocols, all 51 underwent abdominal ultrasonography in our department to detect hepatovascular lesions. They included 27 males (53%) and 24 (47%) females ranging in age from 11 to 86 years (mean 48.5 years). The sample was selected in an arbitrary manner to take maximum advantage of the hospital stay and monitor patients from regions other than our own.Retrospective analysis of the findings from these sonographic examinations revealed hepatic HHT in 27 (53%) of the 51 patients. Nineteen (70%) of these (age range 40-86 years, mean 63) had vascular malformations of various dimensions but no portal hypertension; the other eight (30%) (age range 39-81 years, mean 60) had vascular malformations plus portal hypertension.Our retrospective analysis indicates that a significant number of patients can have unrecognized hepatic involvement; that the appearance of hepatic lesions can be fairly unpredictable, even when the HHT has been diagnosed for years and the patients are already symptomatic; and that the hepatic lesions are frequently progressive. Therefore, regular sonographic follow-up is advisable for patients with HHT.The limitations of this study are related to the small number of patients examined and to the fact that all of them were symptomatic. Further study is therefore needed (especially in asymptomatic patients) to define the indications for hepatic sonography and the optimum examination schedule.Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by the presence of telangiectasias and arteriovenous malformations in various organs and organ systems, including the liver. The most frequent clinical manifestation of HHT is epistaxis.In 2010 (January–December inclusive) 51 patients with HHT were admitted to the ENT ward of our hospital for epistaxis, and in accordance with routine protocols, all 51 underwent abdominal ultrasonography in our department to detect hepatovascular lesions. They included 27 males (53%) and 24 (47%) females ranging in age from 11 to 86 years (mean 48.5 years). The sample was selected in an arbitrary manner to take maximum advantage of the hospital stay and monitor patients from regions other than our own.Retrospective analysis of the findings from these sonographic examinations revealed hepatic HHT in 27 (53%) of the 51 patients. Nineteen (70%) of these (age range 40–86 years, mean 63) had vascular malformations of various dimensions but no portal hypertension; the other eight (30%) (age range 39–81 years, mean 60) had vascular malformations plus <i>portal hypertension</i>.Our retrospective analysis indicates that a significant number of patients can have unrecognized hepatic involvement; that the appearance of hepatic lesions can be fairly unpredictable, even when the HHT has been diagnosed for years and the patients are already symptomatic; and that the hepatic lesions are frequently progressive. Therefore, regular sonographic follow-up is advisable for patients with HHT.The limitations of this study are related to the small number of patients examined and to the fact that all of them were symptomatic. Further study is therefore needed (especially in asymptomatic patients) to define the indications for hepatic sonography and the optimum examination schedule.

Half of all dental restorations fail within 10 years, with secondary caries and restoration fracture being the main reasons. Calcium phosphate (CaP) composites can release Ca and PO(4) ions and remineralize tooth lesions. However, there has been no report on their long-term mechanical durability. The objective of this study was to investigate the wear, thermal-cycling, and water-aging of composites containing amorphous calcium phosphate nanoparticles (NACP). NACP of 112-nm and glass particles were used to fabricate four composites: (1) 0% NACP+75% glass; (2) 10% NACP+65% glass; (3) 15% NACP+60% glass; and (4) 20% NACP+50% glass. Flexural strength and elastic modulus of NACP nanocomposites were not degraded by thermal-cycling. Wear depth increased with increasing NACP filler level. Wear depths of NACP nanocomposites after 4 × 10(5) cycles were within the range for commercial controls. Mechanical properties of all the tested materials decreased with water-aging time. After 2 years, the strengths of NACP nanocomposites were moderately higher than the control composite, and much higher than the resin-modified glass ionomers. The mechanism of strength loss for resin-modified glass ionomer was identified as microcracking and air-bubbles. NACP nanocomposites and control composite were generally free of microcracks and air-bubbles. In conclusion, combining NACP nanoparticles with reinforcement glass particles resulted in novel nanocomposites with long-term mechanical properties higher than those of commercial controls, and wear within the range of commercial controls. These strong long-term properties, plus the Ca-PO(4) ion release and acid-neutralization capability reported earlier, suggest that the new NACP nanocomposites may be promising for stress-bearing and caries-inhibiting restorations.

<h4>Aim</h4>To examine the role of 5-HTTLPR, rs25531 and 5-HTT VNTR Intron 2 variants in subjects with psychotic disorders manifesting suicide ideation and behaviour.<h4>Methods</h4>The study included 519 subsequently hospitalized subjects who were genotyped for 5-HTTLPR, rs25531 and 5-HTT VNTR In2 variants. Clinical assessments included structured psychiatric interview, sociodemographic characteristics, suicide ideation and behaviour (SIBQ), severity of psychopathology (PANSS) and depression (CDSS).<h4>Results</h4>Three subgroups were identified: suicide attempters (N = 161), suicide ideators (N = 174) and subjects who never reported suicide ideation or behaviour (comparative group, N = 184).<h4>Major findings</h4>1) Suicide attempters scored highest on the CDSS, while no differences between the three clinical subgroups were detected in the PANSS scores; 2) Suicide attempters were more frequently the carriers of L(A) allele, while subjects in the comparative group were more frequently the carriers of low expression 5-HTTLPR/5-HTT rs25531 haplotype SL(G); 3) No difference was found between the three clinical groups in the 5-HTT VNTR In2 variants; 4) Subjects with 5-HTTLPR/5-HTT rs25531 intermediate expression haplotype (L(A)L(G,)SL(A)) scored higher on the PANSS general psychopathology subscale; 5) There was no association between suicide attempt or ideation and 5-HTTLPR/In2 or 5-HTTLPR/rs25531/In2 haplotype distribution.<h4>Conclusion</h4>The suicide ideators, attempters and controls did not differ significantly in 5-HTTLPR or 5-HTT VNTR In 2 variants, but 5-HTTLPR/5-HTT rs25531 haplotype might be a useful genetic marker in distinguishing these three clinical groups.

Alzheimer's dementia (AD) and frontotemporal dementias (FTD) are common and their clinical differential diagnosis may be complicated by overlapping symptoms, which is why biomarkers may have an important role to play. Cerebrospinal fluids (CSF) Aβ2-42 and 1-42 have been shown to be similarly decreased in AD, but 1-42 did not display sufficient specificity for exclusion of other dementias from AD. The objective of the present study was to clarify the diagnostic value of Aβ2-42 peptides for the differential diagnosis of AD from FTD. For this purpose, 20 non-demented disease controls (NDC), 22 patients with AD and 17 with FTD were comparatively analysed by a novel sequential aminoterminally and carboxyterminally specific immunoprecipitation protocol with subsequent Aβ-SDS-PAGE/immunoblot, allowing the quantification of peptides 1-38(ox), 2-40 and 2-42 along with Aβ 1-37, 1-38, 1-39, 1-40, 1-40(ox) and 1-42. CSF Aβ1-42 was decreased in AD as compared to NDC, but not to FTD. In a subgroup of the patients analyzed, the decrease of Abeta2-42 in AD was evident as compared to both NDC and FTD. Aβ1-38 was decreased in FTD as compared to NDC and AD. For differentiating AD from FTD, Aβ1-42 demonstrated sufficient diagnostic accuracies only when combined with Aβ1-38. Aβ2-42 yielded diagnostic accuracies of over 85 % as a single marker. These accuracy figures could be improved by combining Aβ2-42 to Aβ1-38. Aβ2-42 seems to be a promising biomarker for differentiating AD from other degenerative dementias, such as FTD.

In the basal ganglia circuitry, the striatum is a highly complex structure coordinating motor and cognitive functions and it is severely affected in Huntington's disease (HD) patients. Transplantation of fetal ganglionic eminence (GE) derived precursor cells aims to restore neural circuitry in the degenerated striatum of HD patients. Pre-clinical transplantation in genetic and lesion HD animal models has increased our knowledge of graft vs. host interactions, and clinical studies have been shown to successfully reduce motor and cognitive effects caused by the disease. Investigating the molecular mechanisms of striatal neurogenesis is a key research target, since novel strategies aim on generating striatal neurons by differentiating embryonic stem cells or by reprogramming somatic cells as alternative cell source for neural transplantation.

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.

Chronic, multi-factorial conditions caused by a complex interaction between genetic and environmental risk factors frequently share common disease mechanisms, as evidenced by an overlap between genetic risk factors for cardiovascular disease (CVD) and Alzheimer's disease (AD). Single nucleotide polymorphisms (SNPs) in several genes including ApoE, MTHFR, HFE and FTO are known to increase the risk of both conditions. The E4 allele of the ApoE polymorphism is the most extensively studied risk factor for AD and increases the risk of coronary heart disease by approximately 40%. It furthermore displays differential therapeutic responses with use of cholesterol-lowering statins and acetylcholinesterase inhibitors, which may also be due to variation in the CYP2D6 gene in some patients. Disease expression may be triggered by gene-environment interaction causing conversion of minor metabolic abnormalities into major brain disease due to cumulative risk. A growing body of evidence supports the assessment and treatment of CVD risk factors in midlife as a preventable cause of cognitive decline, morbidity and mortality in old age. In this review, the concept of pathology supported genetic testing (PSGT) for CVD is described in this context. PSGT combines DNA testing with biochemical measurements to determine gene expression and to monitor response to treatment. The aim is to diagnose treatable disease subtypes of complex disorders, facilitate prevention of cumulative risk and formulate intervention strategies guided from the genetic background. CVD provides a model to address the lifestyle link in most chronic diseases with a genetic component. Similar preventative measures would apply for optimisation of heart and brain health.

<h4>Unlabelled</h4>Hemochromatosis is considered by many to be an uncommon disorder, although the prevalence of HFE (High Iron) 282 Cys → Tyr (C282Y) homozygosity is relatively high in Caucasians. Liver disease is one of the most consistent findings in advanced iron overload resulting from hemochromatosis. Liver clinics are often thought to be ideal venues for diagnosis of hemochromatosis, but diagnosis rates are often low. The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 99,711 primary care participants in North America for iron overload using serum ferritin and transferrin saturation measurements and HFE genotyping. In this HEIRS substudy, serum hepatic transaminases activities (e.g., alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) were compared between 162 C282Y homozygotes and 1,367 nonhomozygotes with serum ferritin levels >300 μg/L in men and >200 μg/L in women and transferrin saturation >45% in women and 50% in men. The probability of being a C282Y homozygote was determined for AST and ALT ranges. Mean ALT and AST activities were significantly lower in C282Y homozygotes than nonhomozygotes. The probability of being a C282Y homozygote increased as the ALT and AST activities decreased.<h4>Conclusion</h4>Patients with hyperferritinemia are more likely to be C282Y homozygotes if they have normal liver transaminase activities. This paradox could explain the low yields of hemochromatosis screening reported by some liver clinics.

Some bacterial plasmids carry antibiotic resistance genes and replicate by an asymmetric, rolling circle mechanism, in which replication of the two strands is not concurrent. Initiation of this replication occurs via an initiator protein that nicks one DNA strand at the double-stranded origin of replication. In this work, RepD protein from the staphylococcal plasmid pC221 carries this function and allows PcrA helicase to bind and begin unwinding the plasmid DNA. This work uses whole plasmid constructs as well as oligonucleotide-based mimics of parts of the origin to examine the initiation reaction. It investigates the phenomenon that nicking, although required to open a single-stranded region at the origin and so allow PcrA to bind, is not required for another function of RepD, namely to increase the processivity of PcrA, allowing it to unwind plasmid lengths of DNA. A kinetic mechanism of RepD initiation is presented, showing rapid binding of the origin DNA. The rate of nicking varies with the structure of the DNA but can occur with a rate constant of >25 s(-1) at 30 °C. The equilibrium constant of the nicking reaction, which involves a transesterification to form a phosphotyrosine bond within the RepD active site, is close to unity.

The development of selectively protected monosaccharide building blocks that can reliably be glycosylated with a wide variety of acceptors is expected to make oligosaccharide synthesis a more routine operation. In particular, there is an urgent need for the development of modular building blocks that can readily be converted into glycosyl donors for glycosylations that give reliably high 1,2-cis-anomeric selectivity. We report here that 1,2-oxathiane ethers are stable under acidic, basic, and reductive conditions making it possible to conduct a wide range of protecting group manipulations and install selectively removable protecting groups such as levulinoyl (Lev) ester, fluorenylmethyloxy (Fmoc)- and allyloxy (Alloc)-carbonates, and 2-methyl naphthyl ethers (Nap). The 1,2-oxathiane ethers could easily be converted into bicyclic anomeric sulfonium ions by oxidization to sulfoxides and arylated with 1,3,5-trimethoxybenzene. The resulting sulfonium ions gave high 1,2-cis-anomeric selectivity when glycosylated with a wide variety of glycosyl acceptors including properly protected amino acids, primary and secondary sugar alcohols and partially protected thioglycosides. The selective protected 1,2-oxathianes were successfully employed in the preparation of a branched glucoside derived from a glycogen-like polysaccharide isolated form the fungus Pseudallescheria boydii , which is involved in fungal phagocytosis and activation of innate immune responses. The compound was assembled by a latent-active glycosylation strategy in which an oxathiane was employed as an acceptor in a glycosylation with a sulfoxide donor. The product of such a glycosylation was oxidized to a sulfoxide for a subsequent glycosylation. The use of Nap and Fmoc as temporary protecting groups made it possible to install branching points.

Several common genetic variants have recently been discovered that appear to influence white matter microstructure, as measured by diffusion tensor imaging (DTI). Each genetic variant explains only a small proportion of the variance in brain microstructure, so we set out to explore their combined effect on the white matter integrity of the corpus callosum. We measured six common candidate single-nucleotide polymorphisms (SNPs) in the COMT, NTRK1, BDNF, ErbB4, CLU, and HFE genes, and investigated their individual and aggregate effects on white matter structure in 395 healthy adult twins and siblings (age: 20-30 years). All subjects were scanned with 4-tesla 94-direction high angular resolution diffusion imaging. When combined using mixed-effects linear regression, a joint model based on five of the candidate SNPs (COMT, NTRK1, ErbB4, CLU, and HFE) explained ≈ 6% of the variance in the average fractional anisotropy (FA) of the corpus callosum. This predictive model had detectable effects on FA at 82% of the corpus callosum voxels, including the genu, body, and splenium. Predicting the brain's fiber microstructure from genotypes may ultimately help in early risk assessment, and eventually, in personalized treatment for neuropsychiatric disorders in which brain integrity and connectivity are affected.

Genome-wide association (GWA) studies of complex diseases including coronary heart disease (CHD) challenge investigators attempting to identify relevant genetic variants among hundreds of thousands of markers being tested. A selection strategy based purely on statistical significance will result in many false negative findings after adjustment for multiple testing. Thus, an integrated analysis using information from the learned genetic pathways, molecular functions, and biological processes is desirable. In this study, we applied a customized method, variable set enrichment analysis (VSEA), to the Framingham Heart Study data (404,467 variants, n=6421) to evaluate enrichment of genetic association in 1395 gene sets for their contribution to CHD. We identified 25 gene sets with nominal P<0.01; at least four sets are previously known for their roles in CHD: vascular genesis (GO:0001570), fatty-acid biosynthetic process (GO:0006633), fatty-acid metabolic process (GO:0006631), and glycerolipid metabolic process (GO:0046486). Although the four gene sets include 170 genes, only three of the genes contain a variant ranked among the top 100 in single-variant association tests of the 404,467 variants tested. Significant enrichment for novel gene sets less known for their importance to CHD were also identified: Rac 1 cell-motility signaling pathway (h_rac1 Pathway, P<0.001) and sulfur amino-acid metabolic process (GO:0000096, P<0.001). In summary, we showed that the pathway-based VSEA can help prioritize association signals in GWA studies by identifying biologically plausible targets for downstream searches of genetic variants associated with CHD.

<h4>Background</h4>Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1-42/Aβ1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1-42 production, decreasing Aβ1-40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q.<h4>Methodology/principal findings</h4>We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1-40, Aβ1-42 and APP intracellular domain productions in vitro.<h4>Conclusion/significance</h4>Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1-42/Aβ1-40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease.

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3' untranslated region (UTR), along with exon seven. Therefore, this 3' UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3'-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites--two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements.

<h4>Background</h4>Organic anion transporters 1 (Oat1) and 3 (Oat3) mediate the transport of organic anions, including frequently prescribed drugs, across cell membranes in kidney proximal tubule cells. In rats, these transporters are known to be male-dominant and testosterone-dependently expressed. The molecular mechanisms that are involved in the sex-dependent expression are unknown. Our aim was to identify genes that show a sex-dependent expression and could be involved in male-dominant regulation of Oat1 and Oat3.<h4>Methodology/principal findings</h4>Promoter activities of Oat1 and Oat3 were analyzed using luciferase assays. Expression profiling was done using a SurePrint G3 rat GE 8 × 60K microarray. RNA was isolated from renal cortical slices of four adult rats per sex. To filter the achieved microarray data for genes expressed in proximal tubule cells, transcription database alignment was carried out. We demonstrate that predicted androgen response elements in the promoters of Oat1 and Oat3 are not functional when the promoters were expressed in OK cells. Using microarray analyses we analyzed 17,406 different genes. Out of these genes, 56 exhibit a sex-dependent expression in rat proximal tubule cells. As genes potentially involved in the regulation of Oat1 and Oat3 expression, we identified, amongst others, the male-dominant hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1), B-cell CLL/lymphoma 6 (BCL6), and polymerase (RNA) III (DNA directed) polypeptide G (Polr3g). Moreover, our results revealed that the transcription factor BCL6 activates promoter constructs of Oat1 and Oat3.<h4>Conclusion</h4>The results indicate that the male-dominant expression of both transporters, Oat1 and Oat3, is possibly not directly regulated by the classical androgen receptor mediated transcriptional pathway but appears to be regulated by the transcription factor BCL6.

<h4>Background</h4>Hepatocellular carcinoma (HCC) is increasing in the U.S. despite a decline in cancer overall. Latinos have higher rates of HCC than the general population according to the Surveillance, Epidemiology, and End Results (SEER) Program. Not included in SEER, Texas Latinos make up one-fifth of the U.S. Latino population. To determine whether HCC incidence differs among U.S. and Texas Latinos, this descriptive study compares HCC incidence from 1995 through 2006 among three Latino populations: U.S. SEER, Texas overall and a South Texas subset. To identify lines of prevention research, we compare prevalence of known HCC risk factors among these Latino groups.<h4>Methods</h4>Data were collected from the U.S. SEER Program, Texas Cancer Registry and Texas Department of State Health Services (TDSHS). Annual age-specific and age-adjusted HCC incidence rates, annual percent changes (APCs) and 95% confidence intervals were calculated as well as prevalence of obesity, diabetes, heavy alcohol use and cigarette smoking.<h4>Results</h4>Of the three Latino groups compared, South Texas Latinos had the highest age-adjusted HCC incidence rates and SEER Latinos had the lowest (10.6/100,000 (10.1-11.1) and 7.5/100,000 (7.2-7.7), respectively). HCC incidence significantly increased over time (APCs>0) among Latinos in all three geographic groups. Between 1995 and 2006, there was an increase in obesity among all three populations, and obesity was highest among South Texas Latinos. Diabetes increased among U.S. Latinos, and Latino women in South Texas had significantly higher diabetes prevalence than U.S. Latino women. Cigarette smoking and heavy alcohol use were similar among groups.<h4>Conclusions</h4>The incidence of HCC among Latinos in South Texas is higher than elsewhere in the United States. Higher rates of HCC among Texas and South Texas Latinos may be associated with greater prevalence of obesity and diabetes, risk factors for HCC that are amenable to intervention.

A recent meta-analysis of genome-wide association (GWA) studies identified 95 loci that influence lipid traits in the adult population and found that collectively these explained about 25-30% of heritability for each trait. Little is known about how these loci affect lipid levels in early life, but there is evidence that genetic effects on HDL- and LDL-cholesterol (HDL-C, LDL-C) and triglycerides vary with age. We studied Australian adults (N = 10,151) and adolescents (N = 2,363) who participated in twin and family studies and for whom we have lipid phenotypes and genotype information for 91 of the 95 genetic variants. Heterogeneity tests between effect sizes in adult and adolescent cohorts showed an excess of heterogeneity for HDL-C (p(Het)<0.05 at 5 out of 37 loci), but no more than expected by chance for LDL-C (1 out of 14 loci), or trigycerides (0 out 24). There were 2 (out of 5) with opposite direction of effect in adolescents compared to adults for HDL-C, but none for LDL-C. The biggest difference in effect size was for LDL-C at rs6511720 near LDLR, adolescents (0.021 ± 0.033 mmol/L) and adults (0.157 ± 0.023 mmol/L), p(Het) = 0.013; followed by ZNF664 (p(Het) = 0.018) and PABPC4 (p(Het) = 0.034) for HDL-C. Our findings suggest that some of the previously identified variants associate differently with lipid traits in adolescents compared to adults, either because of developmental changes or because of greater interactions with environmental differences in adults.

Pork quality is an economically important trait and one of the main selection criteria for breeding in the swine industry. In this genome-wide association study (GWAS), 455 pigs from a porcine Large White × Minzhu intercross population were genotyped using the Illumina PorcineSNP60K Beadchip, and phenotyped for intramuscular fat content (IMF), marbling, moisture, color L*, color a*, color b* and color score in the longissimus muscle (LM). Association tests between each trait and the SNPs were performed via the Genome Wide Rapid Association using the Mixed Model and Regression-Genomic Control (GRAMMAR-GC) approach. From the Ensembl porcine database, SNP annotation was implemented using Sus scrofa Build 9. A total of 45 SNPs showed significant association with one or multiple meat quality traits. Of the 45 SNPs, 36 were located on SSC12. These significantly associated SNPs aligned to or were in close approximation to previously reported quantitative trait loci (QTL) and some were located within introns of previously reported candidate genes. Two haplotype blocks ASGA0100525-ASGA0055225-ALGA0067099-MARC0004712-DIAS0000861, and ASGA0085522-H3GA0056170 were detected in the significant region. The first block contained the genes MYH1, MYH2 and MYH4. A SNP (ASGA0094812) within an intron of the USP43 gene was significantly associated with five meat quality traits. The present results effectively narrowed down the associated regions compared to previous QTL studies and revealed haplotypes and candidate genes on SSC12 for meat quality traits in pigs.

<h4>Aims</h4>  To examine the association of person-specific trajectories of withdrawal symptoms of urge-to-smoke, negative affect, physical symptoms and hunger during the first 7 days after smoking cessation with abstinence at end of treatment (EOT) and at 6 months.<h4>Design</h4>  Hierarchical linear modeling (HLM) was used to model person-specific trajectory parameters (level, slope, curvature and volatility) for withdrawal symptoms.<h4>Setting</h4>  University-based smoking cessation trials.<h4>Participants</h4>  Treatment-seeking smokers in clinical trials of transdermal nicotine versus nicotine spray (n = 514) and bupropion versus placebo (n = 421).<h4>Measurements</h4>  Self-reported withdrawal symptoms for 7 days after the planned quit date, and 7-day point prevalence and continuous abstinence at EOT and 6 months.<h4>Findings</h4>  In regressions that included trajectory parameters for one group of withdrawal symptoms, both urge-to-smoke and negative affect were predictive of abstinence while physical symptoms and hunger were generally not predictive. In stepwise regressions that included the complete set of trajectory parameters across withdrawal symptoms (for urge-to-smoke, negative affect, physical symptoms and hunger), with a single exception only the trajectory parameters for urge-to-smoke were predictive. Area under the receiver operator characteristic curve was 0.594 for covariates alone, and 0.670 for covariates plus urge-to-smoke trajectory parameters.<h4>Conclusions</h4>  Among a number of different withdrawal symptoms (urge-to-smoke, negative affect, physical symptoms and hunger) urge-to-smoke trajectory parameters (level, slope and volatility) over the first 7 days of smoking cessation show the strongest prediction of both short- and long-term relapse. Other withdrawal symptoms increase the predictive ability by negligible amounts.

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

Porphyria cutanea tarda (PCT) is the most common form of porphyria across the world. Unlike other forms of porphyria, which are inborn errors of metabolism, PCT is usually an acquired liver disease caused by exogenous factors, chief among which are excess alcohol intake, iron overload, chronic hepatitis C, oestrogen therapy and cigarette smoking. The pathogenesis of PCT is complex and varied, but hereditary or acquired factors that lead to hepatic iron loading and increased oxidative stress are of central importance. Iron loading is usually only mild or moderate in degree [less than that associated with full-blown haemochromatosis (HFE)] and is usually acquired and/or mutations in HFE. Among acquired factors are excessive alcohol intake and chronic hepatitis C infection, which, like mutations in HFE, decrease hepcidin production by hepatocytes. The decrease in hepcidin leads to increased iron absorption from the gut. In the liver, iron loading and increased oxidative stress leads to the formation of non-porphyrin inhibitor(s) of uroporphyrinogen decarboxylase and to oxidation of porphyrinogens to porphyrins. The treatment of choice of active PCT is iron reduction by phlebotomy and maintenance of a mildly iron-reduced state without anaemia. Low-dose antimalarials (cinchona alkaloids) are also useful as additional therapy or as alternative therapy for active PCT in those without haemochromatosis or chronic hepatitis C. In this review, we provide an update of PCT with special emphasis upon the important role often played by the hepatitis C virus.

Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.

<h4>Objective</h4>To characterize important patterns of genetic testing behavior and reporting in modern electronic medical records (EMRs) at the institutional level.<h4>Materials and methods</h4>Retrospective observational study using EMR data of all 10,715 patients who received genetic testing by physicians trained in a primary care specialty or subspecialty at an academic medical center between January 1, 2008 and December 31, 2010.<h4>Results</h4>Patients had a mean±SD age of 38.3±15.8 years (median 36.1, IQR 30.0-43.8). The proportion of female subjects in the study population was larger than in the general patient population (77.2% vs 55.0%, p<0.001) and they were younger than the male subjects in the study (36.5±13.2 vs 44.6±21.2 years, p<0.001). Approximately 1.1% of all patients received genetic testing. There were 942 physicians who ordered a total of 15,320 genetic tests. By volume, commonly tested genes involved mutations for cystic fibrosis (36.7%), prothrombin (13.7%), Tay-Sachs disease (6.7%), hereditary hemochromatosis (4.4%), and chronic myelogenous leukemia (4.1%). EMRs stored reports as free text with categorical descriptions of mutations and an average length of 269.4±153.2 words (median 242, IQR 146-401).<h4>Conclusions</h4>In this study, genetic tests were often ordered by a diverse group of physicians for women of childbearing age being evaluated for diseases that may affect potential offspring. EMRs currently serve primarily as a storage warehouse for textual reports that could potentially be transformed into meaningful structured data for next-generation clinical decision support. Further studies are needed to address the design, development, and implementation of EMRs capable of managing the critical genetic health information challenges of the future.

Pharmacobehavioral studies in experimental animals, and imaging studies in humans, indicate that serotonergic transmission in the amygdala plays a key role in emotional processing, especially for anxiety-related stimuli. The lateral and basolateral amygdaloid nuclei receive a dense serotonergic innervation in all species studied to date. We investigated interrelations between serotonergic afferents and neuropeptide Y (NPY)-producing neurons, which are a subpopulation of inhibitory interneurons in the rat lateral and basolateral nuclei with particularly strong anxiolytic properties. Dual light microscopic immunolabeling showed numerous appositions of serotonergic afferents on NPY-immunoreactive somata. Using electron microscopy, direct membrane appositions and synaptic contacts between serotonin-containing axon terminals and NPY-immunoreactive cellular profiles were unequivocally established. Double in situ hybridization documented that more than 50 %, and about 30-40 % of NPY mRNA-producing neurons, co-expressed inhibitory 5-HT1A and excitatory 5-HT2C mRNA receptor subtype mRNA, respectively, in both nuclei with no gender differences. Triple in situ hybridization showed that individual NPY mRNA-producing interneurons co-express both 5-HT1A and 5-HT2C mRNAs. Co-expression of NPY and 5-HT3 mRNA was not observed. The results demonstrate that serotonergic afferents provide substantial innervation of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei. Studies of serotonin receptor subtype co-expression indicate a differential impact of the serotonergic innervation on this small, but important, population of anxiolytic interneurons, and provide the basis for future studies of the circuitry underlying serotonergic modulation of emotional stimulus processing in the amygdala.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by cortical and spinal motor neuron dysfunction. Routine magnetic resonance imaging (MRI) studies have previously shown hypointense signal in the motor cortex on T(2)-weighted images in some ALS patients, however, the cause of this finding is unknown. To investigate the utility of this MR signal change as a marker of cortical motor neuron degeneration, signal abnormalities on 3T and 7T MR images of the brain were compared, and pathology was obtained in two ALS patients to determine the origin of the motor cortex hypointensity. Nineteen patients with clinically probable or definite ALS by El Escorial criteria and 19 healthy controls underwent 3T MRI. A 7T MRI scan was carried out on five ALS patients who had motor cortex hypointensity on the 3T FLAIR sequence and on three healthy controls. Postmortem 7T MRI of the brain was performed in one ALS patient and histological studies of the brains and spinal cords were obtained post-mortem in two patients. The motor cortex hypointensity on 3T FLAIR images was present in greater frequency in ALS patients. Increased hypointensity correlated with greater severity of upper motor neuron impairment. Analysis of 7T T(2)(*)-weighted gradient echo imaging localized the signal alteration to the deeper layers of the motor cortex in both ALS patients. Pathological studies showed increased iron accumulation in microglial cells in areas corresponding to the location of the signal changes on the 3T and 7T MRI of the motor cortex. These findings indicate that the motor cortex hypointensity on 3T MRI FLAIR images in ALS is due to increased iron accumulation by microglia.

Spinocerebellar ataxia 17 (SCA17) is caused by expansion of the polyglutamine (polyQ) tract in human TATA-box binding protein (TBP) that is ubiquitously expressed in both central nervous system and peripheral tissues. The spectrum of SCA17 clinical presentation is broad. The precise pathogenic mechanism in SCA17 remains unclear. Previously proteomics study using a cellular model of SCA17 has revealed reduced expression of heat shock 70 kDa protein 5 (HSPA5) and heat shock 70 kDa protein 8 (HSPA8), suggesting that impaired protein folding may contribute to the cell dysfunction of SCA17 (Lee et al., 2009). In lymphoblastoid cells, HSPA5 and HSPA8 expression levels in cells with mutant TBP were also significantly lower than that of the control cells (Chen et al., 2010). As nuclear transcription factor Y (NFY) has been reported to regulate HSPA5 transcription, we focused on if NFY activity and HSPA5 expression in SCA17 cells are altered. Here, we show that TBP interacts with NFY subunit A (NFYA) in HEK-293 cells and NFYA incorporated into mutant TBP aggregates. In both HEK-293 and SH-SY5Y cells expressing TBP/Q(61~79), the level of soluble NFYA was significantly reduced. In vitro binding assay revealed that the interaction between TBP and NFYA is direct. HSPA5 luciferase reporter assay and endogenous HSPA5 expression analysis in NFYA cDNA and siRNA transfection cells further clarified the important role of NFYA in regulating HSPA5 transcription. In SCA17 cells, HSPA5 promoter activity was activated as a compensatory response before aggregate formation. NFYA dysfunction was indicated in SCA17 cells as HSPA5 promoter activity reduced along with TBP aggregate formation. Because essential roles of HSPA5 in protection from neuronal apoptosis have been shown in a mouse model, NFYA could be a target of mutant TBP in SCA17.

Huntington's disease (HD) is a genetic neurodegenerative disease characterized by movement disorders, cognitive decline and neuropsychiatric symptoms. HD is caused by expanded CAG tract within the coding region of Huntingtin protein. Despite major insights into the molecular mechanisms leading to HD, no effective cure is yet available. Mutant Huntingtin (mHtt) has been reported to alter the stability and levels of β-Catenin, a key molecule in cell adhesion and signal transduction in Wingless (Wg)/Wnt pathway. However it remains to establish whether manipulation of Wg/Wnt signaling can impact HD pathology. We here investigated the phenotypic interactions between mHtt and Wg/Wnt signaling by using the power of Drosophila genetics. We provide compelling evidence that reducing Armadillo/β-Catenin levels confers protection and that this beneficial effect is correlated with the inactivation of the canonical Wg/Wnt signaling pathway. Knockdowns of Wnt ligands or of the downstream transcription factor Pangolin/TCF both ameliorate the survival of HD flies. Similarly, overexpression of one Armadillo/β-Catenin destruction complex component (Axin, APC2 or Shaggy/GSK-3β) increases the lifespan of HD flies. Loss of functional Armadillo/β-Catenin not only abolishes neuronal intrinsic but also glia-induced alterations in HD flies. Our findings highlight that restoring canonical Wg/Wnt signaling may be of therapeutic value.

Background. Sensory neuropathy (SN) is one of the most common AIDS-associated neurologic disorders especially in the era of highly active antiretroviral therapy (HAART). The aim of this study was to determine the prevalence of SN among highly-active-antiretroviral-therapy- (HAART-) experienced and HAART-naïve HIV-positive individuals and to investigate the relationship to demographic, clinical, and laboratory factors. Methods. 323 patients with HIV infection (142 on HAART and 181 HAART naïve) were enrolled in a cross-sectional neuropathy screening program. Data was collected using structured questionnaires which contained the brief peripheral neuropathy screening tool of AIDS Clinical Trial Group protocol. Neuropathy was defined by the presence of at least 1 clinical sign in a distal, symmetrical pattern. Patients were classified as symptomatic if they described aching, stabbing, or burning pain, paresthesia, or numbness in a similar distribution. Demographic, clinical, and laboratory details were documented as risk factors. Result. The prevalence of sensory neuropathy was 39.0% (126/323), (of which 29/126 (23%)) were symptomatic. Amongst those on HAART, 60/142 (42.3%) had SN compared to 66/181 (36.5%) HAART-naïve individuals (P = 0.29). On multivariate analyses, the independent associations with SN were increasing age (P = 0.03) and current exposure to stavudine (P = 0.00). Gender (P = 0.99) height (P = 0.07) use of HAART (P = 0.50), duration of HAART treatment (P = 0.10), and lower CD4 count (P = 0.12) were not associated with an increased SN risk. Conclusion. HIV SN remains common despite improved immunologic function associated with HAART and decreased neurotoxic HAART use. In this cross-sectional analysis, age and stavudine-based therapies were the independent risk factors.

Older adults are among the most vulnerable to adverse cognitive effects of psychotropic medications and, therefore, the personalization of psychotropic treatment based on adverse drug reactions in this demographic is of great importance. We examined changes on neuropsychological tests of attention attributable to selective serotonin reuptake inhibitor (SSRI) treatment in anxious older adults. We also examined whether variation in serotonin receptor genes was associated with reduced attentional performance with SSRIs. We examined change from pre- to post-treatment in two attention measures - digit span and coding - in 133 adults aged ≥60 yr with generalized anxiety disorder in a 12-wk trial of escitalopram vs. placebo. We also examined attentional change in relation to genetic variability in four central serotonin receptors: the serotonin transporter and serotonin 1A, 2A and 1B receptors. Digit span scores were significantly lowered in patients receiving escitalopram relative to placebo, indicating reduced attentional performance attributable to the SSRI. Individuals with high-transcription variants in the receptors 5-HTR2A rs6311 and 5-HTR1B rs11568817 had greater reductions in attention with SSRI treatment compared to placebo. We conclude that SSRIs reduce attention in older adults, particularly in those with high-expression genetic variants at the serotonin 2A and 1B receptors. Analysing neuropsychological changes with SSRIs in relation to genetic variation in the serotonin system may be a useful strategy for detecting subgroups of older adults who are more susceptible to side-effects of SSRIs. These results, if confirmed, could lead to the personalization of SSRI use to reduce adverse neurocognitive effects.

Mutations in the haemochromatosis gene (HFE) influence iron status in the general population of Northern Europe, and excess iron is associated with the impairment of spermatogenesis. The aim of this study is to investigate the association between three mutations (C282Y, H63D and S65C) in the HFE gene with idiopathic male infertility in the Chinese Han population. Two groups of Chinese men were recruited: 444 infertile men (including 169 with idiopathic azoospermia) and 423 controls with proven fertility. The HFE gene was detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The experimental results demonstrated that no C282Y or S65C mutations were detected. Idiopathic male infertility was not significantly associated with heterozygous H63D mutation (odds ratio=0.801, 95% confidence interval=0.452-1.421, χ(2)=0.577, P=0.448). The H63D mutation frequency did not correlate significantly with the serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) levels in infertile men (P=0.896, P=0.404 and P=0.05, respectively). Our data suggest that the HFE H63D mutation is not associated with idiopathic male reproductive dysfunction.

<h4>Background</h4>Current models suggest that a variation in the promoter region of the serotonin transporter gene (5-HTTLPR) is associated with altered amygdala reactivity not only towards negative but also towards positive stimuli, which has been neglected in the past. This association may possibly convey an elevated vulnerability for psychopathology like abuse, craving, and relapses. Since appetitive conditioning is a crucial mechanism in the pathogenesis of these psychiatric disorders, the identification of specific factors contributing to interindividual variation is important.<h4>Methods</h4>In the present study (N = 86), an appetitive conditioning paradigm was conducted, in which a neutral stimulus (CS+) was associated with appetitive stimuli, while a second stimulus (CS-) predicted their absence. Subjects were genotyped according to the 5-HTTLPR genotype.<h4>Results</h4>As the main result, we report a significant association between the 5-HTTLPR genotype and hemodynamic responses. Individuals with the s-allele displayed elevated conditioned bilateral amygdala activity in contrast to l/l-allele carriers. Further, increased hemodynamic responses in s-allele carriers were also found in the extended emotional network including the orbitofrontal cortex, the thalamus, and the ventral striatum.<h4>Conclusion</h4>The present findings indicate an association of the 5-HTTLPR and altered conditioned responses in appetitive conditioning. Further, the findings contribute to the ongoing debate on 5-HTTLPR dependent hemodynamic response patterns by emphasizing that s-allele carriers are not exclusively biased towards fearful, but also towards positive stimuli. In conclusion, our results imply that s-allele carriers might be better described as hyper-reactive towards salient stimuli, which may convey vulnerability for the development of psychiatric disorders.

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.

ISWI is an evolutionarily conserved ATPase that catalyzes nucleosome remodeling in different macromolecular complexes. Two mammalian ISWI orthologs, SNF2H and SNF2L, are thought to have specialized functions despite their high sequence similarity. To date, the function of SNF2L in human cells has not been a focus of research. Newly established specific monoclonal antibodies and selective RNA interference protocols have now enabled a comprehensive characterization of loss-of-function phenotypes in human cells. In contrast to earlier results, we found SNF2L to be broadly expressed in primary human tissues. Depletion of SNF2L in HeLa cells led to enhanced proliferation and increased migration. These phenomena were explained by transcriptome profiling, which identified SNF2L as a modulator of the Wnt signaling network. The cumulative effects of SNF2L depletion on gene expression portray the cell in a state of activated Wnt signaling characterized by increased proliferation and chemotactic locomotion. Accordingly, high levels of SNF2L expression in normal melanocytes contrast with undetectable expression in malignant melanoma. In summary, our data document an inverse relationship between SNF2L expression and features characteristic of malignant cells.

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin (Htt). Striatal and cortical neuronal loss are prominent features of this disease. No disease-modifying treatments have been discovered for HD. To identify new therapeutic targets in HD, we screened a kinase inhibitor library for molecules that block mutant Htt cellular toxicity in a mouse HD striatal cell model, Hdh(111Q/111Q) cells. We found that diacylglycerol kinase (DGK) inhibitor II (R59949) decreased caspase-3/7 activity after serum withdrawal in striatal Hdh(111Q/111Q) cells. In addition, R59949 decreased the accumulation of a 513-amino acid N-terminal Htt fragment processed by caspase-3 and blocked alterations in lipid metabolism during serum withdrawal. To identify the diacylglycerol kinase mediating this effect, we knocked down all four DGK isoforms expressed in the brain (β, γ, ε, and ζ) using siRNA. Only the knockdown of the family member, DGKε, blocked striatal Hdh(111Q/111Q)-mediated toxicity. We also investigated the significance of these findings in vivo. First, we found that reduced function of the Drosophila DGKε homolog significantly improves Htt-induced motor dysfunction in a fly model of HD. In addition, we find that the levels of DGKε are increased in the striatum of R6/2 HD transgenic mice when compared with littermate controls. Together, these findings indicate that increased levels of kinase DGKε contribute to HD pathogenesis and suggest that reducing its levels or activity is a potential therapy for HD.

Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.

Bone mineral density (BMD) is the most widely used predictor of fracture risk. We performed the largest meta-analysis to date on lumbar spine and femoral neck BMD, including 17 genome-wide association studies and 32,961 individuals of European and east Asian ancestry. We tested the top BMD-associated markers for replication in 50,933 independent subjects and for association with risk of low-trauma fracture in 31,016 individuals with a history of fracture (cases) and 102,444 controls. We identified 56 loci (32 new) associated with BMD at genome-wide significance (P < 5 × 10(-8)). Several of these factors cluster within the RANK-RANKL-OPG, mesenchymal stem cell differentiation, endochondral ossification and Wnt signaling pathways. However, we also discovered loci that were localized to genes not known to have a role in bone biology. Fourteen BMD-associated loci were also associated with fracture risk (P < 5 × 10(-4), Bonferroni corrected), of which six reached P < 5 × 10(-8), including at 18p11.21 (FAM210A), 7q21.3 (SLC25A13), 11q13.2 (LRP5), 4q22.1 (MEPE), 2p16.2 (SPTBN1) and 10q21.1 (DKK1). These findings shed light on the genetic architecture and pathophysiological mechanisms underlying BMD variation and fracture susceptibility.

Siderophores are best known as small iron binding molecules that facilitate microbial iron transport. In our previous study we identified a siderophore-like molecule in mammalian cells and found that its biogenesis is evolutionarily conserved. A member of the short chain dehydrogenase family of reductases, 3-hydroxy butyrate dehydrogenase (BDH2) catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore. We have shown that depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of cellular iron and mitochondrial iron deficiency. These observations suggest that the mammalian siderophore is a critical regulator of cellular iron homeostasis and facilitates mitochondrial iron import. By utilizing bioinformatics, we identified an iron-responsive element (IRE; a stem-loop structure that regulates genes expression post-transcriptionally upon binding to iron regulatory proteins or IRPs) in the 3'-untranslated region of the human BDH2 (hBDH2) gene. In cultured cells as well as in patient samples we now demonstrate that the IRE confers iron-dependent regulation on hBDH2 and binds IRPs in RNA electrophoretic mobility shift assays. In addition, we show that the hBDH2 IRE associates with IRPs in cells and that abrogation of IRPs by RNAi eliminates the iron-dependent regulation of hBDH2 mRNA. The key physiologic implication is that iron-mediated post-transcriptional regulation of hBDH2 controls mitochondrial iron homeostasis in human cells. These observations provide a new and an unanticipated mechanism by which iron regulates its intracellular trafficking.

<h4>Objective</h4>To determine whether premature infants with invasive Candida infection caused by strains with increased virulence properties have worse clinical outcomes than those infected with less virulent strains.<h4>Study design</h4>Clinical isolates were studied from 2 populations of premature infants, those colonized with Candida spp (commensal; n = 27) and those with invasive candidiasis (n = 81). Individual isolates of C albicans and C parapsilosis were tested for virulence in 3 assays: phenotypic switching, adhesion, and cytotoxicity. Invasive isolates were considered to have enhanced virulence if detected at a level >1 SD above the mean for the commensal isolates in at least one assay. Outcomes of patients with invasive isolates with enhanced virulence were compared with those with invasive isolates lacking enhanced virulence characteristics.<h4>Results</h4>Enhanced virulence was detected in 61% of invasive isolates of C albicans and 42% of invasive isolates of C parapsilosis. All C albicans cerebrospinal fluid isolates (n = 6) and 90% of urine isolates (n = 10) had enhanced virulence, compared with 48% of blood isolates (n = 40). Infants with more virulent isolates were younger at the time of positive culture and had higher serum creatinine levels.<h4>Conclusion</h4>Individual isolates of Candida species vary in their virulence properties. Strains with higher virulence are associated with certain clinical outcomes.

<h4>Aims</h4>Orthostatic hypotension (OH), an independent predictor of mortality and cardiovascular events, strongly correlates with hypertension. Recent genome-wide studies have identified new loci influencing blood pressure (BP) in populations, but their impact on OH remains unknown.<h4>Methods and results</h4>A total of 38 970 men and women of European ancestry from five population-based cohorts were included, of whom 2656 (6.8%) met the diagnostic criteria for OH (systolic/diastolic BP drop ≥ 20/10 mmHg within 3 min of standing). Thirty-one recently discovered BP-associated single nucleotide polymorphisms (SNPs) were examined using an additive genetic model and the major allele as referent. Relations between OH, orthostatic systolic BP response, and genetic variants were assessed by inverse variance-weighted meta-analysis. We found Bonferroni adjusted (P < 0.0016) significant evidence for association between OH and the EBF1 locus (rs11953630, per-minor-allele odds ratio, 95% confidence interval: 0.90, 0.85-0.96; P = 0.001), and nominal evidence (P < 0.05) for CYP17A1 (rs11191548: 0.85, 0.75-0.95; P = 0.005), and NPR3-C5orf23 (rs1173771: 0.92, 0.87-0.98; P= 0.009) loci. Among subjects not taking BP-lowering drugs, three SNPs within the NPPA/NPPB locus were nominally associated with increased risk of OH (rs17367504: 1.13, 1.02-1.24; P = 0.02, rs198358: 1.10, 1.01-1.20; P = 0.04, and rs5068: 1.22, 1.04-1.43; P = 0.01). Moreover, an ADM variant was nominally associated with continuous orthostatic systolic BP response in the adjusted model (P= 0.04).<h4>Conclusion</h4>The overall association between common gene variants in BP loci and OH was generally weak and the direction of effect inconsistent with resting BP findings. These results suggest that OH and resting BP share few genetic components.

Coordinated limb rhythmic movements take place through organized signaling in local spinal cord neuronal networks. The establishment of these circuitries during development is dependent on the correct guidance of axons to their targets. It has previously been shown that the well-known axon guidance molecule netrin-1 is required for configuring the circuitry that provides left-right alternating coordination in fictive locomotion. The attraction of commissural axons to the midline in response to netrin-1 has been shown to involve the netrin-1 receptor DCC (deleted in Colorectal Cancer). However, the role of DCC for the establishment of CPG coordination has not yet been resolved. We show that mice carrying a null mutation of DCC displayed an uncoordinated left-right activity during fictive locomotion accompanied by a loss of interneuronal subpopulations originating from commissural progenitors. Thus, DCC plays a crucial role in the formation of spinal neuronal circuitry coordinating left-right activities. Together with the previously published results from netrin-1 deficient mice, the data presented in this study suggest a role for the most ventral originating V3 interneurons in synchronous activities over the midline. Further, it provides evidence that axon crossing in the spinal cord is more intricately controlled than in previously suggested models of DCC-netrin-1 interaction.

Atrial fibrillation (AF) is the most frequent supraventricular arrhythmia with an approximative prevalence of 1 % in the general population and above 6 % in the elderly. After a first AF diagnosis, the hospitalization rate is markedly increased. Management of a first AF episode is different depending on the clinical status of patients. Practical guidelines developed in collaboration with the European Heart Rhythm Association and the Heart Rhythm Society are available for the management of these patients. A four-step decisional scheme must be followed in the management of a first recent AF episode: need for a short- and long-term anticoagulation, define a rythmologic strategy (rhythm or rate control), select the weapon (drug, device or ablation) and reconsider the strategy if needed. After a first uncomplicated paroxysmal AF episode, guidelines recommend that prescription of antiarrhythmics must be avoided and anticoagulation is optional. After a first persistent AF episode, guidelines recommend to either respect or reduce the arrhythmia. Prescription of antiarrhythmics and anticoagulation is also optional depending on the patient?s condition. In case of the AF reduction decision, anticoagulation must be tailored preliminary to this reduction. AF recurrence rate varies depending on the patient?s condition, and the risk of stroke assessed by the CHA₂DS₂-VASc score might be similarly considered for both paroxysmal and persistent AF.

mRNAs can be targeted to specific neuronal subcellular domains, which enables rapid changes in the local proteome through local translation. This mRNA-based mechanism links extrinsic signals to spatially restricted cellular responses and can mediate stimulus-driven adaptive responses such as dendritic plasticity. Local mRNA translation also occurs in growing axons where it can mediate directional responses to guidance signals. Recent profiling studies have revealed that both growing and mature axons possess surprisingly complex and dynamic transcriptomes, thereby suggesting that axonal mRNA localization is highly regulated and has a role in a broad range of processes, a view that is increasingly being supported by new experimental evidence. Here, we review current knowledge on the roles and regulatory mechanisms of axonal mRNA translation and discuss emerging links to axon guidance, survival, regeneration and neurological disorders.

With a view to identify genomic risk variants in chewing-tobacco associated oral cancer patients, a genome-wide association study was conducted in patients of Indian ethnicity with long term tobacco chewing habit. We analyzed 55 oral cancer patients and 92 healthy controls for single nucleotide polymorphisms, using high throughput microarray Illumina Infinium II Assay platform and Human CNV370k-bead chip containing 370,000 single nucleotide polymorphisms. The PLINK software platform defined 298 SNPs with minor allele frequency of several genes significantly increased in oral cancer patients as compared to the controls (p<0.001). Illumina Genome Viewer Software Version 3.2.9, further delineated 93 SNPs with p-values ranging from 9.3×10(-4) to 1.38×10(-5) and Odd's ratio of 2.18-8.48, associated with 70 genes. Analysis using Kyoto Encyclopedia of Genes and Genome Pathway database, indicated SNP association with several genes including GRIK2, RASGRP3, CAMK4, SYK, RAPTOR, FHIT, DCC, active in signal transduction; MMP2, CNTNAP2, PTPRJ associated with tumor cell migration; and apoptotic gene IRAK3. The data indicates an inherent role for the genetic constitution of individuals in oral carcinogenesis, with the genomic variants contributing to increased risk or susceptibility to oral cancer.

α(v)β(3) integrin is involved in (tumor-induced) angiogenesis and is a promising candidate for the specific visualization of both primary tumors and of their distant metastases. Combination of radioactive and fluorescent imaging labels in a single multimodal, or rather hybrid, RGD-based imaging agent enables integration of pre-, intra-, and postoperative angiogenesis imaging. A hybrid imaging agent targeting the α(v)β(3) integrin--(111)In-MSAP-RGD (MSAP = multifunctional single-attachment-point reagent), which contains a targeting moiety, a pentetic acid (DTPA) chelate, and a cyanine dye--was evaluated for its potential value in combined lesion detection and interventional molecular imaging in a 4T1 mouse breast cancer model. SPECT/CT and fluorescence imaging were used to visualize the tumor in vivo. Tracer distribution was evaluated ex vivo down to the microscopic level. The properties of (111)In-MSAP-RGD were compared with those of (111)In-DTPA-RGD. Biodistribution studies revealed a prolonged retention and increased tumor accumulation of (111)In-MSAP-RGD relative to (111)In-DTPA-RGD. With (111)In-MSAP-RGD, identical features could be visualized preoperatively (SPECT/CT) and intraoperatively (fluorescence imaging). As well as the primary tumor, (111)In-MSAP-RGD also enabled detection and accurate excision of distant metastases in the head and neck region of the mice. Therefore, the hybrid RGD derivative (111)In-MSAP-RGD shows potential in preoperative planning and fluorescence-based surgical intervention.

Vertebrate genome comparisons revealed that there are highly conserved noncoding sequences (HCNSs) among a wide range of species and many of which contain regulatory elements. However, recently emerged sequences conserved in specific lineages have not been well studied. Toward this end, we identified 8,198 primate and 21,128 specific HCNSs as representative ones among mammals from human-marmoset and mouse-rat comparisons, respectively. Derived allele frequency analysis of primate-specific HCNSs showed that these HCNSs were under purifying selection, indicating that they may harbor important functions. We selected the top 1,000 largest HCNSs and compared the lineage-specific HCNS-flanking genes (LHF genes) with ultraconserved element (UCE)-flanking genes. Interestingly, the majority of LHF genes were different from UCE-flanking genes. This lineage-specific set of LHF genes was more enriched in protein-binding function. Conversely, the number of LHF genes that were also shared by UCEs was small but significantly larger than random expectation, and many of these genes were involved in anatomical development as transcriptional regulators, suggesting that certain groups of genes preferentially recruit new HCNSs in addition to old HCNSs that are conserved among vertebrates. This group of LHF genes might be involved in the various levels of lineage-specific evolution among vertebrates, mammals, primates, and rodents. If so, the emergence of HCNSs in and around these two groups of LHF genes developed lineage-specific characteristics. Our results provide new insight into lineage-specific evolution through interactions between HCNSs and their LHF genes.

The Epstein-Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human β-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the β-globin transcript suggesting that EB2 acts by stabilizing SRp20's labile interactions with the RNA.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

The TonB system of proteins is required for the energy-dependent active transport of iron-bound substrates across the outer membrane of gram-negative bacteria. We have identified three TonB systems within the human pathogen Vibrio vulnificus. The TonB1 system contains the TonB1, ExbD1, and ExbB1 proteins, whereas both the TtpC2-TonB2 and TtpC3-TonB3 systems contain an additional fourth protein, TtpC. Here we report that TtpC3, although highly related to TtpC2, is inactive in iron transport, whereas TtpC2 is essential for the function of the TtpC2-TonB2 system in V. vulnificus. This protein, together with TonB2, is absolutely required for both the uptake of endogenously produced iron-bound siderophores as well as siderophores produced from other organisms. Through complementation we show that V. vulnificus is capable of using different TtpC2 proteins from other Vibrio species to drive the uptake of multiple siderophores. We have also determined that aerobactin, a common bacterial siderophore involved in virulence of enteric bacteria, can only be brought into the cell using the TtpC2-TonB2 system, indicating an important evolutionary adaptation of TtpC2 and TonB2. Furthermore, in the absence of TonB1, TtpC2 is essential for a fully virulent phenotype as demonstrated using 50% lethal dose (LD(50)) experiments in mice.

Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT(-/-)) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility, extradimensional strategy set-shifting (EDSS), heavily depends on the medial prefrontal cortex. This region shows functional changes in 5-HTT(-/-) rodents as well. Here we subjected 5-HTT(-/-) rats and their wild-type counterparts to an EDSS paradigm and a supplementary latent inhibition task. Results indicate that 5-HTT(-/-) rats also show improved EDSS, and indicate that reduced latent inhibition may contribute as an underlying mechanism.

<h4>Background</h4>Taenia pisiformis is one of the most common intestinal tapeworms and can cause infections in canines. Adult T. pisiformis (canines as definitive hosts) and Cysticercus pisiformis (rabbits as intermediate hosts) cause significant health problems to the host and considerable socio-economic losses as a consequence. No complete genomic data regarding T. pisiformis are currently available in public databases. RNA-seq provides an effective approach to analyze the eukaryotic transcriptome to generate large functional gene datasets that can be used for further studies.<h4>Methodology/principal findings</h4>In this study, 2.67 million sequencing clean reads and 72,957 unigenes were generated using the RNA-seq technique. Based on a sequence similarity search with known proteins, a total of 26,012 unigenes (no redundancy) were identified after quality control procedures via the alignment of four databases. Overall, 15,920 unigenes were mapped to 203 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Through analyzing the glycolysis/gluconeogenesis and axonal guidance pathways, we achieved an in-depth understanding of the biochemistry of T. pisiformis. Here, we selected four unigenes at random and obtained their full-length cDNA clones using RACE PCR. Functional distribution characteristics were gained through comparing four cestode species (72,957 unigenes of T. pisiformis, 30,700 ESTs of T. solium, 1,058 ESTs of Eg+Em [conserved ESTs between Echinococcus granulosus and Echinococcus multilocularis]), with the cluster of orthologous groups (COG) and gene ontology (GO) functional classification systems. Furthermore, the conserved common genes in these four cestode species were obtained and aligned by the KEGG database.<h4>Conclusion</h4>This study provides an extensive transcriptome dataset obtained from the deep sequencing of T. pisiformis in a non-model whole genome. The identification of conserved genes may provide novel approaches for potential drug targets and vaccinations against cestode infections. Research can now accelerate into the functional genomics, immunity and gene expression profiles of cestode species.

Quantifying the amount and defining the location of metal ions in cells and organisms are critical steps in understanding metal homeostasis and how dyshomeostasis causes or is a consequence of disease. A number of recent advances have been made in the development and application of analytical methods to visualize metal ions in biological specimens. Here, we briefly summarize these advances before focusing in more depth on probes for examining transition metals in living cells with high spatial and temporal resolution using fluorescence microscopy. This article is part of a Special Issue entitled: Cell Biology of Metals.

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A 'pro-cancer' gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment.

<h4>Background</h4>Although several billion corneocytes are shed from human skin daily, metabolic studies from 50 years ago led to the conclusion that corneocyte desquamation had no measurable impact on systemic protein or iron status in humans.<h4>Objective</h4>To measure iron content of internal organs after introducing local genetic changes in epidermis that alter iron metabolism in skin.<h4>Methods</h4>Iron was measured in tissues and blood from groups of animals 7 weeks after weaning in three different mouse models expressing a transgene in epidermis: a hyperproliferation model in which the HPV16 E7 gene causes a 3-fold increase in epidermal turnover; an epidermal iron sink model in which overexpression of the transferrin receptor causes a 3-4 fold increase of iron in epidermis; a systemic hemochromatosis knockout model that has been crossed with the epidermal iron sink model.<h4>Results</h4>In the hemochromatosis model with the iron sink transgene in epidermis, there was a statistically significant reduction in non-heme iron in serum and in the liver and kidney. In all models there was a statistically significant reduction in non-heme iron in the kidney.<h4>Conclusion</h4>Local changes in iron metabolism in epidermis can have a measurable impact on systemic iron metabolism. By implication, disruptions in epidermal homeostasis might affect systemic levels of trace nutrients, and circulating toxins might be remediated by sequestering them in epidermis.

<h4>Objective</h4>Evaluate the efficacy of phototherapy (PT) devices and the outcomes of extremely premature infants treated with those devices.<h4>Study design</h4>This substudy of the National Institute of Child Health and Human Development Neonatal Research Network PT trial included 1404 infants treated with a single type of PT device during the first 24±12 h of treatment. The absolute (primary outcome) and relative decrease in total serum bilirubin (TSB) and other measures were evaluated. For infants treated with one PT type during the 2-week intervention period (n=1223), adjusted outcomes at discharge and 18 to 22 months corrected age were determined.<h4>Result</h4>In the first 24 h, the adjusted absolute (mean (±s.d.)) and relative (%) decrease in TSB (mg dl(-1)) were: light-emitting diodes (LEDs) -2.2 (±3), -22%; Spotlights -1.7 (±2), -19%; Banks -1.3 (±3), -8%; Blankets -0.8 (±3), -1%; (P<0.0002). Some findings at 18 to 22 months differed between groups.<h4>Conclusion</h4>LEDs achieved the greatest initial absolute reduction in TSB but were similar to Spots in the other performance measures. Long-term effects of PT devices in extremely premature infants deserve rigorous evaluation.

Vulvar squamous cell carcinoma (VSCC) is the fourth most common gynecological cancer. Based on etiology VSCC is divided into two subtypes; one related to high-risk human papilloma virus (HPV) and one HPV negative. The two subtypes are proposed to develop via separate intracellular signaling pathways. We investigated a suggested link between HPV infection and relapse risk in VSCC through in-depth protein profiling of 14 VSCC tumor specimens. The tumor proteomes were analyzed by liquid-chromatography tandem mass spectrometry. Relative protein quantification was performed by 8-plex isobaric tags for relative and absolute quantification. Labeled peptides were fractionated by high-resolution isoelectric focusing prior to liquid-chromatography tandem mass spectrometry to reduce sample complexity. In total, 1579 proteins were regarded as accurately quantified and analyzed further. For classification of clinical groups, data analysis was performed by comparing protein level differences between tumors defined by HPV and/or relapse status. Further, we performed a biological analysis on individual tumor proteomes by matching data to known biological pathways. We here present a novel analysis approach that combines pathway alteration data on individual tumor level with multivariate statistics for HPV and relapse status comparisons. Four proteins (signal transducer and activator of transcription-1, myxovirus resistance protein 1, proteasome subunit alpha type-5 and legumain) identified as main classifiers of relapse status were validated by immunohistochemistry (IHC). Two of the proteins are interferon-regulated and on mRNA level known to be repressed by HPV. By both liquid-chromatography tandem mass spectrometry and immunohistochemistry data we could single out a subgroup of HPV negative/relapse-associated tumors. The pathway level data analysis confirmed three of the proteins, and further identified the ubiquitin-proteasome pathway as altered in the high risk subgroup. We show that pathway fingerprinting with resolution on individual tumor level adds biological information that strengthens a generalized protein analysis.

The double-stranded RNA binding protein Staufen1 (Stau1) is involved in diverse gene expression pathways. For Stau1-mediated mRNA decay (SMD) in mammals, Stau1 binds to the 3' untranslated region of target mRNA and recruits Upf1 to elicit rapid mRNA degradation. However, the events downstream of Upf1 recruitment and the biological importance of SMD remain unclear. Here we show that SMD involves PNRC2, decapping activity, and 5'-to-3' exonucleolytic activity. In particular, Upf1 serves as an adaptor protein for the association of PNRC2 and Stau1. During adipogenesis, Stau1 and PNRC2 increase in abundance, Upf1 becomes hyperphosphorylated, and consequently SMD efficiency is enhanced. Intriguingly, downregulation of SMD components attenuates adipogenesis in a way that is rescued by downregulation of an antiadipogenic factor, Krüppel-like factor 2 (KLF2), the mRNA of which is identified as a substrate of SMD. Our data thus identify a biological role for SMD in adipogenesis.

<h4>Background</h4>The Nkx3.2 transcription factor promotes chondrogenesis by forming a positive regulatory loop with a crucial chondrogenic transcription factor, Sox9. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms.<h4>Methodology/principal findings</h4>C3H10T1/2 pluripotent mesenchymal cells were cultured with bone morphogenetic protein 2 (BMP2) to induce endochondral ossification. Overexpression of wild-type Nkx3.2 (WT-Nkx3.2) upregulated glycosaminoglycan (GAG) production and expression of type II collagen α1 (Col2a1) mRNA, and these effects were evident before WT-Nkx3.2-mediated upregulation of Sox9. RNAi-mediated inhibition of Nkx3.2 abolished GAG production and expression of Col2a1 mRNA. Dual luciferase reporter assays revealed that WT-Nkx3.2 upregulated Col2a1 enhancer activity in a dose-dependent manner in C3H10T1/2 cells and also in N1511 chondrocytes. In addition, WT-Nkx3.2 partially restored downregulation of GAG production, Col2 protein expression, and Col2a1 mRNA expression induced by Sox9 RNAi. ChIP assays revealed that Nkx3.2 bound to the Col2a1 enhancer element.<h4>Conclusions/significance</h4>Nkx3.2 promoted primary chondrogenesis by two mechanisms: Direct and Sox9-independent upregulation of Col2a1 transcription and upregulation of Sox9 mRNA expression under positive feedback system.

Huntington's disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. The molecular pathogenesis of HD is complex and many mechanisms and cellular processes have been proposed as potential sites of therapeutic intervention. However, prior to embarking on drug development initiatives, it is essential that therapeutic targets can be validated in mammalian models of HD. Previous studies in invertebrate and cell culture HD models have suggested that inhibition of SIRT2 could have beneficial consequences on disease progression. SIRT2 is a NAD(+)-dependent deacetylase that has been proposed to deacetylate α-tubulin, histone H4 K16 and to regulate cholesterol biogenesis - a pathway which is dysregulated in HD patients and HD mouse models. We have utilized mice in which SIRT2 has been reduced or ablated to further explore the function of SIRT2 and to assess whether SIRT2 loss has a beneficial impact on disease progression in the R6/2 mouse model of HD. Surprisingly we found that reduction or loss of SIRT2 had no effect on the acetylation of α-tubulin or H4K16 or on cholesterol biosynthesis in the brains of wild type mice. Equally, genetic reduction or ablation of SIRT2 had no effect on HD progression as assessed by a battery of physiological and behavioural tests. Furthermore, we observed no change in aggregate load or levels of soluble mutant huntingtin transprotein. Intriguingly, neither the constitutive genetic loss nor acute pharmacological inhibition of SIRT2 affected the expression of cholesterol biosynthesis enzymes in the context of HD. Therefore, we conclude that SIRT2 inhibition does not modify disease progression in the R6/2 mouse model of HD and SIRT2 inhibition should not be prioritised as a therapeutic option for HD.

<h4>Purpose</h4>Myopia, or nearsightedness, is highly prevalent in Asian countries and is considered a serious public health issue globally. High-grade myopia can predispose individuals to myopic maculopathy, premature cataracts, retinal detachment, and glaucoma. A recent study implicated zinc finger protein 644 isoform 1 (ZNF644) variants with non-syndromic high-grade myopia in a Chinese-Asian population. Herein we focused on investigating the role for ZNF644 variants in high-grade myopia in a United States (US) cohort.<h4>Methods</h4>DNA from a case cohort of 131 subject participants diagnosed with high-grade myopia was screened for ZNF644 variants. Spherical refractive error of -≤-6.00 diopters (D) in at least one eye was defined as affected. All coding, intron/exon boundaries were screened using Sanger sequencing. Single nucleotide allele frequencies were determined by screening 672 ethnically matched controls.<h4>Results</h4>Sequencing analysis did not detect previously reported mutations. However, our analysis identified 2 novel single nucleotide variants (c.725C>T, c.821A>T) in 2 high-grade myopia individuals- one Caucasian and one African American, respectively. These variants were not found in normal controls. A rare variant - dbsSNP132 (rs12117237→c.2119A>G) - with a minor allele frequency of 0.2% was present in 6 additional cases, but was also present in 5 controls.<h4>Conclusions</h4>Our study has identified two novel variants in ZNF644 associated with high-grade myopia in a US cohort. Our results suggest that ZNF644 may play a role in myopia development.

Beef is one of the leading sources of protein, B vitamins, iron, and zinc in human food. Beef palatability is based on three general criteria: tenderness, juiciness, and flavor, of which tenderness is thought to be the most important factor. In this study, we found that beef tenderness, measured by the Warner-Bratzler shear force (WBSF), was dramatically increased by acute stress. Microarray analysis and qPCR identified a variety of genes that were differentially expressed. Pathway analysis showed that these genes were involved in immune response and regulation of metabolism process as activators or repressors. Further analysis identified that these changes may be related with CpG methylation of several genes. Therefore, the results from this study provide an enhanced understanding of the mechanisms that genetic and epigenetic regulations control meat quality and beef tenderness.

The association of single-nucleotide polymorphisms (SNPs) in the human tryptophan hydroxylase 2 (TPH2) gene with anxiety traits and depression has been inconclusive. Observed inconsistencies might result from the fact that TPH2 polymorphisms have been studied in a genetically heterogeneous human population. A defined genetic background, control over environmental factors, and the ability to analyze the molecular and neurochemical consequences of introduced genetic alterations constitute major advantages of investigating SNPs in inbred laboratory mouse strains. To investigate the behavioral and neurochemical consequences of a functional C1473G SNP in the mouse Tph2 gene, we generated congenic C57BL/6N mice homozygous for the Tph2 1473G allele. The Arg(447) substitution in the TPH2 enzyme resulted in a significant reduction of the brain serotonin (5-HT) in vivo synthesis rate. Despite decreased 5-HT synthesis, we could detect neither a reduction of brain region-specific 5-HT concentrations nor changes in baseline and stress-induced 5-HT release using a microdialysis approach. However, using a [(35)S]GTP-γ-S binding assay and 5-HT(1A) receptor autoradiography, a functional desensitization of 5-HT(1A) autoreceptors could be identified. Furthermore, behavioral analysis revealed a distinct anxiety phenotype in homozygous Tph2 1473G mice, which could be reversed with chronic escitalopram treatment. Alterations in depressive-like behavior could not be detected under baseline conditions or after chronic mild stress. These findings provide evidence for an involvement of functional Tph2 polymorphisms in anxiety-related behaviors, which are likely not caused directly by alterations in 5-HT content or release but are rather due to compensatory changes during development involving functional desensitization of 5-HT(1A) autoreceptors.

Here we summarize evidence obtained by our group during the last two decades, and contrasted it with a review of related data from the available literature to show that behavioral syndromes involving attention deficit/hyperactivity disorder (ADHD), externalizing disorders, and substance-use disorder (SUD) share similar signs and symptoms (i.e., have a biological basis as common syndromes), physiopathological and psychopathological mechanisms, and genetic factors. Furthermore, we will show that the same genetic variants harbored in different genes are associated with different syndromes and that non-linear interactions between genetic variants (epistasis) best explain phenotype severity, long-term outcome, and response to treatment. These data have been depicted in our studies by extended pedigrees, where ADHD, externalizing symptoms, and SUD segregate and co-segregate. Finally, we applied here a new formal network analysis using the set of significantly replicated genes that have been shown to be either associated and/or linked to ADHD, disruptive behaviors, and SUD in order to detect significantly enriched gene categories for protein and genetic interactions, pathways, co-expression, co-localization, and protein domain similarity. We found that networks related to pathways involved in axon guidance, regulation of synaptic transmission, and regulation of transmission of nerve impulse are overrepresented. In summary, we provide compiled evidence of complex networks of genotypes underlying a wide phenotype that involves SUD and externalizing disorders.

<h4>Background</h4>15-20% of all patients initially diagnosed with colorectal cancer develop metastatic disease and surgical resection remains the only potentially curative treatment available. Current 5-year survival following R0-resection of liver metastases is 28-39%, but recurrence eventually occurs in up to 70%. To date, adjuvant chemotherapy has not improved clinical outcomes significantly. The primary objective of the ongoing LICC trial (L-BLP25 In Colorectal Cancer) is to determine whether L-BLP25, an active cancer immunotherapy, extends recurrence-free survival (RFS) time over placebo in colorectal cancer patients following R0/R1 resection of hepatic metastases. L-BLP25 targets MUC1 glycoprotein, which is highly expressed in hepatic metastases from colorectal cancer. In a phase IIB trial, L-BLP25 has shown acceptable tolerability and a trend towards longer survival in patients with stage IIIB locoregional NSCLC.<h4>Methods/design</h4>This is a multinational, phase II, multicenter, randomized, double-blind, placebo-controlled trial with a sample size of 159 patients from 20 centers in 3 countries. Patients with stage IV colorectal adenocarcinoma limited to liver metastases are included. Following curative-intent complete resection of the primary tumor and of all synchronous/metachronous metastases, eligible patients are randomized 2:1 to receive either L-BLP25 or placebo. Those allocated to L-BLP25 receive a single dose of 300 mg/m2 cyclophosphamide (CP) 3 days before first L-BLP25 dose, then primary treatment with s.c. L-BLP25 930 μg once weekly for 8 weeks, followed by s.c. L-BLP25 930 μg maintenance doses at 6-week (years 1&2) and 12-week (year 3) intervals unless recurrence occurs. In the control arm, CP is replaced by saline solution and L-BLP25 by placebo. Primary endpoint is the comparison of recurrence-free survival (RFS) time between groups. Secondary endpoints are overall survival (OS) time, safety, tolerability, RFS/OS in MUC-1 positive cancers. Exploratory immune response analyses are planned. The primary endpoint will be assessed in Q3 2016. Follow-up will end Q3 2017. Interim analyses are not planned.<h4>Discussion</h4>The design and implementation of such a vaccination study in colorectal cancer is feasible. The study will provide recurrence-free and overall survival rates of groups in an unbiased fashion.<h4>Trial registration</h4>EudraCT Number 2011-000218-20.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef-PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE(65) acidic cluster on the N-terminal domain and W(113) in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef-PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.

<h4>Background</h4>Iron-refractory iron deficiency anaemia (IRIDA) is a rare disorder which was linked to mutations in two genes (SLC11A2 and TMPRSS6). Common polymorphisms within these genes were associated with serum iron levels. We identified a family of Serbian origin with asymptomatic non-consanguineous parents with three of four children presenting with IRIDA not responding to oral but to intravenous iron supplementation. After excluding all known causes responsible for iron deficiency anaemia we searched for mutations in SLC11A2 and TMPRSS6 that could explain the severe anaemia in these children.<h4>Methodology/results</h4>We sequenced the exons and exon-intron boundaries of SLC11A2 and TMPRSS6 in all six family members. Thereby, we found seven known and fairly common SNPs, but no new mutation. We then genotyped these seven SNPs in the population-based SAPHIR study (n = 1,726) and performed genetic association analysis on iron and ferritin levels. Only two SNPs, which were top-hits from recent GWAS on iron and ferritin, exhibited an effect on iron and ferritin levels in SAPHIR. Six SAPHIR participants carrying the same TMPRSS6 genotypes and haplotype-pairs as one anaemic son showed lower ferritin and iron levels than the average. One individual exhibiting the joint SLC11A2/TMPRSS6 profile of the anaemic son had iron and ferritin levels lying below the 5(th) percentile of the population's iron and ferritin level distribution. We then checked the genotype constellations in the Nijmegen Biomedical Study (n = 1,832), but the profile of the anaemic son did not occur in this population.<h4>Conclusions</h4>We cannot exclude a gene-gene interaction between SLC11A2 and TMPRSS6, but we can also not confirm it. As in this case candidate gene sequencing did not reveal causative rare mutations, the samples will be subjected to whole exome sequencing.

Cell migration is a crucial event for tissue organization during development, and its dysregulation leads to several diseases, including cancer. Cells exhibit various types of migration, such as single mesenchymal or amoeboid migration, collective migration and scaffold cell-dependent migration. The migration properties are partly dictated by cell adhesion and its endocytic regulation. While an epithelial-mesenchymal transition (EMT)-mediated mesenchymal cell migration requires the endocytic recycling of integrin-mediated adhesions after the disruption of cell-cell adhesions, an amoeboid migration is not dependent on any adhesions to extracellular matrix (ECM) or neighboring cells. In contrast, a collective migration is mediated by both cell-cell and cell-ECM adhesions, and a scaffold cell-dependent migration is regulated by the endocytosis and recycling of cell-cell adhesion molecules. Although some invasive carcinoma cells exhibit an EMT-mediated mesenchymal or amoeboid migration, other cancer cells are known to maintain cadherin-based cell-cell adhesions and epithelial morphology during metastasis. On the other hand, a scaffold cell-dependent migration is mainly utilized by migrating neurons in normal developing brains. This review will summarize the structures of cell adhesions, including adherens junctions and focal adhesions, and discuss the regulatory mechanisms for the dynamic behavior of cell adhesions by endocytic pathways in cell migration in physiological and pathological conditions, focusing particularly on neural development and cancer metastasis.

Human papillomavirus (HPV) infection is well established as an etiological agent responsible for a number of pathologies affecting the stratified epithelia of skin and anogenital sites. More recently, the infection by (mucosal) high-risk HPV types has also been found to be causally associated with squamous cell carcinoma in the head and neck region (HNSCC), especially in the oropharynx. Intriguingly, HPV-related oropharyngeal squamous cell carcinomas (OPSCC) represent a distinct clinical entity compared to HPV-negative tumors with particular regard to treatment-response and survival outcome. The association between HPV infection and OPSCC may therefore have important implications for the prevention and/or treatment of OPSCC. The improved survival of patients with HPV-related tumors also raises the question, as to whether a better understanding of the underlying differences may help to identify new therapeutic concepts that could be used in targeted therapy for HPV-negative and improved therapy for HPV-positive cancers. This review summarizes the most recent advances in our understanding of the molecular principles of HPV-related OPSCC, mainly based on functional genomic approaches, but also emphasizes the significant role played by the tumor microenvironment, especially the immune system, for improved clinical outcome and differential sensitivity of HPV-related tumors to current treatment options.

<h4>Introduction</h4>Premature ejaculation (PE) is defined as the inability of men to control ejaculation and it is the most prevalent male sexual dysfunction. The neurobiogenesis of ejaculation is very complex and involves the serotoninergic (5-hydroxytryptamine [5-HT]) system. A genetic etiology of PE in humans was stated accounting for around 30%. Recently, genetic polymorphisms located on SLC6A4 gene codifying for 5-HT transporter (5-HTT or serotonin transporter [SERT]), the major regulator of serotonergic neurotransmission, have been linked with the pathogenesis of PE and associated with the clinical response to therapy with contrasting results.<h4>Aim</h4>In order to establish a possible pathogenetic link between PE and SLC6A4 polymorphisms, we analyzed the 5-HTT-linked polymorphic region (5-HTTLPR), rs25531, and STin2 polymorphisms in 121 patients affected by lifelong and acquired PE.<h4>Methods</h4>Polymerase chain reaction (PCR)-based technology followed by restriction fragment length polymorphism (RFLP) analysis.<h4>Main outcome measures</h4>Intravaginal ejaculatory latency time was measured by stopwatch in order to diagnose PE, and the results of the SLC6A4 polymorphisms analysis in PE patients was compared with the control group.<h4>Results</h4>Genotype frequencies for 5-HTTLPR, rs25531, and STin2 for both patients and controls showed no significant deviation from Hardy-Weinberg equilibrium. No statistically significant differences were found in the frequency of SLC6A4 gene polymorphisms in PE patients vs. controls, or in lifelong PE patients vs. controls, or acquired PE patients vs. controls, or lifelong PE vs. acquired PE patients. The obtained data were contrasting with three out of four previously published reports.<h4>Conclusions</h4>The present results indicate that no difference exists in SLC6A4 polymorphisms frequency between PE patients and controls. A comparison with the previously published reports on this field is reported.

Many neurodegenerative disorders, although related by their destruction of brain function, display remarkable cellular and/or regional pathogenic specificity likely due to a deregulated functionality of the mutant protein. However, neurodegenerative disease genes, for example huntingtin (HTT), the ataxins, the presenilins (PSEN1/PSEN2) are not simply localized to neurons but are ubiquitously expressed throughout peripheral tissues; it is therefore paramount to properly understand the earliest precipitating events leading to neuronal pathogenesis to develop effective long-term therapies. This means, in no unequivocal terms, it is crucial to understand the gene's normal function. Unfortunately, many genes are often essential for embryogenesis which precludes their study in whole organisms. This is true for HTT, the β-amyloid precursor protein (APP) and presenilins, responsible for early onset Alzheimer's disease (AD). To better understand neurological disease in humans, many lower and higher eukaryotic models have been established. So the question arises: how reasonable is the use of organisms to study neurological disorders when the model of choice does not contain neurons? Here we will review the surprising, and novel emerging use of the model organism Dictyostelium discoideum, a species of soil-living amoeba, as a valuable biomedical tool to study the normal function of neurodegenerative genes. Historically, the evidence on the usefulness of simple organisms to understand the etiology of cellular pathology cannot be denied. But using an organism without a central nervous system to understand diseases of the brain? We will first introduce the life cycle of Dictyostelium, the presence of many disease genes in the genome and how it has provided unique opportunities to identify mechanisms of disease involving actin pathologies, mitochondrial disease, human lysosomal and trafficking disorders and host-pathogen interactions. Secondly, I will highlight recent studies on the function of HTT, presenilin γ-secretase and Hirano bodies conducted in Dictyostelium. I will then outline the limitations and future directions in using Dictyostelium to study disease, and finally conclude that given the evolutionary conservation of genes between Dictyostelium and humans and the organisms' genetic tractability, that this system provides a fertile environment for discovering normal gene function related to neurodegeneration and will permit translational studies in higher systems.

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.

No abstract available.

Hypoferremia, associated with immune system activation, involves a marked reduction in the levels of circulating iron, coupled with iron sequestration within macrophages. Toll-like receptor (TLR) signaling plays an important role in the development of the hypoferremic response, but how downstream signaling events affect genes involved in iron metabolism is incompletely understood. We investigated the involvement of MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling in the development of hypoferremia. Using MyD88-deficient and TRIF-deficient mice, we show that MyD88 and TRIF signaling pathways are critical for up-regulation by lipopolysaccharide (LPS) of the iron regulator hepcidin. In addition, MyD88 signaling is required for the induction of lipocalin 2 secretion and iron sequestration in the spleen. Activation of TLR4 and TLR3 signaling through LPS and polyinosinic:polycytidylic acid [poly(I:C)] treatments resulted in rapid down-regulation of HFE protein [encoded by the hemochromatosis gene (Hfe)] and ferroportin [encoded by solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1)] expression in the spleen, independent of MyD88 or TRIF signaling and proinflammatory cytokine production. However, lack of MyD88 signaling significantly impaired the hypoferremic response triggered by LPS, indicating that ferroportin and HFE protein down-regulation alone are insufficient to maintain hypoferremia. The extent of the hypoferremic response was found to be limited by initial, basal iron levels. Together, these results suggest that targeting specific TLR signaling pathways by affecting the function of adaptor molecules may provide new strategies to counteract iron sequestration within macrophages during inflammation.

<h4>Background</h4>Idiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile.<h4>Methodology/principal findings</h4>Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF.<h4>Conclusions/significance</h4>Our results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.

Cell-to-cell communication and exchange of materials are vital processes in multicellular organisms during cell development, cell repair, and cell survival. In neuronal and immunological cells, intercellular transmission between neighboring cells occurs via different complex junctions or synapses. Recently, long distance intercellular connections in mammalian cells called tunneling nanotubes (TNTs) have been described. These structures have been found in numerous cell types and shown to transfer signals and cytosolic materials between distant cells, suggesting that they might play a prominent role in intercellular trafficking. However, these cellular connections are very heterogeneous in both structure and function, giving rise to more questions than answers as to their nature and role as intercellular conduits. To better understand and characterize the functions of TNTs, we have highlighted here the latest discoveries regarding the formation, structure, and role of TNTs in cell-to-cell spreading of various signals and materials. We first gathered information regarding their formation with an emphasis on the triggering mechanisms observed, such as stress and potentially important proteins and/or signaling pathways. We then describe the various types of transfer mechanisms, in relation to signals and cargoes that have been shown recently to take advantage of these structures for intercellular transfer. Because a number of pathogens were shown to use these membrane bridges to spread between cells we also draw attention to specific studies that point toward a role for TNTs in pathogen spreading. In particular we discuss the possible role that TNTs might play in prion spreading, and speculate on their role in neurological diseases in general.

Development of the palate comprises sequential stages of growth, elevation, and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis. There is compelling evidence for the vital role of TGFβ-mediated regulation of palate development. We hypothesized that the isoforms of TGFβ regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM) cells were treated with TGFβ1, β2, and β3 for microarray-based gene expression studies in order to identify the roles of TGFβ in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFβ-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them are known to be crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGFβ1, TGFβ2, and TGFβR1). These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFβ treatment in HEPM cells. Our results suggest that both TGFβ1 and TGFβ2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by regulating expression of 234 genes.

The brain changes in response to experience and altered environment. To do that, the nervous system often remodels the structures of neuronal circuits. This structural plasticity of the neuronal circuits appears to be controlled not only by intrinsic factors, but also by extrinsic mechanisms including modification of the extracellular matrix. Recent studies employing a range of animal models implicate that matrix metalloproteinases regulate multiple aspects of the neuronal development and remodeling in the brain. This paper aims to summarize recent advances of our knowledge on the neuronal functions of matrix metalloproteinases and discuss how they might relate in neuronal disease.

Cooperativity between oncogenic mutations is recognized as a fundamental feature of malignant transformation, and it may be mediated by synergistic regulation of the expression of pro- and antitumorigenic target genes. However, the mechanisms by which oncogenes and tumor suppressors coregulate downstream targets and pathways remain largely unknown. Here, we used ChIP coupled to massively parallel sequencing (ChIP-seq) and gene expression profiling in mouse prostates to identify direct targets of the tumor suppressor Nkx3.1. Further analysis indicated that a substantial fraction of Nkx3.1 target genes are also direct targets of the oncoprotein Myc. We also showed that Nkx3.1 and Myc bound to and crossregulated shared target genes in mouse and human prostate epithelial cells and that Nkx3.1 could oppose the transcriptional activity of Myc. Furthermore, loss of Nkx3.1 cooperated with concurrent overexpression of Myc to promote prostate cancer in transgenic mice. In human prostate cancer patients, dysregulation of shared NKX3.1/MYC target genes was associated with disease relapse. Our results indicate that NKX3.1 and MYC coregulate prostate tumorigenesis by converging on, and crossregulating, a common set of target genes. We propose that coregulation of target gene expression by oncogenic/tumor suppressor transcription factors may represent a general mechanism underlying the cooperativity of oncogenic mutations during tumorigenesis.

We previously reported frequent truncating mutations of the RNA-binding protein gene, La ribonucleoprotein domain family, member-7 (LARP7) in gastric cancers (GCs) with frequent microsatellite instability. LARP7 negatively regulates positive transcription elongation factor-b (p-TEFb) by binding to and stabilizing 7sk RNA. p-TEFb has been linked to proliferation and de-differentiation in various tissues. Therefore, we reasoned that loss of LARP7 may contribute to gastric tumorigenesis. In this study, we evaluated LARP7 mRNA expression in 18 GCs, their corresponding non-neoplastic gastric tissues (N(GC)), and 18 normal gastric tissues from healthy individuals (N(N)). We also assessed the effects of transient small interfering (siRNA)-mediated LARP7 knockdown in immortalized non-neoplastic gastric epithelial cells. LARP7 mRNA was significantly decreased in GCs (median 2.5) relative to N(N)s (median 14.9, P<0.01) as well as relative to their corresponding N(GC)s (median 8.1, P<0.01). Transfection of an siRNA directed against LARP7 (anti-LARP7 siRNA) into non-neoplastic gastric epithelial cells decreased 7sk levels by 72% relative to a control siRNA (P<0.01). Furthermore, anti-LARP7 siRNA transfection increased cell proliferation by 23% (P<0.01) and cell migration by 22% (P<0.001) relative to control siRNA transfection. Taken together, these findings suggest that LARP7 downregulation occurs early during gastric tumorigenesis and may promote gastric tumorigenesis via p-TEFb dysregulation.

A previous genome-wide linkage study of alcohol dependence (AD) in multiplex families found a suggestive linkage result for a region on Chromosome 1 near microsatellite markers D1S196 and D1S2878. The ASTN1 gene is in this region, a gene previously reported to be associated with substance abuse, bipolar disorder and schizophrenia. Using the same family data consisting of 330 individuals with phenotypic data and DNA, finer mapping of a 26 cM region centered on D1S196 was undertaken using SNPs with minor allele frequency (MAF) ≥ 0.15 and pair-wise linkage disequilibrium (LD) of r(2) < 0.8 using the HapMap CEU population. Significant FBAT P-values for SNPs within the ASTN1 gene were observed for four SNPs (rs465066, rs228008, rs6668092, and rs172917), the most significant, rs228008, within intron 8 had a P-value of 0.001. Using MQLS, which allows for inclusion of all families, we find three of these SNPs with MQLS P-values < 0.003. In addition, two additional neighboring SNPs (rs10798496 and rs6667588) showed significance at P = 0.002 and 0.03, respectively. Haplotype analysis was performed using the haplotype-based test function of FBAT for a block that included rs228008, rs6668092, and rs172917. This analysis found one block (GCG) over-transmitted and another (ATA) under-transmitted to affected offspring. Linkage analysis identified a region consistent with the association results. Family-based association analysis shows the ASTN1 gene significantly associated with alcohol dependence. The potential importance of the ASTN1 gene for AD risk may be related its role in glial-guided neuronal migration.

Covault et al. [Covault et al. (2007); Biol Psychiatry 61(5): 609-616] reported that the common functional polymorphism, 5-HTTLPR, in the serotonin transporter gene moderated the association between past-year stressful events and daily reports of drinking in a sample of European-American (EA) college students. We examined this effect in college students of African descent. Students recruited at a Historically Black University (n = 564) completed web-based measures of past-year stressful life experiences and daily reports of drinking and heavy drinking over a 30-day period. Participants were genotyped for the tri-allelic 5-HTTLPR polymorphism and dichotomized as low-activity S' allele carriers or high-activity L' homozygotes. Generalized linear models were used to examine the effects of life stress, genotype, and their interaction on the two drinking measures. In students who completed 15 or more daily surveys (n = 393), there was a significant interaction of past-year stressful events, 5-HTTLPR genotype, and gender on the number of drinking days (P = 0.002). Similar findings were obtained in relation to heavy drinking days (P = 0.007). Men showed a main effect of past-year stressful events on both drinking outcomes (P's < 0.001), but no main or moderator effects of genotype. In women, the S' allele moderated the impact of past-year life stressors on the frequency of drinking and heavy drinking days (P's < 0.001). In college students of African descent, past-year stressful events were associated with more frequent drinking and heavy drinking, an effect that was moderated by the 5-HTTLPR polymorphism. However, in contrast to the findings in EA students, in the current sample, 5-HTTLPR moderated the association only among women.

Eating disorder patients show different long-term outcomes, and trait-related alterations of serotonergic function, which might be related with the serotonin transporter (5-HTT) gene. We studied the relationships between 5-HTTLPR polymorphism, eating specific and general psychopathology and the long-term outcome of anorexia nervosa (AN) and bulimia nervosa (BN) patients. We evaluated the distribution of the functional 5-HTTLPR polymorphism in a series of 201 Italian, Caucasian, eating disorder patients (113 with AN and 88 with BN binge/purging (BP subtype) and in 150 Caucasian unrelated controls. Prior to starting an individual cognitive behavior therapy, a clinical assessment was performed by means of the structured clinical interview for DSM-IV axis I disorders and several self-report questionnaires. This assessment was repeated at the end of treatment, 3 years after the end of treatment and 3 years after the first follow-up. Diagnostic changes between AN and BN were frequent (28.3%), and the presence of depressive disorders was associated with a higher rate of diagnostic crossover during the follow-up period. The S-allele of the 5-HTTLPR genotype increases the risk susceptibility for both depressive comorbidity (OR = 4.23; 95% CI, 1.45-12.37) and diagnostic crossover during the follow-up period in AN patients (OR = 5.04; 95% CI, 1.69-14.98). Logistic regression analyses confirmed these findings, when the interaction between genotype and psychiatric comorbidity as predictors of diagnostic instability in AN patients were taken into account. No significant association was found between 5-HTTLPR genotype and recovery. The S-allele of the 5-HTTLPR genotype increases the risk for depressive disorders comorbidity, and moderates the long-term outcome of anorectic patients.

Myelin is essential for the rapidity of saltatory nerve conduction, and also provides trophic support for axons to prevent axonal degeneration. Two critical determinants of myelination are SOX10 and EGR2/KROX20. SOX10 is required for specification of Schwann cells from neural crest, and is required at every stage of Schwann cell development. Egr2/Krox20 expression is activated by axonal signals in myelinating Schwann cells, and is required for cell cycle arrest and myelin formation. To elucidate the integrated function of these two transcription factors during peripheral nerve myelination, we performed in vivo ChIP-Seq analysis of myelinating peripheral nerve. Integration of these binding data with loss-of-function array data identified a range of genes regulated by these factors. In addition, although SOX10 itself regulates Egr2/Krox20 expression, leading to coordinate activation of several major myelin genes by the two factors, there is a large subset of genes that are activated independent of EGR2. Finally, the results identify a set of SOX10-dependent genes that are expressed in early Schwann cell development, but become subsequently repressed by EGR2/KROX20.

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A-RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)-dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.

The specific set of proteins bound at each genomic locus contributes decisively to regulatory processes and to the identity of a cell. Understanding of the function of a particular locus requires the knowledge of what factors interact with that locus and how the protein composition changes in different cell types or during the response to internal and external signals. Proteomic analysis of isolated chromatin segments (PICh) was developed as a tool to target, purify, and identify proteins associated with a defined locus and was shown to allow the purification of human telomeric chromatin. Here we have developed this method to identify proteins that interact with the Drosophila telomere-associated sequence (TAS) repeats. Several of the purified factors were validated as novel TAS-bound proteins by chromatin immunoprecipitation, and the Brahma complex was confirmed as a dominant modifier of telomeric position effect through the use of a genetic test. These results offer information on the efficacy of applying the PICh protocol to loci with sequence more complex than that found at human telomeres and identify proteins that bind to the TAS repeats, which might contribute to TAS biology and chromatin silencing.

Adenomyosis is a common estrogen-dependent disorder of females characterized by a downward extension of the endometrium into the uterine myometrium and neovascularization in ectopic lesions. It accounts for chronic pelvic pain, dysmenorrhea, menorrhagia, and infertility in 8.8-61.5% women worldwide. However, the molecular mechanisms for adenomyosis development remain poorly elucidated. Here, we utilized a two-dimensional polyacrylamide gel electrophoresis/MS-based proteomics analysis to compare and identify differentially expressed proteins in matched ectopic and eutopic endometrium of adenomyosis patients. A total of 93 significantly altered proteins were identified by tandem MS analysis. Further cluster analysis revealed a group of estrogen-responsive proteins as dysregulated in adenomyosis, among which annexin A2, a member of annexin family proteins, was found up-regulated most significantly in the ectopic endometrium of adenomyosis compared with its eutopic counterpart. Overexpression of ANXA2 was validated in ectopic lesions of human adenomyosis and was found to be tightly correlated with markers of epithelial to mesenchymal transition and dysmenorrhea severity of adenomyosis patients. Functional analysis demonstrated that estrogen could remarkably up-regulate ANXA2 and induce epithelial to mesenchymal transition in an in vitro adenomyosis model. Enforced expression of ANXA2 could mediate phenotypic mesenchymal-like cellular changes, with structural and functional alterations in a β-catenin/T-cell factor (Tcf) signaling-associated manner, which could be reversed by inhibition of ANXA2 expression. We also proved that enforced expression of ANXA2 enhanced the proangiogenic capacity of adenomyotic endometrial cells through HIF-1α/VEGF-A pathway. In vivo, we demonstrated that ANXA2 inhibition abrogated endometrial tissue growth, metastasis, and angiogenesis in an adenomyosis nude mice model and significantly alleviated hyperalgesia. Taken together, our data unraveled a dual role for ANXA2 in the pathogenesis of human adenomyosis through conferring endometrial cells both metastatic potential and proangiogenic capacity, which could serve as a potential therapeutic target for the treatment of adenomyosis patients.

<h4>Background</h4>In various animal models pregnancy diets have been shown to affect offspring phenotype. Indeed, the underlying programming of development is associated with modulations in birth weight, body composition, and continual diet-dependent modifications of offspring metabolism until adulthood, producing the hypothesis that the offspring's transcriptome is permanently altered depending on maternal diet.<h4>Methodology/principal findings</h4>To assess alterations of the offspring's transcriptome due to gestational protein supply, German Landrace sows were fed isoenergetic diets containing protein levels of either 30% (high protein--HP) or 12% (adequate protein--AP) throughout their pregnancy. Offspring muscle tissue (M. longissimus dorsi) was collected at 94 days post conception (dpc), and 1, 28, and 188 days post natum (dpn) for use with Affymetrix GeneChip Porcine Genome Arrays and subsequent statistical and Ingenuity pathway analyses. Numerous transcripts were found to have altered abundance at 94 dpc and 1 dpn; at 28 dpn no transcripts were altered, and at 188 dpn only a few transcripts showed a different abundance between diet groups. However, when assessing transcriptional changes across developmental time points, marked differences were obvious among the dietary groups. Depending on the gestational dietary exposure, short- and long-term effects were observed for mRNA expression of genes related to cell cycle regulation, energy metabolism, growth factor signaling pathways, and nucleic acid metabolism. In particular, the abundance of transcripts related to cell cycle remained divergent among the groups during development.<h4>Conclusion</h4>Expression analysis indicates that maternal protein supply induced programming of the offspring's genome; early postnatal compensation of the slight growth retardation obvious at birth in HP piglets resulted, as did a permanently different developmental alteration and responsiveness to the common environment of the transcriptome. The transcriptome modulations are interpreted as the molecular equivalent of developmental plasticity of the offspring that necessitates adaptation and maintenance of the organismal phenotype.

During normal aging or neurodegenerative diseases, neuronal survival and function depend on protein homeostasis, which is regulated by multiple mechanisms, including the microRNA (miRNA) pathway. In different cells types, the absence of Dicer, a key miRNA processing enzyme, leads to neurodegeneration through cell-autonomous and non-cell-autonomous mechanisms. Loss of certain miRNAs also causes neurodegeneration in some model organisms. On the other hand, miRNA expression is misregulated in patients with different neurodegenerative diseases. Thus, the miRNA pathway appears to be essential in the pathogenesis of several age-dependent neurodegenerative conditions; however, our understanding of the underlying mechanism remains rudimentary. The precise causal relationships between specific miRNAs and neurodegeneration in humans need to be further investigated.

In the last few decades great thrust has been put in the area of regenerative neurobiology research to combat brain injuries and neurodegenerative diseases. The recent discovery of neurogenic niches in the adult brain has led researchers to study how to mobilize these cells to orchestrate an endogenous repair mechanism. The brain can minimize injury-induced damage by means of an immediate glial response and by initiating repair mechanisms that involve the generation and mobilization of new neurons to the site of injury where they can integrate into the existing circuit. This review highlights the current status of research in this field. Here, we discuss the changes that take place in the neurogenic milieu following injury. We will focus, in particular, on the cellular and molecular controls that lead to increased proliferation in the Sub ventricular Zone (SVZ) as well as neurogenesis. We will also concentrate on how these cellular and molecular mechanisms influence the migration of new cells to the affected area and their differentiation into neuronal/glial lineage that initiate the repair mechanism. Next, we will discuss some of the different factors that limit/retard the repair process and highlight future lines of research that can help to overcome these limitations. A clear understanding of the underlying molecular mechanisms and physiological changes following brain damage and the subsequent endogenous repair should help us develop better strategies to repair damaged brains.

The objective of this study was to investigate the efficiency of multifunctional poly(ethylene glycol)-based hemoglobin conjugates crosslinked with antioxidant enzymes for their ability to protect an oxygen carrier (hemoglobin) and insulin secreting islets from the combination of hypoxic and free radical stress under simulated transplantation conditions. In this study, RINm5F cells and isolated pancreatic islets were challenged with oxidants (H(2)O(2) or xanthine and xanthine oxidase) and incubated with conjugates (hemoglobin-hemoglobin or superoxide dismutase-catalase-hemoglobin) in normoxia (21% oxygen) or hypoxia (6% or 1% oxygen). Hemoglobin protection, intracellular free radical activity and cell viability in RINm5F cells measured by methemoglobin, dichlorofluorescein-diacetate, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, respectively, showed that cells were better protected by conjugates containing antioxidant enzymes. Insulin secretion from islets and qualitative confocal evaluation of viability showed beta cells were protected by conjugates containing antioxidant enzymes when exposed to induced stress. Our study suggested that antioxidant enzymes play a significant role in hemoglobin protection and thus extended cell protection.

No abstract available.

The LOTUS or OST-HTH domain is a recently discovered motif of about 80 amino acids and is found in several germline-specific proteins including the Tudor domain-containing proteins TDRD5 and TDRD7, which are important for germ cell development. The LOTUS domain is an RNA binding domain but its exact function is unknown. Here, we report the (1)H, (13)C and (15)N resonance assignments for the three LOTUS domains present in mouse TDRD7. These assignments will allow three-dimensional structure determination of the LOTUS domains and mapping of their interaction with RNA, steps toward deciphering the function of TDRD7.

<h4>Background</h4>Chronic alcohol exposure produces neuroadaptation, which increases the risk of cellular excitotoxicity and autonomic dysfunction during withdrawal. The temporal progression and regulation of the gene expression that contributes to this physiologic and behavioral phenotype is poorly understood early in the withdrawal period. Further, it is unexplored in the dorsal vagal complex (DVC), a brainstem autonomic regulatory structure.<h4>Methods</h4>We use a quantitative polymerase chain reaction platform to precisely and simultaneously measure the expression of 145 neuromodulatory genes in more than 100 rat DVC samples from control, chronically alcohol-exposed, and withdrawn rats. To gain insight into the dynamic progression and regulation of withdrawal, we focus on the expression of a subset of functionally relevant genes during the first 48 hours, when behavioral symptoms are most severe.<h4>Results</h4>In the DVC, expression of this gene subset is essentially normal in chronically alcohol-exposed rats. However, withdrawal results in rapid, large-magnitude expression changes in this group. We observed differential regulation in 86 of the 145 genes measured (59%), some as early as 4 hours into withdrawal. Time series measurements (4, 8, 18, 32, and 48 hours after alcohol removal) revealed dynamic expression responses in immediate early genes, γ-aminobutyric acid type A, ionotropic glutamate, and G-protein coupled receptors and the Ras/Raf signaling pathway. Together, these changes elucidate a complex, temporally coordinated response that involves correlated expression of many functionally related groups. In particular, the expression patterns of Gabra1, Grin2a, Grin3a, and Grik3 were tightly correlated. These receptor subunits share overrepresented transcription factor binding sites for Pax-8 and other transcription factors, suggesting a common regulatory mechanism and a role for these transcription factors in the regulation of neurotransmission within the first 48 hours of alcohol withdrawal.<h4>Conclusions</h4>Expression in this gene set is essentially normal in the alcohol-adapted DVC, but withdrawal results in immediate, large-magnitude, and dynamic changes. These data support both increased research focus on the biological ramifications of alcohol withdrawal and enable novel insights into the dynamic withdrawal expression response in this understudied homeostatic control center.

The myotonic dystrophies (DM) are human diseases in which the accumulation of toxic RNA (CUG or CCUG) repeats in the cell causes sequestration of splicing factors, including MBNL1, leading to clinical symptoms such as muscle wasting and myotonia. We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro. We now report transformation of those compounds into structures with activity in vivo. Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG-CUG duplex RNA. Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG-RNA retention. Most importantly, two compounds are able to partially restore splicing in a mouse model of DM1.

Forkhead box protein p1 (Foxp1), a transcription factor showing highly enriched expression in the striatum, has been implicated in central nervous system (CNS) development, but its role in the mature brain is unknown. In order to ascertain functional roles for Foxp1 in the CNS, we have identified gene targets for Foxp1 both in vitro and in vivo using genome-wide expression microarrays and chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-seq) assays. We found that mouse Foxp1 overexpression in striatal cells elicited expression changes of genes related to immune signaling, transcriptional regulation and a manually curated Huntington's disease (HD)-signaling pathway. Similar results were found when the gene expression data set was integrated with Foxp1-binding data determined from ChIP-seq analysis. In vivo lentiviral-mediated overexpression of human FOXP1 in the context of mutant huntingtin (Htt) protein resulted in a robust downregulation of glial cell-associated, immune genes, including those encoding a variety of cytokines and chemokines. Furthermore, Foxp1-induced expression changes were significantly negatively correlated with those changes elicited by mutant Htt protein in several different HD mouse models, and most significantly in post-mortem caudate from human HD subjects. We finally show that Foxp1 interacts with mutant Htt protein in mouse brain and is present in nuclear Htt aggregates in the striatum of R6/1 transgenic mice. These findings implicate Foxp1 as a key repressor of immune signaling in the CNS and suggest that the loss of Foxp1-mediated gene regulation in HD contributes to the immune dysfunction in this disease. We further suggest that Foxp1-regulated pathways might be important mediators of neuronal-glial cell communication.

Observational studies of health conditions and outcomes often combine clinical care data from many sites without explicitly assessing the accuracy and completeness of these data. In order to improve the quality of data in an international multi-site observational cohort of HIV-infected patients, the authors conducted on-site, Good Clinical Practice-based audits of the clinical care datasets submitted by participating HIV clinics. Discrepancies between data submitted for research and data in the clinical records were categorized using the audit codes published by the European Organization for the Research and Treatment of Cancer. Five of seven sites had error rates >10% in key study variables, notably laboratory data, weight measurements, and antiretroviral medications. All sites had significant discrepancies in medication start and stop dates. Clinical care data, particularly antiretroviral regimens and associated dates, are prone to substantial error. Verifying data against source documents through audits will improve the quality of databases and research and can be a technique for retraining staff responsible for clinical data collection. The authors recommend that all participants in observational cohorts use data audits to assess and improve the quality of data and to guide future data collection and abstraction efforts at the point of care.

To elucidate target molecules of white matter development responding to hypothyroidism, global gene expression profiling of cerebral white matter from male rat offspring was performed after maternal exposure to anti-thyroid agents, 6-propyl-2-thiouracil and methimazole, on postnatal day 20. Genes involved in central nervous system development commonly up- or down-regulated among groups treated with anti-thyroid agents. Immunohistochemical distributions of vimentin, Ret proto-oncogene (Ret), deleted in colorectal cancer protein (DCC), and Claudin11 (Cld11) were examined based on the gene expression profile. Immunoreactive cells for vimentin and Ret in the cingulum, and the immunoreactive intensity of Cld11 and DCC in whole white matter were increased by treatment with anti-thyroid agents. Immunoreactive cells for vimentin and Ret were immature astrocytes and oligodendrocytes, respectively. Thus, immunoreactive cells for vimentin and Ret may be quantitatively measurable targets of developmental hypothyroidism in white matter.

Neural stem (NS) cells are a limitless resource, and thus superior to primary neurons for drug discovery provided they exhibit appropriate disease phenotypes. Here we established NS cells for cellular studies of Huntington's disease (HD). HD is a heritable neurodegenerative disease caused by a mutation resulting in an increased number of glutamines (Q) within a polyglutamine tract in Huntingtin (Htt). NS cells were isolated from embryonic wild-type (Htt(7Q/7Q)) and "knock-in" HD (Htt(140Q/140Q)) mice expressing full-length endogenous normal or mutant Htt. NS cells were also developed from mouse embryonic stem cells that were devoid of Htt (Htt(-/-)), or knock-in cells containing human exon1 with an N-terminal FLAG epitope tag and with 7Q or 140Q inserted into one of the mouse alleles (Htt(F7Q/7Q) and Htt(F140Q/7Q)). Compared to Htt(7Q/7Q) NS cells, HD Htt(140Q/140Q) NS cells showed significantly reduced levels of cholesterol, increased levels of reactive oxygen species (ROS), and impaired motility. The heterozygous Htt(F140Q/7Q) NS cells had increased ROS and decreased motility compared to Htt(F7Q/7Q). These phenotypes of HD NS cells replicate those seen in HD patients or in primary cell or in vivo models of HD. Huntingtin "knock-out" NS cells (Htt(-/-)) also had impaired motility, but in contrast to HD cells had increased cholesterol. In addition, Htt(140Q/140Q) NS cells had higher phospho-AKT/AKT ratios than Htt(7Q/7Q) NS cells in resting conditions and after BDNF stimulation, suggesting mutant htt affects AKT dependent growth factor signaling. Upon differentiation, the Htt(7Q/7Q) and Htt(140Q/140Q) generated numerous Beta(III)-Tubulin- and GABA-positive neurons; however, after 15 days the cellular architecture of the differentiated Htt(140Q/140Q) cultures changed compared to Htt(7Q/7Q) cultures and included a marked increase of GFAP-positive cells. Our findings suggest that NS cells expressing endogenous mutant Htt will be useful for study of mechanisms of HD and drug discovery.

This review aims to provide a comprehensive overview of the recent advances made in the field of ionic liquids in peptide chemistry and peptide analytics.

Scientific research has shifted from studies conducted by single investigators to the creation of large consortia. Genetic epidemiologists, for example, now collaborate extensively for genome-wide association studies (GWAS). The effect has been a stream of confirmed disease-gene associations. However, effects on human subjects oversight, data-sharing, publication and authorship practices, research organization and productivity, and intellectual property remain to be examined. The aim of this analysis was to identify all research consortia that had published the results of a GWAS analysis since 2005, characterize them, determine which have publicly accessible guidelines for research practices, and summarize the policies in these guidelines. A review of the National Human Genome Research Institute's Catalog of Published Genome-Wide Association Studies identified 55 GWAS consortia as of April 1, 2011. These consortia were comprised of individual investigators, research centers, studies, or other consortia and studied 48 different diseases or traits. Only 14 (25%) were found to have publicly accessible research guidelines on consortia websites. The available guidelines provide information on organization, governance, and research protocols; half address institutional review board approval. Details of publication, authorship, data-sharing, and intellectual property vary considerably. Wider access to consortia guidelines is needed to establish appropriate research standards with broad applicability to emerging forms of large-scale collaboration.

Species-specific lung tumors in the mouse are induced by a number of chemicals. The underlying cause appears to be a high metabolic activity of mouse lung, due to relatively high abundance of Clara cells in mice compared with humans and the mouse-specific cytochrome P450 isoform 2f2 in the Clara cells. The chemicals are activated to reactive intermediates, leading to local cytotoxicity or mitogenicity resulting in increased cell proliferation and tumors. Rats have lower metabolic activity than mice (already below the threshold needed to cause lung tumors upon lifetime exposure) and activity in humans is lower than in rats. The carcinogenic risk for human lung is low for this mode of action (MOA). Fluensulfone has shown an increased incidence of lung adenomas in mice, but not in rats, at high doses. Fluensulfone is not genotoxic. MOA studies were conducted investigating key events of the postulated MOA. Fluensulfone is extensively metabolized by mouse lung microsomes, whereas no metabolic activity is seen with human lung microsomes. Cyp 2f2 is a major contributor in fluensulfone's metabolism and Cyp 2e1 is not involved. Furthermore, administration of fluensulfone to mice led to an early increase in Clara cell proliferation. The International Programme on Chemical Safety (IPCS) MOA and human relevance framework was used to evaluate the collective data on fluensulfone. We concluded that fluensulfone leads to species-specific mouse lung tumors and that these tumors are likely not relevant to human hazard or risk.

Seminal oxidative stress occurs when there is an increased production of reactive oxygen species (ROS) and/or a decrease of antioxidant activity, promoting impaired sperm function. Peroxiredoxins (PRDX) are abundant in human semen and are important antioxidant enzymes, which act as ROS scavengers and modulators in ROS-dependent signaling. Our aim was to determine whether the levels of PRDX1 and PRDX6 and their oxidation on thiol groups are associated with a decrease in sperm motility and DNA integrity. We evaluated the sperm and seminal PRDX level in men (13 healthy controls, 15 men with clinical varicocele, and 17 men with idiopathic infertility). We assessed conventional semen parameters, sperm DNA integrity (by the sperm chromatin structure assay), lipid peroxidation in seminal plasma and spermatozoa (by the thiobarbituric acid reactive substances assay), and the amount and thiol oxidation of PRDX1 and PRDX6 (by immunoblotting). PRDXs were affected in seminal plasma (lower amounts) and in sperm samples (lower amounts and higher levels of thiol oxidation) characterized by lower sperm motility, higher lipid peroxidation, and sperm DNA damage. The thioloxidation ratio of PRDXs (thiol-oxidized PRDX/total PRDX) correlated negatively with sperm motility (total and progressive) and positively with sperm DNA damage and sperm lipid peroxidation. In conclusion, because of the lower amount of total PRDX1 and PRDX6 and the high thiol oxidation of these PRDXs, very little (less than 20%) protection due to PRDXs remains, and this is associated with impaired sperm function and poor DNA integrity and suggests an important role of PRDXs in the protection of human spermatozoa against oxidative stress.

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of brain tumor-related death in children. DIPG is not surgically resectable, resulting in a paucity of tissue available for molecular studies. As such, tumor biology is poorly understood, and, currently, there are no effective treatments. In the absence of frozen tumor specimens, body fluids--such as cerebrospinal fluid (CSF), serum, and urine--can serve as more readily accessible vehicles for detecting tumor-secreted proteins. We analyzed a total of 76 specimens, including CSF, serum, urine, and normal and tumor brainstem tissue. Protein profiling of CSF from patients with DIPG was generated by mass spectrometry using an LTQ-Orbitrap-XL and database search using the Sequest algorithm. Quantitative and statistical analyses were performed with ProteoIQ and Partek Genomics Suite. A total of 528 unique proteins were identified, 71% of which are known secreted proteins. CSF proteomic analysis revealed selective upregulation of Cyclophillin A (CypA) and dimethylarginase 1 (DDAH1) in DIPG (n = 10), compared with controls (n = 4). Protein expression was further validated with Western blot analysis and immunohistochemical assays using CSF, brain tissue, serum, and urine from DIPG and control specimens. Immunohistochemical staining showed selective upregulation of secreted but not cytosolic CypA and DDAH1 in patients with DIPG. In this study, we present the first comprehensive protein profile of CSF specimens from patients with DIPG to demonstrate selective expression of tumor proteins potentially involved in brainstem gliomagenesis. Detection of secreted CypA and DDAH1 in serum and urine has potential clinical application, with implications for assessing treatment response and detecting tumor recurrence in patients with DIPG.

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions-particularly those expressed with low abundance- is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample.

Pseudomonas aeruginosa, an important opportunistic pathogen of man, exploits numerous factors for initial attachment to the host, an event required to establish bacterial infection. In this paper, we rigorously explore the role of two major bacterial adhesins, type IV pili (Tfp) and flagella, in bacterial adherence to distinct host receptors at the apical (AP) and basolateral (BL) surfaces of polarized lung epithelial cells and induction of subsequent host signaling and pathogenic events. Using an isogenic mutant of P. aeruginosa that lacks flagella or utilizing beads coated with purified Tfp, we establish that Tfp are necessary and sufficient for maximal binding to host N-glycans at the AP surface of polarized epithelium. In contrast, experiments utilizing a P. aeruginosa isogenic mutant that lacks Tfp or using beads coated with purified flagella demonstrate that flagella are necessary and sufficient for maximal binding to heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) at the BL surface of polarized epithelium. Using two different cell-free systems, we demonstrate that Tfp-coated beads show highest binding affinity to complex N-glycan chains coated onto plastic plates and preferentially aggregate with beads coated with N-glycans, but not with single sugars or HS. In contrast, flagella-coated beads bind to or aggregate preferentially with HS or HSPGs, but demonstrate little binding to N-glycans. We further show that Tfp-mediated binding to host N-glycans results in activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and bacterial entry at the AP surface. At the BL surface, flagella-mediated binding to HS activates the epidermal growth factor receptor (EGFR), adaptor protein Shc, and PI3K/Akt, and induces bacterial entry. Remarkably, flagella-coated beads alone can activate EGFR and Shc. Together, this work provides new insights into the intricate interactions between P. aeruginosa and lung epithelium that may be potentially useful in the development of novel treatments for P. aeruginosa infections.

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7)GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.

Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes.

<h4>Background</h4>Spontaneous preterm birth (SPB, before 37 gestational weeks) is a major cause of perinatal mortality and morbidity, but its pathogenesis remains unclear. Studies on SPB have been hampered by the limited availability of markers for SPB in predelivery clinical samples that can be easily compared with gestational age-matched normal controls. We hypothesize that SPB involves aberrant placental RNA expression, and that such RNA transcripts can be detected in predelivery maternal plasma samples, which can be compared with gestational age-matched controls.<h4>Principal findings</h4>Using gene expression microarray to profile essentially all human genes, we observed that 426 probe signals were changed by >2.9-fold in the SPB placentas, compared with the spontaneous term birth (STB) placentas. Among the genes represented by those probes, we observed an over-representation of functions in RNA stabilization, extracellular matrix binding, and acute inflammatory response. Using RT-quantitative PCR, we observed differences in the RNA concentrations of certain genes only between the SPB and STB placentas, but not between the STB and term elective cesarean delivery placentas. Notably, 36 RNA transcripts were observed at placental microarray signals higher than a threshold, which indicated the possibility of their detection in maternal plasma. Among them, the IL1RL1 mRNA was tested in plasma samples taken from 37 women. It was detected in 6 of 10 (60%) plasma samples collected during the presentation of preterm labor (≤32.9 weeks) in women eventually giving SPB, but was detected in only 1 of 27 (4%) samples collected during matched gestational weeks from women with no preterm labor (Fisher exact test, p = 0.00056).<h4>Conclusion</h4>We have identified 36 SPB-associated RNA transcripts, which are possibly detectable in maternal plasma. We have illustrated that the IL1RL1 mRNA was more frequently detected in predelivery maternal plasma samples collected from women resulting in SPB than the gestational-age matched controls.

Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51-121.01 μM within single milked venom samples. This provides for a 6.37-20,965 fold-excess of toxin to induce apparent IC₅₀ for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus.

Imatinib therapy has undoubtedly contributed to the treatment of metastatic gastrointestinal stromal (GIST) tumors that were previously untreatable. However, disease progression during treatment with tyrosine kinase inhibitors remains an issue in clinical practice not fully explained by KIT and PDGFRA mutation status. We investigated the role of three important signaling molecules (insulin-like growth factor 1 receptor [IGF1R], protein kinase C-θ [PKCθ], and Raf kinase inhibitor protein [RKIP]) that have been implicated in GIST pathogenesis as potential biomarkers for prediction of response to imatinib treatment. We retrospectively reviewed 76 patients with metastatic GIST submitted to imatinib treatment between 2002 and 2007, and analyzed 63 of them. Insulin-like growth factor 1, total PKCθ, phosphorylated PKCθ, and RKIP immunohistochemical expression were correlated with objective response to imatinib treatment and progression-free and overall survival. Median follow-up was 31.2 mo (95% confidence interval, 26.3-36.1 mo). There was a statistically significant association between IGF1R expression and type of response to imatinib treatment (P = 0.05)-that is, higher IGF1R expression was related to lower objective response. However, IGF1R higher expression did not affect progression-free and overall survival. Insulin-like growth factor 1, but not PKCθ and RKIP, emerges as a potential biomarker for prediction of response to imatinib treatment in metastatic GISTs. Validation studies are warranted.

This review is focused on the materials and methods used to fabricate closed-loop systems for type 1 diabetes therapy. Herein, we give a brief overview of current methods used for patient care and discuss two types of possible treatments and the materials used for these therapies-(i) artificial pancreases, comprised of insulin producing cells embedded in a polymeric biomaterial, and (ii) totally synthetic pancreases formulated by integrating continuous glucose monitors with controlled insulin release through degradable polymers and glucose-responsive polymer systems. Both the artificial and the completely synthetic pancreas have two major design requirements: the device must be both biocompatible and be permeable to small molecules and proteins, such as insulin. Several polymers and fabrication methods of artificial pancreases are discussed: microencapsulation, conformal coatings, and planar sheets. We also review the two components of a completely synthetic pancreas. Several types of glucose sensing systems (including materials used for electrochemical, optical, and chemical sensing platforms) are discussed, in addition to various polymer-based release systems (including ethylene-vinyl acetate, polyanhydrides, and phenylboronic acid containing hydrogels).

<h4>Background</h4>No disease modifying treatment currently exists for Huntington's disease (HD), a fatal neurodegenerative disorder characterized by the formation of amyloid-like aggregates of the mutated huntingtin protein. Curcumin is a naturally occurring polyphenolic compound with Congo red-like amyloid binding properties and the ability to cross the blood brain barrier. CAG140 mice, a knock-in (KI) mouse model of HD, display abnormal aggregates of mutant huntingtin and striatal transcriptional deficits, as well as early motor, cognitive and affective abnormalities, many months prior to exhibiting spontaneous gait deficits, decreased striatal volume, and neuronal loss. We have examined the ability of life-long dietary curcumin to improve the early pathological phenotype of CAG140 mice.<h4>Results</h4>KI mice fed a curcumin-containing diet since conception showed decreased huntingtin aggregates and increased striatal DARPP-32 and D1 receptor mRNAs, as well as an amelioration of rearing deficits. However, similar to other antioxidants, curcumin impaired rotarod behavior in both WT and KI mice and climbing in WT mice. These behavioral effects were also noted in WT C57Bl/6 J mice exposed to the same curcumin regime as adults. However, neither locomotor function, behavioral despair, muscle strength or food utilization were affected by curcumin in this latter study. The clinical significance of curcumin's impairment of motor performance in mice remains unclear because curcumin has an excellent blood chemistry and adverse event safety profile, even in the elderly and in patients with Alzheimer's disease.<h4>Conclusion</h4>Together with this clinical experience, the improvement in several transgene-dependent parameters by curcumin in our study supports a net beneficial effect of dietary curcumin in HD.

Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C(2)H(2) zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons observed in Bcl11a mutant mice interferes with their correct innervation by cutaneous sensory neurons. To understand the mechanism underlying the innervation deficit, we characterized changes in gene expression in the dorsal horn of Bcl11a mutants and identified dysregulated expression of the gene encoding secreted frizzled-related protein 3 (sFRP3, or Frzb). Frzb mutant mice show a deficit in the innervation of the spinal cord, suggesting that the dysregulated expression of Frzb can account in part for the phenotype of Bcl11a mutants. Thus, our genetic analysis of Bcl11a reveals essential functions of this transcription factor in neuronal morphogenesis and sensory wiring of the dorsal spinal cord and identifies Frzb, a component of the Wnt pathway, as a downstream acting molecule involved in this process.

Small-molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of proapoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of an SMC library was performed using stable EphA4-expressing NIH 3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.

It is well established that autism spectrum disorders (ASD) have a strong genetic component; however, for at least 70% of cases, the underlying genetic cause is unknown. Under the hypothesis that de novo mutations underlie a substantial fraction of the risk for developing ASD in families with no previous history of ASD or related phenotypes--so-called sporadic or simplex families--we sequenced all coding regions of the genome (the exome) for parent-child trios exhibiting sporadic ASD, including 189 new trios and 20 that were previously reported. Additionally, we also sequenced the exomes of 50 unaffected siblings corresponding to these new (n = 31) and previously reported trios (n = 19), for a total of 677 individual exomes from 209 families. Here we show that de novo point mutations are overwhelmingly paternal in origin (4:1 bias) and positively correlated with paternal age, consistent with the modest increased risk for children of older fathers to develop ASD. Moreover, 39% (49 of 126) of the most severe or disruptive de novo mutations map to a highly interconnected β-catenin/chromatin remodelling protein network ranked significantly for autism candidate genes. In proband exomes, recurrent protein-altering mutations were observed in two genes: CHD8 and NTNG1. Mutation screening of six candidate genes in 1,703 ASD probands identified additional de novo, protein-altering mutations in GRIN2B, LAMC3 and SCN1A. Combined with copy number variant (CNV) data, these results indicate extreme locus heterogeneity but also provide a target for future discovery, diagnostics and therapeutics.

The biosynthetic gene cluster for endophenazines, i.e., prenylated phenazines from Streptomyces anulatus 9663, was heterologously expressed in several engineered host strains derived from Streptomyces coelicolor M145. The highest production levels were obtained in strain M512. Mutations in the rpoB and rpsL genes of the host, which result in increased production of other secondary metabolites, had no beneficial effect on the production of phenazines. The heterologous expression strains produced, besides the known phenazine compounds, a new prenylated phenazine, termed endophenazine E. The structure of endophenazine E was determined by high-resolution mass spectrometry and by one- and two-dimensional NMR spectroscopy. It represented a conjugate of endophenazine A (9-dimethylallylphenazine-1-carboxylic acid) and L-glutamine (L-Gln), with the carboxyl group of endophenazine A forming an amide bond to the α-amino group of L-Gln. Gene inactivation experiments in the gene cluster proved that ppzM codes for a phenazine N-methyltransferase. The gene ppzV apparently represents a new type of TetR-family regulator, specifically controlling the prenylation in endophenazine biosynthesis. The gene ppzY codes for a LysR-type regulator and most likely controls the biosynthesis of the phenazine core. A further putative transcriptional regulator is located in the vicinity of the cluster, but was found not to be required for phenazine or endophenazine formation. This is the first investigation of the regulatory genes of phenazine biosynthesis in Streptomyces.

Two aromatic amino acids, Tyr(1) and Phe(3) or Phe(4), are important structural elements in opioid peptides because they interact with opioid receptors. The usefulness of an artificial amino acid residue 2',6'-dimethylphenylalanine (Dmp) was investigated as an aromatic amino acid surrogate for several opioid peptides, including enkephalin, dermorphin, deltorphin, endomorphin, dynorphin A, and nociceptin peptides. In most peptides, substitutions of Phe(3) by a Dmp residue produced analogs with improved receptor-binding affinity and selectivity, while the same substitution of Phe(4) induced markedly reduced receptor affinity and selectivity. Interestingly, replacement of Tyr(1) by Dmp produced analogs with unexpectedly high affinity or produced only a slight drop in receptor affinity and bioactivity for most peptides. Thus, Dmp is also a useful surrogate for the N-terminal Tyr residue in opioid peptides despite the lack of a phenolic hydroxyl group, which is considered necessary for opioid activity. The Dmp(1)-substituted analogs are superior to 2',6'-dimethyltyrosine (Dmt)(1)-substituted analogs for high receptor selectivity since the latter generally have poor receptor selectivity. Thus, Dmp is very useful as an aromatic amino acid surrogate in opioid peptides and may be useful for developing other novel peptide mimetics with high receptor specificity.

<h4>Background</h4>Human olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.<h4>Methods</h4>OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H2O2) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.<h4>Results</h4>The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H2O2 or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H(2)O(2) or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).<h4>Conclusion</h4>Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H2O2 or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.

<h4>Unlabelled</h4><h4>Background</h4>3D domain swapping is a novel structural phenomenon observed in diverse set of protein structures in oligomeric conformations. A distinct structural feature, where structural segments in a protein dimer or higher oligomer were shared between two or more chains of a protein structure, characterizes 3D domain swapping. 3D domain swapping was observed as a key mediator of numerous functional mechanisms and play pathogenic role in various diseases including conformational diseases like amyloidosis, Alzheimer's disease, Parkinson's disease and prion diseases. We report the first study with a focus on identifying functional classes, pathways and diseases mediated by 3D domain swapping in the human proteome.<h4>Methods</h4>We used a panel of four enrichment tools with two different ontologies and two annotations database to derive biological and clinical relevant information associated with 3D domain swapping. Protein domain enrichment analysis followed by Gene Ontology (GO) term enrichment analysis revealed the functional repertoire of proteins involved in swapping. Pathway analysis using KEGG annotations revealed diverse pathway associations of human proteins involved in 3D domain swapping. Disease Ontology was used to find statistically significant associations with proteins in swapped conformation and various disease categories (P-value < 0.05).<h4>Results</h4>We report meta-analysis results of a literature-curated dataset of human gene products involved in 3D domain swapping and discuss new insights about the functional repertoire, pathway associations and disease implications of proteins involved in 3D domain swapping.<h4>Conclusions</h4>Our integrated bioinformatics pipeline comprising of four different enrichment tools, two ontologies and two annotations revealed new insights into the functional and disease correlations with 3D domain swapping. GO term enrichment were used to infer terms associated with three different GO categories. Protein domain enrichment was used to identify conserved domains enriched in swapped proteins. Pathway enrichment analysis using KEGG annotations revealed that proteins with swapped conformations are present in all six classes of KEGG BRITE hierarchy and significantly enriched KEGG pathways were observed in five classes. Five major classes of disease were found to be associated with 3D domain swapping using functional disease ontology based enrichment analysis. Five classes of human diseases: cancer, diseases of the respiratory or pulmonary system, degenerative diseases of the central nervous system, vascular disease and encephalitis were found to be significant. In conclusion, our study shows that bioinformatics based analytical approaches using curated data can enhance the understanding of functional and disease implications of 3D domain swapping.

Although psychiatric disorders such as autism spectrum disorders, schizophrenia and bipolar disorder affect a number of brain regions and produce a complex array of clinical symptoms, basic phenotypes likely exist at the level of single neurons and simple networks. Being highly heritable, it is hypothesized that these disorders are amenable to cell-based studies in vitro. Using induced pluripotent stem cell-derived neurons and/or induced neurons from fibroblasts, limitless numbers of live human neurons can now be generated from patients with a genetic background permissive to the disease state. We predict that cell-based studies will ultimately contribute to our understanding of the initiation, progression and treatment of these psychiatric disorders.

The ability of Rhodococcus rhodochrous (NCIMB 9703) to catalyse the regio- and stereoselective hydroxylation of a range of benzyloxy-substituted heterocycles has been investigated. Incubation of 2-benzyloxytetrahydropyrans with resting cell suspensions of the organism yielded predominantly a mixture of 5-hydroxylated isomers in combined yields of up to 40%. Exposure of the corresponding 2-benzyloxytetrahydrofuran derivatives to the cell suspensions gave predominantly the 4-hydroxylated isomers in yields of up to 26%. Most interestingly, 2-(4-nitrobenzyloxy)tetrahydrofuran and 2-(4-nitrobenzyloxy)tetrahydropyran were transformed in high yields to the 4-hydroxylated and 5-hydroxylated products, respectively.

Increasing evidence has pointed to an important role of SUMOylation in cell cycle regulation, especially for M phase. In the current studies, we have obtained evidence through in vitro studies that the master M phase regulator CDK1/cyclin B kinase phosphorylates the SUMOylation machinery component Ubc9, leading to its enhanced SUMOylation activity. First, we show that CDK1/cyclin B, but not many other cell cycle kinases such as CDK2/cyclin E, ERK1, ERK2, PKA and JNK2/SAPK1, specifically enhances SUMOylation activity. Second, CDK1/cyclin B phosphorylates the SUMOylation machinery component Ubc9, but not SAE1/SAE2 or SUMO1. Third, CDK1/cyclin B-phosphorylated Ubc9 exhibits increased SUMOylation activity and elevated accumulation of the Ubc9-SUMO1 thioester conjugate. Fourth, CDK1/cyclin B enhances SUMOylation activity through phosphorylation of Ubc9 at serine 71. These studies demonstrate for the first time that the cell cycle-specific kinase CDK1/cyclin B phosphorylates a SUMOylation machinery component to increase its overall SUMOylation activity, suggesting that SUMOylation is part of the cell cycle program orchestrated by CDK1 through Ubc9.

In the present study, we studied changes in gene expression induced by chemotherapy (CT) on normal peripheral blood leukocytes (PBLs), at baseline and following three CT cycles, in order to identify which genes were specifically affected and were potentially useful as biomarkers for a personalised prognosis and follow-up. A PBL subtraction cDNA library was constructed from four patients undergoing CT with paclitaxel and carboplatin (PC). mRNA from the PBLs was isolated prior to the patients receiving the first cycle and following the completion of the third cycle. The library was screened and the expression of the identified genes was studied in PBLs obtained from patients suffering from cancer prior to and following three cycles of PC and a reference group of patients undergoing treatment with Adriamycin-cyclophosphamide (AC). From the 1,200 screened colonies, 65 positive clones showed varied expression intensity and were sequenced; 27 of these were mitochondrial DNA and 38 clones (27 different) were coded for cytosolic and nuclear proteins. The genes that were studied in patients undergoing CT were ATM (ataxia-telangiectasia mutated gene), eIF4B (translation initiation factor 4B), MATR3 (Matrin 3), MORC3 (microrchidia 3), PCMTD2 (protein-L-isoaspartate O-methyltransferase), PDCD10 (programmed cell death gene 10), PSMB1 (proteasome subunit type β), RMND5A (required for meiotic nuclear division 5 homologue A), RUNX2 (runt-related transcription factor 2), SACM1L (suppressor of actin mutations 1-like), TMEM66 (transmembrane protein 66) and ZNF644 (zinc finger protein 644). Certain variations were observed in the expression of the genes that are involved in drug resistance mechanisms, some of which may be secondary to non-desirable effects and others of which may cause the undesired effects of CT. The expression of genes with a dynamic cellular role showed a marked positive correlation, indicating that their upregulation may be involved in a specific pattern of cell survival versus apoptosis in response to the cell damage induced by CT. Whether these CT-induced changes are random or directed in a specific selection-evolution manner needs to be elucidated.

The Cold Spring Harbour Asia conference on 'Assembly, Plasticity, Dysfunction and Repair of Neural Circuits' took place in Suzhou, China, in October 2011. Developmental, cell, molecular and systems neuroscientists convened to discuss the establishment, function and plasticity of neural circuits in diverse organisms.

The purpose of this study was to investigate interactions between exposure to supportive family environments and genetic characteristics, which were hypothesized to forecast variations in allostatic load (AL) in a representative sample of 315 rural African American youths. Data on family environments were gathered when youths were 11-13, and genetic data were collected when they were 16, years of age. Data on AL were obtained at the beginning of emerging adulthood, age 19 years. The data analyses revealed that, as predicted, emerging adults exposed to less supportive family environments across preadolescence manifested higher levels of AL when they carried the short (s) allele at the 5-HTTLPR and an allele of DRD4 with seven or more repeats. This is an E(family environment) × G(5-HTTLPR status) × G(DRD4 status) interaction. These data suggest that African American youths carrying genes that confer sensitivity who are exposed to less supportive family environments may be at greater risk for adverse physical health consequences that AL presages.

Thioamide modifications of the peptide backbone are used to perturb secondary structure, to inhibit proteolysis, as photoswitches, and as spectroscopic labels. Thus far, their incorporation has been confined to single peptides synthesized on solid phase. We have generated thioamides in C-terminal thioesters or N-terminal Cys fragments and examined their compatibility with native chemical ligation conditions. Most sequence variants can be coupled in good yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations. Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track conformational changes during aggregation of labeled α-synuclein.

Prognosis of urinary bladder urothelial carcinomas may be challenging; many tumors with similar histopathologic features show significantly different clinical outcomes. CSE1L, the chromosome segregation 1-like protein, is both a cytoplasmic and nuclear protein. We investigated the cytoplasmic/nuclear expression pattern of CSE1L to determine its potential prognostic significance. In immunohistochemical analysis, nonneoplastic urothelium showed faint CSE1L staining, whereas all tumors in the bladder cancer specimens had significant staining for CSE1L (100%, or 38/38). CSE1L cytoplasmic/nuclear staining was defined based on relative staining intensity. A total of 20 (52.6%) of 38 cancer specimens had strong nuclear CSE1L staining, and 44.7.3% (17/38) of the samples had strong cytoplasmic CSE1L staining. Bladder urothelial carcinomas with high CSE1L nuclear staining had a significantly lower overall survival rate (log-rank test, P = .011). CSE1L expression was not correlated with tumor stage, likely reflecting the faultiness of current urothelial carcinoma evaluation methods. Our results suggest that nuclear CSE1L may play an oncogenic role in bladder tumor progression and that immunohistochemical staining of nuclear CSE1L may be useful for the prognosis of bladder urothelial carcinomas.

Global gene expression profiling was performed using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define a gene expression pattern and signaling pathways that are specific for each cell lineage. We have demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. By Gene Ontology, 325 out of 3875 investigated gene sets were scientifically different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison had revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR that are known to be involved in NS cell fates determination. The transcriptional signature also deciphers the role of genes involved in epigenetic modifications. SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, were found specifically up-regulated in our OBNSC but not in hENSC. Differences in gene expression profile of transcripts controlling epigenetic modifications, and signaling pathways might indicate differences in the therapeutic potential of our examined two cell populations in relation to in cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults.

<h4>Purpose</h4>Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare group of tumors with a wide spectrum of clinical behavior. However, there are no known clinically relevant biomarkers to predict metastasis.<h4>Experimental design</h4>To investigate differential gene expression signatures of metastatic vs non-metastatic NETs, we studied cell cycle regulatory genes in 19 metastatic and 22 non-metastatic colorectal NETs by PCR arrays. Immunohistochemistry (IHC) and quantitative real-time RT-PCR were performed to verify the results and another set of 38 GEP-NETs were further studied for validation.<h4>Results</h4>We first delineated six candidate genes for metastasis including ATM, CCND2, RBL2, CDKN3, CCNB1, and GTSE1. ATM was negatively correlated with metastatic NETs (p<0.001) with more than 2-fold change compared to non-metastatic NETs. Overexpression of ATM protein by IHC was strongly correlated with high ATM mRNA levels and low Ki-67 labeling index. Patients with ATM-negativity by IHC showed significantly decreased overall survival than patients with ATM-positivity (median OS, metastatic vs non-metastatic NETs; 2.7 years vs not reached; p = 0.003) and 85.7% of metastatic NETs were ATM-negative. In another validation set of GEP-NETs, decreased mRNA of ATM gene was associated with metastasis and remained significant (p = 0.023).<h4>Conclusions</h4>ATM down-regulation was strongly associated with metastatic NETs when compared with non-metastatic NETs and ATM may be a potential predictive marker for metastasis as well as a novel target in metastatic GEP-NETs.

<h4>Background</h4>Prior to the onset of human labour there is an increase in the synthesis of prostaglandins, cytokines and chemokines in the fetal membranes, particular the amnion. This is associated with activation of the transcription factor nuclear factor kappa B (NFκB). In this study we characterised the level of NFκB activity in amnion epithelial cells as a measure of amnion activation in samples collected from women undergoing caesarean section at 39 weeks gestation prior to the onset of labour.<h4>Methodology/principal findings</h4>We found that a proportion of women exhibit low or moderate NFκB activity while other women exhibit high levels of NFκB activity (n = 12). This activation process does not appear to involve classical pathways of NFκB activation but rather is correlated with an increase in nuclear p65-Rel-B dimers. To identify the full range of genes upregulated in association with amnion activation, microarray analysis was performed on carefully characterised non-activated amnion (n = 3) samples and compared to activated samples (n = 3). A total of 919 genes were upregulated in response to amnion activation including numerous inflammatory genes such cyclooxygenase-2 (COX-2, 44-fold), interleukin 8 (IL-8, 6-fold), IL-1 receptor accessory protein (IL-1RAP, 4.5-fold), thrombospondin 1 (TSP-1, 3-fold) and, unexpectedly, oxytocin receptor (OTR, 24-fold). Ingenuity Pathway Analysis of the microarray data reveal the two main gene networks activated concurrently with amnion activation are i) cell death, cancer and morphology and ii) cell cycle, embryonic development and tissue development.<h4>Conclusions/significance</h4>Our results indicate that assessment of amnion NFκB activation is critical for accurate sample classification and subsequent interpretation of data. Collectively, our data suggest amnion activation is largely an inflammatory event that occurs in the amnion epithelial layer as a prelude to the onset of labour.

The ER-localized chaperone glucose-regulated protein (GRP78) protects neurons against excitotoxicity and apoptosis. Here we show that overexpressing GRP78 protects N2a cells against mutant huntingtin proteins, reduces formation of mutant huntingtin aggregates, inhibits caspase-12 activation and blocks cell death. Our data suggest that GRP78 may be a promising therapeutic target for the treatment of Huntington's disease.

The pathways that control mesenchymal stem cells (MSCs) differentiation are not well understood, and although some of the involved transcription factors (TFs) have been characterized, the role of others remains unclear. We used human MSCs from tibial plateau (TP) trabecular bone, iliac crest (IC) bone marrow and Wharton's jelly (WJ) umbilical cord demonstrating a variability in their mineral matrix deposition, and in the expression levels of TFs including Runx2, Sox9, Sox5, Sox6, STAT1 and Slug, all involved in the control of osteochondroprogenitors differentiation program. Because we reasoned that the basal expression level of some TFs with crucial role in the control of MSC fate may be correlated with osteogenic potential, we considered the possibility to affect the hMSCs behaviour by using gene silencing approach without exposing cells to induction media. In this study we found that Slug-silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression. On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type. Interestingly, we demonstrated a direct in vivo regulatory action of Slug by chromatin immunoprecipitation, showing a specific recruitment of this TF in the promoter of Runx2 and Sox9 genes. As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.

Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ± 1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.

Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR-ΔF508) and the NBCe1-null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr, and Car2, Car3, Car6, and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR-ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild-type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth.

Netrin-1 influences retinal ganglion cell (RGC) axon pathfinding and also participates in the branching and synaptic differentiation of mature RGC axons at their target. To investigate whether netrin also serves as an early target recognition signal in the brain, we examined the dynamic behavior of Xenopus RGC axons soon after they innervate the optic tectum. Time-lapse confocal microscopy imaging of RGC axons expressing enhanced yellow fluorescent protein demonstrated that netrin-1 is involved in early axon branching, as recombinant netrin-1 halted further advancement of growth cones into the tectum and induced back branching. RGC growth cones exhibited differential responses to netrin-1 that depended on the degree of differentiation of the axon and the developmental stage of the tadpole. Netrin-1 decreased the total number of branches on newly arrived RGC growth cones at the target, but increased the dynamic branching of more mature arbors at the later developmental stage. To further explore the response of axonal growth cones to netrin, Xenopus RGC axons were followed in culture by time-lapse imaging. Exposure to netrin-1 rapidly increased the forward advancement of the axon and decreased the size and expanse of the growth cone, while also inducing back branching. Taken together, the differential in vivo and in vitro responses to netrin-1 suggest that netrin alone is not sufficient to induce the cessation of growth cone advancement in the absence of a target but can independently modulate axon branching. Collectively, our findings reveal a novel role for netrin on RGC axon branch initiation as growth cones innervate their target.

Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) is safe and may improve cardiac function and structural remodelling in patients following myocardial infarction (MI). Cardiovascular cell differentiation and paracrine effects to promote endogenous cardiac regeneration, neovascularization, anti-inflammation, anti-apoptosis, anti-remodelling and cardiac contractility, may contribute to MSC-based cardiac repair following MI. However, current evidence indicates that the efficacy of MSC transplantation was unsatisfactory, due to the poor viability and massive death of the engrafted MSCs in the infarcted myocardium. MicroRNAs are short endogenous, conserved, non-coding RNAs and important regulators involved in numerous facets of cardiac pathophysiologic processes. There is an obvious involvement of microRNAs in almost every facet of putative repair mechanisms of MSC-based therapy in MI, such as stem cell differentiation, neovascularization, apoptosis, cardiac remodelling, cardiac contractility and arrhythmias, and others. It is proposed that therapeutic modulation of individual cardiovascular microRNA of MSCs, either mimicking or antagonizing microRNA actions, will hopefully enhance MSC therapeutic efficacy. In addition, MSCs may be manipulated to enhance functional microRNA expression or to inhibit aberrant microRNA levels in a paracrine manner. We hypothesize that microRNAs may be used as novel regulators in MSC-based therapy in MI and MSC transplantation by microRNA regulation may represent promising therapeutic strategy for MI patients in the future.

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the intrahepatic bile duct epithelium, has a poor prognosis and is refractory to conventional chemotherapy and radiation therapy. Thus, there is an urgent need to develop new effective therapeutic strategies for this disease. We previously found that L1 cell adhesion molecule (L1CAM) plays an important role in tumor progression of ICC, and we generated a murine mAb, A10-A3 (IgG1), that binds to the Ig1 domain of L1CAM. In the present study, we further characterized A10-A3, constructed a chimeric A10-A3 antibody (cA10-A3) containing the constant regions of human IgG1, and evaluated the therapeutic potential in a human ICC xenograft nude mice model. The affinities (KD) of A10-A3 and cA10-A3 for soluble L1CAM were 1.8 nM and 1.9 nM, respectively, as determined by competition ELISA. A10-A3 inhibited L1CAM homophilic binding and was slowly internalized into the tumor cells, but it did not significantly inhibit proliferation of ICC cells in vitro. cA10-A3 mediated antibody- dependent cell-mediated cytotoxicity in vitro and displayed anti-tumor activity in the ICC animal model. These results suggest that the humanized A10-A3 antibody may have potential as an anticancer agent for the treatment of ICC.

SOX5 encodes a transcription factor involved in the regulation of chondrogenesis and the development of the nervous system. Despite its important developmental roles, SOX5 disruption has yet to be associated with human disease. We report one individual with a reciprocal translocation breakpoint within SOX5, eight individuals with intragenic SOX5 deletions (four are apparently de novo and one inherited from an affected parent), and seven individuals with larger 12p12 deletions encompassing SOX5. Common features in these subjects include prominent speech delay, intellectual disability, behavior abnormalities, and dysmorphic features. The phenotypic impact of the deletions may depend on the location of the deletion and, consequently, which of the three major SOX5 protein isoforms are affected. One intragenic deletion, involving only untranslated exons, was present in a more mildly affected subject, was inherited from a healthy parent and grandparent, and is similar to a deletion found in a control cohort. Therefore, some intragenic SOX5 deletions may have minimal phenotypic effect. Based on the location of the deletions in the subjects compared to the controls, the de novo nature of most of these deletions, and the phenotypic similarities among cases, SOX5 appears to be a dosage-sensitive, developmentally important gene.

The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Beta thalassemia is known to be characterized by a hypercoagulable state, with prothrombotic factors present and thrombotic event development in a number of patients. The aim of the present study was to evaluate subclinical involvement of the central nervous system (CNS) in young patients with thalassemia intermedia, the use of nonimaging, noninvasive laboratory methods for detecting relevant abnormalities, and the frequency and possible correlation of coagulation abnormalities with CNS lesions. In this cross-sectional study, 24 young patients with thalassemia intermedia were evaluated (mean age 12 ± 4.6 years, range 4.5-20 years). Patients underwent neurological examination, inherited and acquired coagulation defect testing, as well as neurophysiologic and neuroimaging evaluation. Patients aged 6-16 also had intelligence scores measured. With regards to coagulation, a decrease in antithrombin III (ATIII), protein C and protein S activity was found in 4.1, 54.16 and 45.8% of patients, respectively. Increased D-dimers, as well as thrombin-antithrombin complex (TAT) and prothrombin fragment (F1 + 2) values were found in 12.5, 62.5 and 8.33% of patients, respectively. Heterozygosity and homozygosity for the methylenetetrahydrofolate reductase mutation was found in 45.8 and 12.5% of patients, whereas heterozygosity for factor V Leiden and G20210FII was found in 8.33 and 12.5% of patients, respectively, with increased prevalence compared to Greek population. Neuroimaging evaluation was normal in all patients. Neurophysiologic evaluation revealed abnormal findings in 33.3% of patients on electroencephalogram (EEG), 16% on brain auditory-evoked potentials (BAEPs) and 4.12% on somatosensory evoked potentials (SEPs). Visual-evoked potentials (VEPs) were normal in all patients. A statistically significant difference was found between low protein C values, as well as high platelet counts, with abnormal EEG findings (P = 0.004 and P = 0.039, respectively). Transcranial Doppler (TCD) measurements revealed increased peak systolic velocities in anterior and posterior cerebral arteries and in basilar artery in 57, 38 and 41% of patients, respectively, as compared to healthy population values. On the contrary, decreased mean velocities were found both on middle cerebral artery and pars terminalis of internal carotid examination in 28.5% of patients. Patients with pathological findings on TCD study had lower hematocrit (P = 0.049) and younger age (P = 0.001) than patients with normal measurements. With regards to intelligence scores, mean intelligence quotient (IQ) was 100 ± 19.1, with 11.7% of patients demonstrating IQ below 85. The study results confirm the early presence of hemostatic changes in patients with thalassemia intermedia. Additionally, they demonstrate subclinical CNS involvement starting at childhood. For such involvement detection, in addition to neuroimaging, neurophysiological and neuropsychological evaluation is warranted.

The Pneumonia Etiology Research for Child Health (PERCH) project is a 7-country, standardized, comprehensive evaluation of the etiologic agents causing severe pneumonia in children from developing countries. During previous etiology studies, between one-quarter and one-third of patients failed to yield an obvious etiology; PERCH will employ and evaluate previously unavailable innovative, more sensitive diagnostic techniques. Innovative and rigorous epidemiologic and analytic methods will be used to establish the causal association between presence of potential pathogens and pneumonia. By strategic selection of study sites that are broadly representative of regions with the greatest burden of childhood pneumonia, PERCH aims to provide data that reflect the epidemiologic situation in developing countries in 2015, using pneumococcal and Haemophilus influenzae type b vaccines. PERCH will also address differences in host, environmental, and/or geographic factors that might determine pneumonia etiology and, by preserving specimens, will generate a resource for future research and pathogen discovery.

Neuronal disorders, like Huntington's disease (HD), are difficult to study, due to limited cell accessibility, late onset manifestations, and low availability of material. The establishment of an in vitro model that recapitulates features of the disease may help understanding the cellular and molecular events that trigger disease manifestations. Here, we describe the generation and characterization of a series of induced pluripotent stem (iPS) cells derived from patients with HD, including two rare homozygous genotypes and one heterozygous genotype. We used lentiviral technology to transfer key genes for inducing reprogramming. To confirm pluripotency and differentiation of iPS cells, we used PCR amplification and immunocytochemistry to measure the expression of marker genes in embryoid bodies and neurons. We also analyzed teratomas that formed in iPS cell-injected mice. We found that the length of the pathological CAG repeat did not increase during reprogramming, after long term growth in vitro, and after differentiation into neurons. In addition, we observed no differences between normal and mutant genotypes in reprogramming, growth rate, caspase activation or neuronal differentiation. However, we observed a significant increase in lysosomal activity in HD-iPS cells compared to control iPS cells, both during self-renewal and in iPS-derived neurons. In conclusion, we have established stable HD-iPS cell lines that can be used for investigating disease mechanisms that underlie HD. The CAG stability and lysosomal activity represent novel observations in HD-iPS cells. In the future, these cells may provide the basis for a powerful platform for drug screening and target identification in HD.

Transcription of retrotransposons is usually repressed by DNA methylation, but a few elements, such as intracisternal A-particles (IAPs) associated with the Agouti and Axin-fused loci, partially escape this repression mechanism. The levels of this repression are also variable among individuals with an identical genome sequence, generating epigenetically different states of loci or 'epialleles.' In the current study, we tested the existence of additional retrotransposon-derived epialleles in the mouse genome. Using a series of bioinformatic approaches, 143 candidate epialleles were first identified from the mouse genome based on their promoter activity and association with active histone modification marks. Detailed analyses suggest that a subset of these elements showed variable levels of DNA methylation among the individual mice of an isogenic background, revealing their stochastic nature (metastability) of DNA methylation. The analyses also identified two opposite patterns of DNA methylation during development, progressive gaining vs. losing, confirming the dynamic nature of their DNA methylation patterns. qRT-PCR analyses demonstrated that the expression levels of these elements are indeed variable among the individual mice, suggesting functional consequences on their associated endogenous genes. Overall, these data confirm the presence of a number of new retrotransposon-derived epialleles with suggestions of the presence of more, and further identify retrotransposons as a major source of epigenetic variations in the mammalian genome.

Recent clinical trials aimed at improved treatment of AML by administration of vitamin D derivatives showed unremarkable results, suggesting development of vitamin D resistance in patients' AML blasts. Since mechanisms of vitamin D resistance are not clear, we studied 40AF cells, a subline of HL60 cells that can proliferate in the presence of 1α,25-dihydroxyvitamin D₃ (1,25D). We found that mRNA and protein levels of HPK1, an upstream MAP4 kinase, are dramatically increased in 40AF cells, and HPK1 protein is further increased when the 1,25D resistance of 40AF cells is partially reversed by the addition of carnosic acid and p38MAPK inhibitor SB202190 (DCS cocktail). Knockdown of HPK1 reduces 1,25D/DCS-induced differentiation of both 1,25D-sensitive HL60 and U937 cells and 1,25D-resistant 40AF cells, but the effect of HPK1 knockdown on differentiation-associated G 1 arrest is more apparent in the resistant than the sensitive cells. To explain why 40AF and the intrinsically vitamin D-resistant KG-1a cells can proliferate in the presence of vitamin D, we found that the cleaved HPK1 fragment (HPK1-C) level is high in 40AF and KG-1a cells, but when differentiation is induced by DCS, HPK1-C decreases while full-length (FL)-HPK1 increases. Accordingly, inhibition of proteolysis with the pan-caspase inhibitor Q-VD-OPh reduced HPK1 cleavage and enhanced DCS-induced differentiation of 40AF cells. The results indicate that FL-HPK1 is a positive regulator of vitamin D-induced differentiation in AML cells, but the cleaved HPK1 fragment inhibits differentiation. Thus, high HPK1 cleavage activity contributes to vitamin D resistance, and HPK1 has a dual role in AML cell differentiation.

Huntington's disease is an autosomal dominant disorder caused by a mutation in the gene encoding the protein huntingtin on chromosome 4. The mutation is an expanded CAG repeat in the first exon, encoding a polyglutamine tract. If the polyglutamine tract is > 40, penetrance is 100% and death is inevitable. Despite the widespread expression of huntingtin, HD has long been considered primarily as a disease of the striatum. It is characterized by selective vulnerability with dysfunction followed by death of the medium size spiny neuron. Considerable effort is being expended to determine whether striatal damage is cell-autonomous, non-cell-autonomous, requiring cell-cell and region to region communication, or both. We review data supporting both mechanisms. We also attempt to organize the data into common mechanisms that may arise outside the medium, spiny neuron, but ultimately have their greatest impact in the striatum.

<h4>Background</h4>Esophageal cancer consists of two major histologic types: esophageal squamous cell carcinoma (ESCC), predominant globally, and esophageal adenocarcinoma (EAC), which has a higher incidence in westernized countries. Five-year overall survival is 15%. Clinical trials frequently combine histologic types although they are different diseases with distinct origins. In the evolving era of personalized medicine and targeted therapies, we hypothesized that ESCC and EAC have genomic differences important for developing new therapeutic strategies for esophageal cancer.<h4>Methods</h4>We explored DNA copy number abnormalities in 70 ESCCs with publicly available array data and 189 EACs from our group. All data was from single nucleotide polymorphism arrays. Analysis was performed using a segmentation algorithm. Log ratio thresholds for copy number gain and loss were set at ±0.2 (approximately 2.3 and 1.7 copies, respectively).<h4>Results</h4>The ESCC and EAC genomes showed some copy number abnormalities with similar frequencies (eg, CDKN2A, EGFR, KRAS, MYC, CDK6, MET) but also many copy number abnormalities with different frequencies between histologic types, most of which were amplification events. Some of these regions harbor genes for which targeted therapies are currently available (VEGFA, ERBB2) or for which agents are in clinical trials (PIK3CA, FGFR1). Other regions contain putative oncogenes that may be targeted in the future.<h4>Conclusions</h4>Using single nucleotide polymorphism arrays we compared genomic abnormalities in a large cohort of EACs and ESCCs. We report here the similar and different frequencies of copy number abnormalities in ESCC and EAC. These results may allow development of histology-specific therapeutic agents for esophageal cancer.

<h4>Background</h4>Vitamin C is an immune-relevant micronutrient, which is depleted in viral infections and this deficiency seems to play a critical role in the pathogenesis of herpes infections and in the development of postherpetic neuralgia. The objective of this observational multicenter study was to evaluate the utilization, safety and efficacy of intravenously administrated vitamin C in patients with shingles.<h4>Material/methods</h4>Between April 2009 and December 2010 16 general practitioners recorded data of 67 participants with symptomatic herpes zoster who received vitamin C intravenously (Pascorbin® 7.5 g/50 ml) for approximately 2 weeks in addition to standard treatment. The assessment of pain (VAS) and the dermatologic symptoms of shingles such as hemorrhagic lesions and the number of efflorescences were investigated in a follow-up observation phase of up to 12 weeks.<h4>Results</h4>Mean declines of pain scores (VAS), number of affected dermatomes and efflorescences, and the presence of hemorrhagic vesicles between the baseline and follow-up assessments at 2 and 12 weeks were statistically significant. Overall, 6.4% of the participants experienced post-herpetic neuralgia. Common complaints such as general fatigue and impaired concentration also improved during the study. The effects and the tolerability of the treatment were evaluated positively by the physicians. The risk of developing PHN was reduced.<h4>Conclusions</h4>The data presented here provide evidence that concomitant use of intravenously administered ascorbic acid may have beneficial effects on herpes zoster-associated pain, dermatologic findings and accompanying common complaints. To confirm our findings, randomized, placebo-controlled clinical studies are necessary.

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.

The Wnt signaling pathway was originally uncovered as one of the prototype developmental signaling cascades in invertebrates as well as in vertebrates. The first indication that Wnt signaling also plays a role in the adult animal came from the study of the intestine of Tcf-4 (Tcf7L2) knockout mice. The gastrointestinal epithelium continuously self-renews over the lifetime of an organism and is, in fact, the most rapidly self-renewing tissue of the mammalian body. Recent studies indicate that Wnt signaling plays a central role in the biology of gastrointestinal stem cells. Furthermore, mutational activation of the Wnt cascade is the principle cause of colon cancer.

Rapamycin and its derivatives are mTOR inhibitors used in tissue transplantation and cancer therapy. A percentage of patients treated with these inhibitors develop diabetic-like symptoms, but the molecular mechanisms are unknown. We show here that chronic rapamycin treatment in mice led to insulin resistance with suppression of insulin/IGF signaling and genes associated within this pathway, such as Igf1-2, Irs1-2, and Akt1-3. Importantly, skeletal muscle-specific YY1 knockout mice were protected from rapamycin-induced diabetic-like symptoms. This protection was caused by hyperactivation of insulin/IGF signaling with increased gene expression in this cascade that, in contrast to wild-type mice, was not suppressed by rapamycin. Mechanistically, rapamycin induced YY1 dephosphorylation and recruitment to promoters of insulin/IGF genes, which promoted interaction with the polycomb protein-2 corepressor. This was associated with H3K27 trimethylation leading to decreased gene expression and insulin signaling. These results have implications for rapamycin action in human diseases and biological processes such as longevity.

The hemochromatosis (HFE) gene encodes the HFE protein that regulates iron absorption. HFE mutations lead to the hemochromatosis disease of excessive iron absorption. HFE mutations may also influence the sustained virologic response (SVR, long-term virus suppression) in chronic hepatitis C patients treated with interferon-based antiviral therapy. We performed a meta-analysis of all English and Chinese language studies of HFE mutations and SVR in interferon-treated chronic hepatitis C patients indexed in the Medline, PubMed, Embase, and China National Knowledge Infrastructure databases to November 2011. Seven studies involving 605 patients with HFE mutations (homozygous or heterozygous mutation of C282Y, H63D or S65C) and 1279 with wild-type HFE (no mutation of C282Y, H63D or S65C for both alleles) were analyzed. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated with the fixed- or random-effect models. HFE mutations were associated with significantly higher SVR rate (vs. wild-type: OR = 1.56, 95% CI: 1.23-1.97, P < 0.001), indicating that mutation carriers were likely to achieve SVR in response to interferon-based antiviral therapy. Stratification analysis by HFE mutation type revealed that the H63D mutation was associated with a significantly higher SVR rate (OR = 1.60, 95% CI: 1.09-2.34, P = 0.020), while the C282Y mutation was not (OR = 1.19, 95% CI: 0.71-1.98, P = 0.510). Our meta-analysis results indicate that the H63D mutation in HFE is associated with a higher SVR rate in chronic hepatitis C patients treated with interferon-based antiviral therapy.

Glioblastoma is both the most common and lethal primary malignant brain tumor. Extensive multiplatform genomic characterization has provided a higher-resolution picture of the molecular alterations underlying this disease. These studies provide the emerging view that "glioblastoma" represents several histologically similar yet molecularly heterogeneous diseases, which influences taxonomic classification systems, prognosis, and therapeutic decisions.

Arf GTPases are key regulators of both retrograde and anterograde traffic at the Golgi complex. The Golgi-localized Arf activators, Arf-GEFs (guanine exchange factor) of the BIG/GBF family, are poorly understood in terms of both their regulatory and localization mechanisms. We have performed a detailed kinetic characterization of a functional Golgi Arf-GEF, the trans-Golgi network (TGN)-localized Sec7 protein from yeast. We demonstrate that Sec7 is regulated by both autoinhibition and positive feedback. We show that positive feedback arises through the stable recruitment of Sec7 to membranes via its HDS1 domain by interaction with its product, activated Arf1. This interaction mediates localization of Sec7 to the TGN, because deletion of the HDS1 domain or mutation of the HDS1 domain in combination with deletion of Arf1 significantly increases cytoplasmic localization of Sec7. Our results lead us to propose a model in which Arf-GEF recruitment is linked to Golgi maturation via Arf1 activation.

The lymphatic system is crucial for fluid homeostasis, immune responses, and numerous pathological processes. However, the molecular mechanisms responsible for establishing the anatomical form of the lymphatic vascular network remain largely unknown. Here, we show that chemokine signaling provides critical guidance cues directing early trunk lymphatic network assembly and patterning. The chemokine receptors Cxcr4a and Cxcr4b are expressed in lymphatic endothelium, whereas chemokine ligands Cxcl12a and Cxcl12b are expressed in adjacent tissues along which the developing lymphatics align. Loss- and gain-of-function studies in zebrafish demonstrate that chemokine signaling orchestrates the stepwise assembly of the trunk lymphatic network. In addition to providing evidence for a lymphatic vascular guidance mechanism, these results also suggest a molecular basis for the anatomical coalignment of lymphatic and blood vessels.