Gene Literature Dashboard

<h4>Background</h4>Age-related macular degeneration (AMD) is an ocular disease affecting macula - the central part of the retina, resulting in the degeneration of photoreceptors and retinal epithelium and causing severe central vision impairment. The pathophysiology of the disease is not completely known, but a significant role is attributed to genetic factors. The contribution of oxidative stress in AMD as a trigger of the degenerative process is well-established. Iron ions may act as a source of reactive oxygen species; therefore, maintaining iron homeostasis is important for redox balance in the organism. Diversity in iron homeostasis genes may counterpart in unbalanced redox state, and thus be involved in AMD pathophysiology.<h4>Methods</h4>In this work, we searched for an association between some single nucleotide polymorphisms in the divalent metal transporter 1 (DMT1) gene intronic IVS4+44C>A (rs224589) and 3'-UTR c.2044T>C (rs2285230) and environmental factors and AMD. Genotyping was performed using the PCR-RFLP method. DNA was obtained from 436 AMD patients and 168 controls.<h4>Results</h4>We did not find any association between the genotypes of the two polymorphisms and AMD occurrence. However, we observed that AMD patients living in a rural environment and having the CC genotype of the IVS4+44C>A polymorphism had an increased risk of AMD, while individuals with the CA genotype or the A allele had a decreased risk of the disease. Moreover, in male AMD patients the C allele increased the risk of the disease, while the AA genotype decreased it.<h4>Conclusions</h4>These results suggest that the VS4+44C>A polymorphism of the DMT1 gene may interact with place of living and gender to modulate the risk of AMD.

The purpose of this study was to investigate the effect of Raf kinase inhibitor protein (RKIP) on the growth, proliferation, invasion and metastasis of triple-negative breast cancer (TNBC) cells to provide experimental evidence for developing future therapies against human TNBC. The pcDNA3.1-RKIP eukaryotic expression vector was constructed and transfected into the TNBC cell line MDA-MB-231. The alterations of the biological characteristics of RKIP-transfected MDA-MB-231 cells were analyzed using the following approaches: a growth curve, a 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) staining and a cell migration assay. The effects of the RKIP gene on MMP-1 and MMP-2 expression were also examined. The pcDNA3.1 empty vector-transfected and mock-transfected MDA-MB-231 cells were used as control groups. Compared with the empty vector-transfected and mock-transfected cells, the cell growth of RKIP-transfected MDA-MB-231 cells was significantly reduced. The empty vector-transfected group was not significantly different compared with the mock-transfected MDA-MB-231 cells. The results of the MTT and BrdU assays demonstrated that the proliferation of pcDNA3.1-RKIP-transfected cells was significantly reduced compared to the control cells (P < 0.05). The result of the cell migration assay suggested that the cross-membrane migration rate of the pcDNA3.1-RKIP-transfected cells was significantly lower than that of the control MDA-MB-231 cells (P < 0.05). We also demonstrated that RKIP may inhibit MMP-1 and MMP-2 expression in MDA-MB-231 cells. The RKIP gene may play a role in inhibiting cellular proliferation. The RKIP gene may also have some inhibitory effects on the invasiveness and metastatic capability of human TNBC cells.

Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.

Hepatitis C virus (HCV) infection causes chronic hepatitis, which frequently leads to hepatic fibrosis and hepatocellular carcinoma (HCC). Alanine aminotransferase (ALT) is a biomarker of hepatocyte injury and is associated with the progression of hepatic fibrosis. Advanced hepatic fibrosis also predisposes HCV carriers to a risk of HCC. In contrast, some cases with persistent HCV infection have normal ALT levels that persist for a long time, and these HCV carriers have no or mild hepatitis and hepatic fibrosis. These HCV carriers are defined as persistent normal ALT (PNALT) cases and their risk of HCC is low compared to HCV carriers with abnormal ALT. However, there are various definitions of normal ALT and PNALT, and advanced hepatic fibrosis may be missed without a liver biopsy. In addition, there is also a risk of ALT elevation in HCV carriers with PNALT, which increases the risk of progression to hepatic fibrosis and HCC. Most HCV carriers with PNALT have asymptomatic or nonspecific symptoms. HCV carriers with PNALT are also considered to be responsive to interferon-based treatment. Thus, assessment of hepatic fibrosis is important in HCV carriers, and the eradication of HCV infection is more likely in HCV carriers with evidence of hepatic fibrosis, regardless of their ALT levels.

<h4>Background</h4>There is limited data on the natural history of autoimmune hepatitis (AIH) and on the long-term follow-up of AIH patients who have been referred for regular medical attention.<h4>Objectives</h4>We evaluated the clinical presentation and natural history of AIH in a large cohort of type I AIH patients from Iran.<h4>Patients and methods</h4>Between 1997 and 2008, 102 patients were enrolled in the study. Patients were diagnosed using the International Autoimmune Hepatitis Group criteria and were followed up for an average of 60 months. Clinical and biochemical data were gathered from all the patients at both the beginning and the end of the follow-up period. Liver biopsy was performed in all patients before treatment, and the biopsies were performed in 28 patients after treatment.<h4>Results</h4>Biochemical remission was achieved by 80 (79.4%) patients. Of these, 53 (66.5%) showed near-normal liver histology or liver function test results and sonogram. The remaining 27 (33.5%) patients also achieved clinical and biochemical remission, but developed compensated cirrhosis. After a period of remission, 24 patients (32.5%) relapsed. Among the 22 (21.6%) patients who showed ultimate treatment failure, 6 underwent orthotopic liver transplantation and 3 died of liver failure while awaiting a transplant. Sixteen (72.7%) of the 22 patients who did not respond to therapy were non-compliant with medications and had irregular follow-up. The overall 10-year survival rate in the cohort was 96%.<h4>Conclusions</h4>Long-term survival in AIH patients is very good. Prompt diagnosis and appropriate first-line and salvage therapy that includes close follow-up will make liver transplantation a rare necessity in the treatment of this disease.

No abstract available.

PHYLOGENY SUGGESTS THAT THE EVOLUTION OF PLACENTATION IN MAMMALS WAS ACCOMPANIED BY SUBSTANTIAL CHANGES IN THE MAMMALIAN IMMUNE SYSTEM: in particular lymph nodes and CD4 high affinity memory antibody responses co-evolved during the same period. Lymphoid tissue inducer cells (LTi) are members of an emerging family of innate lymphoid cells (ILCs) that are crucial for lymph node development, but our studies have indicated that they also play a pivotal role in the long-term maintenance of memory CD4 T cells in adult mammals through their expression of the tumor necrosis family members, OX40- and CD30-ligands. Additionally, our studies have shown that these two molecules are also key operators in CD4 effector function, as their absence obviates the need for the FoxP3 dependent regulatory T (T(regs)) cells that prevent CD4 driven autoimmune responses. In this perspective article, we summarize findings from our group over the last 10 years, and focus specifically on the role of LTi in thymus. We suggest that like memory CD4 T cells, LTi also play a role in the selection and maintenance of the T(regs) that under normal circumstances are absolutely required to regulate CD4 effector cells.

<h4>Background</h4>Alterations of coagulation profile have been reported in patients with β-thalassaemia major (β-TM).<h4>Method</h4>To investigate this in the paediatric population, we studied haemostatic parameters in pre-transfusion blood samples from 50 non-splenectomised transfusion-dependent children with β-TM (mean age 6±2.5 years) and in blood from 25 healthy controls.<h4>Results</h4>Laboratory evaluation showed thrombocytopenia in 40%, prolongation of prothrombin time (PT) in 12% and prolongation of activated partial thromboplastin time (APTT) in 6% of the patients. Mean values for PT, APTT and platelet count (PC) were all raised in the patient population compared with the controls. The alteration of coagulation status was significant for PT (p value <0.005) and APTT (p value <0.0001). However, the change for PC was not significant (p value <0.05). No significant liner correlation could be identified between PT, APTT, PC of the patients and interval between transfusions (in days) or days since last transfusion.<h4>Conclusion</h4>The findings from this study suggest that a subclinical haemorrhagic tendency exists in patients with β-TM at a very early age. The intrinsic pathway appears to be more affected than the extrinsic pathway.

<h4>Background</h4>Increasing evidence suggests an association between elevated serum aminotransferase levels and metabolic disorders (metabolic syndrome, hyperlipemia and diabetes mellitus). However, the significance of relatively low levels of aminotransferases in relation to metabolic disorders has not been fully investigated in the general population. We investigated the association between serum amiontransferase levels and metabolic disorders using data from a survey in Jilin province, China.<h4>Methods</h4>In 2007, a survey was conducted throughout Jilin, China, covering both urban and rural areas. A total of 3835 people, 18 to 79 years old including 1761 men and 2074 women, underwent real-time ultrasonography, blood tests including aspartate aminotransferase and alanine aminotransferase, and had interviews with a structured questionnaire.<h4>Results</h4>Serum aminotransferase levels within the normal range were associated with metabolic syndrome independent of age, occupation, cultural and educational level, income, body mass index, waist circumference, smoking, and alcohol intake. Compared with the lowest level (<20 IU/L), the adjusted odds ratios for ALT levels of 20-29, 30-39, 40-49 and >50 IU/L were 1.92, 2.50, 2.97, and 3.52 in men, and 1.38, 1.54, 3.06, and 2.62 in women, respectively. Near-normal serum aminotransferase levels associated with hyperlipemia, NAFLD, DM were also found in the study.<h4>Conclusions</h4>Normal to near-normal serum aminotransferase levels are associated with metabolic disorders. Serum ALT levels of 21-25 IU/L for men, and 17-22 IU/L for women are suggested as cutoff levels that detect metabolic disorders affecting the liver.

The interpatient variability in response to asthma controllers is significant and associates with pharmacogenomic variability. The goal of the present study was to identify novel variants that associate with response to common asthma controllers: fluticasone, combination of fluticasone + salmeterol and montelukast with single nucleotide polymorphisms (SNPs) in β2-adrenergic receptor, corticosteroid and leukotriene pathway candidate genes. Participants in a large clinical trial of step-down strategies volunteered for this pharmacogenetic study. A total of 169 SNPs in 26 candidate genes were genotyped in 189 Caucasian participants with asthma who took either fluticasone (100 μg bid), fluticasone propionate (100 μg) + salmeterol (50 μg) (FP/Salm) or montelukast (5 or 10 mg) each night for 16 weeks. Primary outcomes were the slopes of plots of Asthma Control Questionnaire (ACQ) scores versus time following randomization; and the percent change in percent predicted FEV1 (ΔFEV1%pred) from enrollment to the end of the study. Associations between SNPs and outcomes were analyzed using general linear models. False discovery rate and Bonferroni corrections were used to correct for multiple comparisons. In all, 16 SNPs in seven genes were significantly associated with outcomes. For FP/Salm, three SNPs in CHRM2 associated with ACQ slope (P=2.8 × 10⁻⁵), and rs1461496 in HSPA8 associated with ΔFEV1%pred. For fluticasone, five SNPs in CRHR1 (P=1.9 × 10⁻⁴), and three SNPs in COL2A1 associated with ACQ slope and ΔFEV1%pred, respectively. For montelukast, four SNPs in CHRM2 associated with ΔFEV1%pred and predicted an opposite effect compared with fluticasone (P=9 × 10⁻³). The present study indentified several novel SNPs that associate with response to common asthma controllers, and support further pharmacogenomic study and the use of genetic variants to personalize asthma treatment.

In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8-dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8- and RNF168-deficient cells to form 53BP1 foci. We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites.

'Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instability.

<h4>Rationale</h4>MyomiRs miR-499, miR-208a and miR-208b direct cardiac myosin gene expression. Sequence complementarity between miRs and their mRNA targets determines miR effects, but the functional consequences of human myomiR sequence variants are unknown.<h4>Objective</h4>To identify and investigate mutations in human myomiRs in order to better understand how and to what extent naturally-occurring sequence variation can impact miR-mRNA targeting and end-organ function.<h4>Methods and results</h4>Screening of ≈2,600 individual DNAs for myomiR sequence variants identified a rare mutation of miR-499, u17c in the 3' end, well outside the seed region thought to determine target recognition. In vitro luciferase reporter analysis showed that the 3' miR-499 mutation altered suppression of a subset of artificial and natural mRNA targets. Cardiac-specific transgenic expression was used to compare consequences of wild-type and mutant miR-499. Both wild-type and mutant miR-499 induced heart failure in mice, but miR-499 c17 misdirected recruitment of a subset of miR-499 target mRNAs to cardiomyocyte RNA-induced silencing complexes, altering steady-state cardiac mRNA and protein make-up and favorably impacting cardiac function. In vitro analysis of miR-499 target site mutations and modeling of binding energies revealed abnormal miR-mRNA duplex configurations induced by the c17 mutation.<h4>Conclusions</h4>A naturally occurring miR-499 mutation outside the critical seed sequence modifies mRNA targeting and end-organ function. This first description of in vivo effects from a natural human miR mutation outside the seed sequence supports comprehensive studies of individual phenotypes or disease-modification conferred by miR mutations.

<h4>Objective</h4>Prediabetes and type 2 diabetes mellitus (T2DM) are believed to be common and associated with a worse metabolic profile in patients with nonalcoholic fatty liver disease (NAFLD). However, no previous study has systematically screened this population.<h4>Research design and methods</h4>We studied the prevalence and the metabolic impact of prediabetes and T2DM in 118 patients with NAFLD. The control group comprised 20 subjects without NAFLD matched for age, sex, and adiposity. We measured 1) plasma glucose, insulin, and free fatty acid (FFA) concentration during an oral glucose tolerance test; 2) liver fat by magnetic resonance spectroscopy (MRS); 3) liver and muscle insulin sensitivity (euglycemic insulin clamp with 3-[(3)H]glucose); and 4) indexes of insulin resistance (IR) at the level of the liver (HIR(i)= endogenous glucose production × fasting plasma insulin [FPI]) and adipose tissue (Adipo-IR(i)= fasting FFA × FPI).<h4>Results</h4>Prediabetes and T2DM was present in 85% versus 30% in controls (P < 0.0001), all unaware of having abnormal glucose metabolism. NAFLD patients were IR at the level of the adipose tissue, liver, and muscle (all P < 0.01-0.001). Muscle and liver insulin sensitivity were impaired in patients with NAFLD to a similar degree, whether they had prediabetes or T2DM. Only adipose tissue IR worsened in T2DM and correlated with the severity of muscle (r = 0.34; P < 0.001) and hepatic (r = 0.57; P < 0.0001) IR and steatosis by MRS (r = 0.35; P < 0.0001).<h4>Conclusions</h4>Patients with NAFLD may benefit from early screening for T2DM, because the prevalence of abnormal glucose metabolism is much higher than previously appreciated. Regardless of glucose tolerance status, severe IR is common. In patients with T2DM, adipose tissue IR appears to play a major role in the severity of NAFLD.

IgLON family is a subgroup of the immunoglobulin superfamily cell adhesion molecules and composed of limbic system-associated protein (LAMP), opioid binding cell adhesion molecule (OBCAM), neurotrimin (Ntm) and Kilon. In the present study, we investigated the overexpression of LAMP, OBCAM, Ntm and Kilon on the proliferation and cell size of type-1 astrocytes in vitro. Quantitative analysis using bromodeoxyuridine immunocytochemistry revealed that the expression of OBCAM had greater inhibitory effect on astrocytic proliferation as compared with LAMP, Ntm and Kilon ones. OBCAM overexpression increased the cell size of astrocytes as compared with the control. The treatment of FGF-2 had greater proliferative effect on OBCAM-transfected astrocytes as compared with the control. These results suggest that OBCAM is more potent regulator for controlling the proliferation and cell size of astrocytes as compared with other IgLON proteins possibly through FGF-2 receptor-mediated pathway.

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.

Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila and mouse models, we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that, as found in SIOD patients, deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice, although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.

While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs.

Growing evidence suggests that Huntington's disease (HD), a neurodegenerative movement disorder caused by the mutant huntingtin (htt) with an expanded polyglutamine (polyQ) repeat, is associated with the altered intracellular trafficking and synaptic function. GABA(A) receptors, the key determinant of the strength of synaptic inhibition, have been found to bind to the huntingtin associated protein 1 (HAP1). HAP1 serves as an adaptor linking GABA(A) receptors to the kinesin family motor protein 5 (KIF5), controlling the transport of GABA(A) receptors along microtubules in dendrites. In this study, we found that GABA(A)R-mediated synaptic transmission is significantly impaired in a transgenic mouse model of HD expressing polyQ-htt, which is accompanied by the diminished surface expression of GABA(A) receptors. Moreover, the GABA(A)R/HAP1/KIF5 complex is disrupted and dissociated from microtubules in the HD mouse model. These results suggest that GABA(A)R trafficking and function is impaired in HD, presumably due to the interference of KIF5-mediated microtubule-based transport of GABA(A) receptors. The diminished inhibitory synaptic efficacy could contribute to the loss of the excitatory/inhibitory balance, leading to increased neuronal excitotoxicity in HD.

<h4>Objective</h4>To investigate the differential protein profile of human lung squamous carcinoma (HLSC) and normal bronchial epithelium (NBE) and provide preliminary results for further study to explore the carcinogenic mechanism of HLSC.<h4>Methods</h4>Laser capture microdissection (LCM) was used to purify the target cells from 10 pairs of HLSC tissues and their matched NHBE, respectively. A stable-isotope labeled strategy using iTRAQ, followed by 2D-LC/Q-STAR mass spectrometry, was performed to separate and identify the differential expression proteins.<h4>Results</h4>A total of 96 differential expression proteins in the LCM-purified HLSC and NBE were identified. Compared with NBE, 49 proteins were upregulated and 47 proteins were downregulated in HLSC. Furthermore, the expression levels of the differential proteins including HSPB1, CKB, SCCA1, S100A8, as well as S100A9 were confirmed by western blot and tissue microarray and were consistent with the results of quantitative proteomics.<h4>Conclusion</h4>The different expression proteins in HLSC will provide scientific foundation for further study to explore the carcinogenic mechanism of HLSC.

Chronic inflammation is a well-recognized risk factor for development of human cancer in several tissues, including large bowel. Inflammatory bowel disease, including ulcerative colitis and Crohn's disease, is a longstanding inflammatory disease of intestine with increased risk for colorectal cancer development. Several molecular events involved in chronic inflammatory process may contribute to multistep carcinogenesis of human colorectal cancer in the inflamed colon. They include overproduction of reactive oxygen and nitrogen species, overproduction and upregulation of productions and enzymes of arachidonic acid biosynthesis pathway and cytokines, and intestinal immune system dysfunction. In this paper, I will describe several methods to induce colorectal neoplasm in the inflamed colon. First, I will introduce a protocol of a novel inflammation-associated colon carcinogenesis in mice. In addition, powerful tumor-promotion/progression activity of dextran sodium sulfate in the large bowel of Apc(Min/+) mice will be described. Finally, chemoprevention of inflammation-associated colon carcinogenesis will be mentioned.

<h4>Background</h4>In rat, the first 18-24 h after partial hepatectomy (PH) are characterized by an acute-phase reaction, after which liver regeneration predominates. Interleukin-6 (IL-6) induces the iron hormone hepcidin, which blocks iron uptake and may compromise iron uptake in the growing liver. The expressions of hepcidin and the iron-regulatory pathway of hepcidin gene expression during the late phase of liver regeneration are unknown.<h4>Aim</h4>To characterize the expression pattern of hepcidin and the iron-sensing pathway of hepcidin regulation during liver regeneration.<h4>Methods</h4>Rats were subjected to PH or sham operation. Liver weights, number of S-phase nuclei, and serum levels of iron and IL-6 were determined. Messenger-RNA levels of hepcidin, ferritin, hemojuvelin, transferrin receptor 1 and 2, HFE, divalent metal transporter 1, ferroportin, and ceruloplasmin were determined with qPCR at different time points. Protein levels of STAT3 and SMAD4 were determined with western blot.<h4>Results</h4>During the acute-phase response, IL-6 release induced STAT3 protein and hepcidin mRNA, whereas mRNA levels of proteins in the iron-sensing pathway (HFE, hemojuvelin, and transferrin receptor 2) decreased. The mRNA levels of proteins involved in cellular iron uptake were increased and cellular iron export unchanged. During liver regeneration >24 h after PH, gene expressions in the iron-sensing pathway were continuously suppressed and hepcidin mRNA levels declined 3-7 days after surgery.<h4>Conclusions</h4>Hepcidin gene expression peaks during the acute-phase response, but a sustained down-regulation of the iron-sensing pathway of hepcidin regulation gradually reduces hepcidin gene expression until regeneration is complete, thereby promoting iron mobilization to the regenerating liver.

The structural maintenance of chromosomes (Smc) proteins regulate nearly all aspects of chromosome biology and are critical for genomic stability. In eukaryotes, six Smc proteins form three heterodimers--Smc1/3, Smc2/4, and Smc5/6--which together with non-Smc proteins form cohesin, condensin, and the Smc5/6 complex, respectively. Cohesin is required for proper chromosome segregation. It establishes and maintains sister-chromatid cohesion until all sister chromatids achieve bipolar attachment to the mitotic spindle. Condensin mediates chromosome condensation during mitosis. The Smc5/6 complex has multiple roles in DNA repair. In addition to their major functions in chromosome cohesion and condensation, cohesin and condensin also participate in the cellular DNA damage response. Here we review recent progress on the functions of all three Smc complexes in DNA repair and their cell cycle regulation by posttranslational modifications, such as acetylation, phosphorylation, and sumoylation. An in-depth understanding of the mechanisms by which these complexes promote DNA repair and genomic stability may help us to uncover the molecular basis of genomic instability in human cancers and devise ways that exploit this instability to treat cancers.

The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting.

Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt-whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network.

How does one deal with unfair behaviors? This subject has long been investigated by various disciplines including philosophy, psychology, economics, and biology. However, our reactions to unfairness differ from one individual to another. Experimental economics studies using the ultimatum game (UG), in which players must decide whether to accept or reject fair or unfair offers, have also shown that there are substantial individual differences in reaction to unfairness. However, little is known about psychological as well as neurobiological mechanisms of this observation. We combined a molecular imaging technique, an economics game, and a personality inventory to elucidate the neurobiological mechanism of heterogeneous reactions to unfairness. Contrary to the common belief that aggressive personalities (impulsivity or hostility) are related to the high rejection rate of unfair offers in UG, we found that individuals with apparently peaceful personalities (straightforwardness and trust) rejected more often and were engaged in personally costly forms of retaliation. Furthermore, individuals with a low level of serotonin transporters in the dorsal raphe nucleus (DRN) are honest and trustful, and thus cannot tolerate unfairness, being candid in expressing their frustrations. In other words, higher central serotonin transmission might allow us to behave adroitly and opportunistically, being good at playing games while pursuing self-interest. We provide unique neurobiological evidence to account for individual differences of reaction to unfairness.

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.

Rest in the fruit fly Drosophila melanogaster has key characteristics of mammalian sleep and is thus considered as a fly version of sleep. Drosophila sleep has been studied extensively, with the aim of gaining fundamental insights into the evolutionarily conserved functions of sleep as well as the mechanisms that regulate it. An interesting question that has not yet been addressed is whether fly sleep can be classified into distinct sleep types, each having particular biological roles - like rapid eye movement (REM) and non-REM sleep in birds and mammals. Typically, Drosophila sleep displays a bimodal pattern, consisting of distinct daytime and nighttime components. Notably, daytime and nighttime sleep differ with respect to several qualities, such as sleep-bout lengths and arousal thresholds. In this short review, we describe several genetic and environmental factors that differentially affect daytime and nighttime sleep, highlighting the observations suggesting the notion that these temporally distinct components of Drosophila sleep may have unique biological functions and be regulated by different homeostatic regulatory mechanisms.

Genetic association studies are now routinely used to identify single nucleotide polymorphisms (SNPs) linked with human diseases or traits through single SNP-single trait tests. Here we introduced partial least squares path modeling (PLSPM) for association between single or multiple SNPs and a latent trait that can involve single or multiple correlated measurement(s). Furthermore, the framework naturally provides estimators of polygenic effect by appropriately weighting trait-attributing alleles. We conducted computer simulations to assess the performance via multiple SNPs and human obesity-related traits as measured by body mass index (BMI), waist and hip circumferences. Our results showed that the associate statistics had type I error rates close to nominal level and were powerful for a range of effect and sample sizes. When applied to 12 candidate regions in data (N = 2,417) from the European Prospective Investigation of Cancer (EPIC)-Norfolk study, a region in FTO was found to have stronger association (rs7204609∼rs9939881 at the first intron P = 4.29×10(-7)) than single SNP analysis (all with P>10(-4)) and a latent quantitative phenotype was obtained using a subset sample of EPIC-Norfolk (N = 12,559). We believe our method is appropriate for assessment of regional association and polygenic effect on a single or multiple traits.

Genomes of higher eukaryotes are mosaics of segments with various structural, functional, and evolutionary properties. The availability of whole-genome sequences allows the investigation of their structure as "texts" using different statistical and computational methods. One such method, referred to as Compositional Spectra (CS) analysis, is based on scoring the occurrences of fixed-length oligonucleotides (k-mers) in the target DNA sequence. CS analysis allows generating species- or region-specific characteristics of the genome, regardless of their length and the presence of coding DNA. In this study, we consider the heterogeneity of vertebrate genomes as a joint effect of regional variation in sequence organization superimposed on the differences in nucleotide composition. We estimated compositional and organizational heterogeneity of genome and chromosome sequences separately and found that both heterogeneity types vary widely among genomes as well as among chromosomes in all investigated taxonomic groups. The high correspondence of heterogeneity scores obtained on three genome fractions, coding, repetitive, and the remaining part of the noncoding DNA (the genome dark matter--GDM) allows the assumption that CS-heterogeneity may have functional relevance to genome regulation. Of special interest for such interpretation is the fact that natural GDM sequences display the highest deviation from the corresponding reshuffled sequences.

Studies comparing chronologically "young" versus "old" humans document age-related decline of classical immunological functions. However, older adults aged ≥65 years have very heterogeneous health phenotypes. A significant number of them are functionally independent and are surviving well into their 8(th)-11(th) decade life, observations indicating that aging or old age is not synonymous with immune incompetence. While there are dramatic age-related changes in the immune system, not all of these changes may be considered detrimental. Here, we review evidences for novel immunologic processes that become elaborated with advancing age that complement preserved classical immune functions and promote immune homeostasis later in life. We propose that elaboration such of late life immunologic properties is indicative of beneficial immune remodeling that is an integral component of successful aging, an emerging physiologic construct associated with similar age-related physiologic adaptations underlying maintenance of physical and cognitive function. We suggest that a systems approach integrating immune, physical, and cognitive functions, rather than a strict immunodeficiency-minded approach, will be key towards innovations in clinical interventions to better promote protective immunity and functional independence among the elderly.

Iron overload is considered as a significant cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. The presence of hemochromatosis gene (HFE) mutations might exacerbate iron toxicity in the post-transplant setting. This prospective study was planned to evaluate the genetic spectrum of HFE mutations in Turkish patients undergoing HSCT and the impact of HFE genotype on transplant morbidity and mortality. HFE genotypes of 102 patients [median age, 27.5 years (16-64 years); male/female, 73:29], who underwent allogeneic HSCT, were analyzed. Twenty-two patients were heterozygous and 1 patient was homozygous for the H63D mutation, while the C282Y mutation was observed in none of our patients. The frequency of invasive fungal infections (IFI) was significantly higher in H63D-mutated patients (p=0.004). H63D mutation was identified as an independent risk factor for the development of IFI (p=0.006, OR=0.554, SE=0.208), without an impact on overall survival and transplant-related mortality. The multifactorial iron-overloaded state in HSCT recipients might affect the phenotypic expression of HFE mutations and alter the severity of clinical presentation. The impact of HFE genotype on iron parameters and transplant-related morbidity and mortality should be validated with further studies.

Severe trauma-related bleeding is associated with high mortality. Standard coagulation tests provide limited information on the underlying coagulation disorder. Whole-blood viscoelastic tests such as rotational thromboelastometry or thrombelastography offer a more comprehensive insight into the coagulation process in trauma. The results are available within minutes and they provide information about the initiation of coagulation, the speed of clot formation, and the quality and stability of the clot. Viscoelastic tests have the potential to guide coagulation therapy according to the actual needs of each patient, reducing the risks of over- or under-transfusion. The concept of early, individualized and goal-directed therapy is explored in this review and the AUVA Trauma Hospital algorithm for managing trauma-induced coagulopathy is presented.

Genes selectively expressed in the striatum may be involved in the preferential vulnerability of striatal neurons to Huntington's disease (HD). Here, we investigated whether perturbations of Capucin expression, which is enriched in the striatum and downregulated in Huntington's disease models, could modify the neurotoxicity induced by the injection of a lentiviral vector encoding a short N-terminal fragment of mutant Huntingtin (mHtt) into the mouse striatum. Neither constitutive Capucin deficiency in knockout mice nor lentiviral vector-mediated Capucin overexpression in the striatum of adult wild type mice significantly modified vulnerability to the mHtt fragment in vivo, suggesting that Capucin has no impact on mHtt toxicity.

<h4>Objective</h4>For the examination of psychological stress effects on coagulation, the Dill and Costill correction (DCC) for hemoconcentration effects has been used to adjust for stress-induced plasma volume changes. Although the correction is appropriate for adjusting concentrations of various large blood constituents, it may be inappropriate for time-dependent or functional coagulation assays. Two new plasma reconstitution techniques for correcting hemoconcentration effects on stress-induced changes in coagulation were compared with the DCC.<h4>Methods</h4>Blood was collected from 31 men during baseline, the Trier Social Stress Test (TSST), and after 20-minute recovery. For the reconstitution techniques, TSST plasma samples were reconstituted with either baseline plasma or physiological saline equal to the amount of plasma lost during stress.<h4>Results</h4>Uncorrected activated partial thromboplastin time (APTT) decreased, whereas fibrinogen, factor VIII clotting activity (FVIII:C), D-dimer and prothrombin time (PT%) increased significantly during the TSST. The DCC produced a significantly greater decrease in APTT during stress compared to uncorrected APTT, a significant decrease in PT% compared to uncorrected PT%, and stress D-dimer and fibrinogen and FVIII:C being no different than baseline. APTT, fibrinogen, D-dimer and PT% after saline reconstitution were not different from baseline, whereas FVIII:C after saline reconstitution remained elevated. APTT, PT%, fibrinogen and D-dimer after plasma reconstitution were no different from uncorrected values, whereas FVIII:C remained significantly elevated.<h4>Conclusions</h4>The observed changes in coagulation are likely in part a consequence of stress and hemoconcentration, but the DCC seems to be an inappropriate hemoconcentration correction technique of time-dependent assays. The saline reconstitution technique may be more biologically relevant when examining stress-hemoconcentration effects on coagulation.

We report the successful control of bleeding in two patients who underwent post-cardiotomy extracorporeal circulatory support (ECMO) and then developed life-threatening bleeding due to severe coagulopathy. After the failure of conventional techniques, bleeding control was achieved using Celox Gauze (MedTrade Products Ltd, Cheshire, UK) packed on the sternal edges and pericardial cavity.

Huntington's disease (HD) is a fatal, genetic, neurodegenerative disorder characterized by deficits in motor and cognitive function. Here, we have quantitatively characterized motor deficiencies and dopamine release dynamics in transgenic HD model rats. Behavioral analyses were conducted using a newly-developed force-sensing runway and a previously-developed force-plate actometer. Gait disturbances were readily observed in transgenic HD rats at 12 to 15months of age. Additionally, dopamine system challenge by ip injection of amphetamine also revealed that these rats were resistant to the expression of focused stereotypy compared to wild-type controls. Moreover, dopamine release, evoked by the application of single and multiple electrical stimulus pulses applied at different frequencies, and measured using fast-scan cyclic voltammetry at carbon-fiber microelectrodes, was diminished in transgenic HD rats compared to age-matched wild-type control rats. Collectively, these results underscore the potential contribution of dopamine release alterations to the expression of motor impairments in transgenic HD rats.

The absolute stereostructures of the components of symplocin A (3), a new N,N-dimethyl-terminated peptide from the Bahamian cyanobacterium Symploca sp., were assigned from spectroscopic analysis, including MS, 2D NMR, and Marfey's analysis. The complete absolute configuration of symplocin A, including the unexpected D-configurations of the terminal N,N-dimethylisoleucine and valic acid residues, was assigned by chiral-phase HPLC of the corresponding 2-naphthacyl esters, a highly sensitive, complementary strategy for assignment of N-blocked peptide residues where Marfey's method is ineffectual or other methods fall short. Symplocin A exhibited potent activity as an inhibitor of cathepsin E (IC(50) 300 pM).

<h4>Background</h4>Geldanamycin (GA), a benzoquinone ansamycin antibiotic has been shown in vitro to possess anti-plasmodial activity. Pharmacological activity of this drug is attributed to its ability to inhibit PfHSP90. The parasite growth arrest has been shown to be due to drug-induced blockage of the transition from ring to trophozoite stage. To further evaluate the consequences of this pharmacodynamic feature, the anti-malarial activity of GA analogs with enhanced drug properties in a Plasmodium-infected animal model have been evaluated for their capacity to induce clearance of the parasite. In the process, a hypothesis was subsequently tested regarding the susceptibility of the cured animals to malaria reflected in an attenuated parasite load that may be evoked by a protective immune response in the host.<h4>Methods</h4>Six weeks old Swiss mice were infected with a lethal Plasmodium yoelii (17XL) strain. On appearance of clinical symptoms of malaria, these animals were treated with two different GA derivatives and the parasite load was monitored over 15-16 days. Drug-treated animals cured of the parasite were then re-challenged with a lethal dose of P. yoelii 17XL. Serum samples from GA cured mice that were re-challenged with P. yoelii 17XL were examined for the presence of antibodies against the parasite proteins using western blot analysis.<h4>Results</h4>Treatment of P. yoelii 17XL infected mice with GA derivatives showed slow recovery from clinical symptoms of the disease. Blood smears from drug treated mice indicated a dominance of ring stage parasites when compared to controls. Although, P. yoelii preferentially invades normocytes (mature rbcs), in drug-treated animals there was an increased invasion of reticulocytes. Cured animals exhibited robust protection against subsequent infection and serum samples from these animals showed antibodies against a vast majority of parasite proteins.<h4>Conclusions</h4>Treatment with GA derivatives blocked the transition from ring to trophozoite stage presumably by the inhibition of HSP90 associated functions. Persistence of parasite in ring stage leads to robust humoral immune response as well as a shift in invasion specificity from normocytes to reticulocyte. It is likely that the treatment with the water-soluble GA derivative creates an attenuated state (less virulent with altered invasion specificity) that persists in the host system, allowing it to mount a robust immune response.

<h4>Purpose</h4>Return of individual genetic results to research participants, including participants in archives and biorepositories, is receiving increased attention. However, few groups have deliberated on specific results or weighed deliberations against relevant local contextual factors.<h4>Methods</h4>The Electronic Medical Records and Genomics (eMERGE) Network, which includes five biorepositories conducting genome-wide association studies, convened a return of results oversight committee to identify potentially returnable results. Network-wide deliberations were then brought to local constituencies for final decision making.<h4>Results</h4>Defining results that should be considered for return required input from clinicians with relevant expertise and much deliberation. The return of results oversight committee identified two sex chromosomal anomalies, Klinefelter syndrome and Turner syndrome, as well as homozygosity for factor V Leiden, as findings that could warrant reporting. Views about returning findings of HFE gene mutations associated with hemochromatosis were mixed due to low penetrance. Review of electronic medical records suggested that most participants with detected abnormalities were unaware of these findings. Local considerations relevant to return varied and, to date, four sites have elected not to return findings (return was not possible at one site).<h4>Conclusion</h4>The eMERGE experience reveals the complexity of return of results decision making and provides a potential deliberative model for adoption in other collaborative contexts.

Individuals with severe, sporadic disorders of infantile onset represent an important class of disease for which discovery of the underlying genetic architecture is not amenable to traditional genetic analysis. Full-genome sequencing of affected individuals and their parents provides a powerful alternative strategy for gene discovery. We performed whole-genome sequencing (WGS) on a family quartet containing an affected proband and her unaffected parents and sibling. The 15-year-old female proband had a severe epileptic encephalopathy consisting of early-onset seizures, features of autism, intellectual disability, ataxia, and sudden unexplained death in epilepsy. We discovered a de novo heterozygous missense mutation (c.5302A>G [p.Asn1768Asp]) in the voltage-gated sodium-channel gene SCN8A in the proband. This mutation alters an evolutionarily conserved residue in Nav1.6, one of the most abundant sodium channels in the brain. Analysis of the biophysical properties of the mutant channel demonstrated a dramatic increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation. Current-clamp analysis in hippocampal neurons transfected with p.Asn1768Asp channels revealed increased spontaneous firing, paroxysmal-depolarizing-shift-like complexes, and an increased firing frequency, consistent with a dominant gain-of-function phenotype in the heterozygous proband. This work identifies SCN8A as the fifth sodium-channel gene to be mutated in epilepsy and demonstrates the value of WGS for the identification of pathogenic mutations causing severe, sporadic neurological disorders.

Range expansions cause a series of founder events. We show that, in a one-dimensional habitat, these founder events are the spatial analog of genetic drift in a randomly mating population. The spatial series of allele frequencies created by successive founder events is equivalent to the time series of allele frequencies in a population of effective size ke, the effective number of founders. We derive an expression for ke in a discrete-population model that allows for local population growth and migration among established populations. If there is selection, the net effect is determined approximately by the product of the selection coefficients and the number of generations between successive founding events. We use the model of a single population to compute analytically several quantities for an allele present in the source population: (i) the probability that it survives the series of colonization events, (ii) the probability that it reaches a specified threshold frequency in the last population, and (iii) the mean and variance of the frequencies in each population. We show that the analytic theory provides a good approximation to simulation results. A consequence of our approximation is that the average heterozygosity of neutral alleles decreases by a factor of 1-1/(2ke) in each new population. Therefore, the population genetic consequences of surfing can be predicted approximately by the effective number of founders and the effective selection coefficients, even in the presence of migration among populations. We also show that our analytic results are applicable to a model of range expansion in a continuously distributed population.

Endogenous retrotransposons have caused extensive genomic variation within mammalian species, but the functional implications of such mobilization are mostly unknown. We mapped thousands of endogenous retrovirus (ERV) germline integrants in highly divergent, previously unsequenced mouse lineages, facilitating a comparison of gene expression in the presence or absence of local insertions. Polymorphic ERVs occur relatively infrequently in gene introns and are particularly depleted from genes involved in embryogenesis or that are highly expressed in embryonic stem cells. Their genomic distribution implies ongoing negative selection due to deleterious effects on gene expression and function. A polymorphic, intronic ERV at Slc15a2 triggers up to 49-fold increases in premature transcriptional termination and up to 39-fold reductions in full-length transcripts in adult mouse tissues, thereby disrupting protein expression and functional activity. Prematurely truncated transcripts also occur at Polr1a, Spon1, and up to ∼5% of other genes when intronic ERV polymorphisms are present. Analysis of expression quantitative trait loci (eQTLs) in recombinant BxD mouse strains demonstrated very strong genetic associations between the polymorphic ERV in cis and disrupted transcript levels. Premature polyadenylation is triggered at genomic distances up to >12.5 kb upstream of the ERV, both in cis and between alleles. The parent of origin of the ERV is associated with variable expression of nonterminated transcripts and differential DNA methylation at its 5'-long terminal repeat. This study defines an unexpectedly strong functional impact of ERVs in disrupting gene transcription at a distance and demonstrates that ongoing retrotransposition can contribute significantly to natural phenotypic diversity.

Ubiquitination is a post-translational modification in which one or more ubiquitin molecules are covalently linked to lysine residues of target proteins. The ubiquitin system plays a key role in the regulation of protein degradation, which contributes to cell signaling, vesicular trafficking, apoptosis, and immune regulation. Bacterial and viral pathogens exploit the cellular ubiquitin system by encoding their own proteins to serve their survival and replication in infected cells. Recent studies have revealed that Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the ubiquitin system of infected cells to facilitate cell proliferation, anti-apoptosis, and evasion from immunity. This review summarizes recent developments in our understanding of the molecular mechanisms used by KSHV to interact with the cellular ubiquitin machinery.

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

No abstract available.

Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting immune reconstitution. This feature makes IL-7 an ideal candidate for therapeutic development. As with other cytokines, signaling through the IL-7 receptor induces the JAK/STAT pathway. However, the broad scope of IL-7 regulatory targets likely necessitates the use of other signaling components whose identities remain poorly defined. To this end, we used an IL-7 dependent T-cell line to examine how expression of the glycolytic enzyme, Hexokinase II (HXKII) was regulated by IL-7 in a STAT5-independent manner. Our studies revealed that IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Gel shifts showed that the AP-1 complex induced by IL-7 contained JunD but not c-Fos or c-Jun. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. Because others had shown that JunD was a negative regulator of cell growth, we performed a bioinformatics analysis to uncover possible JunD-regulated gene targets. Our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism. One of these growth promoters was the oncogene, Pim-1. Pim-1 is an IL-7-induced protein that was inhibited when the activities of JNK or JunD were blocked, showing that in IL-7 dependent T-cells JunD can promote positive signals transduced through Pim-1. This was confirmed when the IL-7-induced proliferation of CD8 T-cells was impaired upon JunD inhibition. These results show that engagement of the IL-7 receptor drives a signal that is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth stimulation through the expression of metabolic and signaling factors like HXKII and Pim-1.

<h4>Background</h4>Despite a greater burden of risk factors, atrial fibrillation (AF) is less common among African Americans than European-descent populations. Genome-wide association studies (GWAS) for AF in European-descent populations have identified three predominant genomic regions associated with increased risk (1q21, 4q25, and 16q22). The contribution of these loci to AF risk in African American is unknown.<h4>Methodology/principal findings</h4>We studied 73 African Americans with AF from the Vanderbilt-Meharry AF registry and 71 African American controls, with no history of AF including after cardiac surgery. Tests of association were performed for 148 SNPs across the three regions associated with AF, and 22 SNPs were significantly associated with AF (P<0.05). The SNPs with the strongest associations in African Americans were both different from the index SNPs identified in European-descent populations and independent from the index European-descent population SNPs (r(2)<0.40 in HapMap CEU): 1q21 rs4845396 (odds ratio [OR] 0.30, 95% confidence interval [CI] 0.13-0.67, P = 0.003), 4q25 rs4631108 (OR 3.43, 95% CI 1.59-7.42, P = 0.002), and 16q22 rs16971547 (OR 8.1, 95% CI 1.46-45.4, P = 0.016). Estimates of European ancestry were similar among cases (23.6%) and controls (23.8%). Accordingly, the probability of having two copies of the European derived chromosomes at each region did not differ between cases and controls.<h4>Conclusions/significance</h4>Variable European admixture at known AF loci does not explain decreased AF susceptibility in African Americans. These data support the role of 1q21, 4q25, and 16q22 variants in AF risk for African Americans, although the index SNPs differ from those identified in European-descent populations.

Quinolinic acid (QUIN), an endogenous metabolite of the kynurenine pathway, is involved in several neurological disorders, including Huntington's disease, Alzheimer's disease, schizophrenia, HIV associated dementia (HAD) etc. QUIN toxicity involves several mechanisms which trigger various metabolic pathways and transcription factors. The primary mechanism exerted by this excitotoxin in the central nervous system (CNS) has been largely related with the overactivation of N-methyl-D-aspartate receptors and increased cytosolic Ca(2+) concentrations, followed by mitochondrial dysfunction, cytochrome c release, ATP exhaustion, free radical formation and oxidative damage. As a result, this toxic pattern is responsible for selective loss of middle size striatal spiny GABAergic neurons and motor alterations in lesioned animals. This toxin has recently gained attention in biomedical research as, in addition to its proven excitotoxic profile, a considerable amount of evidence suggests that oxidative stress and energetic disturbances are major constituents of its toxic pattern in the CNS. Hence, this profile has changed our perception of how QUIN-related disorders combine different toxic mechanisms resulting in brain damage. This review will focus on the description and integration of recent evidence supporting old and suggesting new mechanisms to explain QUIN toxicity.

Huntington's disease (HD) is an inheritable neurological disorder caused by an abnormal expansion of the polyglutamine tract in the N-terminus of the protein huntingtin (htt). Mutant htt (mhtt) leads to selective neurodegeneration that preferentially affects striatal medium spiny neurons. Although mhtt is also expressed in astrocytes, whether and how astrocyte derived mhtt contributes to the neurodegeneration in HD remains largely unknown. In this study, a glia HD model, using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 aa (htt552) in rat primary cortical astrocytes, was generated. The influence of htt552 on the protein level of brain-derived neurotrophic factor (BDNF) in astrocytes was evaluated. Immunofluorescence showed that htt552-100Q formed aggregates in some astrocytes. These mhtt aggregates sequestered clathrin immunoreactivities and dispersed the Golgi complex. ELISA and immunofluorescence demonstrated an increase in BDNF levels in the astrocytes expressing htt552-100Q. Western blot analysis showed that there was an increase in pro-BDNF, but a decrease in mature BDNF in the astrocytes expressing htt552-100Q. Furthermore, medium collected from astrocytes expressing htt552-100Q showed a lower level of mature BDNF and less activity in supporting neurite development of primary cortical neurons. These results suggest that aggregates formed by mutant htt552 affect processing and secretion of the BDNF in astrocytes, which might contribute to the neuronal dysfunction and degeneration in HD.

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.

The aim of this study was to assess the frequencies of three hemochromatosis gene (HFE) mutations in ethnic Roma/Gypsies in Slovakia. A cohort of 367 individuals representing general population and not preselected for health status was genotyped by TaqMan real-time PCR assay for C282Y, H63D and S65C mutations in HFE gene. A unique genetic profile was revealed: C282Y is found in the highest frequency of all Central European countries (4.90%), while the frequency of H63D mutation (4.09%) is lower than any reported in Europe so far. S65C mutation was not present in the cohort. These mutation frequencies can be explained rather by gene influx and genetic isolation than by genetic inheritance from a former Roma/Gypsy homeland.

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

<h4>Background</h4>A contemporary view of the cancer genome reveals extensive rearrangement compared to normal cells. Yet how these genetic alterations translate into specific proteomic changes that underpin acquiring the hallmarks of cancer remains unresolved. The objectives of this study were to quantify alterations in protein expression in two HER2+ cellular models of breast cancer and to infer differentially regulated signaling pathways in these models associated with the hallmarks of cancer.<h4>Results</h4>A proteomic workflow was used to identify proteins in two HER2 positive tumorigenic cell lines (BT474 and SKBR3) that were differentially expressed relative to a normal human mammary epithelial cell line (184A1). A total of 64 (BT474-184A1) and 69 (SKBR3-184A1) proteins were uniquely identified that were differentially expressed by at least 1.5-fold. Pathway inference tools were used to interpret these proteins in terms of functionally enriched pathways in the tumor cell lines. We observed "protein ubiquitination" and "apoptosis signaling" pathways were both enriched in the two breast cancer models while "IGF signaling" and "cell motility" pathways were enriched in BT474 and "amino acid metabolism" were enriched in the SKBR3 cell line.<h4>Conclusion</h4>While "protein ubiquitination" and "apoptosis signaling" pathways were common to both the cell lines, the observed patterns of protein expression suggest that the evasion of apoptosis in each tumorigenic cell line occurs via different mechanisms. Evidently, apoptosis is regulated in BT474 via down regulation of Bid and in SKBR3 via up regulation of Calpain-11 as compared to 184A1.

Since its discovery in the early 1990s the deleted in colorectal cancer (DCC) gene, located on chromosome 18q21, has been proposed as a tumour suppressor gene as its loss is implicated in the majority of advanced colorectal and many other cancers. DCC belongs to the family of netrin 1 receptors, which function as dependence receptors as they control survival or apoptosis depending on ligand binding. However, the role of DCC as a tumour suppressor remains controversial because of the rarity of DCC-specific mutations and the presence of other tumour suppressor genes in the same chromosomal region. Here we show that in a mouse model of mammary carcinoma based on somatic inactivation of p53, additional loss of DCC promotes metastasis formation without affecting the primary tumour phenotype. Furthermore, we demonstrate that in cell cultures derived from p53-deficient mouse mammary tumours DCC expression controls netrin-1-dependent cell survival, providing a mechanistic basis for the enhanced metastatic capacity of tumour cells lacking DCC. Consistent with this idea, in vivo tumour-cell survival is enhanced by DCC loss. Together, our data support the function of DCC as a context-dependent tumour suppressor that limits survival of disseminated tumour cells.

Neurodegeneration of the striatum in Huntington disease (HD) is characterized by loss of medium-spiny neurons, huntingtin nuclear inclusions, reactive gliosis, and iron accumulation. Neuroimaging allows in vivo detection of the macro- and micro-structural changes that occur from presymptomatic stages of the disease (preHD). The aim of our study was to evaluate the reliability of multimodal imaging as an in vivo biomarker of vulnerability and development of the disease and to characterize macro- and micro-structural changes in subcortical nuclei in HD. Macrostructure (T1-weighted images), microstructure (diffusion tensor imaging), and iron content (R 2* relaxometry) of subcortical nuclei and medial temporal lobe structures were evaluated by a 3 T scanner in 17 preHD carriers, 12 early-stage patients and 29 matched controls. We observed a volume reduction and microstructural changes in the basal ganglia (caudate, putamen, and globus pallidus) and iron accumulation in the globus pallidus in both preHD and symptomatic subjects; all these features were significantly more pronounced in patients, in whom degeneration extended to the other subcortical nuclei (i.e., thalamus and accumbens). Mean diffusivity (MD) was the most powerful predictor in models explaining more than 50% of the variability in HD development in the caudate, putamen, and thalamus. These findings suggest that the measurement of MD may further enhance the well-known predictive value of striatal volume to assess disease progression as it is highly sensitive to tissue microimpairment. Multimodal imaging may detect brain changes even in preHD stages.

Genome-wide association studies (GWAS) have become a preferred method to identify new genetic susceptibility loci. This technique aims to understanding the molecular etiology of common diseases, but in many cases, it has led to the identification of loci with no obvious biological relevance. Herein, we show that previously unrecognized sequence homologies have caused single-nucleotide polymorphism (SNP) microarrays to incorrectly associate a phenotype to a given locus when in fact the linkage is to another distant locus. Using genetic differences between male and female subjects as a model to study the effect of one specific genomic region on the whole SNP microarray, we provide strong evidence that the use of standard methods for GWAS can be misleading. We suggest a new systematic quality control step in the biological interpretation of previous and future GWAS.

<h4>Context</h4>Few studies have examined morbidity and mortality associated with hepatobiliary disease in diabetes. Most have used administrative databases and/or have had limited/incomplete data including recognized risk factors for hepatobiliary disease.<h4>Objective</h4>The objective of the study was to explore the relationship between type 2 diabetes and hepatobiliary disease in well-characterized patients with detailed risk factor data including viral hepatitis status and hemochromatosis genotype.<h4>Design</h4>This was a community-based longitudinal observational study.<h4>Setting</h4>The study was conducted in an urban Australian community.<h4>Patients</h4>The study included 1294 patients of mean ± SD aged 64.1 ± 11.3 yr and 5156 age-, gender-, and ZIP code-matched nondiabetic controls.<h4>Main outcome measures</h4>Prevalent and incident hepatobiliary disease and hepatobiliary disease-related death were measured. Competing risks proportional hazard models provided independent associates of these end points.<h4>Results</h4>During 13,705 patient-years (mean 11.5 yr), 144 patients had an initial hepatobiliary disease-related hospitalization/cancer registration vs. 403 controls during 63,937 person-years of follow-up, an incidence rate ratio of 1.66 (95% confidence interval 1.37-2.02). Incident hepatobiliary disease was associated with a lower glycosylated hemoglobin and higher urinary albumin to creatinine ratio. Nearly half of the patients (49.9%) died during follow-up [crude mortality ratio vs. nondiabetic controls 1.97 (1.16-3.32)], and 21 (3.3%) from hepatobiliary disease including two cases of cirrhosis attributable to nonalcoholic steatohepatitis. Hepatobiliary disease-related death was independently predicted by prior hepatobiliary disease, hepatitis C seropositivity, retinopathy, and peripheral neuropathy; higher educational level and higher fasting serum glucose were protective.<h4>Conclusions</h4>Hepatobiliary disease and associated mortality are increased in type 2 diabetes. Multiple factors including fatty infiltration, microangiopathy, and direct glucotoxicity are likely to contribute, but hospitalization and death due to cirrhosis from nonalcoholic steatohepatitis appear uncommon.

Sigma-1 receptors are associated with Alzheimer's disease, major depressive disorders, and schizophrenia. These receptors show progrowth/antiapoptotic properties via their chaperoning functions to counteract ER (endoplasmic reticulum) stress, to block neurodegeneration, and to regulate neuritogenesis. The sigma-1 receptor knock out mouse offered an opportunity to assess possible mechanisms by which the sigma-1 receptor modulates cellular oxidative stress. Nuclear magnetic resonance (NMR) metabolomic screening of the WT (wild type) and sigma-1 KO (knockout) livers was performed to investigate major changes in metabolites that are linked to oxidative stress. Significant changes in protein levels were also identified by two-dimensional (2D) gel electrophoresis and mass spectrometry. Increased levels of the antioxidant protein peroxiredoxin 6 (Prdx6), and the ER chaperone BiP (GRP78) compared to WT littermates were detected. Oxidative stress was measured in WT and sigma-1 KO mouse liver homogenates, in primary hepatocytes and in lung homogenates. Furthermore, sigma-1 receptor mediated activation of the antioxidant response element (ARE) to upregulate NAD(P)H quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1) mRNA expression in COS cells was shown by RT PCR. These novel functions of the sigma-1 receptor were sensitive to well-known sigma ligands via their antagonist/agonist properties.

<h4>Background</h4>Despite its estimated high heritability, the genetic architecture leading to differences in cognitive performance remains poorly understood. Different cortical regions play important roles in normal cognitive functioning and impairment. Recently, we reported on sets of regionally enriched genes in three different cortical areas (frontomedial, temporal and occipital cortices) of the adult rat brain. It has been suggested that genes preferentially, or specifically, expressed in one region or organ reflect functional specialisation. Employing a gene-based approach to the analysis, we used the regionally enriched cortical genes to mine a genome-wide association study (GWAS) of the Norwegian Cognitive NeuroGenetics (NCNG) sample of healthy adults for association to nine psychometric tests measures. In addition, we explored GWAS data sets for the serious psychiatric disorders schizophrenia (SCZ) (n = 3 samples) and bipolar affective disorder (BP) (n = 3 samples), to which cognitive impairment is linked.<h4>Principal findings</h4>At the single gene level, the temporal cortex enriched gene RAR-related orphan receptor B (RORB) showed the strongest overall association, namely to a test of verbal intelligence (Vocabulary, P = 7.7E-04). We also applied gene set enrichment analysis (GSEA) to test the candidate genes, as gene sets, for enrichment of association signal in the NCNG GWAS and in GWASs of BP and of SCZ. We found that genes differentially expressed in the temporal cortex showed a significant enrichment of association signal in a test measure of non-verbal intelligence (Reasoning) in the NCNG sample.<h4>Conclusion</h4>Our gene-based approach suggests that RORB could be involved in verbal intelligence differences, while the genes enriched in the temporal cortex might be important to intellectual functions as measured by a test of reasoning in the healthy population. These findings warrant further replication in independent samples on cognitive traits.

Nutritional management is essential for patients with inborn errors of metabolism, such as urea cycle disorders (UCDs). Lack of appetite is common in these patients and can lead to underconsumption of calories, catabolism, and subsequently loss of metabolic control. The etiology of anorexia in these patients is largely unexplored. The neuroendocrine hormone peptide tyrosine tyrosine (PYY), secreted postprandially from endocrine cells of the ileum and colon, induces feelings of satiety and decreases food intake. While plasma PYY levels have been characterized in a number of populations, they have not been examined in UCD patients. In a retrospective study, plasma PYY concentrations were measured in UCD (n=42) patients and controls (n=28) via an ELISA to determine if levels of this anorexigenic hormone are altered in this patient population. Median PYY levels were significantly higher in UCD patients compared to controls (p=3.5×10(-5)). Body mass index was significantly associated with increased PYY levels in controls (p=0.02), while UCD diagnosis subtype was associated with PYY levels (p=1×10(-3)) in cases. Median PYY levels were significantly lower in ornithine carbamoyltransferase deficient patients compared with all other UCD subtypes (p=9×10(-3)), but significantly higher compared to controls (p=1.6×10(-3)). Overall, this study demonstrates that UCD cases have increased PYY levels compared to controls, suggesting that regulation of PYY may be altered in these patients. These observations may lead to a better understanding of the development of anorexia in UCD patients.

High levels of profibrinogenic cytokine transforming factor beta (TGF-β), metalloprotease (MMP2), and tissue inhibitor of matrix metalloprotease 1 (TIMP1) contribute to fibrogenesis in hepatitis C virus (HCV) infection and in alcohol-induced liver disease (ALD). The aim of our study was to correlate noninvasive serum markers in ALD and HCV patients with various degrees of inflammation and fibrosis in their biopsies. Methods. Serum cytokines levels in HCV-infected individuals in the presence or absence of ALD were measured. Student's-t-test with Bonferroni correction determined the significance between the groups. Results. Both tumor-necrosis-factor- (TNF)-α and TGF-β levels increased significantly with the severity of inflammation and fibrosis. TGF-β levels increased significantly in ALD patients versus the HCV patients. Proinflammatory cytokines' responses to viral and/or toxic injury differed with the severity of liver inflammation. A combination of these markers was useful in predicting and diagnosing the stages of inflammation and fibrosis in HCV and ALD. Conclusion. Therapeutic monitoring of TGF-β and metalloproteases provides important insights into fibrosis.

<h4>Background</h4>Next-generation DNA sequencing is opening new avenues for genetic association studies in common diseases that, like deep vein thrombosis (DVT), have a strong genetic predisposition still largely unexplained by currently identified risk variants. In order to develop sequencing and analytical pipelines for the application of next-generation sequencing to complex diseases, we conducted a pilot study sequencing the coding area of 186 hemostatic/proinflammatory genes in 10 Italian cases of idiopathic DVT and 12 healthy controls.<h4>Results</h4>A molecular-barcoding strategy was used to multiplex DNA target capture and sequencing, while retaining individual sequence information. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples) and enriched for target DNA sequences. Sequencing was performed on ABI SOLiD-4 platforms. We produced > 12 gigabases of raw sequence data to sequence at high coverage (average: 42X) the 700-kilobase target area in 22 individuals. A total of 1876 high-quality genetic variants were identified (1778 single nucleotide substitutions and 98 insertions/deletions). Annotation on databases of genetic variation and human disease mutations revealed several novel, potentially deleterious mutations. We tested 576 common variants in a case-control association analysis, carrying the top-5 associations over to replication in up to 719 DVT cases and 719 controls. We also conducted an analysis of the burden of nonsynonymous variants in coagulation factor and anticoagulant genes. We found an excess of rare missense mutations in anticoagulant genes in DVT cases compared to controls and an association for a missense polymorphism of FGA (rs6050; p = 1.9 × 10(-5), OR 1.45; 95% CI, 1.22-1.72; after replication in > 1400 individuals).<h4>Conclusions</h4>We implemented a barcode-based strategy to efficiently multiplex sequencing of hundreds of candidate genes in several individuals. In the relatively small dataset of our pilot study we were able to identify bona fide associations with DVT. Our study illustrates the potential of next-generation sequencing for the discovery of genetic variation predisposing to complex diseases.

Heart failure (HF) is a complex disease involving multiple changes including cardiomyocyte hypertrophy (growth). Here we performed a set of screens in different HF and hypertrophy models to identify differentially expressed genes associated with HF and/or hypertrophy. Hypertensive Ren2 rats and animals with postmyocardial infarction (post-MI) HF were used as in vivo HF models, and neonatal rat cardiomyocytes treated with hypertrophy inducing hormones phenylephrine, endothelin-1, and isoproterenol were used as in vitro models. This combined approach revealed a robust set of genes that were differentially expressed both in vitro and in vivo. This included known genes like NPPA (ANP) and FHL1, but also novel genes not previously associated with hypertrophy/HF. Among these are PTGIS, AKIP1, and Dhrs7c, which could constitute interesting targets for further investigations. We also identified a number of in vivo specific genes and these appeared to be enriched for fibrosis, wounding, and stress responses. Therefore a number of novel genes within this in vivo specific list could be related to fibroblasts or other noncardiomyocytes present in the heart. We also observed strong differences between the two HF rat models. For example KCNE1 was strongly upregulated in Ren2, but not in post-MI HF rats, suggesting possible etiology-specific differences. Moreover, Gene Ontology analysis revealed that genes involved in fatty acid oxidation were specifically down regulated in the post-MI group only. Together these results show that combining multiple models, both in vivo and in vitro, can provide a robust set of hypertrophy/HF-associated genes. Moreover it provides insight in the differences between the different etiology models and neurohormonal effects.

Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP-LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation.

Lifelong self-renewal of the adult intestinal epithelium requires the activity of stem cells located in mucosal crypts. Lgr5 and Bmi1 are two molecular markers of crypt-cell populations that replenish all lineages over time and hence function as stem cells. Intestinal stem cells require Wnt signaling, but the understanding of their cellular niche is incomplete. Lgr5-expressing crypt base columnar cells (CBCs) reside deep in the crypt, mingled among mature Paneth cells that are well positioned for short-range signaling. Partial lineage ablation previously had implied that Paneth cells are nonessential constituents of the stem-cell niche, but recently their absence was reported to interfere with Lgr5(+) CBCs, resurrecting an appealing idea. However, previous mouse models failed to remove Paneth cells completely or permanently; defining the intestinal stem-cell niche requires clarity with respect to the Paneth cell role. We find that Lgr5(+) cells with stem-cell activity cluster in future crypts early in life, before Paneth cells develop. We also crossed conditional Atoh1(-/-) mice, which lack Paneth cells entirely, with Lgr5(GFP) mice to visualize Lgr5(+) CBCs and to track their stem-cell function. In the sustained absence of Paneth cells, Lgr5(+) CBCs occupied the full crypt base, proliferated briskly, and generated differentiated progeny over many months. Gene expression in fluorescence-sorted Lgr5(+) CBCs reflected intact Wnt signaling despite the loss of Paneth cells. Thus, Paneth cells are dispensable for survival, proliferation, and stem-cell activity of CBCs, and direct contact with Lgr5-nonexpressing cells is not essential for CBC function.

The sequencing of the human genome led to many insights into gene organization and structure. One interesting observation was the high frequency of bidirectional promoters characterized by two protein encoding genes whose promoters are arranged in a divergent or "head-to-head" configuration with less than 2000 base pairs of intervening sequence. Computational estimates published by various groups indicate that nearly 10% of the coding gene promoters are arranged in such a manner and the extent of this bias is a unique feature of mammalian genomes. Moreover, as a class, head-to-head promoters appear to be enriched in specific categories of gene function. Here we review the structure, composition, genomic properties and functional classifications of genes controlled by bidirectional promoters and explore the biological implication of these features. This article is part of a Special Issue entitled: Chromatin in time and space.

Squamous cell carcinoma of the oral region (OSCC) is one of the most common and highly aggressive malignancies worldwide, despite the fact that significant results have been achieved during the last decades in its detection, prevention and treatment. Although many efforts have been made to define the molecular signatures that identify the clinical outcome of oral cancers, OSCC still lacks reliable prognostic molecular markers. Scientific evidence indicates that transition from normal epithelium to pre-malignancy, and finally to oral carcinoma, depends on the accumulation of genetic and epigenetic alterations in a multistep process. Unlike genetic alterations, epigenetic changes are heritable and potentially reversible. The most common examples of such changes are DNA methylation, histone modification, and small non-coding RNAs. Although several epigenetic changes have been currently linked to OSCC initiation and progression, they have been only partially characterized. Over the last decade, it has been demonstrated that especially aberrant DNA methylation plays a critical role in oral cancer. The major goal of the present paper is to review the recent literature about the epigenetic modifications contribution in early and later phases of OSCC malignant transformation; in particular we point out the current evidence of epigenetic marks as novel markers for early diagnosis and prognosis as well as potential therapeutic targets in oral cancer.

Numerous studies have suggested that the different histological subtypes of ovarian carcinoma (i.e. clear cell, endometrioid, mucinous, and serous) have distinct clinical histories and characteristics; however, most studies that have aimed to determine biomarker have not performed comprehensive analyses based on subtype specificity. In the present study, we performed two-dimensional gel electrophoresis-based differential proteomic analysis of the different histological subtypes of ovarian carcinoma using tissue specimens from 39 patients. Seventy-seven protein spots (55 unique proteins) were found to be up- or downregulated in a subtype-specific manner. The most significant difference was observed for: (i) annexin-A4 (ANXA4) and phosphoserine aminotransferase (PSAT1), which are expressed strongly in clear cell carcinoma; (ii) cellular retinoic acid-binding protein 2 (CRABP2), which is expressed specifically in serous carcinoma; and (iii) serpin B5 (SPB5), which is upregulated in mucinous carcinoma. Validation of these candidates by western blotting using a 34 additional test sample set resulted in an expression pattern that was consistent with the screening and revealed that differential expression was independent of cancer stage or tumor grade within each subtype. Thus, the present study reinforces the notion that ovarian cancer subtypes can be clearly delineated on a molecular basis into four histopathological groups, and we propose that ANXA4, PSAT1, CRABP2, and SPB5 are candidate subtype-specific biomarkers that can help define the basis of tumor histology at a molecular level.

<h4>Background</h4>Acute myeloid leukaemia (AML) with nucleophosmin-1 (NPM1) mutation is a major subtype of AML. The NPM1 mutation induces a myeloproliferative disorder, but evidence indicates that other insults are necessary for the development of AML. We utilised microRNA microarrays and functional assays to determine if microRNA dysregulation could be involved in the pathogenesis of in NPM1 mutated (NPM1mut)-AML.<h4>Results</h4>We used a stringent locked nucleic acid (LNA) based microRNA microarray platform to profile bone marrow samples of patients with normal karyotype AML. A panel of five microRNAs dichotomised AML patients according to their NPM1 mutational status. miR-10a, let-7b and let-7c were significantly over-expressed, while miR-130a and miR-335 were under-expressed in NPM1mut-AML when compared to NPM1wildtype-AML. Of these, miR-10a is the most differentially expressed in NPM1mut-AML versus NPM1wildtype-AML (> 10 fold higher as confirmed by qRT-PCR). To investigate the functions of miR-10a, the OCI-AML3 cell line was utilised, which is the only commercially available cell line bearing NPM1mut. OCI-AML3 cells were firstly demonstrated to have a similarly high miR-10a expression to primary NPM1mut-AML patient samples. Inhibition of miR-10a expression by miRCURY LNA Inhibitors (Exiqon) in these cells resulted in increased cell death as assessed by MTS, cell cycle and Annexin-V assays and reduced clonogenic capacity, indicative of an involvement in leukaemic cell survival. In silico filtering of bioinformatically predicted targets of miR-10a identified a number of potential mRNA targets with annotated functions in haematopoiesis, cell growth and apoptosis. Lucferase reporter assays confirmed a number of these putative tumorogenic genes that are miR-10a suppressible including KLF4 and RB1CC1. This provides a potential mechanism for the pathogenic role of miR-10a in NPM1mut-AML.<h4>Conclusions</h4>This study provides, for the first time, in vitro evidence of a pro-survival role of miR-10a in NPM1mut-AML, that it may contribute to the pathogenesis of NPM1mut-AML and identifies putative tumorogenic targets.

Alcoholism is a significant public health problem. A picture of the genetic architecture underlying alcohol-related phenotypes is emerging from genome-wide association studies and work on genetically tractable model organisms.

Myosin X (Myo X), also known as MYO10, is an unconventional actin-based motor protein that plays an important role in filopodium formation. Its intra-filopodia movement, an event tightly associated with the function of Myo X, has been extensively studied. However, how the motor activity of Myo X and the direction of its movements are regulated remains largely unknown. In our previous study, we demonstrated that DCC (for 'deleted in colorectal carcinoma') and neogenin (neogenin 1, NEO1 or NGN), a family of immunoglobin-domain-containing transmembrane receptors for netrins, interact with Myo X and that DCC is a cargo of Myo X to be delivered to the neurites of cultured neurons. Here, we provide evidence for DCC and neogenin as regulators of Myo X. DCC promotes movement of Myo X along basal actin filaments and enhances Myo-X-mediated basal filopodium elongation. By contrast, neogenin appears to suppress Myo X movement on the basal side, but increases its movement towards the apical and dorsal side of a cell, promoting dorsal filopodium formation and growth. Further studies have demonstrated that DCC, but not neogenin, enhances integrin-mediated tyrosine phosphorylation of focal adhesion kinase and basal F-actin reorganization, providing a cellular mechanism underlying their distinct effects on Myo X. These results thus demonstrate differential regulatory roles on Myo X activity by its cargo proteins, DCC and neogenin, revealing different cellular functions of DCC and neogenin.

The immediate complication after implantation of mechanical circulatory support (MCS) is more often bleeding than thromboembolic events. We report an unusual case of massive thrombus formation following systemic thrombolytic therapy which happened twice after implantation of MCS. Various mechanisms may contribute to this severe complication, but attention should be paid to patients who receive MCS after systemic thrombolysis because of the secondary hypercoagulability induced by this therapy.

<h4>Purpose</h4>The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response.<h4>Experimental design</h4>Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated.<h4>Results</h4>Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAF(V600E/K)), whereas eight of 17 (47%) tumors were BRAF wild type (BRAF(WT)). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAF(WT), of which 1 carried c-KIT(L576P) and another N-RAS(Q61R) mutation, while only 2 (29%) of the responding tumors were BRAF(V600E/K). Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11.<h4>Conclusions</h4>Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles.

<h4>Purpose</h4>Recent studies suggest that intrinsic breast cancer subtypes may differ in their responsiveness to specific chemotherapy regimens. We examined this hypothesis on NCIC.CTG MA.5, a clinical trial randomizing premenopausal women with node-positive breast cancer to adjuvant CMF (cyclophosphamide-methotrexate-fluorouracil) versus CEF (cyclophosphamide-epirubicin-fluorouracil) chemotherapy.<h4>Experimental design</h4>Intrinsic subtype was determined for 476 tumors using the quantitative reverse transcriptase PCR PAM50 gene expression test. Luminal A, luminal B, HER2-enriched (HER2-E), and basal-like subtypes were correlated with relapse-free survival (RFS) and overall survival (OS), estimated using Kaplan-Meier plots and log-rank testing. Multivariable Cox regression analyses determined significance of interaction between treatment and intrinsic subtypes.<h4>Results</h4>Intrinsic subtypes were associated with RFS (P = 0.0005) and OS (P < 0.0001) on the combined cohort. The HER2-E showed the greatest benefit from CEF versus CMF, with absolute 5-year RFS and OS differences exceeding 20%, whereas there was a less than 2% difference for non-HER2-E tumors (interaction test P = 0.03 for RFS and 0.03 for OS). Within clinically defined Her2(+) tumors, 79% (72 of 91) were classified as the HER2-E subtype by gene expression and this subset was strongly associated with better response to CEF versus CMF (62% vs. 22%, P = 0.0006). There was no significant difference in benefit between CEF and CMF in basal-like tumors [n = 94; HR, 1.1; 95% confidence interval (CI), 0.6-2.1 for RFS and HR, 1.3; 95% CI, 0.7-2.5 for OS].<h4>Conclusion</h4>HER2-E strongly predicted anthracycline sensitivity. The chemotherapy-sensitive basal-like tumors showed no added benefit for CEF over CMF, suggesting that nonanthracycline regimens may be adequate in this subtype although further investigation is required.

In colorectal cancer (CRC), chromosomal instability (CIN) is typically studied using comparative-genomic hybridization (CGH) arrays. We studied paired (tumor and surrounding healthy) fresh frozen tissue from 86 CRC patients using Illumina's Infinium-based SNP array. This method allowed us to study CIN in CRC, with simultaneous analysis of copy number (CN) and B-allele frequency (BAF)--a representation of allelic composition. These data helped us to detect mono-allelic and bi-allelic amplifications/deletion, copy neutral loss of heterozygosity, and levels of mosaicism for mixed cell populations, some of which can not be assessed with other methods that do not measure BAF. We identified associations between CN abnormalities and different CRC phenotypes (histological diagnosis, location, tumor grade, stage, MSI and presence of lymph node metastasis). We showed commonalities between regions of CN change observed in CRC and the regions reported in previous studies of other solid cancers (e.g. amplifications of 20q, 13q, 8q, 5p and deletions of 18q, 17p and 8p). From Therapeutic Target Database, we identified relevant drugs, targeted to the genes located in these regions with CN changes, approved or in trials for other cancers and common diseases. These drugs may be considered for future therapeutic trials in CRC, based on personalized cytogenetic diagnosis. We also found many regions, harboring genes, which are not currently targeted by any relevant drugs that may be considered for future drug discovery studies. Our study shows the application of high density SNP arrays for cytogenetic study in CRC and its potential utility for personalized treatment.

Temporal lobe epilepsy (TLE) is usually regarded as a polygenic and complex disorder. To understand its genetic component, numerous linkage analyses of familial forms and association studies of cases versus controls have been conducted since the middle of the nineties. The present paper lists genetic findings for TLE from the initial segregation analysis to the most recent results published in May 2011. To date, no genes have been clearly related to TLE despite many efforts to do so. However, it is vital to continue replication studies and collaborative attempts to find significant results and thus determine which gene variant combination plays a definitive role in the aetiology of TLE.

<h4>Background</h4>Emerging evidence suggests that excess iron may be detrimental for brain development. However, little is known regarding the association between neonatal iron overload and subsequent neurodevelopment during infancy in vulnerable premature infants.<h4>Aims</h4>To evaluate the association between neonatal iron overload and neurodevelopment in premature infants.<h4>Study design</h4>Prospective cohort study.<h4>Subjects</h4>24-32 weeks gestational age infants who had serum ferritin (SF) measured at 34-35 weeks post-menstrual age (PMA) and did not meet exclusion criteria: SF<76 ng/ml, toxoplasmosis, syphilis, rubella, cytomegalovirus, herpes infections, chromosomal disorders, or craniofacial anomalies were eligible. In addition, infants with sepsis or elevated C-reactive protein within 10 days before their SF measurement were excluded.<h4>Outcome measures</h4>Infants were evaluated for neurodevelopmental outcome at 8-12 months of age and were deemed to have neurodevelopmental impairment if they had one or more of the following: mental developmental index<70, abnormal neurological examination, bilateral blindness, bilateral deafness, or required occupational, physical, or speech therapy.<h4>Results</h4>95 infants were studied. 70 had normal iron status (SF 76-400 ng/ml) while 25 were deemed to have iron overload (SF >400 ng/ml) at 34-35 weeks PMA. There was a marginal increase in neurodevelopmental impairment among infants with iron overload compared to infants with normal iron status (64% vs. 41%, p=0.05). However, after controlling for confounders, iron overload was not associated with neurodevelopmental impairment (Adjusted OR 0.71, 95% CI, 0.21-2.5).<h4>Conclusion</h4>Modest neonatal iron overload is not associated with neurodevelopmental impairment during infancy in premature infants.

Defining the regulatory molecular networks involved in patterning the developing anterior endoderm is essential to understand how the pancreas, liver, stomach, and duodenum are discretely specified from each other. In this study, we analyzed the expression and function of the double-stranded RNA-binding protein Staufen2 in Xenopus laevis endoderm. We found that staufen2 was broadly expressed within the developing endoderm beginning at gastrulation becoming localized to the anterior endoderm at later stages. Through morpholino-mediated knockdown, we demonstrate that Staufen2 function is required for proper formation of the stomach, liver, and pancreas. We define that its function is required during gastrulation for proper patterning of the dorsal-ventral axis and that it acts to regulate expression of BMP signaling components.

Elevated levels of DnaA cause excessive initiation, which leads to an increased level of double-strand breaks that are proposed to arise when newly formed replication forks collide from behind with stalled or collapsed forks. These double-strand breaks are toxic in mutants that are unable to repair them. Using a multicopy suppressor assay to identify genes that suppress this toxicity, we isolated a plasmid carrying a gene whose function had been unknown. This gene, carried by the cryptic rac prophage, has been named rcbA for its ability to reduce the frequency of chromosome breaks. Our study shows that the colony formation of strains bearing mutations in rep, recG, and rcbA, like recA and recB mutants, is inhibited by an oversupply of DnaA and that a multicopy plasmid carrying rcbA neutralizes this inhibition. These and other results suggest that rcbA helps to maintain the integrity of the bacterial chromosome by lowering the steady-state level of double-strand breaks.

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase-mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.

N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptor, which play a central role in learning, memory, and synaptic development. NMDARs are assembled as tetramers composed of two GluN1 subunits and two GluN2 or GluN3 subunits. Although NMDARs are widely expressed throughout the central nervous system, their number, localization, and subunit composition are strictly regulated and differ in a cell- and synapse-specific manner. The brain area, developmental stage, and level of synaptic activity are some of the factors that regulate NMDARs. Molecular mechanisms that control subunit-specific NMDAR function include developmental regulation of subunit transcription/translation, differential trafficking through the secretory pathway, posttranscriptional modifications such as phosphorylation, and protein-protein interactions. The GluN2A and GluN2B subunits are highly expressed in cortex and hippocampus and confer many of the distinct properties on endogenous NMDARs. Importantly, the synaptic NMDAR subunit composition changes from predominantly GluN2B-containing to GluN2A-containing NMDARs during synaptic maturation and in response to activity and experience. Some of the molecular mechanisms underlying this GluN2 subunit switch have been recently identified. In addition, the balance between synaptic and extrasynaptic NMDARs is altered in several neuronal disorders. Here, the authors summarize the recent advances in the identification of NMDAR subunit-specific regulatory mechanisms.

Dysregulation of synaptic development and function has been implicated in the pathophysiology of neurodegenerative disorders and mental disease. A neurotrophin that has an important function in neuronal and synaptic development is brain-derived neurotrophic factor (BDNF). In this communication, we examined the effects of lead (Pb(2+)) exposure on BDNF-tropomyosin-related kinase B (TrkB) signaling during the period of synaptogenesis in cultured neurons derived from embryonic rat hippocampi. We show that Pb(2+) exposure decreases BDNF gene and protein expression, and it may also alter the transport of BDNF vesicles to sites of release by altering Huntingtin phosphorylation and protein levels. Combined, these effects of Pb(2+) resulted in decreased concentrations of extracellular mature BDNF. The effect of Pb(2+) on BDNF gene expression was associated with a specific decrease in calcium-sensitive exon IV transcript levels and reduced phosphorylation and protein expression of the transcriptional repressor methyl-CpG-binding protein (MeCP2). TrkB protein levels and autophosphorylation at tyrosine 816 were significantly decreased by Pb(2+) exposure with a concomitant increase in p75 neurotrophin receptor (p75(NTR)) levels and altered TrkB-p75(NTR) colocalization. Finally, phosphorylation of Synapsin I, a presynaptic target of BDNF-TrkB signaling, was significantly decreased by Pb(2+) exposure with no effect on total Synapsin I protein levels. This effect of Pb(2+) exposure on Synapsin I phosphorylation may help explain the impairment in vesicular release documented by us previously (Neal, A. P., Stansfield, K. H., Worley, P. F., Thompson, R. E., and Guilarte, T. R. (2010). Lead exposure during synaptogenesis alters vesicular proteins and impairs vesicular release: Potential role of N-Methyl-D-aspartate receptor (NMDAR) dependent BDNF signaling. Toxicol. Sci. 116, 249-263) because it controls vesicle movement from the reserve pool to the readily releasable pool. In summary, the present study demonstrates that Pb(2+) exposure during the period of synaptogenesis of hippocampal neurons in culture disrupts multiple synaptic processes regulated by BDNF-TrkB signaling with long-term consequences for synaptic function and neuronal development.

A functional polymorphism within the serotonin transporter gene (5-HTTLPR) has been reported to modulate emotionality and risk for affective disorders. The short (S) allele has less functional efficacy than the long (L) allele and has been associated with enhanced emotional reactivity. One possible contributing factor to the high emotionality in S carriers may be inefficient use of cognitive strategies such as reappraisal to regulate emotional responses. The aim of the present study was to test whether the 5-HTTLPR genotype modulates the neural correlates of emotion regulation. To determine neural differences between S and L allele carriers during reappraisal of negative emotions, 15 homozygous S (S'/S') and 15 homozygous L (L'/L') carriers underwent functional magnetic resonance imaging (fMRI), while performing an instructed emotion regulation task including downregulation, upregulation and passive viewing of negative emotional pictures. Compared to L'/L' allele carriers, subjects who carry the S'/S' allele responded with lower posterior insula and prefrontal brain activation during passive perception of negative emotional information but showed greater prefrontal activation and anterior insula activation during down- and upregulation of negative emotional responses. The current results support and extend previous findings of enhanced emotionality in S carriers by providing additional evidence of 5-HTTLPR modulation of volitional emotion regulation.

Over the past decade, non-coding RNAs (ncRNAs) have become a new paradigm of gene regulation. ncRNAs are classified into two major groups based on their size: long non-coding RNAs (lncRNAs) and small non-coding RNAs (including microRNAs, piRNAs, snoRNAs, and endogenous siRNAs). Here we review the recently emerging role of ncRNAs in mammary development, tumorigenesis, and metastasis, with the focus being on microRNAs (miRNAs) and lncRNAs. These findings shed new light on normal development and malignant progression, and suggest the potential for using ncRNAs as new biomarkers of breast cancer and targets for treatment.

The mechanism by which hydroxyapatite (HA)-coated titanium promotes bone-implant integration is largely unknown. Furthermore, refining the fabrication of nano-structured HA to the level applicable to the mass production process for titanium implants is challenging. This study reports successful creation of nanopolymorphic crystalline HA on microroughened titanium surfaces using a combination of flame spray and low-temperature calcination and tests its biological capability to enhance bone-implant integration. Sandblasted microroughened titanium implants and sandblasted + HA-coated titanium implants were subjected to biomechanical and histomorphometric analyses in a rat model. The HA was 55% crystallized and consisted of nanoscale needle-like architectures developed in various diameters, lengths, and orientations, which resulted in a 70% increase in surface area compared to noncoated microroughened surfaces. The HA was free from impurity contaminants, with a calcium/phosphorus ratio of 1.66 being equivalent to that of stoichiometric HA. As compared to microroughened implants, HA-coated implants increased the strength of bone-implant integration consistently at both early and late stages of healing. HA-coated implants showed an increased percentage of bone-implant contact and bone volume within 50 μm proximity of the implant surface, as well as a remarkably reduced percentage of soft tissue intervention between bone and the implant surface. In contrast, bone volume outside the 50 μm border was lower around HA-coated implants. Thus, this study demonstrated that the addition of pure nanopolymorphic crystalline HA to microroughened titanium not only accelerates but also enhances the level of bone-implant integration and identified the specific tissue morphogenesis parameters modulated by HA coating. In particular, the nanocrystalline HA was proven to be drastic in increasing osteoconductivity and inhibiting soft tissue infiltration, but the effect was limited to the immediate microenvironment surrounding the implant.

Studies in simple model organisms have yielded crucial insights into the genetic and molecular aspects of longevity. FOXO, which is most notable for its association with longevity, and its upstream regulators such as sirtuins have received particular attention in translational research because these genes modulate cell survival in several models of neurodegenerative diseases. There is a large amount of knowledge on the pathways that regulate FOXO activity and genes that may be regulated by FOXO. However, for the same reason that the FOXO network is a complex stress response system, its therapeutic potential to develop disease-modifying strategies requires further examination. Although the FOXO network contains druggable genes such as sirtuins and AMPK, whether they should be activated or inhibited and whether protection against the early or late phases of neuronal cell decline might require opposite therapeutic strategies remains unclear. Additionally, the mode of action of small compound molecules believed to act on FOXO network targets was questioned. This review recapitulates essential facts and questions about the promises of FOXO and its interactors in neurodegenerative disease.

Genome-wide association studies (GWAS) have identified 19 risk variants associated with colorectal cancer. As most of these risk variants reside outside the coding regions of genes, we conducted cis-expression quantitative trait loci (cis-eQTL) analyses to investigate possible regulatory functions on the expression of neighboring genes. Forty microsatellite stable and CpG island methylator phenotype-negative colorectal tumors and paired adjacent normal colon tissues were used for genome-wide SNP and gene expression profiling. We found that three risk variants (rs10795668, rs4444235 and rs9929218, using near perfect proxies rs706771, rs11623717 and rs2059252, respectively) were significantly associated (FDR q-value ≤0.05) with expression levels of nearby genes (<2 Mb up- or down-stream). We observed an association between the low colorectal cancer risk allele (A) for rs10795668 at 10p14 and increased expression of ATP5C1 (q = 0.024) and between the colorectal cancer high risk allele (C) for rs4444235 at 14q22.2 and increased expression of DLGAP5 (q = 0.041), both in tumor samples. The colorectal cancer low risk allele (A) for rs9929218 at 16q22.1 was associated with a significant decrease in expression of both NOL3 (q = 0.017) and DDX28 (q = 0.046) in the adjacent normal colon tissue samples. Of the four genes, DLGAP5 and NOL3 have been previously reported to play a role in colon carcinogenesis and ATP5C1 and DDX28 are mitochondrial proteins involved in cellular metabolism and division, respectively. The combination of GWAS findings, prior functional studies, and the cis-eQTL analyses described here suggest putative functional activities for three of the colorectal cancer GWAS identified risk loci as regulating the expression of neighboring genes.

<h4>Purpose</h4>Serotonin and the serotonin transporter have been implicated in the development of pulmonary hypertension (PH). Selective serotonin reuptake inhibitors (SSRIs) may have a role in PH treatment, but the effects of SSRI use on right ventricular (RV) structure and function are unknown. We hypothesized that SSRI use would be associated with RV morphology in a large cohort without cardiovascular disease (N = 4114).<h4>Methods</h4>SSRI use was determined by medication inventory during the Multi-Ethnic Study of Atherosclerosis baseline examination. RV measures were assessed via cardiac magnetic resonance imaging. The cross-sectional relationship between SSRI use and each RV measure was assessed using multivariable linear regression; analyses for RV mass and end-diastolic volume (RVEDV) were stratified by sex.<h4>Results</h4>After adjustment for multiple covariates including depression and left ventricular measures, SSRI use was associated with larger RV stroke volume (RVSV) (2.75 mL, 95% confidence interval [CI] 0.48-5.02 mL, p = 0.02). Among men only, SSRI use was associated with greater RV mass (1.08 g, 95% CI 0.19-1.97 g, p = 0.02) and larger RVEDV (7.71 mL, 95% 3.02-12.40 mL, p = 0.001). SSRI use may have been associated with larger RVEDV among women and larger RV end-systolic volume in both sexes.<h4>Conclusions</h4>SSRI use was associated with higher RVSV in cardiovascular disease-free individuals and, among men, greater RV mass and larger RVEDV. The effects of SSRI use in patients with (or at risk for) RV dysfunction and the role of sex in modifying this relationship warrant further study.

YAC128 Huntington's disease (HD) transgenic mice accumulate less manganese (Mn) in the striatum relative to wild-type (WT) littermates. We hypothesized that Mn and mutant Huntingtin (HTT) would exhibit gene-environment interactions at the level of neurochemistry and neuronal morphology. Twelve-week-old WT and YAC128 mice were exposed to MnCl(2)-4H(2)O (50 mg/kg) on days 0, 3 and 6. Striatal medium spiny neuron (MSN) morphology, as well as levels of dopamine (DA) and its metabolites (which are known to be sensitive to Mn-exposure), were analyzed at 13 weeks (7 days from initial exposure) and 16 weeks (28 days from initial exposure). No genotype-dependent differences in MSN morphology were apparent at 13 weeks. But at 16 weeks, a genotype effect was observed in YAC128 mice, manifested by an absence of the wild-type age-dependent increase in dendritic length and branching complexity. In addition, genotype-exposure interaction effects were observed for dendritic complexity measures as a function of distance from the soma, where only YAC128 mice were sensitive to Mn exposure. Furthermore, striatal DA levels were unaltered at 13 weeks by genotype or Mn exposure, but at 16 weeks, both Mn exposure and the HD genotype were associated with quantitatively similar reductions in DA and its metabolites. Interestingly, Mn exposure of YAC128 mice did not further decrease DA or its metabolites versus YAC128 vehicle exposed or Mn exposed WT mice. Taken together, these results demonstrate Mn-HD disease-toxicant interactions at the onset of striatal dendritic neuropathology in YAC128 mice. Our results identify the earliest pathological change in striatum of YAC128 mice as being between 13 to 16 weeks. Finally, we show that mutant HTT suppresses some Mn-dependent changes, such as decreased DA levels, while it exacerbates others, such as dendritic pathology.

<h4>Objective</h4>Secondhand smoke (SS) induces chronic infection of endothelial cells by Chlamydia pneumoniae (Cpn) in vitro. We investigated the in vivo effect on atherosclerosis following exposure to SS and infection with Cpn both independently and in combination in ApoE-/- mice.<h4>Methods and results</h4>Plaques were largest in the combined SS+Cpn-exposed mice with 12-57% greater cross-sectional area compared with all other groups (P<0.03). Quantitative RT-PCR (qRT-PCR) from aortic roots revealed a synergistic upregulation of both OX40L (CD134L) and MyD88 in SS+Cpn mice (P<0.05). This upregulation occurred despite decreased numbers of macrophage, dendritic cell, CD4 T cell and smooth-muscle-cell infiltrates as determined by quantitative IHC and qRT-PCR. To elucidate whether enhanced apoptosis correlated with reduced plaque cellularity, area of Tdt-mediated dUTP nick labeling positive (TUNEL+) cells and expression of key bridging molecules necessary for efferocytosis (Mertk, Tgm2, FasL and C1qa) were examined. In SS+Cpn mice, there was an increase of the area of TUNEL+ cells in plaque cores (P<0.001) and a downregulation of efferocytosis gene expression (P<0.05). Systemic expression of cytokines in sera (Luminex) showed no differences between groups, suggesting that focal disease mechanisms within the plaque predominated.<h4>Conclusions</h4>The combination of SS exposure and Cpn infection enhanced atherosclerosis more than either variable did independently by activating inflammatory cells and by promoting growth and maturation of lesions via defective phagocytic clearance and accumulation of apoptotic cells.

Neuroendocrine, metabolic and autonomic nervous system dysfunctions are prevalent among patients with Huntington's disease (HD) and may underlie symptoms such as depression, weight loss and autonomic failure. Using post-mortem paraffin-embedded tissue, we assessed the integrity of the major neuropeptide populations in the paraventricular nucleus (PVN)-the hypothalamic neuroendocrine and autonomic integration center-in HD patients. The number corticotropin-releasing hormone, cocaine- and amphetamine-regulated transcript, arginine vasopressin and oxytocin immunoreactive (ir) neurons did not differ between HD patients and control subjects. However, the significant positive correlation between arginine vasopressin and oxytocin ir neurons in control subjects (P = 0.036) was absent in patients. Corticotropin-releasing hormone mRNA levels were 68% higher in HD patients (P = 0.046). Thyrotropin-releasing hormone mRNA levels did not differ between HD patients and control subjects, although a negative correlation with disease duration was present in the former (P = 0.036). These findings indicate that the PVN is largely unaffected in HD patients. However, our findings suggest that hypothalamic-pituitary-thyroid axis activity may alter during the course of the disease and that autonomic nervous system dysfunction might partly arise from an imbalance between arginine vasopressin and oxytocin neurons in the PVN.

The gastrin-releasing peptide receptor (BB2r) has shown great promise for tumor targeting due to the increase of the receptor expression in a variety of human cancers including prostate, breast, small-cell lung, and pancreatic cancer. From clinical investigations, prostate cancer has been shown to be among the most hypoxic of the cancers investigated. Many solid tumors contain regions of hypoxia due to poor organization and efficiency of the vasculature. However, hypoxia is typically not present in normal tissue. Nitroimidazoles, a thoroughly investigated class of hypoxia selective drugs, have been shown to be highly retained in hypoxic tissues. The purpose of this study is to determine if the incorporation of hypoxia trapping moieties into the structural paradigm of BB2r-targeted peptides will increase the retention time of the agents in prostate cancer tumors. The present work involves the design, syntheses, purification, and in vitro investigation of hypoxia enhanced (111)In-BB2r-targeted radioconjugates. A total of four BB2r-targeted conjugates (1-4) were synthesized and coupled with increasing numbers of 2-nitroimidazoles, a hypoxia trapping moiety. Conjugates were radiolabeled with (111)In and purified by HPLC prior to in vitro studies. Receptor saturation assays under both normoxic and hypoxic conditions showed that the BB2r receptor expression on the PC-3 human prostate cancer cell line was not significantly affected by oxygen levels. Competitive binding assays revealed that incorporation of 2-nitroimidazoles had a detrimental effect to BB2r binding when adequate spacer groups, between the hypoxia trapping agent and the pharmacophore, were not employed. All of the 2-nitroimidazole containing BB2r-targeted agents exhibited significantly higher longitudinal retention in PC-3 cells under hypoxic conditions compared to the analogous normoxic studies. Protein association analysis revealed a 3-fold increase in binding of a 2-nitroimidazole containing BB2r-targeted agent under hypoxic relative to normoxic conditions. The positive nature of these results indicate that further exploration into the potential of hypoxia selective trapping agents for BB2r-targeted agents, as well as other targeted compounds, is warranted.

Cutaneous melanoma originates from pigment producing melanocytes or their precursors and is considered the deadliest form of skin cancer. For the last 40 years, few treatment options were available for patients with late-stage melanoma. However, remarkable advances in the therapy field were made recently, leading to the approval of two new drugs, the mutant BRAF inhibitor vemurafenib and the immunostimulant ipilimumab. Although these drugs prolong patients' lives, neither drug cures the disease completely, emphasizing the need for improvements of current therapies. Our knowledge about the complex genetic and biological mechanisms leading to melanoma development has increased, but there are still gaps in our understanding of the early events of melanocyte transformation and disease progression. In this review, we present a summary of the main contributing factors leading to melanocyte transformation and discuss recent novel findings and technologies that will help answer some of the key biological melanoma questions and lay the groundwork for novel therapies.

<h4>Background</h4>Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia and type 2 diabetes mellitus. However, other risk factors for the development of NAFLD are unclear. With the association between periodontal disease and the development of systemic diseases receiving increasing attention recently, we conducted this study to investigate the relationship between NAFLD and infection with Porphyromonas gingivalis (P. gingivalis), a major causative agent of periodontitis.<h4>Methods</h4>The detection frequencies of periodontal bacteria in oral samples collected from 150 biopsy-proven NAFLD patients (102 with non-alcoholic steatohepatitis (NASH) and 48 with non-alcoholic fatty liver (NAFL) patients) and 60 non-NAFLD control subjects were determined. Detection of P. gingivalis and other periodontopathic bacteria were detected by PCR assay. In addition, effect of P. gingivalis-infection on mouse NAFLD model was investigated. To clarify the exact contribution of P. gingivalis-induced periodontitis, non-surgical periodontal treatments were also undertaken for 3 months in 10 NAFLD patients with periodontitis.<h4>Results</h4>The detection frequency of P. gingivalis in NAFLD patients was significantly higher than that in the non-NAFLD control subjects (46.7% vs. 21.7%, odds ratio: 3.16). In addition, the detection frequency of P. gingivalis in NASH patients was markedly higher than that in the non-NAFLD subjects (52.0%, odds ratio: 3.91). Most of the P. gingivalis fimbria detected in the NAFLD patients was of invasive genotypes, especially type II (50.0%). Infection of type II P. gingivalis on NAFLD model of mice accelerated the NAFLD progression. The non-surgical periodontal treatments on NAFLD patients carried out for 3 months ameliorated the liver function parameters, such as the serum levels of AST and ALT.<h4>Conclusions</h4>Infection with high-virulence P. gingivalis might be an additional risk factor for the development/progression of NAFLD/NASH.

Little is known regarding the molecular phenotype of kidneys with AKI because biopsies are performed infrequently. However, all kidney transplants experience acute injury, making early kidney transplants an excellent model of acute injury, provided the absence of rejection, because donor kidneys should not have CKD, post-transplant biopsies occur relatively frequently, and follow-up is excellent typically. Here, we used histopathology and microarrays to compare indication biopsies from 26 transplants with acute injury with 11 pristine protocol biopsies of stable transplants. Kidneys with acute injury showed increased expression of 394 transcripts associated with the repair response to injury, including many epithelium-like injury molecules tissue, remodeling molecules, and inflammation molecules. Many other genes also predicted the phenotype, including the acute injury biomarkers HAVCR1 and IL18. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score in kidneys with acute injury correlated with reduced graft function, future renal recovery, brain death, and need for dialysis, but not with future graft loss. In contrast, histologic features of acute tubular injury did not correlate with function or with the molecular changes. Thus, the transcripts associated with repair of injury suggest a massive coordinated response of the kidney parenchyma to acute injury, providing both an objective measure for assessing the severity of injury in kidney biopsies and validation for many biomarkers of AKI.

A broad array of evidence indicates that T lymphocytes make significant contributions to vascular inflammation in the setting of atherosclerotic disease, hypertension, autoimmune vasculitis, and other disorders. Experimental data show that costimulatory and coinhibitory pathways involving molecules of the B7-CD28 and TNF-TNFR families regulate T cell responses that promote vascular disease. Antigen presenting cells (APCs) display both peptide-major histocompatibility complex antigen and costimulators or coinhibitors to T cells. Two major types of APCs, dendritic cells (DCs) and macrophages, are present in significant numbers in the walls of arteries affected by atherosclerosis and arteritis, and some DCs are present in normal arteries. Costimulatory and coinhibitory molecules expressed by these vascular APCs can contribute to the activation or inhibition of effector T cells within the arterial wall. Vascular DCs may also be involved in transport of antigens to secondary lymphoid organs, where they activate or tolerize naïve T cells, depending on the balance of costimulators and coinhibitors they express. Costimulatory blockade is already an approved therapeutic approach to treat autoimmune disease and prevent transplant rejection. Preclinical models suggest that costimulatory blockade may also be effective in treating vascular disease. Experiential data in mice show that DCs pulsed with the appropriate antigens and treated in a way that reduces costimulatory capacity can reduce atherosclerotic disease, presumably by inducing T cell tolerance. Progress in treating vascular disease by immune modulation will require a more complete understanding of the functions of different costimulatory and coinhibitory pathways and the different subsets of vascular APCs involved.

We previously have shown that cue-induced alcohol craving and propensity for higher drinking are modulated by allelic differences in SLC6A4 associated with serotonin transporter (5-HTT) expression level alterations. In an independent study, we characterized another polymorphism, SNP rs1042173, in 3'-untranslated region (3'-UTR) of the same gene, which also altered 5-HTT expression levels; the T allele of rs1042173 was associated with lower mRNA and protein levels. In subsequent analyses, the TT genotype was found to be associated with higher drinking intensity in alcohol-dependent (AD) individuals of Caucasian descent. Building upon these findings, we hypothesized that the low-expressing TT genotype associated with intense drinking would predict higher craving for alcohol in AD individuals. In this pilot study, we sought to test our hypothesis by examining 34 Hispanic AD volunteers (mean age, 34.8 years) for rs1042173 genotype-based [i.e., TT versus TG/GG (Gx)] differences in subjective response to alcohol. We employed a human laboratory paradigm and analyzed the data using a linear mixed-effects model (SAS® PROC MIXED) to assess treatment, cue procedures, and genotype main effects as well as the two-way interaction effects between them. On subjective "urge to drink" and "crave for a drink," we found a significant main effect of the cue experiment (p ≤ 0.01) and an interaction effect between genotype and cue effects (p < 0.05). TT genotype was associated with higher urge to drink (p = 0.002) and crave for a drink (p = 0.005) when exposed to alcohol cue. Our results not only support the hypothesis that rs1042173 is a genetic marker for cue-induced alcohol craving among AD males but also are suggestive of a neurobiological mechanism associated with the rs1042173-TT genotype that triggers a disproportionate craving in response to alcohol consumption, which in turn may lead to more intense drinking. Future studies with larger sample sizes are needed to characterize the interactive effects of the serotonin transporter-linked polymorphic region (5'-HTTLPR)-L-allele reported in our previous study and of the rs1042173-TT genotype on cue-induced alcohol craving.

<h4>Background</h4>Careful characterization of the phenotype and genotype of Huntington disease (HD) can foster better understanding of the condition.<h4>Methods</h4>We conducted a cohort study in the United States, Canada, and Australia of members of families affected by HD. We collected demographic and clinical data, conducted the Unified Huntington's Disease Rating Scale and Mini-Mental State Examination, and determined Huntingtin trinucleotide CAG repeat length. We report primarily on cross-sectional baseline data from this recently completed prospective, longitudinal, observational study.<h4>Results</h4>As of December 31, 2009, 2,318 individuals enrolled; of these, 1,985 (85.6%) were classified into six analysis groups. Three groups had expanded CAG alleles (36 repeats or more): individuals with clinically diagnosed HD [n = 930], and clinically unaffected first-degree relatives who had previously pursued [n = 248] or not pursued [n = 112] predictive DNA testing. Three groups lacked expanded alleles: first-degree relatives who had previously pursued [n = 41] or not pursued [n = 224] genetic testing, and spouses and caregivers [n = 430]. Baseline mean performance differed across groups in all motor, behavioral, cognitive, and functional measures (p<0.001). Clinically unaffected individuals with expanded alleles weighed less (76.0 vs. 79.6 kg; p = 0.01) and had lower cognitive scores (28.5 vs. 29.1 on the Mini Mental State Examination; p = 0.008) than individuals without expanded alleles. The frequency of "high normal" repeat lengths (27 to 35) was 2.5% and repeat lengths associated with reduced penetrance (36 to 39) was 2.7%.<h4>Conclusion</h4>Baseline analysis of COHORT study participants revealed differences that emerge prior to clinical diagnosis. Longitudinal investigation of this cohort will further characterize the natural history of HD and genetic and biological modifiers.<h4>Trial registration</h4>Clinicaltrials.gov NCT00313495.

The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91-94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.

<h4>Introduction</h4>Nonalcoholic fatty liver disease (NAFLD) can be seen as a manifestation of overnutrition. The muscle is a central player in the adaptation to energy overload, and there is an association between fatty-muscle and -liver. We aimed to correlate muscle morphology, mitochondrial function and insulin signaling with NAFLD severity in morbid obese patients.<h4>Methods</h4>Liver and deltoid muscle biopsies were collected during bariatric surgery in NAFLD patients. NAFLD Activity Score and Younossi's classification for nonalcoholic steatohepatitis (NASH) were applied to liver histology. Muscle evaluation included morphology studies, respiratory chain complex I to IV enzyme assays, and analysis of the insulin signaling cascade. A healthy lean control group was included for muscle morphology and mitochondrial function analyses.<h4>Results</h4>Fifty one NAFLD patients were included of whom 43% had NASH. Intramyocellular lipids (IMCL) were associated with the presence of NASH (OR 12.5, p<0.001), progressive hepatic inflammation (p = 0.029) and fibrosis severity (p = 0.010). There was a trend to an association between IMCL and decreased Akt phosphorylation (p = 0.059), despite no association with insulin resistance. In turn, hepatic steatosis (p = 0.015) and inflammation (p = 0.013) were associated with decreased Akt phosphoryation. Citrate synthase activity was lower in obese patients (p = 0.047) whereas complex I (p = 0.040) and III (p = 0.036) activities were higher, compared with controls. Finally, in obese patients, complex I activity increased with progressive steatosis (p = 0.049) and with a trend with fibrosis severity (p = 0.056).<h4>Conclusions</h4>In morbid obese patients, presence of IMCL associates with NASH and advanced fibrosis. Muscle mitochondrial dysfunction does not appear to be a major driving force contributing to muscle fat accumulation, insulin resistance or liver disease. Importantly, insulin resistance in muscle might occur at a late point in the insulin signaling cascade and be associated with IMCL and NAFLD severity.

Interferon-gamma (IFN-γ) inhibits intracellular replication of Francisella tularensis in human monocyte-derived macrophages (HMDM) and in mice, but the mechanisms of this protective effect are poorly characterized. We used genome-wide RNA interference (RNAi) screening in the human macrophage cell line THP-1 to identify genes that mediate the beneficial effects of IFN-γ on F. tularensis infection. A primary screen identified ∼200 replicated candidate genes. These were prioritized according to mRNA expression in IFN-γ-primed and F. tularensis-challenged macrophages. A panel of 20 top hits was further assessed by re-testing using individual shRNAs or siRNAs in THP-1 cells, HMDMs and primary human lung macrophages. Six of eight validated genes tested were also found to confer resistance to Listeria monocytogenes infection, suggesting a broadly shared host gene program for intracellular pathogens. The F. tularensis-validated hits included 'druggable' targets such as TNFRSF9, which encodes CD137. Treating HMDM with a blocking antibody to CD137 confirmed a beneficial role of CD137 in macrophage clearance of F. tularensis. These studies reveal a number of important mediators of IFN-γ activated host defense against intracellular pathogens, and implicate CD137 as a potential therapeutic target and regulator of macrophage interactions with Francisella tularensis.

Cartilage defects represent a common problem in orthopaedic practice. Predisposing factors include traumas, inflammatory conditions, and biomechanics alterations. Conservative management of cartilage defects often fails, and patients with this lesions may need surgical intervention. Several treatment strategies have been proposed, although only surgery has been proved to be predictably effective. Usually, in focal cartilage defects without a stable fibrocartilaginous repair tissue formed, surgeons try to promote a natural fibrocartilaginous response by using marrow stimulating techniques, such as microfracture, abrasion arthroplasty, and Pridie drilling, with the aim of reducing swelling and pain and improving joint function of the patients. These procedures have demonstrated to be clinically useful and are usually considered as first-line treatment for focal cartilage defects. However, fibrocartilage presents inferior mechanical and biochemical properties compared to normal hyaline articular cartilage, characterized by poor organization, significant amounts of collagen type I, and an increased susceptibility to injury, which ultimately leads to premature osteoarthritis (OA). Therefore, the aim of future therapeutic strategies for articular cartilage regeneration is to obtain a hyaline-like cartilage repair tissue by transplantation of tissues or cells. Further studies are required to clarify the role of gene therapy and mesenchimal stem cells for management of cartilage lesions.

The succinate receptor (also known as GPR91) is a G protein-coupled receptor that is closely related to the family of P2Y purinoreceptors. It is expressed in a variety of tissues, including blood cells, adipose tissue, the liver, retina, and kidney. In these tissues, this receptor and its ligand succinate have recently emerged as novel mediators in local stress situations, including ischemia, hypoxia, toxicity, and hyperglycemia. Amongst others, the succinate receptor is involved in recruitment of immune cells to transplanted tissues. Moreover, it was shown to play a key role in the development of diabetic retinopathy. However, most prominently, the role of locally increased succinate levels and succinate receptor activation in the kidney, stimulating the systemic and local renin-angiotensin system, starts to unfold: the succinate receptor is a key mediator in the development of hypertension and possibly fibrosis in diabetes mellitus and metabolic syndrome. This makes the succinate receptor a promising drug target to counteract or prevent cardiovascular and fibrotic defects in these expanding disorders. Recent development of SUCNR1-specific antagonists opens novel possibilities for research in models for these disorders and may eventually provide novel opportunities for the treatment of patients.

<h4>Significance</h4>Among different forms of oxidative stress, lipid peroxidation comprises the interaction of free radicals with polyunsaturated fatty acids, which in turn leads to the formation of highly reactive electrophilic aldehydes. Among these, the most abundant aldehydes are 4-hydroxy-2-nonenal (HNE) and malondialdehyde, while acrolein is the most reactive. HNE is considered a robust marker of oxidative stress and a toxic compound for several cell types. Proteins are particularly susceptible to modification caused by HNE, and adduct formation plays a critical role in multiple cellular processes.<h4>Recent advances</h4>With the outstanding progress of proteomics, the identification of putative biomarkers for neurodegenerative disorders has been the main focus of several studies and will continue to be a difficult task.<h4>Critical issues</h4>The present review focuses on the role of lipid peroxidation, particularly of HNE-induced protein modification, in neurodegenerative diseases. By comparing results obtained in different neurodegenerative diseases, it may be possible to identify both similarities and specific differences in addition to better characterize selective neurodegenerative phenomena associated with protein dysfunction. Results obtained in our laboratory and others support the common deregulation of energy metabolism and mitochondrial function in neurodegeneration.<h4>Future directions</h4>Research towards a better understanding of the molecular mechanisms involved in neurodegeneration together with identification of specific targets of oxidative damage is urgently required. Redox proteomics will contribute to broaden the knowledge in regard to potential biomarkers for disease diagnosis and may also provide insight into damaged metabolic networks and potential targets for modulation of disease progression.

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein's activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests to determine whether specific proteins colocalize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein colocalization with specific lipid species.

We surveyed gene-gene interactions (epistasis) in human body mass index (BMI) in four European populations (n<1200) via exhaustive pair-wise genome scans where interactions were computed as F ratios by testing a linear regression model fitting two single-nucleotide polymorphisms (SNPs) with interactions against the one without. Before the association tests, BMI was corrected for sex and age, normalised and adjusted for relatedness. Neither single SNPs nor SNP interactions were genome-wide significant in either cohort based on the consensus threshold (P=5.0E-08) and a Bonferroni corrected threshold (P=1.1E-12), respectively. Next we compared sub genome-wide significant SNP interactions (P<5.0E-08) across cohorts to identify common epistatic signals, where SNPs were annotated to genes to test for gene ontology (GO) enrichment. Among the epistatic genes contributing to the commonly enriched GO terms, 19 were shared across study cohorts of which 15 are previously published genome-wide association loci, including CDH13 (cadherin 13) associated with height and SORCS2 (sortilin-related VPS10 domain containing receptor 2) associated with circulating insulin-like growth factor 1 and binding protein 3. Interactions between the 19 shared epistatic genes and those involving BMI candidate loci (P<5.0E-08) were tested across cohorts and found eight replicated at the SNP level (P<0.05) in at least one cohort, which were further tested and showed limited replication in a separate European population (n>5000). We conclude that genome-wide analysis of epistasis in multiple populations is an effective approach to provide new insights into the genetic regulation of BMI but requires additional efforts to confirm the findings.

<h4>Background</h4>Enteroviruses (EVs) are the leading cause of aseptic meningitis, which is the most frequent central nervous system infection worldwide. We aimed to characterize the EVs involved in an aseptic meningitis outbreak in Korea in 2008. In Korea, Echovirus type 30 (E30) and E6 have been associated with outbreaks and frequent meningitis.<h4>Methods</h4>During 2008, through nationwide surveillance, we collected specimens from 758 patients with aseptic meningitis-related clinical manifestations. The detection of EVs from specimens was subjected to a diagnostic real-time RT-PCR in the 5' NCR. A semi-nested polymerase chain reaction (PCR) to amplify sequences from the VP1 region and sequence comparison with reference strains registered in Genbank was performed for the genotype determination.<h4>Results</h4>Most patients (98%) in this outbreak were children < 15 years of age. The temporal distribution of the E6 and E30 epidemics showed an obvious seasonal pattern during the short period from June to July. A large majority of the EV-positive patients experienced fever, headache, vomiting, and neck stiffness. Some patients also showed cold symptoms, sore throat, altered mental status, and seizures. We did not observe a higher fatality rate in children with E6 or E30 infection. Most of the patients recovered uneventfully. In most cases, the cerebrospinal fluid (CSF) profile was studied, and generally showed a higher than normal white blood cell count (≥ 5/mm(3)). We detected EVs from 513 patients (67.68%) and identified the EV genotype in 287 patients. E30 (n = 155, 50.4%) and E6 (n = 95, 33.1%) were the predominant genotypes. E9, E1, E7, E16, coxsackievirus A3, 4, 6, coxsackievirus B1, 3, and 10 were also identified. According to phylogenetic analysis, E30 belonged to subgroup 4b, and E6, to the C4 subgroup.<h4>Conclusions</h4>Conclusively, aseptic meningitis was the most common manifestation in children with either echovirus 30 or 6 infection. Identification of E6 and E30 as the prominent EVs in the 2008 outbreak in South Korea shows the potential of EVs to cause a serious disease in an unpredictable (fashion. Our findings provide new) insights into the clinical and virological features of the aseptic meningitis outbreak caused by E30 and E6.

Metals can have a number of detrimental or beneficial effects in the cell, but first they must get in. Organisms have evolved transport mechanisms to get metals that are required, or essential into the cell. Nonessential metals often enter the cell through use of the machinery provided for essential metals. Much work has been done to advance our understanding of how these metals are transported across plasma and organelle membranes. This review provides an overview of essential and nonessential metal transport and homeostatic processes.

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.

HIV-1 Nef is essential for AIDS pathogenesis, but this viral protein is not targeted by antiviral strategies. The functions of Nef are largely related to perturbations of intracellular trafficking and signaling pathways through leucine-based and polyproline motifs that are required for interactions with clathrin-associated adaptor protein complexes and SH3 domain-containing proteins, such as the phagocyte-specific kinase Hck. We previously described a single-domain antibody (sdAb) targeting Nef and inhibiting many, but not all, of its biological activities. We now report a further development of this anti-Nef strategy through the demonstration of the remarkable inhibitory activity of artificial Nef ligands, called Neffins, comprised of the anti-Nef sdAb fused to modified SH3 domains. The Neffins inhibited all key activities of Nef, including Nef-mediated CD4 and major histocompatibility complex class I (MHC-I) cell surface downregulation and enhancement of virus infectivity. When expressed in T lymphocytes, Neffins specifically inhibited the Nef-induced mislocalization of the Lck kinase, which contributes to the alteration of the formation of the immunological synapse. In macrophages, Neffins inhibited the Nef-induced formation of multinucleated giant cells and podosome rosettes, and it counteracted the inhibitory activity of Nef on phagocytosis. Since we show here that these effects of Nef on macrophage and T cell functions were both dependent on the leucine-based and polyproline motifs, we confirmed that Neffins disrupted interactions of Nef with both AP complexes and Hck. These results demonstrate that it is possible to inhibit all functions of Nef, both in T lymphocytes and macrophages, with a single ligand that represents an efficient tool to develop new antiviral strategies targeting Nef.

A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their full-length sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed.

Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis, were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of M. tuberculosis and purified protein derivative-induced IFN-γ, TNF-α, IL-1β, and IL-6 was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M. tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes including inhibition of expression of many NF-κB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosis-induced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB) pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity.

Acanthocytes, abnormal thorny red blood cells (RBC), are one of the biological hallmarks of neuroacanthocytosis syndromes (NA), a group of rare hereditary neurodegenerative disorders. Since RBCs are easily accessible, the study of acanthocytes in NA may provide insights into potential mechanisms of neurodegeneration. Previous studies have shown that changes in RBC membrane protein phosphorylation state affect RBC membrane mechanical stability and morphology. Here, we coupled tyrosine-phosphoproteomic analysis to topological network analysis. We aimed to predict signaling sub-networks possibly involved in the generation of acanthocytes in patients affected by the two core NA disorders, namely McLeod syndrome (MLS, XK-related, Xk protein) and chorea-acanthocytosis (ChAc, VPS13A-related, chorein protein). The experimentally determined phosphoproteomic data-sets allowed us to relate the subsequent network analysis to the pathogenetic background. To reduce the network complexity, we combined several algorithms of topological network analysis including cluster determination by shortest path analysis, protein categorization based on centrality indexes, along with annotation-based node filtering. We first identified XK- and VPS13A-related protein-protein interaction networks by identifying all the interactomic shortest paths linking Xk and chorein to the corresponding set of proteins whose tyrosine phosphorylation was altered in patients. These networks include the most likely paths of functional influence of Xk and chorein on phosphorylated proteins. We further refined the analysis by extracting restricted sets of highly interacting signaling proteins representing a common molecular background bridging the generation of acanthocytes in MLS and ChAc. The final analysis pointed to a novel, very restricted, signaling module of 14 highly interconnected kinases, whose alteration is possibly involved in generation of acanthocytes in MLS and ChAc.

Genome-wide association studies (GWAS) have identified multiple common variants associated with body mass index (BMI). In this study, we tested 23 genotyped GWAS-significant SNPs (p-value<5*10-8) for longitudinal associations with BMI during childhood (3-17 years) and adulthood (18-45 years) for 658 subjects. We also proposed a heuristic forward search for the best joint effect model to explain the longitudinal BMI variation. After using false discovery rate (FDR) to adjust for multiple tests, childhood and adulthood BMI were found to be significantly associated with six SNPs each (q-value<0.05), with one SNP associated with both BMI measurements: KCTD15 rs29941 (q-value<7.6*10-4). These 12 SNPs are located at or near genes either expressed in the brain (BDNF, KCTD15, TMEM18, MTCH2, and FTO) or implicated in cell apoptosis and proliferation (FAIM2, MAP2K5, and TFAP2B). The longitudinal effects of FAIM2 rs7138803 on childhood BMI and MAP2K5 rs2241423 on adulthood BMI decreased as age increased (q-value<0.05). The FTO candidate SNPs, rs6499640 at the 5 '-end and rs1121980 and rs8050136 downstream, were associated with childhood and adulthood BMI, respectively, and the risk effects of rs6499640 and rs1121980 increased as birth weight decreased. The best joint effect model for childhood and adulthood BMI contained 14 and 15 SNPs each, with 11 in common, and the percentage of explained variance increased from 0.17% and 9.0*10(-6)% to 2.22% and 2.71%, respectively. In summary, this study evidenced the presence of long-term major effects of genes on obesity development, implicated in pathways related to neural development and cell metabolism, and different sets of genes associated with childhood and adulthood BMI, respectively. The gene effects can vary with age and be modified by prenatal development. The best joint effect model indicated that multiple variants with effects that are weak or absent alone can nevertheless jointly exert a large longitudinal effect on BMI.

With the incessant challenge of exposure to the air we breathe, lung tissue suffers the highest levels of oxygen tension and thus requires robust antioxidant defenses. Furthermore, following injury or infection, lung tissue faces the additional challenge of inflammation-induced reactive oxygen and nitrogen species (ROS/RNS). Little is known about the identity or distribution of lung antioxidant enzymes under normal conditions or during infection-induced inflammation. Using a mouse model of influenza (H1N1 influenza virus A/PR/8/34 [PR8]) in combination with bioinformatics, we identified seven lung-abundant antioxidant enzymes: Glutathione peroxidase 3 (Gpx3), Superoxide dismutase 3 (Sod3), Transferrin (Tf), peroxyredoxin6 (Prdx6), glutathione S-transferase kappa 1 (Gstk1), Catalase (Cat), and Glutathione peroxidase 8 (Gpx8). Interestingly, despite the demand for antioxidants during inflammation, influenza caused depletion in two key antioxidants: Cat and Prdx6. As Cat is highly expressed in Clara cells, virus-induced Clara cell loss contributes to the depletion in Cat. Prdx6 is also reduced due to Clara cell loss, however there is a coincident increase in Prdx6 levels in the alveoli, resulting in only a subtle reduction of Prdx6 overall. Analogously, Gpx3 shifts from the basement membranes underlying the bronchioles and blood vessels to the alveoli, thus maintaining balanced expression. Taken together, these studies identify key lung antioxidants and reveal their distribution among specific cell types. Furthermore, results show that influenza depletes key antioxidants, and that in some cases there is coincident increased expression, consistent with compensatory expression. Given that oxidative stress is known to be a key risk factor during influenza infection, knowledge about the antioxidant repertoire of lungs, and the spatio-temporal distribution of antioxidants, contributes to our understanding of the underlying mechanisms of influenza-induced morbidity and mortality.

Working memory (WM) is a limited capacity system that permeates nearly all levels of cognition, ranging from perceptual awareness to intelligence. Through behavioral, electrophysiological, and neuroimaging work, substantial gains have been made in understanding this capacity-limited system. In the current work, we examined genetic contributions to the storage capacity of WM. Multiple studies have demonstrated a link between the serotonin transporter-linked polymorphic region (5-HTTLPR) and cognition, where better performance is observed in individuals possessing a copy of the short (s) variant of the polymorphism compared with individuals homozygous for the long (l) variant. We predicted the same profile in WM performance, such that estimated capacities of l/l carriers should be smaller than s/s and s/l carriers. To measure WM capacity, we implemented a change detection task, which requires observers to actively maintain the color and spatial location of briefly presented squares over a short retention interval. In line with our prediction, we observed similar WM performance between s/s and s/l groups, and these individuals performed better than the l/l group. We then discuss the distribution of the serotonin transporter system and parallels between WM and attention to provide insight into how variation in the 5-HTT polymorphism could lead to individual differences in the storage capacity of WM.

<h4>Background</h4>An array of experimental models have been developed in the small model organisms C. elegans, S. cerevisiae and D. melanogaster for the study of various neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and expanded polyglutamine diseases as exemplified by Huntington's disease (HD) and related ataxias. Genetic approaches to determine the nature of regulators of the disease phenotypes have ranged from small scale to essentially whole genome screens. The published data covers distinct models in all three organisms and one important question is the extent to which shared genetic factors can be uncovered that affect several or all disease models. Surprisingly it has appeared that there may be relatively little overlap and that many of the regulators may be organism or disease-specific. There is, however, a need for a fully integrated analysis of the available genetic data based on careful comparison of orthologues across the species to determine the real extent of overlap.<h4>Results</h4>We carried out an integrated analysis using C. elegans as the baseline model organism since this is the most widely studied in this context. Combination of data from 28 published studies using small to large scale screens in all three small model organisms gave a total of 950 identifications of genetic regulators. Of these 624 were separate genes with orthologues in C. elegans. In addition, 34 of these genes, which all had human orthologues, were found to overlap across studies. Of the common genetic regulators some such as chaperones, ubiquitin-related enzymes (including the E3 ligase CHIP which directly links the two pathways) and histone deacetylases were involved in expected pathways whereas others such as the peroxisomal acyl CoA-oxidase suggest novel targets for neurodegenerative disease therapy<h4>Conclusions</h4>We identified a significant number of overlapping regulators of neurodegenerative disease models. Since the diseases have, as an underlying feature, protein aggregation phenotypes it was not surprising that some of the overlapping genes encode proteins involved in protein folding and protein degradation. Interestingly, however, some of the overlapping genes encode proteins that have not previously featured in targeted studies of neurodegeneration and this information will form a useful resource to be exploited in further studies of potential drug-targets.

Although the morphological and physiological changes involved in pregnancy in live-bearing reptiles are well studied, the genetic mechanisms that underlie these changes are not known. We used the viviparous African Ocellated Skink, Chalcides ocellatus, as a model to identify a near complete gene expression profile associated with pregnancy using RNA-Seq analyses of uterine transcriptomes. Pregnancy in C. ocellatus is associated with upregulation of uterine genes involved with metabolism, cell proliferation and death, and cellular transport. Moreover, there are clear parallels between the genetic processes associated with pregnancy in mammals and Chalcides in expression of genes related to tissue remodeling, angiogenesis, immune system regulation, and nutrient provisioning to the embryo. In particular, the pregnant uterine transcriptome is dominated by expression of proteolytic enzymes that we speculate are involved both with remodeling the chorioallantoic placenta and histotrophy in the omphaloplacenta. Elements of the maternal innate immune system are downregulated in the pregnant uterus, indicating a potential mechanism to avoid rejection of the embryo. We found a downregulation of major histocompatability complex loci and estrogen and progesterone receptors in the pregnant uterus. This pattern is similar to mammals but cannot be explained by the mammalian model. The latter finding provides evidence that pregnancy is controlled by different endocrinological mechanisms in mammals and reptiles. Finally, 88% of the identified genes are expressed in both the pregnant and the nonpregnant uterus, and thus, morphological and physiological changes associated with C. ocellatus pregnancy are likely a result of regulation of genes continually expressed in the uterus rather than the initiation of expression of unique genes.

<h4>Background</h4>Cardiosphere-derived cells (CDCs) reduce scarring after myocardial infarction, increase viable myocardium, and boost cardiac function in preclinical models. We aimed to assess safety of such an approach in patients with left ventricular dysfunction after myocardial infarction.<h4>Methods</h4>In the prospective, randomised CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial, we enrolled patients 2-4 weeks after myocardial infarction (with left ventricular ejection fraction of 25-45%) at two medical centres in the USA. An independent data coordinating centre randomly allocated patients in a 2:1 ratio to receive CDCs or standard care. For patients assigned to receive CDCs, autologous cells grown from endomyocardial biopsy specimens were infused into the infarct-related artery 1·5-3 months after myocardial infarction. The primary endpoint was proportion of patients at 6 months who died due to ventricular tachycardia, ventricular fibrillation, or sudden unexpected death, or had myocardial infarction after cell infusion, new cardiac tumour formation on MRI, or a major adverse cardiac event (MACE; composite of death and hospital admission for heart failure or non-fatal recurrent myocardial infarction). We also assessed preliminary efficacy endpoints on MRI by 6 months. Data analysers were masked to group assignment. This study is registered with ClinicalTrials.gov, NCT00893360.<h4>Findings</h4>Between May 5, 2009, and Dec 16, 2010, we randomly allocated 31 eligible participants of whom 25 were included in a per-protocol analysis (17 to CDC group and eight to standard of care). Mean baseline left ventricular ejection fraction (LVEF) was 39% (SD 12) and scar occupied 24% (10) of left ventricular mass. Biopsy samples yielded prescribed cell doses within 36 days (SD 6). No complications were reported within 24 h of CDC infusion. By 6 months, no patients had died, developed cardiac tumours, or MACE in either group. Four patients (24%) in the CDC group had serious adverse events compared with one control (13%; p=1·00). Compared with controls at 6 months, MRI analysis of patients treated with CDCs showed reductions in scar mass (p=0·001), increases in viable heart mass (p=0·01) and regional contractility (p=0·02), and regional systolic wall thickening (p=0·015). However, changes in end-diastolic volume, end-systolic volume, and LVEF did not differ between groups by 6 months.<h4>Interpretation</h4>We show intracoronary infusion of autologous CDCs after myocardial infarction is safe, warranting the expansion of such therapy to phase 2 study. The unprecedented increases we noted in viable myocardium, which are consistent with therapeutic regeneration, merit further assessment of clinical outcomes.<h4>Funding</h4>US National Heart, Lung and Blood Institute and Cedars-Sinai Board of Governors Heart Stem Cell Center.

<h4>Introduction</h4>The small molecule 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid (NST732) is a member of the ApoSense family of compounds, capable of selective targeting, binding and accumulation within cells undergoing apoptotic cell death. It has application in molecular imaging and blood clotting particularly for monitoring antiapoptotic drug treatments. We are investigating a fluorine-18-radiolabeled analog of this compound for positron emission tomography studies.<h4>Methods</h4>We prepared the tosylate precursor methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(tosyloxymethyl)butanoate (4) to synthesize fluorine-18-labeled NST732. Fluorination reaction of the tosylate precursor in 1:1 acetonitrile:dimethylsulfoxide with tetrabutyl ammonium fluoride proceeds through an aziridine intermediate (4A) to afford two regioisomers: 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-fluorobutanoate (5) and methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoate (6). Acid hydrolysis of the fluoromethylbutanoate (6) isomer produced NST732. As the fluorination reaction of the tosylate precursor proceeds through an aziridine intermediate (4A) and the fluorination conceivably could be done directly on the aziridine, we have separately prepared an aziridine precursor (4A). Fluorine-18 labeling of the aziridine precursor (4A) was performed with [(18)F]tetrabutyl ammonium fluoride to afford the same two regioisomers (5 and 6). The [18F]2-((5-dimethylamino)naphthalene-1-sulfonamido)methyl)-2-fluorobutanoic acid (NST732) was then obtained by the hydrolysis of corresponding [18F]-labeled ester (6) with 6 N hydrochloric acid.<h4>Results</h4>Two regioisomers obtained from the fluorination reaction of aziridine were easily separated by high-performance liquid chromatography. The total radiochemical yield was 15%±3% (uncorrected, n=18) from the aziridine precursor in a 70-min synthesis time with a radiochemical purity>99%.<h4>Conclusion</h4>Fluorine-18-labeled ApoSense compound [18F]NST732 is prepared in moderate yield by direct fluorination of an aziridine precursor.

Mutations leading to expansion of a poly-glutamine track in Huntingtin (Htt) cause Huntington's disease (HD). Signs of endoplasmic reticulum (ER) stress have been recently reported in animal models of HD, associated with the activation of the unfolded protein response (UPR). Here we have investigated the functional contribution of ER stress to HD by targeting the expression of two main UPR transcription factors, XBP1 and ATF4 (activating transcription factor 4), in full-length mutant Huntingtin (mHtt) transgenic mice. XBP1-deficient mice were more resistant to developing disease features, associated with improved neuronal survival and motor performance, and a drastic decrease in mHtt levels. The protective effects of XBP1 deficiency were associated with enhanced macroautophagy in both cellular and animal models of HD. In contrast, ATF4 deficiency did not alter mHtt levels. Although, XBP1 mRNA splicing was observed in the striatum of HD transgenic brains, no changes in the levels of classical ER stress markers were detected in symptomatic animals. At the mechanistic level, we observed that XBP1 deficiency led to augmented expression of Forkhead box O1 (FoxO1), a key transcription factor regulating autophagy in neurons. In agreement with this finding, ectopic expression of FoxO1 enhanced autophagy and mHtt clearance in vitro. Our results provide strong evidence supporting an involvement of XBP1 in HD pathogenesis probably due to an ER stress-independent mechanism involving the control of FoxO1 and autophagy levels.

The cytotoxic and antiproliferative properties of many natural sesquiterpene-quinones and -hydroquinones from sponges offer promising opportunities for the development of new drugs. A review dealing with different strategies for obtaining bioactive terpenyl quinones/hydroquinones is presented. The different synthetic approches for the preparation of the most relevant quinones/hydroquinones are described.

Background. Osteoporosis is a chronic disease that can significantly impact numerous aspects of health and wellness. The individual consequences of osteoporosis can be devastating, often resulting in substantial loss of independence and sometimes death. One of the few illnesses with greater disease burden than low bone mineral density (BMD) is major depressive disorder (MDD). Both depression and antidepressant use have been identified as secondary causes of osteoporosis. The objective of this paper is to review and summarize the current findings on the relationship between antidepressant use and BMD. Methods. Relevant sources were identified from the Pubmed and MEDLINE databases, citing articles from the first relevant publication to September 1st, 2010. Results. 2001 articles initially met the search criteria, and 35 studies were thoroughly reviewed for evidence of an association between SSRI use and BMD, and 8 clinical studies were detailed and summarized in this paper. Conclusions. Current findings suggest a link between mental illness and osteoporosis that is of clinical relevance. Additional longitudinal studies and further research on possible mechanisms surrounding the association between SSRI use on bone metabolism need to be conducted. Treatment algorithms need to recognize this association to ensure that vulnerable populations are screened.

Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.

Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species.

Nuclear deformed epidermal autoregulatory factor-1 (NUDR/Deaf-1) and five prime repressor element under dual repression (Freud-1) are novel transcriptional regulators of the 5-HT(1A) receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17β-Estradiol (17βE(2)) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17βE(2)-induced downregulation of the 5-HT(1A) receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10 nM of 17βE(2) for 3 or 48 h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by western blotting. 17βE(2) treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT(1A) receptor showed significant decreases. Upon withdrawal of 17βE(2), protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT(1A) receptor protein by 17βE(2), which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory/homeostatic responses to changes in 5-HT(1A) receptor induced by 17βE(2). These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT(1A) receptor expression.

Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of Escherichia coli thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50-70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high-affinity ACP binding as elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers 3 weeks postinjection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.

A series of 4-(methylsulfonyl)aniline derivatives were synthesized in order to obtain new compounds as a potential anti-inflammatory agents with expected selectivity against COX-2 enzyme. In vivo acute anti-inflammatory activity of the final compounds 11-14 was evaluated in rat using an egg-white induced edema model of inflammation in a dose equivalent to 3 mg/Kg of diclofenac sodium. All tested compounds produced significant reduction of paw edema with respect to the effect of propylene glycol 50% v/v (control group). Moreover, the activity of compounds 11 and 14 was significantly higher than that of diclofenac sodium (at 3 mg/Kg) in the 120-300 minute time interval, while compound 12 expressed a comparable effect to that of diclofenac sodium in the 60-240 minute time interval time, and compound 13 showed a comparable effect to that of diclofenac sodium at all experimental times. The result of this study indicates that the incorporation of the 4-(methylsulfonyl)aniline pharamacophore into naproxen, indomethacine, diclofenac and mefanamic acid maintained their anti-inflammatory activity and may increase selectivity towards the COX-2 enzyme which will be confirmed in the future by assessing COX-2: COX-1 inhibitory ratios using a whole blood assay.

Most glandular tissues comprise polarized epithelial cells organized around a single central lumen. Although there is active research investigating the molecular networks involved in the regulation of lumenogenesis, little is known about the extracellular factors that influence lumen formation and maintenance. Using a three-dimensional culture system of epithelial endometrial cells, we have revealed a new role for pro-inflammatory cytokines such as TNFα and IL1α in the formation and, more importantly, maintenance of a single central lumen. We also studied the mechanism by which glucocorticoids repress TNFα and IL1α expression. Interestingly, regulation of pro-inflammatory cytokine expression and subsequent lumen formation is mediated by estrogen receptor α (ERα) but not by the glucocorticoid receptor. Finally, we investigated the signaling pathways involved in the regulation of lumen formation by pro-inflammatory cytokines. Our results demonstrate that activation of the ERK/MAPK signaling pathway, but not the PI3K/Akt signaling pathway, is important for the formation and maintenance of a single central lumen. In summary, our results suggest a novel role for ERα-regulated pro-inflammatory cytokine expression in lumen formation and maintenance.

<h4>Purpose</h4>Esophageal adenocarcinoma (EAC) is a lethal malignancy that can develop from the premalignant condition, Barrett's esophagus (BE). Currently, there are no validated simple methods to predict which patients will progress to EAC. A better understanding of the genetic mechanisms driving EAC tumorigenesis is needed to identify new therapeutic targets and develop biomarkers capable of identifying high-risk patients that would benefit from aggressive neoadjuvant therapy. We employed an integrative genomics approach to identify novel genes involved in EAC biology that may serve as useful clinical markers.<h4>Experimental design</h4>Whole genome tiling-path array comparative genomic hybridization was used to identify significant regions of copy number alteration in 20 EACs and 10 matching BE tissues. Copy number and gene expression data were integrated to identify candidate oncogenes within regions of amplification and multiple additional sample cohorts were assessed to validate candidate genes.<h4>Results</h4>We identified RFC3 as a novel, candidate oncogene activated by amplification in approximately 25% of EAC samples. RFC3 was also amplified in BE from a patient whose EAC harbored amplification and was differentially expressed between nonmalignant and EAC tissues. Copy number gains were detected in other cancer types and RFC3 knockdown inhibited proliferation and anchorage-independent growth of cancer cells with increased copy number but had little effect on those without. Moreover, high RFC3 expression was associated with poor patient outcome in multiple cancer types.<h4>Conclusions</h4>RFC3 is a candidate oncogene amplified in EAC. RFC3 DNA amplification is also prevalent in other epithelial cancer types and RFC3 expression could serve as a prognostic marker.

Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.

In this review we put the spotlight on crosslinked polymer nanogels, a promising platform that has the characteristics of an "ideal" drug delivery vehicle. Some of the key aspects of drug delivery vehicle design like stability, response to biologically relevant stimuli, passive targeting, active targeting, toxicity and ease of synthesis are discussed. We discuss several delivery systems in this light and highlight some examples of systems, which satisfy some or all of these design requirements. In particular, we point to the advantages that crosslinked polymeric systems bring to drug delivery. We review some of the synthetic methods of nanogel synthesis and conclude with the diverse applications in drug delivery where nanogels have been fruitfully employed.

Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals.

KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene-gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.

Traditional proteomics analysis is plagued by the use of arbitrary thresholds resulting in large loss of information. We propose here a novel method in proteomics that utilizes all detected proteins. We demonstrate its efficacy in a proteomics screen of 5 and 7 liver cancer patients in the moderate and late stage, respectively. Utilizing biological complexes as a cluster vector, and augmenting it with submodules obtained from partitioning an integrated and cleaned protein-protein interaction network, we calculate a Proteomics Signature Profile (PSP) for each patient based on the hit rates of their reported proteins, in the absence of fold change thresholds, against the cluster vector. Using this, we demonstrated that moderate- and late-stage patients segregate with high confidence. We also discovered a moderate-stage patient who displayed a proteomics profile similar to other poor-stage patients. We identified significant clusters using a modified version of the SNet approach. Comparing our results against the Proteomics Expansion Pipeline (PEP) on which the same patient data was analyzed, we found good correlation. Building on this finding, we report significantly more clusters (176 clusters here compared to 70 in PEP), demonstrating the sensitivity of this approach. Gene Ontology (GO) terms analysis also reveals that the significant clusters are functionally congruent with the liver cancer phenotype. PSP is a powerful and sensitive method for analyzing proteomics profiles even when sample sizes are small. It does not rely on the ratio scores but, rather, whether a protein is detected or not. Although consistency of individual proteins between patients is low, we found the reported proteins tend to hit clusters in a meaningful and informative manner. By extracting this information in the form of a Proteomics Signature Profile, we confirm that this information is conserved and can be used for (1) clustering of patient samples, (2) identification of significant clusters based on real biological complexes, and (3) overcoming consistency and coverage issues prevalent in proteomics data sets.

Gallbladder cancer (GC) is a relatively uncommon cancer with higher incidence in certain areas including Japan. Because of the difficulty in diagnosis, prognosis of GC is very poor. To identify genetic determinants of GC, we conducted a genome-wide association study (GWAS) in 41 GC patients and 866 controls. Association between each single-nucleotide polymorphism (SNP) with GC susceptibility was evaluated by multivariate logistic regression analysis conditioned on age and gender of subjects. SNPs that showed suggestive association (P<1 × 10(-4)) with GC were further examined in 30 cases and 898 controls. SNP rs7504990 in the DCC (deleted in colorectal cancer, 18q21.3) that encodes a netrin 1 receptor achieved a combined P-value of 7.46 × 10(-8) (OR=6.95; 95% CI=3.43-14.08). Subsequent imputation analysis identified multiple SNPs with similarly strong associations in an adjacent genomic region, where loss of heterozygosity was reported in GC and other cancers. Reduced expression of DCC was indicated to be associated with the poorly differentiated histological type, increased proliferation and metastasis through loss of adhesiveness. However, due to the limited sample size investigated here, further replication study and functional analysis would be necessary to further confirm the result of the association.

Zinc, iron, and manganese are essential trace elements that serve as catalytic or structural components of larger molecules that are indispensable for life. The three metal ions possess similar chemical properties and have been shown to compete for uptake in a variety of tissues, suggesting that they share common transport proteins. Two likely candidates are the recently identified transmembrane proteins ZIP14 and ZIP8, which have been shown to mediate the cellular uptake of a number of divalent metal ions including zinc, iron, manganese, and cadmium. Although knockout and transgenic mouse models are beginning to define the physiologic roles of ZIP14 and ZIP8 in the handling of zinc and cadmium, their roles in the metabolism of iron and manganese remain to be defined. Here we review similarities and differences in ZIP14 and ZIP8 in terms of structure, metal transport, tissue distribution, subcellular localization, and regulation. We also discuss potential roles of these proteins in the metabolism of zinc, iron, manganese, and cadmium as well as recent associations with human diseases.

<h4>Purpose</h4>Hereditary haemochromatosis is a common genetic disorder involving dysregulation of iron absorption. There is some evidence to suggest that abnormal iron absorption and metabolism may influence the status of other important trace elements. In this study, the effect of abnormal HFE genotypes and associated iron overload on the status of other trace elements was examined.<h4>Methods</h4>Dietary data and blood samples were collected from 199 subjects (mean age = 55.4 years; range = 21-81 years). Dietary intakes, serum selenium, copper and zinc concentrations and related antioxidant enzymes (glutathione peroxidase and superoxide dismutase) in subjects with normal HFE genotype (n = 118) were compared to those with abnormal HFE genotype, with both normal iron status (n = 42) and iron overload (n = 39).<h4>Results</h4>For most dietary and biochemical variables measured, there were no significant differences between study groups. Red cell GPx was significantly higher in male subjects with normal genotypes and normal iron status compared to those with abnormal genotypes and normal iron status (P = 0.03) or iron overload (P = 0.001). Red cell GPx was also highest in normal women and significantly lower in the abnormal genotype and normal iron group (P = 0.016), but not in the iron overload group (P = 0.078).<h4>Conclusion</h4>Although it may not be possible to exclude a small effect between the genotype groups on RBC GPx, overall, haemochromatosis genotypes or iron overload did not appear to have a significant effect on selenium, copper or zinc status.

<h4>Background and purpose</h4>In clinical practice, durability of occlusion following coil embolization is superior in densely packed, compared with loosely packed, aneurysms. In a rabbit model, we probed, by using proteomics tools, the biologic mechanisms associated with densely packed and completely occluded aneurysms, compared with loosely packed and incompletely occluded aneurysms, to explore the biologic mechanisms of intra-aneurysmal healing following embolization.<h4>Materials and methods</h4>Elastase-induced, saccular aneurysms were created in 24 rabbits. Aneurysms were allowed to mature, after which aneurysms were either densely (packing attenuation >25%) or loosely (packing attenuation <20%) packed with platinum coils by endovascular means. After 2 weeks (n = 6 for both groups) and 4 weeks (n = 6 for both groups) of implantation, aneurysm samples harboring coils were harvested. Soluble proteins were extracted from the necks and domes of aneurysms, and proteins were studied using proteomics and bioinformatics tools.<h4>Results</h4>In dome tissue, 128 proteins at 2 weeks, and 8 proteins at 4 weeks, were differentially expressed in densely packed, compared with loosely packed, aneurysms. In the neck tissue, 2 proteins at 4 weeks were differentially expressed in densely packed aneurysms. Specific pathway analysis revealed that compared with loosely packed aneurysms, densely packed aneurysms were associated with up-regulation of cell-to-cell signaling and cell adhesion at 2 weeks. Conversely, at 4 weeks, densely packed aneurysms showed a decrease in the expression of structural proteins compared with loosely packed aneurysms.<h4>Conclusions</h4>These findings may focus efforts on specific targets aimed at improving the long-term healing of intracranial, saccular aneurysms.

Published guidelines suggest that research results and incidental findings should be offered to study participants under some circumstances. Although some have argued against the return of results in research, many cite an emerging consensus that there is an ethical obligation to return at least some results; the debate quickly turns to issues of mechanics (e.g., which results? who discloses? for how long does the obligation exist?). Although commentators are careful to distinguish this as an ethical rather than legal obligation, we worry that return of results may unjustifiably become standard of care based on this growing "consensus," which could quickly lead to a legal (negligence-based) duty to offer and return individualized genetic research results. We caution against this and argue in this essay that the debate to date has failed to give adequate weight to a number of fundamental ethical and policy issues that should undergird policy on return of research results in the first instance, many of which go to the fundamental differences between research and clinical care. We confine our comments to research using data from large biobanks, the topic of the guidelines proposed in this symposium issue.

<h4>Purpose</h4>With the advent of whole-genome sequencing made clinically available, the number of incidental findings is likely to rise. The false-positive incidental findings are of particular clinical concern. We provide estimates on the size of these false-positive findings and classify them into four broad categories.<h4>Methods</h4>Whole-genome sequences (WGS) of nine individuals were scanned with several comprehensive public annotation databases and average estimates for the number of findings. These estimates were then evaluated in the perspective of various sources of false-positive annotation errors.<h4>Results</h4>At present there are four main sources of false-positive incidental findings: erroneous annotations, sequencing error, incorrect penetrance estimates, and multiple hypothesis testing. Of these, the first two are likely to be addressed in the near term. Conservatively, current methods deliver hundreds of false-positive incidental findings per individual.<h4>Conclusion</h4>The burden of false-positives in whole-genome sequence interpretation threatens current capabilities to deliver clinical-grade whole-genome clinical interpretation. A new generation of population studies and retooling of the clinical decision-support approach will be required to overcome this threat.

Hepatocellular carcinoma (HCC) is a common cancer worldwide that primarily develops in cirrhosis resulting from chronic infection by hepatitis B virus and hepatitis C virus, alcoholic injury, and to a lesser extent from genetically determined disorders such as hemochromatosis. HCC has recently been linked to non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of obesity and related metabolic disorders such as diabetes. This association is alarming due to the globally high prevalence of these conditions and may contribute to the rising incidence of HCC witnessed in many industrialized countries. There is also evidence that NAFLD acts synergistically with other risk factors of HCC such as chronic hepatitis C and alcoholic liver injury. Moreover, HCC may complicate non-cirrhotic NAFLD with mild or absent fibrosis, greatly expanding the population potentially at higher risk. Major systemic and liver-specific molecular mechanisms involved include insulin resistance and hyperinsulinemia, increased TNF signaling pathways, and alterations in cellular lipid metabolism. These provide new targets for prevention, early recognition, and effective treatment of HCC associated with NAFLD. Indeed, both metformin and PPAR gamma agonists have been associated with lower risk and improved prognosis of HCC. This review summarizes current evidence as it pertains to the epidemiology, pathogenesis, and prevention of NAFLD-associated HCC.

The development of animal models of Huntington disease (HD) has enabled studies that help define the molecular aberrations underlying the disease. The BACHD and YAC128 transgenic mouse models of HD harbor a full-length mutant huntingtin (mHTT) and recapitulate many of the behavioural and neuropathological features of the human condition. Here, we demonstrate that while BACHD and YAC128 animals exhibit similar deficits in motor learning and coordination, depressive-like symptoms, striatal volume loss and forebrain weight loss, they show obvious differences in key features characteristic of HD. While YAC128 mice exhibit significant and widespread accumulation of mHTT striatal aggregates, these mHTT aggregates are absent in BACHD mice. Furthermore, the levels of several striatally enriched mRNA for genes, such as DARPP-32, enkephalin, dopamine receptors D1 and D2 and cannabinoid receptor 1, are significantly decreased in YAC128 but not BACHD mice. These findings may reflect sequence differences in the human mHTT transgenes harboured by the BACHD and YAC128 mice, including both single nucleotide polymorphisms as well as differences in the nature of CAA interruptions of the CAG tract. Our findings highlight a similar profile of HD-like behavioural and neuropathological deficits and illuminate differences that inform the use of distinct endpoints in trials of therapeutic agents in the YAC128 and BACHD mice.

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients.

Many techniques for the surface modification of titanium and its alloys have been proposed from the viewpoint of improving bioactivity. This paper contains an overview of surface treatment methods, including coating with hydroxyapatite (HAp), an osteoconductive compound. There are two types of coating methods: pyroprocessing and hydroprocessing. In this paper, hydroprocessing for coating on the titanium substrate with HAp, carbonate apatite (CO(3)-Ap), a CO(3)-Ap/CaCO(3) composite, HAp/collagen, and a HAp/gelatin composite is outlined. Moreover, evaluation by implantation of surface-modified samples in rat tibiae is described.

<h4>Unlabelled</h4>The most common inborn error of bile acid metabolism is 3β-hydroxy-Δ(5)-C(27)-steroid oxidoreductase (3β-HSD) deficiency, a disorder that usually presents in early childhood with hepatic dysfunction. Timely diagnosis of this disorder is crucial because it can be effectively treated with primary bile acid replacement. Here we describe a 24-year-old woman from Iran with cirrhosis of unknown etiology. Her sister and a first cousin died of cirrhosis (ages 19 and 6 years) and another 32-year-old first cousin had a self-limited liver disorder in childhood that resolved at age 9 years. The family history suggested that the affected family members were homozygous for a mutant allele inherited identical-by-descent. A genome-wide analysis of 2.4 million single nucleotide polymorphisms was performed to identify regions of homozygosity that were present in the proband and the 32-year-old first cousin, but not in a healthy relative. One of these regions contained the gene encoding 3β-HSD (HSD3B7). Sequence analysis of HSD3B7 revealed that the proband and her 32-year-old cousin were homozygous for a frameshift mutation (c.45_46del AG, p.T15Tfsx27) in exon 1. The diagnosis of 3β-HSD deficiency was confirmed by documenting high levels of 3β-hydroxy-Δ(5) bile acids in the serum of the proband and the 32-year-old first cousin using mass spectrometry. To our knowledge, the 32-year-old relative in this family represents the oldest asymptomatic patient with this disorder.<h4>Conclusion</h4>This study highlights the clinical utility of homozygosity mapping in diagnosing autosomal recessive metabolic disorders. This family illustrates the wide variation in expressivity that occurs in 3β-HSD deficiency and underscores the need to consider a bile acid synthetic defect as a possible cause of liver disease in adults.

Activation of TLR signaling in the innate immune cells is critical for the elimination of invading microorganisms. However, uncontrolled activation may lead to autoimmune and inflammatory diseases. In this article, we report the identification of tripartite motif (TRIM) 38 as a negative feedback regulator in TLR signaling by targeting TNFR-associated factor 6 (TRAF6). TRIM38 was induced by TLR stimulation in an NF-κB-dependent manner in macrophages. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of NF-κB and MAPKs, and enhanced expression of proinflammatory cytokines, whereas overexpression of TRIM38 has an opposite effect. As an E3 ligase, TRIM38 bound to TRAF6 and promoted K48-linked polyubiquitination, which led to the proteasomal degradation of TRAF6. Consistently, knockdown of TRIM38 expression resulted in higher protein level of TRAF6 in primary macrophages. Our findings defined a novel function for TRIM38 to prevent excessive TLR-induced inflammatory responses through proteasomal degradation of TRAF6.

Tissues from 98 human hepatocellular carcinomas (HCCs) obtained from hepatic resections were subjected to somatic copy number variation (CNV) analysis. Most of these HCCs were discovered in livers resected for orthotopic transplantation, although in a few cases, the tumors themselves were the reason for the hepatectomies. Genomic analysis revealed deletions and amplifications in several genes, and clustering analysis based on CNV revealed five clusters. The LSP1 gene had the most cases with CNV (46 deletions and 5 amplifications). High frequencies of CNV were also seen in PTPRD (21/98), GNB1L (18/98), KIAA1217 (18/98), RP1-1777G6.2 (17/98), ETS1 (11/98), RSU1 (10/98), TBC1D22A (10/98), BAHCC1 (9/98), MAML2 (9/98), RAB1B (9/98), and YIF1A (9/98). The existing literature regarding hepatocytes or other cell types has connected many of these genes to regulation of cytoskeletal architecture, signaling cascades related to growth regulation, and transcription factors directly interacting with nuclear signaling complexes. Correlations with existing literature indicate that genomic lesions associated with HCC at the level of resolution of CNV occur on many genes associated directly or indirectly with signaling pathways operating in liver regeneration and hepatocyte growth regulation.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expansion of a CAG/polyglutamine repeat for which there are no disease modifying treatments. In recent years, transcriptional dysregulation has emerged as a pathogenic process that appears early in disease progression and has been recapitulated across multiple HD models. Altered histone acetylation has been proposed to underlie this transcriptional dysregulation and histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), have been shown to improve polyglutamine-dependent phenotypes in numerous HD models. However potent pan-HDAC inhibitors such as SAHA display toxic side-effects. To better understand the mechanism underlying this potential therapeutic benefit and to dissociate the beneficial and toxic effects of SAHA, we set out to identify the specific HDAC(s) involved in this process. For this purpose, we are exploring the effect of the genetic reduction of specific HDACs on HD-related phenotypes in the R6/2 mouse model of HD. The study presented here focuses on HDAC3, which, as a class I HDAC, is one of the preferred targets of SAHA and is directly involved in histone deacetylation. To evaluate a potential benefit of Hdac3 genetic reduction in R6/2, we generated a mouse carrying a critical deletion in the Hdac3 gene. We confirmed that the complete knock-out of Hdac3 is embryonic lethal. To test the effects of HDAC3 inhibition, we used Hdac3(+/-) heterozygotes to reduce nuclear HDAC3 levels in R6/2 mice. We found that Hdac3 knock-down does not ameliorate physiological or behavioural phenotypes and has no effect on molecular changes including dysregulated transcripts. We conclude that HDAC3 should not be considered as the major mediator of the beneficial effect induced by SAHA and other HDAC inhibitors in HD.

We previously demonstrated that black bean (BB) and soy flour (SF)-based diets inhibit azoxymethane (AOM)-induced colon cancer. The objective of this study was to identify genes altered by carcinogen treatment in normal-appearing colonic mucosa and those attenuated by bean feeding. Ninety-five male F344 rats were fed control (AIN) diets upon arrival. At 4 and 5 weeks, rats were injected with AOM (15 mg/kg) or saline and one week later administered an AIN, BB-, or SF-based diet. Rats were sacrificed after 31 weeks, and microarrays were conducted on RNA isolated from the distal colonic mucosa. AOM treatment induced a number of genes involved in immunity, including several MHC II-associated antigens and innate defense genes (RatNP-3, Lyz2, Pla2g2a). BB- and SF-fed rats exhibited a higher expression of genes involved in energy metabolism and water and sodium absorption and lower expression of innate (RatNP-3, Pla2g2a, Tlr4, Dmbt1) and cell cycle-associated (Cdc2, Ccnb1, Top2a) genes. Genes involved in the extracellular matrix (Col1a1, Fn1) and innate immunity (RatNP-3, Pla2g2a) were induced by AOM in all diets, but to a lower extent in bean-fed animals. This profile suggests beans inhibit colon carcinogenesis by modulating cellular kinetics and reducing inflammation, potentially by preserving mucosal barrier function.

Chronic pancreatitis is characterized by inflammation, fibrosis, pain, and loss of exocrine function of the pancreas. We aimed to identify differentially expressed proteins in the ePFT-collected pancreatic fluid from individuals with chronic pancreatitis (CP; n = 9) and controls with chronic abdominal pain not associated with the pancreas (NP; n = 9). Using GeLC-MS/MS techniques, we identified a total of 1391 different proteins in 18 pancreatic fluid samples. Of these proteins, 257 and 413 were identified exclusively in the control and chronic pancreatitis cohorts, respectively, and 721 were identified in both cohorts. Spectral counting and statistical analysis thereof revealed an additional 38 and 77 proteins that were up- or down-regulated, respectively, in the pancreatic fluid from individuals with chronic pancreatitis. As expected, gene ontology analysis illustrated that the largest percentage of differentially regulated proteins was secreted/extracellular in origin. In addition, proteins that were down-regulated with statistical significance in the chronic pancreatitis cohort were determined to have biological function of proteases, corresponding to the canonical pancreatic insufficiency associated with chronic pancreatitis. Proteins enriched in the pancreatic fluid of chronic pancreatitis patients had roles in fibrosis, inflammation, and pain, whereas digestive enzymes were significantly less abundant. Our workflow provided a mass spectrometry-based approach for the further study of the pancreatic fluid proteome, which may lead to the discovery potential biomarkers of chronic pancreatitis.

Osteophilic modular nanostructured multilayers containing hydroxyapatite nanoparticles complexed with a natural polymer chitosan create an osteoconductive surface for mesenchymal stem cells (MSCs). Coupled with the sustained release of physiological amounts of osteoinductive bone morphogenetic protein over several days from degradable poly(β-amino ester) based multilayers, this single coating results in a synergistic accelerated and upregulated differentiation of MSCs into osteoblasts laying down new bone tissue on orthopedic implants.

<h4>Background</h4>Human papillomavirus (HPV) infection has been demonstrated in the sperm of a large percentage of sexually active males and is associated with an impairment of sperm parameters, with a particular negative impact on sperm motility, suggesting a possible role in male infertility. Conventional sperm selection techniques have a low efficiency in removing HPV.<h4>Methods</h4>Evaluation of sperm parameters, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling test to evaluate DNA fragmentation and fluorescence in situ hybridization or immunohistochemistry for HPV were performed on semen samples from infected patients (n= 22), control subjects (n= 13) and on pooled control sperm samples incubated with HPV16-L1 (HPV capsid), before and after direct swim-up and modified swim-up (with added Heparinase-III). Moreover, cytofluorimetry for HPV detection was performed in pooled sperm pre- and post-incubation with HPV 16-L1 before and after direct and modified swim-up. Statistical analysis was performed with a two-tailed Student's t-test.<h4>Results</h4>Direct swim-up reduces the number of HPV-infected sperm by ~24% (P< 0.01), while modified swim-up is able to remove completely HPV DNA both from naturally and artificially infected sperm. Enzymatic treatment with Heparinase-III tended to decrease sperm motility, viability and DNA integrity but the effects were not significant.<h4>Conclusions</h4>This study shows that Heparinase-III treatment seems not to affect spermatozoa in vitro and suggests that this treatment should be investigated further as a means of preparing sperm from patients who are infected with HPV in order to reduce the risk of HPV infection when using assisted reproduction techniques.

Mycobacterium tuberculosis infection claims approximately 2 million lives per year, and improved efficacy of the BCG vaccine remains a World Health Organization priority. Successful vaccination against M. tuberculosis requires the induction and maintenance of T cells. Targeting molecules that promote T-cell survival may therefore provide an alternative strategy to classic adjuvants. We show that the interaction between T-cell-expressed OX40 and OX40L on antigen-presenting cells is critical for effective immunity to BCG. However, because OX40L is lost rapidly from antigen-presenting cells following BCG vaccination, maintenance of OX40-expressing vaccine-activated T cells may not be optimal. Delivering an OX40L:Ig fusion protein simultaneously with BCG provided superior immunity to intravenous and aerosol M. tuberculosis challenge even 6 months after vaccination, an effect that depends on natural killer 1.1(+) cells. Attenuated vaccines may therefore lack sufficient innate stimulation to maintain vaccine-specific T cells, which can be replaced by reagents binding inducible T-cell costimulators.

Dyslipidemia has been associated with type 2 diabetes, but it remains unclear whether dyslipidemia plays a causal role in type 2 diabetes. We aimed to examine the association between the genetic predisposition to dyslipdemia and type 2 diabetes risk. The current study included 2,447 patients with type 2 diabetes and 3,052 control participants of European ancestry from the Nurses' Health Study and the Health Professionals Follow-up Study. Genetic predisposition to dyslipidemia was estimated by three genotype scores of lipids (LDL cholesterol, HDL cholesterol, and triglycerides) on the basis of the established loci for blood lipids. Linear relation analysis indicated that the HDL cholesterol and triglyceride genotype scores, but not the LDL cholesterol genotype score, were linearly related to elevated type 2 diabetes risk. Each point of the HDL cholesterol and triglyceride genotype scores was associated with a 3% (odds ratio [OR] 1.03 [95% CI 1.01-1.04]) and a 2% (1.02 [1.00-1.04]) increased risk of developing type 2 diabetes, respectively. The ORs were 1.39 (1.17-1.65) and 1.19 (1.01-1.41) for type 2 diabetes by comparing extreme quartiles of the HDL cholesterol genotype score and triglyceride genotype score, respectively. In conclusion, genetic predisposition to low HDL cholesterol or high triglycerides is related to elevated type 2 diabetes risk.

<h4>Background</h4>Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain.<h4>Methods and results</h4>Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes, and a number of wig-1 regulated genes were identified that potentially links wig-1 various signaling pathways and diseases.<h4>Conclusion</h4>Antisense oligonucleotides can effectively reduce wig-1 levels in mouse liver and brain, which results in specific changes in gene expression for pathways relevant to both the nervous system and cancer.

The high prevalence of smoking in schizophrenia of European background may be related to smoking's reducing clinical symptoms and medication side effects. Because smoking prevalence and its associations with clinical phenotypes are less well characterized in Chinese than European patients with schizophrenia, we assessed these smoking behaviors using clinician-administered questionnaires and the Fagerstrom Test for Nicotine Dependence (FTND) in 776 Chinese male schizophrenia and 560 control subjects. Patients also were rated on the Positive and Negative Symptom Scale (PANSS), the Simpson and Angus Extrapyramidal Symptom Rating Scale (SAES), and the Abnormal Involuntary Movement Scale (AIMS). We found that the schizophrenia patients had a higher lifetime incidence of smoking (79% vs 63%), were more likely to be heavy smokers (61% vs 31%), and had lower smoking cessation rates (4% vs 9%) (all p<0.0001) than controls. Among the schizophrenia patients smoking prevalence increased with age, with the largest difference from controls in the age cohort of 55-75 years: 75% vs 46% (p<0.0001). Among the schizophrenia smokers 73% started to smoke before the onset of their illness by an average of 7.6 years. The patients with schizophrenia who were current smokers scored significantly lower on the PANSS negative symptom subscore (p<0.005), and on the SAES symptom scale (p<0.04; Bonferroni corrected p>0.05) than the non-smoking patients. These results suggest that Chinese males with schizophrenia smoke more frequently than the general population. Further, smokers with schizophrenia may display fewer negative symptoms and possibly less parkinsonism than non-smokers with schizophrenia.

<h4>Background</h4>NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides.<h4>Methodology/principal findings</h4>The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs.<h4>Conclusions/significance</h4>These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

Hexahomotrioxacalix[3]arenes, commonly called oxacalix[3]arenes, were first reported in 1962. Since then, their chemistry has been expanded to include numerous derivatives and complexes. This review describes the syntheses of the parent compounds, their derivatives, and their complexation behaviour towards cations. Extraction data are presented, as are crystal structures of the macrocycles and their complexes with guest species. Applications in fields as diverse as ion selective electrode modifiers, fluorescence sensors, fullerene separations and biomimetic chemistry are described.

Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic β-neurexins (Nrxn) to post-synaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.

Astaxanthin at 0.01 or 0.05% of the diet was supplied to diabetic rats for 12 wk. Astaxanthin intake significantly increased its deposit in plasma, and retained glutathione content, reduced the production of reactive oxygen species, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 in blood and kidney of diabetic rats (P < 0.05). Astaxanthin treatments also significantly decreased plasma levels of C-reactive protein and von Willebrand factor in diabetic rats (P < 0.05). Astaxanthin intake at 0.05% significantly diminished plasminogen activator inhibitor-1 and factor VII activities, enhanced antithrombin-III and protein C activities in circulation (P < 0.05). These results support that astaxanthin could attenuate diabetes associated coagulatory, oxidative, and inflammatory stress.

<h4>Background</h4>Prostatitis is an inflammation of the prostate gland which affects approximately 10% of men. Despite its frequency, diagnosing prostatitis and monitoring patient response to treatment remains frustrating. As the prostate contributes a substantial percentage of proteins to seminal plasma, we hypothesized that a protein biomarker of prostatitis might be found by comparing the seminal plasma proteome of patients with and without prostatitis.<h4>Results</h4>Using mass spectrometry, we identified 1708 proteins in the pooled seminal plasma of 5 prostatitis patients. Comparing this list to a previously published list of seminal plasma proteins in the pooled seminal plasma of 5 healthy, fertile controls yielded 1464 proteins in common, 413 found only in the control group, and 254 found only in the prostatitis group. Applying a set of criteria to this dataset, we generated a high-confidence list of 59 candidate prostatitis biomarkers, 33 of which were significantly increased in prostatitis as compared to control, and 26 of which were decreased. The candidates were analyzed using Gene Ontology and Ingenuity Pathway analysis to delineate their subcellular localizations and functions.<h4>Conclusions</h4>Thus, in this study, we identified 59 putative biomarkers in seminal plasma that need further validation for diagnosis and monitoring of prostatitis.

<h4>Introduction</h4>Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.<h4>Methods</h4>A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.<h4>Results</h4>Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER+/HER2+, a metastatic variant), and HR5 (ER+/HER2+, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.<h4>Conclusion</h4>Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

The generation and maintenance of a plethora of neuronal subtypes is essential for normal brain function. Nevertheless, little is known about the molecular mechanisms that maintain the defining characteristics of neurons following their initial postmitotic specification. Using conditional gene ablation in mice, we demonstrate here that the homeodomain protein LIM homeobox (Lhx)7 is essential for maintaining the morphological and molecular characteristics of cholinergic interneurons of the striatum. Lhx7-depleted cholinergic interneurons extinguish expression of several subtype-specific markers, including choline acetyl transferase and Isl1, and are respecified into Lhx6-expressing mature GABAergic interneurons. Additional expression studies support a model where Lhx7 controls the choice between cholinergic or GABAergic identity by gating a cross inhibitory regulation between Isl1 and Lhx6. By demonstrating that the switch between alternative striatal interneuron fates depends on persistent activity of a single transcription factor, we provide evidence that the intrinsic plasticity of mammalian forebrain neuronal subtypes is maintained after the initial specification and lineage commitment and possibly throughout life.

<h4>Background & aims</h4>Gestational alloimmune liver disease is the main cause of the neonatal hemochromatosis phenotype, wherein severe neonatal liver disease is associated with iron overload and extrahepatic tissue siderosis. How fetal liver disease produces extrahepatic siderosis is not known. We hypothesized that fetal liver injury causes deficient hepcidin production and poor regulation of placental iron flux. Under the resulting conditions of iron overload, the tissue pattern of extrahepatic siderosis is determined by the normal expression of proteins involved in the import of non-transferrin-bound iron and the export of cellular iron.<h4>Methods</h4>Liver and extrahepatic tissues from infants with gestational alloimmune liver disease were examined and compared to normal age-appropriate tissues.<h4>Results</h4>Serum iron indices indicate iron overload and excess non-transferrin bound iron in gestational alloimmune liver disease. The diseased liver showed significantly reduced hepcidin, hemojuvulin, and transferrin gene expression compared to the normal fetal and neonatal liver. Those extrahepatic tissues that are typically involved in pathological siderosis in neonatal hemochromatosis, whether from normal or diseased newborns, consistently expressed solute carrier family 39 (zinc transporter), member 14 (ZIP14) for non-transferrin-bound iron uptake and expressed little ferroportin for iron export.<h4>Conclusions</h4>Excess non-transferrin-bound iron in gestational alloimmune liver disease may result from fetal liver injury that causes reduced synthesis of key iron regulatory and transport proteins. Whereas, the pattern of extrahepatic siderosis appears to be determined by the normal capacity of various tissues to import non-transferrin-bound iron and not export cellular iron.

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.

Whether induction of low-level neurogenesis in normally non-neurogenic regions of the adult brain mimics aspects of developmental neurogenesis is currently unknown. Previously, we and others identified that biophysically induced, neuron subtype-specific apoptosis in mouse neocortex results in induction of neurogenesis of limited numbers of subtype-appropriate projection neurons with axonal projections to either thalamus or spinal cord, depending on the neuron subtype activated to undergo targeted apoptosis. Here, we test the hypothesis that developmental genes from embryonic corticogenesis are re-activated, and that some of these genes might underlie induction of low-level adult neocortical neurogenesis. We directly investigated this hypothesis via microarray analysis of microdissected regions of young adult mouse neocortex undergoing biophysically activated targeted apoptosis of neocortical callosal projection neurons. We compared the microarray results identifying differentially expressed genes with public databases of embryonic developmental genes. We find that, following activation of subtype-specific neuronal apoptosis, three distinct sets of normal developmental genes are selectively re-expressed in neocortical regions of induced neurogenesis in young adult mice: (1) genes expressed by subsets of progenitors and immature neurons in the developing ventricular and/or subventricular zones; (2) genes normally expressed by developmental radial glial progenitors; and (3) genes involved in synaptogenesis. Together with previous results, the data indicate that at least some developmental molecular controls over embryonic neurogenesis can be re-activated in the setting of induction of neurogenesis in the young adult neocortex, and suggest that some of these activate and initiate adult neuronal differentiation from endogenous progenitor populations. Understanding molecular mechanisms contributing to induced adult neurogenesis might enable directed CNS repair.

Myelination of axons by oligodendrocytes in the central nervous system is essential for normal neuronal functions. The failure of remyelination due to injury or pathological insults results in devastating demyelinating diseases. Oligodendrocytes originate in restricted regions of the embryonic ventral neural tube. After migration to populate all areas of the brain and spinal cord, oligodendrocyte precursors undergo a temporally well-defined series of molecular and structural changes, ultimately culminating in the cessation of proliferation, and the elaboration of a highly complex myelin sheath. The emergence of microRNAs (miRNAs) as potent regulators of gene expression at the posttranscriptional level has broad implications in all facets of cell biology. Recent studies have demonstrated a critical role of miRNAs in oligodendrocyte development, including cell proliferation, differentiation, and myelin formation. In this review, we will highlight and discuss the recent understanding of functional links of miRNAs to regulatory networks for central myelination, as well as perspectives on the role of miRNAs in demyelinating diseases.

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by progressive atrophy of the striatum, cerebral cortex, and white matter tracks. Major pathological hallmarks of HD include neuronal loss, primarily in the striatum, and dendritic anomalies in surviving striatal neurons. Although many mouse models of HD have been generated, their success at reproducing all pathological features of the disease is not fully known. Previously, we demonstrated extensive striatal neuronal loss and striatal atrophy at 20-26 months of age in a knock-in (KI) mouse model of HD. To further investigate this model, which carries a human exon 1 with ∼119 CAG repeats inserted into the mouse gene (initially 140 repeats), we have examined whether these mice exhibit the atrophy and neuronal anomalies characteristic of HD. Stereological analyses revealed no changes in the striatal volume of male and female homozygote mice at 4 months, however striatal atrophy was already present at 12 months in both sexes. Analysis of cortical and corpus callosum volume in male homozygotes revealed a loss in corpus callosum volume by 20-26 months. At this later age, the surviving striatal neurons displayed extensive loss of spines in distal branch orders that affected both immature and mature spines. Mirroring late stage HD striatal neuronal morphology, the striatal neurons at this late age also showed reduced dendritic complexity, as revealed by Sholl analysis. Tyrosine hydroxylase immunoreactivity was also decreased in the striatum of 20-26 month old KI mice, suggesting an alteration in striatal inputs. These data further indicate that CAG140 homozygote KI mice exhibit HD-like pathological features and are a useful model to test the effects of early and/or sustained administration of novel neuroprotective treatments.

Lupus is less common in men than women, and the reason is incompletely understood. Current evidence indicates that lupus flares when genetically predisposed individuals encounter environmental agents that trigger the disease, and that the environmental contribution is mediated at least in part by T cell DNA demethylation. We hypothesized that lupus disease activity is directly related to total genetic risk and inversely related to T cell DNA methylation levels in each patient. Since women are predisposed to lupus in part because of their second X chromosome, we also hypothesized that men would require a greater genetic risk, a greater degree of autosomal T cell DNA demethylation, or both, to achieve a lupus flare equal in severity to women. Genetic risk was determined by genotyping men and women with lupus across 32 confirmed lupus susceptibility loci. The methylation status of two autosomal genes known to demethylate in T cells in proportion to disease activity, KIR2DL4 (KIR) and PRF1, was measured by bisulfite sequencing. Lupus disease activity was determined by the SLEDAI. Interactions between genetic score, T cell DNA demethylation, and the SLEDAI score were compared between the men and women by regression analysis. Combining the degree of DNA demethylation with the genetic risk score for each patient demonstrated that the (genetic risk)/(DNA methylation) ratio increased directly with disease activity in both men and women with lupus. Importantly, men required a greater (genetic risk)/(DNA methylation) ratio to achieve a SLEDAI score equivalent to women (P = 0.010 for KIR and P = 0.0054 for PRF1). This difference was not explained by a difference in the genetic risk or T cell DNA demethylation alone, suggesting a genetic-epigenetic interaction. These results suggest that genetic risk and T cell DNA demethylation interact in lupus patients to influence the severity of lupus flares, and that men require a higher genetic risk and/or greater degree of T cell DNA demethylation to achieve a lupus flare equal in severity to women.

MicroRNAs (miRNAs) are a class of short RNA molecules that play an important role in post-transcriptional gene regulation. Computational prediction of the miRNA target sites in mRNA is crucial for understanding the mechanism of miRNA-mRNA interactions. We here develop a new computational model that allows us to treat a variety of miRNA-mRNA kissing interactions, which have been ignored in the currently existing miRNA target prediction algorithms. By including all the different inter- and intra-molecular base pairs, this new model can predict both the structural accessibility of the target sites and the binding affinity (free energy). Applications of the model to a test set of 105 miRNA-gene systems show a notably improved success rate of 83/105. We found that although the binding affinity alone predicts the miRNA repression efficiency with a high success rate of 73/105, the structure in the seed region can significantly influence the miRNA activity. The method also allows us to efficiently search for the potent miRNA from a pool of miRNA candidates for any given gene target. Furthermore, extension of the method may enable predictions of the three-dimensional (3D) structures of miRNA/mRNA complexes.

TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position -59 to -509, cAMP-CRP binds exclusively in a region centered at position -122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between "feast" and "famine"). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms.

Many chronic pulmonary diseases are associated with pulmonary hypertension (PH) and pulmonary vascular remodeling, which is a term that continues to be used to describe a wide spectrum of vascular abnormalities. Pulmonary vascular structural changes frequently increase pulmonary vascular resistance, causing PH and right heart failure. Although rat models had been standard models of PH research, in more recent years the availability of genetically engineered mice has made this species attractive for many investigators. Here we review a large amount of data derived from experimental PH reports published since 1996. These studies using wild-type and genetically designed mice illustrate the challenges and opportunities provided by these models. Hemodynamic measurements are difficult to obtain in mice, and right heart failure has not been investigated in mice. Anatomical, cellular, and genetic differences distinguish mice and rats, and pharmacogenomics may explain the degree of PH and the particular mode of pulmonary vascular adaptation and also the response of the right ventricle.

The therapeutic benefit of the small heat shock protein αB-crystallin (HspB5) in animal models of multiple sclerosis and ischemia is proposed to arise from its increased capacity to bind proinflammatory proteins at the elevated temperatures within inflammatory foci. By mass spectral analysis, a common set of ∼70 ligands was precipitated by HspB5 from plasma from patients with multiple sclerosis, rheumatoid arthritis, and amyloidosis and mice with experimental allergic encephalomyelitis. These proteins were distinguished from other precipitated molecules because they were enriched in the precipitate as compared with their plasma concentrations, and they exhibited temperature-dependent binding. More than half of these ligands were acute phase proteins or members of the complement or coagulation cascades. Consistent with this proposal, plasma levels of HspB5 were increased in patients with multiple sclerosis as compared with normal individuals. The combination of the thermal sensitivity of the HspB5 combined with the high local concentration of these ligands at the site of inflammation is proposed to explain the paradox of how a protein believed to exhibit nonspecific binding can bind with some relative apparent selectivity to proinflammatory proteins and thereby modulate inflammation.

Huntington's disease (HD) is an inherited neurodegenerative disorder that causes neurological pathology in the basal ganglia and related circuitry. A key site of HD pathology is striatum, the principal basal ganglia input structure; striatal pathology likely changes basal ganglia output but no existing studies address this issue. In this report, we characterize single-neuron activity in the substantia nigra reticulata (SNr) of awake, freely behaving 140 CAG knock-in (KI) mice at 16-40 weeks. KI mice are a well characterized model of adult HD and are mildly symptomatic in this age range. As the primary basal ganglia output nucleus in rodents, the SNr receives direct innervation from striatum, as well as indirect influence via polysynaptic inputs. We analyzed 32 single neurons recorded from KI animals and 44 from wild-type (WT) controls. We found increased burst rates, without a concordant change in spike discharge rate, in KI animals relative to WTs. Furthermore, although metrics of burst structure, such as the inter-spike interval in bursts, do not differ between groups, burst rate increases with age in KI, but not WT, animals. Our findings suggest that altered basal ganglia output is a physiological feature of early HD pathology.

<h4>Background</h4>NURR1 (also named as NR4A2) is a member of the steroid/thyroid hormone receptor family, which can bind to DNA and modulate expression of target genes. Previous studies have shown that NURR1 is essential for the nigral dopaminergic neuron phenotype and function maintenance, and the defects of the gene are possibly associated with Parkinson's disease (PD).<h4>Results</h4>In this study, we used new born Nurr1 knock-out mice combined with Affymetrix genechip technology and real time polymerase chain reaction (PCR) to identify Nurr1 regulated genes, which led to the discovery of several transcripts differentially expressed in the nigro-striatal pathway of Nurr1 knock-out mice. We found that an axon genesis gene called Topoisomerase IIβ (Top IIβ) was down-regulated in Nurr1 knock-out mice and we identified two functional NURR1 binding sites in the proximal Top IIβ promoter. While in Top IIβ null mice, we saw a significant loss of dopaminergic neurons in the substantial nigra and lack of neurites along the nigro-striatal pathway. Using specific TOP II antagonist ICRF-193 or Top IIβ siRNA in the primary cultures of ventral mesencephalic (VM) neurons, we documented that suppression of TOP IIβ expression resulted in VM neurites shortening and growth cones collapsing. Furthermore, microinjection of ICRF-193 into the mouse medial forebrain bundle (MFB) led to the loss of nigro-striatal projection.<h4>Conclusion</h4>Taken together, our findings suggest that Top IIβ might be a down-stream target of Nurr1, which might influence the processes of axon genesis in dopaminergic neurons via the regulation of TOP IIβ expression. The Nurr1-Top IIβ interaction may shed light on the pathologic role of Nurr1 defect in the nigro-striatal pathway deficiency associated with PD.

Links between substance use habits, obesity, stress and the related cardiovascular outcomes can be, in part, because of loci with pleiotropic effects. To investigate this hypothesis, we performed genome-wide mapping in 119 multigenerational families from a population in the Saguenay-Lac-St-Jean region with a known founder effect using 58,000 single-nucleotide polymorphisms and 437 microsatellite markers to identify genetic components of the following factors: habitual alcohol, tobacco and coffee use; response to mental and physical stress; obesity-related traits; and heart rate (HR) and blood pressure (BP) measures. Habitual alcohol and/or tobacco users had attenuated HR responses to mental stress compared with non-users, whereas hypertensive individuals had stronger HR and systolic BP responses to mental stress and a higher obesity index than normotensives. Genetic mappings uncovered numerous shared genes among substance use, stress response, obesity and hemodynamic traits, including CAMK4, CNTN4, DLG2, FHIT, GRID2, ITPR2, NOVA1 and PRKCE, forming network of interacting proteins, sharing synaptic function and display higher and patterned expression profiles in brain-related tissues; moreover, pathway analysis of shared genes pointed to long-term potentiation. Subgroup genetic mappings uncovered additional shared synaptic genes, including CAMK4, CNTN5 and DNM3 (hypertension-specific); CNTN4, DNM3, FHIT and ITPR1 (sex-specific), having protein interactions with genes driven from general analysis. In summary, consistent with the observed phenotypic correlations, we found substantial overlap among genomic determinants of these traits in synapse, which supports the notion that the neural synapse may be a shared interface behind substance use, stress, obesity, HR, BP as well as the observed sex- and hypertension-specific genetic differences.

Once a liver offer has been refused locally and regionally, it is offered nationally. We characterized nationally (n = 1567) versus locally (n = 19 893) placed grafts from adult, nonfulminant, deceased donor liver transplants (LT) from 2/1/05 to 1/31/10. Donors of nationally versus locally placed livers differed by age (50 vs. 42 years), positive HCV antibody (11 vs. 2%) and death from stroke (51 vs. 42%) (p < 0.001 for all). Recipients of nationally versus locally placed livers differed by LT-MELD (20 vs. 24), rates of ascites (35 vs. 37%), encephalopathy (12 vs. 15%), hepatocellular (17 vs. 24%) and nonhepatocellular exceptions (6 vs. 11%) (p ≤ 0.03 for all). Six (5%) centers utilized 64% of the nationally placed grafts while 43 (38%) centers accepted zero during the 5-year period; all high volume centers used ≥1. Compared to local distribution, transplantation with a nationally placed liver was associated with a similar adjusted risk of graft (HR, 0.99; 95% CI, 0.86-1.14) and patient (HR, 0.98; 95% CI, 0.84-1.14; p = 0.77) survival. In conclusion, utilization of nationally placed livers is highly concentrated in very few centers, with no increased adjusted risk of graft loss. These findings provide the foundation for a more informed discussion about changing our current liver allocation and distribution policies.

Congenital mirror movements (CMM) are characterized by involuntary movements of one side of the body that mirror intentional movements on the opposite side. CMM reflect dysfunctions and structural abnormalities of the motor network and are mainly inherited in an autosomal-dominant fashion. Recently, heterozygous mutations in DCC, the gene encoding the receptor for netrin 1 and involved in the guidance of developing axons toward the midline, have been identified but CMM are genetically heterogeneous. By combining genome-wide linkage analysis and exome sequencing, we identified heterozygous mutations introducing premature termination codons in RAD51 in two families with CMM. RAD51 mRNA was significantly downregulated in individuals with CMM resulting from the degradation of the mutated mRNA by nonsense-mediated decay. RAD51 was specifically present in the developing mouse cortex and, more particularly, in a subpopulation of corticospinal axons at the pyramidal decussation. The identification of mutations in RAD51, known for its key role in the repair of DNA double-strand breaks through homologous recombination, in individuals with CMM reveals a totally unexpected role of RAD51 in neurodevelopment. These findings open a new field of investigation for researchers attempting to unravel the molecular pathways underlying bimanual motor control in humans.

Although, the nucleolus has been observed for almost 200 years in neurons, studies that directly address the neuronal roles of this subnuclear structure have appeared only recently. The aim of this review is to discuss recent progress and identify some critical questions that remain to be answered. As expected for the cellular center of ribosome biogenesis, the nucleolus is essential for the growth of developing neurons, including neurite morphogenesis and long-term maintenance of mature neurons. In addition, the nucleolus contributes to neuronal stress responses, including the regulation of apoptosis. Hence, disrupted neurodevelopment or neurodegeneration are among the likely consequences of nucleolar dysfunction. Conversely, the presence of active nucleoli may determine the potential for neurorepair.

The HMGN family of proteins binds to nucleosomes without any specificity for the underlying DNA sequence. They affect the global and local structure of chromatin, as well as the levels of histone modifications and thus play a role in epigenetic regulation of gene expression. This review focuses on the recent studies that provide new insights on the interactions between HMGN proteins, nucleosomes, and chromatin, and the effects of these interactions on epigenetic and transcriptional regulation. This article is part of a Special Issue entitled: Chromatin in time and space.

It has long been assumed that DNA sequences and corresponding RNA transcripts are almost identical; a recent discovery, however, revealed widespread RNA-DNA differences (RDDs), which represent a largely unexplored aspect of human genome variation. It has been speculated that RDDs can affect disease susceptibility and manifestations; however, almost nothing is known about how RDDs are related to disease. Here, we show that RDDs are rarer in proto-oncogenes than in tumor suppressor genes; the number of RDDs in coding exons, but not in 3'UTR and 5'UTR, is significantly lower in the former than the latter, and this trend is especially pronounced in non-synonymous RDDs, i.e., those cause amino acid changes. A potential mechanism is that, unlike proto-oncogenes, the requirement of tumor suppressor genes to have both alleles affected to cause tumor 'buffers' these genes to tolerate more RDDs.

<h4>Background</h4>There is emerging evidence that clinical and neuro-pathological manifestations of Huntington's disease (HD) may occur in individuals with intermediate length cytosine-adenine-guanine (CAG) repeats (27-35 CAG repeats) in the Huntingtin (HTT) gene. We aim to further define the clinical characteristics of individuals who possess CAG repeat lengths in this range.<h4>Methods</h4>Data from the Cooperative Huntington's Observational Research Trial (COHORT) were analyzed. Participants were categorized according to the number of CAG repeats into normal (≤26), intermediate (27-35) and HD (≥36) groups. The motor, cognitive and behavioral scores on the Unified Huntington's Disease Rating Scale (UHDRS) were compared between the intermediate and normal groups.<h4>Results</h4>Of 1985 individuals affected by HD or with a family history of HD who were genotyped, 50 (2.5%) had their larger CAG repeat in the intermediate range. There were statistically significant differences in scores of some motor, cognitive, and behavioral domains of UHDRS at baseline between normal and intermediate length CAG repeats. Furthermore, a significantly greater number of subjects with CAG repeats in the intermediate range reported at least one suicide attempt compared to the normal group.<h4>Discussion</h4>Our findings of motor, cognitive and behavioral abnormalities in individuals with intermediate CAG repeats suggest the presence of subtle, but relevant, disease manifestations in patients with intermediate CAG repeats. These results have important implications for the pathogenesis of the disease and genetic counseling.

Despite significant improvements in therapeutic protocols, head and neck squamous cell carcinoma (HNSCC) remains a major health problem worldwide. The 5-year post-therapeutic survival rate is among the lowest of the major cancers with loco-regional relapse being the main cause of death. Moreover, in most instances, the quality of life of the afflicted patient is severely compromised. The poor prognosis for HNSCC is primarily due to disease detection at advanced stages. Accordingly, development of early detection and preventive strategies are essential. Recent advances in our understanding of the molecular biology and etiology of HNSCC should facilitate development of improved intervention and therapeutic approaches. The present review discusses the potential role of such factors for developing preventive and early diagnostic strategies for HNSCC management.

Heparin and HS (heparan sulfate) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS binding to VEGF (vascular endothelial growth factor) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that the VEGF binding affinity likely depends on the specific structural features of these oligosaccharides, including their degree of sulfation, sugar-ring stereochemistry and conformation. Notably, the unique 3-O-sulfo group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue-specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs.

<h4>Background and purpose</h4>Inflammation and reactive oxygen species are associated with the promotion of various cancers. The use of non-steroidal anti-inflammatory drugs (NSAIDs) in cancer prevention treatments has been promising in numerous cancers. We report the evaluation of NSAIDs chemically modified by the addition of a redox-active nitroxide group. TEMPO-aspirin (TEMPO-ASA) and TEMPO-indomethacin (TEMPO-IND) were synthesized and evaluated in the lung cancer cell line A549.<h4>Experimental approaches</h4>We evaluated physico-chemical properties of TEMPO-ASA and TEMPO-IND by electron paramagnetic resonance and cyclic voltammetry. Superoxide dismutase-like properties was assayed by measuring cytochrome c reduction and anti-inflammatory effects were assayed by measuring production of prostaglandin E(2) (PGE(2) ) and leukotriene B(4) (LTB(4) ). MTT proliferation assay and clonogenic assay were evaluated in the A549 lung carcinoma cell line. Maximum tolerated doses (MTD) and acute ulcerogenic index were also evaluated in in vivo.<h4>Key results</h4>MTD were: TEMPO (140 mg·kg(-1) ), ASA (100 mg·kg(-1) ), indomethacin (5 mg·kg(-1) ), TEMPO-ASA (100 mg·kg(-1) ) and TEMPO-IND (40 mg·kg(-1) ). While TEMPO-ASA was as well tolerated as ASA, TEMPO-IND showed an eightfold improvement over indomethacin. TEMPO-IND showed markedly less gastric toxicity than the parent NSAID. Both TEMPO-ASA and TEMPO-IND inhibited production of PGE(2) and LTB(4) in A549 cells with maximum effects at 100 µg·mL(-1) or 10 µg·mL(-1) respectively.<h4>Conclusions and implications</h4>The nitroxide-NSAIDs retained superoxide scavenging capacity of the parent nitroxide and anti-inflammatory effects, inhibiting cyclooxygenase and 5-lipoxygenase enzymes. These redox-modified NSAIDs might be potential drug candidates, as they exhibit the pharmacological properties of the parent NSAID with antioxidant activity decreasing NSAID-associated toxicity.

Redox changes are observed in neurodegenerative diseases, ranging from increased levels of reactive oxygen/nitrogen species and disturbance of antioxidant systems, to nitro-oxidative damage. By reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides, peroxiredoxins (Prdxs) represent a major potential protective barrier against nitro-oxidative insults in the brain. While recent works have investigated the putative role of Prdxs in neurodegenerative disorders, less is known about their expression in the healthy brain. Here we used immunohistochemistry to map basal expression of Prdxs throughout C57BL/6 mouse brain. We first confirmed the neuronal localization of Prdx2-5 and the glial expression of Prdx1, Prdx4, and Prdx6. Then we performed an in-depth analysis of neuronal Prdx distribution in the brain. Our results show that Prdx2-5 are widely detected in the different neuronal populations, and especially well expressed in the olfactory bulb, in the cerebral cortex, in pons nuclei, in the red nucleus, in all cranial nerve nuclei, in the cerebellum, and in motor neurons of the spinal cord. In contrast, Prdx expression is very low in the dopaminergic neurons of substantia nigra pars compacta and in the CA1/2 pyramidal cells of hippocampus. This low basal expression may contribute to the vulnerability of these neurons to nitro-oxidative attacks occurring in Parkinson's disease and Alzheimer's disease. In addition, we found that Prdx expression levels are unevenly distributed among neurons of a determined region and that distinct regional patterns of expression are observed between isoforms, reinforcing the hypothesis of the nonredundant function of Prdxs.

Tyrosine kinase fusion genes represent an important class of oncogenes associated with leukaemia and solid tumours. They are produced by translocations and other chromosomal rearrangements of a subset of tyrosine kinase genes, including ABL, PDGFRA, PDGFRB, FGFR1, SYK, RET, JAK2 and ALK. Based on recent findings, this review discusses the common mechanisms of activation of these fusion genes. Enforced oligomerization and inactivation of inhibitory domains are the two key processes that switch on the kinase domain. Activated tyrosine kinase fusions then signal via an array of transduction cascades, which are largely shared. In addition, the fusion partner provides a scaffold for the recruitment of proteins that contribute to signalling, protein stability, cellular localization and oligomerization. The expression level of the fusion protein is another critical parameter. Its transcription is controlled by the partner gene promoter, while translation may be regulated by miRNA. Several mechanisms also prevent the degradation of the oncoprotein by proteasomes and lysosomes, leading to its accumulation in cells. The selective inhibition of the tyrosine kinase activity by adenosine-5'-triphosphate competitors, such as imatinib, is a major therapeutic success. Imatinib induces remission in leukaemia patients that are positive for BCR-ABL or PDGFR fusions. Recently, crizotinib produced promising results in a subtype of lung cancers with ALK fusion. However, resistance was reported in both cases, partially due to mutations. To tackle this problem, additional levels of therapeutic interventions are suggested by the complex mechanisms of fusion tyrosine kinase activation. New approaches include allosteric inhibition and interfering with oligomerization or chaperones.

<h4>Objective</h4>Fibromyalgia (FM) represents a complex disorder that is characterized by widespread pain and tenderness and is frequently accompanied by additional somatic and cognitive/affective symptoms. Genetic risk factors are known to contribute to the etiology of the syndrome. The aim of this study was to examine >350 genes for association with FM, using a large-scale candidate gene approach.<h4>Methods</h4>The study group comprised 496 patients with FM (cases) and 348 individuals with no chronic pain (controls). Genotyping was performed using a dedicated gene array chip, the Pain Research Panel, which assays variants characterizing >350 genes known to be involved in the biologic pathways relevant to nociception, inflammation, and mood. Association testing was performed using logistic regression.<h4>Results</h4>Significant differences in allele frequencies between cases and controls were observed for 3 genes: GABRB3 (rs4906902; P = 3.65 × 10(-6)), TAAR1 (rs8192619; P = 1.11 × 10(-5)), and GBP1 (rs7911; P = 1.06 × 10(-4)). These 3 genes and 7 other genes with suggestive evidence for association were examined in a second, independent cohort of patients with FM and control subjects who were genotyped using the Perlegen 600K platform. Evidence of association in the replication cohort was observed for TAAR1, RGS4, CNR1, and GRIA4.<h4>Conclusion</h4>Variation in these 4 replicated genes may serve as a basis for development of new diagnostic approaches, and the products of these genes may contribute to the pathophysiology of FM and represent potential targets for therapeutic action.

<h4>Objective</h4>Several confirmed genetic susceptibility loci for lupus have been described. To date, no clear evidence for genetic epistasis in lupus has been established. The aim of this study was to test for gene-gene interactions in a number of known lupus susceptibility loci.<h4>Methods</h4>Eighteen single-nucleotide polymorphisms tagging independent and confirmed lupus susceptibility loci were genotyped in a set of 4,248 patients with lupus and 3,818 normal healthy control subjects of European descent. Epistasis was tested by a 2-step approach using both parametric and nonparametric methods. The false discovery rate (FDR) method was used to correct for multiple testing.<h4>Results</h4>We detected and confirmed gene-gene interactions between the HLA region and CTLA4, IRF5, and ITGAM and between PDCD1 and IL21 in patients with lupus. The most significant interaction detected by parametric analysis was between rs3131379 in the HLA region and rs231775 in CTLA4 (interaction odds ratio 1.19, Z = 3.95, P = 7.8 × 10(-5) [FDR ≤0.05], P for multifactor dimensionality reduction = 5.9 × 10(-45)). Importantly, our data suggest that in patients with lupus, the presence of the HLA lupus risk alleles in rs1270942 and rs3131379 increases the odds of also carrying the lupus risk allele in IRF5 (rs2070197) by 17% and 16%, respectively (P = 0.0028 and P = 0.0047, respectively).<h4>Conclusion</h4>We provide evidence for gene-gene epistasis in systemic lupus erythematosus. These findings support a role for genetic interaction contributing to the complexity of lupus heritability.

Despite the well established central pathophysiology of Huntington's disease (HD), less is known about systemic impairments that are emerging as significant contributors to the morbidity of this neurodegenerative condition. Given the evidence of neuroendocrine dysfunction in HD patients and the pro-neural properties of sex-hormones, we explored the therapeutic potential of hormone therapy in the HD R6/1 mouse model (HD mice). HD mice over-express exon-1 of the defective human HD gene and replicate many of the clinical behavioural, biochemical and physiological impairments. Seven-week-old HD and wild-type littermate mice had either saline (control) or testosterone (treatment; 160μg/day over 90days) pellets implanted s.c. and were subsequently subjected to behavioural, molecular and cellular analysis. Separate mice were used to establish a decrease in serum testosterone concentrations in HD mice at 12weeks of age. Baseline serum testosterone was significantly reduced in control 19-week-old HD mice, whereas treatment significantly raised serum testosterone in both wild-type and HD mice. Testosterone treatment had a limited effect on the development of rotarod deficiencies in HD mice and no effect on progressive body weight loss or the development of central mutant huntingtin-containing aggregates. Testosterone treatment induced hypo-locomotion in both genotypes. Deficits in hippocampal-dependent cognition and neurogenesis were not rescued in testosterone-treated HD mice. By contrast, wild-type-treatment mice experienced significantly increased neuronal survival and differentiation. Testosterone treatment in HD mice did rescue androgen receptor levels in the hippocampus and testes, significantly improved severe testicular atrophy and restored spermatogenesis. We conclude that chronic testosterone provides systemic efficacy in treating spermatogenesis deficits and testicular atrophy but not central cellular and behavioural pathologies in R6/1 HD mice.

LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is an autosomal recessive white matter disorder with slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction. Magnetic resonance imaging shows characteristic abnormalities in the cerebral white matter and specific brain stem and spinal cord tracts. LBSL is caused by mutations in the gene DARS2, which encodes mtAspRS (mitochondrial aspartyl-tRNA synthetase). The selective involvement of specific white matter tracts in LBSL is striking since this protein is ubiquitously expressed. Almost all LBSL patients have one mutation in intron 2 of DARS2, affecting the splicing of the third exon. Using a splicing reporter construct, we find cell-type-specific differences in the sensitivity to these mutations: the mutations have a larger effect on exon 3 exclusion in neural cell lines, especially neuronal cell lines, than in non-neural cell lines. Furthermore, correct inclusion of exon 3 in the normal mtAspRS mRNA occurs less efficiently in neural cells than in other cell types, and this effect is again most pronounced in neuronal cells. The combined result of these two effects may explain the selective vulnerability of specific white matter tracts in LBSL patients.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, immune cell activation, and fibrosis of the skin and internal organs. Over the past few years, a role for genetics in the susceptibility for SSc has been established. This review aims to provide an update on the progress made in the past year or so within the field of SSc genetics research. This year has been of particular interest due to the publication of a large genome-wide association study, further investigations into gene-gene interactions, and the tendency to validate genetic results in functional models.

HD (Huntington's disease) is caused by an expanded polyQ (polyglutamine) repeat in the htt (huntingtin protein). GABAergic medium spiny neurons in the striatum are mostly affected in HD. However, mhtt (mutant huntingtin)-induced molecular changes in these neurons remain largely unknown. The present study focuses on the effect of mhtt on the subcellular localization of GAD (glutamic acid decarboxylase), the enzyme responsible for synthesizing GABA (γ-aminobutyric acid). We report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus of mhtt or full-length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal htt; however, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of HIP14 (huntingtin-interacting protein 14), the enzyme responsible for palmitoylating GAD65 in vivo, could rescue GAD65 palmitoylation and vesicle-associated GAD65 trafficking. Taken together, our data support the idea that GAD65 palmitoylation is important for the delivery of GAD65 to inhibitory synapses and suggest that impairment of GAD65 palmitoylation by mhtt may lead to altered inhibitory neurotransmission in HD.

<h4>Background</h4>The role of cholesteryl ester transfer protein (CETP) in the metabolism of high-density lipoprotein cholesterol (HDL-C) is well studied but still controversial. More recently, genome-wide association studies and meta-analyses reported the association of a promoter variant (rs3764261) with HDL-C in Caucasians and other ethnic groups. In this study, we have examined the role of genetic variation in the promoter region of CETP with HDL-C, CETP activity, coronary artery disease (CAD), CAD risk factors, and the interaction of genetic factors with environment in a unique diabetic cohort of Asian Indian Sikhs.<h4>Methods and results</h4>We genotyped four variants; three tagging single nucleotide polymorphisms from promoter (rs3764261, rs12447924, rs4783961) and one intronic variant (rs708272 Taq1B) on 2431 individuals from the Sikh Diabetes study. Two variants (rs3764261 and rs708272) exhibited a strong association with HDL-C in both normoglycemic controls (β=0.12; P=9.35×10 for rs3764261; β=0.10, P=0.002 for rs708272) and diabetic cases (β=0.07, P=0.016 for rs3764261; β=0.08, P=0.005 for rs708272) with increased levels among minor homozygous 'AA' carriers. In addition, the same 'A' allele carriers in rs3764261 showed a significant decrease in systolic blood pressure (β=-0.08, P=0.002) in normoglycemic controls. Haplotype analysis of rs3764261, rs12447924, rs4783961, and rs708272 further revealed a significant association of 'ATAA' haplotype with an increased HDL-C (β=2.71, P=6.38×10) and 'CTAG' haplotype with decreased HDL-C levels (β=-1.78, P=2.5×10). Although there was no direct association of CETP activity and CETP polymorphisms, low CETP activity was associated with an increased risk to CAD (age, BMI, and sex-adjusted odds ratio=2.2; 95% confidence interval: 1.4-3.4; P=0.001) in this study. Our data revealed a strong interaction of rs3764261 and rs708272 for affecting the association between CETP activity and HDL-C levels (P=2.2×10 and P=4.4×10, respectively).<h4>Conclusion</h4>Our results, in conjunction with earlier reports confirm low CETP activity to be associated with higher CAD risk. Although there was no direct association of CETP activity with CETP polymorphisms, our findings revealed a significant interaction between CETP variants and CETP activity for affecting HDL-C levels. These results urge a deeper evaluation of the individual genetic variation in the CETP before implementing pharmaceutical intervention of blocking CETP for preventing CAD events.

Blood pressure has a significant genetic component, but less than 3% of the observed variance has been attributed to genetic variants identified to date. Candidate gene studies of rare, monogenic hypertensive syndromes have conclusively implicated several genes altering renal sodium balance, and studies of essential hypertension have inconsistently implicated over 50 genes in pathways affecting renal sodium balance and other functions. Genome-wide linkage scans have replicated numerous quantitative trait loci throughout the genome, and over 50 single nucleotide polymorphisms (SNPs) have been replicated in multiple genome-wide association studies. These studies provide considerable evidence that epistasis and other interactions play a role in the genetic architecture of blood pressure regulation, but candidate gene studies have limited scope to test for epistasis, and genome-wide studies have low power for both main effects and interactions. This review summarizes the genetic findings to date for blood pressure, and it proposes focused, pathway-based approaches involving epistasis, gene-environment interactions, and next-generation sequencing to further the genetic dissection of blood pressure and hypertension.

The generation of functional neural progenitor cells (NPCs) independent of donor brain tissue and embryonic tissues is of great therapeutic interest with regard to regenerative medicine and the possible treatment of neurodegenerative disorders. Traditionally, NPCs are derived through the differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, the induction of NPCs from ESCs and iPSCs is a complicated process that increases the risk of neoplasia and undesired cell types. This process can be circumvented through the direct conversion of somatic cells from one cell type to another by ectopic expression of specifically defined transcription factors. Using gene expression profiling and parental cells from E/Nestin:EGFP transgenic mice as a monitoring system, we tested nine factors with the potential to directly convert fibroblasts into NPCs. We found that five of these factors can directly convert adult dermal fibroblasts into NPC-like cells (iNPCs), and the resulting iNPCs possessed similar properties as primary NPCs including proliferation, self-renewal and differentiation. Significantly, iNPCs also exhibit chemotactic properties similar to those of primary NPCs. These provide an important alternative strategy to generate iNPCs for cell replacement therapy of neurodegenerative diseases.

Hereditary haemochromatosis (HH) is a common autosomal recessive disorder of iron overload in Caucasian populations. Clinical manifestations usually occur in individuals homozygous for the C282Y mutation in the HFE gene product and who have developed significant iron loading. Current screening methods can detect affected individuals either prior to or early during disease evolution, enabling early introduction of phlebotomy treatment that can normalise life expectancy. Evaluation of possible iron overload, via measurement of serum transferrin saturation and ferritin level, is the most appropriate initial test for those subjects presenting clinically for evaluation. HFE genotyping, when combined with serum biochemical measurements, defines the presence of likely iron overload and the underlying genetic disorder and is the preferred initial screening modality for families of an affected individual. Definitive proof of iron overload requires measurement of hepatic iron concentration or total iron burden via therapeutic phlebotomy; elevated serum ferritin level alone is not adequate. We now recognise that the natural history of HH is not as discrete as previously believed, because genetic and environmental modifiers of disease penetrance are increasingly identified as influencing the clinical expression of HH. In fact, a minority of C282Y homozygotes develop classical 'iron overload disease', although it has recently emerged that the disorder may predispose to breast and colorectal cancer. Uncertainties as to the true clinical impact of the condition at a population level lead to current recommendations of cascade screening of families of affected patients, case-finding in high-risk groups, such as patients with clinical manifestations consistent with the diagnosis, and a high level of clinical awareness in the community to facilitate early diagnosis. Generalised population screening is not presently recommended.

Protons are important signals for neuronal function. In the central nervous system (CNS), proton concentrations change locally when synaptic vesicles release their acidic contents into the synaptic cleft, and globally in ischemia, seizures, traumatic brain injury, and other neurological disorders due to lactic acid accumulation. The finding that protons gate a distinct family of ion channels, the acid-sensing ion channels (ASICs), has shed new light on the mechanism of acid signaling and acidosis-associated neuronal injury. Accumulating evidence has suggested that ASICs play important roles in physiological processes such as synaptic plasticity, learning/memory, fear conditioning, and retinal integrity, and in pathological conditions such as brain ischemia, multiple sclerosis, epileptic seizures, and malignant glioma. Thus, targeting these channels may lead to novel therapeutic interventions for neurological disorders. The goal of this review is to provide an update on recent advances in our understanding of the functions of ASICs in the CNS.

Protocadherin 17 (PCDH17) is a member of the cadherin superfamily, but little is known about its functions. We focused on it as a candidate tumor suppressor gene (TSG) and hypothesized that PCDH17 may be susceptible to promoter methylation and gene silencing. Genistein has been reported to upregulate mRNA expression in many TSGs. We further tried to determine whether genistein could increase transcriptional PCDH17 by promoter demethylation. Gastric cancer cell line AGS and normal gastric cell line Ges-1 were treated with genistein and 5-aza-2'-deoxycytidine (5Aza-C). Fluorescence-activated cell sorting was performed to analyze cell cycle and apoptosis, and cell proliferation was examined by cell viability assay. PCDH17 mRNA expression in pairs of gastric cancer and normal tissue samples and cell lines were determined by quantitative real-time polymerase chain reaction. Bisulfite-modified polymerase chain reaction, cloning and sequencing were used to examine promoter methylation. We found genistein has an anti-proliferative effect on cancer cell growth through induction of cell cycle arrest. PCDH17 mRNA expression was down-regulated in cancer tissues, and PCDH17 promoter in cancer tissues was hyper-methylated in comparison with normal ones. Genistein and 5Aza-C induced PCDH17 mRNA expression in AGS, but not in Ges-1. Furthermore, genistein and 5Aza-C treatment significantly decreased promoter methylation in putative methylation target regions in AGS, reactivating PCDH17 expression. These results suggest that silencing of PCDH17 expression through promoter hypermethylation leads to loss of its tumor-suppressive activity. Genistein showed similar effects to that of 5Aza-C. Our results indicate that genistein is a novel, advantageous therapeutic agent for treating gastric cancer.

Dioscin has been shown to promote anticancer activity against several forms of cancers. However, its detailed molecular mechanisms have not been clearly clarified.In this study, we demonstrate that dioscin induces apoptosis in cancer cells through the induction of oxidative stress. Treatment with cancer cells in vitro with dioscin resulted in rapid generation of reactive oxygen species (ROS) and the induction of mitochondrial pathway apoptosis in human esophageal cancer cell line Kyse510. Inhibition of oxidative stress by the antioxidant N-acetylcysteine blocked the induction of apoptosis by dioscin, indicating that ROS generation is the primary mechanism responsible for the proapoptotic activity of dioscin. Proteomic analysis and protein gel blotting further revealed peroxiredoxins 1 and 6 (PRDX 1 and 6), which are implicated in ROS metabolism and apoptosis, were associated with the anticancer effects of dioscin. Meanwhile, overexpression of PRDX 1 and 6 significantly blocked the elevated ROS and apoptosis induced by dioscin. In conclusion, we suggest that PRDX1 and PRDX6 are key targets in the process of dioscin-induced apoptosis that involves intracellular elevated ROS.

A limitation to many polymer-based drug delivery systems is the lack of ability to customize a particular polymer composition for tailoring drug release kinetics to a specific clinical application. In this study, we investigated the structure-property effects of conjugating various hydrophobic biocompatible side chains to poly(glycerol-co-caprolactone) copolymers with the goal of achieving prolonged and controlled release of a chemotherapeutic agent. The choice of side chain significantly affected the resulting polymer properties including thermal transitions, relative crystallinity (ΔH(f)), and hydrophobicity. Drug-loaded films cast from solutions of polymer and 10-hydroxycamptothecin demonstrated prolonged release from four to over seven weeks depending upon side chain structure without initial burst release behavior. Use of the stearic acid-conjugated poly(glycerol-co-caprolactone) films afforded substantial anticancer activity in vitro for at least 50 days when exposed to fresh cultures of A549 human lung cancer cells over 24 h intervals, correlating well with the measured drug release kinetics.

Autophagy is a dynamic cellular pathway involved in the turnover of proteins, protein complexes, and organelles through lysosomal degradation. The integrity of postmitotic neurons is heavily dependent on high basal autophagy compared to non-neuronal cells as misfolded proteins and damaged organelles cannot be diluted through cell division. Moreover, neurons contain the specialized structures for intercellular communication, such as axons, dendrites and synapses, which require the reciprocal transport of proteins, organelles and autophagosomes over significant distances from the soma. Defects in autophagy affect the intercellular communication and subsequently, contributing to neurodegeneration. The presence of abnormal autophagic activity is frequently observed in selective neuronal populations afflicted in common neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. These observations have provoked controversy regarding whether the increase in autophagosomes observed in the degenerating neurons play a protective role or instead contribute to pathogenic neuronal cell death. It is still unknown what factors may determine whether active autophagy is beneficial or pathogenic during neurodegeneration. In this review, we consider both the normal and pathophysiological roles of neuronal autophagy and its potential therapeutic implications for common neurodegenerative diseases.

microRNA-122 (miR-122) was one of the first examples of a tissue-specific miRNA. It is highly expressed in liver, where it constitutes 70% of the total miRNA pool. miR-122 expression is specific to the vertebrate lineage, where the sequence of the mature miRNA is completely conserved. miR-122 is a target for extensive study due to its association with cholesterol metabolism and hepatocellular carcinoma, and its important role in promoting hepatitis C virus (HCV) replication. This review will discuss the biogenesis and function of miR-122.

<h4>Purpose</h4>To report a series of patients with Streptococcus endophthalmitis after injection with intravitreal bevacizumab prepared by the same compounding pharmacy.<h4>Design</h4>Noncomparative consecutive case series.<h4>Methods</h4>Medical records and microbiology results of patients who presented with endophthalmitis after injection with intravitreal bevacizumab between July 5 and July 8, 2011, were reviewed.<h4>Results</h4>Twelve patients were identified with endophthalmitis, presenting 1 to 6 days after receiving an intravitreal injection of bevacizumab. The injections occurred at 4 different locations in south Florida. All patients received bevacizumab prepared by the same compounding pharmacy. None of the infections originated at the Bascom Palmer Eye Institute, Miami, Florida, although 9 patients presented to its tertiary-care ophthalmic emergency room for treatment, and 3 additional patients were seen in consultation. All patients were treated initially with a vitreous tap and injection; 8 patients subsequently received a vitrectomy. Microbiology cultures for 10 patients were positive for Streptococcus mitis/oralis. Seven unused syringes of bevacizumab prepared by the compounding pharmacy at the same time as those prepared for the affected patients also were positive for S. mitis/oralis. After 4 months of follow-up, all but 1 patient had count fingers or worse visual acuity, and 3 required evisceration or enucleation. Local, state, and federal health department officials have been investigating the source of the contamination.<h4>Conclusions</h4>In this outbreak of endophthalmitis after intravitreal bevacizumab injection, Streptococcus mitis/oralis was cultured from the majority of patients and from all unused syringes. Visual outcomes were generally poor. The most likely cause of this outbreak was contamination during syringe preparation by the compounding pharmacy.

Cerebral small vessel disease (SVD) is an important cause of stroke, cognitive decline and vascular dementia (VaD). It is associated with diffuse white matter abnormalities and small deep cerebral ischemic infarcts. The molecular mechanisms involved in the development and progression of SVD are unclear. As hypertension is a major risk factor for developing SVD, Spontaneously Hypertensive Rats (SHR) are considered an appropriate experimental model for SVD. Prior work suggested an imbalance between the number of blood microvessels and astrocytes at the level of the neurovascular unit in 2-month-old SHR, leading to neuronal hypoxia in the brain of 9-month-old animals. To identify genes and pathways involved in the development of SVD, we compared the gene expression profile in the cortex of 2 and 9-month-old of SHR with age-matched normotensive Wistar Kyoto (WKY) rats using microarray-based technology. The results revealed significant differences in expression of genes involved in energy and lipid metabolisms, mitochondrial functions, oxidative stress and ischemic responses between both groups. These results strongly suggest that SHR suffer from chronic hypoxia, and therefore are unable to tolerate ischemia-like conditions, and are more vulnerable to high-energy needs than WKY. This molecular analysis gives new insights about pathways accounting for the development of SVD.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the neurotropic human polyomavirus JC (JCV) lytic infection of oligodendrocytes. PML was first described as a complication of lymphoproliferative disorders more than 50 years ago and emerged as a major complication of human immunodeficiency virus (HIV) infection in the 1980s. Despite the ubiquity of this virus, PML is rare and always seen in association with underlying immunosuppressive condition, such as HIV infection, autoimmune diseases, cancer, and organ transplantation. JCV remains quiescent in the kidneys, where it displays a stable archetypal non-coding control region (NCCR). Conversely, rearranged JCV NCCR, including tandem repeat patterns found in the brain of PML patients, have been associated with neurovirulence. The specific site and mechanism of JCV NCCR transformation is unknown. According to one model, during the course of immunosuppression, JCV departs from its latent state and after entering the brain, productively infects and destroys oligodendrocytes. Although the majority of PML cases occur in severely immunesuppressed individuals, PML has been increasingly diagnosed in patients treated with biological therapies such as monoclonal antibodies (mAbs) that modulate immune system functions: in fact, CD4+ and CD8+ T lymphopenia, resulting from this immunomodulatory therapy, are the primary risk factor. Furthermore, JCV reactivation in nonpermissive cells after treatment with mAbs, such as intestinal epithelial cells in Crohn's disease patients, in association with other host tumor-inducing factors, could provide valid information on the role of JCV in several malignancies, such as colorectal cancer.

Long-term therapy with systemic corticosteroids is not recommended in the treatment of chronic obstructive pulmonary disease (COPD). However, experience demonstrates that some patients receive low dose therapy. Our objective was to describe the demographic, physiologic and radiologic characteristics of COPD patients treated with chronic systemic corticosteroids. We analyzed COPD subjects with GOLD I-IV disease in the COPDGene® study. Subjects were divided into 2 groups based on whether they reported using chronic oral steroids or not; 1264 subjects were included. Fifty-eight (4.5%) reported chronic systemic corticosteroid use. There were no differences in age, race, co-morbid conditions (other than asthma), or body mass index between the groups. There was a greater proportion of GOLD III (41% vs. 26%) and IV (41% vs. 13%) subjects in the group using chronic systemic corticosteroids. This group used more respiratory medications, required more oxygen (2.31 ± 0.21 vs. 0.59 ± 0.05 L/min; p < 0.0001), and walked less distance (245.4 ± 17.4 vs. 367.2 ± 3.9 meters; p < 0.0001). They reported more total (1.7 ± 0.16 vs. 0.62 ± 0.03; p < 0.0001) and severe exacerbations per year (0.41 ± 0.05 vs. 0.18 ± 0.01; p < 0.0001). BODE (5.0 ± 0.3 vs. 2.6 ± 0.1; p < 0.0001), MMRC (3.31 ± 0.19 vs. 1.90 ± 0.04; p < 0.0001) and SGRQ scores (54.9 ± 2.9 vs 53.3 ± 0.6; p < 0.0001) were higher. They also had a higher percentage of emphysema (22.4 ± 1.9 vs. 14.0 ± 0.4;%, p = <0.0001) on CT scan. COPD patients that report using chronic systemic corticosteroids have more severe clinical, physiologic, and radiographic disease.

Childhood neglect has been cited as a risk factor for later substance abuse and criminal behavior. However, a large body of literature shows that a substantial percentage of neglected and abused individuals do not go on to abuse substances or engage in criminal behavior. The current study investigates whether a genetic variant (serotonin transporter linked polymorphic region [5-HTTLPR]) in the 5-hydroxytryptamine (5-HTT) gene moderates the effect of childhood neglect on alcohol use problems, marijuana use, and criminal behavior. Data from the National Longitudinal Study of Adolescent Health shows that 5-HTTLPR conditions the effect of neglect on marijuana use for females, but not for males. Findings also reveal a significant gene-environment correlation between 5-HTTLPR and neglect for females only. These results suggest that 5-HTTLPR is associated with an increased risk of neglect for females, and it also increases neglected females' risk of abusing marijuana.

The influence of five monoamine candidate genes on depressive symptom trajectories in adolescence and young adulthood were examined in the Add Health genetic sample. Results indicated that, for all respondents, carriers of the dopamine receptor D4 5-repeat allele were characterized by distinct depressive symptom trajectories across adolescence and early adulthood. Similarly, for males, individuals with the monoamine oxidase A 3.5-repeat allele exhibited unique depressive symptom trajectories. Specifically, the trajectories of those with the dopamine receptor D4 5-repeat allele were characterized by rising levels in the transition to adulthood, while their peers were experiencing a normative drop in depressive symptom frequency. Conversely, males with the monoamine oxidase A 3.5-repeat allele were shown to experience increased distress in late adolescence. An empirical method for examining a wide array of allelic combinations was employed, and false discovery rate methods were used to control the risk of false positives due to multiple testing. Special attention was given to thoroughly interrogate the robustness of the putative genetic effects. These results demonstrate the value of combining dynamic developmental perspectives with statistical genetic methods to optimize the search for genetic influences on psychopathology across the life course.