Circadian Gene Circuitry Predicts Hyperactive Behavior in a Mood Disorder Mouse Model

SummaryBipolar disorder, also known as manic-depressive illness, causes swings in mood and activity levels at irregular intervals. Such changes are difficult to predict, and their molecular basis remains unknown. Here, we use infradian (longer than a day) cyclic activity levels in αCaMKII (Camk2a) mutant mice as a proxy for such mood-associated changes. We report that gene-expression patterns in the hippocampal dentate gyrus could retrospectively predict whether the mice were in a state of high or low locomotor activity (LA). Expression of a subset of circadian genes, as well as levels of cAMP and pCREB, possible upstream regulators of circadian genes, were correlated with LA states, suggesting that the intrinsic molecular circuitry changes concomitant with infradian oscillatory LA. Taken together, these findings shed light onto the molecular basis of how irregular biological rhythms and behavior are controlled by the brain

Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line

BACKGROUND: Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line. METHODS: Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed. RESULTS: In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours. CONCLUSION: We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription. These effects together suggest that Wee1 inhibitor may thus be a potentially useful anti-cancer therapy

A community intervention trial of multimodal suicide prevention program in Japan: A Novel multimodal Community Intervention program to prevent suicide and suicide attempt in Japan, NOCOMIT-J

<p>Abstract</p> <p>Background</p> <p>To respond to the rapid surge in the incidence of suicide in Japan, which appears to be an ongoing trend, the Japanese Multimodal Intervention Trials for Suicide Prevention (J-MISP) have launched a multimodal community-based suicide prevention program, NOCOMIT-J. The primary aim of this study is to examine whether NOCOMIT-J is effective in reducing suicidal behavior in the community.</p> <p>Methods/DesignThis study is a community intervention trial involving seven intervention regions with accompanying control regions, all with populations of statistically sufficient size. The program focuses on building social support networks in the public health system for suicide prevention and mental health promotion, intending to reinforce human relationships in the community. The intervention program components includes a primary prevention measures of awareness campaign for the public and key personnel, secondary prevention measures for screening of, and assisting, high-risk individuals, after-care for individuals bereaved by suicide, and other measures. The intervention started in July 2006, and will continue for 3.5 years. Participants are Japanese and foreign residents living in the intervention and control regions (a total of population of 2,120,000 individuals).</p> <p>Discussion</p> <p>The present study is designed to evaluate the effectiveness of the community-based suicide prevention program in the seven participating areas.</p> <p>Trial registration</p> <p>UMIN Clinical Trials Registry (UMIN-CTR) UMIN000000460.</p

Variants of C-C Motif Chemokine 22 (CCL22) Are Associated with Susceptibility to Atopic Dermatitis: Case-Control Studies

Atopic dermatitis (AD) is a common inflammatory skin disease caused by multiple genetic and environmental factors. AD is characterized by the local infiltration of T helper type 2 (Th2) cells. Recent clinical studies have shown important roles of the Th2 chemokines, CCL22 and CCL17 in the pathogenesis of AD. To investigate whether polymorphisms of the CCL22 gene affect the susceptibility to AD, we conducted association studies and functional studies of the related variants. We first resequenced the CCL22 gene and found a total of 39 SNPs. We selected seven tag SNPs in the CCL22 gene, and conducted association studies using two independent Japanese populations (1st population, 916 cases and 1,032 controls; 2nd population 1,034 cases and 1,004 controls). After the association results were combined by inverse variance method, we observed a significant association at rs4359426 (meta-analysis, combined P = 9.6×10−6; OR, 0.74; 95% CI, 0.65–0.85). Functional analysis revealed that the risk allele of rs4359426 contributed to higher expression levels of CCL22 mRNA. We further examined the allelic differences in the binding of nuclear proteins by electrophoretic mobility shift assay. The signal intensity of the DNA-protein complex derived from the G allele of rs223821, which was in absolute LD with rs4359426, was higher than that from the A allele. Although further functional analyses are needed, it is likely that related variants play a role in susceptibility to AD in a gain-of-function manner. Our findings provide a new insight into the etiology and pathogenesis of AD

Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling

PURPOSE: Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC) development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif. METHODS: We investigated the TCF-4J and K isoform pair characterized by the presence (K) or absence (J) of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells) and K (K cells) to grow as solid tumors in nude mice was explored. RESULTS: TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells) revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells). The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL) protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors. CONCLUSIONS: Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions

A prospective compound screening contest identified broader inhibitors for Sirtuin 1

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified

Augmentation of Reverse Transcription by Integrase through an Interaction with Host Factor, SIP1/Gemin2 Is Critical for HIV-1 Infection

There has been accumulating evidence for the involvement of retroviral integrase (IN) in the reverse transcription of viral RNA. We previously identified a host factor, survival motor neuron-interacting protein 1 (SIP1/Gemin2) that binds to human immunodeficiency virus type 1 (HIV-1) IN and supports HIV-1 infection apparently at reverse transcription step. Here, we demonstrated that HIV-1 IN together with SIP1 augments reverse transcriptase (RT) activity by enhancing the assembly of RT on viral RNA in vitro. Synthetic peptides corresponding to the binding motifs within IN that inhibited the IN-SIP1 interaction abrogated reverse transcription in vitro and in vivo. Furthermore, knockdown of SIP1 reduced intracellular stability and multimer formation of IN through proteasome-mediated degradation machinery. Taken together, SIP1 appears to stabilize functional multimer forms of IN, thereby promoting the assembly of IN and RT on viral RNA to allow efficient reverse transcription, which is a prerequisite for efficient HIV-1 infection

Lack of association between the CARD10 rs6000782 polymorphism and type 1 autoimmune hepatitis in a Japanese population

Background: Previous genome-wide association studies have evaluated the impact of common genetic variants and identified several non-HLA risk loci associated with autoimmune liver diseases. More recent genome-wide association studies and replication analyses reported an association between variants of the CARD10 polymorphism rs6000782 and risk of type 1 autoimmune hepatitis (AIH). In this case-control study, we genotyped 326 Japanese AIH patients and 214 control subjects. Results: Genomic DNA from 540 individuals of Japanese origin, including 326 patients with type-1 AIH and 214 healthy controls, was analyzed for two single nucleotide polymorphisms (SNPs) in the CARD10 gene. We selected CARD10 rs6000782 SNPs and genotyped these using PCR-RFLP method and direct sequencing. The Chi square test revealed that the rs6000782 variant alle (c) was not associated with the susceptibility for AIH in a Japanese population [p = 0.376, odds ratio (OR) 1.271, 95 % confidence interval (CI) 0.747-2.161] in an allele model. Our data also showed that CARD10 rs6000782 variants were not associated with AIH or with the clinical parameters of AIH. Conclusions: In this study we examined an association between rs6000782 SNPs in the CARD10 gene and type-1 AIH. Results showed no significant association of rs62000782 with type-1 AIH in a Japanese population. This study demonstrated no association between CARD10 rs6000782 variants and AIH in a Japanese population

Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation