Project description:The aim of this study was to analyze critically the potential usefulness of selected DNA methylation biomarkers in supporting conventional histological diagnostic tests for PCa. The selection of potential biomarkers was conducted by microarray profiling of DNA methylation on prostate tissues extracted from the gland after total radical prostatectomy. DNA methylation profiles of 16 prostate samples without carcinoma and 16 matched pairs of samples with and without cancer cells isolated from prostates containing prostate carcinoma
Project description:The aim of this study was to analyze critically the potential usefulness of selected RNA biomarkers in supporting conventional histological diagnostic tests for PCa. The selection of potential biomarkers was conducted by microarray profiling of gene expression on prostate tissues extracted from the gland after total radical prostatectomy. Gene expression profiles of 16 prostate samples without carcinoma and 16 matched pairs of samples with and without cancer cells isolated from prostates containing prostate carcinoma
Project description:Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. We carried out a microarray analysis to examine global changes in gene expression in bovine theca cells in response to treatment with BMP6 alone and in combination with LH. There was a major effect of BMP6 treatment on the gene expression profile with a much weaker effect of LH. None of these differences in response to LH treatment was found to be statistically significant after applying Benjamini-Hochberg correction. BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. Theca interna cells (TC) were isolated from adult bovine ovaries obtained from the slaughterhouse. TC pooled from approximately 50 individual 4-6mm follicles were plated out in in 24-well plates (0.5 x106 viable cells/ml/well) and cultured for 6 days under defined serum-free conditions with treatments present on days 3-6 inclusive. TC were treated for 4 days with BMP6 (10 ng/ml) under both basal and LH-stimulated conditions (± 160 pg LH/ml). These dose-levels of BMP6 and LH were chosen because they elicited optimal responses in our previous studies. The experiment was replicated four times with TC harvested from different batches of ovaries. Total RNA was isolated using the Ribopure RNA isolation kit (Ambion) according to the manufacturer’s instructions. RNA yield and quality were evaluated by spectrophotometry at 260/280 nm and agarose gel electrophoresis before submitting 5 μg of each RNA sample (n = 16 comprising 4 biological replicates x 4 treatment conditions) to an accredited Affymetrix service provider for microarray analysis using the bovine genome array GeneChip (n=16 arrays).
Project description:The overall goal of these experiments was to determine how human endothelial cells respond to pathogenic Leptospira interrogans. Leptospira interrogans causes leptospirosis, the most widespread zoonotic infection in the world. A hallmark of leptospirosis is widespread endothelial damage, which in severe cases leads to hemorrhage. In these experiments, we infected two endothelial cell lines with pathogenic Leptospira interrogans serovar Canicola strain Ca12-005, and as controls, with the non-pathogenic Leptospira biflexa serovar Patoc strain Pfra. As additional controls, uninfected cells were also included in the analyses. The cell line used fhere was a microvascular endothelial line, HMEC (Ades et al, 1992. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 99:683-690); due to loss of the original analysis files, only raw data files are provided. Infection times were performed at a multiplicity of infection (# bacteria/endothelial cell) of 10 for either 1 hour or 3 hours, after which RNA was harvested and reverse transcribed. Labeled cDNAs were used to probe HEEBO arrays purchased from Microarrays Inc. (Nashville, TN). In each of three biological replicate experiments, for each time point, three comparisons were made. First, the L. interrogans-infected cells were compared to the L. biflexa-infected cells. Second, the L. Interrogans-infected cells were compared to the uninfected cells. Third, the L. biflexa-infected cells were compared to the uninfected cells. A second endothelial cell line,
Project description:Transposable elements (TEs) have been active in the mammalian genome for hundreds of millions of years and each TE has had a distinct period of transpositional activity, followed by inactivation. Mice carrying reporter transgenes can be used to model transcriptional silencing at TEs. A mutagenesis screen for modifiers of epigenetic gene silencing produced a line with a null mutation in Trim33. Heterozygous mutants displayed increased expression of the reporter transgene. ChIP-seq of Trim33 in testis revealed 9,109 peaks, mostly at promoters, across the mouse genome. Trim33 was enriched at many RLTR10B elements that are among the youngest retrotransposons in the mouse genome. RNA-seq revealed that testis of mice haploinsufficient for Trim33 had altered expression of a small group of genes. The gene with the most significant increase, Nmnat3, was found to be transcribed from an upstream RLTR10B element. We show that Trim33 is involved in repressing RLTR10B elements in mouse testis. Examination of Trim33 binding using ChIP sequencing and the result of Trim33 haploinsufficiency using RNA sequencing, in mouse testis
Project description:PBDEs are widely used in consumer and household products as flame retardants. Many studies have shown that PBDEs could disrupt thyroid hormone homeostasis and adversely affect brain development. Here, we explored the toxical effects of BDE209 on HEK 293 cells and found that BDE209 may have a role in nucleosome remodeling. Many gene sets involved in cancer are enriched at the BDE209-treated sample. This indicates the carcinogenicity of BDE209. Interestingly, the impacts of BDE209 dissoved in DMSO on gene expression are more pronounced than the simple additive effects of BDE209 and DMSO alone. Gene expression profiles of human embryonic kidney 293 cells (HEK 293) cultured in normal medium, and medium containing BDE209 or DMSO were generated by deep sequencing, using Illumina HighSeq2000, respectively..
Project description:Amyotrophic Lateral Sclerosis (ALS) is generally a late onset neurodegenerative disease. Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene accounts for approximately 20% of familial ALS and 2% of all ALS cases. Although a number of hypothesis have been proposed to explain mutant SOD1 toxicity, the molecular mechanisms of the disease remain unclear. SOD1 linked ALS is thought to function in a non-cell autonomous manner such that the motoneurons are critical for the onset and glia contribute to the progress of the disease. To dissect the roles of motoneurons and glia, we used the Gal4-UAS system to determine gene expression changes following the expression of mutant human SOD1 (G85R) selectively in either motoneurons or glia, and concurrently in motoneurons and glia of flies. We conducted a microarray on young (5 days old) and old (45 days old) flies expressing G85R in these cell types and identified a number of genes involved in a variety of processes. The candidate genes identified by this screen may help elucidate the individual and combined contributions of motoneurons and glial cells in ALS. We used microarrays to evaluate the transcriptional profile of 5 day old and 45 day old flies expressing mutant human SOD1 (G85R) in a tissue specific manner in motoneurons, glia, and together in motoneurons and glia and compared the expression to flies expressing wild-type drosophila SOD1 controls. The Gal4-UAS system was used to drive tissue expression of either mutant human SOD1 (G85R) or wild-type drosophila SOD1 (dSOD1) in flies. Flies containing either the motoneuronal driver, D42-Gal4, the glial driver, M1B-Gal4, or the combined motoneuronal and glial drivers, D42+M1B-Gal4 were crossed to flies containing either mutant human SOD1, UAS-G85R, or wild-type drosophila SOD1, UAS-dSOD1, as a control. Adult male progeny were collected within 24 hours after eclosion and aged to 5 (5d) and 45 (45d) days old. Groups of 10 flies were maintained in vials of cornmeal agar food and transferred to fresh food every 5-7 days. For each Gal4-UAS line and each age, 3 biological replicates consisting of 40 whole flies were flash frozen in liquid nitrogen and used to isolate total RNA, for a total of 36 samples.
Project description:BACKGROUND: We analysed critically the potential usefulness of RNA- and DNA-based biomarkers in supporting conventional histological diagnostic tests for prostate carcinoma (PCa) detection. METHODS: Microarray profiling of gene expression and DNA methylation was performed on 16 benign prostatic hyperplasia (BPH) and 32 cancerous and non-cancerous prostate samples extracted by radical prostatectomy. The predictive value of the selected biomarkers was validated by qPCR-based methods using tissue samples extracted from the 58 prostates and, separately, using 227 prostate core biopsies. RESULTS: HOXC6, AMACR and PCA3 expression showed the best discrimination between PCa and BPH. All three genes were previously reported as the most promising mRNA-based markers for distinguishing cancerous lesions from benign prostate lesions; however, none were sufficiently sensitive and specific to meet the criteria for a PCa diagnostic biomarker. By contrast, DNA methylation levels of the APC, TACC2, RARB, DGKZ and HES5 promoter regions achieved high discriminating sensitivity and specificity, with area under the curve (AUCs) reaching 0.95-1.0. Only a small overlap was detected between the DNA methylation levels of PCa-positive and PCa-negative needle biopsies, with AUCs ranging between 0.854 and 0.899. CONCLUSIONS: DNA methylation-based biomarkers reflect the prostate malignancy and might be useful in supporting clinical decisions for suspected PCa following an initial negative prostate biopsy.
Project description:In Xenopus laevis, a number of studies identified vegetal factors that specify the germ line, endoderm and dorsal axis, but there are few studies demonstrating roles for animal-enriched maternal mRNAs. Therefore, we carried out a microarray analysis to identify novel maternal transcripts enriched in animal blastomeres. We sought to maximize differences between animal and vegetal samples. To that end, we dissected 8-cell embryos into animal blastomeres and vegetal blastomeres, and further dissected the vegetal blastomeres into vegetal-most halves (VP) and equatorial regions (discarded).