ABSTRACT: To discover the fission yeast prolyl hydroyxlome. Five biological replicates of WT and Ofd1D cells were cultured. The whole cell lysates were analysed by TMT mass spectrometry for prolyl hydroxylation
Project description:We treated Jurkat cells for 48 hr with a sublethal dose of FK866 (5 nM) and DMSO (Mock, control treatment). RNA samples for microarrays derived from fractionated samples by sucrose gradient (sub-polysomes, polysomes), giving us the chance to perform an analysis among polysomal/subpolysomal distribution in treated or untreated cells and the possibility to identify the multi-level gene expression regulation effects of FK866. We are interested to find differentially expressed genes, in the early phase of cell response to FK866, and genes that account for a specific post-transcriptional regulation exerted by the cell in response to the drug. Keywords: polysomal profiling, translatome profiling, polysomal RNA, subpolysomal RNA, translational profiling, polysome profiling, post-transcriptional regulation, FK866, translational efficiency. Gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from total RNAs (derived from the sum of all the fractions in the polysomal gradient). Polysomal RNA, subpolysomal RNA and total RNA were isolated from Jurkat cells treated with FK866 5 nM or DMSO (mock, control treatment) for 48 hr. Cells lysates were collected from control cells (mock) and from treated cells (FK866). All experiments were run in biological quadruplicates.
Project description:Heligmosomoides polygyrus is a natural intestinal parasite of mice which exerts wide ranging modulatory effects on the immune system. This experiment was designed to investigate its abillity to modify intestinal epithelial cells, which form part of its natural niche. We tested gene expression in vitro, in differentiating organoids of small intestinal origin, exposed to cytokines and the released products of the parasite, termed HpES.
Project description:We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover. s4U metabolic labeling of RNA in 293T cells, followed by biochemical enrichment of labeled RNA with two biotinylation reagents, RNAs >200nt and miRNAs in separate experiments
Project description:Genome-wide translational profiling of rng3-65 compared to wild type cells. We used sucrose gradients to separate RNAs according to the number of associated ribosomes (a surrogate for translational efficiency). Preparation of the extracts and fractionation was carried out as described in Lackner et al, 2007 (Mol Cell 26(1):145-55). The fractions were pooled into four groups (1 closest to the top, i.e. not associated with ribosomes and 4 closest to the bottom, i.e., associated with polysomes). RNA was extracted from the pools and the corresponding pools from wild type and mutant cells were directly compared using DNA microarrays. Changes in translation are expected to alter the number of ribosomes associated with specific transcripts, and therefore result in a redistribution of the RNAs across the different fractions.
Project description:Current human reproductive risk assessment methods rely on semen and serum hormone analyses, which are not easily comparable to the histopathological endpoints and mating studies used in animal testing. Because of these limitations, there is a need to develop universal evaluations that reliably reflect male reproductive function. We hypothesized that toxicant-induced testicular injury can be detected in sperm using mRNA transcripts as indicators of insult. To test this, we exposed adult male Fischer 344 rats to low doses of model testicular toxicants and classically characterized the testicular injury while simultaneously evaluating sperm mRNA transcripts from the same animals. Overall, this study aimed to: 1) identify sperm transcripts altered after exposure to the model testicular toxicant, 2,5-hexanedione (HD) using microarrays; 2) expand on the HD-induced transcript changes in a comprehensive time course experiment using qRT-PCR arrays; and 3) test these injury indicators after exposure to another model testicular toxicant, carbendazim (CBZ). Microarray analysis of HD-treated adult Fischer 344 rats identified 128 altered sperm mRNA transcripts when compared to control using linear models of microarray analysis (q < 0.05). All transcript alterations disappeared after 3 months of post-exposure recovery. In the time course experiment, time-dependent alterations were observed for 12 candidate transcripts selected from the microarray data based upon fold change and biological relevance, and 8 of these transcripts remained significantly altered after the 3-month recovery period (p < 0.05). In the last experiment, 8 candidate transcripts changed after exposure to CBZ (p < 0.05). The two testicular toxicants produced distinct molecular signatures with only 4 overlapping transcripts between them, each occurring in opposite directions. Overall, these results suggest that sperm mRNA transcripts are indicators of low dose toxicant-induced testicular injury in the rat. Rats were exposed to sub-chronic low doses of the Sertoli cell toxicant 2,5-hexanedione (HD) or water (control for HD) for 3 months. Some rats in each group underwent 3 months of post-exposure recovery.
Project description:Intestinal epithelial cells (IECs) were isolated from the colon of Villin-CreERT2, Rnf20-flox and Rnf40-flox mice two weeks upon the Tamoxifen-induced, intestinal knockout of Rnf20 and Rnf40. ChIP-seq for H3K4me3 was performed using snap-frozen IECs.
Project description:Intestinal epithelial cells (IECs) were isolated from the colon of Villin-CreERT2, Rnf20-flox and Rnf40-flox mice two weeks upon the Tamoxifen-induced, intestinal knockout of Rnf20 and Rnf40. RNA was isolated from snap-frozen IECs to perform mRNA-seq.
Project description:Background: Hypoxia is a potent molecular signal for cellular metabolism, mitochondrial function, and migration. Conditions of low oxygen tension trigger regulatory cascades mediated via the highly conserved HIF-1 α post-translational modification system. In the adaptive immune response, B cells (Bc) are activated and differentiate under hypoxic conditions within lymph node germinal centers, and subsequently migrate to other compartments. During migration, they traverse through changing oxygen levels, ranging from 1-5% in the lymph node to 5-13% in the peripheral blood. Interestingly, the calcineurin inhibitor cyclosporine A is known to stimulate prolyl hydroxylase activity, resulting in HIF-1 α destabilization and may alter Bc responses directly. Over 60% of patients taking calcineurin immunosuppressant medications have hypo-gammaglobulinemia and poor vaccine responses, putting them at high risk of infection with significantly increased morbidity and mortality. Results: We demonstrate that oxygen tension is a previously unrecognized Bc regulatory switch, altering CXCR4 and CXCR5 chemokine receptor signaling in activated Bc through HIF-1 α expression, and controlling critical aspects of Bc migration. Our data demonstrate that calcineurin inhibition hinders this oxygen regulatory switch in primary human Bc. Conclusion: This previously unrecognized effect of calcineurin inhibition directly on human Bc has significant and direct clinical implications.
Project description:Case-control study for the analysis of the gene expression profile of epithelial cells microdissected from normal breast tissues obtained from 17 parous and 7 nulliparous women free of breast pathology (controls), and 39 parous and 8 nulliparous women with history of breast cancer (cases). Keywords: genetic modifications Four-condition experiment: nulliparous case, nulliparous control, parous case and parous control labeled with Cy5 and Universal human reference used as a common reference labeled with Cy3. Moderated t statistic was used as the basic statistic for significance analysis; it was computed for each probe and for each contrast. False discovery rate was controlled using the Benjamini and Hochberg. All genes with P value below a threshold of 0.05 were selected as differentially expressed, maintaining the proportion of false discoveries in the selected group below the threshold value, in this case 5%. Breast 11 parous control HuII, Breast 28 parous case HuII, and Breast 62 nulliparous control HuIII excluded: raw data is missing
Project description:Here we quantitatively describe the influence of cell growth rate and amino acid metabolic context on gene expression in the eukaryal model organism Saccharomyces cerevisiae. We show that growth rate and metabolic cues regulate ~70% of the yeast transcriptome and proteome, thereby exerting gene expression control in a global manner. We find that the growth rate-dependent differential gene expression largely reflects changing availabilities of the mRNA and protein synthesis machineries, while metabolic cues influences gene expression through the availabilities of amino acids and nucleotides. Genes in central carbon metabolism, however, are regulated independently of these global physiological controls, demonstrating distinct mechanisms to control their expression levels.