ABSTRACT: We deep sequenced chromatin-associated RNAs (CARs) from human fibroblast (HF) cells. This resulted in the identification of 141 intronic regions and 74 intergenic regions harbouring CARS. Overall design: We purified CARs from normal HFs by isolating soluble chromatin after MNase treatment, followed by separation of chromatin fragments of different lengths on a sucrose gradient. CARs were converted into double-stranded cDNAs and sequenced using the Illumina Genome Analyzer I.
Project description:We deep sequenced chromatin-associated RNAs (CARs) from human fibroblast (HF) cells. This resulted in the identification of 141 intronic regions and 74 intergenic regions harbouring CARS. We purified CARs from normal HFs by isolating soluble chromatin after MNase treatment, followed by separation of chromatin fragments of different lengths on a sucrose gradient. CARs were converted into double-stranded cDNAs and sequenced using the Illumina Genome Analyzer I.
Project description:Human T cells isolated from healthy donors were transduced with non-tonically signaling CARs or tonically signaling CARs, each with CD28z or 4-1BB costimulatory domains Human T cells isolated from healthy donors were transduced with non-tonically signaling CARs or tonically signaling CARs, each with CD28z or 4-1BB costimulatory domains
Project description:In the fungi Fusarium fujikuroi and Fusarium oxysporum, the induction of carotenogenesis by light and its deregulation in carS mutants, affected in a protein of the RING-finger family, are achieved on transcription of the structural genes of the pathway, some of them organized in a cluster. We have carried out global RNAseq transcriptomics analyses to investigate the relationship between the regulatory effects of light and the carS mutation. Either illumination or the absence of a functional carS gene exert wide effects on the transcriptome of F. fujikuroi, with a predominance of activated over repressed genes, and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in the carS mutant, indicating that the deregulation produced by the carS mutation affects the light response of many genes. In addition, light and CarS strongly influence the expression of some genes associated with stress responses. The effects of light and carS mutation on the F. oxysporum transcriptome were partially coincident with those in F. fujikuroi, indicating conservation of the objectives of their regulatory mechanisms. In conclusion, the CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in the wild-type strains of the two Fusarium species investigated, indicating regulatory connections between the control by light and by the CarS protein. Overall design: To check the effect of light in the F. fujikuroi and F. oxysporum transcriptomes we compared dark-grown cultures with parallel cultures exposed for one hour to light. To examine the effect of the carS mutation, in F. fujikuroi we used a formerly investigated carS mutant (SG39), and as an additional control, we used SG256, a stable carS complementing strain (SG256) obtained from SG39 by replacement of the carS mutant allele by a wild type allele. In F. oxysporum, the effect of the carS mutation was inferred from the analysis of two independent carS mutants, SX2 and SX3. Each strain and condition has two independent experimental replicates.
Project description:To define chromatin structure changes along the yeast genome using microarrays. Nucleosomal DNA from WT and delta isw2 yeast were hybridized and differences in signals were calculated. Keywords: Nucleosomal DNA hybridization Overall design: Nucleosomal DNA was harvested from WT and delta isw2 yeast (2 biological replicates of each) then fragmented into ~50bp lengths and then hybridized onto the tilling microarrays. WT signal was the compared to the mutant signal to find regions of chromatin structure change.
Project description:In vivo high frequency stimulated of left dentate gyrus was performed on anaesthetised rats followed by RNA-seq to study long-term potentiation. Both left and right dentate gyrus was collected, sequenced and compared against each other for naive rats and for rats 30 min, 2 hours, and 5 hours post-HFS.
Project description:Chronic high-fat/high-sugar (HFS) feeding is linked to the development of insulin resistance as well as arterial wall and adipose tissue inflammation in nonhuman primates. These changes are significantly reduced when the animals are fed a HFS diet supplemented with resveratrol (RSV) for two years. Herein, we evaluated the occurrence of cerebral cortex injury in these HFS-fed middle-aged male rhesus monkeys and investigated the possibility of brain protection by RSV treatment. HFS caused a reduction in capillary density in the cerebral cortex that was preempted by RSV supplementation. The patterns of cDNA microarray analysis revealed upregulation of markers of oxidative stress, inflammation and apoptosis in the cortical cortex of HFS-fed monkeys, which was reversed by RSV. The underlying mechanism of RSV action included its ability to prevent the HFS-mediated NF-kappa-B activation and loss in mitochondrial aldehyde dehydrogenase 2 expression. We conclude that RSV may confer neuroprotection against HFS-mediated cerebral vascular dysfunction and activation of inflammatory pathways. Overall design: Twenty-four adult (7-13 years old) male rhesus monkeys (Macaca mulatta) were maintained on standard NIH monkey chow (Purina Mills) during baseline assessment, and then quasi-randomized (such that average age and bodyweight did not differ significantly between groups) to one of three two-year dietary regimens: Four animals were fed a healthy standard diet (SD) with 13% Kcal in fat and less than 5% sucrose by weight, ten animals were fed a high fat and sugar (HFS) diet with 42% Kcal in fat and 27% sucrose by weight (Harlan, Teklad), and another ten animals (RESV) were fed the same HFS diet plus a flavored primate treat (Bioserve) containing 40 mg and 240 mg resveratrol (RSV) twice daily, during year 1 and 2, respectively. Monkeys in non-RSV groups received placebo treats.
Project description:This study addresses the molecular mechanisms underlying the action of subthalamic nucleus high frequency stimulation (STN-HFS) in the treatment of Parkinson’s disease and its interaction with levoDOPA (L-DOPA), focusing on the striatum. The objectives were 1) to identify the molecular signature of STN-HFS action at striatal level, associated with its efficient antiparkinsonian action, and 2) to investigate the molecular substrates of the interaction between the two treatments in order to evidence possible genes involved in dyskinesia. Striatal gene expression profile was assessed in rats with nigral DOPAmine neuron lesion, either treated or not, using agilent microarrays and qPCR verification. The treatments consisted in anti-akinetic STN-HFS (5 days), chronic L-DOPA treatment inducing dyskinesia (LIDs) or the combination of the two treatments that exacerbated LIDs. STN-HFS modulated 71 genes with functional or biochemical annotation, including genes sharing the GO terms regulation of growth, regulation of apoptosis, extracellular region. Ttr, Igf2, Sostdc1 and Nr4A3 (Nor-1), are among the 5 genes showing the highest specific upregulation. Down-regulated genes include Prkcd, Sirt5 and Bbc3. These results show that genes involved in neuroprotection and/or neurogenesis are key components of STN-HFS action in the striatum. STN-HFS and LDOPA treatment share very few common gene regulation features suggesting that the molecular substrates underlying their striatal action are mostly different. In addition to genes already reported to be associated with LIDs (Pdyn, Trh, Grm4/mGlu4, Cnr1/CB1), the comparison between DOPA and DOPA/STN-HFS identifies immunity-related genes: C1s, Rt1-Da and Irf7a, as potential players in L-DOPA side effects. Total RNA was extracted from striatal tissue from four groups of 3 animals bearing 6-hydroxyDOPAmine (6-OHDA)-induced lesion of the nigrostriatal DA pathway: lesion alone without any subsequent treatment (L), L-DOPA treatment for 19 days (D), STN-HFS for 5 days (S) and combination of L-DOPA and STN-HFS (DS).
Project description:In the yeast genome, a large proportion of nucleosomes occupy well-defined positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genomic context remains controversial. Through genome-wide, quantitative analysis of nucleosome positioning and high-resolution mutagenenesis of mononucleosomal DNA, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes. This effect is equally detected in transcribed and non-transcribed regions, suggesting the existence of sequence elements contributing to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open up the possibility of designing coding and non-coding DNA molecules capable of directing their own nucleosomal organization. Overall design: Samples from mononucleosomal DNA from S. pombe strains (h- 972) and S. cerevisiae (W303) harbouring different mutant sequences, were sequenced (Illumina NextSeq 500 platform) using the pair-end read protocol
Project description:Investigation of 5' transcripts using strand specific sequencing for T. maritima under logphase, maltose minimal media growth conditions Two replicate samples were sequenced after isolating total RNA of cultures grown in maltose minimal media. Cells were harvested in logphase growth.
Project description:Investigation of 5' transcripts using strand specific sequencing for T. maritima under logphase, maltose minimal media growth conditions Overall design: Two replicate samples were sequenced after isolating total RNA of cultures grown in maltose minimal media. Cells were harvested in logphase growth.