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Project description:RNAseq analyses of WT MDCKII cells
| PRJNA952242 | ENA
Project description:Stably transfected cell models are routinely used to examine drug-transporter interactions. In one such model of bcrp1-transfected MDCKII cells, we observed a significant enhancement of organic cation intracellular accumulation. Therefore, our goal was to further explore the expression and functional consequences of this cation transport system. Transport assays were carried out in wild-type and bcrp1-transfected MDCKII cells to examine uptake of [3H]-prazosin (bcrp1 positive control), [3H]-agmatine, [3H]-TEA, and [14C]-choline. RT-PCR was employed to determine the mRNA levels of bcrp1 and OCT2/OCT3. Western blots were used to evaluate corresponding protein levels. Accumulation studies determined a significant increase in the uptake of the organic cations agmatine, TEA, and choline in bcrp1-transfected cells when compared to wild-type cells. Directional transport of [3H]-agmatine showed a significantly greater apical (A) to basolateral (B) than B-to-A flux in both cell types. In spite of this, the A-to-B flux was significantly lower in bcrp1-transfected cells. RT-PCR revealed 10-fold higher OCT2 mRNA levels in bcrp1-transfected cells, with no changes in OCT3. OCT2 protein expression was approximately 3.5-fold higher in bcrp1-transfected cells. The upregulation of OCT2 in bcrp1-transfected MDCKII cells contributed to a significant enhancement in the uptake of several organic cations. These results are consistent with the endogenous expression of OCT2 in the kidney tubule, and may be related to the expression and function of bcrp1. Our findings illustrate the importance of understanding how endogenous transporters, which may compete for common substrates, may be influenced by the overexpression and enhanced function of recombinant transport systems.
| S-EPMC2814896 | biostudies-literature
Project description:In bovine mammary glands, the ABCG2 transporter actively secretes xenobiotics into dairy milk. This can have significant implications when cattle are exposed to pesticide residues in feed. Recent studies indicate that the fungicide prochloraz activates the aryl hydrocarbon receptor (AhR) pathway, increasing bovine ABCG2 (bABCG2) gene expression and efflux activity. This could enhance the accumulation of bABCG2 substrates in dairy milk, impacting pesticide risk assessment. We therefore investigated whether 13 commonly used pesticides in Europe are inducers of AhR and bABCG2 activity. MDCKII cells expressing mammary bABCG2 were incubated with pesticides for up to 72 h. To reflect an in vivo situation, applied pesticide concentrations corresponded to the maximum residue levels (MRLs) permitted in bovine fat or muscle. AhR activation was ascertained through CYP1A mRNA expression and enzyme activity, measured by qPCR and 7-ethoxyresorufin-Ο-deethylase (EROD) assay, respectively. Pesticide-mediated increase of bABCG2 efflux activity was assessed using the Hoechst 33342 accumulation assay. For all assays, the known AhR-activating pesticide prochloraz served as a positive control, while the non-activating tolclofos-methyl provided the negative control. At 10-fold MRL concentrations, chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron, and tebuconazole significantly increased CYP1A1 mRNA levels, CYP1A activity, and bABCG2 efflux activity compared to the vehicle control. In contrast, dimethoate, dimethomorph, glyphosate, iprodione, methiocarb and thiacloprid had no impact on AhR-mediated CYP1A1 mRNA levels, CYP1A activity or bABCG2 efflux. In conclusion, the MDCKII-bABCG2 cell model proved an appropriate tool for identifying AhR- and bABCG2-inducing pesticides. This provides an in vitro approach that could reduce the number of animals required in pesticide approval studies.
| S-EPMC7413513 | biostudies-literature
Project description:Bordetella pertussis regulates the production of its virulence factors by the two-component system BvgAS. In the virulence phase, BvgS phosphorylates BvgA, which then activates the transcription of virulence-activated genes (vags). In the avirulence phase, such as during growth in the presence of MgSO4, BvgA is not phosphorylated and the vags are not expressed. Instead, a set of virulence-repressed genes (vrgs) is expressed. Here, we performed transcriptome sequencing (RNAseq) analyses on B. pertussis cultivated with or without MgSO4 and on a BvgA-deficient Tohama I derivative. We observed that 146 genes were less expressed under modulating conditions or in the BvgA-deficient strain than under the nonmodulating condition, while 130 genes were more expressed. Some of the genes code for proteins with regulatory functions, suggesting a BvgA/S regulation cascade. To determine which genes are directly regulated by BvgA, we performed chromatin immunoprecipitation sequencing (ChIPseq) analyses. We identified 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Among the former, 32 are in BvgA-regulated putative promoter regions. Some vags, such as dnt and fhaL, contain no BvgA-binding site, suggesting indirect BvgA regulation. Unexpectedly, BvgA also bound to some vrg putative promoter regions. Together, these observations indicate an unrecognized complexity of BvgA/S biology.IMPORTANCEBordetella pertussis, the etiological agent of whooping cough, remains a major global health problem. Despite the global usage of whole-cell vaccines since the 1950s and of acellular vaccines in the 1990s, it still is one of the most prevalent vaccine-preventable diseases in industrialized countries. Virulence of B. pertussis is controlled by BvgA/S, a two-component system responsible for upregulation of virulence-activated genes (vags) and downregulation of virulence-repressed genes (vrgs). By transcriptome sequencing (RNAseq) analyses, we identified more than 270 vags or vrgs, and chromatin immunoprecipitation sequencing (ChIPseq) analyses revealed 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Some vags, such as dnt and fhaL, do not contain a BvgA-binding site, suggesting indirect regulation. In contrast, several vrgs and some genes not identified by RNAseq analyses under laboratory conditions contain strong BvgA-binding sites, indicating previously unappreciated complexities of BvgA/S biology.
| S-EPMC7253368 | biostudies-literature
Project description:At genome-wide level, loss of OsChz1 causes mis-regulation of thousands of genes and broad alterations of nucleosome occupancy as well as reduction of H2A.Z-enrichment within chromatin along the gene body and at TSS. While OsChz1 associates with chromatin regions enriched of repressive histone marks (H3K27me3 and H4K4me2), its loss does not affect the genome landscape of DNA methylation.
2020-10-15 | GSE155268 | GEO
Project description:BackgroundRNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming - to allow analysis of a broader range of transcripts - remains challenging.ResultsWe here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps.ConclusionT-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.
| S-EPMC10111750 | biostudies-literature
Project description:It is well understood how proteins regulate cell fate, both in normal development and disease. However, a substantial fraction of the genome is transcribed in a cell type- specific manner, producing long non-coding RNAs (lncRNA) rather than protein- coding transcripts. Here we systematically characterize transcriptional dynamics (both mRNA and lncRNA) during hematopoiesis and in hematological malignancies. We present de novo assembled transcriptome models and expression values for hematopoietic lncRNAs. We found lncRNAs to be regulated during differentiation and misregulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. With this approach, we identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the c-MYC oncogene.
2017-09-18 | GSE90059 | GEO
Project description:We found two DNA binding proteins, CRP and PepA, associate at the membrane. This study was performed to determine if their interaction affects transcription activity..
2022-10-24 | GSE203646 | GEO
Project description:ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned >50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG's). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNFα-induced protein 2 (TNFAIP2) as a major network "hub" gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF's involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNFα signaling may be helpful in ALS patients.
| S-EPMC4972368 | biostudies-literature
Project description:Estradiol Timecourse of MDA-MB-231ER+ cells containing a WT-ER and DBDmut-ER Cells were treated with 10 nM estradiol in a timecouse ranging from 1 hour to 24 hour treatments.
| S-ECPF-GEOD-22593 | biostudies-other