ABSTRACT: Comparison of whole cell lystates of swimming (motile) and adherent (stationary) Crithidia fasciculata. Cf-C1 strain parasites grown in standard Brain-Heart Infusion medium supplemented with Hemin and cultured at 28 ºC.
Project description:In cell senescence, cultured cells cease proliferating and acquire aberrant gene expression patterns. MicroRNAs (miRNAs) modulate gene expression through translational repression or mRNA degradation, and have been implicated in senescence. We have used deep sequencing to carry out a comprehensive survey of miRNA expression and its involvement in cell senescence. Informatic analysis of small RNA sequence datasets from young and senescent IMR90 human fibroblasts identifies many known miRNAs, and a small number of novel miRNAs, that are regulated (either up or down) with cell senescence. Comparison with mRNA expression profiles revealed potential mRNA targets of the senescence-regulated miRNAs. The target mRNAs are enriched for genes involved in biological processes associated with cell senescence. This result greatly extends existing information on the role of miRNAs in cell senescence, and is consistent with miRNAs having a causal role in the process. Comprehensive survey of miRNA from young and senescent IMR90 fibroblasts using deep sequencing
Project description:Cytotoxic CD8+ T cells can effectively kill target cells by producing cytokines, chemokines and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and pre-select potent killer cells are lacking. Human CD8+ T cells can be divided into IFNGand IL-2 producing cells. Unbiased RNA-sequencing and proteomics analysis on cytokine-producing fixed CD8+ T cells revealed that IL-2+ T cells produce helper cytokines, and that IFNG+ T cells produce cytotoxic molecules. IFNG+ cytotoxic T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, the cytotoxic features of CD29+ T cells were maintained during cell culture, suggesting a stable phenotype. Pre-selecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that selecting for CD29+ T cells could boost the anti-tumoral activity of T cell therapeutics.
Project description:To identify proteins interacting with Merlin in response to calcium signaling, Flag-tagged Merlin was stably transduced into LN229 cells. These cells and empty vector-transduced LN229 cells were treated with DMSO or thapsigargin for 15 minutes and subjected to immunoprecipitation followed by Mass Spectrometry.
Project description:Analysis of the ovarian cancer cell line OVCAR-5. A standard trypsin digest was carried out on the OVCAR-5 cell lysates which were then analysed in the un-fractionated and fractionated forms. Fractionation was completed using a peptide IEF separation method. All samples were analysed by nano-LC-ESI-MS/MS using a QTOF.
Project description:The earliest stages of Huntington’s disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin protein (HTT). To explore the hypothesis DNA methylation may be altered in cells expressing mutated HTT, we use reduced-representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. Based on the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and at later stages, cognitive decline in Huntington’s patients. mRNA-seq in STHdhQ7/Q7 and STHdhQ111/Q111 cells
Project description:A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodellling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodellling complex, which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodelller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodelller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed.
Project description:Background & Aims: MicroRNAs have been shown to offer great potential in the diagnosis of cancer. We aimed to identify microRNAs in peripheral blood mononuclear cells (PBMCs) for diagnosing pancreatic cancer (PC). Methods: PBMCs microRNA expression was investigated in three independent cohorts including 352 participants (healthy, benign pancreatic/peripancreatic diseases (BPD), and PC). First, we used sequencing technology to identify differentially expressed microRNAs in 60 PBMCs samples for diagnosing PC. Quantitative reverse-transcriptase polymerase chain reaction assay was then applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed using an independent cohort. Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. Results: We found that PBMCs miR-27a-3p could efficiently discriminate PC from BPD (AUC=0.840; 95% CI, 0.787 to 0.885; sensitivity=82.2%, specificity=76.7%). A panel composed of PBMCs miR-27a-3p and serum CA19-9 provided a high diagnostic accuracy in differentiating PC from BPD in the clinical setting (AUC=0.886; 95% CI, 0.837 to 0.923; sensitivity=85.3%, specificity=81.6%). The satisfactory diagnostic performance of the panel persisted regardless of disease status (AUCs for tumour-node-metastasis stages?,?, and ? were 0.881, 0.884, and 0.893, respectively). Conclusion: PBMCs miR-27a-3p could be a potential marker for PC screening. A panel composed of PBMCs miR-27a-3p and serum CA19-9 has considerable clinical value in diagnosing early-stage PC. Therefore, patients who would have otherwise missed the curative treatment window can benefit from optimal therapy. Examination of different MicroRNA profiles in 3 types of PBMCs samples
Project description:The yeast cyclic-AMP dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the catalytic subunit, tpk1, reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ regulator (nej1), co-function in the same pathway. Chromatin immunoprecipitation and resection data suggest that the recruitment of repair proteins and DNA resection is influenced by tpk1 deletion. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair, in addition to nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (the human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.
Project description:The yeast cyclic-AMP dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the catalytic subunit, tpk1, reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ regulator (nej1), co-function in the same pathway. Chromatin immunoprecipitation and resection data suggest that the recruitment of repair proteins and DNA resection is influenced by tpk1 deletion. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair, in addition to nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (the human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.
Project description:DYT-TOR1A dystonia is a movement disorder characterized by involuntary muscle contractions. Despite being the most common monogenetic form of dystonia, its pathophysiolofy remains unclear. With a reduced penetrance of about 30%, there is a suggestion that extragenetic factors are needed to develeop a dystonic phenotype. In the present study, we induced a sciatic nerve crush injury in a genetically predisposed DYT-TOR1A mouse model (DYT1KI) to evoke a dystonic phenotype. Subsequently, we employed a multi-omic approach to uncover novel pathophysiological pathways associated with DYT-TOR1A dystonia. Utilizing a deep-learning-based characterization of the dystonic phenotype, we observed that nerve-injured DYT1KI animals exhibited significantly more dystonia-like movement (DLM) compared to naive DYT1KI animals, with noticeable effects as early as two weeks post-surgery. Moreover, nerve-injured DT1KI mice displayed significantly more DLM than their wildtype (wt) counterparts starting 6 weeks post-injury. In the cerebellum of nerve-injured wt mice, multi-omice analysis indicated regulatory changes in translation-related processes, a phenomenon not observed in the cerebellum of nerve-injured DYT1KI mice; instead, these changes were localized to the cortex and striatum. Our findings suggest a failure of translational compensatory mechanisms in the cerebellum of phenotypic DY1KI mice displaying DLM, while dysregulations in translation in the cortex and striatum likely contribute to the promotion of the dystonic phenotype.