Project description:MicroRNAs are important negative regulators of protein coding gene expression, and have been studied intensively over the last few years. To this purpose, different measurement platforms to determine their RNA abundance levels in biological samples have been developed. In this study, we have systematically compared 12 commercially available microRNA expression platforms by measuring an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples, and synthetic spikes from homologous microRNA family members. We developed novel quality metrics in order to objectively assess platform performance of very different technologies such as small RNA sequencing, RT-qPCR and (microarray) hybridization. We assessed reproducibility, sensitivity, quantitative performance, and specificity. The results indicate that each method has its strengths and weaknesses, which helps guiding informed selection of a quantitative microRNA gene expression platform in function of particular study goals.
Project description:The human dataset includes the gene expression profile of CD4+ T cells isolated from blood of healthy controls and plated on TCP in RPMI-1640 containing 10% FCS, Penicillin-Streptomycin (50,000 units-50 mg) and L-glutamine (2 mM). Cells were stimulated for 4 days with 20 ng/ml of IL-1beta, 100 IU/ml of IL-2, 20 ng/ml of IL-6, 20 ng/ml IL-23 plus anti-CD2/3/28 beads at a ratio of 1 bead per 10 cells. RNA samples were isolated using the RNeasy Mini Kit (Qiagen) with on-column DNA digestion. The transcriptional profile was evaluated in three different donors using the HT12v4.1 BeadChip arrays from Illumina. Total RNA obtained from CD4+ T cells exposed to Th17-promoting cytokines.
Project description:PurposeAmongst the epigenetically targeted therapies, targeting of the histone deacetylases (HDACs) has yielded numerous drugs for clinical use in hematological malignancies, but none as yet for acute lymphocytic leukemia (ALL). Single agent activity of HDAC inhibitors (HDACi) has been elusive in ALL, and has prompted study of combinatorial strategies. Because several HDACi raise levels of intracellular oxidative stress, we evaluated combinations of two structurally distinct HDACi with the redox active compound adaphostin in ALL.MethodsThe HDACi vorinostat and entinostat were tested in combination with adaphostin in human ALL cell lines. DNA fragmentation, caspase activation, mitochondrial disruption and levels of intracellular peroxides, superoxide and glutathione were measured in cells treated with the HDACi/adaphostin combinations. Antioxidant blockade of cell death induction and gene expression profiling of cells treated with vorinostat/adaphostin versus entinostat/adaphostin combinations were evaluated.ResultsBoth combinations synergistically induced apoptotic DNA fragmentation, which was preceded by an increase in superoxide levels, a reduction in mitochondrial membrane potential, and an increase in caspase-9 activation. The antioxidant N-acetylcysteine (NAC) blocked superoxide generation and prevented reduction of mitochondrial membrane potential. NAC decreased DNA fragmentation and caspase activity in cells treated with adaphostin and vorinostat, but not in those treated with adaphostin and entinostat. Gene expression arrays revealed differential regulation of several redox genes prior to cell death induction.ConclusionsA redox modulatory agent, adaphostin, enhances efficacy of two HDACi, vorinostat or entinostat, but via different mechanisms indicating a point of divergence in the mechanisms of synergy between the two distinct HDACi and adaphostin.
Project description:H. seropedicae wild-type or ntrC mutant were grown on three different nitrogen conditions: nitrogen limiting, ammonium shock and nitrate shock.
Project description:TGFB2-AS1 is a long non-coding RNA which is induced by ΤGFβ signaling. In order to assess the importance of TGFB2-AS1 on the regulation of gene expression, we performed an AmpliSeq transcriptomic array in human keratinocytes (HaCaT), which stably over-express TGFB2-AS1 or control pcDNA3 empty vector. In addition, cells were stimulated with TGFβ1 for 24 hours, in order to observe the effects of TGFB2-AS1 on gene expression, downstream of TGFβ signaling. RNA from the following four conditions was used in this experiment: 1) pcDNA3, 2) pcDNA3+TGFβ1, 3) pcDNA3-TGFB2-AS1, 4) pcDNA3-TGFB2-AS1+TGFβ1. Biological triplicates were used per condition.
Project description:Naïve and activated T-cells has a different response to antigenic challenge. We examine whether a cytokine like IL-6 induces different responses through the Jak-STAT pathway to affect the functional characteristics of a given CD4 T‑cell subset. We isolated naïve and effector memory (Tem) CD4 T-cells to investigated STAT1 and STAT3 binding after 1-hour treatment with 20ng/ml IL-6 in the presence of anti-CD3/CD28.
Project description:To define the role of MAGE-A1 in melanoma growth and metastasis, we performed RNA-seq analysis on MAGE-A1 overexpression (OE) and knockdown (KD) models in A375 human melanoma cell line. Our results revealed that overexpression of MAGE-A1 dramatically promoted proliferation, migration, and invasion of human melanoma cells in vitro and down-regulated of MAGE-A1 inhibited tumor cell proliferation and invasion. Furthermore, MAGE-A1 exerts its tumor promoting activity via activating including ERK-MAPK signaling pathway by RNA-seq analysis. mRNA profiles of MAGE-A1 over expression (OE), knockdown (KD), pcDNA-vector control, and pRNAT-scramble control in A375 cell line were generated using Ion torrent
Project description:Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we present a novel MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is scalable and accurately identifies sites of differential DNA methylation. Additionally, we demonstrate that the high-throughput data derived from MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450K Beadchip array. We applied this integrative approach to further investigate the role of DNA methylation in alternative splicing and to profile 5-mC and 5-hmC variants of DNA methylation in normal human brain tissue that we observed localize over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions. MeDIP-Seq on Ion Torrent Platform in HCT116 and Human Brain
Project description:Pathogenic bacteria must swiftly adapt to dynamic infection environments in order to survive and colonize in the host. 1-Deoxy-D-xylulose-5-phosphate synthase (DXPS) is thought to play a critical role in bacterial adaptation during infection and is a promising drug target. DXPS utilizes a thiamine diphosphate (ThDP) cofactor to catalyze the decarboxylative condensation of pyruvate and D-glyceraldehyde-3-phosphate (D-GAP) to form DXP, a precursor to isoprenoids and B vitamins. DXPS follows a ligand-gated mechanism in which pyruvate reacts with ThDP to form a long-lived lactyl-ThDP (LThDP) adduct which is coordinated by an active-site network of residues. D-GAP binding ostensibly disrupts this network to activate LThDP for decarboxylation. Our lab previously reported trihydroxybenzaldoximes inhibitors which are competitive with respect to D-GAP, and uncompetitive with respect to pyruvate, suggesting they bind after E-LThDP complex formation. Here, we conducted mechanistic studies to determine if these compounds inhibit DXPS by preventing LThDP activation or if they act as inducers of LThDP activation. We discovered that the catechol moiety of the trihydroxybenzaldoxime scaffold undergoes oxidation under alkaline aerobic conditions, and inhibitory potency is reduced under oxygen restriction. Leveraging long range 1 H- 15 N HSQC NMR and electrochemical measurements, we demonstrated that the oxidized form of the trihydroxybenzaldoxime induces LThDP decarboxylation. The oxime moiety accepts electrons from the resulting carbanion, resulting in formation of acetyl-ThDP which hydrolyzes to form acetate. SAR studies revealed that the catechol attenuates the redox activity of the oxime moiety, and under aerobic conditions these compounds are oxidized and thus act as redox cycling inhibitors of DXPS. Further exploration of redox active DXPS probes may provide new insights for inhibition strategies and selective probe development.
Project description:Analysis of CGTH-W-1 follicular thyroid carcinoma cells transcriptome following 48 hrs siRNA-mediated depletion of PROX1. PROX1 is a homeobox transcription factor. PROX1 depletion decreases migratory ability, motility and invasivness and induces profound cytoskeleton changes of CGTH-W-1 cells. Results provide insight into the role of PROX1 in the thyroid cancer. Three biological replicates for a given condition