Project description:Long non-coding RNAs (lncRNAs) are recently characterized players that are involved in the regulatory circuitry of self-renewal in human embryonic stem cells (hESCs). However, the specific roles of lncRNAs in this circuitry are poorly understood. Here, we determined that growth-arrest-specific transcript 5 (GAS5), which is a known tumor suppressor and growth arrest gene, is abundantly expressed in the cytoplasm of hESCs and essential for hESC self-renewal. GAS5 depletion in hESCs significantly impaired their pluripotency and self-renewal ability, whereas GAS5 overexpression in hESCs accelerated the cell cycle, enhanced their colony formation ability and increased pluripotency marker expression. By RNA sequencing and bioinformatics analysis, we determined that GAS5 activates NODAL-SMAD2/3 signaling by sustaining the expression of NODAL, which plays a key role in hESC self-renewal but not in somatic cell growth. Further studies indicated that GAS5 functions as a competing endogenous RNA (ceRNA) to protect NODAL mRNA against degradation and that GAS5 transcription is directly controlled by the core pluripotency transcriptional factors (TFs). Taken together, we suggest that the core TFs, GAS5 and NODAL-SMAD2/3 form a feed-forward loop to maintain the hESC self-renewal process. These findings are specific to ESCs and did not occur in the somatic cell lines we tested; therefore, our findings also provide evidence that the functions of lncRNAs vary in different biological contexts. We analyzed long non-coding RNAs in two hESC cell lines (X-01 and H1), and found GAS5 is highly expressed and functional in maintaining hESC self-renewal. We generate stable overexpressed or knockdown hESC cell lines using lentiviral approach. We transfected cells initialy after passage, and lentiviruses are added with daily medium change for three days (at a final concentration of 10^5 IU/ml). Puromycin is added for selection and supplied with daily medium change. Stable cell lines are established after two passages and verified under fluorescence scope. Total RNAs and miRNAs are extracted separately of all three cell lines (LV-NC, LV-GAS5 and LV-shGAS5) and put to sequencing.
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:Cell toxicity may result in organ dysfunction and cause severe health problem. Recent studies revealed many toxicants may induced the over production of Nitric oxide, reactive oxygen species and the subsequent oxidative stress, cause cell toxicity. Mitochondrion dysfunction maybe the subsequent consequence of oxidative stress and has been recognized as another contributing factor in cell toxicity. Besides, oxidative products induced by some toxicants may also produce the compounds that damage cell DNA, leading to toxicity. Especially, the significance of nanoparticle induced cell toxicity was disclosed recently and attract more concern. The mechanism mainly includes inflammation, oxidative stress and DNA damage. On the other side, some biomarkers of cell toxicity including autophagy, cytokines, miRNA has been identified. The understanding of these phenomenon may enable us to clarify the cell toxicity mechanism then contribute to cell toxicity protection, disease treatment and drug side effect prevention.
Project description:We report the effects of a nickel stress on the transcriptome of Escherichia coli coli cells, evaluated by RNA-Seq experiments Comparison between wild type E. coli grown in miminal media in the presence or not of 50 µM NiCl2 during 10 min - 2 conditions compared with 3 replicates each
Project description:In the present study we created and analyzed cardiomyocytes from two separate iPSC clones from the fibroblasts of five different female individuals and two male individuals, using footprint-free Sendai virus RNA-seq of iPSC cardiomyocytes, Ampli-seq of heart left ventricle and iPSC cardiomyocytes (with and without drug treatment) and Exome-seq of patient fibroblasts.
Project description:Somatic mutations can lead to the transformation of healthy cells into malignant cells and allow their evasion from immune surveillance. To uncover genes that play a role in the detection and lysis of tumor cells by natural killer (NK) cells, a B lymphoblastoid cell line was subjected to a genome-wide CRISPR screen. Deletion of genes for death receptor and ligands for NK activation receptors was found in cells that survived incubation with primary NK cells. Among the top hits that facilitated NK evasion was SPPL3, a gene for an endoprotease that cleaves transmembrane glycosyl transferases. SPPL3-deficient cells accumulated glycosyl transferases, such as MGAT5, and displayed increased N-glycosylation. Binding of NK receptors NKG2D and CD2 to their corresponding ligands MICB and CD58, and binding of rituximab to CD20, was disrupted. Inhibition of N-glycan maturation restored receptor binding and sensitivity to NK cells. To investigate the cause of this resistant phenotype, a secondary CRISPR screen using a glycosylation-focused library was performed in SPPL3-deficient cells. This screen identified transferases that promote the formation of highly branched N-glycans and N-acetyl-lactosamine (LacNAc) extensions as key regulators that prevent killing. A significant enrichment of poly-LacNAc-containing tetra-antennary species was confirmed by glycoproteomic analysis. These findings provide a basis for understanding why SPPL3 deletions have been linked to cancer.
Project description:Rapid advances in the fields of DNA-, RNA-, polypeptide-sequencing and metabolomics have markedly enhanced our understanding of fundamental biological processes. In contrast to other post-translational modifications (PTMs), the most abundant PTM, glycosylation, remains largely unexplored at the proteome scale because of a scarcity of technologies for profiling the immensely complex glycoproteome. We developed a novel quantitative approach for the identification of intact glycopeptides from comparative proteomic data-sets, allowing us to not only identify complex sugar structures but also to directly map them to the corresponding glycosylation sites in the associated proteins at the proteome scale. Applying this method to human and murine embryonic stem cells, we were able to nearly double the number of all experimentally confirmed glycoproteins, including the identification of completely unknown glycosylation sites, multiple glycosylated stemness factors and the elucidation of evolutionary conserved core as well as species-specific glycoproteins in embryonic stem cells. Specificity of our method was confirmed in sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Since ablation of fucosylation confers cellular resistance towards the bioweapon ricin (see accompanying paper), our method also allowed us to directly identify the proteins that carry the terminal fucosylation code for ricin toxicity; genetic mutations in these proteins functionally confirmed their role in ricin killing. This novel comparative and high-throughput glycoproteomics platform should allow for entirely novel insights in protein glycosylation and sugar modifications involved in nearly all biological systems.
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: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