Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).
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:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:We used ATLAS-seq to comprehensively map the genomic location of LINE-1 elements belonging to the youngest and potentially polymorphic subfamily (L1HS-Ta). This was performed in a panel of 12 human primary or transformed cell lines (BJ, IMR90, MRC5, H1, K562, HCT116, HeLa S3, HepG2, MCF7, HEK-293, HEK-293T, 2102Ep). In brief, ATLAS-seq relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of L1HS-Ta element junctions, and Ion Torrent sequencing using single-end 400 bp read chemistry. A notable aspect of ATLAS-seq is that we can obtain both L1 downstream and upstream junctions (3'- and 5'-ATLAS-seq libraries, respectively), for full-length L1 elements. Note that a 10-nt sample-specific barcode has been removed at the 5' end of the reads in the .fastq files upon demultiplexing. This was achieved using cutadapt v1.9.2.dev0 (with the following parameters: -e 0.1 -q 10 -m 25 -g <barcode_name>=^<barcode_sequence>)
Project description:Human microbiota-associated (HMA) mice are an important model to study the relationship between liver diseases and intestinal microbiota. We describe a new method to humanize conventional mice based on bowel cleansing with polyethylene glycol followed by fecal microbiota transplantation (FMT) from a human donor. Four successive bowel cleansings were sufficient to empty the intestine and decrease the microbiota by 90%. We then compared four different strategies based on the frequency of FMT over four weeks: (1) twice a week; (2) once a week; (3) two FMTs; (4) one FMT. We were able to transfer human bacteria to mice, irrespective of the strategy used. We detected human bacteria after four weeks, even if only one FMT was performed, but there was a shift of the microbiota over time. FMT twice a week for four weeks was too frequent and perturbed the stability of the newly formed ecosystem. FMT once a week appears to be the best compromise as it allowed engraftment of Faecalibacterium, and a higher diversity of bacteria belonging to the Bacteroidales order. Our easy to establish HMA mouse model could be used as an alternative to classical HMA mice to study the relationship between the liver and the microbiota.
Project description:The mammalian intestinal tract is heavily colonized with a dense, complex, and diversified microbial populations. In healthy individuals, an array of epithelial antimicrobial agents is secreted in the gut to aid intestinal homeostasis. Enterochromaffin cells (EC) in the intestinal epithelium are a major source of chromogranin A (CgA), which is a pro-hormone and can be cleaved into many bioactive peptides that include catestatin (CST). This study was carried out to evaluate the possible impact of CST on gut microbiota in vivo using a mouse model. The CST (Human CgA352-372) or normal saline was intrarectally administered in C57BL/6 male mice for 6 days and then sacrificed. Feces and colonic mucosa tissue samples were collected, DNA was extracted, the V4 region of bacterial 16S rRNA gene was amplified and subjected to MiSeq Illumina sequencing. The α-diversity was calculated using Chao 1 and β-diversity was determined using QIIME. Differences at the genus level were determined using partial least square discriminant analysis (PLS-DA). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict functional capacity of bacterial community. CST treatment did not modify bacterial richness in fecal and colonic mucosa-associated microbiota; however, treatment significantly modified bacterial community composition between the groups. Also, CST-treated mice had a significantly lower relative abundance of Firmicutes and higher abundance of Bacteroidetes, observed only in fecal samples. However, at lower phylogenetic levels, PLS-DA analysis revealed that some bacterial taxa were significantly associated with the CST-treated mice in both fecal and colonic mucosa samples. In addition, differences in predicted microbial functional pathways in both fecal and colonic mucosa samples were detected. The results support the hypothesis that CST treatment modulates gut microbiota composition under non-pathophysiological conditions, however, the result of this study needs to be further validated in a larger experiment. The data may open new avenues for the development of a potential new line of antimicrobial peptides and their use as therapeutic agents to treat several inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or other health conditions.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. Recently, disorders of metabolism are thought to be the center of many diseases such as OPLL. Advanced glycation end product (AGE) are accumulated in many extracellular matrixes such as ligament fibers, and it can functions as cellular signal through its receptor (RAGE), contributing to various events such as atherosclerosis or oxidative stress. However, its role in OPLL formation is not yet known. Therefore, we performed high-through-put RNA sequencing on primary posterior longitudinal ligament cells treated with different doses of AGEs (1µM, 5µM and negative control), with or without BMP2 (1µM). mRNA profiles of Primary human posterior longitudinal ligament cells stimulated with various stimuli (Control, 1µM AGE-BSA, 5µM AGE-BSA, 1µM AGE-BSA with BMP2, 5µM AGE-BSA with BMP2) were generated by deep sequencing on Ion Proton
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 aim of this study was to decipher the mechanisms by which the major human milk oligosaccharide (HMO), 2’-fucosyllactose (2’FL), can affect body weight and fat mass gain upon high-fat diet (HFD) feeding in mice. In particular, we wanted to elucidate whether 2’FL metabolic effects are linked with changes in intestinal mucus production and secretion, mucin glycosylation and degradation, as well as with the modulation of the gut microbiota, fecal proteome and endocannabinoid (eCB) system.
Project description:In this study, RNA-Seq was used to reveal the differences of molecular pathways in hepatopancreas of O. niloticus adapated to water with salinity of 8 or 16 practical salinity (psu), respectively, with fish at freshwater as the control,. Significantly changed pathways were mainly related to lipid metabolism, glucose utilization, protein consumption, osmotic regulation, signal transduction and immunology. Based on the tendencies from freshwater to 8 or 16 psu, the differentially expressed gene unions were categorized into eight unique models, which were further classified into three categories which were constant-change (either keep increasing or decreasing), change-then-stable and stable-then-change. In constant-change category, steroid biosynthesis, steroid hormone biosynthesis, fat digestion and absorption, complement and coagulation cascades were extremely significantly affected by ambient salinity (P < 0.01), indicating that these pathways play pivotal roles in molecular response to salinity acclimation from freshwater to saline water in O. niloticus, and should be the main research focus in the future. In change-then-stable category, ribosome, oxidative phosphorylation, peroxisome proliferator-activated receptors (PPAR) signaling pathway, fat digestion and absorption changed significantly with ambient increasing salinity (P < 0.01), showing these pathways were sensitive to environmental salinity variation, but had a response threshold, and would stop changing once salinity exceeds the threshold. In stable-then-change category, protein export, protein processing in endoplasmic reticulum, tight junction, thyroid hormone synthesis, antigen processing and presentation, glycolysis/gluconeogenesis and glycosaminoglycan biosynthesis - keratan sulfate were the top changed pathways (P < 0.01), suggesting that these pathways were not sensitive to salinity variation, but these pathways will respond significantly under salinity exceeding a certain level. The pathways and genes reported in this study laid on a solid foundation for future studies in understanding the underlying mechanism for salinity adaptation of freshwater fish. Examination of 3 different salinities treated hepatopancreas in Nile tilapia