Project description:To demonstrate that the P. multistriata gene MRP3 is responsible for sex determination, we overexpressed it in a mating type minus strain. The transgenic strain generated displayed sex reversal and behaved like a strain of the opposite mating type. In this study, we compared the gene expression profile of the wild type versus the transformed strain.
Project description:In this study, we have investigated the physiological consequences of PHB (poly(3-hydroxybutyrate)) synthesis in H. seropedicae by characterising the trancriptional changes in a mutant strain lacking phaC1 gene which codes for the PHA synthase enzyme essential for the last step in the synthesis of PHB. To do this experiment both wild type (SmR1) and phaC1 mutant were cultured on Nfb-Malate-HP media suplemented with 20 mM of ammonium chloride under 30 Celsius degrees and 120 rpm shaking rate until the late log-phase (O.D600 = 1.0), when the peak of PHB production is observed. The cultures obtained as above were harvested for RNA extraction and transcriptome analysis.
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:Malaria in pregnancy remains a substantial public health concern in malaria-endemic areas. Accumulation of maternal immune cells in the placenta and increased levels of inflammatory cytokines caused by sequestration of Plasmodium falciparum-infected erythrocytes (IE) in the placental intervillous blood spaces have been associated to poor neonatal outcomes, including low birth weight due to fetal growth restriction. However, little is known about the molecular changes occurring in the placenta in past-stages of P. falciparum infection when the hemozoin pigment is present in the absence of parasites. We conducted an integrated proteome, phosphoproteome and glycoproteome analysis in P. falciparum infected and non-infected placentas aiming to find molecular changes occurring in past-stage infection. A total of 2946 proteins, 1733 glycosites and 4100 phosphosites were identified and quantified in this study, disclosing overrepresented processes related to oxidative stress, protein folding and regulation of apoptosis, as well as AKT and ERK signaling pathways activation, which together with clinical data and literature-based information were further correlated to an increased apoptosis in infected placentas. This study showed apoptosis-related mechanisms associated with past-stage of malaria infection that can be further explored as therapeutic target against adverse pregnancy outcomes in placental malaria.
Project description:Purpose: Study transcriptome differences between biofilm, planktonic and stationary cultures. Methods: Total mRNA from in vitro cultures was extracted and sequenced using Ion Torrent PGM sequencer. Results: Characteristic transcriptomic profile was observed for biofilm, planktonic and stationary cultures. Biofilm and planktonic were similar biological states. Conclusions: Results suggest that H. parasuis F9 has more active metabolism during biofilm or planktonic growth when compared to stationary culture. Some identified membrane-related genes could play an important role in biofilm life. RNA profiles of 36 hours biofilm or planktonic cultures were generated and compared with stationary culture profile.
Project description:We addressed the integrated analysis of mRNA and miRNA expression levels of Tg6799 AD model mice at 4 month and 8 months of age. Total 8 gene cluster modules for co-expression network were predicted from transcriptome data and 6 modules were show relation with AD or aging. We constructed early stage AD network using data integration between mRNA and miRNA profiles and predicted miRNAs strongly involved in module regulation. We found that ARRDC3 showed AD mutation dependent changes of expression and was related metabolic dysfunction in early stage AD. These results demonstrate that candidate genes on the simultaneous profiling of mRNA and miRNA expressions in genome wide can be used for the understanding of non-coding RNA related gene expression in early stage AD. We suggested that our results could be future candidate to be developed as early biomarkers in progressive AD pathology. This result can be used for the further application in neurodegenerative diseases. Tg6799 transgenic mice were purchased from The Jackson Laboratory (USA) and were housed under a 12h light-dark cycle with free access to food and water. Female Tg6799 mice are maintained until 4 months and 8 months of age (for littermate control: LM and mutant subjects: MT). RNA samples were isolated from hippocampus of mice using TRI-Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. Gene expression was analyzed with GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA), which is comprised of over 45,000 probe sets representing approximately 28,700 well-characterized mouse genes. The Ion Total RNA-Seq Kit v2 (Lifetechnologies, USA) was used for the preparation of micro RNA libraries according to the manufacturer's instructions. Total numbers of subject used are as followed: 1) LM 4 months : MT 4 months : LM 8 months : MT 8 months (2:4:2:4) for screening mRMA and miRNA, 2) LM 4 months : MT 4 months : LM 8 months : MT 8 months (4:4:4:4) for expression verification.
Project description:The advent of high-throughput sequencing has allowed genome wide profiling of histone modifications by Chromatin ImmunoPrecipitation (ChIP) followed by sequencing (ChIP-seq). In this assay the histone mark of interest is enriched through a chromatin pull-down assay using an antibody for the mark. Due to imperfect antibodies and other factors, many of the sequenced fragments do not originate from the histone mark of interest, and are referred to as background reads. Background reads are not uniformly distributed and therefore control samples are usually used to estimate the background distribution at any given genomic position. The Encyclopedia of DNA Elements (ENCODE) Consortium guidelines suggest sequencing a whole cell extract (WCE, or “input”) sample, or a mock ChIP reaction such as an IgG control, as a background sample. However, for a histone modification ChIP-seq investigation it is also possible to use a Histone H3 (H3) pull-down to map the underlying distribution of histones. In this paper we generated data from a hematopoietic stem and progenitor cell population isolated from mouse foetal liver to compare WCE and H3 ChIP-seq as control samples. The quality of the control samples is estimated by a comparison to pull-downs of histone modifications and to expression data. We find minor differences between WCE and H3 ChIP-seq, such as coverage in mitochondria and behaviour close to transcription start sites. Where the two controls differ, the H3 pull-down is generally more similar to the ChIP-seq of histone modifications. However, the differences between H3 and WCE have a negligible impact on the quality of a standard analysis. WCE and histone H3 ChIP-seq samples are compared to H3K27me3 ChIP-seq and RNA-seq.
Project description:Asthma is a complex syndrome associated with episodic decompensations provoked by aeroaller-gen exposures. The underlying pathophysiological states driving exacerbations are latent in the resting state and do not adequately inform biomarker-driven therapy. A better understanding of the pathophysiological pathways driving allergic exacerbations is needed. We hypothesized that disease-associated pathways could be identified in humans by unbiased metabolomics of bron-choalveolar fluid (BALF) during the peak inflammatory response provoked by a bronchial aller-gen challenge. We analyzed BALF metabolites in samples from 12 volunteers who underwent segmental bronchial antigen provocation (SBP-Ag). Metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by pathway analysis and cor-relation with airway inflammation. SBP-Ag induced statistically significant changes in 549 fea-tures that mapped to 72 uniquely identified metabolites. From these features, two distinct induci-ble metabolic phenotypes were identified by the principal component analysis, partitioning around medoids (PAM) and k-means clustering. Ten index metabolites were identified that in-formed the presence of asthma-relevant pathways, including unsaturated fatty acid produc-tion/metabolism, mitochondrial beta oxidation of unsaturated fatty acid, and bile acid metabolism. Pathways were validated using proteomics in eosinophils. A segmental bronchial allergen chal-lenge induces distinct metabolic responses in humans, providing insight into pathogenic and pro-tective endotypes in allergic asthma.
Project description:Genome-wide identification of transcription factor (TF) binding sites in the genome of the fission yeast Schizosaccharomyces pombe. The ChIP-nexus method was used. TFs included were: Cbf11-TAP and Cbf12-TAP (and their DBM mutants with impaired DNA binding), TAP-Mga2, and Fkh2-TAP (as an irrelevant control TF). IPs from an untagged WT strain were also analyzed. Cbf11-related IPs were performed from exponential cultures, while Cbf12-related IPs were performed from stationary cultures. YES complex medium was used for all cultivations.