Transcriptome comparison of S. pyogenes strains MGAS2221 and 2221ΔFasX following exposure to human plasma
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ABSTRACT: The fasX gene encodes a small regulatory RNA in the group A Streptococcus (GAS). To determine the FasX regulon during GAS exposure to human plasma we compared parental strain MGAS2221 with isogenic fasX mutant strain 2221ΔFasX. Gene expression was analyzed 0, 15, and 60 minutes post-plasma exposure. GAS strains were grown in THY broth to mid-exponential phase, corresponding to an O.D.600 of 0.5. Two 16ml aliquots of each GAS culture were pelleted by centrifugation and the supernatant removed. Each GAS cell pellet was resuspended in 16ml of pre-warmed human plasma. Thus, four GAS-plasma suspensions were created, two each for each of the GAS strains, with one of the two using plasma from person A, and the other two using plasma from person B. Aliquots were recovered from each of the four GAS-plasma suspensions at T = 0, 15, and 60 mins. For the first two time-points 4.5ml of GAS-plasma suspension were added to 9ml of RNAprotect (Qiagen Inc.). For the final time-point, 4ml was added to 8ml of RNAprotect. Following addition of RNAprotect, samples were incubated at room temperature for 5 mins before centrifuging. Supernatant was removed and the pellets quick frozen in liquid nitrogen and stored at -80 °C until ready to use. RNA isolation, cDNA synthesis, and use of our custom Affymetrix microarray was as previously described (Perez et al., 2009)
Project description:The rivR gene encodes a putative transcriptional regulator, while the rivX gene encodes a putative small regulatory RNA. To determine the RivRX regulon during exponential phase growth in THY broth we compared parental strain MGAS2221 with isogenic rivRX mutant strain 2221?rivRX. GAS strains were grown in THY broth to mid-exponential phase, corresponding to an O.D.600 of 0.5. A 4.5ml aliquot of each GAS culture was added to 9ml of RNAprotect (Qiagen Inc.). Following addition of RNAprotect, samples were incubated at room temperature for 5 mins before centrifuging. Supernatant was removed and the pellets quick frozen in liquid nitrogen and stored at -80 °C until ready to use. RNA isolation, cDNA synthesis, and use of our custom Affymetrix microarray was as previously described (Perez et al., 2009)
Project description:We used RNA-Seq to assess how the presence/absence of the RD2 pathogenicity island influences global gene expression in a serotype M28 GAS isolate following exposure to human plasma for 15 mins and for 60 mins
Project description:Platelets contain abundant miRNAs, however, the biogenesis pathway of miRNAs in anucleate platelets is unclear. Platelet-rich plasma was diluted in washing buffer and the platelet suspension was centrifuged to isolated pure platelets.Finally, platelets were recovered in suspension buffer at the concentration of 4–5×10^8 platelets/ml. Aliquots of the platelet suspensions were activated in the presence of 2.5 mM CaCl2 with 0ng/ml or 1 ng/ml thrombopoietin (TPO)
Project description:Interventions: Experimental group:OralSanghuangporusbaumii tablets (15g per person per day, 200ml of boiling water for 15min, filter out the supernatant, then 200ml of boiling water for 15min, filter out the supernatant, combine the two supernatants for oral use), continuous treatment for 2 weeks;Positive control group:The patient was treated with thymofaxine (1.6 mg subcutaneously twice a week) for 2 consecutive weeks.
Primary outcome(s): Immunocompromised control ratio;Immune function index;Adverse reactions;Symptom score
Study Design: Parallel
Project description:We have employed whole genome microarray expression profiling to identify genes that are differentially expressed after a single gas plasma exposure in two different head and neck cancer cell lines. Additionally, we aimed to identify genes correlating with cellular adaptation upon repetitive oxidative stress wherefore two different head and neck cancer celllines were exposed to gas plasma weekly in eight treatment cycles.
Project description:200,000 worms were grown to the young adult stage, collected, and washed. Worms were homogenized in lysis buffer (10mM Tris, pH 7.5, 138mM NaCl, 2mM CaCl2, and 0.1% NP-40). After homogenization, 125U/mL Benzonase nuclease was added to homogenates and samples were incubated for 30 mins at room temperature with gentle rocking. Samples were spun at 21,000g for 5 mins, followed by resuspension of the pellet in lysis buffer. Soluble proteins were removed from the pellet with repeated washes in lysis buffer until A280 of the supernatant was equal to A280 of lysis buffer. Resulting pellet was resuspended in ddH2O, spun at 21,000g for 5 mins, and supernatant was transferred to a collection tube. Water resuspensions and recoveries were continued until A280 of H2O supernatants was 0. Recovered water washes were pooled and amyloids salted out by adding NaCl to 200mM final concentration, followed by 1-3 days incubation at 4oC. Precipitated amyloids were recovered by centrifuging at 21,000g for 30 mins, followed by resuspension in 50-100uL ddH2O. Amyloids were digested (trypsin and chymotrypsin) and prepared for mass spectrometry as described in the methods files.
Project description:Large cohort: exRNA composition of platelet-depleted plasma from a cohort of 173 female and 63 male healthy volunteers of diverse races and ages, collected at two visits spaced two weeks apart; 4 formerly-collected aliquots from study subject P12, who displayed a stable neuroendocrine miRNA signature in plasma; 20 plasma samples of pregnant women spanning all trimesters T1 to T3 of pregnancy, with an expected placenta miRNA contribution. P12 Family: exRNA from two replicates of platelet-depleted plasma from three unstudied P12 family members and a sample from P12 collected four years after the initial discovery of the phenotype, along with samples of two healthy volunteers. Plasma subfractions: exRNA of samples from P12 and his family, two healthy male volunteers, two non-pregnant, and nine pregnant women, separated by differential ultracentrifugation into 10,000 × g and 100,000 × g pellets, and corresponding 10,000 × g and 100,000 × g supernatants.
Project description:This experiment used ChIP-seq technology to create a genome-wide profile of histone marks in normal human pancreatic islets. In the current work we analyzed two histone marks associated with gene expression (H3K4me3, H3K4me1) and marks associated with gene repression (H3K27me3). Each mark was anayzed using samples obtained from four donors (n=4). Chromatin Immunoprecipitations (ChIPs) for histone marks were performed using specific anti-histone antibodies. Enrichment of each sample was calulated with respect to its individual input using qPCR. Samples were sequenced with Solexa and sequenced DNA from both Input (n=4) and ChIP (n = 4) samples were aligned to the NCBI Genome Build 36.1 Hg18 to determine regions that were enriched for binding by modified histones. ArrayExpress Release Date: 2010-02-17 Publication Title: Genome-wide analysis of histone modifications in human pancreatic islets Publication Author List: Bhandare R, Schug J, Le Lay J, Fox A, Smirnova O, Liu C, Naji A, Kaestner KH Person Roles: submitter Person Last Name: Bhandare Person First Name: Reena Person Email: bhandare@mail.med.upenn.edu Person Phone: Person Address: 771 Clinical Research Building, 415 Curie Blvd, Philadelphia, PA, 19104 Person Affiliation: Department of Genetics, University of Pennsylvania
Project description:As part of the ENCODE consortium the GENCODE project is producing a reference gene set through manual and automated gene prediction. Selected transcript models are verified experimentally by RT-PCR amplification followed by sequencing. This is batch IV, based on chromosome 3, 4 and 5 annotations from Gencode 4 (January 2010). The sequences in this submission were obtained from two different RNA amplifications of the same original RNA for each tissue (called cdna1 and cdna2 in the assay names). In addition testis cDNA1 and lung cDNA2 were sequenced twice (rep1, rep2), and sequences originated from a new dilution of brain and testis cDNA1 (newdil) have also been included. ArrayExpress Release Date: 2011-08-22 Publication Title: GENCODE: producing a reference annotation for ENCODE Publication Author List: Harrow J, Denoeud F, Frankish A, Reymond A, Chen CK, Chrast J, Lagarde J, Gilbert JG, Storey R, Swarbreck D, Rossier C, Ucla C, Hubbard T, Antonarakis SE, Guigo R Person Roles: submitter Person Last Name: Gonzalez Person First Name: Jose Person Mid Initials: M Person Email: jmg@sanger.ac.uk Person Phone: -498006 Person Address: Wellcome Trust Genome Campus, Hinxton, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: investigator Person Last Name: Hubbard Person First Name: Tim Person Mid Initials: Person Email: th@sanger.ac.uk Person Phone: -498055 Person Address: Wellcome Trust Genome Campus, Hinxton, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: investigator Person Last Name: Reymond Person First Name: Alexandre Person Mid Initials: Person Email: Alexandre.Reymond@unil.ch Person Phone: Person Address: Lausanne, Switzerland Person Affiliation: University of Lausanne For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
Project description:Bronchoalveolar lavage fluid was collected from three clinically normal horses by infusing 300 ml of warm saline through a 300 cm foley catheter that was passed through the nasal passage into a terminal bronchus of the lung. BALF was retrieved by manual aspiration using a 60 cc syringe, placed immediately on ice, transported to the laboratory, and centrifuged (600xg 10 minutes 4oC). Aliquots of cell free BALF supernatant were frozen at -80C for subsequent proteomic analysis. Proteomic analysis was performed using a composite sample consisting of 100 g of BALF protein from each of three horses. Triplicate aliquots of the composite (75 g of protein each) were subjected to proteomic analysis as described (McCarthy et al. 2006) except that we did not perform differential detergent fractionation and we used one dimensional liquid chromatography (1D LC) nanospray ionization and not electrospray ionization.