Transcription profiling of Streptococcus mutans grown in a biofilm with or without the presence of Veillonella parvula
ABSTRACT: Streptococcus mutans was grown for 48 h in a biofilm in the absence (single species) and in the presence (dual species) of Veillonella parvula. In addition V. parvula single species 48 h biofilms were grown, to be used as a control. RNA was harvested from all types of biofilms and the transcript levels of the two types of biofilms containing S. mutans were compared with the use of S. mutans microarrays. V. parvula RNA was hybridized to S. mutans microarrays as a control for possible cross-hybridisation.
INSTRUMENT(S): G2505A DNA microarray scanner [Agilent]
Project description:Analysis of intra- and inter variation using multiple biopsies taken from the same knee. The biopsies were obtained from thirteen different patients; seven by orthopedic surgery and six by rheumatic arthroscopy. Orthopedic samples (patients 1-7 Three synovial tissue specimens were obtained from random sites of the synovium for each patient. Biopsy III of patient 7 was not used due to bad RNA quality. For patients 1-3 each biopsy was divided into three parts (1/3 of a biopsy is denoted subbiopsy). In total this resulted in 39 specimens from the orthopedic patients (nine biopsies from patients 1-3 and three biopsies from each of patients 4-7). For patient 1-3, pt1 b1.BA means patient 1 biopsy 1 subbiopsy B Technical replicate A. For patient 4-7, pt4 b1A means patient 4 biopsy 1 technical replicate A. Arthroscopic samples (patients 8-13) Arthroscopic biopsies were taken at the site of inflammation close to cartilage (cc) or not close to cartilage (ncc), defined as less or more than 1.5 cm away from cartilage, respectively. Multiple biopsies were taken from two sites in patients 8, 11, 12, 13 and from four sites in patients 9 and 10. pt12_ncc_1A means patient 12 not close to cartilage biopsy 1 technical replicate.
Project description:Comparison at two different timepoints of expression profiles of wild_type Bacillus and a deletion mutant of the PlcR regulator. Additional processed data can be found in the FTP directory for this experiment <a href="ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1472/">ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1472/</a>
Project description:The purpose of this experiment was to do investigatge the transcriptional effect of anti-TNF treatment in patients suffering from rheumatoid arthritis. Two series of hybridizations were performed. Each hybridization was done with a technical replicate (the same amount of RNA taken from the same amplified RNA aliquot labeled in two separate reactions and hybridized onto two separate arrays). In the first series of hybridizations RNA exptracted from biopsies taken before treatment was hybridized in a common reference design to enable comparisons between patients. In the second series RNA from biopsies taken after and before treatment were hybridized on the same chip in a direct design to enable investigation of the effect of treatment. 10 patients (8 females and 2 males, median age 54, range 25-69) meeting the American College of Rheumatology (ACR) criteria for RA were recruited for this study. All patients received infliximab (an antibody directed against TNF-alpha) infusions 3mg/kg at week 0 and than after 2 weeks and 6 weeks. Five of these patients were receiving prednisolon and all ten patients were treated with methotrexate to a maximum of 20 mg per week. Synovial biopsies were obtained during arthroscopy from all patients before and after a median of 9 weeks of treatment (with one exception, patient 7 where the second biopsy was obtained during open surgery). Biopsies were taken at the site of inflammation close to cartilage (cc) or not close to cartilage (ncc), defined as less or more than 1.5 cm away from cartilage, respectively. One biopsy was taken before and after treatment from patient 5, 7, 9 and 10. Multiple biopsies were retrieved before and/or after treatment from patient 1, 2, 3, 4, 6 and 7. Due to small amounts of RNA available for patient 9 it was only used in the second series of hybridizations. The average biopsy weight was 22 mg. The ethical committee at the Karolinska Hospital approved all experiments on human cells and tissues. Informed consent was obtained from all study subjects.
Project description:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community. Dual-species biofilms of S. mutans and C. albicans and single-species biofilms of S. mutans were cultivated in 24-well microtitre plates in YNBB medium. Transcriptional profiles of S. mutans in single- and dual-species biofilms were analysed at early (6 h) and late (10 h) logarithmic phase of the biofilm growth, as well as after 24 h when biofilms entered stationary phase. Transcriptional profiles of S. mutans grown in the dual-species biofilms were compared to profiles obtained for single-species biofilm from the same time point. Three biological and one to two technical replicas were used in the microarray study. RNA samples were labeled with Cy3 or Cy5 using the ULS fluorescent labeling kit (Kreatech, Germany). Seven hundred nanograms of Cy3 or Cy5 labeled RNA after fragmentation were hybridized to the microarray at 65°C for 17 h using the Agilent hybridization chamber according to the manufacturer's instructions. The arrays were scanned using the Agilent DNA microarray scanner and the raw data were extracted using Agilent Feature Extraction software (v. 10.7).
Project description:In order to study the physiological consequences of a high-copper diet on hepatic gene expression, 6 mM CuCl2 was added to the drinking water for a period of 1 month. After this period, livers of seven control mice and eight copper-treated mice were isolated and were subjected to microarray analysis and copper measurements. The hepatic gene expression profile of copper-treated mice was compared to non-treated mice using a pooled reference.
Project description:Nine time points for microarray analysis were chosen to study early and late transcriptional responses in copper metabolism upon copper overload in HepG2 cells. Samples of copper-treated cells were hybridized using non-treated samples as a reference.