Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To this end, a large marinerT7 transposon mutant library (~28,000 independent transposon mutants) was grown under iron-limiting conditions, achieved by sequestration of iron by 30 M-BM-5M Desferal (DF30), and under control growth conditions (brain heart infusion broth, DF0). Mutants were recovered at exponential- and the early-stationary growth phase and used for the generation of mutant-specific cDNA probes that were hybridized to custom-designed NimbleGen GAF microarrays. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology. cDNA probes generated from mutants recovered during the exponential growth phase, or early-stationary phase under challenge (iron-limiting, DF30) or control conditions (DF0). Hybridization on GAF 4x72K custom design Nimblegen GAF arrays for read-out.
Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To further characterize the functional consequences of the gene deletions (GAF identified genes), we determined transcriptome profiles of the wild-type and directed mutant strains during exponential growth in BHI medium. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology. M. catarrhalis BBH18 directed mutants (n = 3) and their parental wild-type BBH18 strain (n = 4) were expanded to mid-log phase (OD620nm, 1.2 to 1.4) and RNA was isolated as previously described (de Vries et al., 2010 J Bacteriol 192: 3574-3583). Total RNA was labeled according to standard Nimblegen gene expression array protocols and hybridized to 4x72K custom design Nimblegen arrays for read-out.
Project description:Investigation of the effect of atpme3-1 mutant on whole gene expression level hypocotyls of 96 hours-old plantlets were analyzed for this study A 12plex high density chip was used. Six or 5 biological replicates for each condition. Each replicate was obtained separately from the others and each replicate represents about 300 hypocotyles.
Project description:Full transcriptomes of the Botrytis cinerea wild-type strain B0510 and the null-mutants deltaBcVEL1 and deltaBcLAE1, cultured onto solid grape juice medium with cellophane overlays , were compared to identify BcVEL1 or/and BcLAE1-dependent genes. The Botrytis cinerea wild-type strain and the null-mutants deltaBcVEL1 and BcLAE1 were cultured for 48h onto solid grape juice medium with cellophane overlays. 4 replicates were performed. The 12 total-RNA samples (3 strains* 4 replicates) were used for hybridization on NimbleGen 4plex gene expression arrays (20,885 gene models from Botrytis cinerea with three 60-mer probes per gene).
Project description:Full transcriptomes of the Botrytis cinerea wild-type strain B0510 and the null-mutant deltaBcLTF2, cultured in continuous darkness or submitted to a light stimulation, were compared to identify light- or/and BcLTF2-dependent genes. The Botrytis cinerea wild-type strain and the null-mutant deltaBcLTF2 were cultured for 54h in continuous darkness ; then, cultures were exposed for 60 min to light before harvest (L) or were harvested after additional 60 min darkness (D). 4 replicates were performed. The 12 total-RNA samples (4 conditions* 3 replicates) were used for hybridization on NimbleGen 4plex gene expression arrays (20,885 gene models from Botrytis cinerea with three 60-mer probes per gene).
Project description:Full transcriptomes of the Botrytis cinerea wild-type strain B0510 and the null-mutant deltaBcLTF1, cultured in continuous darkness or submitted to a light stimulation, were compared to identify light- or/and BcLTF1-dependent genes. The Botrytis cinerea wild-type strain and the null-mutant deltaBcLTF1 were cultured for 52h in continuous darkness ; then, cultures were exposed for 60 min to light before harvest (L) or were harvested after additional 60 min darkness (D). 4 replicates were performed. The 16 total-RNA samples (4 conditions* 4 replicates) were used for hybridization on NimbleGen 4plex gene expression arrays (20,885 gene models from Botrytis cinerea with three 60-mer probes per gene).
Project description:Total RNA was extracted from the striatum of vehicle- or L-DOPA-treated FRL and FSL rats. After quality control, RNA samples were submitted to the Gene Expression Service Workflow of Roche NimbleGen, which included hybridization with a Roche NimbleGen Rat Gene Expression 12x135K Array (Design Name 100718_Rat_HX12_expr), array scanning, data extraction and processing. Normalized gene expression data (\\"Normalized Calls\\") provided by Roche NimbleGen were analyzed using ANAIS [Simon and Biot, Bioinformatics 2010 Oct; 26(19):2468-9] via the \\"Norm genes\\" function. Genes were considered differentially expressed if their transcript abundance (scanned signal intensity) was ≥1.5-fold higher or lower in at least one experimental group compared to striatum of vehicle-treated FRL rats. The resulting list of 6040 genes was imported into Microsoft Excel, where only those genes with ANAIS-computed ANOVA p-values <0.05 were retained.
Project description:Expression of the extensive arsenal of virulence factors by Streptococcus pyogenes are controlled by many regulators, of which covR/S is one of the best characterized and can influence ~15% of the genome. Animal models have established that mutants of CovR/S arise spontaneously in vivo resulting in highly invasive organisms. We analyzed a pharyngeal and a blood isolate of S. pyogenes recovered from the same individual 13 days apart. The two isolates varied in many phenotypic properties including speB production, which were reflected in transcriptome analyses. Pulsed field gel electrophoresis, multilocus sequence typing, and partial sequencing of some key genes failed to show any differences except for an 11-base insert in the covS gene in the blood isolate. These results showing that pharyngeal and blood isolates from a single individual which differ by a simple insertion, provide evidence for the model that regulatory gene mutations allow S. pyogenes to invade different niches in the body. A chip study using total RNA recovered from two separate wild-type cultures of group A Streptococcus, Streptococcus pyogenes UH322 and UH328. Each chip measures the expression level of 1865 genes replicated twice from 7 fully sequenced strains of Streptococcus pyogenes (M1_GAS NC_002737; MGAS10394 NC_006086; MGAS315 NC_004070; MGAS5005 NC_007297; MGAS6180 NC_007296; MGAS8232 NC_003485; SSI-1 NC_004606 with fourteen 24-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:This dataset encompassing the profiles of 150 lung cancer tumors was developed to serve as test dataset in the SBV IMPROVER Diagnostic Signature Challenge (sbvimprover.com). The aim of this subchallenge was to verify that it is possible to extract a robust diagnostic signature from gene expression data that can identify stages of different types of lung cancer. Participants were asked to develop and submit a classifier that can stratify lung cancer patients in one of four groups M-bM-^@M-^S Stage 1 of Adenocarcinoma (AC Stage 1), Stage 2 of Adenocarcinoma (AC Stage 2), Stage 1 of Squamous cell carcinoma (SCC Stage 1) or Stage 2 of Squamous cell carcinoma (SCC Stage 2). The classifier could be built by using any publicly available gene expression data with related histopathological information and was tested on the independent dataset described here. 150 non-small cell lung cancer tumors (adenocarcinoma, AC and squamous cell carcinoma, SCC) of stages I and II were collected by surgical resection from patients who have provided consent. Adenosquamous and large cell tumor samples were excluded. The number of smokers and non-smokers was balanced: there were 41 AC1 (adenocarcinoma stage I), 36 AC2, 34 SCC1, and 39 SCC2 samples. Study pathologists at each of the seven sites (Lebanon, Republic of Moldova, Romania, Russian Federation, Ukraine, Vietnam and United States of America) reviewed both the tumor permanent sections and the frozen sections of the samples. Clinical information was also collected about tumor staging, history of prior cancers, lymph node involvement by lymph node sampling/dissection, smoking history, age, gender.
Project description:Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infectious process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus development. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes up-regulated during infection of MB are enriched in functional categories related to necrotrophy such as degradation of plant cell wall, proteolysis, membrane transport, reactive oxygen species generation and detoxification. Quantitative-PCR on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but stops at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathways switch during berry ripening. In response to B. cinerea infection, VB activated a burst of reactive oxygen species (ROS), the salicylate (SA)-dependent defense pathway, the synthesis of the resveratrol phytoalexin and cell-wall strengthening. In opposite, infected MB activated the jasmonate (JA)-dependent pathway which does not stop the fungal necrotrophic process. Grapevine berries at veraison (VB) and harvesting stages (MB) were inoculated with Botrytis cinerea B05-10 and samples were taken at 24h and 48h post-inoculation. An additional uninfected control sample taken at 0h post-inoculation was included in the experimental design. 3 replicates per sample were performed. The total-RNA samples were labeled and used for hybridization on NimbleGen 12plex Vitis vinifera gene expression array.