Exposure of microaerobic Campylobacter jejuni to 10 micromolar NOC-5 & NOC-7
Ontology highlight
ABSTRACT:
ORGANISM(S): Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819
SUBMITTER: Robert Poole
PROVIDER: E-GEOD-38114 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached 0.010 mM NOC-5 & NOC-7 was added to one of the cultures. After a 15 minute exposure samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure, 30 ml samples of both treated and untreated cells were mixed immediately on ice with 3.56 ml 100% ethanol and 185 M-BM-5l phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. Equivalent amounts of RNA (15 M-NM-<g) from control and test cultures were used as template for synthesis of labelled cDNA. Labelling was done by using dCTP nucleotide analogues containing either Cy3 or Cy5 fluorescent dyes. RNA was primed with 9 M-NM-<g pd(N)6 random hexamers (Amersham Biosciences). For annealing, the mixture was incubated for 10 min at 65oC and then 10 min at room temperature. Each reaction mixture (0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP, 0.1 mM DTT (Invitrogen) and 1 mM Cy3-dCTP or Cy5-dCTP, total volume 25ul) was incubated for 3 h at 42 oC with 200 U of Superscript III RNase-H Reverse Transcriptase (Invitrogen). The reaction was terminated by the addition of 5ul 1 mM NaOH and heating the tube to 65 oC for 10 min to hydrolyse the RNA. Then it was neutralised with 5ul 1 M HCl and 1 M TE (pH 8). Purification of cDNA was done with a PCR purification kit (Qiagen). The cDNA was eluted and resuspended in 30 M-NM-<l elution buffer (Qiagen, supplied in kit). Each slide set (control slide and dye-swap) was prepared as follows: For control slide Cy3-dCTP labelled control cDNA was mixed with Cy5-dCTP labelled test cDNA. For dye-swap slide Cy5-dCTP labelled control cDNA was mixed with Cy3-dCTP labelled test cDNA. This is made for compensate possible differences in the labelled nucleotides incorporation. The slides used were C. jejuni OciChipM-BM-. arrays from Ocimum Biosolutions. The cDNA mixture for each slide was dried by evaporation for approximately 35 min in a SPD 121P SpeedVacM-BM-. (Thermo Savant, Waltham, MA, USA) The dry cDNA was resuspended in pre-warmed (42M-BM-:C) salt-based hybridisation buffer (Ocimum Biosolutions) and was heated to 95 M-BM-0C for 3 min and then placed on ice for 3 min. The spoted area of the slide (located with an array finder) was enclosed within a gene frame (MWG/Ocimum). The cDNA suspension was distributed through the inner space of the gene frame and enclosed with an air-tight coverslip. The slides were incubated for 16-24 hours at 42M-BM-: C in sealed MWG hybridisation chambers shaken in a water bath. After incubation, gene frames and coverslips were removed and slides washed sequentially in 2x, 1x, 0.2x y 0.1x SSC buffer, by shaking for 5 minutes at 80 rpm at 37M-BM-: C pre-warmed buffer. (2x buffer was supplemented with 1% SDS). Then the slides were dried by centrifugation at 250 x g for 5 min. Slides were scanned using an Affymetrix 428 scanner. The processing of images and quantification of the microarrays signal was done using software from Biodiscovery Inc (Imagene, version 4.0 and Genesight, version 4.0). Spots with signal intensity lower than background or other significant blemishes were eliminated from subsequent processing. Mean values from each channel were then log2 transformed and normalised using the Subtract by Mean method to remove intensity-dependent effects in the log2 (ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalised values. Significance analysis of the data used the StudentM-bM-^@M-^Ys t test to determine the probability that the average of the experimental replicates was significantly different from the average of the control replicates. p-values for the data were calculated by treating each slide as a repeat using Genesight 4. Genes differentially regulated M-bM-^IM-% 2-fold and displaying a p-value of M-bM-^IM-$ 0.05 were defined as being statistically significant and differentially transcribed.
2012-05-22 | E-GEOD-38115 | biostudies-arrayexpress
Project description:Two batch cultures of wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 500 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10% Oxygen, 10% Carbon dioxide, 80% Nitrogen) from Don Whitley Scientific, Ltd, which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached, a custom-made diffusion capsule (as described in Pirt, 1971) containing chicken caecal contents was placed for 10, 30, or 60 minutes. After the exposure, samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen's RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Batch cultures of wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 500 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10% Oxygen, 10% Carbon dioxide, 80% Nitrogen) from Don Whitley Scientific, Ltd, which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached, a custom-made diffusion capsule (as described in Pirt, 1971) containing chicken caecal contents was placed for 10, 30, or 60 minutes. After the exposure, 30 ml samples of both treated and untreated cells were mixed immediately on ice with 3.56 ml 100% ethanol and 185 M-BM-5l phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. Equivalent amounts of RNA (15 M-NM-<g) from control and test cultures were used as template for synthesis of labelled cDNA. Labelling was done by using dCTP nucleotide analogues containing either Cy3 or Cy5 fluorescent dyes. RNA was primed with 9 M-NM-<g pd(N)6 random hexamers (Amersham Biosciences). For annealing, the mixture was incubated for 10 min at 65oC and then 10 min at room temperature. Each reaction mixture (0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP, 0.1 mM DTT (Invitrogen) and 1 mM Cy3-dCTP or Cy5-dCTP, total volume 25ul) was incubated for 3 h at 42 oC with 200 U of Superscript III RNase-H Reverse Transcriptase (Invitrogen). The reaction was terminated by the addition of 5ul 1 mM NaOH and heating the tube to 65 oC for 10 min to hydrolyse the RNA. Then it was neutralised with 5ul 1 M HCl and 1 M TE (pH 8). Purification of cDNA was done with a PCR purification kit (Qiagen). The cDNA was eluted and resuspended in 30 M-NM-<l elution buffer (Qiagen, supplied in kit). Each slide set (control slide and dye-swap) was prepared as follows: For the control slide, Cy3-dCTP labelled control cDNA was mixed with Cy5-dCTP labelled test cDNA. For the dye-swap slide, Cy5-dCTP labelled control cDNA was mixed with Cy3-dCTP labelled test cDNA. This is made to compensate for possible differences in the labelled nucleotides incorporation. The slides used were C. jejuni OciChipM-BM-. arrays from Ocimum Biosolutions. The cDNA mixture for each slide was dried by evaporation for approximately 35 min in a SPD 121P SpeedVacM-BM-. (Thermo Savant, Waltham, MA, USA). The dry cDNA was resuspended in pre-warmed (42M-BM-:C) salt-based hybridisation buffer (Ocimum Biosolutions) and was heated to 95 M-BM-0C for 3 min and then placed on ice for 3 min. The spotted area of the slide (located with an array finder) was enclosed within a gene frame (MWG/Ocimum). The cDNA suspension was distributed through the inner space of the gene frame and enclosed with an air-tight coverslip. The slides were incubated for 16-24 hours at 42M-BM-: C in sealed MWG hybridisation chambers shaken in a water bath. After incubation, gene frames and coverslips were removed and slides washed sequentially in 2x, 1x, 0.2x y 0.1x SSC buffer, by shaking for 5 minutes at 80 rpm at 37M-BM-: C pre-warmed buffer. (2x buffer was supplemented with 1% SDS). Then the slides were dried by centrifugation at 250 x g for 5 min. Slides were scanned using an Affymetrix 428 scanner. The processing of images and quantification of the microarrays signal was done using software from Biodiscovery Inc. (ImaGene, version 4.0 and GeneSight, version 4.0). Spots with signal intensity lower than background or other significant blemishes were eliminated from subsequent processing. Mean values from each channel were then log2 transformed and normalised using the Subtract by Mean method to remove intensity-dependent effects in the log2 (ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalised values. Significance analysis of the data used the Student's t test to determine the probability that the average of the experimental replicates was significantly different from the average of the control replicates. p-values for the data were calculated by treating each slide as a repeat using Genesight 4. Genes differentially regulated M-bM-^IM-% 2-fold and displaying a p-value of M-bM-^IM-$ 0.05 were defined as being statistically significant and differentially transcribed.
2012-08-06 | E-GEOD-39917 | biostudies-arrayexpress
Project description:Although LIPUS has been shown to enhance fracture healing, the underlying mechanism of LIPUS remains to be fully elucidated. Here, to understand the molecular mechanism underlying cellular responses to LIPUS, we investigated gene expression profiles in mouse MC3T3-E1 preosteoblast cells using a GeneChipM-BM-. system. The mouse preosteoblast MC3T3-E1 cells were treated with LIPUS (30 mW/cm2) for 20 min, followed by culturing for 24 h at 37M-KM-^ZC. Mock-treated cells served as the control. Total RNA samples were prepared from the cells, and the quality of the RNA was analyzed using a Bioanalyzer 2100. Gene expression was monitored by an Affymetrix GeneChipM-BM-. system with a Mouse Genome 430 2.0 array. Sample preparation for array hybridization was carried out as described in the manufacturer's instructions.
2013-03-26 | E-GEOD-45487 | biostudies-arrayexpress
Project description:The advent of M-bM-^@M-^XomicsM-bM-^@M-^Y techniques bear significant potential for the assessment of the microbiological stability of foods. This requires the integration of molecular data with their implication for cellular physiology. Here we performed a comparative physiological and transcriptional analysis of Bacillus subtilis stressed with three different weak organic acids: the commonly used food preservatives sorbic- and acetic- acid, plus the well-known uncoupler carbonyl cyanide-m-chlorophenyl hydrazone (CCCP). The concentration of each compound needed to cause a similar reduction of the growth rate negatively correlated with their membrane solubility, and positively with the concentration of undissociated acid. Intracellular acidification was demonstrated by expressing a pH-sensitive GFP derivative. The largest drop in intracellular pH was observed in CCCP-stressed cells and was accompanied by the transcriptional induction of the general stress response (GSR) and SigM regulon, responses known to be induced by acidification. The GSR was induced by acetate, but not by sorbate in mildly-stressed cells. Microarray analysis further revealed that all three acids activate transcriptional programs normally seen upon nutrient limitation and cause diverse responses indicative of an adaptation of the cell envelope. Based on the responses observed and the utilized pH measurements, the inhibitory effect of sorbic acid seems to be more focused on the cell membrane than that of acetic acid or CCCP. Time-series of B. subtilis response to 25 mM potassium acetate and 0.85 M-BM-5M CCCP during controlled growth in batch-fermentors in defined minimal medium (pH 6.4). Samples for microarrays were taken from both the treated and control cultures at 0, 10, 20, 30, 40, and 50 min after addition of acetate and CCCP. Two biologically independent experiments were performed.
2014-04-11 | E-GEOD-55051 | biostudies-arrayexpress
Project description:Although an appropriate range of fluoride is thought to be safe and effective, excessive fluoride intake results in toxic effects in either hard tissues of teeth and skeleton or soft tissues of kidney, lung and brain. It is also well known that fluoride at a millimolar range elicits the complex cellular responses such as enzyme activity, signal transduction and apoptosis in many kinds of cells. However, its toxic effects are still unclear. In this study, to identify genes involved in apoptosis induced by sodium fluoride (NaF) in rat oral epithelial ROE2 cells, global-scale gene expression analysis was carried out using a GeneChipM-BM-. system. NaF (2 mM) significantly induced apoptosis accompaning chromatin condensation and caspase-3 activation. Total RNA samples were prepared from the NaF-treated cells, and quality of the RNA was analyzed using a Bioanalyzer 2100. Gene expression was monitored by an Affymetrix GeneChipM-BM-. system with a Rat Genome 230 2.0 array. Sample preparation for array hybridization was carried out as described in the manufacturerM-bM-^@M-^Ys instructions.
2014-01-09 | E-GEOD-53937 | biostudies-arrayexpress
Project description:Escherichia coli possesses >65 small proteins of <50 amino acids, many of which are uncharacterized. We have identified a new small protein, MntS, involved in manganese homeostasis. Manganese is a critical micronutrient, serving as an enzyme cofactor and protecting against oxidative stress. Yet manganese is toxic in excess and little is known about its function in cells. Bacteria carefully control intracellular manganese levels using the transcription regulator MntR. Before this work, mntH, which encodes a manganese importer, was the only gene known to respond to manganese via MntR repression in E. coli K12. We demonstrated that mntS is another member of the MntR manganese regulon. We also identified yebN, which encodes a putative manganese efflux pump, as the first gene positively regulated by MntR in Enterobacteria. Since MntS is expressed when manganese levels are low, causes manganese sensitivity when overexpressed, and binds manganese, we propose that MntS may be a manganese chaperone. This study reveals new factors involved in manganese regulation and metabolism and expands our knowledge of how small proteins function. Two E. coli strains, MG1655 (wild type) and GSO458 (Delta-mntR) were grown to OD600 ~ 0.5 in M9 glucose media at 37 M-BM-:C and treated with 10 microM MnCl2. In the first experiment, this incubation with 10 microM MnCl2 was for 60 min and in the second experiment, it was for 10 min. RNA was extracted using the hot phenol method and cDNA prepared and hybridized according the manufacturer's instructions (Affymetrix).
2011-12-08 | E-GEOD-25318 | biostudies-arrayexpress
Project description:This was an accompanying experiment. Previously, the expression of genes hsp70A and hsp70B had been established as being tetrapyrrole-dependent (Kropat et al., 1997, 2000). Since both are well-known heat-shock proteins, we performed a one-step heat shock treatment by shifting the temperatures from 23M-BM-0C to 42M-BM-0C for 45 min. Moreover, this experiment served as an additional global control to further test the reliability of the microarray and our general conditions since the heat shock response in general is quite well investigated. 3988 responding genes were identified. Among them are 22 genes known to be regulated by HS, selected examples are: the heat shock proteins 22A, 22B and 22C, here leading the list of top-regulated genes on place 1 with a FC of 91297 (22A), place 3 with a FC of 45023 (22B) and place 5 with a FC of 6570 (22C). Other examples are several DnaJ-like proteins or the ClpB chaperone, from the Hsp100 family. In total the analysis comprises 8 samples. For heat shock, a 20 ml culture with ~4 M-CM-^W 106 cells per ml in a 100 ml Erlenmeyer flask was incubated at 42M-BM-0C for 45 min in the light with shaking. Control cultures were incubated in the light at 23M-BM-0C (von Gromoff et al., 1989; Kropat et al., 2000). For harvest, cultures were chilled immediately by pouring them onto crushed ice, centrifuged (5 min, 3000 g, 4M-BM-0C) and resuspended in 1 ml TAP. This procedure was repeated three times, resulting in 3 biological replicates.
2011-03-02 | E-GEOD-20859 | biostudies-arrayexpress
Project description:In this study transcriptional start sites (TSS) of E. coli were determined for several growth conditions To detect the complement of transcripts expressed from E. coli, we collected two independent biological replicates (B1 and B2 samples) from MG1655 wild type strain grown to exponential (OD 600 ~0.4) or stationary phase (OD 600 ~2.0) in LB medium (samples LB 0.4 and LB 2.0, respectively) as well as grown to exponential phase (OD 600 ~0.4) in M63 minimal glucose medium (sample M63 0.4). For all six samples, total RNA was extracted and subjected to differential RNA-seq (dRNA-seq) library preparation for primary transcriptome analysis as described previously (Sharma et al., 2010). Specifically, prior to cDNA library construction half of each RNA sample was treated with 5M-bM-^@M-^Y terminator exonuclease (+TEX samples), which degrades RNAs containing a 5M-bM-^@M-^Y-mono-phosphate (5M-bM-^@M-^Y-P) and, thus, enriching enriches for primary transcripts containing 5M-bM-^@M-^Y-tri-phosphates (5M-bM-^@M-^Y-PPP). The other half of each sample was left untreated (-TEX samples) and thus contains both primary transcripts (5M-bM-^@M-^Y-PPP) and processed RNAs (5M-bM-^@M-^Y-P).
2014-10-18 | E-GEOD-55199 | biostudies-arrayexpress
Project description:Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we have explored the use of short fragment libraries (85-110bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. These short fragment libraries were enriched for 1.69 Mb of exonic sequences, using custom 244K microarrays, and sequenced using AB/SOLiD. High enrichment specificity (60 M-bM-^@M-^S 75%) was obtained at 67-213x average coverage, with 77-92% and 90-98% of targeted regions covered with more than 25% and 10% of the average coverage, respectively. As a more appropriate measure of the evenness of coverage, which is relatively independent of sequencing depth, we introduce the evenness of coverage parameter E. E values up to 75% were achieved. To verify the accuracy of SNP/mutation detection we evaluated 384 known non-reference SNPs in the targeted regions. At ~ 200x average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls while 3.6% of targeted regions were marked as unsurveyed. A total of 1197 new variants were detected. Verification revealed only 8 false positive calls, resulting in an overall false positive rate of less than 1 per ~200,000 bp (0.0005%, equivalent to an overall phred score of 55). 4 samples + capture design file
2010-02-01 | E-GEOD-18542 | biostudies-arrayexpress
Project description:The use of in vitro cell culture systems has been of central importance for research of physiology, pharmacology, and toxicology, and functions at the cellular and molecular levels. We have developed an immortalized oral epithelial cell line ROE2 from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen. Here, to identify genes involved in ROE2 cell differentiation, global-scale gene expression analysis was carried out using a GeneChipM-BM-. system. ROE2 cells, an immortalized oral epithelial cell line from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen, were cultured for 0, 3, 6 and 9 days at 37M-KM-^ZC. Total RNA samples were prepared from the cells, and quality of the RNA was analyzed using a Bioanalyzer 2100. Gene expression was monitored by an Affymetrix GeneChipM-BM-. system with a Rat Genome 230 2.0 array. Sample preparation for array hybridization was carried out as described in the manufacturer's instructions.
2013-02-22 | E-GEOD-44560 | biostudies-arrayexpress