Nutritional condition dependant differential tolerance to hydrogen peroxide stress in Escherichia coli
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ABSTRACT: Escherichia coli culture was subjected to two different types of nutritional scenarios, abundant carbon/ nitrogen sources and scarce carbon/nitrogen medium. Study revealed that scarce medium adapted culture were more tolerant to hydrogen peroxide than abundant medium.
Project description:Model topology is divided into two compartments, cell programming and performance testing. The cell programming compartment is split into history and pre-treatment. History ( History I or H1: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask after that. History II or H2: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask for 45 min. From this flask, 0.1O.D./ml transferred to rich medium and grown for 4 hours. From this,0.1O.D./ml transferred to fresh rich medium. History III or H3: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask for 45 min. From this flask, 0.1O.D./ml transferred to starvation medium and grown for 4 hours. From this,0.1O.D./ml transferred to fresh starvation medium. Pre-treatment (Pre-treatment 1(T1): 2.5g glucose/litre 5mM NH4Cl. Pre-treatment 2 (T2): 2.5g glucose/litre 0.25mM NH4Cl. Pre-treatment 3 (T3): - 0.25g glucose/litre 5mmM NH4Cl. Pre-treatment 4 (T4): 0.25g glucose/litre 0.25mM NH4Cl). Each pre-treatment given for 2.5 hours.The culture nomenclature indicates the adaptive path followed, for example, H1T1 indicates the culture has encountered history I (H1) and then transferred to pre-treatment 1 (T1).RNA was extracted for selected combinations. Performance testing : Performance testing describes the type of analysis done which is the growth pattern study onto three substrates, glucose, succinate and pyruvate. This performance testing revealed specific history-pretreatment combinations to be better suited for growth on certain substrate and some not suited for growth. The samples were harvested for RNA isolation at peak growth points and named worst_glucose, best_glucose,worst_succinate, best_succinate, worst_pyruvate and best_pyruvate according to the growth shown after testing all 12 history-pretreatment combinations. The differences in physiology were studied in details using microarray analysis of 13 samples including 3 history samples, 4 pre-treatment samples and 6 samples at performance testing level. RNA extraction was done using Qiagen RNeasy minikit (Germany). Standard Affymetrix protocol was followed for hybridization on Affymetrix E. coli Genome 2.0 Array.
Project description:RNA pools were labeled and hybridized to an Affymetrix HG-U133 Plus 2.0 array (Affymetrix, USA). Results derived from untreated OCI-AML cells (sample) were compared to results from OCI-AML treated with TAT-2 (1 mg/mL) for 3 h by M-^Scomparative analysisM-^T with GCOS software. Data were then analyzed using GenePicker software, using a fold change cutoff >1.5 and a p-value of 0.05.
Project description:The goal was to screen for the expressed genes in Semi-Circular Canal Duct (SCCD) that are related to ion transport and its regulation. The objective was to discover which genes changed expression levels in response to glucocorticoids. Keywords: drug response Primary cultures were incubated for 24 hours in the presence or absence of 100 nM dexamethasone. Four independent cultures from each group were processed for total RNA and submitted for gene array analysis. Although two Samples out of four in each group were done at different time points, we found that there was not much variation between the datasets (by cluster analysis).
Project description:The objective of the study was to find cardiac GATA-4 target genes by overexpressing GATA-4 transcription factor in the left ventricle by adenoviral gene transfer. Gene expression profiles three days after GATA-4 gene transfer were compared with those of Lac Z –treated animals by screening Affymetrix Rat Expression Set 230_2.0 Arrays (there are 5 samples in both group). Strain:Sprague-Dawley; Gender, Male; Weight 250-300g; tissue, left ventricle.
Project description:Avian Pathogenic Escherichia coli (APEC) are a group of extra-intestinal E. coli that infect poultry, and are able to cause a variety of diseases, systemic or localized, collectively designated as colibacillosis. Colibacillosis is the most common bacterial illness in poultry production, resulting in significant economic losses world-wide. Despite of its importance, pathogenicity mechanisms of APEC strains remain not completelly elucidated and available vaccines are not fully effectives. In order to better understand which genes could be related to pathogenicity in different APEC isolated, a microarray analyses of two APEC strains representing: Swollen Head Syndrome and Omphalitis was carried out. We used the microarray methodology to evaluate the expression profile of two different APEC strains
Project description:Cancer cells utilize a unique form of aerobic glycolysis, called the Warburg effect, to efficiently produce the macromolecules required for proliferation. Here we show that a metabolic program related to the Warburg effect is used during normal Drosophila development and regulated by the fly ortholog of the Estrogen-Related Receptor (ERR) family of nuclear receptors. dERR null mutants die as second instar larvae with abnormally low ATP levels, diminished triacylglyceride stores, and elevated levels of circulating sugars. Metabolomic profiling revealed that the pathways affected in these mutants correspond to those used in the Warburg effect. The expression of active dERR protein in mid-embryogenesis triggers a coordinate switch in gene expression that drives a metabolic program supporting the dramatic growth that occurs during larval development. This study suggests that mammalian ERR family members may promote cancer by directing a metabolic state that supports proliferation. Drosophila larvae were staged at a mid-second instar time point and hand sorted for developmental progression. Individual pools of isogenic animals were collected for each replicate. Three replcates were assayed for each genotype. The two genotypes assayed were a control wild type strain (w1118) and a transheteroallelic combination of err mutant alleles (err1/err2). Labled RNA was then hybridized onto Affymetrix microarrays.
Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA. Cell culture conditions and media Human fibroblast cell strains (WS: AG05229, AG12795, AG12797; BS: GM02932, GM03402, GM16891; RTS: AG18371, AG18375, AG05013; Normal/Wild-type: AG04054, AG06310, AG09975) were obtained from the Coriell Repository (Camden, NJ), from donors matched for gender and of similar ages, and were at similar passage levels. Cells were cultured in MEM supplemented with Earle’s salts, 20% fetal bovine serum, 1x penicillin/streptomycin, and 1x fungizone in 3% O2 at 37oC and harvested for RNA extraction during active growth and at ~ 80% confluence. GeneChip microarray expression Total RNA from the 12 fibroblast cell strains was isolated by extraction with TRIzol (Invitrogen) and purified using the RNeasy system (Qiagen). Total RNA was amplified by in vitro transcription using the Ovation RNA Amplification System V2 (NuGen). The resultant cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen), and then purified using QIAquick columns (Qiagen), as specified by the Ovation System manual. Labeled probe was hybridized to Affymetrix U133A 2.0 GeneChips, and ultimately scanned using an Axon GenePix array scanner. Statistical analysis of microarray expression experiment The output files were normalized by Robust Multiarray Average (RMA), using the R package GCRMA and gene expression levels were log2-transformed.
Project description:The molecular mechanism defining susceptibility of normal cells to oncogenic transformation may be a valuable therapeutic target. We characterized the cell of origin and its critical pathways in MN1 leukemias. Common myeloid (CMP), but not granulocyte-macrophage progenitors (CMP) could be transformed by constitutively overexpressed MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that leukemogenicity of MN1 required the MEIS1/abdB-like HOX protein complex. Colocalization studies by ChIP-seq identified common chromatin targets of MN1 and MEIS1 that were associated with open chromatin and transcriptional activation. Transcriptional repression of MEIS1 target sites in established MN1 leukemias had antileukemic activity. As MN1 relies on but can not activate expression of MEIS1/abdB-like HOX proteins, transcriptional activity of these genes determines which cell is the cell of origin in MN1 leukemia. We have showed at the single cell level that CMPs, but not GMPs, are susceptible to MN1-induced transformation. To identify transcriptional differences between CMPs and GMPs that may explain this difference in susceptibilities to MN1 transformation we produced gene expression profiles (two biological replicates in each experimental arm) of bone marrow cells from MN1 leukemic mice and mature myeloid bone marrow cells (Gr1+/CD11b+) from healthy mice and compared those to already published gene expression profiles of CMPs and GMPs (Krivtsov, A.V., et al. (2006). Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442, 818-822).
Project description:We have developed a novel spontaneous model of epithelial-to-mesenchymal transition (EMT) which involves four phenotypically distinct clones derived from a primary tumour-derived human prostate cancer cell line (OPCT-1), and its use to explore relationships between EMT and the generation of cancer stem cells (CSCs) in prostate cancer. Expression of epithelial (E-Cadherin) and mesenchymal markers (vimentin, fibronectin) revealed that two of the four clones were incapable of spontaneously activating EMT, whereas the others contained large populations of EMT-derived, vimentin-positive cells having spindle-like morphology. One of the two EMT-positive clones exhibited aggressively and stem cell-like characteristics, whereas the other was non-aggressive and had no stem cell phenotype. One of the two EMT-negative clones exhibited aggressive stem cell-like properties, whereas the other was the least aggressive of all clones. To understand this phenotypic differences between the clones we have conducted a microarray expression profiling experiment using Affymetrix platform.
Project description:We explored the transcriptional response to parasitoid attack in Drosophila larvae at nine time points following parasitism, hybridizing five biologic replicates per time point to whole-genome microarrays for both parasitized and control larvae. We found significantly different expression profiles for 159 probe sets (representing genes), and we classified them into 16 clusters based on patterns of co-expression. A series of functional annotations were nonrandomly associated with different clusters, including several involving immunity and related functions. We also identified nonrandom associations of transcription factor binding sites for three main regulators of innate immune responses (GATA/srp-like, NF-kappaB/Rel-like and Stat), as well as a novel putative binding site for an unknown transcription factor. The appearance or absence of candidate genes previously associated with insect immunity in our differentially expressed gene set was surveyed