Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Profiling of human PBMC exposed to M. leprae

ABSTRACT: Leprosy is major public health problem in developing countries. M. leprae infects human macrophages and Schwann cells leading to a chronic disease. Leprosy studies are constrained due to the lack of in vitro growth system for M. leprae. Moreover, the overall pathogenesis is poorly understood although the expression of some pro-inflammatory cytokines is associated with nerve damage that occurs during the infection. PBMC may participate of the host response to M. leprae infection leading to the inflammatory profile. We aimed to investigate the global human gene expression profile of human PBMC exposed to M. leprae for 4 H. Due to the reduced supply of M. leprae our study was restricted to three chips, including a dye-swap set. Our analysis revealed gene expression differences in cell receptors, NF-kB signaling and oxidative stress response. The results were confirmed by Real-time PCR and flow-cytometry and point to new insights into the molecular events resulted from host-pathogen interaction We analyzed 3 oligo-human chips, 10K elements each, spotted in duplicates. Two chips were replicas and one a dye-swap

ORGANISM(S): Homo sapiens

SUBMITTER: Ulisses Lopes

PROVIDER: E-GEOD-5765 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A mouse hepatoma cell line (Hepa1-6 cells, ATCC) was grown in T-25 tissue culture flasks (Corning) at 37ÂºC in 5% CO2 under two conditions. The first condition was the control, which contained DMEM medium (Gibco BRL) with 25 mM glucose, 4 mM glutamine, 1% penicillin-streptomycin (Sigma), and 10% CPSR-3 (Sigma). The second condition was identical to the first, however the glutamine concentration was altered periodically. Once the cells had grown to confluency, the experiment began at 9:30 PM by exchanging the medium in all flasks. The control flasks received the same medium, containing 4 mM glutamine, while the experimental flasks received identical medium with no glutamine. Twenty-four hours later, the medium was exchanged again, however this time all flasks received medium containing 4 mM glutamine. The first medium exchange was repeated at 48 hours into the experiment, while the second was repeated at 72 hours into the experiment. Hence the glutamine concentration of the experimental flasks oscillated between 0 mM and 4 mM glutamine over 96 hours, while the glutamine concentration of the control flasks remained constant at 4 mM glutamine. At regular intervals, the flasks were sampled. Total RNA was isolated, medium samples were retained, and isotopic measurements were performed from flasks fed different labelled compounds. All samples taken during the experiment were analyzed. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60ÂºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42ÂºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ÂºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples from identical time points that were to be compared on DNA microarrays were then mixed. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65ÂºC. Following concentration, the sample was resuspended in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), 1 uL was removed for quantitation, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 ÂºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride, 239 mL 1-methyl-2-pyrrolidinone, and 10.7 mL of 1 M boric acid, pH 8.0. They were cleaned by rinsing in 250 mL of milli-Q water (X2), followed by 250 mL of 95% ethanol. They were finally dried using filtered air and stored in the dark at room temperature. Keywords = mouse, liver, hepatoma, cell culture, glutamine, carbon metabolism

Project description:Sugar is an important resource for energy generation and developmental regulation in plants, and sucrose starvation causes enormous changes in cellular morphology, enzyme activities and gene expression. Genome-wide gene expression profiling provides a comprehensive knowledge into gene expression under nutrients depletion and senescence, however, that of monocot model plant rice under sucrose depletion is still under investigation. Here, the time-course monitoring of gene expression profiles in sucrose-starved rice (Oryza sativa cv Tainung67) suspension cells was investigated by 21495 probes-containing Agilent rice chip. In sucrose-starved cells, the induced vacuolar biogenesis was coincided with the significantly upregulated expression of genes encoding H+-pyrophosphatase, ï?¤-TIP, one putative ï?¡-TIP, several vacuolar proteases and proteinase inhibitors, and one OsATG3. To survey the overall metabolic adaptations under sucrose depletion the genes significantly alternating expression level were incorporated into multiple metabolic pathways. The majority of genes encoding enzymes involved in biosynthesis and degradation pathways of various macromolecules were comprehensively down- and upregulated, respectively, by sucrose starvation. Transcriptional regulation of gene expression is important for the physiological adaptations to environmental stress and many transcription factors, including bZIPs, NACs, and WRKY showed significant increase in transcript levels under sucrose starvation. Concurrently, statistical analysis reveals that their corresponding consensus cis-elements, such as ABA-responsive element, CACG, ACI, ACII and CTTATCC, are frequently found in the promoter regions of many Suc starvation-upregulated genes. Particle bombardment-mediated transient promoter activity assays further showed that the CTTATCC, derived form TATCCA, and the AC motifs, are the promising sucrose starvation responsive activators in sucrose-starved rice suspension cells. Experiment Overall Design: We used Agilent rice gene chips to investigate the gene expression profiling in rice cells after periods of 12, 24 and 48 h Suc starvation. To eliminate the effect of osmotic stress on alternation of gene expression, an additional set of control, 88 mM Sorbitol in MS medium verse Suc-free samples for 12 h Suc starvation, was also applied for array analysis.

Project description:Cows were selected from two groups of 12 cows enrolled in a large experiment to assess the effects of ad libitum or restricted intake of moderate-energy diets during the entire dry period on pre-partum metabolism and post-partum metabolism and performance. A corn silage-based diet (26% of diet dry matter) providing 1.59 Mcal/kg during the far-off dry period (first 5 wk of an 8-wk dry period) or providing 1.61 Mcal/kg during the close-up dry period (last 3 wk of the dry period) was fed for ad libitum or restricted intake. Four multiparous Holstein cows were randomly selected from the ad libitum and restricted intake groups. A 7,872-element cDNA microarray spotted in duplicate on amino silane-coated glass slides (Everts et al. 2005, Vet. Immunol. Immunopathol. 105:235-245) was used for transcript profiling. Annotation was based on similarity searches (January, 2005) using sequential BLASTN and TBLASTX against human and mouse UniGene databases and the human genome. The 7,872-element array represents more than 6,300 unique genes. Gene Ontology (GO) terms were parsed from LocusLink to functionally annotate cDNA sequences. RNA (20 ug) from liver tissue and a reference standard derived from a mixture of tissues not including liver were used to make aminoallyl-labeled cDNA followed by incorporation of Cy3-ester and Cy5-ester (Amersham, Piscataway, NJ). Each experimental sample was co-hybridized with the reference standard, which allowed the treatment of fluorescence ratios as measurements of relative expression across all samples and time points. Probes were hybridized to the array for 2 days at 42 C. All samples were hybridized to duplicate slides for a total of 4 spots per cDNA element. A total of 106 microarrays were run to complete the experiment. Slides were scanned for both dye channels with a Scanarray 4000 (GSI-Lumonics, Billerica, MA) dual-laser confocal scanner and images were processed using GenePix 4.0 (Axon Instruments, Inc., CA).

Project description:Approximately half of M. lepraeâ??s transcriptome consists of inactive gene products. This has an impact on overall energy and resource consumption without potential benefit to this organism. However, multiple translational â??silencingâ?? mechanisms are present, reducing additional energy and resource expenditure required for protein production from these transcripts. The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials should increase our understanding of their impact on M. leprae physiology. To address this, M. leprae was purified from the granulomatous hind footpad tissue of four individual nu/nu nude mice six months post-infection. M. leprae whole genome DNA microarrays representing the 1,614 annotated ORFs and 1,133 identified pseudogenes, were obtained from the Leprosy Research Support and Maintenance of an Armadillo Colony Post-Genome Era, Part I: Leprosy Research Support Contract (NO1 AI-25469) at Colorado State University. To validate 20% of genes positive by microarray analysis, RT-PCR was performed. Results of this study Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational silencing of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno sequences. Transcribed pseudogenes also contained multiple in-frame stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational silencing mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts. The overall design of this study was to identify the transcriptome of M. leprae in the granulomatous tissue of the mouse hind foot pad 6 months post infection.

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Profiling of human PBMC exposed to M. leprae

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