Expression data from Caenorhabditis elegans fed with 13L cocoa peptide
ABSTRACT: Cocoa protein content is a very interesting source for isolation of antioxidant bio-peptides, which can be used for the prevention of age-related diseases. We use microarrays to study the global genome expression of C. elegans fed with a peptide (13L) isolated from cocoa. Wild type strain N2 of C. elegans was fed with 1 µg/mL of 13L peptide or in Nematode Growth medium (MGM, control fed) until reach young adult stage. Worm population were age-synchronized. RNA was isolated from each populations (control and treated) using RNAasy Kit (Qiagen) and hybridizated on Affymetrix microarrays.
Project description:Modulation of gut microbiota through probiotic supplementation is an interesting strategy to prevent obesity We use microarrays to study the global genome expression of C. elegans fed with the probiotic strain Bifidobacterium animalis sbsp. lactis CECT 8145 Wild type strain N2 of C. elegans was cutured in Nematode Growth medium (NGM, control fed) or NGM with a bacterial lawn fed of the strain B. animalis subsp. lactis CECT 8145, until reach young adult stage. Worm population were age-synchronized. RNA was isolated from each populations (control and treated) using RNAasy Kit (Qiagen) and hybridizated on Affymetrix microarrays.
Project description:We have previously reported that tyrosol (TYR), one of the main phenols in extra virgin olive oil (EVOO), promotes lifespan extension in the nematode Caenorhabditis elegans, also inducing a stronger resistance to thermal and oxidative stress in this animal model. Although the influence of several longevity-related genes in these effects has been reported by our group, we decided to perform a whole genome DNA-microarray approach in order to identify other genes and molecular pathways further involved in TYR effects on C. elegans longevity. Microarray analysis identified 208 differentially expressed genes (206 overexpressed and 2 underexpressed) when comparing TYR-treated nematodes with non-treated controls. Many of these genes seem linked to processes such as regulation of growth, transcription, reproduction, lipid metabolism and body morphogenesis. Data obtained by microarray was validated by qRT-PCR analysis of selected genes. Our results confirm that several important cellular mechanisms related to longevity are influenced by TYR treatment in this animal model. Moreover, we detected an interesting overlap between the expression pattern elicited by TYR and those induced by other dietary polyphenols known to extend lifespan in C. elegans, such as quercetin and tannic acid. C. elegans were maintained on Nematode Growth Medium (NGM) and E. coli OP50 as described . For microarray experiments, fer15(b26) nematodes were synchronized by hypochlorite treatment and raised at 25°C on NGM plates containing either 250 μM TYR (n=3) or vehicle (control; n=3). At the fourth day of adulthood, nematodes were collected from the plates in M9 buffer, washed 3 times and pelleted by centrifugation for RNA isolation. After centrifugation, worms were resuspended in 350 μl of RLT/BME buffer, flash frozen in liquid nitrogen and thawed at 37 °C three times for disruption and total RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer recommended protocol. Final volume of isolated RNA was 50 μl per biological sample. RNA quality was analyzed with the 2100 Bioanalyzer (Agilent Technologies) using the RNA 6000 nano kit. All RNA samples were of sufficient quality for gene array analysis with RIN>7. A total amount of 50 ng of RNA was used as the template for cDNA synthesis and in vitro transcription to synthesized biotin-modified aRNA using the GeneChip® 3’ IVT Express Kit (Affymetrix, 901228). aRNA was purified from unincorporated nucleotides and other reaction components using the RNeasy Mini Kit (Qiagen). A total of 15 µg of biotin-labeled aRNA was fragmented following the instructions described in the Affymetrix manual (P/N 702646 Rev.8) and hybridized to C. elegans GeneChip® Genome Arrays (Affymetrix, 900383). They were processed and scanned using Affymetrix instrumentation and with hybridization, washing and scanning parameters provided by the manufacturer. Computational and statistical analyses were carried out using the R software (http://www.r-project.org/) and the appropriate Bioconductor packages (http://www.bioconductor.org/) run under R. In order to remove all the possible sources of variation of a non-biological origin between arrays, densitometry values between arrays were normalized using the RMA (robust multiarray) normalization function implemented in the Bioconductor affylmGUI. Statistically significant differences between groups were identified using the rank product non-parametric test implemented in the Bioconductor Rank-Prod package. Those genes showing a corrected (FDR) p-value < 0.05 were selected as significant.
Project description:Ionizing radiation-induced changes to the redox balance does not only represent a risk for the cellular homeostasis, but can also induce a drastic modulation in the overall signalling system of cells. In the current study, effects of chronic exposure to ionizing gamma radiation were assessed in the radioresistant nematode Caenorhabditis elegans in order to understand whether antioxidant defences (AODs) could ameliorate radical formation, or if increased ROS levels would cause oxidative damage. This analysis was accompanied by phenotypical as well as molecular investigations, via assessment of reproductive capacity and somatic growth and differential gene expression through RNA sequencing. The use of a fluorescent reporter strain (sod1::gfp) and two ratiometric biosensors (Hyper and Grx1-roGFP2) demonstrated increased ROS production (H2O2) and activation of AODs (SOD1 and GPx) in vivo. The data indicate that at dose-rates ≤10 mGy/h defence mechanisms were able to prevent the manifestation of oxidative stress. In contrast, at dose-rates ≥40 mGy/h the constant formation of radicals induced a redox shift, which lead to oxidative damage responses, including changes in mitochondrial metabolism and functions, protein degradation, lipid metabolism, collagen synthesis and modulation of transcription. Moreover, genotoxic effects were among the most over-represented functions affected by chronic gamma irradiation, as indicated by differential regulation of genes involved in DNA damage, DNA repair, cell-cycle checkpoints, chromosome segregation and chromatin remodelling. Ultimately, the exposure to gamma radiation caused reprotoxic effects, with >20% reduction in the number of offspring per adult hermaphrodite at dose-rates ≥40 mGy/h, accompanied by the down-regulation of more than 300 genes related to reproductive system, meiotic functions and gamete development and fertilization.
Project description:The response of the nematode C. elegans to Y. pestis infection was evaluated by gene expression profiling. A synchronized population of nematodes were exposed to Y. pestis KIM5 for 24h. Transcript levels from Y. pestis-treated animals were compared with animals maintained on relatively nonpathogenic E. coli OP50 for 24h. Three independent RNA isolations were performed following exposure to either Y. pestis KIM5 or E. coli OP50. Exposures to the different pathogens were performed in parallel for each replicate isolation.
Project description:In the current study a systematic investigation of life stage, tissue and cell dependent sensitivity to ionizing radiation in the nematode Caenorhabditis elegans was conducted. This revealed that individuals that have reached the post-mitotic L4 stage showed no significant effects with respect to mortality, morbidity or reproduction when subjected to either acute dose ≤6 Gy(1500 mGy/h) or chronic exposure ≤4 Gy( ≤ 100 mGy/h). In contrast, chronic exposure from embryo to young adult stage caused a dose and dose rate dependent reprotoxicitiy with 43% reduction in total brood size at 6.7Gy (107 mGy/h). Systematic targeted irradiation of developmental stages showed that exposure during L1 to young L4 was sufficient to induce reprotoxic effects. Exposure during these stages was associated with a dose rate dependent genotoxic effects on gonads with 1.7 to 3.2 fold increase in germ cell apoptosis in larvae subjected to 40-100 mGy/h, respectively. Importantly, exposure to gamma radiation significantly impaired spermatogenesis in a dose rate dependent manner. The observed reduction in the number of spermatids accounted for xx% of the reprotoxic effects, thus signifying spermatids as the most radiosensitive cell type in C. elegans. Molecular responses analyzed by RNAseq of nematodes irradiated from L1 to L4 stage revealed a significant enrichment of genes related to both male and hermaphrodite reproductive processes. Gene network analysis identified adverse genotoxic effects related to down-regulation of genes required for spindle formation and sperm meiosis/maturation, including smz-1, smz-2 and htas-1. The expression of a subset of 28 set-17 regulated Major Sperm Proteins (MSP) required for spermatids production was correlated to the reduction in reproduction and the number of spermatids, thus corroborating the impairment of spermatogenesis as the major cause of gamma radiation induced life-stage dependent reprotoxic effect. Furthermore, the progeny of irradiated nematodes showed significant embryonal DNA damage that was associated with persistent effect on somatic growth. Unexpectedly, these nematodes did however maintain much of their reproductive capacity in spite of the reduced growth.
Project description:Recent research has highlighted that the polyphenols Quercetin (Q) and Tannic acid (TA) are capable of extending the lifespan of C. elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to Quercetin or Tannic acid concentrations that are non-effective (in lifespan extension), lifespan extending or toxic. The global transcriptome was compared in wild type nematodes raised in the presence of 0, 50, 100, and 200 µM Quercetin (Q) or 0, 100, 200, and 300 µM Tannic acid (TA).
Project description:Hsp90 is a molecular chaperone involved in the regulation and maturation of kinases and transcription factors. In C. elegans Hsp90 participates in the development of fertility, maintenance of muscle structure and the regulation of stress response and dauer state. To understand the consequences of Hsp90-depletion we studied Hsp90 RNAi-treated nematodes by DNA microarrays and mass spectrometry. We find that upon development of phenotypes the levels of chaperones and cofactors are increased, while specific proteins related to the innate immune response are specifically depleted. We further uncover Hsp90-dependent signatures of gonad development and larval development. Using reporter strains for the responsive genes skr-5, dod-24 and clec-60, we confirm an influence on the innate immune response in intestinal tissues, while alterations to development become evident in strains reporting on the presence of ZIP-8, CAV-1 and SEPA-1 in the gonad arms and very early embryos. These observations add molecular details to the Hsp90-dependent processes during development and stress responses and imply that multiple interactions of this chaperone system are relevant for the nematode’s life and development.
Project description:Transcriptional profile of C. elegans comparing control vs. nematodes treated with 0.5mg/l of Diazinon (DZN) at 24°C. Toxicant was added to agar and nematode culture on petri dishes for 72h before harvesting. One condition experiment with six biological replicates in a dye swap design.