Project description:To characterize the ecological interactions among S. cerevisiae strains coming from the same geographical area, we examined the fitness of two natural isolates from San Giovese grapes, alone or in competition, in synthetic wine must (SWM). We performed genome-wide analyses in order to identify the genes involved in yeast competition and cooperation. 2 samples and 4 replicates.
Project description:To characterize the ecological interactions among S. cerevisiae strains coming from the same geographical area, we examined the fitness of two natural isolates from San Giovese grapes, alone or in competition, in synthetic wine must (SWM). We performed genome-wide analyses in order to identify the genes involved in yeast competition and cooperation. 2 samples and 2 replicates.
Project description:Variance in microarray studies has been widely discussed as a critical topic of the identification of differentially expressed gene; however, few studies have addressed the influence of estimating variance. To break intra- and inter-individual variance in clinical studies down to three levels: technical, anatomic, and individual, we designed experiments and algorithms to investigate three forms of variances. As a case study, a group of “inter-individual variable genes” were identified to exemplify the influence of underestimated variance on the statistical and biological aspects in identification of differentially expressed genes. Our results showed that inadequate estimation of variance inevitably led to the inclusion of non-statistically significant genes into those listed as significant, thereby interfering with the correct prediction of biological functions. Applying a higher cutoff value of fold changes in the selection of significant genes reduce/eliminate the effects of underestimated variance. Our data demonstrates that an appropriate evaluation of variance is critical in selecting significant genes of differential expression. If the estimation of precise variance has not been adequately considered in the experimental design, using a higher fold change criteria is one possible solution to overcome the difficulties associated with the identification of significant genes, but it paid by losing the number of selected genes. A total of 11 normal placenta tissues obtained from 9 healthy individuals with term pregnancies, whom underwent cesarean section without labor pain. The first sample group (G1) was composed of samples 1 to 9 of nine individuals. The second sample group (G2) contained 8-1, 8-2 and 8-3, which were three different placental tissues taken from the same individual. The third sample group (G3) consisted of two technical replicates, 8-3_1 and 8-3_2, using the identical RNA pool. Sample 8-3 is estimated by sample 8-3_1 and 8-3_2. Sample 8 is estimated by sample 8-1, 8-2 and 8-3.
Project description:Human multipotent mesenchymal stem cells (MSCs), isolated based on their adherence to plastic, show poor growth and differentiation and frequently contain contaminating cells, which limits to clarify their own characteristics. In this study, the identification of two cell surface markers, LNGFR and Thy-1, allowed the prospective isolation of highly purified clonogenic human MSCs. Furthermore, single cell sorting assays followed by in vitro expansion revealed three distinct MSC subpopulations: rapidly-, moderately- and slowly proliferating MSC clones (RPCs, MPCs and SPCs, respectively). While RPCs exhibited robust multilineage differentiation and self-renewal potency, MPCs and SPCs contained a majority of senescent cells and exhibited frequent genome errors. Single cell sorting assays followed by in vitro expansion revealed three distinct MSC subpopulations: rapidly-, moderately- and slowly proliferating MSC clones (RPCs, MPCs and SPCs, respectively). Sample: RPC, MPC and SPC (n=3).
Project description:In a study to elucidate the genetic defects in patients with X-linked intellectual disability (XLID) we performed X chromosome-specific BAC-array-CGH and identified 0.33 to 1.0 Mb nonrecurrent copy number gains at Xp11.22 in affected males of unrelated XLID families. All aberrations segregate with the disease in the families and the carrier mothers show a nonrandom X-inactivation. Affected males suffered from mild to moderate ID. Tiling Xp11.22 region-specific oligo-array (ChrX:52.50 - 54.50 Mb) revealed that all aberrations had different start and stop sites. The twofold copy number gain included up to 20 genes but only the HUWE1 gene is located in the minimal common region of overlap in these families. Moreover, expression analysis revealed about twofold increased HUWE1 mRNA levels in affected patients when compared to control individuals. Breakpoint analysis revealed Non-homologous end-joining (4 cases), serial replication slippage (1 case) and non-allelic homologous recmbination (one case) as the potential recombination mechanism. For duplication mapping and exact copy number analysis in all four families, differentially-labeled patient versus male control DNA samples were hybridized onto a custom designed 4x44k oligo-array (Agilent Technologies) that covers the repeat-masked region 52.50 Mb to 54.50 Mb at tiling resolution.
Project description:Fluorosurfactants are the key components in aqueous film forming foams (AFFF). They provide these fire fighting agents with the required low surface tension and they enable film formation on top of lighter fuels to prevent burn back. Development of effective and environmentally acceptable PFOS alternatives is one of the most important priorities in the fire fighting foam industry. DuPontTM offers the fluorosurfactant mixtures Forafac®1157 and Forafac®1157N for the formulation of AFFFs which are alternatives to the persistent and toxic perfluorooctane sulphonate (PFOS). Ecotoxicological testing of these inadequately documented mixtures is necessary to include them in AFFF hazard and risk assessment. Juvenile turbot (Scophthalmus maximus) was exposed for 14 days to 0.5 and 1.5 mg/l of the fluorosurfactant mixtures used in Forafac®1157 and Forafac®1157N. In a first transcriptomics experiment, microarray analysis revealed differentially expressed gene transcripts which were mainly involved in digestion and in the immune system. This discovery-driven screening approach offered the basis for new hypotheses that were tested in two subsequent experiments in which food intake, energy reserves, growth and a set of haematological parameters were examined. Additionally, effects of the two mixtures were compared to those of PFOS. Based on the results of this study, the mode of action of Forafac®1157N was the activation of the acute phase reaction resulting in increased leukocyte concentrations and the inhibition of growth due to the high energetic cost of toxicant exposure. For Forafac®1157, evidences of immunosuppression were found on the transcriptional level and the altered differential leukocyte profiles indicated that stress was induced in these fish. However, food intake, energy reserves and growth were not compromised, even at high exposure concentrations, which was in contrast to the effects seen after PFOS exposure. Taking into account that Forafac®1157 appeared to be less toxic than PFOS, this mixture could be considered as a more environmentally acceptable PFOS alternative for the use in AFFFs. Juvenile turbot with a mean weight of 7.21 ± 1.91 g and a mean length of 7.34 ± 0.33 cm were exposed to nominal concentrations of 0 mg/L; 0.5 mg/L and 1.5 mg/L for both Forafac®1157 and Forafac®1157N during 14 days. Three different 45 L aquaria per exposure condition were used resulting in 3 tank replicates. Each replicate aquarium contained six turbot. After decapitation, the liver was dissected, homogenized in liquid nitrogen and stored at -80 °C until further analysis. The liver homogenates of the 6 fish per aquarium of exposure experiment 1 were pooled prior to RNA extraction. After RNA extraction, fluorescently labelled cRNA was constructed with each sample labelled with Cy3 as well as with Cy5.An Agilent custom 15k oligonucleotide microarray (Agilent Technologies) was used to measure gene transcription levels. The microarray platform, constructed by ACUIGEN (University de Santiago de compostela, Spain), contained 4 305 turbot-specific oligonucleotide fragments originating from an immune-related EST turbot database. Two probe volumes (Cy3 vs. Cy5) corresponding with 300 ng cRNA were mixed and applied onto every microarray. The hybridization design comprised one separate n+2 A-optimal design for each of the Forafac® mixtures.
Project description:Microarray analysis with 40,000 cDNA gene chip arrays determined differential gene expression profiles (GEPs) in CD34+ marrow cells from myelodysplastic syndrome (MDS) patients compared to normal individuals. Using focused bioinformatics analyses, we found 1175 genes significantly differentially expressed by MDS vs Normal, requiring a minimum of 39 genes to separately classify these patients. Major GEP differences were demonstrated between Normal and MDS patients and between several MDS subgroups: (1) those whose disease remained stable (sMDS) and those who subsequently transformed (tMDS) to acute myeloid leukemia (AML); (2) between del(5q) and other MDS patients. A 6-gene ‘poor risk’ signature was defined which was associated with AML transformation and provided additive prognostic information for IPSS Intermediate-1 patients. Over-expression of genes generating ribosomal proteins and for other signaling pathways was demonstrated in the tMDS patients. Comparison of del(5q) to the remaining MDS patients showed 1924 differentially expressed genes, with under-expression of 1014 genes, 11 of which were within the 5q31-32 Commonly Deleted Region. These data demonstrated (1) GEPs distinguishing MDS patients from normal and between those with differing clinical outcomes (tMDS vs sMDS) and cytogenetics [eg, del(5q)] ; and (2) molecular criteria refining prognostic categorization and associated biologic processes in MDS. Gene expression profiles from CD34+ cells of 35 MDS subjects and 6 Normals were compared.
Project description:In a study to elucidate the genetic defects in patients with X-linked mental retardation (XLMR) we performed X chromosome-specific BAC-array-CGH and identified a 0.33 Mb inherited recurrent copy number gain at Xq28 in affected males of four unrelated XLMR families. All aberrations segregate with the disease in the families and the carrier mothers show a nonrandom X-inactivation. Tiling Xq28 region-specific oligo-array revealed that all aberrations start at the same position (153.218 Mb) and end between 153.530 and 154.542 Mb. The copy number gain is complex in nature but always included 18 genes of which three, RPL10, ATP6AP1 and GDI1, are highly expressed in brain. From these, the copy number of GDI1 correlated with the severity of clinical features since it was duplicated in one family with nonsyndromic moderate MR, triplicated in males from two families with mild MR and additional features, while in a fourth family with a severe syndromic form of MR, it was present in four copies. Moreover, expression analysis revealed copy number-dependent increased mRNA levels in affected patients compared to control individuals. Interestingly, the breakpoint junction regions suggested a yet unknown recombination mechanism between two adjacent but different sets of low copy repeats. For duplication mapping and exact copy number analysis in all four families, differentially-labeled patient versus male control DNA samples were hybridized onto a custom designed 4x44k oligo-array (Agilent Technologies) that covers the repeat-masked region 152.70 Mb to 153.65 Mb at tiling resolution.
Project description:The widespread commercial use of TiO2-NPs, such as in medical stents, has led to extensive investigations on their toxicity and biological impact but lacks a detailed study on their effects. Proteomics and transcriptomics are key methodologies to unravel cellular dynamics, macromolecular interactions and biological response to NP exposure through the protein or gene identification and quantification. We focus on integration of several Omics strategies that could offer a more complex understanding of the impact. In this study, our goal was to gain insight into the cellular response after the exposure to sublethal concentration of TiO2-ultra small nanoparticles (USNPs) and NPs by analyzing the modification on the genomic and proteomic expression in human microdermal endothelial cells (HMEC-1). We investigated the correlation between the NP size and the cellular response to the exposure. We evaluated if multiOmics approaches could thus reveal more extensive information on the potential cellular responses to NPs exposure.
Project description:In Drosophila, the accessory gland proteins (Acps) secreted from the male accessory glands (MAGs) and transferred along with sperm into the female reproductive tract have been implicated in triggering postmating behavioral changes, including refractoriness to subsequent mating and propensity to egg laying. Recently, Acps have been found also in Anopheles, suggesting similar functions. Understanding the mechanisms underlying transcriptional regulation of Acps and their functional role in modulating Anopheles postmating behavior may lead to the identification of novel vector control strategies to reduce mosquito populations. We identified heat-shock factor (HSF) binding sites within the Acp promoters of male Anopheles gambiae and discovered three distinct Hsf isoforms; one being significantly up-regulated in the MAGs after mating. Through genome-wide transcription analysis of Hsf-silenced males, we observed significant down-regulation in 50% of the Acp genes if compared to control males treated with a construct directed against an unrelated bacterial sequence. Treated males retained normal life span and reproductive behavior compared to control males. However, mated wild-type females showed a ∼46% reduction of egg deposition rate and a ∼23% reduction of hatching rate (∼58% combined reduction of progeny). Our results highlight an unsuspected role of HSF in regulating Acp transcription in A. gambiae and provide evidence that Acp down-regulation in males leads a significant reduction of progeny, thus opening new avenues toward the development of novel vector control strategies. Total RNA from 4-d-old dsLacZ-treated controls and HSF-silenced males was extracted using TRIzol reagent (Life Technologies), following protocols set according to manufacturer’s instructions. RNA preparation, labelling, hybridization and data analysis were performed by the Oxford Gene Technology (OGT) company. In particular, RNA was further cleaned up using the RNeasy Mini Kit (Qiagen) followed by ethanol precipitation. Sample passing the purity metrics (260/280 and 260/230 ratio) were considered for labeling and hybridization procedures. Labeling was done with cyanine 3 (dsHSF1 and dsHSF123) and cyanine 5 (control dsLacZ). Samples and controls were hybridized in triplicates to the Agilent Mosquito Gene expression arrays (4x44,000, Agilent Technologies, Santa Clara, CA, USA). Data analysis was performed by OGT according to a standardized procedure, as implemented by the software applications Feature Extraction (version 22.214.171.124.), and Genespring GX (version 11.0). Expression data was first normalised using linear and loess normalization in Feature Extraction, then it was imported into Genespring where it was converted into normalised log ratios. The distribution of the data between and within the treatments was checked via boxplots and hierarchical clustering to confirm consistency. Intensity measures from spots flagged as being of poor quality were excluded. Then, the control probes were removed for the statistical analysis: t-tests were carried out for each condition with null hypothesis of log ratio being equal to zero. This was done on the normalised log ratios from the 3 technical replicates. Probes meeting a cut-off of p<0.05 with Benjamini-Hochberg multiple testing correction and with a fold change cutoff greater than 2 were considered statistically and biologically relevant and were used to generate lists of differentially expressed targets.