Drosophila gene expression in response to the botanical insecticide Piper nigrum
ABSTRACT: This experiment was used to determine the effect of a botanical insecticide upon gene expression profiles in Drosophila melanogaster. Adult female Drosophila (oregon-R strain) were treated with an ethylacetate extract of Piper nigrum (Piperaceae) seeds formulated in 99% ethanol. Treatment was topical, using a Potter's tower to administer a total of 2 mL of a 0.9mg/mL concentration. Control treatment was identical except flies were treated with 99% ethanol as a solvent control. Gene expression was studied four hours post-treatment. The design is a direct comparison between total RNA of treated and untreated insects. Sample size is two and two reverse label hybridizations were done. Each sample is a pool of 240 insects which received the same treatment simultaneously.
Project description:This experiment was intended to determine if topical exposure to ethanol led to significant changes in gene expression in adult Drosophila melanogaster. It was determined that topical exposure to ethanol did not lead to a strong differential expression of genes in Drosophila after 4 hours. The effect of topical treatment with 99% ethanol was compared to the effect of a water treatment which was used as a control and the differential expression between these two treatments was assessed four hours after treatment. Two biological replicates of each treatment were compared for a total of two microarray hybridizations
Project description:Enteric pathogens have developed several resistance mechanisms to survive the antimicrobial action of bile. We investigated the transcriptional profile of Vibrio cholerae O1 El Tor strain C6706 under virulence gene inducing conditions, in the presence and absence of bile. Microarray analysis revealed that the expression of 119 genes was affected by bile. The mRNA levels of genes encoding proteins involved in transport was increased in the presence of bile, whereas mRNA levels of genes encoding proteins involved in pathogenesis and chemotaxis was decreased. This study identified genes encoding transcriptional regulators from the TetR family (vexR and breR) and multidrug efflux pumps from the RND superfamily (vexB and vexD [here renamed breB]) that were induced in response to bile. Further analysis regarding vexAB and breAB expression in the presence of various antimicrobial compounds established that vexAB was induced in the presence of bile, SDS or novobiocin, whereas induction of breAB was specific to bile. BreR is a direct repressor of the breAB promoter and is able to autoregulate its own expression, as demonstrated by transcriptional and electrophoretic mobility shift assays (EMSA). Expression of breR and breAB is induced in the presence of the bile salts cholate, deoxycholate or chenodeoxycholate and EMSA showed that deoxycholate is able to abolish formation of BreR-PbreR complexes. We propose that deoxycholate is able to interact with BreR and induce a conformational change that will interfere with its DNA binding ability resulting in breAB and breR expression. These results provide new insight into a transcriptional regulator and a transport system that likely play an essential role in the ability of Vibrio cholerae to resist the action of bile in the host. Keywords: Vibrio cholerae response to bile Three independent experiments were performed for the microarray analysis. From each experiment, RNA was obtained from four different time points and was prepared for hybridization, therefore, a total of 3 slides were analyzed per time point. The growth conditions were as follows; C6706 str2 was grown 15 h in LB medium at 30 degrees C with aeration. The cultures were then diluted 100-fold into AKI medium with, or without, 0.4% crude bile (high bile concentration) and were grown at 37degrees C for 3.5 h stationary followed by 2 h with aeration to induce virulence gene expression. Then the cultures were diluted 100-fold into AKI medium with, or without, 0.02% crude bile (low bile concentration) and were grown at 37 degrees C for 2 h stationary. Samples were obtained from 4 different time points: 2, 4, and 5.5 h from the high bile cultures, and at 2 h from the low bile cultures.
Project description:Endothelial responses to LPS using endothelial cells freshly isolated from Tie2 GFP animals vs. cultured cell line. This SuperSeries is composed of the following subset Series: GSE1042: Cardiac endothelial in vivo responses to LPS GSE1043: In vitro LPS response of SVEC cells
Project description:Oxygen lack of various severity can force many organisms to enter into recoverable hypometabolic states. To better understand how organisms cope with oxygen deprivation, our lab had previously shown that when challenged with anoxia, both the nematode Caenorhabditis elegans and embryos of the zebrafish Danio rerio enter into suspended animation, where all life processes that can be observed by light microscopy reversibly halt, pending restoration of oxygen. Here, we show that both sporulating and vegetative cells of the budding yeast Saccharomyces cerevisiae also enter into a similar state of suspended animation when made anoxic on a non-fermentable carbon source. Transcriptional profiling using cDNA microarrays shows upregulation of aerobic metabolism genes in carbon monoxide (CO)-induced anoxia, but not nitrogen (N2) gas-induced anoxia, consistent with the known oxygen-mimetic effects of CO. Our results lead us to propose a model for oxygen-regulated gene expression in yeast where two oxygen-sensitive mechanisms operate simultaneously, such that treatment with N2 results in both mechanisms signaling a lack of oxygen, while treatment with CO results in one sensing mechanism signaling a lack of oxygen, while the other signals an abundance of oxygen. Cells were pregrown on glucose media. Cells were then plated onto nylon membranes on acetate solid media and made anoxic using either pure nitrogen or carbon monoxide. Cells were collected at 15, 30, 45, 60, 120 minutes and 24 hours after initiation of gas exposure. Reference samples were derived from cells on acetate in room air for corresponding time point. Six CO-treated samples were compared to room air references and six nitrogen-treated samples were similarly compared to room air references.
Project description:Yeast cells can be affected during their growth to several stress conditions. One of the most known and characterised is the osmotic stress and most of the studies about osmotic sterss response in yeast have been focused on salt or sorbitol stress. However, during yeast growth in industrially relevant processes (for instance throughout alcoholic fermentation on the must to produce alcoholic beverages) the osmotic stress is mainly due to the high sugar(in particular glucose) concentration (200-250 g/L). In this study we want to know the transcriptional response of the Saccharomyces cerevisiae when it was grown in a medium with high glucose concentration. For this aim we have grown yeast in YP medium containing 2% of glucose in cultures overnight and after that we diluted this cultures to an OD600 of 0.1 in two differents mediums: YP containing 2% or 20% of glucose.One hour later of inoculation we collect the cells and quikly frozen in liquid nitrogen. We extracted the total mRNA of the cells and after that we did the microarrays, comparing cells were grown in YP2 media against the cells were grown in YP20 media.
Project description:The HIVEP/ZAS/Schnurri genes encode large zinc finger proteins that regulate gene expression through DNA binding to the kappaB motif. The ZAS proteins have also been shown to associate with signaling molecules to regulate the TNFa and TGFb signaling pathways. Because ZAS3 transcript and protein expression is rapidly abrogated in primary lymphocytes upon tissue culture, we have generated a ZAS3-null mouse model to study the physiological function of ZAS3. Mice with targeted disruption of ZAS3 are viable with life span comparable to controls. Additionally, the gross anatomy and histology of the major organs, proliferation rates of splenocytes and thymocytes, and the diversity of the TCRb chains of ZAS3 mice are comparable to wild-type. However, compared to wild-type mice, there are decreased CD3+CD4+ and CD3+CD4+CD69+ thymocyte populations. Additionally, CD44hi/CD62Llo subset and CD25 and CD69 expression are increased in splenocytes of ZAS3 mice. Microarray analysis validated by real time PCR revealed changes in the expression level of specific transcripts in ZAS3-null thymus, including several proteins in the G-protein coupled olfactory receptor family. Furthermore, EMSA shows that disruption of ZAS3 results in varying changes in binding activities towards NF-kB and AP1 binding sites. Other phenotypes observed in the ZAS3 mice include generalized increased bone density in older animals and infertility in females. As with ZAS2/shn-2 mice, disruption of ZAS3 is not lethal and does not grossly affect development or homeostasis. We propose that the ZAS proteins likely have overlapping functions, which may be uncovered with deletion of multiple ZAS genes in a single animal. Experiment Overall Design: Five independent microarray hybridizations were performed with thymocytes isolated from sex- and age-matched mutant and wild-type littermates at 6-8 weeks of age. The Agilent Whole Mouse Genome Oligo Microarray (G4121A) containing over 20,000 60-mer oligonucleotide probes representing mouse genes, ESTs and EST clusters was used (Agilent, Cincinnati, OH). Probe labeling and hybridization were preformed by the Microarray Core Faculty (Columbus Children’s Research Institute) following the manufacturer's protocols. Briefly, cDNA probes were synthesized with Superscript III, oligo-dT primers and dNTPs supplemented with amino-allyl-UTP to improve hybridization characteristics and stability. After purification, cDNA samples from mutant or control mice were labeled with Cy3 or Cy5, respectively, and hybridized to the microarray for 14 hours at 48o C. Slide image was acquired using an Affymetrix 428 scanner with gain settings set so that 95% of spots were below saturation to yield the maximum dynamic range within an experiment. Images were converted into “.gpr” files using GenePix software (Axon, Union City, CA). Data were analyzed using GeneTraffic 2.6 software (Iobion, La Jolla, CA). Lowess-global normalization was applied to all experiments. Flagging parameters were set as spot intensity lower than the intensity of local spot background, spot intensity lower than average background, and raw spot intensity less than 100. Flagged spots were not included in normalization or aggregate calculations. Only genes that were induced or repressed 1.5-fold in at least four of the five independent analyses and hybridization were targeted for potential further study.
Project description:Pharmaceuticals are pseudo persistent aquatic pollutants with unknown effects at environmentally relevant concentrations. Atlantic salmon (Salmo salar) were exposed to Acetaminophen: 54.77 ± 34.67; Atenolol: 11.08 ± 7.98 and Carbamazepine: 7.85 ± 0.13 µg•L-1 for 5 days. After Acetaminophen treatment, 19 proteins were differently expressed, of which 11 were significant with respect to the control group (eight up-regulated and three down-regulated). After Atenolol treatment, 7 differently expressed proteins were obtained in comparison with the control, of which 6 could be identified (four up-regulated and 2 down-regulated). Carbamazepine exposure resulted in 15 differently expressed proteins compared with the control, with 10 of them identified (seven up-regulated and three down-regulated). Out of these, 3 features were common between Acetaminophen and Carbamazepine and one between Carbamazepine and Atenolol. One feature was common across all treatments. Principal component analysis and heat map clustering showed a clear grouping of the variability due to the applied treatments. The obtained data suggest (1) that exposure to environmentally relevant concentrations of the pharmaceuticals alters the hepatic protein expression profile of the Atlantic salmon; and (2) the existence of treatment specific processes that may be useful for biomarker development.
Project description:Breast Cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC-associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot study, using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE)-based mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)-based MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her mil. Statistically different gel spots were picked for protein digestion followed by nanoLC-MS/MS analysis. The upregulated proteins in BC versus control are alpha-amylase, gelsolin isoform a precursor, alpha-2-glycoprotein 1 zinc isoform CRA_b partial, apoptosis-inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease.