A multiomics study on the metabolism of Ca. Accumulibacter phosphatis
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ABSTRACT: A metabolomics and whole cell lysate shotgun proteomics study was performed to investigate the glycolytic modes in Ca. Accumulibacter phosphatis.
Project description:Mating is a complex process that causes many behavioral and physiological changes, but the factors triggering these changes and the underlying molecular processes are not well characterized. Honey bee queens provide a convenient system for dissecting these factors (e.g., physical manipulation, insemination volume, insemination substance) via instrumental insemination. We examined the effects of carbon dioxide (CO2), a commonly used anesthetic in instrumental insemination that causes changes similar to those observed after mating, and physical manipulation, which presumably mimics the act of copulation, on the brain transcriptional changes in honey bee queens. We found significant gene overlap between our study and previous mating studies in honey bee queens and Drosophila. This suggests that molecular pathways regulating the mating process are conserved across different mating regimes of honey bees as well as across insect orders.
Project description:Protein complexes are responsible for the bulk of activities within the cell, but how their behavior and abundance varies across tumors remains poorly understood. By combining proteomic profiles of breast tumors with a large-scale protein-protein interaction network, we have identified a set of 285 high-confidence protein complexes whose subunits have highly correlated protein abundance across tumor samples. We used this set to identify complexes that are reproducibly under- or over-expressed in specific breast cancer subtypes. We found that mutation or deletion of one subunit of a co-regulated complex was often associated with a collateral reduction in protein expression of additional complex members. This collateral loss phenomenon was typically evident from proteomic, but not transcriptomic, profiles suggesting post- transcriptional control. Mutation of the tumor suppressor E-cadherin (CDH1) was associated with a collateral loss of members of the adherens junction complex, an effect we validated using an engineered model of E-cadherin loss.
Project description:The human hair proteome offers new insight in the field of human identification. Hair often contains DNA that has been degraded due to the process of cornification. The smaller fragments of DNA present a challenge for obtaining useful information concerning the identity of the donor. Proteomic genotyping offers an alternative approach which starts with protein, a biomolecule abundant in the hair shaft. Nonsynonymous single nucleotide polymorphisms may be detected in the sequence of these proteins, allowing the inference of SNP genotypes. Population genetics-based information from these genotypes may be used to calculate random match probability or even infer ancestry. The current challenge of this research is to optimize processing chemistry in order to maximize genetic information from a single human hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 hrs of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 hrs of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times for disulfide reduction and protein digestion produced random match probabilities of up to 1 in 624 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original processing method.
Project description:Sexual reproduction in flowering plants involves intimate interactions between the growing pollen tube and the female reproductive structure. These interactions start immediately after pollen landing on the stigma and continue during the pollen tube journey through the style and the ovary. Thus, well before fertilization, genes in the gynoecium are affected by the growing pollen tubes. Genes activated at a distance in the ovary before pollen tubes arrival represent one class of such genes. Using a global transcriptomic approach, expression profiles obtained from compatible (SC), incompatible (SI), semi-compatible (SeC) and interspecific (IS) pollinations revealed that these pollinations are perceived differently from a distance in the ovary. As the pollen tubes grew along the style, more and more genes became specific for each pollination type, although even early on when no difference could be observed in pollen tube growth rates, each pollination type already displayed its specific signature. Wounding experiments as well as methyl jasmonate treatment were also conducted to determine if transmitting tissue cell death caused by pollen tube growth in the style could also activate gene expression at distance in the ovary. Our data suggest that pollen tube growth in the style is at least partially perceived as a wounding aggression, and that a SI pollination is more akin to a wound response than the other pollination type tested, suggesting similarities in the signaling pathways controlling pollen recognition and stress responses. More importantly, our transcriptomic analysis reveals a highly specific recognition of various pollination types that is relayed from a distance to the ovary ahead of fertilization. To determine on a more global scale if interorgan communication during pollen-pistil interactions is a widespread phenomenon and does not only involve few specialized genes, we used a 7.7K cDNA microarray comprising ~6500 ovule-derived unigenes in duplicata from Solanum chacoense, a self-incompatible wild potato species (Germain et al., 2005), to conduct gene expression analyses through cDNA microarray hybridizations. Profiles obtained from compatible (SC), incompatible (SI), semi-compatible (SeC) and interspecific (IS) pollinations as well as from wounding treatments were all analyzed using the same minimal expression level critera (p < 0.05, fold change ≥± 1.5). Four independent biological replicates were produced from each time points. Ovules were collected at 6, 24 and 48 hours after each treatment and used for RNA extraction and probe preparation . compatible (SC), incompatible (SI), semi-compatible (SeC) and interspecific (IS) pollinations as well as from wounding treatments mRNA preparations were individually hybridized against unpollinated ovule mRNAs . All data was substracted from the touch control. Althought Methanol treated ovules RNA were hybridized against unpollinated ovule mRNAs from plant in closed chamber. To estimate reproducibility and to produce control data for statistical analysis, a large number of unfertilized ovules were isolated and separated between seven independent control groups. RNA from randomly selected pairs of control was hybridized on six microarrays.
Project description:The yeast transcriptomic response to quercetin, a naturally-occurring flavonol with antioxidant, anticancer and anti-ageing activities, was evaluated by differential gene expression analysis using a microarray containing probes for S. cerevisiae ORFeome. Samples obtained from BY4741 strain cells treated with 300uM quercetin were compared to control samples (obtained from cells incubated with vehicle) on dual-color microarray experiments. Three independent biological replicates and the respective dye-swap hybridizations were combined, in a total of 6 microarray hybridizations.
Project description:We developed a novel sample preparation method by combining Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (PEPPI-MS) fractionation with Anion-Exchange disc-assisted Sequential sample Preparation (AnExSP) purification, and evaluated its performance in TDP analysis by comparing it with a conventional PEPPI workflow using Methanol-Chloroform-Water (MCW) precipitation.
Project description:Nitrobenzothiazinones (BTZs) are undergoing late-stage development as a novel class of potent antituberculotic drug candidates with two compounds in clinical phases. BTZs inhibit decaprenylphosphoryl-β-D-ribose oxidase 1 (DprE1), a key enzyme in cell wall biosynthesis of mycobacteria. Their mechanism of action involves an in-situ reduction of the nitro moiety to a reactive nitroso intermediate capable of covalent binding to Cys387 in the catalytic cavity. The electron-deficient nature of the aromatic core is a key driver for the formation of hydride-Meisenheimer complexes (HMC) as main metabolites in vivo. To mimic the electrophilic character of the nitroso moiety, bioisosteric replacement against electrophilic warheads was attempted to reduce HMC formation without compromising covalent reactivity. Herein, we synthesized and characterized a set of various covalent warheads covering different reaction principles. Covalent inhibition was confirmed for all antimycobacterial compounds by enzymatic inhibition assays and protein mass spectrometry analysis.
Project description:Transcriptome analysis in natural Saccharomyces cerevisiae as function of fermentation stage. Strains used were the reference strain S288C, two (06L3FF02 and 06L6FF20) isolates from the Bairrada wine region, Portugal, three (Lalvin EC-1118, Lalvin ICV D254 and AEB Fermol Rouge) wine yeast obtained commercially and one (J940047) isolate from a human patient. Fermentation was carried out in synthetic must MS300, in semi-anaerobic conditions. Cells were harvested at six time-points during fermentation: early exponential growth (T1), mid-exponential growth (T2), diauxic shift (T3), early stationary growth (T4) Mid-stationary growth (T5) and end of fermentation (T6). Hybridizations were carried out using a common reference design, using RNA obtained from S288C at T2, in dye-swap replicates, and four self-self hybridizations were performed using the common reference sample for control of the experiment background, in a total of 88 hybridizations.
Project description:Tissue specific microarray data is obtained by generating a whole-organism extract and subsequent co-immune precipitation of mRNA with the poly-A binding protein FLAG::PAB-1 specifically expressed in body wall muscle cells (Roy et al. 2002). Different lines were compared: SV911 (control), SV912 (CYE-1/CDK-2AF), and SV985 (CYD-1/CDK-4). As reference total RNA was used.
Project description:A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes. 2 Biological replicate experimens conducted 1 month apart. In the first there were 2 dye-swapped duplicates (total 4) of VC+MeOH versus MeOH only. In the second experiment there was one set of dye swapped arrays. Thus 6 arrays were performed including biological replicates, dye swapped replicates and technical duplicates.