Project description:Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors and gels derived from alkoxide precursors and incubated under constant light for 24 hours prior to RNA extraction. Cultures suspended in liquid media were also exposed to 500 mM salt stress and incubated under identical conditions, for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change >/= 1.5 and P-value < 0.01), the expression of 1143 genes were significantly altered by encapsulation in alkoxide derived gels, and only 243 genes were common to both encapsulation chemistries. Additional qRT-PCR analyses of four select genes; ggpS, cpcG2, slr5055, and sll5057, confirmed microarray results. These results illustrate that encapsulation stress is quite different than salt stress in terms of gene expression response. Furthermore, a number of hypothetical and unknown proteins associated with encapsulation and alcohol stress have been identified, with implications for improving encapsulation protocols and rationally engineering microorganisms for direct biofuel production. 16 samples; 4 biological replicates each of 4 treatments
Project description:MicroRNAs are important negative regulators of protein coding gene expression, and have been studied intensively over the last few years. To this purpose, different measurement platforms to determine their RNA abundance levels in biological samples have been developed. In this study, we have systematically compared 12 commercially available microRNA expression platforms by measuring an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples, and synthetic spikes from homologous microRNA family members. We developed novel quality metrics in order to objectively assess platform performance of very different technologies such as small RNA sequencing, RT-qPCR and (microarray) hybridization. We assessed reproducibility, sensitivity, quantitative performance, and specificity. The results indicate that each method has its strengths and weaknesses, which helps guiding informed selection of a quantitative microRNA gene expression platform in function of particular study goals.
Project description:Asthma is a complex syndrome associated with episodic decompensations provoked by aeroaller-gen exposures. The underlying pathophysiological states driving exacerbations are latent in the resting state and do not adequately inform biomarker-driven therapy. A better understanding of the pathophysiological pathways driving allergic exacerbations is needed. We hypothesized that disease-associated pathways could be identified in humans by unbiased metabolomics of bron-choalveolar fluid (BALF) during the peak inflammatory response provoked by a bronchial aller-gen challenge. We analyzed BALF metabolites in samples from 12 volunteers who underwent segmental bronchial antigen provocation (SBP-Ag). Metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by pathway analysis and cor-relation with airway inflammation. SBP-Ag induced statistically significant changes in 549 fea-tures that mapped to 72 uniquely identified metabolites. From these features, two distinct induci-ble metabolic phenotypes were identified by the principal component analysis, partitioning around medoids (PAM) and k-means clustering. Ten index metabolites were identified that in-formed the presence of asthma-relevant pathways, including unsaturated fatty acid produc-tion/metabolism, mitochondrial beta oxidation of unsaturated fatty acid, and bile acid metabolism. Pathways were validated using proteomics in eosinophils. A segmental bronchial allergen chal-lenge induces distinct metabolic responses in humans, providing insight into pathogenic and pro-tective endotypes in allergic asthma.
Project description:Genome-wide identification of transcription factor (TF) binding sites in the genome of the fission yeast Schizosaccharomyces pombe. The ChIP-nexus method was used. TFs included were: Cbf11-TAP and Cbf12-TAP (and their DBM mutants with impaired DNA binding), TAP-Mga2, and Fkh2-TAP (as an irrelevant control TF). IPs from an untagged WT strain were also analyzed. Cbf11-related IPs were performed from exponential cultures, while Cbf12-related IPs were performed from stationary cultures. YES complex medium was used for all cultivations.
Project description:Lysine methylation on histone tails impacts genome regulation and cell fate determination in many developmental processes. Apicomplexa intracellular parasites cause major diseases and they have developed complex life cycles with fine-tuned differentiation events. Yet, apicomplexa genomes have few transcription factors and little is known about their epigenetic control systems. Tick-borne Theileria apicomplexa species have relatively small, compact genomes and a remarkable ability to transform leukocytes in their bovine hosts. Here we report enriched H3 lysine 18 monomethylation (H3K18me1) on the gene bodies of repressed genes in Theileria macroschizonts. Differentiation to merozoites (merogony) led to decreased H3K18me1 in parasite nuclei. Pharmacological manipulation of H3K18 acetylation or methylation impacted parasite differentiation and expression of stage-specific genes. Finally, we identified a parasite SET-domain methyltransferase (TaSETup1) that can methylate H3K18 and represses gene expression. Thus, H3K18me1 emerges as an important epigenetic mark which controls gene expression and stage differentiation in Theileria parasites.
Project description:Elucidation of complex molecular networks requires integrative analysis of molecular features and changes at different levels of information flow and regulation. Accordingly, high throughput functional genomics tools such as transcriptomics, proteomics, metabolomics and lipidomics have emerged to provide system-wide investigations. Unfortunately, analysis of different types of biomolecules requires specific sample extraction procedures in combination with specific analytical instrumentation. The most efficient extraction protocols often only cover a restricted type of biomolecules due to their different physicochemical properties. Therefore, several sets/aliquots of samples are needed for extracting different molecules. Here we adapted a bi-phasic fractionation method to extract proteins, metabolites and lipids from the same sample (3-in-1) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-omics. To demonstrate utility of the improved method, we used bacteria-primed Arabidopsis leaves to generate multi-omics datasets from the same sample. In total, we were able to analyze 1849 proteins, 1967 metabolites and 424 lipid species in single samples. The molecules cover a wide range of biological and molecular processes. Our results have shown the clear advantages of the multi-omics method, including sample conservation, high reproducibility and tight correlation between different types of biomolecules.
Project description:We have developed a strategy for the detailed structural characterization of complex proteoglycan-derived glycosaminoglycans. Chondroitin/dermatan sulfate isolated from rat INS-1 832/13 insulinoma cells known to produce primarily one proteoglycan was used to evaluate and demonstrate the efficacy of the strategy.
Project description:In order to identify how MnTE-2-PyP affects p300 association to chromatin genome-wide, we performed a p300 chromatin Immunoprecipitation assay followed by Next Generation Sequencing on PC3 cells treated with or without MnTE-2-PyP one hour post-irradiation (Figure 3A). Based on the called peaks near genes, we predicted that HIF-1βand CREB transcription factors were associating DNA less in the presence of MnTE-2-PyP. DNA was ChIP-Fixed from Pc3 cells treated with 20 Gy radiation and with and without T2E drug. There are 2 biological replicates of PC3 untreated cells and 3 biological replicates of PC3 cells treated with MnTE-2-PyP. There are two corresponding input samples for the biological replicates.