Project description:We demonstrate the feasibility of total RNA-SIP in experiments where microbes from a hydrocarbon-contaminated aquifer were studied in microcosms with 13C-labelled-toluene to understand their adaptation to the simultaneous availability of low levels of different electron acceptors. SIP successfully resolved the involvement of microaerobic vs. aerobic and anaerobic populations. Under microoxic, nitrate-amended conditions hydrocarbon degradation was actually stimulated, but transcripts of denitrification showed no signs of 13C-labelling. The expression of distinct oxygenase-based catabolic pathways for toluene degradation was clearly apparent in 13C-labelled mRNA. We discuss how these direct insights into the gene expression and adaptation mechanisms within complex degrader communities can guide more integrated approaches in monitoring and restoration of contaminated sites.
Project description:Transcriptional responses in ovariectomized mouse uterine tissue to estradiol (E2) and diethylstilbestrol (DES), known long-acting estrogens, and propyl pyrazole triol (PPT), an ER-alpha selective estrogen, were profiled. Profiles were used together with those from other estrogens to derive a biomarker panel.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site. 2 samples examined from the different electron-acceptors (sulphate or ferric iron) incubates at the time point of maximal toluene degradation.
Project description:The Virochip microarray (version 4.0) was used to detect viruses in patients from North America with unexplained influenza-like illness at the onset of the 2009 H1N1 pandemic. We used metagenomics-based technologies (the Virochip microarray) and deep sequencing to analyze nasal swab samples from individuals with 2009 H1N1 infection. This Series includes the Virochip microarray data only.
Project description:Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we performed in-depth measurement of metabolism in intact human liver tissue ex vivo using global 13C tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Cultured liver tissue exhibited normal anatomical structure and retained canonical liver functions such as glucose production, albumin and VLDL synthesis at near-physiological rates. Isotope tracing with a highly 13C-labeled medium generated 13C enrichment in hundreds of compounds, allowing qualitative assessment of a wide range of metabolic pathways within a single experiment. This confirmed well-known features of liver metabolism, but also revealed unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Metabolic flux analysis identified glycogenolysis as the main source of glucose production, which could be suppressed by pharmacological inhibition of glycogen phosphorylase. Glucose production ex vivo also correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes. Moreover, liver tissue responded to postprandial levels of nutrients and insulin by suppressing glucose production and increasing nutrient uptake. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.
Project description:We analyzed the transcriptional response of the actinomycete Rhodococcus aetherivorans I24 to biphenyl and polychlorinated biphenyls (PCBs). This species has not been extensively exposed to PCBs, as it was first isolated from a toluene contaminated aquifer, rather than a site contaminated with polychlorinated hydrocarbons. Using a microarray targeting 3524 genes, we assessed gene expression in minimal medium supplemented with various substrates (e.g. PCBs) and in both PCB-contaminated and non-contaminated sediment slurries. Relative to the reference condition (minimal medium supplemented with glucose), 408 genes were up-regulated in the various treatments. In medium and in sediment, PCBs elicited the up-regulation of a common set of 100 genes, including chaperones (groEL), a superoxide dismutase (sodA), alkyl hydroperoxide reductase protein C (ahpC), and a catalase/peroxidase (katG). Analysis of the R. aetherivorans I24 genome sequence identified orthologs of many of the genes in the canonical biphenyl pathway, but very few of these genes were up-regulated in response to PCBs or biphenyl. This study is one of the first which utilizes microarrays to assess the transcriptional response of a soil bacterium to a pollutant under conditions which more closely resemble the natural environment. Our results indicate that the transcriptional response of R. aetherivorans I24 to PCBs, in both medium and sediment, is primarily directed towards reducing oxidative stress, rather than catabolism. In addition, the identification of numerous genes expressed in contaminated soil specifically may have implications for the development of biosensors. Finally, comparative genomic and transcriptomic analyses suggest that the mere presence of orthologs of the required enzymes may not be sufficient to confer a vigorous biphenyl/PCB metabolism.
Project description:There is great medical need for estrogens having favorable pharmacological profiles, supporting desirable activities for menopausal women such as metabolic and vascular protection but lacking stimulatory activities on the breast or uterus. Here, we report the development of structurally novel estrogens with favorable target tissue-selective estrogenic activity. Through a process of structural alteration of the hormone estradiol that preserves essential chemical and physical features of estradiol but greatly moderates its binding affinity for the estrogen receptors (ERs), we obtained Pathway Preferential Estrogens (PaPEs) capable of having interaction with ER that is sufficient to activate the extranuclear-initiated signaling pathway preferentially over the direct nuclear-initiated pathway. PaPE modulate a pattern of gene regulation and cellular and biological processes that result in essentially no stimulation of reproductive and mammary tissues and breast cancer cells, but have a favorable pattern of activity on metabolic tissues and the vasculature. The structural permutation process represents a novel approach to govern the balance in utilization of extranuclear vs. nuclear pathways of ER action to obtain tissue-selective/non-nuclear pathway-preferential estrogens, which should prove to be beneficial for postmenopausal hormone replacement. The approach may also have broad applicability for other members of the nuclear hormone receptor superfamily.
Project description:There is great medical need for estrogens having favorable pharmacological profiles, supporting desirable activities for menopausal women such as metabolic and vascular protection but lacking stimulatory activities on the breast or uterus. Here, we report the development of structurally novel estrogens with favorable target tissue-selective estrogenic activity. Through a process of structural alteration of the hormone estradiol that preserves essential chemical and physical features of estradiol but greatly moderates its binding affinity for the estrogen receptors (ERs), we obtained Pathway Preferential Estrogens (PaPEs) capable of having interaction with ER that is sufficient to activate the extranuclear-initiated signaling pathway preferentially over the direct nuclear-initiated pathway. PaPE modulate a pattern of gene regulation and cellular and biological processes that result in essentially no stimulation of reproductive and mammary tissues and breast cancer cells, but have a favorable pattern of activity on metabolic tissues and the vasculature. The structural permutation process represents a novel approach to govern the balance in utilization of extranuclear vs. nuclear pathways of ER action to obtain tissue-selective/non-nuclear pathway-preferential estrogens, which should prove to be beneficial for postmenopausal hormone replacement. The approach may also have broad applicability for other members of the nuclear hormone receptor superfamily. 24 samples; inhibitor and time course experiments