Project description:Primary objectives: The primary objective is to investigate circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Primary endpoints: circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Project description:Concerted metabolic shifts give new insights into the syntrophic mechanism between propionate-fermenting Pelotomaculum thermopropionicum and hydrogenotrophic Methanocella conradii
Project description:The anaerobic biodegradation of organic matter is accomplished by sequential syntrophic catabolism by microbes in different niches. Pelotomaculum thermopropionicum is a representative syntrophic bacterium that catalyzes the intermediate bottleneck step in the anaerobic-biodegradation process, whereby volatile fatty acids (VFAs) and alcohols produced by upstream fermenting bacteria are converted to acetate, hydrogen, and carbon dioxide (substrates for downstream methanogenic archaea). To reveal genomic features that contribute to our understanding of the ecological niche and evolution of P. thermopropionicum, we sequenced its 3,025,375-bp genome and performed comparative analyses with genomes of other community members available in the databases. In the genome, 2920 coding sequences (CDSs) were identified. These CDSs showed a distinct distribution pattern in the functional categories of the Clusters of Orthologous Groups database, which is considered to reflect the niche of this organism. P. thermopropionicum has simple catabolic pathways, in which the propionate-oxidizing methylmalonyl-CoA pathway constitutes the backbone and is linked to several peripheral pathways. Genes for most of the important catabolic enzymes are physically linked to those for PAS-domain-containing regulators, suggesting that the catabolic pathways are regulated in response to environmental conditions and/or global cellular situations rather than specific substrates. Comparative analyses of codon usages revealed close evolutionary relationships between P. thermopropionicum and other niche members, while it was distant from phylogenetically related sugar-fermenting bacteria. These analyses suggest that P. thermopropionicum has evolved as a syntrophy specialist by interacting with niche-associated microbes.
Project description:In the syntrophic interaction between fermentative bacteria (Pelotomaculum thermopropionicum) and methanogenic archaea (methanogens: Methanothemobacter thermautotrophicus), reducing equivalents (e.g., H2) produced by fermentative bacteria should efficiently be consumed by methanogens in order for the fermentation of volatile fatty acids (VFA, e.g., butyrate, propionate, and acetate) to be thermodynamically feasible. It has been known that physical approximation (e.g., coaggregation) between VFA-fermenting syntrophic bacteria (syntrophs) and hydrogenotrophic methanogens is necessary for efficient H2 transfer between them. Our previous study has shown that, at an early exponential growth phase of syntrophic coculture, cells of Pelotomaculum thermopropionicum (syntroph) were connected to cells of Methanothermobacter thermautotrophicus (methanogen) via unidentified extracellular filamentous appendages, after which they started to coaggregate, suggesting that the filamentous appendages may have been important for their syntrophic interaction. The filamentous appendages seemed to specifically connect these syntrophic partners, since such pairwise connection has been observed neither in single-species cultures (monocultures) nor in mixtures with other microbes.<br>We found that P. thermopropionicum has putative gene clusters for flagellum and pilus, while no extracellular filament gene was identified in the M. thermautotrophicus genome. So we examined transcriptome responses of M. thermautotrophicus to the contact with flagellar filament protein (FliC) and flagellar cap protein (FliD) of P. thermopropionicum.
Project description:In the syntrophic interaction between fermentative bacteria (Pelotomaculum thermopropionicum) and methanogenic archaea (methanogens: Methanothemobacter thermautotrophicus), reducing equivalents (e.g., H2) produced by fermentative bacteria should efficiently be consumed by methanogens in order for the fermentation of volatile fatty acids (VFA, e.g., butyrate, propionate, and acetate) to be thermodynamically feasible. It has been known that physical approximation (e.g., coaggregation) between VFA-fermenting syntrophic bacteria (syntrophs) and hydrogenotrophic methanogens is necessary for efficient H2 transfer between them. Our previous study has shown that, at an early exponential growth phase of syntrophic coculture, cells of Pelotomaculum thermopropionicum (syntroph) were connected to cells of Methanothermobacter thermautotrophicus (methanogen) via unidentified extracellular filamentous appendages, after which they started to coaggregate, suggesting that the filamentous appendages may have been important for their syntrophic interaction. The filamentous appendages seemed to specifically connect these syntrophic partners, since such pairwise connection has been observed neither in single-species cultures (monocultures) nor in mixtures with other microbes. <br> We found that P. thermopropionicum has putative gene clusters for flagellum and pilus, while no extracellular filament gene was identified in the M. thermautotrophicus genome. So we examined transcriptome responses of M. thermautotrophicus to the contact with flagellar filament protein (FliC) and flagellar cap protein (FliD) of P. thermopropionicum.
Project description:Pelotomaculum thermopropionicum is a syntrophic propionate-oxidizing bacterium that catalyses the intermediate bottleneck step of the anaerobic-biodegradation process. As it thrives on a very small energy conserved by propionate oxidation under syntrophic association with a methanogen, its catabolic pathways and regulatory mechanisms are of biological interest. In this study, we constructed high-density oligonucleotide microarrays for P. thermopropionicum, and used them to analyse global transcriptional responses of this organism to different growth substrates (propionate, ethanol, propanol and lactate) in co-culture with a hydrogenotrophic methanogenic archaeon, Methanothermobacter thermautotrophicus (by reference to fumarate monoculture). We found that a substantial number of genes were upregulated in the syntrophic co-cultures irrespective of growth substrates (including those related to amino-acid and cofactor metabolism), suggesting that these processes were influenced by the syntrophic partner. Expression of the central catabolic pathway (the propionate-oxidizing methylmalonyl-CoA pathway) was found to be substrate-dependent and was largely stimulated when P. thermopropionicum was grown on propionate and lactate. This finding was supported by results of growth tests, revealing that syntrophic propionate oxidation was largely accelerated by supplementation with lactate. These results revealed that P. thermopropionicum has complex regulatory mechanisms that alter its metabolism in response to the syntrophic partner and growth substrates.
Project description:The study is intended to collect specimens to support the application of genome analysis technologies, including large-scale genome sequencing. This study will ultimately provide cancer researchers with specimens that they can use to develop comprehensive catalogs of genomic information on at least 50 types of human cancer. The study will create a resource available to the worldwide research community that could be used to identify and accelerate the development of new diagnostic and prognostic markers, new targets for pharmaceutical interventions, and new cancer prevention and treatment strategies. This study will be a competitive enrollment study conducted at multiple institutions.
Project description:Obligate anaerobic bacteria fermenting volatile fatty acids in syntrophic association with methanogenic archaea share the intermediate bottleneck step in organic-matter decomposition. These organisms (called syntrophs) are biologically significant in terms of their growth at the thermodynamic limit and are considered to be the ideal model to address bioenergetic concepts. We conducted genomic and proteomic analyses of the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum to obtain the genetic basis for its central catabolic pathway. Draft sequencing and subsequent targeted gap closing identified all genes necessary for reconstructing its propionate-oxidizing pathway (i.e., methylmalonyl coenzyme A pathway). Characteristics of this pathway include the following. (i) The initial two steps are linked to later steps via transferases. (ii) Each of the last three steps can be catalyzed by two different types of enzymes. It was also revealed that many genes for the propionate-oxidizing pathway, except for those for propionate coenzyme A transferase and succinate dehydrogenase, were present in an operon-like cluster and accompanied by multiple promoter sequences and a putative gene for a transcriptional regulator. Proteomic analysis showed that enzymes in this pathway were up-regulated when grown on propionate; of these enzymes, regulation of fumarase was the most stringent. We discuss this tendency of expression regulation based on the genetic organization of the open reading frame cluster. Results suggest that fumarase is the central metabolic switch controlling the metabolic flow and energy conservation in this syntroph.