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:Pelagic aggregates function as hotspots for microbial activity and biological carbon pumps for exporting OM fixed by photoautotrophs to sediments in lakes and oceans. In iron-rich (ferruginous) lakes, photoferrotrophic or chemolithoautotrophic bacteria appear to contribute to CO2 fixation by oxidizing reduced iron which leads to the formation of iron-rich pelagic aggregates called iron-snow. In acidic lakes, iron snow is colonized mainly by acidophilic iron-cycling microbes that can trigger interspecies aggregation mechanisms. However, the significance of iron oxidizers in carbon fixation, their general role in iron snow functioning, and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis with a 13CO2 metabolic labeling approach to determine general metabolic activities. Protein-based stable isotope probing (protein-SIP) was used to trace the 13CO2 incorporation in iron snow microcosms over time under both oxic and anoxic conditions. Analysis of our mRNA-derived metatranscriptome data identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6%-85.7%) in iron snow encoding a variety of ecologically relevant pathways, including carbon fixation, polysaccharide biosynthesis, and flagellar-based motility. We did not detect transcriptional activity for carbon fixation from archaea or eukaryotes. The largest numbers of expressed genes (3008, 2991, 2936) matched to the genomes of our previously obtained iron snow isolates (Acidithrix sp. C25, Acidiphilium sp. C61, Acidocella sp. C78) separately. 13CO2 incorporation studies identified Leptospirillum and Ferrovum, as the main active chemolithoautotrophs under oxic conditions, and Ferrovum was the main active organism under anoxic conditions as well. Small amounts of labeled 13C (Relative isotope abundance: 1.0%-5.3%) were found in the heterotrophic Acidiphilium and Acidocella. Overall, our data show that iron oxidizers play an important role in the formation of iron minerals and CO2 fixation, but the majority of fixed C apparently did not reach other iron snow microbes. This finding suggests that most of the fixed C will be directly exported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.
Project description:DNA methylation is an epigenetic mark that has a crucial role in regulating gene expression. Aberrant DNA methylation results in severe diseases in humans, such as cancer, autoimmune disease, atherosclerosis, and cardiovascular diseases. Whole-genome bisulfite sequencing and methylated DNA immunoprecipitation are available to study DNA methylation changes, but they are typically used on a few samples at a time. Here, we developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), that can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis.