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:The pluripotency maintenance of pluripotent stem cells (PSCs) cultured in vitro requires the suitable microenvironment, which is commonly provided by the feeder layer. However, the preparation of feeder layer is time consuming and labor exhaustive. More importantly, the feeder cells treated with mitomycin or gamma-ray irradiation brings heterologous contamination to stem cells. The feeder-free PSC cultures are associated with high costs because of the requirement for additional supplements and special media. In this study, we characterize the pluripotency and metabolic status of bovine ESCs-F7 (classic bESCs lines, abbreviated as F7), which were cultured on methanol fixed mouse embryonic fibroblasts (MT-MEFs) or mitomycin C treated MEFs (1M-MEFs). MT-MEFs could be reused several times and were highly resistant to digestive enzymes. The relative expression levels of pluripotent markers were different between F7 cultured on MT-MEFs (marked as MT-F7) and those cultured on the 1M-MEFs (1M-F7). The long-term cultured MT-F7 cells formed embryoid bodies in vitro, showing the ability to differentiate into endodermal, ectodermal, and mesodermal germ layers like 1M-F7. RNA-sequencing analysis showed that the replacement of the feeder layer from 1M-MEFs to MT-MEFs lead to a novel steady transition of the F7, which included alteration of the expression patterns of genes that regulate pluripotency and metabolism. Further, the long-term cultured bovine expanded pluripotent stem cells (bEPSCs) on MT-MEFs (MT-bEPSCs) formed classical colonies, maintained pluripotency, and demonstrated elevated level of metabolic activity. In conclusion, this study demonstrated that methanol-fixed MEFs were efficient feeder layer that maintain the unique pluripotency and the distinctive metabolic characteristics of the bPSCs cultured in vitro.
Project description:Escherichia coli is the leading cause of catheter-associated urinary tract infections, caused by biofilm formation on implanted biomaterial surfaces. Understanding the genes that cause cellular adhesion to diverse biomaterial surfaces may aid in the design of targeting anti-biofouling chemicals to prevent these biofilm infections, but our current knowledge on such surfaces is limited. Here, we incorporate a platform of six biomaterials of varying hydrophilicities and stiffnesses and a comprehensive genome-wide CRISPR interference (CRISPRi) library to elucidate genotype-phenotype relationships for cellular adhesion to physicochemically varied biomaterial surfaces in E. coli MG1655. After characterization of the biomaterials and CRISPRi tool, we designed a CRISPRi library of 34,315 unique designs targeting 99.0% of the genome in MG1655 (up to 8 designs per gene). We then performed pooled selections for adhesion to each biomaterial surface, elucidating over 400 novel gene hits to each biomaterial surface. Data analysis revealed greater correlations between biomaterials of the same hydrophilicity rather than stiffness, in addition to more gene hits associated with decreased adhesion across all six surfaces than increased adhesion. Monoclonal verification of select designs from the library exhibited strong correlations between results from the pooled selections and individual measurements for adhesion to each gel for most designs. The results from this study provide comprehensive gene sets for cellular adhesion to physicochemically diverse biomaterial surfaces that may be potential gene targets for the design of targeting anti-biofouling agents and offer insight that could be used for the design of “smart” biomaterials, both with potential to prevent biofilm infections.
Project description:The transcriptome of P. aeruginosa PA01 biofilm cells was compared to the associated suspended culture, upon growth on reverse osmosis membrane coupon under limited nutrient conditions. Experiment Overall Design: Cells were collected from the RO unit and from the membrane coupon after 20 hours of growth and biofouling of the membrane. A defined media was used, resembeling secondary wastewaters.