Project description:We combined high-resolution tiling microarrays and 5'-end RNA sequencing to obtain a genome-wide map of transcription start sites (TSSs) for Shewanella oneidensis MR-1. To test the reliability of these TSSs, we compared our result to those from differential RNA sequencing (dRNA-seq), which discriminates primary and processed ends of transcripts. We found that our identified TSSs tend to have significantly more mapped reads in the TEX(+) sample than the TEX(-) sample. Overall, the dRNA-seq results support the validity of our predictions for TSS. S. oneidensis MR-1 was grown to mid-log phase in Luria-Bertani broth (LB) or defined lactate minimal medium, and total RNA was isolated and used for differential RNA-sequencing (dRNA-seq) by next-generation sequencing, which is used to verify genome-wide transcription start sites. For dRNA-seq, total RNA was partially treated with Terminator Exonuclease (TEX) to digest processed RNA and thereby enrich for primary transcript ends.
Project description:This experiment was performed to measure differences in pathway-level evolution of cis-regulation between closely related Kluyveromyces species. Sequencing of mRNA as well as DNA was performed in hybrids of Kl. lactis with two other species, Kl. wickerhamii and Kl. marxianus. Cells were grown overnight for DNA sequencing and for 4-8h to an OD600=0.7-1.0 for mRNA sequencing. DNA and mRNA reads were mapped to hybrid genomes, multimapping reads discarded, and allele-specific expression ratio for each gene was calculated after first normalizing mRNA reads to the number of DNA reads for each gene in each species. We found that the ribosomal protein genes showed pathway-level allele-specific expression differences between species in both interspecies hybrids.
Project description:While distinct NK-like CD57+ and PD-1+ CD8+ exhausted T cell populations (Tex) were both linked to beneficial immunotherapy response in autoimmune Type 1 Diabetes (T1D) patients, relationships between these cell types are poorly understood. We show that PD-1+ and CD57+ Tex populations in this context were epigenetically similar, but CD57+ Tex cells displayed unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also showed reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex showed unappreciated gene expression heterogeneity and shared clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.
Project description:Background: KRAS mutation is one of the most common oncogenic drivers in NSCLC, however, the response to immunotherapy is heterogeneous owing to the distinct co-occurring genomic alterations. KRAS/LKB1 co-mutated lung adenocarcinoma displays poor response to PD-1 blockade whereas the mechanism remains undetermined. Methods: We explored the specific characteristics of tumor microenvironment (TME) in KL tumors using syngeneic KRASG12DLKB1-/-(KL) and KRASG12DTP53-/- (KP) lung cancer mouse models. The impact of focal adhesion kinase (FAK) inhibitor on KL lung tumors was investigated in vitro and in vivo through evaluation of both KL cell lines and KL lung cancer mouse models. Results: We identified KL tumors as “immune-cold” tumors with excessive extracellular matrix (ECM) collagen deposition that formed a physical barrier to block the infiltration of CD8+T cells. Mechanistically, abundant activated cancer-associated fibroblasts (CAFs) resulted from FAK activation contributed to the formation of the unique TME of KL tumors. FAK inhibition with a small molecular inhibitor could remodel the TME by inhibiting CAFs activation, decreasing collagen deposition and further facilitating the infiltration of anti-tumor immune cells, including CD8+ T cells, DC cells and M1-like macrophages into tumors, hence, converting “immune-cold” KL tumors into “immune-hot” tumors. The combined FAK inhibitor and PD-1 blockade therapy synergistically retarded primary and metastatic tumor growth of KL tumors.Conclusions: Our study identified FAK as a promising intervention target for KL tumors and provided basis for the combination of FAK inhibitor with PD-1 blockade in the management of KL lung cancers.
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:We combined high-resolution tiling microarrays and 5'-end RNA sequencing to obtain a genome-wide map of transcription start sites (TSSs) for Shewanella oneidensis MR-1. To test the reliability of these TSSs, we compared our result to those from differential RNA sequencing (dRNA-seq), which discriminates primary and processed ends of transcripts. We found that our identified TSSs tend to have significantly more mapped reads in the TEX(+) sample than the TEX(-) sample. Overall, the dRNA-seq results support the validity of our predictions for TSS.