Project description:Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser and Thr residues, is found on most solid tumors yet rarely detected in adult tissues: featuring it one of the most distinctive signatures of cancers. Although it is prevalent in cancers, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites will spur the development of the new vaccines, diagnostics, and therapeutics of cancers. Here, we report a novel technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn. This exquisite Gal(13C6)-Tn structure is recognized by a human-gut-bacterial enzyme, called OpeRATOR, that specifically cleaves N-termini of the Gal(13C6)-Tn-occupied Ser and Thr residues to yield site-containing glycopeptides. The enzymes C1GalT1 and OpeRATOR could be used concurrently in one-pot. The effectiveness of EXoO-Tn was evaluated by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. Bioinformatic analysis of the identified site-specific Tn-glycoproteins revealed conserved motif, cellular localization, relative position in proteins, and mapped site-specific Tn-glycoproteome in different studies. Given the significance of Tn in cancers, EXoO-Tn is anticipated to have broad utilities in clinical study of cancers.
Project description:Naive CD4 T (TN) cells show some degree of heterogeneity. However, mechanisms that alter TN cells are still largely unknown, and physiological importance in the phenotypic alteration of TN cells is unclear. Thus, to reveal the mechanisms that alter TN cell characteristics, we performed transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) on TN cells from different secondary lymphoid organs with different strength of tonic TCR stimulation.
Project description:Light dependent gene expression in D. shibae wildtype compared to the gene expression in the transposonmutants D. shibae Dshi_1135::Tn, D. shibae ppaA::Tn and D. shibae ppsR::Tn. Dinoroseobacter shibae DFL12T (DSM 16493T) and the transposonmutants D. shibae Dshi_1135::Tn, D. shibae ppaA::Tn and D. shibae ppsR::Tn were grown in artificial saltwater minimal medium (SWM) in baffled flasks shaking at 180 rpm and 30 °C and incubation was performed under light, dark and bluelight conditions. D. shibae wild type and mutant strain were grown under aerobic conditions up to the mid exponential growth phase (OD578 nM 0.5).
Project description:T and CAR T cells were generated from mock or CD19CAR+ Tn/mem-derived induced pluripotent stem cells (Tn/mem-iPS) by 3D-organoid culture system. We performed RNA deep sequencing analysis to better elucidate their phenotype and compare their gene expression profiles among groups of Tn/mem-iPSC derived T/ CAR T cells, PBMC derived T/CAR T cells, PBMC derived NK cells and T-iPSC cells, as well as CD4 and CD8 subsets of iPSC derived or PBMC derived CAR T cells.