Project description:Glutamicibacter arilaitensis grown in large scale liquid Cheese Curd media and extracted with Amberlite XAD16, back extracted with MeOH:DCM 50:50, separated on SPE column to afford 7 fractions labeled A through G based on increasing polarity (A = 10% MeOH, F = 100% MeOH and G = 100% EtoAC. Fractions E(60% MeOH), D(80% MeOH), and F(100% MeOH) run on a qTOF and submitted here. suffix 1 refers to treatment of cultures with desferrioxamine and suffix PLAIN means untreated. JB182new_20170614 is a crude extract from a subsequent small scale growup without desferrioxamine.
Project description:We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata, on cell survival of colon (HCT116) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. Here, we found that the extract of Cotyledon orbiculata, a South African medicinal plant, had an anti-proliferative effect in cancer cells, mediated by apoptosis induced by alternative splicing of hnRNPA2B1 and BCL2L1.
Project description:In order to get mechanistic insights into the cardiomyogenic and differentiation-promoting activity of Crataegus spp. Extract WS®1442 and the identified bioactive subfractions (MeOH eluate), we have employed whole genome microarray expression profiling after 6 hours and 24 hours of treatment. We found that stress-associated genes are affected as well as specific signaling pathways such as TGFbeta, FGF, BDNF and retinoic acid.
Project description:Mycobacterium tuberculosis causes tuberculosis (TB) which remains one of the world’s deadliest diseases. As mycobacteria are protected by its thick lipid cell wall, the host has developed immune systems targeting their unique lipids. Among them is an unconventional T cell subset called mycolipid-specific T cells restricted by common antigen-presenting molecules, such as CD1. Here, we identified novel mycolipid-specific T cells using a method combining bulk T cell stimulation with crude lipids and single cell-TCR-RNA-sequencing-based clonotypic analysis. In human PBMCs, a CD4+ T cell clone with cytotoxic signature, Y-50, was proliferated in response to mycobacterial crude lipid extract. Activity-based purification of using reporter cells reconstituted with Y-50 TCR identified trehalose monomycolate (TMM) as an antigen. CD1b was necessary and sufficient for the presentation of TMM to be recognized by Y-50. Y-50 TCRab chains displayed characteristic features, such as positively-charged CDR3a and extra-long CDR3b. Mutagenesis and structural analysis of TCRab suggested that these unique CDR3 properties were required for the recognition of TMM presented by CD1b. CD1b tetramer loaded with TMM could detect T cells with similar characteristics to Y-50 in uninfected healthy donors, but their frequency was significantly higher in PBMCs from TB patients. In this study, we identified pre-set unconventional T cells specific for a mycobacterial adjuvant, TMM.
Project description:Untargeted metabolomics (LC-MS/MS) of water samples (SPE on Strata XL Phenomonex) and of macroalgal whole tissues (50% MeOH/DCM) and surface extracts (MeOH). Positive and negative ionization mode
Project description:One of the central goals for the development of cell-free (CF) gene expression systems is to ensure reproducible expression of proteins at high quality and yield. Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols have been developed and optimized reaction conditions have been established. One of the crucial steps during the preparation of cell extract-based expression systems, however, is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here, we demonstrate for an E. coli based system that a lysis procedure combining incubation of the cells with lysozyme with a gentle sonication step results in highly active cell extracts. As examples for the capabilities of our extract, we demonstrate the production of several fluorescent proteins as well as the production and assembly of T7 bacteriophages. Compared to other cell-free expression systems, our method achieved the highest phage titers in our plaque assays. Stateof-the-art quantitative proteomics revealed that enzymes of the energy regeneration pathway were enriched in our extract compared to a widely used commercial cell extract. On the other hand, ribosomal proteins were found to be more abundant in the commercial product.