ABSTRACT: Conventional culture methods with commercially available media unveil the presence of novel culturable bacteria Whole genome sequencing of novel bacterial isolates
Project description:Conventional culture methods with commercially available media unveil the presence of novel culturable bacteria 16S metagenomics of healthy Japanese individual derived-gut microbiome
Project description:In the research field of extracellular vesicles (EVs), the use of EV-depleted fetal bovine serum (FBS) for in vitro studies is highly recommended to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or bought commercially, nevertheless these depletion methods do not guarantee an RNA-free preparation. In this study we have addressed the RNA contamination issue in FBS, ultracentrifuged EV-depleted FBS, commercially available EV-depleted FBS, and also from our recently developed filtration based EV depleted FBS. Commercially available serum-free, xeno-free defined media were also screened for RNA contamination.
Project description:Rationale: Tuberculosis has a devastating impact on global health by claiming nearly 1.4 million lives each year. During infection Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, produces heterogenous populations some of which don’t produce colonies on agar but grow in liquid media and often require supplementation with culture supernatants or recombinant Resuscitation-promoting factor, thus defined as differentially culturable bacilli. Objectives: to evaluate whether exposure to nitric oxide (NO), a well-known host defence molecule, alters mycobacterial growth phenotypes and drives generation of Rpf-dependent differentially culturable bacilli. Methods: a novel NO donor was synthesised and tested against Mtb and Mycobacterium bovis BCG in vitro, followed by growth assays, flow cytometry analysis and assessment of transcriptomic responses. Resuscitation-promoting factor (Rpf) inhibitors were used to characterise the role of Rpf proteins in the reactivation of NO-treated mycobacteria. Mycobacterial phenotypes were also investigated during infection of THP-1 macrophages activated with retinoic acid and vitamin D3. Measurements and Main Results: differentially culturable mycobacteria were generated after exposure to the novel NO donor or during infection of activated THP-1 cells. Resuscitation of these differentially culturable bacilli was largely abolished by specific Rpf inhibitors. Transcriptomic analysis revealed redox-associated stress signatures mediated by SigH and SigF, with significant down-regulation of ribosome and cell wall architecture genes, including rpfA, rpfB and rpfE, and induction of genes involved in response to thiol stress, sulphur metabolism and iron acquisition. Conclusion: Our study provides mechanistic insights into the generation of Rpf-dependent Mtb during tuberculosis and outlines a critical role of NO in this process.
Project description:hCMEC/D3 cell line can be cultured in 2 distinct commercially available media. The objective here was to determine the differences such media induced.
Project description:Library preparation for whole genome bisulphite sequencing (WGBS) is challenging due to side effects of the bisulphite treatment, which leads to extensive DNA damage. Recently, a new generation of methods for bisulphite sequencing library preparation have been devised. They are based on initial bisulphite treatment of the DNA, followed by adaptor tagging of single stranded DNA fragments, and enable WGBS using low quantities of input DNA. In this study, we present a novel approach for quick and cost effective WGBS library preparation that is based on splinted adaptor tagging (SPLAT) of bisulphite-converted single-stranded DNA. Moreover, we validate SPLAT against three commercially available WGBS library preparation techniques, two of which are based on bisulphite treatment prior to adaptor tagging and one is a conventional WGBS method.
Project description:In this work, we established a bacteria-infected diabetic mouse model by spraying MRSA bacteria on the wound of diabetic mice, and then used a novel bioelectronic wound dressing to treat the wound, with commercially available Tegaderm film as a control. After 20 days of therapy, we collected wound tissue from each group for proteomic analysis using the LC-MS/MS methodology and data independent acquistion (DIA) mode.
2023-02-18 | PXD040252 |
Project description:Oilfield microbial community response to commercially available minerals