Phosphoproteomics of Mycobacterium tuberculosis DosR, DosS, and DosT
Ontology highlight
ABSTRACT: Mass Spectrometry based Phosphoproteomics was carried out on M. tuberculosis wild type strain H37Rv and the isogenic mutant strains MtbΔdosR, MtbΔdosS, and MtbΔdosT that were cultured in aerobic as well as hypoxic conditions. During later the oxygen was gradually deprived in cultures that were kept static.
Project description:Analysis of the transcriptome of the wild-type strain BY4741 and its isogenic derivative ixr1 null, grown in aerobic, hypoxic conditions and after a hypoxic shift
Project description:Atmospheric oxygen tension is increasingly recognized to be hyperoxic for the maintenance of in vitro cell cultures. Oxygen has broad implications on energy metabolism, cellular growth and many regulatory functions. Primary airway epithelial cells (AECs) differentiated at air-liquid interface (ALI) are the gold standard for preclinical assessment of cystic fibrosis transmembrane conductance regulatory (CFTR) modulator efficacy in CF. The fidelity of CF AECs cultured at atmospheric oxygen tension rather than at reduced oxygen tension which more closely reflect the in vivo environment has not been studied to date. This study examined the impact of ambient (21%) and low (2%) oxygen tension on the expansion and differentiation of nasal epithelial cells (hNECs) derived from 11 participants (8 with CF and 3 non-CF). hNECs expanded under normoxic and chronic hypoxic conditions demonstrated epithelial cobblestone morphology and similar proliferation rate. hNECs differentiated at hypoxia demonstrated poorer differentiation capacity (significantly thinner epithelium and lower TEER) and a shift from ciliated to secretory epithelial phenotype. Hypoxic differentiated hNECs had significantly shorter cilia length, slower beating frequency but had improved cilia coordination. CFTR functional response is altered in hypoxic differentiated hNECs. This study highlights the need to reconsider the oxygen tension used in CF primary cell cultures so as to preserve the characteristics and functional response of the cell models as we progress towards personalised medicine in CF.
Project description:Atmospheric oxygen tension is increasingly recognized to be hyperoxic for the maintenance of in vitro cell cultures. Oxygen has broad implications on energy metabolism, cellular growth and many regulatory functions. Primary airway epithelial cells (AECs) differentiated at air-liquid interface (ALI) are the gold standard for preclinical assessment of cystic fibrosis transmembrane conductance regulatory (CFTR) modulator efficacy in CF. The fidelity of CF AECs cultured at atmospheric oxygen tension rather than at reduced oxygen tension which more closely reflect the in vivo environment has not been studied to date. This study examined the impact of ambient (21%) and low (2%) oxygen tension on the expansion and differentiation of nasal epithelial cells (hNECs) derived from 11 participants (8 with CF and 3 non-CF). hNECs expanded under normoxic and chronic hypoxic conditions demonstrated epithelial cobblestone morphology and similar proliferation rate. hNECs differentiated at hypoxia demonstrated poorer differentiation capacity (significantly thinner epithelium and lower TEER) and a shift from ciliated to secretory epithelial phenotype. Hypoxic differentiated hNECs had significantly shorter cilia length, slower beating frequency but had improved cilia coordination. CFTR functional response is altered in hypoxic differentiated hNECs. This study highlights the need to reconsider the oxygen tension used in CF primary cell cultures so as to preserve the characteristics and functional response of the cell models as we progress towards personalised medicine in CF.
Project description:To reveal the transcriptional profiles of Actinobacillus pleuropneumoniae under biofilm and planktonic growth, we established a biofilm-forming culture method and constructed a mutant strain Δpga with defect in biofilm formation. Wild-type and Δpga mutant strains of Actinobacillus pleuropneumoniae strain 4074 were cultured in bottles with shaking for planktonic (WT_PK) and in microplates in static status for biofilm (WT_BF, Δpga), respectively. The bacteria in logarithmic growth period of different culture groups were collected for RNA seq.
Project description:To test the effects of hypoxia on transcription in Caulobacter crescentus, we cultured cells in a New Brunswick bioreactor under controlled conditions. Prior to innoculation, the medium was bubbled with laboratory air at maximum flow and stirred at 300 rpm for 2 hours. After this period, the medium was considered saturated with air and the oxygen probe was set to 100%. Untreated cultures were grown in air-saturated complex medium at 30 degrees C to OD660=0.5 at pH=7 (continuous air-bubbling; 300 rpm stirring). At cell harvest in aerated culture, the dissolved oxygen probe remained above 98%. To subject cells to hypoxia, culture at OD660=0.5, pH=7 was sparged continuously with nitrogen gas; the dissolved oxygen level as measured by the gas probe dropped from 100% to 0% over the course of 5 minutes under this condition. Hypoxic cultures were continually stirred and bubbled with nitrogen for another 20 minutes after the dissolved gas probe read 0%. Hypoxic cells were then harvested for RNA isolation.
Project description:relied on two-dimensional (2D) static cultures which neglect the dynamic,three-dimensional (3D) nature of the biophysical tumour microenvironment (TME), especially the role and impact of interstitial fluid flow (IFF). To address this, we undertook a transcriptome-wide analysis of the impact of IFF-like perfusion flow using a spheroid-on-chip microfluidic platform using cell line MCF7 (pleural effusion of metastatic breast adenocarcinoma). IFF-like perfusion flow allows 3D cancer spheroids to be integrated into extracellular matrices (ECM)-like hydrogels and exposed to continuous perfusion, to mimic IFF in the TME. Importantly, we have performed these studies both in experimental (normoxia) and pathophysiological (hypoxia) oxygen conditions. Our data indicated that gene expression was altered by flow when compared to static conditions, and for the first time showed that these gene expression patterns differed in different oxygen tensions, reflecting a differential role of spheroid perfusion inIFF-like flow in tumour-relevant hypoxic conditions in the biophysical TME. We were also able to identify factors primarily linked with IFF-like conditions which are linked with prognostic value in cancer patients and therefore could correspond to a potential novel biomarker of IFF in cancer. This study therefore highlights the need to consider relevant oxygen conditions when studying the impact of flow in cancer biology, as well as demonstrating the potential of microfluidic models of flow to identify IFF-relevant tumour biomarkers.
Project description:Purpose:The oxygen-regulated genes FNR and ARCA were combined with Komagataeibacter xylinus CGMCC 2955 to provide a new perspective for the study of the mechanism of oxygen environment on BC synthesis. Methods:The FNR and Arca overexpressing strains and the control strains were fermented under different partial oxygen pressures. The bacterial cellulose membrane in the logarithmic period of fermentation was enzymolyzed, and the bacteria were collected for transcriptome analysis.Sequencing was performed with Illumina and transcriptome analysis was performed on the bacteria under different conditions. Results:Transcriptome sequencing was performed using Illumina high-throughput sequencing technology on K. xylinus cultured under different oxygen tensions. The differentially expressed genes in the arcA overexpressing strains were mainly in the sulfur metabolism, two-component system, purine metabolism, and amino acid metabolism pathways compared to the control strains. Analysis showed that the arcA overexpression strain activated the sulfur metabolic pathway in K. xylinus. Due to the insufficient oxygen electron acceptors in the hypoxia, sulfate acted as the final electron acceptor and enhanced the growth ability of the strain. Through global regulation of the pathways of bacterial growth and metabolism as well as BC synthesis under low oxygen conditions, the arcA gene has enabled the strain to reach new levels of BC production. This study lays the foundation for further investigation of the mechanism of the effect of oxygen on BC synthesis in K. xylinus.
Project description:Transcriptome analysis of murine foetal NSCs (E14) after short-term (48 hours) and long-term (13 days) hypoxic (3% oxygen) culture compared to normoxic culture (21% oxygen) We focused on whole-transcriptome analyses using gene chip microarrays to compare expression profiles of NSCs cultured at hypoxic conditions to those of normoxic cells. Therefore, we used NSCs derived from the mesencephalon and the cortex and cultured them for short- and long-term at hypoxia/normoxia.
Project description:Senescent is an irreversible form of cell cycle arrest initiated by damaged cell constituents and subsequent pro-oncogenic signaling. Replicative senescence in vitro can be considered a model for human aging. When fibroblasts are cultured under atmospheric oxygen conditions of 20%, typical of normal tissue culture procedure, fibroblasts generally reach their replicative capacity at 50-60 population doublings (PDs). When fibroblasts are cultured under normal physiological oxygen conditions of 3%, PDs increase about 30% relative to atmospheric levels. Hence while oxygen is a requirement for normal aerobic respiration, it can contribute to the total amount of oxidative stress to which cells are exposed to, leading to a long-term adverse effect in vitro. Inasmuch, cultures maintained under hyperoxic and hypoxic conditions provide a convenient model system for assessing the relationship between oxygen/oxidative stress and senescence. We used microarrays to profile the changes in global gene expression during aging and senescence of Imr90 cells under growth oxygen conditions of 3% and 20%.
Project description:Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots and are known for their peculiar aromas and flavors. Axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation prompts searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) has been isolated and cultured, and its transcriptome has been analyzed under different in vitro culture conditions. The results show that the best T. borchii SP1 growth was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22ºC. The transcriptome analysis of this strain cultured in different media indicated that most of the gene transcription effort is due to a limited number of genes (20% of genes account for 80% of the transcription), that the transcription profile of the central metabolism genes was similar in the different conditions analyzed with a transcription signal detected for around 80% of the annotated genes. The gene expression profile suggests that T. borchii uses a fermentative rather than respiratory metabolism, even in aerobic conditions. Finally, there is a reduced expression of genes belonging to secondary metabolite clusters, whereas there is a significative transcription of those involved in producing volatile aromatic compounds.