Project description:Heterogeneity of host cells as well as bacteria residing within them has been known to induce drug tolerance in pathogens. In Mycobacterium tuberculosis, particularly, drug tolerance within host is a major hurdle in the path to attain a sterlising cure. Reports have shown how residence of Mtb within macrophages makes the pathogen refractory to anti-TB therapy. However, the mechanisms responsible for induction of tolerance to antibiotics, particularly in resting macrophages where Mtb continues to actively replicate, is yet to be deciphered. Reports have suggested that heterogeneity induced in Mtb by cues sensed in the host environment could be a contributing factor to drug tolerance. With regard to this, differences in redox physiology of intra-macrophage Mtb (mid-point potential of the major cytosolic redox buffer in Mtb, mycothiol- EMSH) have been shown to play a role in influencing antibiotic-mediated killing. In this study, we have attempted to analyse the trascriptomic profiles of individual redox sub-populations of Mtb from within macrophages- viz., the EMSH-reduced bacteria with mid-point potential ranging between -285 and -310 mV, shown to be most refractory to killing by front-line anti-TB antibiotics inside macrophages, as well as EMSH-basal bacteria with mid-point potential ranging between -270 and -280 mV, with a much higher degree of antibiotic susceptibility within macrophages. Analysing gene expression level differences between these bacterial sub-populations would help in providing a mechanistic understanding of drug tolerance in Mtb, bred from phenotypic heterogeneity in the pathogen.
Project description:RNA was isolated from mammary glands from 55 day old control mice, mice overexpressing the miR-200b/200a/429 cluster in mammary epithelial cells (MTB-200ba429) mice overexpressing the IGF-IR transgene in mammary epithelial cells (MTB-IGFIR), and mice overexpressing both the miR-200b/200a/429 cluster and the IGF-IR transgene in mammary epithelial cells (MTB-IGFIRba429)
Project description:Iron-sulfur (Fe-S) cluster containing proteins are a subset of proteins with crucial functions in the maintenance of cellular physiology throughout all kingdoms of life. The systems involved in the biogenesis and repair of Fe-S clusters hence plays important role in fine-tuning the availability and functionality of Fe-S proteins. Two of the systems known in bacteria are, Isc and Suf. Compared to the facultative anaerobe, E. coli, which codes for the two multi-genic Fe-S biogenesis systems; Mtb Fe-S biogenesis machinery is skewed with a multi-genic Suf system (sufRBDCSUT) and a single gene of Isc system (iscS). Several Fe-S proteins are deployed by Mtb to maintain cellular homeostasis and survival in a hostile host environment. Hence, we determine the transcriptome of Mtb on depletion of the two key enzymes of Fe-S biogenesis- IscS and sufS, that could help understand the role and regulation between the two systems in the human pathogen Mtb.
Project description:Genome-wide expression data can provide important insights into normal and pathological cellular processes. However, the ability to use gene expression data to quantitatively assess the activation state of a given signaling pathway or transcriptional network in a sensitive and specific manner remains an important unmet goal. We now describe a computational algorithm, energy-paired scoring (EPS), that satisfies these criteria by predicting pathway activity using gene-gene interactions within a pathway signature in a manner analogous to the estimation of energy generated by two charged particles, as described by Coulomb’s law. We demonstrate the ability of EPS to: quantitatively assess pathway activation levels in vivo and in vitro; accurately estimate the extent of pathway inhibition achieved by gene knockdown; sensitively detect crosstalk between endogenous signaling pathways in vivo; and accurately identify compounds capable of inhibiting selected signaling pathways. Our findings indicate that EPS can accurately predict pathway activity over a wide dynamic range based upon gene expression data sets derived from multiple profiling platforms, as well as different species, tissues and cell types in both in vitro and in vivo contexts Four timepoints (0h, 24h, 48h and 96h) with 3 replicates per timepoint of doxycycline induction for MTB (Control), MTB/TAN, MTB/TOM and MTB/TWNT1
Project description:Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study is to compare gene expression (RNA-seq) profile between Mtb infected Fth+/+ and Fth-/- mice at 9 weeks post Mtb infection. Gene expression analysis demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo
Project description:COPE is a biology driven protocol with 2 independent, multicentric, two-arm non-comparative randomized (2:1) phase II trials in 2 distinct populations: colorectal cancer patients and non-small-lung cancer patients.
For each phase II trial, patient will be randomized between two arms with two patients randomized in arm A for one patient randomized in arm B:
* Arm A (Experimental - initial MTB providing therapeutic recommendation based on tumor sequencing and then follow-up combining standard imaging and ctDNA analysis)
* Arm B (Standard - initial MTB providing therapeutic recommendation based on tumor sequencing and then follow-up based on standard imaging).
Project description:ILCs were sorted from the lungs of Mtb infected mice at 5 and 14 days post infection and subjected to sc-RNA sequencing to determine gene signature profile
Project description:Respiratory ATP-synthesis is at present the only known mechanism for ATP synthesis in Mtb. This makes Mtb particularly vulnerable to inhibition of respiratory ATP synthase inhibitors such as TMC207, a novel compound for treatment of tuberculosis. We now provide first evidence that Mtb possesses a pathway that is fermentative in nature that could compensate lack of respiratory ATP synthesis. We identified acetate as a fermentation product in Mtb. Production of acetate was mediated by phosphotransacetylase (Pta) and acetate kinase (AckA). In acetate fermenting Mtb cultures, ATP levels remained stable despite inhibition of respiratory ATP synthase. Deletion of the PtaAckA pathway in Mtb decreased ATP content and impaired survival. This study provides evidence that in Mtb substrate level phosphorylation can compensate lack of oxidative phosphorylation, and thus facilitates survival of Mtb in the absence of respiration. Acetate fermentation contributes to adaptation to respiration-limiting conditions, and plays an important role in the emerging field of fermentative metabolism of Mtb. We performed DNA microarray analysis to validate the reduction of oxygen concentration by comparing aerobic and hypoxic cultures. RNA was prepared from Mtb after two days of cultivation in aerobic and in hypoxic cultures. At each condition, Mtb were cultured in medium supplemented with glycerol and glucose. Labelled cDNA from three independent experiments was subjected to array analysis.