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: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: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.
Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.