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Translatome Hypoxia-Acidosis, TMT-pulse SILAC

ABSTRACT: Cells were grown in light (R0K0) SILAC media (AthenaES) for 7 days, they were then incubated in light media for 24 hours in respective condition (normoxia neutral (NN) pH: 7.4; hypoxia neutral (HN)1% O2, pH: 7.4; hypoxia acidosis (HA), 1% O2, pH=6) and pulsed with heavy (R10K8) SILAC media (AthenaES) for 16 hr (TMT-pSILAC) following treatment.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Brain Glioblastoma Multiforme

SUBMITTER: Nathan Balukoff

LAB HEAD: Stephen Lee

PROVIDER: PXD015643 | Pride | 2020-09-28

REPOSITORIES: Pride

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Project description:Background: Penicillins inhibit cell wall synthesis; therefore, H. pylori must be dividing for these antibiotics to be effective. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens. Aim: To determine the effect of acidity on bacterial growth and the bactericidal efficacy of ampicillin. Methods: H. pylori were incubated in dialysis chambers suspended in 1.5L of media at various pHs with 5mM urea, with or without ampicillin, for 4, 8 or 16 hours, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined. The bacterial load of H. pylori infected gerbils was determined with and without profound acid inhibition. Results: At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division genes and penicillin-binding proteins. In the presence of ampicillin, viability and survival declined at pH 4.5 and 7.4 but not at pH 3.0. Profound acid inhibition of H. pylori infected gerbils increased the antral bacterial load >3 fold, showing that elevation of intragastric pH stimulated growth. Conclusions: Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes at pH 3.0. Therefore, at pH 3.0, the pH at the gastric surface, the bacteria are non-dividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 hours/day should enhance the efficacy of triple therapy and of amoxicillin, even allowing dual therapy for H. pylori eradication. There were a total of three biological replicates, each containing one control (incubated at pH 7.4) and one acidic (pH 3.0) sample, whose RNA were extracted on different dates. Microarray studies performed on these six samples were done in triplicate. One of the triplicates for two of the biological control triplicates were removed from the data set due to poor hybridization

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Translatome Hypoxia-Acidosis, TMT-pulse SILAC

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