Kostyuk, A. | Search bioRxiv

bioRxiv2021

Monitoring oxidative inflammatory processes in live cells and tissue with Hypocrates, a genetically encoded biosensor for hypochlorite

Hypochlorous acid, an aggressive oxidant, is important in immune defense against pathogens. The current lack of tools to monitor the dynamics of hypochlorous acid in live cells and tissue hinders a better understanding of inflammatory processes. We engineered a genetically encoded biosensor, Hypocrates, for the visualization of hypochlorous acid. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from E. coli. We determined sensitivity, selectivity, reaction rates, and the X-ray structure of this ratiometric redox biosensor, and tested the response of Hypocrates in HeLa Kyoto cells at varying hypochlorite concentrations. By combining Hypocrates with the biosensor HyperRed, we visualized the dynamics of hypochlorous acid and hydrogen peroxide in a zebrafish tail fin injury model.

immunology↗

bioRxiv2020

A Low-Cost Mass Spectrometry-Based Approach for Quantifying Purines in Placental Extracts

Hyperhomocysteinemia is a medical condition characterized by an abnormally high level of homocysteine in the blood associated with multiple human pathologies including preeclampsia - the most feared complication of pregnancy, with placenta playing the central role in the pathogenesis of preeclampsia. The developing placenta is highly sensitive to different adverse factors but its response to hyperhomocysteinemia is not fully clear. Previously we have demonstrated the activation of reactions of methionine cycle and the transsulfuration pathway in placental explants cultivated with homocysteine. The reactions of the methionine cycle are tightly connected with reactions of the folate cycle, encompassing reactions of de novo purine biosynthesis, which are crucial for the developing placenta, as they support rapid ATP generation to maintain energy status and increased biosynthesis of macromolecules. The sensitivity of de novo purine biosynthesis to hyperhomocysteinemia is not known. The aim of this study was to evaluate the impact of homocysteine on placental de novo purine biosynthesis. MethodsWe developed a simplified method to measure the level of all and newly formed purines by HPLC/ESI-MS, using a stable isotope glycine to label newly synthesized purines. The developed method proved to be highly sensitive, interday repeatable and intraday reproducible. We applied a method for placental explants from the first and third trimesters of gestation and MCF7 cells cultivated with 20 M and 40 M homocysteine corresponding to its concentrations at mild and intermediate hyperhomocysteinemia. ResultsThe developed method proved to be highly sensitive, interday repeatable and intraday reproducible. The content of total purines in placental explants from the first trimester of gestation was around 9.0 mol/g wet tissues at specified conditions of cultivation. The newly formed purines comprised around 1 % of total purines, decreased steadily in explants cultivated with 20 M and 40 M homocysteine, and reached the values characteristic for explants from third trimester cultivated without homocysteine, 4.0 mol/g wet tissues. The effect of homocysteine reproduced with MCF7 cells. ConclusionHomocysteine in concentrations characteristic of mild and intermediate hyperhomocysteinemia induces the down regulation of de novo purine biosynthesis in placental explants, and implies the shift of metabolic pathway to homocysteine remethylation and transsulfuration at the expense of de novo purine biosynthesis.

molecular biology↗