Tankyrase1/2 inhibitor XAV-939 reverts EMT and suggests that PARylation partially regulates aerobic activities in human hepatocytes and HepG2 cells.

The maintenance of a highly functional metabolic epithelium in vitro is challenging. Metabolic impairments in primary human hepatocytes (PHHs) over time is primarily due to epithelial-to-mesenchymal transitioning (EMT). The immature hepatoma cell line HepG2 was used as an in vitro model to explore strategies for enhancing the hepatic phenotype. The phenotypic characterization includes measuring the urea cycle, lipid storage, tricarboxylic acid-related metabolites, reactive oxygen species, endoplasmic reticulum calcium efflux, mitochondrial membrane potentials, oxygen consumptions rate, and CYP450 biotransformation capacity. Expression studies were performed with transcriptomics, co-immunoprecipitation and proteomics. CRISPR/Cas9 was also employed to genetically engineer HepG2 cells. After confirming that PHHs develop an EMT phenotype, expression of tankyrase1/2 was found to increase over time. EMT was reverted when blocking tankyrases1/2-dependent poly-ADP-ribosylation (PARylation) activity, by biochemical and genetic perturbation. Wnt/ß-catenin inhibitor XAV-939 blocks tankyrase1/2 and treatment elevated several oxygen-consuming reactions (electron-transport chain, OXHPOS, CYP450 mono-oxidase activity, phase I/II xenobiotic biotransformation, and prandial turnover), suggesting that cell metabolism was enhanced. Glutathione-dependent redox homeostasis was also significantly improved in the XAV-939 condition. Oxygen consumption rate and proteomics experiments in tankyrase1/2 double knockout HepG2 cells then uncovered PARylation as master regulator of aerobic-dependent cell respiration. Furthermore, novel tankyrase1/2-dependent PARylation targets, including mitochondrial DLST, and OGDH, were revealed. This work exposed a new mechanistic framework by linking PARylation to respiration and metabolism, thereby broadening the current understanding that underlies these vital processes. XAV-939 poses an immediate and straightforward strategy to improve aerobic activities, and metabolism, in (immature) cell cultures.

Staphylococcus epidermidis biofilm assembly and self-dispersion: bacteria and matrix dynamics.

Staphylococcus epidermidis, despite being a commensal of human skin and mucosa, is a major nosocomial pathogen implicated in device-associated infections. The dissemination of infection to other body sites is related to biofilm dispersal. This study focused on the dispersion stage of S. epidermidis CIP 444 biofilm, with the assessment of biofilm matrix composition in a time-dependent experiment (7 days extended) with 3 independent repetitions, using confocal laser scanning microcopy (CLSM) in association with ZEN 3.4 blue edition, COMSTAT, and ImageJ software. SYTO-9, propidium iodide (PI), DID'OIL, FITC, and calcofluor white M2R (CFW) were used to stain biofilm components. The results indicated that the biomass of dead cells increased from 15.18 ± 1.81 µm3/µm2 (day 3) to 23.15 ± 6.075 µm3/µm2 (day 4), along with a decrease in alive cells' biomass from 22.75 ± 2.968 µm3/µm2 (day 3) to 18.95 ± 5.713 µm3/µm2 (day 4). When the intensities were measured after marking the biofilm components, in a 24-h-old biofilm, polysaccharide made up the majority of the investigated components (52%), followed by protein (18.9%). Lipids make up just 11.6% of the mature biofilm. Protein makes up the largest portion (48%) of a 4-day-old biofilm, followed by polysaccharides (37.8%) and lipids (7.27%). According to our findings, S. epidermidis CIP 444 dispersion occurred on day 4 of incubation, and new establishment of the biofilm occurred on day 7. Remarkable changes in biofilm composition will pave the way for a new approach to understanding bacterial strategies inside biofilms and finding solutions to their impacts in the medical field.