Biology subjects

Sackett, D. L.

Publications and source records attributed to Sackett, D. L..

3 recordsLinked to original sources

bioRxiv2023

High resolution spatial investigation of intracellular oxygen in muscle cells.

Molecular oxygen (O2) is one of the most functionally relevant metabolites. O2 is essential for mito-chondrial aerobic respiration. Changes in O2 affect muscle metabolism and play a critical role in the maintenance of skeletal muscle mass, with lack of sufficient O2 resulting in detrimental loss of muscle mass and function. How exactly O2 is used by muscle cells is less known, mainly due to the lack of tools to address O2 dynamics at the cellular level. Here we discuss a new imaging method for the real time quantification of intracellular O2 in muscle cells based on a genetically encoded O2-responsive sensor, Myoglobin-mCherry. We show that we can spatially resolve and quantify intracellular O2 concentration in single muscle cells and that the spatiotemporal O2 gradient measured by the sensor is linked to, and reflects, functional metabolic changes occurring during the process of muscle differentiation. HighlightsO_LIReal time quantitation of intracellular oxygen with spatial resolution C_LIO_LIIdentification of metabolically active sites in single cells C_LIO_LIOxygen metabolism is linked to muscle differentiation C_LI

cell biology↗

bioRxiv2022

Histone deacetylase inhibitor induces acetyl-CoA depletion leading to lethal metabolic stress in RAS-pathway activated cells

RAS-mutant cancers are among the most refractory to treatment. Apart from new G12C genotype targeted therapies, strategies to kill RAS-mutant cells by directly targeting RAS or its downstream effectors have been mostly unsuccessful, mainly due to pathway redundancy and heterogeneities in RAS-induced phenotypes. Here we identified a RAS-phenotype that can be targeted by the histone deacetylase inhibitor (HDACi) romidepsin. We showed that the hyperacetylation induced by romidepsin depleted acetyl-CoA, the cell donor substrate for acetylation, and led to metabolic stress and death in KRAS-activated cells. Elastic net analysis on transcriptomics from a 608-cell panel confirmed that HDACi sensitivity was linked to a difference in profiles in two pathways involved in acetyl-CoA metabolism. The analysis of a clinical dataset confirmed that perturbation of the two acetyl-CoA pathways were correlated with HDACi sensitivity in patients treated with belinostat. Our analysis suggests the potential utility of a RAS-associated acetyl-CoA phenotype to sharpen treatment choices for RAS-activated tumors.

cancer biology↗

bioRxiv2020

A Highly Sensitive Fluorogenic Assay for the Detection of Nephrotoxin-Induced Oxidative Stress in Live Cells and Renal Tissue

A common manifestation of drug toxicity is kidney injury or nephrotoxicity. Nephrotoxicity frequently leads to termination of clinical trials and drug withdrawals, which jeopardize biomedical progress. Efficient preclinical screening platforms capable of detecting mild signs of kidney damage, which may elicit considerable toxic response in vivo, are essential. A common manifestation of chemical toxicity is oxidative modification of cellular biomolecules. Therefore, we have developed a facile biomolecule carbonyl detection assay that well surpasses the sensitivity of the standard assays in identifying modest forms of renal injury. Using a novel fluorogenic sensor, TFCH, we have demonstrated the applicability of the assay in live kidney cells and in renal tissue. This robust assay can help inform preclinical decisions to recall unsafe drug candidates. Application of this assay in identifying and analyzing diverse pathologies is envisioned.

biochemistry↗