A Proposed Methodology to Deal with the Impact of In Vitro Cellular Matrix on the Analytical Performances of a Targeted Metabolomic LC-HRMS Method.

Performances of metabolomic methods have been widely studied on biological matrices such as serum, plasma, and urine; but much less on in vitro cell extracts. While the impact of cell culture and sample preparation on results are well-described, the specific effect of the in vitro cellular matrix on the analytical performance remains uncertain. The aim of the present work was to study the impact of this matrix on the analytical performance of an LC-HRMS metabolomic method. For this purpose, experiments were performed on total extracts from two cell lines (MDA-MB-231 and HepaRG) using different cell numbers. Matrix effects, carryover, linearity, and variability of the method were studied. Results showed that the performances of the method depend on the nature of the endogenous metabolite, the cell number, and the nature of the cell line. These three parameters should, therefore, be considered for the processing of experiments and the interpretation of results depending on whether the study focuses on a limited number of metabolites or aims to establish a metabolic signature.

CLN3 deficiency leads to neurological and metabolic perturbations during early development.

Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3 Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.

Molecular ruler mechanism and interfacial catalysis of the integral membrane acyltransferase PatA.

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity "ruler." We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl­coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors.