Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex

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Combining reverse genetics and nuclear magnetic resonance‐based metabolomics unravels trypanosome‐specific metabolic pathways

Numerous eukaryotes have developed specific metabolic traits that are not present in extensively studied model organisms. For instance, the procyclic insect form of Trypanosoma brucei, a parasite responsible for sleeping sickness in its mammalian‐specific bloodstream form, metabolizes glucose into excreted succinate and acetate through pathways with unique features. Succinate is primarily produced from glucose‐derived phosphoenolpyruvate in peroxisome‐like organelles, also known as glycosomes, by a soluble NADH‐dependent fumarate reductase only described in trypanosomes so far. Acetate is produced in the mitochondrion of the parasite from acetyl‐CoA by a CoA‐transferase, which forms an ATP‐producing cycle with succinyl‐CoA synthetase. The role of this cycle in ATP production was recently demonstrated in procyclic trypanosomes and has only been proposed so far for anaerobic organisms, in addition to trypanosomatids. We review how nuclear magnetic resonance spectrometry can be used to analyze the metabolic network perturbed by deletion (knockout) or downregulation (RNAi) of the candidate genes involved in these two particular metabolic pathways of procyclic trypanosomes. The role of succinate and acetate production in trypanosomes is discussed, as well as the connections between the succinate and acetate branches, which increase the metabolic flexibility probably required by the parasite to deal with environmental changes such as oxidative stress.     

On the use of aptamer microarrays as a platform for the exploration of human prothrombin/thrombin conversion

Microarrays are particular biosensors with multiple grafted probes that are generally used for parallel and simultaneous detection of various targets. In this study, we used microarrays with aptamer probes in order to follow up the different biomolecular interactions of a single enzyme, the thrombin protein, involved in the complex coagulation cascade. More precisely, thanks to label-free surface plasmon resonance imaging, we were able to monitor in real time an important step in the firing of the coagulation cascade in situ—the enzymatic transformation of prothrombin into thrombin, catalyzed by factor Xa. We were also able to appraise the influence of other biochemical factors and their corresponding inhibiting or enhancing behaviors on thrombin activation. Our study opens the door for the development of a complete microarray-based platform not only for the whole coagulation cascade analysis but also for novel drug screening assays in pharmacology.     

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.     

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

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Gene electrotransfer: from biophysical mechanisms to in vivo applications: Part 1- Biophysical mechanisms

Electropulsation is one of the nonviral methods successfully used to deliver genes into living cells in vitro and in vivo. This approach shows promise in the field of gene and cellular therapies. The present review focuses on the processes supporting gene electrotransfer in vitro. In the first part, we will report the events occurring before, during, and after pulse application in the specific field of plasmid DNA electrotransfer at the cell level. A critical discussion of the present theoretical considerations about membrane electropermeabilization and the transient structures involved in the plasmid uptake follows in a second part.     

Stimulating Effect of External Myo-Inositol on the Expression of Mutant Forms of Aquaporin 2

Myo-inositol (MI; hexahydroxycyclohexane, C₆H₆O₁₂) is a small neutral molecule used as a compatible osmolyte in the kidney medulla. At high concentrations, MI appears to act as a chemical chaperone and was shown to promote plasma membrane expression of the impaired cystic fibrosis chloride channel (Δ508-CFTR). In the present study, we measured whether MI could increase expression of two human aquaporin 2 (AQP2) mutants which were recently identified as causing nephrogenic diabetes insipidus (NDI). Both proteins (D150E and G196D) were expressed in Xenopus laevis oocytes, but only D150E displayed an increase in oocyte water permeability (P _f). Adding 5 mM MI to the bathing solution for 24 h produced a 50% increase in the D150E-associated P _f, while it had no effect on noninjected oocytes or on oocytes expressing wt-AQP2 or G196D. Western blots performed on purified plasma membrane preparations confirmed that MI increased the amount of D150E present at the plasma membrane, while G196D was always undetectable. X. laevis oocytes are remarkably impermeable to MI, and the effect of MI on D150E expression does not require the presence of intracellular MI. The effect of external MI was dose-dependent (K _0.5 was 130 μM) and specific with respect to other forms of inositols. Further studies on a second group of AQP2 mutants causing NDI showed that K228E activity was similarly stimulated by MI, while V71M, A70D and S256L were not. It is concluded that physiological concentrations of extracellular MI can stimulate the expression of a specific subgroup of AQP2 mutants.