Reshaping the intestinal microbiota by the ingestion of fiber, such as pectin, improves alcohol-induced liver lesions in mice by modulating bacterial metabolites, including indoles, as well as bile acids (BAs). In this context, we aimed to elucidate how oral supplementation of pectin affects BA metabolism in alcohol-challenged mice receiving feces from patients with alcoholic hepatitis. Pectin reduced alcohol liver disease. This beneficial effect correlated with lower BA levels in the plasma and liver but higher levels in the caecum, suggesting that pectin stimulated BA excretion. Pectin modified the overall BA composition, favoring an augmentation in the proportion of hydrophilic forms in the liver, plasma, and gut. This effect was linked to an imbalance between hydrophobic and hydrophilic (less toxic) BAs in the gut. Pectin induced the enrichment of intestinal bacteria harboring genes that encode BA-metabolizing enzymes. The modulation of BA content by pectin inhibited farnesoid X receptor signaling in the ileum and the subsequent upregulation of Cyp7a1 in the liver. Despite an increase in BA synthesis, pectin reduced BA serum levels by promoting their intestinal excretion. In conclusion, pectin alleviates alcohol liver disease by modifying the BA cycle through effects on the intestinal microbiota and enhanced BA excretion.

Flow cytometry is a relevant tool to meet the requirements of academic and industrial research projects aimed at estimating the features of a bacterial population (e.g., quantity, viability, activity). One of the remaining challenges is now the safe assessment of bacterial viability while minimizing the risks inherent to existing protocols. In our core facility at the Paris‐Saclay University, we have addressed this issue with two objectives: measuring bacterial viability in biological samples and preventing bacterial contamination and chemical exposure of the staff and cytometers used on the platform. Here, we report the development of a protocol achieving these two objectives, including a viability labeling step before bacteria fixation, which removes the risk of biological exposure, and the decrease of the use of reagents such as propidium iodide (PI), which are dangerous for health (CMR: carcinogenic, mutagenic, and reprotoxic). For this purpose, we looked for a non‐CMR viability dye that can irreversibly label dead bacteria before fixation procedures and maintain intense fluorescence after further staining. We decided to test on the bacteria, eFluor Fixable Viability dyes, which are usually used on eukaryotic cells. Since the bacteria had size and granularity characteristics very similar to those associated with flow cytometry background signals, a step of bacterial DNA labeling with SYTO or DRAQ5 was necessarily added to differentiate them from the background. Three marker combinations (viability‐DNA) were tested on LSR Fortessa and validated on pure bacterial populations (Gram⁺, Gram⁻) and polybacterial cultures. Any of the three methods can be used and adapted to the needs of each project and allow users to adapt the combination according to the configuration of their cytometer. Having been tested on six bacterial populations, validated on two cytometers, and repeated at least two times in each evaluated condition, we consider this method reliable in the context of these conditions. The reliability of the results obtained in flow cytometry was successfully validated by applying this protocol to confocal microscopy, permeabilization, and also to follow cultures over time. This flow cytometry protocol for measuring bacterial viability under safer conditions also opens the prospect of its use for further bacterial characterization.

This study investigates the potential of pulmonary delivery of siRNA as an emergency therapy for acute lung injury (ALI). To obtain a quick anti‐inflammatory response, TNF‐α, a critical pro‐inflammatory cytokine, is knocked down for its early involvement in inflammatory responses. Therefore, TNF‐α siRNA‐lipid nanoparticles (LNPs) are designed and characterized for cellular uptake in lipopolysaccharide (LPS)‐activated RAW264.7 murine macrophages, primary alveolar macrophages, and neutrophils from a murine ALI model. Intracellular trafficking and siRNA cytoplasmic release are evaluated in untreated and LPS‐activated RAW264.7 cells. LPS‐activated cells exhibit a fast and strong uptake of LNPs, including in primary cells. In RAW264.7 cells, significant endosomal escape and siRNA cytoplasmic release are observed after 16 h. In vit ro efficacy studies reveal consistent TNF‐α inhibition across pre‐, co‐, and post‐incubation protocols, confirming the versatility of siRNA‐LNPs in preventive or curative conditions. After intratracheal administration of TNF‐α siRNA‐LNPs in a murine ALI model, the distribution of LNPs demonstrates an accumulation in immune cells, including macrophages and neutrophils, reducing TNF‐α and IL‐6 levels, indicating a rapid anti‐inflammatory effect. This work underscores the efficacy of TNF‐α siRNA‐LNPs in treating lung inflammatory diseases like ALI and highlights the importance of optimizing LNP distribution and delivery timing to enhance therapeutic outcomes.

Trichomonas vaginalis, a protozoan parasite specific to the human genital tract, is one of the most common sexually transmitted pathogens. Its pathogenicity is strongly associated with its expression of a broad array of proteases triggering cytotoxic effects in host epithelial cells. Vaginal microbiota-associated Lactobacillus, including those of L. gasseri in particular, can counteract T. vaginalis pathogenesis, but the mechanisms involved have yet to be clarified. T. vaginalis strain G3 (Tv G3) cytotoxicity was assessed by examining cell morphology, cell detachment, and fluorescent labeling of the F-actin cytoskeleton and immunolabeling of vinculin-position focal adhesions (FAs) by confocal laser scanning electron microscopy on confluent cervicovaginal epithelial HeLa cell monolayers. The inhibitory effects of bacterial cells and secreted products of L. gasseri ATCC 9857 and KS 120.1 on the Tv G3 viability and parasite deleterious effects on HeLa cells were investigated. Pre-adhering L. gasseri cells delayed but did not inhibit Tv G3-induced cell detachment, F-actin cytoskeleton disorganization and the disappearance of vinculin-positive focal FAs. L. gasseri KS 120.1 secretion products had a rapid parasiticide activity by killing time- and concentration-dependent Tv G3 parasites after direct contact. By killing Tv G3 parasites already associated with the epithelial cells, secretion products have abolished parasite-induced cell detachment. Our findings suggest that vagina microbiota-associated L. gasseri creates a physical barrier and exerts pharmacological-type mechanisms to counteract the deleterious cytotoxic effects of T. vaginalis.

BACKGROUND: Pulmonary hypertension is a severe disease with high mortality rates due to right ventricular (RV) failure. The molecular and cellular processes involved in RV remodeling, including Ca ²⁺ handling, remain elusive due to the lack of relevant animal models. In this study, we aim to understand better the pathophysiological mechanisms involved in RV failure. METHODS: We used the chronic thromboembolic pulmonary hypertension (CTEPH) pig model, which leads to progressive RV hypertrophy and dysfunction. Cellular, molecular unbiased global transcriptional profiling and biochemical analyses were performed on RV cardiomyocytes from CTEPH and Sham-operated pigs. RESULTS: CTEPH pigs replicated the hemodynamics and histological changes of human CTEPH features. Transcriptome analysis in Sham and CTEPH pigs revealed molecular RV remodeling close to human patients with pulmonary arterial hypertension with decompensated RV function and notably identified changes in genes involved in Ca ²⁺ signaling. At the cellular level, CTEPH myocytes presented reduced L-type Ca ²⁺ current in association with reduced mRNA of CACNA1C . Furthermore, CTEPH myocytes showed lower [Ca ²⁺ ] _i transients, decreased sarcoplasmic reticulum Ca ²⁺ content, and decreased cell shortening, related to reduced SERCA2a (Sarco/endoplasmic reticulum Ca ²⁺ -ATPase isoform 2a) protein expression. Moreover, CTEPH cardiomyocytes exhibited reduced Ca ²⁺ spark occurrence, which relied on smaller RyR2 (ryanodine receptor 2) clusters and T-tubule disorganization. Finally, these alterations in Ca ²⁺ homeostasis were also associated with an increased store-operated Ca ²⁺ entry and the de novo expression of the Ca ²⁺ sensor protein STIM1L (stromal interaction molecule 1 long isoform) in CTEPH myocytes as well as in RV from human patients with pulmonary arterial hypertension. CONCLUSIONS: Our data reveal cellular Ca ²⁺ cycling remodeling that participates in the pathogenesis of RV dysfunction and may constitute therapeutic targets to limit the development of RV dysfunction.

Cystathionine-β-synthase (CBS) domains are found in proteins of all living organisms and have been proposed to play a role as energy sensors regulating protein activities through their adenosyl ligand binding capacity. In plants, members of the CBSX protein family carry a stand-alone pair of CBS domains. In Arabidopsis (Arabidopsis thaliana), CBSX1 and CBSX2 are targeted to plastids where they have been proposed to regulate thioredoxins (TRXs). TRXs are ubiquitous cysteine thiol oxido-reductases involved in the redox-based regulation of numerous enzymatic activities as well as in the regeneration of thiol-dependent peroxidases. In Arabidopsis, 10 TRX isoforms have been identified in plastids and divided into five sub-types. Here, we show that CBSX2 specifically inhibits the activities of m-type TRXs toward two chloroplast TRX-related targets. By testing activation of NADP-malate dehydrogenase and reduction of 2-Cys peroxiredoxin, we found that TRXm1/2 inhibition by CBSX2 was alleviated in the presence of AMP or ATP. We also determined, by pull-down assays, a direct interaction of CBSX2 with reduced TRXm1 and m2 that was abolished in the presence of adenosyl ligands. In addition, we report that, compared with wild-type plants, the Arabidopsis T-DNA double mutant cbsx1 cbsx2 exhibits growth and chlorophyll accumulation defects in cold conditions, suggesting a function of plastidial CBSX proteins in plant stress adaptation. Together, our results show an energy-sensing regulation of plastid TRX m activities by CBSX, possibly allowing a feedback regulation of ATP homeostasis via activation of cyclic electron flow in the chloroplast, to maintain a high energy level for optimal growth.