Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10⁻⁸ M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.

Ventilator-associated pneumonia (VAP) remains a significant complication among critically ill patients, with associated mortality approaching 50%. Despite the implementation of established preventive strategies, additional interventions are urgently needed to further reduce the incidence of VAP. Probiotic therapy has emerged as a promising adjunctive approach; the benefits of probiotic therapy may be more pronounced in critically ill patients without pre-existing infections. The PROACT study will evaluate the prophylactic role of probiotics in a critically ill population with acute brain injury to reduce the VAP incidence, while also exploring microbiological endpoints and mortality to refine patient selection criteria. The PROACT study is a prospective, randomized, double-blind, placebo-controlled, multicenter trial designed to evaluate the efficacy of probiotic prophylaxis in adult critically ill patients requiring invasive mechanical ventilation following acute brain injury (e.g., head trauma, ischemic or hemorrhagic stroke). Patients with suspected pulmonary aspiration or pre-existing pulmonary infections at enrollment are excluded to reduce confounding. Participants are randomized in a 1:1 ratio to receive either a placebo (glucose polymer) or a probiotic formulation containing Lactobacillus acidophilus LA-5, Lactobacillus plantarum, Bifidobacterium lactis BB12, and Saccharomyces boulardii. The assigned intervention is administered twice daily via nasogastric tube and oropharyngeal application for up to 30 days or until intensive care unit (ICU) discharge. The primary endpoint is the incidence of VAP, as defined by current international guidelines, in the intention-to-treat (ITT) population. Secondary endpoints include the incidence of VAP in the modified intention-to-treat (mITT) population, catheter-related bloodstream infections, occurrence of sepsis and septic shock, 30-day all-cause mortality, ICU length of stay, and duration of mechanical ventilation. The study is powered at 80% (α = 0.05) to detect a clinically meaningful difference based on effect size estimates based on the PROVAP study, requiring a total sample size of 208 patients. All participating centers implement standard VAP prevention bundles as part of routine care. By evaluating the efficacy of probiotic therapy in preventing VAP and the impact on mortality among critically ill brain-injured patients, this trial has the potential to generate high-quality evidence supporting the incorporation of probiotics into standard VAP prevention protocols. The findings may have significant implications for clinical practice guidelines and public health policy related to infection control and microbiome-targeted interventions in the intensive care setting. Registered at ClinicalTrials.gov (identifier: [NCT06092554]). Registered on 2023-10-15.

Background : Ventilator-associated pneumonia (VAP) remains a significant complication among critically ill patients, with associated mortality approaching 50%. Despite the implementation of established preventive strategies, additional interventions are urgently needed to further reduce the incidence of VAP. Probiotic therapy has emerged as a promising adjunctive approach; the benefits of probiotic therapy may be more pronounced in critically ill patients without pre-existing infections. The PROACT study will evaluate the prophylactic role of probiotics in a critically ill population with acute brain injury to reduce the VAP incidence, while also exploring microbiological endpoints and mortality to refine patient selection criteria. Methods : The PROACT study is a prospective, randomized, double-blind, placebo-controlled, multicenter trial designed to evaluate the efficacy of probiotic prophylaxis in adult critically ill patients requiring invasive mechanical ventilation following acute brain injury (e.g. head trauma, ischemic or hemorrhagic stroke). Patients with suspected pulmonary aspiration or pre-existing pulmonary infections at enrollment are excluded to reduce confounding. Participants are randomized in a 1:1 ratio to receive either a placebo (glucose polymer) or a probiotic formulation containing Lactobacillus acidophilus LA-5, Lactobacillus plantarum , Bifidobacterium lactis BB12, and Saccharomyces boulardii . The assigned intervention is administered twice daily via nasogastric tube and oropharyngeal application for up to 30 days or until Intensive Care Unit (ICU) discharge. The primary endpoint is the incidence of VAP, as defined by current international guidelines, in the intention-to-treat (ITT) population. Secondary endpoints include the incidence of VAP in the modified intention-to-treat (mITT) population, catheter-related bloodstream infections, occurrence of sepsis and septic shock, 30-day all-cause mortality, ICU length of stay and duration of mechanical ventilation. The study is powered at 80% (α=0.05) to detect a clinically meaningful difference based on effect size estimates based on the PROVAP study, requiring a total sample size of 186 patients. All participating centers implement standard VAP prevention bundles as part of routine care. Discussion : By evaluating the efficacy of probiotic therapy in preventing VAP and the impact on mortality among critically ill brain-injured patients, this trial has the potential to generate high-quality evidence supporting the incorporation of probiotics into standard VAP prevention protocols. The findings may have significant implications for clinical practice guidelines and public health policy related to infection control and microbiome-targeted interventions in the intensive care setting. Trial Registration Registered at ClinicalTrials.gov (identifier: [NCT06092554]). Registered on 2023-10-15.