Biology subjects

Bouchet, A.

Publications and source records attributed to Bouchet, A..

4 recordsLinked to original sources

bioRxiv2026

Ohmline lipid platform: a dual antimicrobial and nanocarrier strategy to potentiate antibiotic efficacy

The global rise of antimicrobial resistance has significantly reduced the effectiveness of conventional antibiotics, highlighting the urgent need for alternative and complementary therapeutic strategies. Nanotechnology-based drug delivery systems, particularly lipid nanoparticles, have emerged as promising tools to enhance antibiotic efficacy while limiting toxicity and resistance development. In this study, we evaluated the antimicrobial activity and drug carrier potential of Ohmline, a novel alkyl-ether glycolipid capable of self-assembling into nanotubes and lipid nanoparticles. First, a wide range of Gram-positive and Gram-negative bacteria were used to test Ohmline nanotubes antibacterial activity. All examined strains were partially inhibited, with a more noticeable effect on Gram-positive bacteria. Then, the synergistic potential of Ohmline combined with commercially available antibiotics (ampicillin, ceftriaxone, and ciprofloxacin) was evaluated using two different approaches: binary mixtures of Ohmline nanotubes and antibiotics and microfluidically produced Ohmline:DMPC (75:25) nanoparticles with the antibiotics encapsulated. Binary formulations demonstrated strong, strain-dependent synergistic effects at sub-MIC antibiotic concentrations, particularly against Enterococcus faecalis and Citrobacter braakii. Notably, antibiotic encapsulation within Ohmline nanoparticles further enhanced antimicrobial efficacy compared to non-encapsulated combinations, achieving near-complete growth inhibition in E. faecalis and significant inhibition in Klebsiella pneumoniae and C. braakii. Overall, our findings demonstrate that Ohmline possesses intrinsic antibacterial activity and acts as an effective lipid nanocarrier that potentiates antibiotic action. The dual functionality of Ohmline supports its potential as a versatile building block for next-generation antimicrobial formulations.

microbiology↗

bioRxiv2025

NKCC1 as a signaling hub regulating KCC2 stability, chloride homeostasis, and seizure susceptibility

Chloride homeostasis relies on the dynamic balance between the neuronal co-transporters NKCC1 and KCC2. We reveal an unexpected mechanism by which NKCC1 governs KCC2 membrane stability. NKCC1 clusters recruits SPAK and PP1 to dynamically trap KCC2, compensating for its lack of a direct SPAK-binding site. Single-particle tracking shows that these NKCC1-rich assemblies operate as signaling hubs, enabling either SPAK-driven KCC2 phosphorylation and its membrane destabilization or PP1-mediated dephosphorylation of SPAK and KCC2 membrane stabilization. Peptides that activate SPAK by engaging NKCC1s PP1-binding motif lower KCC2 surface levels and reduce chloride extrusion, whereas a SPAK-inhibiting peptide prevents SPAK recruitment to NKCC1, stabilizes KCC2 in membrane clusters, and enhances chloride extrusion. An optimized peptide analog preserves KCC2 clustering under hyperexcitable conditions, reduces seizure frequency and severity in PTZ-induced epilepsy, and suppresses ictal activity in human epileptic tissue. These findings identify NKCC1-KCC2 coupling as a central regulatory axis for inhibitory signaling, and position our peptides as promising therapeutic candidates to restore chloride homeostasis in epilepsy and other disorders marked by impaired KCC2 membrane stability.

neuroscience↗

bioRxiv2025

Discovery of T-dioxygenases in bacteriophages and identification of a subclass that is dependent on a regulator protein for oxidation

5-Methylpyrimidine dioxygenases (5mYOXs) are iron (II)/2-oxoglutarate-dependent enzymes that catalyze the oxidation of DNA 5-methylpyrimidines. Key members include mammalian ten-eleven translocation (TET) dioxygenases and J-base binding proteins (JBP) from trypanosomes, which oxidize 5-methylcytosine (5mC) and thymine (T) on DNA, respectively, and are essential in gene regulation. Using sequence similarity networks and genome mining, we highlight functional predictions within the 5mYOX superfamily and identify thousands of bacteriophage-derived sequences, often found in operons encoding DNA modification machinery and binding proteins. Through high-throughput in vivo functional assays, we confirm that T oxidation occurs in bacterial viruses, establishing the presence of T dioxygenases outside of eukaryotes. We provide the first evidence for a 5mYOX subclass that is inactive unless co-expressed with a regulator protein, and through structural modeling, show that this regulator bears homology to the bacterial partition protein B (ParB). We propose a model of interaction between ParB and 5mYOX that includes complex formation, with 5mYOX binding DNA and ParB utilizing CTP, as in bacteria, to form an optimal structural configuration for productive oxidation and possibly migration on the DNA. We show these enzymes retain key catalytic residues found in TET/JBP enzymes and employ AlphaFold2-guided mutagenesis to identify clade-specific features critical for T oxidation, including a variable insertion important for ParB-independent activity and a conserved C-terminal extension essential for T oxidation in ParB-dependent homologs. These findings uncover modular determinants, regulatory mechanisms, and the evolutionary diversity of T-dioxygenases, expanding the functional landscape of the 5mYOX superfamily. SignificanceThis study demonstrates that enzymatic oxidation of thymidine (T) by 5-methylpyrimidine dioxygenases (5mYOXs) occurs in nature outside eukaryotes. Through bioinformatics and in vivo screening, we elucidate minimal T-dioxygenases that share conserved catalytic properties with eukaryotic 5mYOXs, yet possess unique domains, lineage-specific inserts, and/or regulators essential for activity. We uncovered two distinct, bacteriophage-encoded T-dioxygenase subclasses: one requiring a partition protein B (ParB)-like regulator for oxidation and another independent of it. This regulator is predicted to structurally resemble bacterial segregation ParB, which utilizes CTP binding and hydrolysis to migrate along DNA, suggesting novel mechanisms for T oxidation in phages. Our identification of subclass-specific features provides a foundation for further investigation of substrate selectivity, oxidation regulation, and 5mYOX divergence across domains of life.

biochemistry↗

bioRxiv2024

Positive Allosteric Modulation of the α5-GABAA receptors prevents neuronal atrophy and cognitive decline independently of tau tangle accumulation in the PS19 mouse model

BackgroundDysregulated Tau phosphorylation (p-Tau) is a hallmark of neurodegenerative disorders such as Alzheimers disease (AD) or frontotemporal dementia (FTD), resulting in neurofibrillary tangle accumulation, neuronal atrophy and cognitive impairment. Impaired somatostatin (SST) expression and reduced SST-expressing GABAergic neurons significantly contributes to AD-related pathophysiology and correlates with cognitive impairment. SST+ interneurons inhibit the dendrites of excitatory neurons in cortical layers and hippocampus, primarily through 5-GABA-A receptors, regulating cognitive function. Leveraging a newly developed small molecule that targets the 5-GABA-A receptors via positive allosteric modulation (5-PAM), we assessed its effects on p-Tau-related neuronal morphology, cognitive deficits and protein expression. MethodsIn the PS19 transgenic mouse model, we administered the 5-PAM, GL-II-73, either acutely or chronically at 3 and 6 months, corresponding to early and advanced stage of p-Tau accumulation. Golgi staining analyzed dendritic morphology and spine density in mice chronically exposed to 5-PAM. Western blotting was used to quantify p-tau and Tau expression. Spatial working memory was assessed using the Y-maze. ResultsChronic treatment at 3 and 6 months mitigated p-Tau-induced loss of spine density and reduced dendritic length. 5-PAM treatment did not affect p-tau levels. 5-PAM effectively reversed spatial working memory deficits induced by p-tau accumulation both acutely and chronically. Conclusions5-GABA-A receptor positive allosteric modulation displayed neurotrophic (spine and dendritic pathology) and procognitive (working memory) effects in the PS19 model, independently of p-Tau burden. This suggests a novel therapeutic strategy for p-Tau-related pathologies with both symptomatic and disease-modifying potential.

neuroscience↗