Biology subjects

Valerio, M.

Publications and source records attributed to Valerio, M..

4 recordsLinked to original sources

bioRxiv2026

3D Printed X-ray Compatible Microfluidics for Online Characterization of Hexosomes: A Synchrotron SAXS-on-Chip Study with Molecular Dynamics Insights

Online structural characterization during microfluidic lipid self-assembly is important for understanding and controlling the formation of nonlamellar liquid crystalline nanodispersions. Here, we report a 3D-printed, X-ray-compatible hydrodynamic flow-focusing microfluidic chip with variable channel dimensions, integrated with synchrotron small-angle X-ray scattering (SAXS), for position-resolved SAXS-on-chip monitoring of Ca2+-triggered hexosome formation. Hexosomes were produced under continuous flow by mixing ethanolic solutions of docosahexaenoic acid monoglyceride (MAG-DHA), the negatively charged phosphatidylglycerol DOPG, and -tocopherol with Ca2+-containing PIPES buffer. Online SAXS-on-chip measurements detected three Bragg reflections characteristic of the internal inverse hexagonal (H2) phase on a tens-of-milliseconds residence-time scale, revealing rapid structural evolution during microfluidic mixing. Complementary ex situ SAXS identified the DOPG/Ca2+ molar ratio as a key parameter modulating the direct vesicle-to-hexosome transformation and the compactness of the internal H2 nanostructures. Dynamic light scattering showed that the flow-rate ratio modulated nanoparticle size, yielding hexosomes with mean hydrodynamic diameters in the range of approximately 120-175 nm and polydispersity index values down to 0.14 at a total flow rate of 200 {micro}L min-1. Cryo-TEM revealed coexistence of hexosomes and vesicular nanostructures, highlighting morphological heterogeneity, while Coarse-Grained Molecular Dynamics simulations supported a central role of Ca2+-DOPG association in promoting a direct lamellar-H2 phase transition. Overall, this work shows that 3D-printed SAXS-compatible microfluidics can integrate continuous production with online structural characterization, providing a basis for future formulation and process optimization of drug-loaded cubosomes, hexosomes, and related nonlamellar liquid crystalline nanodispersions.

biophysics↗

bioRxiv2026

When lipids embrace RNA: pH-driven dynamics and mechanisms of LNP-mediated siRNA delivery

We present a comprehensive analysis of an efficient lipid nanoparticle (LNP) formulation that exhibits strong nucleic-acid delivery and potent inhibition of targeted RNAs. Using a combination of coarse-grained and atomistic molecular dynamics simulations, we characterized the pH-dependent structure of both unloaded and RNA-loaded LNPs, elucidated the mechanism of RNA encapsulation, and used these models to propose a plausible mechanism of endosomal escape. Consistent with prior experimental and computational studies, our simulations reproduce an inverted-hexagonal-type morphology in RNA-loaded LNPs, in which a hydrated core provides a polar environment suitable for accommodating RNA, whose charge is neutralized mainly by ionizable lipids that remain protonated near the RNA even at high pH, thereby bridging the RNA with the surrounding lipid environment. This structural picture is consistently observed across our multiscale simulations, with smaller self-assembled LNP mimetics reproducing the same local organization at both coarse-grained and atomistic resolution. A potential mechanism of endosomal escape emerges spontaneously from the simulations, involving stalk formation between the LNP and the endosomal membrane, followed by the opening of a water-filled pore that permits slow RNA diffusion, in line with the low efficiency and slow kinetics reported for endosomal escape. The rate-limiting step of endosomal escape arises from persistent electrostatic coupling between RNA and protonated ionizable lipids maintained by the immediate RNA-lipid environment, hindering cargo disengagement even after pore formation. This delayed release is consistent with the experimentally observed time-dependent inhibitory activity of the loaded LNP. Together, these results highlight the importance of local pK and protonation effects near the RNA, which are not captured by global apparent pK measurements.

biophysics↗

bioRxiv2026

Extending the MAD Toolbox: New Polymer Builder and Enhanced Martini Database

The MArtini Database (MAD) web server (https://mad.ens-lyon.fr/) has been updated with new tools, models, and capabilities to support a broader range of molecular systems for the Martini coarse-grained (CG) force field. The most notable addition is the MAD:Polymer Builder, enabling the automated construction of CG polymer models with varying architectures and complexities. The server now incorporates the latest developments in Martini 3, providing enhanced control within the MAD:Molecule Builder over the conversion of all-atom structures into CG representations, including the implementation of G[o]Martini 3 for protein complexes and water-protein interaction biases. The MAD:Polymer Builder and MAD:Molecule Builder are both adapted to work with intrinsically disordered proteins and domains. Substantial progress has been made in expanding the MAD:Database, making a growing library of Martini-ready compounds readily accessible across the entire MAD ecosystem. These advances position MAD as a comprehensive and evolving platform for the preparation of diverse systems in CG molecular simulations.

bioinformatics↗

bioRxiv2021

Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics. One-Sentence SummaryAmino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme

immunology↗