Biology subjects

Villa, R.

Publications and source records attributed to Villa, R..

5 recordsLinked to original sources

bioRxiv2026

Environmental monitoring for pathogen detection in zebrafish housing systems using molecular techniques: A 3Rs principles-based approach

Health monitoring is essential to ensure laboratory animal welfare and the reliability of experimental data in zebrafish research facilities. Conventional surveillance strategies based on resident or sentinel fish have limitations in terms of sensitivity and animal use, highlighting the need for alternative approaches consistent with the 3Rs principles. In this perspective, the present study evaluated the use of sludge collected from recirculating aquaculture systems as an environmental matrix for molecular health monitoring. Because sludge accumulates microorganisms and organic material from the entire system, it represents a promising sample for pathogen detection. Following an initial environmental surveillance phase to detect pathogens present in the system, the study aimed to optimise a molecular protocol for sludge analysis. To this end, four commercial DNA extraction kits were evaluated to assess their effectiveness in recovering bacterial DNA from sludge. Their performance was analysed by quantitative PCR in terms of extraction efficiency, repeatability, and limit of detection. The results highlighted the strengths and limitations of each DNA extraction protocol and confirmed the suitability of sludge as a non-lethal matrix for pathogen detection. These findings support the implementation of environmental monitoring as a practical and cost-effective alternative to sentinel-based surveillance, improving pathogen detection while reducing animal use in accordance with the 3Rs principles and Directive 2010/63/EU.

molecular biology↗

bioRxiv2026

Molecular mechanism of M1-mediated influenza A virus assembly

Influenza A virus assembly is orchestrated by matrix protein 1 (M1), which engages ribonucleoproteins, mediates glycoprotein incorporation, and scaffolds lipid envelopment, but how these activities are performed and coordinated is unclear. We determined high-resolution structures of M1 within virions, virus-like particles, and in vitro. We found that M1 binds phosphatidylinositol 4,5-bisphosphate at the membrane, oligomerizes through an electrostatic interface, and switches between three alternative conformational states in different regions of the virion. The M1 conformer at the front associates with ribonucleoproteins and initiates budding. In the body of the virion, two conformers alternate to create an extended filament with seam-like discontinuities. Enrichment of the seam-prone conformation at the virion rear induces envelope closure and exposes a binding site for the cytoplasmic tail of neuraminidase (NA) to promote virus release. Conformational switching within a polarized M1 lattice thereby couples organization of the viral components with shaping of the virion architecture.

microbiology↗

bioRxiv2025

Low-temperature integration of printed electrochemical sensors into OSTE+ micrfluidics for oxygen monitoring and sensing

Real-time monitoring of biological events is essential for advancing in drug testing, disease modeling, and precision medicine. However, conventional analytical methods often lack the spatiotemporal resolution to capture dynamic cellular responses, particularly in Organ-on-a-Chip (OoC) platforms and microphysiological systems (MPS). While progress has been made in sensor development, integrating sensors into microfluidic devices remains technically challenging. Materials commonly used in microfluidics, such as polydimethylsiloxane (PDMS), are poorly suited for stable sensor integration due to low metal adhesion. Off-stoichiometry thiol-ene-epoxy (OSTE+) polymers present a promising alternative due to the presence of unreacted thiol groups. These groups enable dual functionalities: strong metal affinity for direct for robust sensor integration, and oxygen scavenging essential for microscale oxygen level control. However, their compatibility with conventional sensor fabrication methods, particularly those requiring elevated temperatures, is limited. This work introduces a low-temperature fabrication method based on inkjet printing and photonic curing, suitable for thermally sensitive substrates. Gold and silver nanoparticle inks were directly sintered onto OSTE+ using photonic curing, producing functional electrochemical sensors for spatially resolved and real-time oxygen detection. Unlike conventional thermal sintering, which is incompatible with many microfluidic materials, photonic curing preserves the chemical reactivity and mechanical properties of the polymer without requiring surface treatment or adhesive layers. To demonstrate functionality, sensors were integrated into microfluidic devices for continuous monitoring of dissolved oxygen. Real-time oxygen depletion driven by the materials intrinsic scavenging capability was successfully quantified. These findings support the compatibility of sensor integration into OSTE+ polymers enhancing the real-time monitoring capabilities in OoC and MPS systems. HighlightsO_LIInkjet-printed sensors are integrated into OSTE+ using a low-temperature protocol. C_LIO_LIPhotonic curing enables sintering of gold and silver nanoparticle inks without damaging the polymer substrate. C_LIO_LIOxygen scavenging is monitored in real time using integrated electrochemical sensors. C_LIO_LITunable oxygen gradients are achieved in OSTE+ microfluidics for organ-on-chip models. C_LI

bioengineering↗

bioRxiv2022

Epithelial monolayer development and tight junction assembly on nanopillar arrays

Nanostructured materials provide an outstanding opportunity to both stimulate and measure cellular processes. In the context of tight junctions, it was previously reported that transient application of a nanotopographic surface over the apical brush border membrane of epithelial monolayers triggers redistribution of ZO-1, claudins, and F-actin that increases paracellular macromolecular flux. In excitable tissues, nanomaterials have been used to apply and measure electrical signals, such action potentials. As a first step towards translating these technologies for use in analysis of epithelial function, we sought to culture monolayers composed of transporting epithelia over nanopillar arrays without perturbing cellular structure or function. Madin-Darby Canine kidney I (MDCK I) cells were cultured on collagen-coated silicon chips with [~]1 m diameter nanopillar arrays. Fluorescence and scanning electron microscopy were used to assess the impact of height on nanopillar-epithelial interactions. Monolayers formed over and were largely unaffected by short nanopillars. These nanopillars were located beneath basal epithelial surfaces and were not preferentially located within lateral intercellular spaces or beneath ZO-1-containing junctions. In contrast, tall nanopillars that exceeded cell height disrupted MDCK I monolayer growth. Cells interacted with, encircled, and extended cytoplasm over the top of tall nanopillars, and dense ZO-1 and F-actin accumulations occasionally surrounded apical membranes adjacent to nanopillars. Finally, when grown over arrays composed of nanopillars 1 - 2 m shorter than cells, MDCK I frequently grew between nanopillars. As a result, nanopillars were more commonly present within lateral intercellular spaces beneath junctions. Apical complex structure was intact, as assessed by fluorescence microscopy of ZO-1, occludin, claudin-2, F-actin, and E-cadherin. Apical microvilli were also unaffected. We therefore show that conditions can be defined to allow growth of mature, correctly assembled epithelial monolayers with nanopillars localized to lateral intercellular spaces. This sets the stage for application of nanotechnologies for perturbation and analysis of epithelial biology.

cell biology↗

bioRxiv2021

Full bandwidth electrophysiology of seizures and epileptiform activity enabled by flexible graphene micro-transistor depth neural probes

Mapping the entire frequency bandwidth of neuronal oscillations in the brain is of paramount importance for understanding physiological and pathological states. The ability to record simultaneously infraslow activity (<0.1 Hz) and higher frequencies (0.1-600 Hz) using the same recording electrode would particularly benefit epilepsy research. However, commonly used metal microelectrode technology is not well suited for recording infraslow activity. Here we use flexible graphene depth neural probes (gDNP), consisting of a linear array of graphene microtransistors, to concurrently record infraslow and high frequency neuronal activity in awake rodents. We show that gDNPs can reliably record and map with high spatial resolution seizures, post-ictal spreading depolarisation, and high frequency epileptic activity through cortical laminae to the CA1 layer of the hippocampus in a mouse model of chemically-induced seizures. We demonstrate functionality of chronically implanted devices over 10 weeks by recording with high fidelity spontaneous spike-wave discharges and associated infraslow activity in a rat model of absence epilepsy. Altogether, our work highlights the suitability of this technology for in vivo electrophysiology research, in particular, to examine the contributions of infraslow activity to seizure initiation and termination.

neuroscience↗