Biology subjects

Brun, J.

Publications and source records attributed to Brun, J..

8 recordsLinked to original sources

bioRxiv2024

Fibrotic-like collagen matrices as innovative 3D in vitro models for investigating the impact of pathological ECM on muscle regeneration in muscular dystrophies

Muscular dystrophies are characterized by impaired skeletal muscle contraction due to genetic mutations. Beyond the disruption of muscle function, the extracellular matrix (ECM) surrounding muscle fibers becomes fibrotic, further impeding tissue repair and function. In this study, we designed both healthy and fibrotic matrices to examine the impact of persistent fibrosis on muscle cell behavior. Fibrotic matrices were synthesized by 3D printing of dense collagen solutions in air, followed by slow gelation to yield non-porous, isotropic hydrogels. These matrices were subsequently crosslinked using EDC/NHS chemistry, resulting in a Youngs modulus of approximately 50 kPa. The behavior of C2C12 myoblasts cultured within the fibrotic matrices was compared to cells grown in healthy matrices. Our results showed that the fibrotic matrix had a detrimental effect on cell behavior. Myoblasts were unable to differentiate into mature myotubes, exhibited poor alignment, and suffered from hypoxia. Furthermore, these cells failed to proliferate, secreted inflammatory cytokines, and were unable to remodel their ECM. Using matrices possessing a single characteristic of the fibrotic matrix (i.e stiff or non porous), the predominant factor for each feature of the cell phenotype was determined. These findings underscore the detrimental effects of a fibrotic persistent ECM on muscle homeostasis. The development of these two distinct 3D muscle models, one representing healthy muscle and the other fibrotic, offers a valuable tool for investigating the pathophysiology of muscular dystrophy. HighlightsO_LIStiff and non-porous dense collagen matrices synthesized by 3D printing recapitulate the key characteristics of fibrosis observed in muscular dystrophies C_LIO_LIFibrotic matrices negatively impact myoblast proliferation and differentiation into mature myocytes C_LIO_LIFibrotic matrices impede the 3D organotypic organization of myocytes C_LIO_LIThis 3D pathological model of skeletal muscle offers a valuable tool for investigating how persistent fibrosis influences the progression of muscular dystrophy C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/630059v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c2403corg.highwire.dtl.DTLVardef@1074147org.highwire.dtl.DTLVardef@14eb055org.highwire.dtl.DTLVardef@11ac419_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Sclerostin blockade inhibits bone resorption through PDGF receptor signaling in osteoblast lineage cells

While sclerostin-neutralizing antibodies (Scl-Ab) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Ab control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anti-catabolic effect of Scl-Ab could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effect on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin co-activates PDGFRs independently of Wnt/{beta}-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in pre-osteoblasts. In turn, Scl-Ab prevents sclerostin-mediated co-activation of PDGFR signaling and consequent M-CSF up-regulation in pre-osteoblast cultures, thereby inhibiting osteoclast activity in pre-osteoblast/osteoclast co-culture assays. These results provide a new potential mechanism explaining the dissociation between anabolic and anti-resorptive effects of long-term Scl-Ab.

pharmacology and toxicology↗

bioRxiv2023

Cooperativity and induced oligomerisation control the interaction of SARS- CoV-2 with its cellular receptor and patient-derived antibodies

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. One-Sentence SummaryMultivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.

biophysics↗

bioRxiv2022

Anisotropic dense collagen hydrogels possessing two ranges of porosity to create the adequate microenvironment for muscle bundles: a step towards skeletal muscle modeling

Despite the crucial role of the extracellular matrix (ECM) in the organotypic organization and function of skeletal muscles, most 3D models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy, and porosity. Here, a novel 3D in vitro model of muscle extracellular matrix was developed to differentiate myogenic cells (C2C12 line) into myotubes and reproduce their natural cell/cell and cell/matrix interactions. An anisotropic hydrogel mimicking the perimysium was obtained thanks to unidirectional 3D printing of dense collagen with aligned collagen fibrils. The space between the different layers was tuned to generate an intrinsic porosity (100 {micro}m) suitable for nutrient and oxygen diffusion. By modulating the gelling conditions, the mechanical properties of the construct reached those measured in the physiological muscle ECM. The addition of large channels (600 {micro}m) by molding permitted to create a second range of porosity suitable for cell colonization without altering the physical properties of the hydrogel. C2C12 cells embedded in Matrigel(R), seeded within the channels, organized in 3D, and differentiated into multinucleated mature myotubes. This organization reproduced the global muscular bundles, i.e., the endomysium encompassing myotubes. These results show that porous and anisotropic dense collagen hydrogels colonized with myoblasts are promising biomaterials to model skeletal muscle. Table of contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=39 SRC="FIGDIR/small/496716v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@bae7e8org.highwire.dtl.DTLVardef@cf453corg.highwire.dtl.DTLVardef@1ca69a2org.highwire.dtl.DTLVardef@1dd13cd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA novel extracellular matrix-like hydrogel increases the physiological relevance of the skeletal muscle model. C_LIO_LIPorous and anisotropic dense collagen hydrogels mimic the muscle ECM physical properties. C_LIO_LIUnidirectional printing of dense collagen creates a porous and anisotropic scaffold in a single step. C_LIO_LIAnisotropic dense collagen hydrogels promote C2C12 differentiation into myotubes and their 3D organization. C_LI

bioengineering↗

bioRxiv2021

Human basigin (CD147) does not directly interact with SARS-CoV-2 spike glycoprotein

Basigin, or CD147, has been reported as a co-receptor used by SARS-CoV-2 to invade host cells. Basigin also has a well-established role in Plasmodium falciparum malaria infection of human erythrocytes where it is bound by one of the parasites invasion ligands, reticulocyte binding protein homolog 5 (RH5). Here, we sought to validate the claim that the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein can form a complex with basigin, using RH5-basigin as a positive control. Using recombinantly expressed proteins, size exclusion chromatography and surface plasmon resonance, we show that neither RBD nor full-length spike glycoprotein bind to recombinant human basigin (either expressed in E. coli or mammalian cells). Given the immense interest in SARS-CoV-2 therapeutic targets, we would caution the inclusion of basigin in this list on the basis of its reported direct interaction with SARS-CoV-2 spike glycoprotein. ImportanceReducing the mortality and morbidity associated with COVID-19 remains a global health priority. Critical to these efforts is the identification of host factors that are essential to viral entry and replication. Basigin, or CD147, was previously identified as a possible therapeutic target based on the observation that it may act as a co-receptor for SARS-COV-2, binding to the receptor binding domain of the spike protein. Here, we show that there is no direct interaction between the RBD and basigin, casting doubt on its role as a co-receptor and plausibility as a therapeutic target.

biochemistry↗

bioRxiv2020

Analysis of SARS-CoV-2 spike glycosylation reveals shedding of a vaccine candidate

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, which may have implications for the resulting immune response and future vaccine design.

biochemistry↗

bioRxiv2020

COVID Moonshot: Open Science Discovery of SARS-CoV-2 Main Protease Inhibitors by Combining Crowdsourcing, High-Throughput Experiments, Computational Simulations, and Machine Learning

We report the results of the COVID Moonshot, a fully open-science, crowd sourced, structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. We discovered a non-covalent, non-peptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>840 ligand-bound X-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2,400 compounds) for this campaign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.

biochemistry↗

bioRxiv2018

Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2-1 m) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

biophysics↗