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Glioblastoma (GBM) is the most aggressive type of primary brain tumours. Anti-angiogenic therapies (AAT), such as bevacizumab, have been developed to target the tumour blood supply. However, GBM presents mechanisms of escape from AAT activity, including a speculated direct effect of AAT on GBM cells. Furthermore, bevacizumab can alter the intercellular communication of GBM cells with their direct microenvironment. Extracellular vesicles (EVs) have been recently described as main acts in the GBM microenvironment, allowing tumour and stromal cells to exchange genetic and proteomic material. Herein, we examined and described the alterations in the EVs produced by GBM cells following bevacizumab treatment. Interestingly, bevacizumab that is able to neutralise GBM cells-derived VEGF-A, was found to be directly captured by GBM cells and eventually sorted at the surface of the respective EVs. We also identified early endosomes as potential pathways involved in the bevacizumab internalisation by GBM cells. Via MS analysis, we observed that treatment with bevacizumab induces changes in the EVs proteomic content, which are associated with tumour progression and therapeutic resistance. Accordingly, inhibition of EVs production by GBM cells improved the anti-tumour effect of bevacizumab. Together, this data suggests of a potential new mechanism of GBM escape from bevacizumab activity.

Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this model

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems

Summary Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems., Graphical Abstract, Highlights • CryoET and ssNMR give complementary information about proteins in native membranes • One sample can be prepared for both methods without the use of detergents • Hybrid method shows differences between purified and native preparations of YidC • Sample preparation reduces costs and time and suggests new strategy for assignment, Membrane proteins are often treated with detergents, which can affect structure and activity. Baker et al. apply a hybrid method to bacterial membrane proteins in native membranes without detergent, using solid-state NMR spectroscopy and electron cryotomography. They find that the structure and function of YidC differ with and without detergent.

Materials and methods. (DOCX 22 kb)

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Le Grapevine fanleaf virus (GFLV) et l'Arabis mosaic virus (ArMV) sont deux agents responsables de la maladie du court-noué de la vigne. Ces deux virus, appartenant au genre Nepovirus de la famille des Comoviridae, sont transmis spécifiquement par deux espèces distinctes de nématodes ectoparasites, Xiphinema index et Xiphinema diversicaudatum respectivement, selon un mode de transmission semi-persistant, non circulant et non multipliant. Des expériences antérieures ont montré que seule la protéine de capside (CP) du GFLV détermine la spécificité de transmission par X. index [1, 2]. La capacité du virus à être transmis spécifiquement par son vecteur implique probablement des déterminants viraux situés à la surface de la particule virale. L'objectif de notre étude est de caractériser les domaines de la capside du GFLV participant à cette spécificité. A partir de la structure cristallographique du Tobacco ringspot virus [3], un modèle tridimensionnel de la particule virale de GFLV a été construit. Cinq zones, de 6 à 12 acides aminés, exposées à la surface externe du modèle et divergentes entre le GFLV et l’ArMV, ont été identifiées. Par mutagenèse dirigée, ces zones de la CP du GFLV ont été substituées par leurs équivalents ArMV. Parmi les constructions recombinantes obtenues, seules deux ont abouti à des virus chimériques infectieux. Tous les mutants non infectieux se sont avérés incapables de protéger les ARN viraux de l’action de Rnases et sont donc très probablement déficients dans l’encapsidation du virus. L’évaluation de la transmissibilité des virus chimériques infectieux, par X. index et X. diversicaudatum, a permis d’étudier l’implication de deux zones de la CP dans la transmission. Les résultats suggèrent que l’une d’entre elles intervient dans l’interaction avec le nématode. Afin de compléter cette étude, une analyse structurale des particules virales de GFLV et d’ArMV est en cours. La reconstruction de la structure 3D de ces deux virus est réalisée à partir d'images prises en cryomicroscopie électronique à transmission, de particules virales purifiées, hydratées-congelées. Ces deux approches permettront d’affiner la cartographie des domaines de surface de la CP impliqués dans la transmission du GFLV et de l’ArMV, par leurs vecteurs respectifs.

International audience