Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

The discovery of heavy-chain-only antibodies (HCAbs) in camelids and sharks led to the rise of a new research field in which single-domain antibodies are used for various applications. Single-domain antibodies are the antigen-binding fragments derived from HCAbs showing several beneficial properties (e.g., small size, specificity, stability under extreme conditions, cost-effective production, and ease of engineering). Importantly, they are stable in reducing cytoplasmic environment, which allows their use as an intrabody to target a wide range of intracellular targets. In this chapter, we discuss both the therapeutic potential of camelid single-domain antibodies (nanobodies) and their use as a research tool with the main focus on its intracellular employment. Targeting intracellular proteins using nanobodies as a therapeutic per se is, up to now, limited due to its incapacity to traverse the cellular membrane. They can however serve as a stepping stone to small compound development, since they directly target a resident, endogenous protein, similar to how a conventional drug acts. In addition, nanobodies are highly adaptable tools and possess interesting properties for more fundamental research objectives like the elucidation of protein function, the tracking and visualization of endogenous proteins in an in vivo setting, and the assessment of protein-protein interactions

Comment opérationnaliser et évaluer la prise en compte du concept ‘FAIR' dans le partage des données: Vers une grille simplifiée d’évaluation du respect des critères FAIR.

National audienceIndexed identifier ? Identification Are each data/dataset identified by an indexed and independant identifier ? Persistent metadata / data link ? Metadata traceability Are the metadata linked to the dataset through a persistent identifier? Metadata & authority linked ? Metadata traceability Are the metadata of each dataset linked to a unique authority (responsible for the datasets at a given time)? Unique, global, persistent ID? Identification Are the data identifiers unique, global and persistent ? Are the data identifiers unique, global and persistent ? Datasets linked to authority ? Metadata traceability Are all datasets linked to an authority (legal entity) through a unique and persistent identifier over time (e.g. institution, association or established body)? In case of a legal reuse restriction (such as personal data, state and public security, national defense secret, confidentiality of external relations, information systems security, secrets in industrial and commercial matters) , is the restriction properly justified?SHARC (SHAring Reward & Credit) est un groupe d’intérêt scientifique interdisciplinaire créé dans le cadre de RDA (Research Data Alliance) dans le but de faciliter le partage des données de recherche (et des ressources) par la valorisation de l’ensemble des activités pré-requises à ce partage, tout au long du cycle de vie des données. Dans ce cadre, un sous-groupe de travail SHARC élabore des grilles d’évaluation des chercheurs afin de mesurer leur niveau de prise en compte des principes FAIR dans la gestion de leurs données.La grille d’évaluation présentée dans ce poster est destinée à être complétée par tout scientifique produisant et / ou utilisant des données. Il s'agit d'un résumé d'une grille d'évaluation plus étendue conçue pour un partage optimal des données (non encore mise en œuvre pour le moment par la plupart des scientifiques).L'évaluation est basée sur les critères de conformité FAIR. Pour remplir cet objectif, la grille affiche le minimum de critères qui doivent absolument être appliqués par les chercheurs pour attester de leur pratique FAIR. Ces critères sont organisés en 5 groupes: «Motivations de partage»; "Trouvable", "Accessible", "Interopérable" et "Réutilisable". Pour chaque critère, 4 degrés d’évaluation sont proposés ("Jamais / Non évaluable"; "Si obligatoire"; "Parfois"; "Toujours"). Au moins un degré mais un seul doit être sélectionné par critère. L'évaluation doit être effectuée pour chaque catégorie F / A / I / R; L'évaluation finale est la somme de chaque degré coché rapportée au nombre total de critères dans chaque catégorie F / A / I / R. Des règles d'interprétation prenant en compte les «motivations du partage» sont proposées

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Nanobodies targeting cortactin proline rich, helical and actin binding regions downregulate invadopodium formation and matrix degradation in SCC-61 cancer cells

Cortactin is a multidomain actin binding protein that activates Arp2/3 mediated branched actin polymerization. This is essential for the formation of protrusive structures during cancer cell invasion. Invadopodia are cancer cell-specific membrane protrusions, specialized at extracellular matrix degradation and essential for invasion and tumor metastasis. Given the unequivocal role of cortactin at every stage of invadopodium formation, it is considered an invadopodium marker and potential drug target. We used cortactin nanobodies to examine the role of cortactin domain-specific function at endogenous protein level. Two cortactin nanobodies target the central region of cortactin with high specificity. One nanobody interacts with the actin binding repeats whereas the other targets the proline rich region and was found to reduce EGF-induced cortactin phosphorylation. After intracellular expression as an intrabody, they are both capable of tracing their target in the complex environment of the cytoplasm, and disturb cortactin functions during invadopodia formation and extracellular matrix degradation. These data illustrate the use of nanobodies as a research tool to dissect the role of cortactin in cancer cell motility. This information can contribute to the development of novel therapeutics for tumor cell migration and metastasis

Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion

Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp) 2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds

The effect of high and low frequency cortical stimulation with a fixed or a Poisson distributed interpulse interval on cortical excitability in rats

Neurostimulation is a promising treatment for refractory epilepsy. We studied the effect of cortical stimulation with different parameters in the rat motor cortex stimulation model. High intensity simulation (threshold for motor response - 100 mu A), high frequency (130 Hz) stimulation during 1 h decreased cortical excitability, irrespective of the interpulse interval used (fixed or Poisson distributed). Low intensity (10 mu A) and/or low frequency (5Hz) stimulation had no effect. Cortical stimulation appears promising for the treatment of neocortical epilepsy if frequency and intensity are high enough

Comment évaluer la FAIRness pour améliorer les processus de récompense du partage de la donnée? Une étape au travers d'une grille d'évaluation plus exhaustive

Le poster a été déposé dans ZenodoInternational audienceThe SHARC (SHAring Reward & Credit) interest group (IG) is an interdisciplinary group set up in the framework of RDA (Research Data Alliance) to improve crediting and rewarding mechanisms in the sharing process throughout the data life cycle. Notably, one of the objectives is to promote data sharing activities in research assessment schemes at national and European levels. To this aim, the RDA-SHARC IG is developing assessment grids using criteria to establish if data are compliant to the FAIR principles (findable /accessible / interoperable / reusable).The grid is aiming to be extensive, generic and trans-disciplinary. It is meant to be used by evaluators to assess the quality of the sharing practice of the researcher/scientist over a given period, taking into account the means & support available over that period. The grid displays a mind-mapped tree-graph structure based on previous works on FAIR data management (Reymonet et al., 2018; Wilkinson et al., 2016; Wilkinson et al., 2018; and E.U.Guidelines about FAIRness Data Management Plans). The criteria used are based on the work from FORCE 11*, and the Open Science Career Assessment Matrix designed by the EC Working group on Rewards under Open science. The criteria are organised in 5 clusters: ‘Motivations for sharing’; ‘Findable’, ‘Accessible’, ‘Interoperable’ and ‘Reusable’. For each criterion, 4 graduations are proposed (‘Never / Not Assessable’; ‘If mandatory’; ‘Sometimes’; ‘Always’). Only one value must be selected per criterion. Evaluation should be done by cluster; the final overall assessment will be based on the sum of the number of each ticked value / total number of criteria in each cluster; the ‘motivations for sharing’ should be appreciated qualitatively in the final interpretation. The final goals are to develop a graduated assessment of the researcher FAIRness literacy and help identifying needs to build FAIRness guidelines to improve the sharing capacity of researchers

Operationalizing and evaluating the FAIRness concept for a good quality of data sharing in Research: the RDA-SHARC-IG (SHAring Rewards and Credit Interest Group)

National audienceThe RDA-SHARC (SHAring Reward & Credit) interest group is an interdisciplinary volunteer member-based group set up as part of RDA (Research Data Alliance) to unpack and improve crediting and rewarding mechanisms in the sharing process throughout the data life cycle. Background and objectives of this group are reported here. Notably, one of the objectives is to promote the inclusion of data sharing activities in the research (& researchers) assessment scheme at national and European levels. To this aim, the RDA-SHARC-IG is developing two assessment grids using criteria to establish if data are compliant to the F.A.I.R principles (findable /accessible / interoperable / reusable) based on previous works on FAIR data management (Reymonet et al., 2018; Wilkinson et al., 2018; and E.U.Guidelines*): 1/ The self-assessment grid to be used by a scientist as a ‘checklist’ to identify her/his own activities and to pinpoint the hurdles that hinder efficient sharing and reuse of his/her data by all potential users. 2/ The two-level grid (quick/extensive) to be used by the evaluator to assess the quality of the researcher/scientist sharing practice, over a given period, taking into account the means & support available over that period. Assessment criteria are classified according their importance with regards to FAIRness (essential / recommended / desirable) meanwhile good practices are recommended for critical steps. To implement a highly fair assessment of the sharing process, appropriate criteria must be selected in order to design optimal generic assessment grids. This process requires participation, time and input from volunteer scientists data producers/users from various fields

EV-TRACK : transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice