The Collagen V Homotrimer [α1(V)]3 Production Is Unexpectedly Favored over the Heterotrimer [α1(V)]2α2(V) in Recombinant Expression Systems

Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [α1(V)]3 homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V α-chains showed that, contrary to fibroblasts, collagen V α-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly

FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1

The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention

Rôle du collagène V dans l'élaboration de matrice extracellulaire (production recombinante de la protéine, interactions moléculaires et tests fonctionnels in vivo)

Le collagène V est faiblement représenté dans les tissus. Sa discrète présence tissulaire ne le confine cependant pas à un rôle insignifiant. De fait, des mutations sur ses gènes sont la cause chez l homme de l apparition du syndrome d Ehlers-Danlos classique, caractérisé par une hyperétirabilité de la peau et une hypermobilité des articulations. Le projet de recherche qui m'a été confié visait à définir le rôle exact du collagène V dans l'élaboration de matrices extracellulaires fonctionnelles c'est-à-dire dont les propriétés biomécaniques et physiologiques sont préservées. Le développement de deux approches s est avéré incontournable : (1) caractériser les interactions avec d'autres composants matriciels, en particulier les protéoglycannes, et définir leurs implications dans la fibrillogénèse, (2) générer des modèles murins par transgénèse pour mieux cerner le rôle de ce collagène in vivo au cours du développement. Un rôle isoforme- et tissu-spécifique a été dégagé pour ce collagèneAlthough being a quantitatively minor component of connective tissues, collagen V plays a crucial role in matrix organization. Mutations in human collagen V genes have been shown to be responsible for the classic type Ehlers-Danlos syndrome (EDS) which is characterised by skin laxity and joint hypermobility. The aim of my research project was to provide new insights on the role of collagen V in the elaboration of a functional extracellular matrix for which biomechanical and physiological properties are preserved. Two approaches that combine in vitro and in vivo experiments have been carried out : (1) interactions between collagen V and matrix components, namely the proteoglycans, were analyzed in order to determine their implication in fibrillogenesis regulation, (2) a functional analysis by generating transgenic mice has been performed to decipher the role of this collagen during development and in EDS pathophysiology. An isoform- and tissue-specific function has been drawn for this collagenLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Les collagènes du derme : au-delà de leurs propriétés structurales

La matrice extracellulaire est un réseau complexe de macromolécules comme les collagènes, les protéoglycannes et l'élastine qui s'imbriquent fortement les unes aux autres et qui, par l'intermédiaire d'interactions avec les cellules environnantes, vont permettre le maintien de la cohésion tissulaire. C'est grâce à ces nombreuses connexions que se mettent en place la plupart des évènements importants pour les grands programmes cellulaires, à savoir la migration, la prolifération, la différenciation et l'apoptose. La peau, et plus particulièrement le derme, est un exemple parfait de cette grande variété de molécules dont l'organisation et la composition doivent être parfaitement maintenues afin d'assurer les propriétés biomécaniques et fonctionnelles de ce tissu. Les collagènes sont les composants les plus abondants des matrices extracellulaires et représentent une famille de protéines de 27 membres différents. Mais ce sont surtout les collagènes fibrillaires I, III et V, les collagènes FACITs XII, XIV et XVI ainsi que le collagène de type VI qui sont les plus représentés dans le derme. Ces molécules sont à présent très bien caractérisées du point de vue de leur structure, mais leur fonction demeure toujours un peu floue. De nombreux gènes ont déjà été identifiés comme étant responsables de pathologies du tissu conjonctif et plus particulièrement de la peau. Les gènes codant le collagène V sont, par exemple, la cible de nombreuses mutations conduisant au syndrome d'Ehlers-Danlos classique qui est une maladie qui atteint principalement la peau et la structure du derme. Cependant, il semble clair qu'il est très difficile d'établir un lien réel entre génotype et phénotype pas plus qu'un lien entre la localisation et la fonction, pour ces molécules. Ce sont sans doute des études in vivo, dont de nombreuses sont d'ailleurs en cours de réalisation, qui devraient apporter des réponses plus claires à toutes ces interrogations

The governance of ASGM in Guinea and Côte d'Ivoire

International audienceIn Guinea and Côte d’Ivoire, the development of artisanal and small-scale gold mining (ASGM) is linked to the emergence and intensification of labor migration directed toward mining areas. Four forms of gold mining mobility can be distinguished: short-term mobility, long-term mobility, transitional mobility, and circular mobility. Based on ethnographic data collection at several gold mining sites in both countries, this chapter aims to illustrate how ASGM migrations are shaped by systems of territorial governance in mining areas. This governance takes various forms: collective at the village level in Guinea, and more individualized in Côte d’Ivoire, where governance is linked to landed property. Since the 2010s, ASGM has been transformed and mechanized, while maintaining an informal status. This evolution is a new context that leads to a more disputed local governance in Guinea and to the reinforcement of the land issue in Côte d’Ivoire. These joint developments reflect the strong links between mobility and territorial governance, two sides of the same coin in the development of gold mining

Iatrogénie médicamenteuse : contribution à l’uniformisation de la terminologie en langue française pour la pratique de soins et la recherche clinique

Si de nombreuses initiatives ont déjà précisé la terminologie relative à la iatrogénie médicamenteuse en langue anglaise, des définitions consensuelles n’ont pas encore été formalisées en langue française. A partir d’un groupe de réflexion multidisciplinaire, nous proposons une terminologie se démarquant de celle utilisée pour la pharmacovigilance ou la gestion des risques. Nous recommandons d’éviter l’utilisation du terme « événement indésirable », trop imprécis, et de préférer le terme « d’événement iatrogène médicamenteux » (EIM) à celui « d’effet indésirable ». Nous proposons de classer les EIM en « effet indésirable stricto sensu » versus « participation iatrogène à une situation pathologique multifactorielle » pour prendre en compte l’intrication fréquente des causes médicamenteuses et non médicamenteuses. L’association systématique entre les notions d’erreur et d’évitabilité est également discutable, et nous suggérons d’évaluer « l’atténuabilité » des EIM plutôt que leur « évitabilité ». Le « non-respect de l’information sur le médicament par le patient » devrait être distingué de la « prescription hors autorisation de mise sur le marché » et de « l’usage détourné »

Orthogonal scaffold of magnetically aligned collagen lamellae for corneal stroma reconstruction

The creation of 3D scaffolds that mimic the structure of physiological tissue required for normal cell function is a major bioengineering challenge. For corneal stroma reconstruction this necessitates the creation of a stroma-like scaffold consisting of a stack of orthogonally disposed sheets of aligned collagen fibrils. This study demonstrates that such a scaffold can be built up using magnetic alignment. By allowing neutralized acid-soluble type I collagen to gel in a horizontal magnetic field (7 T) and by combining a series of gelation-rotation-gelation cycles, a scaffold of orthogonal lamellac composed of aligned collagen fibrils has been formed. Although initially dilute, the gels can be concentrated without noticeable loss in orientation. The gels are translucent but their transparency can be greatly improved by the addition of proteoglycans to the gel-forming solution. Keratocytes align by contact guidance along the direction of collagen fibrils and respect the orthogonal design of the collagen template as they penetrate into the bulk of the 3D matrix. The scaffold is a significant step towards the creation of a corneal substitute with properties resembling those of native corneal stroma. (c) 2007 Elsevier Ltd. All rights reserved

Tissue engineering of the cornea: orthogonal scaffold of magnetically aligned collagen lamellae for corneal stroma reconstruction

International audiencexx

p.L1612P, a novel voltage-gated sodium channel Nav1.7 mutation inducing a cold sensitive paroxysmal extreme pain disorder.

BACKGROUND: Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. METHODS: Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. RESULTS: The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady-state inactivation curve (V1/2 from -61.8 ± 4.5 mV to -30.9 ± 2.2 mV, n = 4 and 7, P < 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady-state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. CONCLUSIONS: The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD

The collagen V homotrimer [alpha1(V)](3) production is unexpectedly favored over the heterotrimer [alpha1(V)](2)alpha2(V) in recombinant expression systems.

Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [alpha1(V)](2)alpha2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proalpha1(V) and proalpha2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [alpha1(V)](3) homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proalpha2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V alpha-chains showed that, contrary to fibroblasts, collagen V alpha-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly