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A new approach to the synthesis of the (-)-205B alkaloid is described in this paper. This work is characterised by the development of an efficient chirality transfer through a silyl tethered intramolecular alkylation reaction, an unprecedented tandem highly selective iridium catalyzed partial reduction of lactam coupled with an acid promoted aza-Prins reaction, and an almost complete stereochemical control in Shenvi's radical hydrogen atom transfer on an exocyclic methylene. The second part of this work demonstrates the positive allosteric behavior of this natural alkaloid toward α7 nAChRs, in contrast to the reported inhibitory effect of the unnatural enantiomer.

Summary Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering., Graphical abstract, Highlights • GPCR regulation of diverse potassium channels through N-terminal fusions • Fusion to the viral PBCV-1 Kcv channel requires a specific linker and receptor • Tetrameric Kcv and dimeric mTREK-1 channels fusions respond to GPCR ligands • The engineered fusions broaden the properties of the channels' electrical signals, Motivation For biosensing applications, electrical signals are attractive since they can be recorded by micro- or nano-electronic systems for developing miniaturized detection devices in biomedical or environmental fields. One of the main limiting steps is the design of sensing elements that specifically recognize ligands such as biomarkers or chemical compounds. Ligand-gated ion channels are natural biosensors that specifically recognize ligands and generate an electrical signal. They have naturally evolved to be finely tuned by regulatory domains or proteins resulting in an appropriate electrical signal. The objective of this work is to leverage these natural biosensors by engineering diverse artificial ion channels that have the desired properties for biosensing or basic research applications. The main challenge is to design de novo allosteric regulations between ion channels and physiologically unrelated membrane proteins. Previously, different G protein-coupled receptors were successfully coupled to a specific ion channel, Kir6.2. In this work, we demonstrate that other ion channels (viral and murine) with distinct oligomerization properties can also be fused to GPCRs via their N-terminal domains. These results open avenues for diversifying engineered ligand-gated ion channels., Ligand-gated ion channels are natural biosensors that generate highly sensitive and fast electrical signals. García-Fernández et al. broaden the regulatory properties of electrical signal output by fusing different potassium channels to GPCRs. Functional fusion to tetrameric and dimeric channels demonstrates the versatility of this approach.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has shown that vaccine preparedness is critical to anticipate a fast response to emergent pathogens with high infectivity. To rapidly reach herd immunity, an affordable, easy to store and versatile vaccine platform is thus desirable. We previously designed a non-infectious adenovirus-inspired nanoparticle (ADDomer), and in the present work, we efficiently decorated this original vaccine platform with glycosylated receptor binding domain (RBD) of SARS-CoV-2. Cryo-Electron Microscopy structure revealed that up to 60 copies of this antigenic domain were bound on a single ADDomer particle with the symmetrical arrangements of a dodecahedron. Mouse immunization with the RBD decorated particles showed as early as the first immunization a significant anti-coronavirus humoral response, which was boosted after a second immunization. Neutralization assays with spike pseudo-typed-virus demonstrated the elicitation of strong neutralization titers. Remarkably, the existence of pre-existing immunity against adenoviral-derived particles enhanced the humoral response against SARS-CoV-2. This plug and play vaccine platform revisits the way of using adenovirus to combat emergent pathogens while potentially taking advantage of the adenovirus pre-immunity.

National audience; In this paper, we present how education can be involved in innovative CAS-based solutions and precision agriculture. We focus on a student project dedicated to plant growth monitoring using the Internet of Things (IoT). The objective was to make the students aware of the need for electronic solutions to sustain food production. They were proposed to design and deploy a network of connected devices using LoRa technology to monitor plants in our engineering school, ENSEIRB-MATMECA. We choose a deployment in LoRa technology in order to master the deployment of the network from the beginning to the end. Their work was divided into 3 parts: sensors, radiofrequency circuitry and data management.

EnglishThrough this article, we present the first elements of knowledge resulting from an experiment on the school climate that takes place in 10 junior high schools of the Nantes agglomeration, from 2017 to 2021. We present the experimentation which is currently in course in Loire-Atlantique (1). We present our questions about the school climate under the particular prism of the living spaces in the schools and the well-being of the students (2). Then we present the findings that emerge in terms of the layout of reception and living spaces for schoolchildren, and the educational experience of adolescents in these spaces (3). An analysis addresses the various issues (educational, pedagogical and social) to which it refers, and describes some tools, approaches and inspiring practices in the direction of improving the quality of school climate and the well-being of junior high schools students (4). francaisA travers cet article, nous presentons les premiers elements de connaissance issus d’une experimentation sur le climat scolaire qui a lieu dans 10 colleges de l’agglomeration nantaise, de 2017 a 2021. Nous revenons en introduction sur l’experimentation qui est actuellement en cours en Loire-Atlantique (1). Nous presentons nos questionnements autour du climat scolaire sous le prisme particulier des espaces de vie dans les etablissements et du bien-etre des eleves (2). Sont ensuite presentes les constats qui ressortent en termes d’amenagement des espaces d’accueil et de vie des collegiens, et concernant le vecu scolaire des adolescents au sein de ces espaces (3). Une analyse sur l’appropriation des lieux d’accueil et de vie par les adolescents aborde les differents enjeux (educatifs, pedagogiques et sociaux) auquel elle fait reference, et decrit quelques outils, demarches et pratiques inspirantes dans le sens d’une amelioration du climat scolaire et du bien-etre des collegiens (4).

The present work deals with the study of the electrical behavior of cross-linked polyethylene (XLPE) used for HVDC cable insulation. The aim is to better understand the influences of electrical and thermal stresses on the insulating material in order to provide useful information for designing HVDC cables. This study was carried out on Rogowski samples made of XLPE insulation with semiconductive electrodes, aged for more than 3 years (1220 days) at three different temperatures (70, 80 and 90 °C) under two DC electric fields (30 and 60 kV/mm). Dielectric loss factor, volume resistivity and space charge accumulation were measured. Results are analyzed and cross-correlated, in order to propose possible ageing kinetics.

The absolute dating of paintings is crucial for tackling the problem of fake art. Investigations to authenticate paintings rely on an advanced knowledge of art history and a collection of scientific techniques. Radiocarbon dating is the only technique that gives access to an absolute time scale, but its application is limited to organic materials such as wood, canvas or natural binder. Extending absolute dating to inorganic pigments would make it possible to overcome the lack of available materials for dating easel and mural paintings. Here, we present a novel technique permitting paintings that contain inorganic pigment to be radiocarbon dated. We report results obtained on lead white that was the major white pigment used from Antiquity to the 20th century. We demonstrate that its manufacture is the key point for an absolute and reliable dating. We report an unprecedented use of 14C to date 14th to 16th century wall paintings. Since lead white was extensively used by the greatest artists, we anticipate that this study will open new avenues for detecting forgeries on the art market and for museums.

International audience; Intrinsically disordered proteins (IDPs) are flexible biomolecules whose essential functions are defined by their dynamic nature. Nuclear magnetic resonance (NMR) spectroscopy is ideally suited to the investigation of this behavior at atomic resolution. NMR relaxation is increasingly used to detect conformational dynamics in free and bound forms of IDPs under conditions approaching physiological, although a general framework providing a quantitative interpretation of these exquisitely sensitive probes as a function of experimental conditions is still lacking. Here, measuring an extensive set of relaxation rates sampling multiple-time-scale dynamics over a broad range of crowding conditions, we develop and test an integrated analytical description that accurately portrays the motion of IDPs as a function of the intrinsic properties of the crowded molecular environment. In particular we observe a strong dependence of both short-range and long-range motional time scales of the protein on the friction of the solvent. This tight coupling between the dynamic behavior of the IDP and its environment allows us to develop analytical expressions for protein motions and NMR relaxation properties that can be accurately applied over a vast range of experimental conditions. This unified dynamic description provides new insight into the physical behavior of IDPs, extending our ability to quantitatively investigate their conformational dynamics under complex environmental conditions, and accurately predicting relaxation rates reporting on motions on time scales up to tens of nanoseconds, both in vitro and in cellulo.

International audience

Herbovore livestock farming systems and climatic changes in france : conclusions from a series of studies from 2006 to 2009 (the acta/ mires project). Four partners are involved in the ACTA project called “Study of the sensitiveness to climatic changes of the large scale farms and herbivore livestock farming systems” ; these are Arvalis, Inra (national institute for agronomic research), the French Livestock Institute and Meteo France. As part of this programme, simulations based on the STICS farming model were achieved in order to estimate consequences of climatic changes on biomass production of lucerne, corn and grasses. Mid-term (2020-2049) and long-term (2070-2099) basic climatic data to implement such simulations were transmitted by Meteo France according to two socio-demographic scenari (A2 and B1) provided by the 4th report of the GIEC 4 group (government experts). Those simulations include the C0² enrichment effect of atmosphere on photosynthesis and transpiration. Moreover, through the calculation of numerous agro-climatic indicators, we looked into the availability and accessibility to those fodder resources and into the frequency of some climatic risks. Leaning on livestock systems having features of rather different areas regarding the extent and the impact of climatic change, we took into consideration direct consequences of climatic change on fodder farming achieved in those systems, in order to explore the evolution of those particular systems’ balances and to evaluate adapting ways enabling to come back to satisfying and secure balances. These works underline adaptability of those systems to have production levels steady in a near future but put forward less favorable breaking downs in a distant future., Dans le cadre du projet ACTA «Étude de la sensibilité des systèmes de grandes cultures et d’élevages herbivores aux changements climatiques», associant quatre partenaires : ARVALIS, l’INRA, l’Institut de l’Élevage et Météo France, et à partir des données climatiques simulées par Météo-France à moyen (2020-2049) et long terme (2070-2099) selon deux scénarios socio-démographiques (A2 et B1) issus du 4ème rapport du Groupe d’experts intergouvernemental (GIEC 4), des simulations utilisant le modèle de culture STICS (Simulateur multidisciplinaire pour les cultures standard) ont été réalisées pour évaluer les conséquences du changement climatique sur la production de biomasse de la luzerne, du maïs et de prairies à base de graminées. Ces simulations intègrent l’effet de l’enrichissement de l’atmosphère en CO2 sur la photosynthèse et la transpiration. Par ailleurs, au travers du calcul de nombreux indicateurs agro-climatiques, l’étude s’est penchée sur la disponibilité et l’accessibilité à ces ressources fourragères, et sur la fréquence de certains aléas climatiques. En prenant appui sur des systèmes d’élevage caractéristiques de zones bien différentes quant à l’ampleur et l’impact du changement climatique, les conséquences directes du changement climatique sur les cultures fourragères pratiquées dans ces systèmes ont été intégrées afin d’explorer l’évolution des équilibres propres à ces systèmes et évaluer les voies d’adaptation permettant de revenir à des équilibres satisfaisants et sécurisés. Ces travaux soulignent les capacités adaptatives de ces systèmes à maintenir leurs niveaux de production dans le futur proche mais ils mettent en avant des ruptures moins favorables à plus long terme., Moreau Jean-Christophe. Les systèmes d’élevage d’herbivores face au changement climatique en France : quelques conclusions d’une série d’études menées de 2006 à 2009 (projet Acta/Mires). In: Bulletin de l'Académie Vétérinaire de France tome 168 n°2, 2015. pp. 133-138.

Ion channel-coupled receptors (ICCRs) are original man-made ligand-gated ion channels created by fusion of G protein-coupled receptors (GPCRs) to the inward-rectifier potassium channel Kir6.2. GPCR conformational changes induced by ligand binding are transduced into electrical current by the ion channel. This functional coupling is closely related to the length of the linker region formed by the GPCR C-terminus (C-ter) and Kir6.2 N-terminus (N-ter). Manipulating the GPCR C-ter length allows to finely tune the channel regulation, both in amplitude and sign (opening or closing Kir6.2). In this work, we demonstrate that the primary sequence of the channel N-terminal domain is an additional parameter for the functional coupling with GPCRs. As for all Kir channels, a cluster of basic residues is present in the N-terminal domain of Kir6.2 and is composed of 5 arginines which are proximal to the GPCR C-ter in the fusion proteins. Using a functional mapping approach, we demonstrate the role of specific arginines (R27 and R32) for the function of ICCRs, indicating that the position and not the cluster of positively-charged arginines is critical for the channel regulation by the GPCR. Following observations provided by molecular dynamics simulation, we explore the hypothesis of interaction of these arginines with acidic residues, and using site-directed mutagenesis, we identified aspartate D307 and glutamate E308 residues as critical for the function of ICCRs. These results demonstrate the critical role of the N-terminal and C-terminal charged residues of Kir6.2 for its allosteric regulation by the fused GPCR.

International audience; Bacterial pathogens recruit circulating proteins to their own surfaces, coopting the host protein functions as a mechanism of virulence. Particular attention has focused on the binding of plasminogen (Plg) to bacterial surfaces, as it has been shown that this interaction contributes to bacterial adhesion to host cells, invasion of host tissues and evasion of the immune system. Several bacterial proteins are known to serve as receptors for Plg including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytoplasmic enzyme that appears on the cell surface in this moonlighting role. Although Plg typically binds to these receptors via several lysine-binding domains, the specific interactions that occur have not been documented in all cases. However, identification of the relevant residues could help define strategies for mitigating the virulence of important human pathogens, like Streptococcus pneumoniae (Sp). To shed light on this question, we have described a combination of peptide-spot array screening, competition and SPR assays, high-resolution crystallography and mutational analyses to characterize the interaction between SpGAPDH and Plg. We identified three SpGAPDH lysire residues that were instrumental in defining the kinetic and thermodynamic parameters of the interaction. Altogether, the integration of the data presented in this work allows us to propose a structural model for the molecular interaction of the SpGAPDH-Plg complex.

Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications.

International audience; Power transmission through power cables installed in ventilated tunnels is increasingly used worldwide and offers a complex thermal environment. Established correlations currently in use have been deduced from a nonfully developed turbulent flow and the effect of the proximity to a tunnel wall is not accounted for. This paper details the experimental and numerical investigations conducted on a mock-up of the heat transfer from a single cable in a fully developed turbulent airflow, with emphasis on the effect of the cable spacing from the tunnel wall. Nusselt numbers have been compared for different spacing and Reynolds numbers. The overall heat transfer is found to be meaningfully lower than in previous studies as only a third of the heat transfer is found, possible explanation of these differences are given in this paper. A threshold value is observed for the heat transfer while the cable rating depreciation is more gradual. The radiative heat exchange is found to be up to 30% of the total heat loss. An improved cooling law for the case of a single cable in tunnel taking into account the proximity of the wall has been developed from this study and confronted to the experimental and simulated data.

Absolute dating of mortars is crucial when trying to pin down construction phases of archaeological sites and historic stone buildings to a certain point in time or to confirm, but possibly also challenge, existing chronologies. To evaluate various sample preparation methods for radiocarbon (14C) dating of mortars as well as to compare different dating methods, i.e.14C and optically stimulated luminescence (OSL), a mortar dating intercomparison study (MODIS) was set up, exploring existing limits and needs for further research. Four mortar samples were selected and distributed among the participating laboratories: one of which was expected not to present any problem related to the sample preparation methodologies for anthropogenic lime extraction, whereas all others addressed specific known sample preparation issues. Data obtained from the various mortar dating approaches are evaluated relative to the historical framework of the mortar samples and any deviation observed is contextualized to the composition and specific mineralogy of the sampled material.

New insights into calreticulins are obtained by studying parasite species and combining a dissection strategy, X-ray crystallography and SAXS., Calreticulin (CRT) is a multifaceted protein, initially discovered as an endoplasmic reticulum (ER) chaperone protein, that is essential in calcium metabolism. Various implications in cancer, early development and immunology have been discovered more recently for CRT, as well as its role as a dominant ‘eat-me’ prophagocytic signal. Intriguingly, cell-surface exposure/secretion of CRT is among the infective strategies used by parasites such as Trypanosoma cruzi, Entamoeba histolytica, Taenia solium, Leishmania donovani and Schistosoma mansoni. Because of the inherent flexibility of CRTs, their analysis by X-ray crystallography requires the design of recombinant constructs suitable for crystallization, and thus only the structures of two very similar mammalian CRT lectin domains are known. With the X-ray structures of two distant parasite CRTs, insights into species structural determinants that might be harnessed to fight against the parasites without affecting the functions of the host CRT are now provided. Moreover, although the hypothesis that CRT can exhibit both open and closed conformations has been proposed in relation to its chaperone function, only the open conformation has so far been observed in crystal structures. The first evidence is now provided of a complex conformational transition with the junction reoriented towards P-domain closure. SAXS experiments also provided additional information about the flexibility of T. cruzi CRT in solution, thus complementing crystallographic data on the open conformation. Finally, regarding the conserved lectin-domain structure and chaperone function, evidence is provided of its dual carbohydrate/protein specificity and a new scheme is proposed to interpret such unusual substrate-binding properties. These fascinating features are fully consistent with previous experimental observations, as discussed considering the broad spectrum of CRT sequence conservations and differences.

International audience; Power transmission through power cables installed in ventilated tunnels has been increasingly used worldwide and offers a complex thermal environment. Established correlations currently in use have been deduced from a non-fully developed turbulent flow. This paper details the experimental investigation of the heat transfer from a single cable in a fully developed turbulent air flow, with emphasis on the effect of the cable spacing from the tunnel wall. Nusselt numbers have been compared for different spacing and velocities. The overall heat transfer is found to be meaningfully lower than in previous studies and no threshold spacing value for the average heat transfer to decrease is clearly found. Thermal radiation is found to greatly impact the cable cooling profile.

International audience; Complement C1q is a soluble pattern recognition molecule comprising six heterotrimeric subunits assembled from three polypeptide chains (A-C). Each heterotrimer forms a collagen-like stem prolonged by a globular recognition domain. These recognition domains sense a wide variety of ligands, including pathogens and altered-self components. Ligand recognition is either direct or mediated by immunoglobulins or pentraxins. Multivalent binding of C1q to its targets triggers immune effector mechanisms mediated via its collagen-like stems. The induced immune response includes activation of the classical complement pathway and enhancement of the phagocytosis of the recognized target. We report here, the first production of a single-chain recombinant form of human C1q globular region (C1q-scGR). The three monomers have been linked in tandem to generate a single continuous polypeptide, based on a strategy previously used for adiponectin, a protein structurally related to C1q. The resulting C1q-scGR protein was produced at high yield in stably transfected 293-F mammalian cells. Recombinant C1q-scGR was correctly folded, as demonstrated by its X-ray crystal structure solved at a resolution of 1.35 Å. Its interaction properties were assessed by surface plasmon resonance analysis using the following physiological C1q ligands: the receptor for C1q globular heads, the long pentraxin PTX3, calreticulin, and heparin. The 3D structure and the binding properties of C1q-scGR were similar to those of the three-chain fragment generated by collagenase digestion of serum-derived C1q. Comparison of the interaction properties of the fragments with those of native C1q provided insights into the avidity component associated with the hexameric assembly of C1q. The interest of this functional recombinant form of the recognition domains of C1q in basic research and its potential biomedical applications are discussed.

The Grotte Cosquer (southeastern France) is a Paleolithic painted cave only accessible by a deep-water dive. The cave has yielded numerous Paleolithic engravings and drawings, which were produced from wood charcoal. This article presents new radiocarbon dates obtained on samples collected in 2012 directly on 17 parietal representations and at the soil surface, and discusses the 14C results obtained since the discovery of the cave in 1992. A total of 41 samples were dated with ages ranging from 33,000 to 20,000 cal BP. They show that the cave was intermittently decorated over about 10,000 yr.

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

K(ATP) channels are metabolic sensors and targets of potassium channel openers (KCO; e.g., diazoxide and pinacidil). They comprise four sulfonylurea receptors (SUR) and four potassium channel subunits (Kir6) and are critical in regulating insulin secretion. Different SUR subtypes (SUR1, SUR2A, SUR2B) largely determine the metabolic sensitivities and the pharmacological profiles of K(ATP) channels. SUR1- but not SUR2-containing channels are highly sensitive to metabolic inhibition and diazoxide, whereas SUR2 channels are sensitive to pinacidil. It is generally believed that SUR1 and SUR2 are incompatible in channel coassembly. We used triple tandems, T1 and T2, each containing one SUR (SUR1 or SUR2A) and two Kir6.2Delta26 (last 26 residues are deleted) to examine the coassembly of different SUR. When T1 or T2 was expressed in Xenopus laevis oocytes, small whole-cell currents were activated by metabolic inhibition (induced by azide) plus a KCO (diazoxide for T1, pinacidil for T2). When coexpressed with any SUR subtype, the activated-currents were increased by 2- to 13-fold, indicating that different SUR can coassemble. Consistent with this, heteromeric SUR1+SUR2A channels were sensitive to azide, diazoxide, and pinacidil, and their single-channel burst duration was 2-fold longer than that of the T1 channels. Furthermore, SUR2A was coprecipitated with SUR1. Using whole-cell recording and immunostaining, heteromeric channels could also be detected when T1 and SUR2A were coexpressed in mammalian cells. Finally, the response of the SUR1+SUR2A channels to azide was found to be intermediate to those of the homomeric channels. Therefore, different SUR subtypes can coassemble into K(ATP) channels with distinct metabolic sensitivities and pharmacological profiles.

Cardiac ATP-sensitive potassium (KATP) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory – but not inhibitory – transduction processes. These results indicate that, within the KATP channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation.

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.

Artificial media are used in physiological studies of microalgae to maintain consistent conditions from one experiment to another and these media must be adapted to the needs of the organism studied. The artificial medium, in this case named diatom artificial medium (DAM), was designed to maintain long-term cultures of Haslea ostrearia and 19 other planktonic microalgae, and to allow physiological studies related to metal metabolism. The biomass and biochemical composition of H. ostrearia grown in the DAM and in a modified Provasoli medium were compared to assess the suitability of this new artificial medium for the culture of this diatom. The DAM provided sufficient nutrients to allow H. ostrearia to grow as efficiently as in the enriched seawater medium, without negative impact on metabolism. The DAM was tested with 19 other microalgae in order to widen its potential use, and 18 of the 19 showed a good adaptation to this medium. The chemical speciation of metals (Cd, Cu, Pb, Zn) was assessed using a speciation mathematical model. The presence of EDTA resulted in the total complexation of the trace metals implying that they were present in a sole chemical species in the DAM.

Background A 53-year-old female presented with a 10-year history of paroxysmal atrial fibrillation (AF), precipitated by activity and refractory to medical therapy. In the absence of traditional risk factors for disease, a genetic defect in electrical homeostasis underlying stress-induced AF was explored. Investigations Echocardiography, cardiac perfusion stress imaging, invasive electrophysiology with isoproterenol provocation, genomic DNA sequencing of KATP channel genes, exclusion of mutation in 2,000 individuals free of AF, reconstitution of channel defect with molecular phenotyping, and verification of pathogenic link in targeted knockout. Diagnosis KATP channelopathy caused by missense mutation (Thr1547Ile) of the ABCC9 gene conferring predisposition to adrenergic AF originating from the vein of Marshall. Management Disruption of arrhythmogenic gene–environment substrate at the vein of Marshall by radiofrequency ablation.

International audience; ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K(+) channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels.

The CERN n-TOF facility opens new opportunities due to an efficient neutron production for all energies between 1 eV and more than 200 MeV. The neutrons are produced by spallation of a lead target ...

International audience; Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain.

Reference EPFL-CONF-201047View record in Web of Science Record created on 2014-08-29, modified on 2016-08-09

K(ATP) channels incorporate a regulatory subunit of the ATP-binding cassette (ABC) transporter family, the sulfonylurea receptor (SUR), which defines their pharmacology. The therapeutically important K(+) channel openers (e.g. pinacidil, cromakalim, nicorandil) act specifically on the SUR2 muscle isoforms but, except for diazoxide, remain ineffective on the SUR1 neuronal/pancreatic isoform. This SUR1/2 dichotomy underpinned a chimeric strategy designed to identify the structural determinants of opener action, which led to a minimal set of two residues within the last transmembrane helix of SUR. Transfer of either residue from SUR2A to SUR1 conferred opener sensitivity to SUR1, while the reverse operation abolished SUR2A sensitivity. It is therefore likely that these residues form part of the site of interaction of openers with the channel. Thus, openers would target a region that, in other ABC transporters, is known to be tightly involved with the binding of substrates and other ligands. This first glimpse of the site of action of pharmacological openers should permit rapid progress towards understanding the structural determinants of their affinity and specificity.

The pharmacological phenotype of ATP-sensitive potassium (K ATP ) channels is defined by their tissue-specific regulatory subunit, the sulfonylurea receptor (SUR), which associates with the pore-forming channel core, Kir6.2. The potassium channel opener diazoxide has hyperglycemic and hypotensive properties that stem from its ability to open K ATP channels in pancreas and smooth muscle. Diazoxide is believed not to have any significant action on cardiac sarcolemmal K ATP channels. Yet, diazoxide can be cardioprotective in ischemia and has been found to bind to the presumed cardiac sarcolemmal K ATP channel-regulatory subunit, SUR2A. Here, in excised patches, diazoxide (300 μM) activated pancreatic SUR1/Kir6.2 currents and had little effect on native or recombinant cardiac SUR2A/Kir6.2 currents. However, in the presence of cytoplasmic ADP (100 μM), SUR2A/Kir6.2 channels became as sensitive to diazoxide as SUR1/Kir6.2 channels. This effect involved specific interactions between MgADP and SUR, as it required Mg 2+ , but not ATP, and was abolished by point mutations in the second nucleotide-binding domain of SUR, which impaired channel activation by MgADP. At the whole-cell level, in cardiomyocytes treated with oligomycin to block mitochondrial function, diazoxide could also activate K ATP currents only after cytosolic ADP had been raised by a creatine kinase inhibitor. Thus, ADP serves as a cofactor to define the responsiveness of cardiac K ATP channels toward diazoxide. The present demonstration of a pharmacological plasticity of K ATP channels identifies a mechanism for the control of channel activity in cardiac cells depending on the cellular ADP levels, which are elevated under ischemia.

Ion Channel-Coupled Receptors (ICCRs) are artificial ligand-gated ion channels created by genetic fusion of G protein-coupled receptors (GPCRs) to a K+ inward rectifier channel (Kir6.2) such that the channel is a direct reporter of the receptor conformational changes. This concept has been validated with 4 prototypical GPCRs: the M2 muscarinic, the D2L dopaminergic, the β2 adrenergic and the opsin receptors (Moreau et al., Nature Nanotech 3:620 2008, Caro et al. PLoS ONE 6:e18226 2011, Caro et al. PLoS ONE 7:e43766 2012). Voltage-clamp recordings showed that ICCRs detect the agonist- and antagonist-bound states of the receptor via direct physical coupling. This GPCR-channel communication proceeds without any involvement of G proteins and the electrical signal amplitude is correlated with the ligand concentration.The intrinsic instability of the GPCRs has proved a challenge to crystallographic studies. A successful approach, introduced in 2007 by Cherezov et al (Science. 318:1258) and subsequently applied to obtain 12 GPCR structures, consists in the insertion of the T4 phage lysozyme domain in the 3rd intracellular loop of the receptors. However, this modification abolishes G protein binding and prohibits related functional assays. Current characterization of crystallization-optimized GPCR(T4L) is performed by radiolabeled ligand assays or localized FRET techniques. Requiring no biochemical steps, ICCRs are an alternative tool to functionally characterize modified GPCRs that are unable to bind or activate G proteins and not amenable to most GPCR functional assays. We demonstrate here the validity of this tool with 3 different GPCRs (M2-muscarinic, β2-adrenergic and oxytocin receptors). The final application of this study would be the integration of this technology in the current crystallographic platforms dedicated to GPCR structure determination or for structure-function studies independent of G protein interaction.

Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.

Only a few astrophysical points and synchronisms listed in texts provide anchor points for the absolute chronology of Ancient Egypt. At first we will show how we can re-calculate some of these anchor points by using Sothic dating based on the arcus visionis method, and modelling lunar dates using a Bayesian approach. Then, we will discuss two radiocarbon studies carried out on short-lived Egyptian materials held at the Louvre Museum that could be attributed to particular reigns or other precise periods. Using a Bayesian approach, these dates were combined with the known order of succession and the lengths of reigns. Sothic and lunar dates were integrated as priors in the model. This approach has led to a new proposal for the absolute chronology of Egypt's 18th Dynasty.

International audience; Near Boulmane city, in the limestone formation of El Minchare, the Ifri Oussaïd cave open over the Middle Atlas plateau at 2 075 m high. From 2005 to 2007 excavations provided charcoal associated to bone remains mainly belonging to Ursus arctos, at last 156 identiied bones from ive bears. Paleontological studies describe bear of small size (Ouchaou, 2008). Datations of charcoal from stalagmitic loors above bear bone layer give ages between 5 220 ± 30 and 5 865 ± 30 yrs 14 C BP, while the collagen of a radius bone was dated at 7 300 ± 40 yrs 14 C BP. This date, taking in account the local climatic pattern, conirms the presence of Ursus arctos at the beginning of the Holocene period in North Africa but also point out to the importance of environmental changes in the Middle Atlas and their potential importance for the extinction of Ursus arctos.; Dans le Moyen Atlas, près de Boulmane les formations calcaires d'El Minchare abritent une grotte profonde (Ifri Oussaïd) s'ouvrant au-dessus du plateau à 2 075 m d'altitude. Les fouilles de ce site ont livré, de 2005 à 2007, des charbons associés à plusieurs ossements dont au moins 156 restes d'ours brun (Ursus arctos) appartenant à cinq individus. L'étude paléontologique atteste d'un ours de petite taille (Ouchaou, 2008). Les niveaux à charbons de bois scellés dans de petits planchers stalagmitiques situés au-dessus des restes ont donné des dates allant de 5 220 ± 30 à 5 865 ± 30 ans 14 C BP. La datation obtenue directement sur un radius d'ours est de 7 300 ± 40 ans 14 C BP. Cette date, replacée dans le contexte climatique local, conirme la présence d'Ursus arctos au début de l'Holocène moyen en Afrique du Nord, mais souligne aussi l'ampleur des changements environnementaux dans le Moyen Atlas depuis cette époque et leur inluence potentielle sur la disparition d'Ursus arctos de cette région.

International audience; Early Neolithic sedentary villagers started cultivating wild cereals in the Near East 11,500 y ago [Pre-Pottery Neolithic A (PPNA)]. Recent discoveries indicated that Cyprus was frequented by Late PPNA people, but the earliest evidence until now for both the use of cereals and Neolithic villages on the island dates to 10,400 y ago. Here we present the recent archaeological excavation at Klimonas, which demonstrates that established villagers were living on Cyprus between 11,100 and 10,600 y ago. Villagers had stone artifacts and buildings (including a remarkable 10-m diameter communal building) that were similar to those found on Late PPNA sites on the mainland. Cereals were introduced from the Levant, and meat was obtained by hunting the only ungulate living on the island, a small indigenous Cypriot wild boar. Cats and small domestic dogs were brought from the mainland. This colonization suggests well-developed maritime capabilities by the PPNA period, but also that migration from the mainland may have occurred shortly after thebeginning of agriculture.

Summary Birch (Betula pendula Roth) micropropagated plantlets were inoculated with 7 different isolates of the mycorrhizal fungus Paxillus involutus Batsch. Based on the level of fungal ergosterol measured in roots at the end of the “mycorrhiza formation stage”, strain P0 was chosen as the reference strain. Electrophoretic analysis of in vivo labeled proteins extracted from mycorrhizal roots 96 h post-inoculation with this strain, noninoculated roots, and free-living mycelium, revealed that specific polypeptides were synthesized during ectomycorrhiza formation. To examine hypothetical similarity between some of these polypeptides and defense proteins, parts of corresponding putative genes of birch were isolated. Partial sequencing of one clone have shown that it contained a portion of the gene for phenylalanine ammonialyase.

Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K(+)-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K(+) channel Kir6.2 and muscarinic M(2) or dopaminergic D(2) receptors. Here, we extend the concept to the distinct, longer β(2)-adrenergic receptor which, unlike M(2) and D(2) receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β(2) constructs. We then demonstrate how engineering of both receptor and channel can produce β(2)-Kir6.2 ICCRs. Specifically, removal of 62-72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β(2) ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β(2) receptor.

The Bay of Bourgneuf, France, is a main site of shellfish production. In the marshes along the bay, oyster intensive rearing and fattening need the mass production of microalgae. Salt groundwaters, available in this region, support a large part of this production for aquaculture. Studies carried out by local authorities have brought to the fore the accumulation of lead (Pb) in several samples of algal pastes derived from cultures using salt groundwater. The aim of this study was to compare growth, nutritional value and metal (Cd, Cu, Pb and Zn) uptake of four microalgae grown in two salt groundwaters or in enriched coastal seawater. Cultures of microalgae used in aquaculture (Haslea ostrearia, Phaeodactylum tricornutum, Skeletonema costatum and Tetraselmis suecica) were realised at the laboratory under controlled experimental conditions. Results indicated that salt groundwaters provided cultures with, at least, an equal biomass and a nutritional value similar to cultures grown in enriched seawater. There was no difference regarding metal accumulation whatever the culture medium, except when S. costatum was grown in one of the salt groundwater in which case its cadmium levels were higher and could be above the French guideline level. These observations questioned on the bioavailability of metals in salt groundwaters. It also underlines the specificity of metal uptake and accumulation by microalgae., La baie de Bourgneuf (France) est un important site de production conchylicole. Dans les marais littoraux, la culture intensive et l'affinage d'huîtres nécessitent la production en masse de microalgues. Les eaux souterraines salées, facilement exploitables dans cette région, sont majoritairement utilisées dans la production des microalgues pour l'aquaculture en baie de Bourgneuf. Des études ont été menées sur les cultures de microalgues sur ces eaux souterraines salées et ont mis en évidence une accumulation de plomb (Pb) supérieure à la norme alimentaire en vigueur dans différents échantillons de pâte d'algue d'Haslea ostrearia issus de ces cultures. L'objectif de cette étude est de comparer la croissance, la composition biochimique globale et l'accumulation des métaux (Cd, Cu, Pb, Zn) pour quatre microalgues, utilisées en aquaculture, cultivées sur des eaux souterraines salées provenant de deux forages différents et sur un milieu enrichi à base d'eau de mer. Les cultures sont réalisées au laboratoire dans des conditions expérimentales contrôlées. Les eaux souterraines salées permettent la production de cultures d'une biomasse et d'une qualité biochimique au moins équivalentes à celles réalisées sur le milieu enrichi à base d'eau de mer. Les teneurs en métaux dans les microalgues ne sont pas différentes pour les algues cultivées sur les différents milieux, à l'exception de Skeletonema costatum qui, cultivée sur une des eaux souterraines, peut présenter des teneurs supérieures à la norme alimentaire en vigueur. Ces observations soulèvent des questions sur la biodisponibilité des métaux dans les eaux souterraines salées et soulignent la spécificité de l'accumulation des métaux dans les microalgues.

The human ATP-binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), confers resistance to a broad range of anti-cancer agents and transports a variety of organic anions. At present, essentially no structural data exists for MRP1 that might be used to elucidate its mechanism of transport. Consequently, we have applied a modeling strategy incorporating crystal and indirect structural data from other ABC transporters to construct a model of the transmembrane domains of the core region of MRP1 that includes the amino acid side chains. Three conserved Trp residues and one non-conserved Tyr residue, shown previously to be of functional importance (Koike, K., Oleschuk, C. J., Haimeur, A., Olsen, S. L., Deeley, R. G., and Cole, S. P. C. (2002) J. Biol. Chem. 277, 49495-49503), were found to line the "pore" in our model proximal to the membrane cytosol interface. A fifth aromatic residue (Phe594) was identified that, with the Trp and Tyr residues, completed a ring or "basket" of aromatic amino acids and, accordingly, we postulated that it would also be of functional importance. To test this idea, MRP1-Phe594 mutants were expressed in human embryonic kidney cells, and their properties were examined using membrane vesicles. Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4. On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate- and substitution-specific changes were observed. These studies provide some structural insight into a possible substrate binding/transport site of MRP1 at the beginning of a putative substrate translocation pathway and demonstrate the usefulness of modeling for directing structure-function analyses of this transporter.

Inspired by the natural example of the K-ATP channel in which an ion channel (Kir6.2) is regulated by an associated, unrelated membrane protein, the ABC protein SUR, we have engineered Ion-Channel Coupled Receptors (ICCRs) by physical coupling of G-protein coupled receptors (GPCRs) with Kir6.2.A first ICCR was constructed using the muscarinic M2 receptor and Kir6.2. Our strategy consisted of creating various fusion proteins by linking the receptor C-terminus to the channel N-terminus and progressively removing residues from either termini until functional coupling was achieved, i.e., agonist binding to the receptor modified channel activity. The fusion proteins were heterologously expressed in Xenopus oocytes and characterized with the two-electrode voltage-clamp and patch-clamp techniques.Successful coupling was achieved with limited deletions of the channel N-terminal with an optimum of 25 residues. The optimal construct was reversibly upregulated by the M2 agonist, acetylcholine. To further establish proof-of-concept, a second ICCR was obtained by using the dopaminergic D2 receptor. This ICCR was also regulated by D2 agonists and antagonists although, unexpectedly, the D2 ICCR responses were the opposite of those of the M2 ICCR, i.e., agonists caused channel downregulation.We observed that, i) agonist modulation of Kir6.2 was concentration-dependent and saturatable, ii) agonist effects were abolished by receptor antagonists, iii) the GPCRs within the fusion remained functional as verified by their capacity to activate coexpressed G-protein-activated Kir3 channels. iv) receptor-mediated responses were independent of G-protein activation because they persisted in the presence of pertussis toxin, and v) ICCRs remained functional in cell-free, outside-out patch conditions.ICCRs could be useful tools for the study of GPCR activation and K+ channel gating and could also serve as biosensors for drug screening and diagnostics.Ref :Moreau et al, Nature Nanotechnology. 2008, in press.

It is possible to achieve functional coupling between a receptor and an ion channel by covalent linkage so that ligand binding to the receptor modifies channel gating. This was demonstrated with the inward rectifier potassium channel Kir6.2 (the pore subunit of the KATP channel) and the muscarinic M2 and dopaminergic D2 G-protein coupled receptors (GPCRs) [Moreau et al., 2008, Nature Nanotech].To extend this concept of Ion-Channel Coupled Receptor (ICCR), we designed new contructs by engineering fusion between Kir6.2 and 3 GPCRs: the s2 adrenergic, cannabinoid 1 (CB1) and dopaminergic D3 receptors. The receptor C-ter and channel N-ter extremities were pared to promote efficient coupling as in M2 and D2 ICCRs and joined covalently. The fusions were heterologously expressed in Xenopus oocytes and characterized by the two-electrode voltage clamp technique. Construct names ‘G-Kxx-yy’ indicate the GPCR name (G), the residues clipped off from the GPCR C-ter (xx) and from the Kir6.2 N-ter (yy).A D3-based ICCR, D3-K0-25, behaved like the D2-based ICCR, showing channel inhibition upon dopamine application. Two s2-based ICCRs were successfully constructed, s2-K62-25 and s2-K73-25. Only when co-expressed with TMD0 (the Kir6.2-anchoring domain of SUR, the regulatory subunit of the KATP channel) to augment surface expression, these two ICCRs were reversibly activated by the agonist isoproterenol and inhibited by the antagonist alprenolol. Similarly, a CB1-based ICCR, CB1-K0-25, was activated by the agonist W102, an effect that was enhanced by the presence of TMD0.Thus, the ICCR concept is readily applicable to class A GPCRs. Besides their obvious interest in drug screening, the new ICCRs should be valuable tools to investigate the intermolecular events involved in the modulation of Kir6.2 gating and the nature of the GPCR conformational changes evoked by their ligands.

[Departement_IRSTEA]Territoires [TR1_IRSTEA]DTAM; Rangeland-based livestock systems have to deal with the significant instability and uncertainty of the agricultural context (policy changes, volatility of input prices, etc.), and especially of the climatic context. Thus, they are particularly concerned by adaptive management strategies. To support the development of such strategies, we developed a board game including a computer model called "Rangeland Rummy". It is to be used by groups of farmers and agricultural consultants in the context of short workshops (about 3 h). Rangeland Rummy builds upon five types of material object: (i) a game board; (ii) a calendar stick indicating the starting date of the game board; (iii) sticks marked with the feed resources available for combinations of vegetation types and their management practices; (iv) cards to define animal groups and their feeding requirements throughout the year; (v) cards related to types of feed that can be attributed to animal groups throughout the year. Using these material objects, farmers collectively design a rangeland-based livestock system. This system is immediately evaluated using a computer model, i.e. a spreadsheet providing graphs and indicators providing information on, among other things, the extent to which quantitative and qualitative animal feeding requirements are covered across the year. Playing the game thus consists in collectively and iteratively designing and evaluating rangeland-based livestock systems, while confronting the players with new contextual challenges (e.g. interannual variability of weather, volatility of input prices) or new farmers' objectives (e.g. being self-sufficient for animal feeding). An example of application of Rangeland Rummy with 3 farmers in southern France is reported. Applications show that it tends to develop farmers' adaptive capacity by stimulating their discussions and the exchange of locally-relevant knowledge on management strategies and practices in rangeland-based livestock systems.

International audience; Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.