Purification of Synaptosome Populations Using Fluorescence-Activated Synaptosome Sorting

International audienceFor several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and some functional features of the cells in the central nervous system. Indeed, brain tissue contains a complex intermingled network of neuronal, glial, and vascular cells. To reduce this complexity biochemists have optimized fractionation protocols that enrich in specific compartments such as synapses (called “synaptosomes”) and synaptic vesicles, for example. However, recently, these approaches suffered from a lack of specificity and purity. In a recent effort, we extended the conventional synaptosome preparation to purify fluorescent synaptosomes on a cell sorter. We could prove that our method allows for the steep enrichment in fluorescent excitatory VGLUT1venus synaptosomes containing the presynaptic element and the tip of the post-synaptic element and a strong depletion in neuronal and glial contaminants. Here, we propose a detailed procedure for the implementation of Fluorescence Activated Synaptosome Sorting

Methylation Tolerance-Based Functional Assay to Assess Variants of Unknown Significance in the MLH1 and MSH2 Genes and Identify Patients With Lynch Syndrome

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Developmental Genes Have Pleiotropic Effects on Plant Morphology and Source Capacity, Eventually Impacting on Seed Protein Content and Productivity in Pea1[W][OA]

Increasing pea (Pisum sativum) seed nutritional value and particularly seed protein content, while maintaining yield, is an important challenge for further development of this crop. Seed protein content and yield are complex and unstable traits, integrating all the processes occurring during the plant life cycle. During filling, seeds are the main sink to which assimilates are preferentially allocated at the expense of vegetative organs. Nitrogen seed demand is satisfied partly by nitrogen acquired by the roots, but also by nitrogen remobilized from vegetative organs. In this study, we evaluated the respective roles of nitrogen source capacity and sink strength in the genetic variability of seed protein content and yield. We showed in eight genotypes of diverse origins that both the maximal rate of nitrogen accumulation in the seeds and nitrogen source capacity varied among genotypes. Then, to identify the genetic factors responsible for seed protein content and yield variation, we searched for quantitative trait loci (QTL) for seed traits and for indicators of sink strength and source nitrogen capacity. We detected 261 QTL across five environments for all traits measured. Most QTL for seed and plant traits mapped in clusters, raising the possibility of common underlying processes and candidate genes. In most environments, the genes Le and Afila, which control internode length and the switch between leaflets and tendrils, respectively, determined plant nitrogen status. Depending on the environment, these genes were linked to QTL of seed protein content and yield, suggesting that source-sink adjustments depend on growing conditions

Transinteractome analysis reveals distinct niche requirements for isotype‐based plasma cell subsets in the bone marrow

International audienceBone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR +) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype

P-Body Purification Reveals the Condensation of Repressed mRNA Regulons

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Comment favoriser les services écosystémiques assurés par la biodiversité des paysages agricoles ? Intérêts d’un réseau de sites d’observation à long terme

Atteindre l’objectif affiché de réduction du niveau d’usage de produits phytosanitaires tout en maintenant les niveaux de production des cultures implique la mise en place de stratégies de protection des cultures alternatives à la lutte chimique. L’adoption de pratiques plus favorables à la biodiversité dans les agroécosystèmes pourrait contribuer à augmenter les services de régulation biologique des ravageurs de culture. Il est établi que la biodiversité des milieux cultivés est modulée par des facteurs naturels et anthropiques agissant à différentes échelles spatiales, allant de la parcelle agricole au paysage, mais la compréhension des mécanismes écologiques qui produisent ces effets reste limitée. Pour produire des connaissances sur les déterminants des niveaux de régulations biologiques, il est nécessaire d’avoir recours à une approche à long terme qui mobilise à la fois une description des systèmes de cultures et une caractérisation écologique du contexte paysager. Pour répondre à cet enjeu a été constitué SEBIOPAG (http://sebiopag.inra.fr), un réseau pour l’étude des Services Ecosystémiques assurés par la BIOdiversité dans les Paysages Agricoles qui appartient au réseau national des observatoires de recherche sur la biodiversité (ECOSCOPE). Les objectifs sont de suivre sur le long terme, et dans des zones paysagères contrastées (i) les services écosystémiques rendus par la biodiversité, (ii) les facteurs qui les influencent (composition et structure du paysages, pratiques agricoles) et (iii) leur évolution dans le contexte des changements globaux (utilisation des terres, climatiques,…).Le réseau SEBIOPAG est constitué de cinq sites : Avignon, Dijon, Chizé, Rennes et Toulouse. En 2013, danschaque site, le paysage a été décrit et cartographié à l’aide de trois descripteurs : la proportion de cultures,l’hétérogénéité et la longueur des interfaces entre cultures et éléments semi-naturels. Puis, dans chaque site, 20 parcelles d’exploitants ont été choisies le long des trois gradients paysagers et d’un gradient d’intensité de pratiques. Les premières mesures (exposition de cartes de prédation) ont été effectuées en 2014 et portent sur une évaluation du potentiel de régulation biologique de trois espèces cibles : Acyrthosiphon pisum (pucerons), Ephestia kuehniella (lépidoptère) et Viola arvensis (adventice)

The Nucleoside Diphosphate Kinase D (NM23-H4) Binds the Inner Mitochondrial Membrane with High Affinity to Cardiolipin and Couples Nucleotide Transfer with Respiration*

Nucleoside diphosphate kinase (NDPK/Nm23), responsible for intracellular di- and triphosphonucleoside homeostasis, plays multiple roles in cellular energetics, signaling, proliferation, differentiation and tumor invasion. The only human NDPK with a mitochondrial targeting sequence is NDPK-D, the NME4 gene product, which is a peripheral protein of mitochondrial membranes. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. Surface plasmon resonance analysis of the interaction using recombinant NDPK-D and model liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin. Mutation of the central arginine (Arg-90) in a surface-exposed basic RRK motif unique to NDPK-D strongly reduced interaction with anionic phospholipids. Due to its symmetrical hexameric structure, NDPK-D was able to cross-link anionic phospholipid-containing liposomes, suggesting that NDPK-D could promote intermembrane contacts. Latency assays with isolated mitochondria and antibody binding to mitoplasts indicated a dual orientation for NDPK-D. In HeLa cells, stable expression of wild type but not of the R90D mutant led to membrane-bound enzyme in vivo. Respiration was significantly stimulated by the NDPK substrate TDP in mitochondria containing wild-type NDPK-D, but not in those expressing the R90D mutant, which is catalytically equally active. This indicates local ADP regeneration in the mitochondrial intermembrane space and a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on its membrane-bound state

A highly virulent variant of HIV-1 circulating in the Netherlands

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence