Signaling by EphrinB1 and Eph Kinases in Platelets Promotes Rap1 Activation, Platelet Adhesion, and Aggregation via Effector Pathways that Do Not Require Phosphorylation of EphrinB1

We have previously shown that platelets express 2 receptor tyrosine kinases, EphA4 and EphB1, and the Eph kinase ligand, ephrinB1m and proposed that transcellular Eph/ephrin interactions made possible by the onset of platelet aggregation promote the further growth and stability of the hemostatic plug. The present study examines how this might occur. The results show that clustering of either ephrinB1 or EphA4 causes platelets to adhere to immobilized firinogen via αIIbβ3. Adhesion occurs more slowly than with adenosine diphosphate (ADP) abd requires phosphatidylinositol 3 (PI3)—kinase and protein kinase C activity but not ephrinB1 phosphorylation. By itself, Eph and ephrin signaling is insufficient to cause aggregation or the binding of soluble fibrinogen, but it can potentiate aggregation initiated by a Ca++ ionophore or by agonists for thrombin and thromboxane receptors. It also enhances Rap1 activation without requiring ADP secretion, ephrinB1 phosphorylation, or the activation of PI3-kinase and Src. From this we conclude that (1) Eph/ephrin signaling enhances the ability of platelet agonists to cause aggregation provided that those agonists can increase cytosolic Ca++; (2) this is accomplished in part by activating Rap1; and (3) these effects require not phosphotyrosine-based interactions with the ephrinB1 cytoplasmic domain

Faire gras à Molène: dairy products and ruminant fats detected by lipid and isotopic analysis of pottery dating to the Final Neolithic-Early Bronze Age from the island site of Beg ar Loued (Molène, western Brittany, France): Faire gras à Molène : produits laitiers et graisses de ruminants détectés par l’analyse lipidique et isotopique des céramiques du Néolithique final et de l’âge du Bronze ancien du site insulaire de Beg ar Loued (Molène, Bretagne occidentale, France)

The subsistence strategies of early farming communities have been highlighted since the beginning of the Neolithic, thanks to numerous studies on lipid residues from ceramic vessels conducted in various parts of continental Europe. However, after the Early Neolithic, evidence of subsistence strategies along the northern Atlantic coast are still lacking, especially for island contexts. This paper presents the results of lipid residue analysis of 129 potsherds from Beg ar Loued (Molène, France), an island site dating primarily to the Early Bronze Age (c. 2700-2600 to 1800 BCE). Aiming to understand the use of vessels, vessel treatment and culinary practices on the settlement, analyses of visible charred residues, sherds and ceramic surfaces/coating layers were carried out using chromatographic (n = 174) and isotopic techniques (n = 24) after lipid extraction by solvent (n = 174) or acid methanolysis (n = 31). The results demonstrate the extensive use of terrestrial products (ruminant carcass and dairy) in pottery, including occasional plant products (with possible mixtures of different waxes), while the detection of aquatic products is limited. Thus, combined with evidence from faunal remains at the site, the results indicate that terrestrial resources like ruminant meat and dairy products were preferentially processed in vessels, and aquatic products mostly without the use of ceramics. These findings demonstrate the significance of lipid residue analysis for studying the role of pottery in food production and consumption at sites along the Atlantic coast. Les stratégies de subsistance des premières communautés agricoles ont été mises en évidence depuis le début du Néolithique grâce à de nombreuses études sur les résidus lipidiques des récipients en céramique menées dans diverses parties de l’Europe continentale. En revanche, très peu de données sont disponibles pour la fin du Néolithique et le début de l’âge du Bronze sur la côte atlantique, en particulier en contexte insulaire. Cet article présente les résultats de l’analyse de résidus lipidiques provenant de 129 fragments de poteries de Beg ar Loued (Molène, France), un site insulaire dont les principaux vestiges datent de l’âge du Bronze ancien (c. 2700-2600 à 1800 BCE). Dans le but de comprendre l’utilisation des récipients, les pratiques culinaires sur ce site et d’appréhender les techniques de finition des céramiques, des analyses de résidus visibles carbonisés, de tessons, et de surfaces/couches d’engobe ont été effectuées via des analyses chromatographiques (n = 174) et isotopiques (n = 24), après extraction des lipides par solvant (n = 174) ou méthanolyse acide (n = 31). Les résultats démontrent l’utilisation extensive de produits terrestres (carcasses de ruminants et produits laitiers), comprenant occasionnellement des produits végétaux (avec un mélange probable de différentes cires), alors que la détection des produits aquatiques est faible. Comparés aux données fauniques, ces résultats indiquent donc que les produits terrestres, tels que la viande de ruminant et les produits laitiers, sont transformés en utilisant des récipients en céramiques, tandis que les produits aquatiques semblent de préférence exploités sans avoir recours à une poterie. Ces résultats démontrent l’importance de l’analyse des résidus lipidiques pour connaître le rôle des récipients en céramique dans la production et la consommation d’aliments sur les sites de la côte atlantique

Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils

Predictors of mortality and short-term physical and cognitive dependence in critically ill persons 75 years and older: a prospective cohort study

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to identify predictors of 3-month mortality in critically ill older persons under medical care and to assess the clinical impact of an ICU stay on physical and cognitive dependence and subjective health status in survivors.</p> <p>Methods</p> <p>We conducted a prospective observational cohort study including all older persons 75 years and older consecutively admitted into ICU during a one-year period, except those admitted after cardiac arrest, All patients were followed for 3 months or until death. Comorbidities were assessed using the Charlson index and physical dependence was evaluated using the Katz index of Activity of Daily Living (ADL). Cognitive dependence was determined by a score based on the individual components of the Lawton index of Daily Living and subjective health status was evaluated using the Nottingham Health Profile (NHP) score.</p> <p>Results</p> <p>One hundred patients were included in the analysis. The mean age was 79.3 ± 3.4 years. The median Charlson index was 6 [IQR, 4 to 7] and the mean ADL and cognitive scores were 5.4 ± 1.1 and 1.2 ± 1.4, respectively, corresponding to a population with a high level of comorbidities but low physical and cognitive dependence. Mortality was 61/100 (61%) at 3 months. In multivariate analysis only comorbidities assessed by the Charlson index [Adjusted Odds Ratio, 1.6; 95% CI, 1.2-2.2; <it>p </it>< 0.003] and the number of organ failures assessed by the SOFA score [Adjusted Odds Ratio, 2.5; 95% CI, 1.1-5.2; <it>p </it>< 0.02] were independently associated with 3-month mortality. All 22 patients needing renal support after Day 3 died. Compared with pre-admission, physical (<it>p </it>= 0.04), and cognitive (<it>p </it>= 0.62) dependence in survivors had changed very little at 3 months. In addition, the mean NHP score was 213.1 <b>± </b>132.8 at 3 months, suggesting an acceptable perception of their quality of life.</p> <p>Conclusions</p> <p>In a selected population of non surgical patients 75 years and older, admission into the ICU is associated with a 3-month survival rate of 38% with little impact on physical and cognitive dependence and subjective health status. Nevertheless, a high comorbidity level (ie, Charlson index), multi-organ failure, and the need for extra-renal support at the early phase of intensive care could be considered as predictors of death.</p

Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments

Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson–Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange

The neuropeptide 26RFa in the human gut and pancreas: potential involvement in glucose homeostasis

International audienc

Mesoscopic models for DNA stretching under force: new results and comparison to experiments

Single molecule experiments on B-DNA stretching have revealed one or two structural transitions, when increasing the external force. They are characterized by a sudden increase of DNA contour length and a decrease of the bending rigidity. It has been proposed that the first transition, at forces of 60--80 pN, is a transition from B to S-DNA, viewed as a stretched duplex DNA, while the second one, at stronger forces, is a strand peeling resulting in single stranded DNAs (ssDNA), similar to thermal denaturation. But due to experimental conditions these two transitions can overlap, for instance for poly(dA-dT). We derive analytical formula using a coupled discrete worm like chain-Ising model. Our model takes into account bending rigidity, discreteness of the chain, linear and non-linear (for ssDNA) bond stretching. In the limit of zero force, this model simplifies into a coupled model already developed by us for studying thermal DNA melting, establishing a connexion with previous fitting parameter values for denaturation profiles. We find that: (i) ssDNA is fitted, using an analytical formula, over a nanoNewton range with only three free parameters, the contour length, the bending modulus and the monomer size; (ii) a surprisingly good fit on this force range is possible only by choosing a monomer size of 0.2 nm, almost 4 times smaller than the ssDNA nucleobase length; (iii) mesoscopic models are not able to fit B to ssDNA (or S to ss) transitions; (iv) an analytical formula for fitting B to S transitions is derived in the strong force approximation and for long DNAs, which is in excellent agreement with exact transfer matrix calculations; (v) this formula fits perfectly well poly(dG-dC) and $\lambda$-DNA force-extension curves with consistent parameter values; (vi) a coherent picture, where S to ssDNA transitions are much more sensitive to base-pair sequence than the B to S one, emerges.Comment: 14 pages, 9 figure

Early T Cell Signalling Is Reversibly Altered in PD-1+ T Lymphocytes Infiltrating Human Tumors

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL