Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli

NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM: KCl to 3 M: KCl), (2) anion over cation selectivity (V (reversal) = +16 mV in 250 mM: KCl || 1 M: KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 ?M: to 100 mM: in 250 mM: KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM: to 5 mM: in 250 mM: KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar-KCl bridges. A suitable baseline solution for NanC is 250 mM: KCl adjusted to pH 7.0 without buffer

Tyrosine Kinase Syk Non-Enzymatic Inhibitors and Potential Anti-Allergic Drug-Like Compounds Discovered by Virtual and In Vitro Screening

In the past decade, the spleen tyrosine kinase (Syk) has shown a high potential for the discovery of new treatments for inflammatory and autoimmune disorders. Pharmacological inhibitors of Syk catalytic site bearing therapeutic potential have been developed, with however limited specificity towards Syk. To address this topic, we opted for the design of drug-like compounds that could impede the interaction of Syk with its cellular partners while maintaining an active kinase protein. To achieve this challenging task, we used the powerful potential of intracellular antibodies for the modulation of cellular functions in vivo, combined to structure-based in silico screening. In our previous studies, we reported the anti-allergic properties of the intracellular antibody G4G11. With the aim of finding functional mimics of G4G11, we developed an Antibody Displacement Assay and we isolated the drug-like compound C-13, with promising in vivo anti-allergic activity. The likely binding cavity of this compound is located at the close vicinity of G4G11 epitope, far away from the catalytic site of Syk. Here we report the virtual screen of a collection of 500,000 molecules against this new cavity, which led to the isolation of 1000 compounds subsequently evaluated for their in vitro inhibitory effects using the Antibody Displacement Assay. Eighty five compounds were selected and evaluated for their ability to inhibit the liberation of allergic mediators from mast cells. Among them, 10 compounds inhibited degranulation with IC50 values ≤10 µM. The most bioactive compounds combine biological activity, significant inhibition of antibody binding and strong affinity for Syk. Moreover, these molecules show a good potential for oral bioavailability and are not kinase catalytic site inhibitors. These bioactive compounds could be used as starting points for the development of new classes of non-enzymatic inhibitors of Syk and for drug discovery endeavour in the field of inflammation related disorders

Be careful with triage in emergency departments: interobserver agreement on 1,578 patients in France

<p>Abstract</p> <p>Background</p> <p>For several decades, emergency departments (EDs) utilization has increased, inducing ED overcrowding in many countries. This phenomenon is related partly to an excessive number of nonurgent patients. To resolve ED overcrowding and to decrease nonurgent visits, the most common solution has been to triage the ED patients to identify potentially nonurgent patients, i.e. which could have been dealt with by general practitioner. The objective of this study was to measure agreement among ED health professionals on the urgency of an ED visit, and to determine if the level of agreement is consistent among different sub-groups based on following explicit criteria: age, medical status, type of referral to the ED, investigations performed in the ED, and the discharge from the ED.</p> <p>Methods</p> <p>We conducted a multicentric cross-sectional study to compare agreement between nurses and physicians on categorization of ED visits into urgent or nonurgent. Subgroups stratified by criteria characterizing the ED visit were analyzed in relation to the outcome of the visit.</p> <p>Results</p> <p>Of 1,928 ED patients, 350 were excluded because data were lacking. The overall nurse-physician agreement on categorization was moderate (kappa = 0.43). The levels of agreement within all subgroups were variable and low. The highest agreement concerned three subgroups of complaints: cranial injury (kappa = 0.61), gynaecological (kappa = 0.66) and toxicology complaints (kappa = 1.00). The lowest agreement concerned two subgroups: urinary-nephrology (kappa = 0.09) and hospitalization (kappa = 0.20). When categorization of ED visits into urgent or nonurgent cases was compared to hospitalization, ED physicians had higher sensitivity and specificity than nurses (respectively 94.9% versus 89.5%, and 43.1% versus 30.9%).</p> <p>Conclusions</p> <p>The lack of physician-nurse agreement and the inability to predict hospitalization have important implications for patient safety. When urgency screening is used to determine treatment priority, disagreement might not matter because all patients in the ED are seen and treated. But using assessments as the basis for refusal of care to potential nonurgent patients raises legal, ethical, and safety issues. Managed care organizations should be cautious when applying such criteria to restrict access to EDs.</p

Plant-Type Trehalose Synthetic Pathway in Cryptosporidium and Some Other Apicomplexans

The trehalose synthetic pathway is present in bacteria, fungi, plants and invertebrate animals, but is absent in vertebrates. This disaccharide mainly functions as a stress protectant against desiccation, heat, cold and oxidation. Genes involved in trehalose synthesis have been observed in apicomplexan parasites, but little was known about these enzymes. Study on trehalose synthesis in apicomplexans would not only shed new light into the evolution of this pathway, but also provide data for exploring this pathway as novel drug target.We have observed the presence of the trehalose synthetic pathway in Cryptosporidium and other apicomplexans and alveolates. Two key enzymes (trehalose 6-phosphate synthase [T6PS; EC 2.4.1.15] and trehalose phosphatase [TPase; EC 3.1.3.12] are present as Class II bifunctional proteins (T6PS-TPase) in the majority of apicomplexans with the exception of Plasmodium species. The enzyme for synthesizing the precursor (UDP-glucose) is homologous to dual-substrate UDP-galactose/glucose pyrophosphorylases (UGGPases), rather than the "classic" UDP-glucose pyrophosphorylase (UGPase). Phylogenetic recontructions indicate that both T6PS-TPases and UGGPases in apicomplexans and other alveolates are evolutionarily affiliated with stramenopiles and plants. The expression level of T6PS-TPase in C. parvum is highly elevated in the late intracellular developmental stage prior to or during the production of oocysts, implying that trehalose may be important in oocysts as a protectant against environmental stresses. Finally, trehalose has been detected in C. parvum oocysts, thus confirming the trehalose synthetic activity in this parasite.A trehalose synthetic pathway is described in the majority of apicomplexan parasites including Cryptosporidium and the presence of trehalose was confirmed in the C. parvum oocyst. Key enzymes in the pathway (i.e., T6PS-TPase and UGGPase) are plant-type and absent in humans and animals, and may potentially serve as novel drug targets in the apicomplexans

Functional and informatics analysis enables glycosyltransferase activity prediction

The elucidation and prediction of how changes in a protein result in altered activities and selectivities remain a major challenge in chemistry. Two hurdles have prevented accurate family-wide models: obtaining (i) diverse datasets and (ii) suitable parameter frameworks that encapsulate activities in large sets. Here, we show that a relatively small but broad activity dataset is sufficient to train algorithms for functional prediction over the entire glycosyltransferase superfamily 1 (GT1) of the plant Arabidopsis thaliana. Whereas sequence analysis alone failed for GT1 substrate utilization patterns, our chemical–bioinformatic model, GT-Predict, succeeded by coupling physicochemical features with isozyme-recognition patterns over the family. GT-Predict identified GT1 biocatalysts for novel substrates and enabled functional annotation of uncharacterized GT1s. Finally, analyses of GT-Predict decision pathways revealed structural modulators of substrate recognition, thus providing information on mechanisms. This multifaceted approach to enzyme prediction may guide the streamlined utilization (and design) of biocatalysts and the discovery of other family-wide protein functions

Football : la pratique, la carrière, les groupes

Football: Practice, Career and Teams. Sportsmen, like other social actors, describe their practices, characterize their own skills ant the abilities of their critics: journalists, managers and spectators. The aim of this article is to examine what is not seen by the public, despite of the football’s high profile media image. If we take the point of view of players, the play will be described as a closed field with secret rules, informal codes and control of aggressiveness and physical risks. The player career is studied as a long run work, a way of life, a set of profits and sanctions. Trough the observation of the football world during many years, the author, a former player, aims to give signification to actions from the players experience and their analysis of situations on the pitch. He describes some developments of social and ethnic relations in a sport which was an important way of integration in France to many immigrants.RÉSUMÉ: Les sportifs, comme les autres acteurs sociaux, définissent leur pratique, évaluent leurs compétences et leurs juges: journalistes, dirigeants, public. Cet article cherche à apprécier, dans le football, ce que ne peut saisir le profane ou le non spécialiste malgré sa médiatisation. Si l’on prend le point de vue des joueurs, le jeu est perçu comme relativement fermé avec ses règles implicites, ses codes de conduite informels, sa gestion de l’agressivité et des risques. La carrière du joueur est étudiée comme un travail, un mode de vie, un système de récompenses et de sanctions. À travers l’observation de ce milieu sur une longue durée, l’auteur, ancien pratiquant, tente de restituer l’expérience des joueurs et leur analyse des situations de match ou d’apprentissage. Il esquisse une description des rapports ethniques et de classes dans un sport qui fut, en tant que loisir, un moyen d’intégration pour les fils d’étrangers.Peneff Jean. Football : la pratique, la carrière, les groupes. In: Sociétés contemporaines N°37, 2000. pp. 121-141

La face cachée d'Urgences. Le feuilleton de la télévision

Jean Peneff: The Hidden Side of the Television Series Emergency Room i Emergency-room services have just been become the focal point of an entertainment-oriented television series. The 60-episode - television series broadcast over, three years was produced by a pair of famous authors, a novelist and a filmmaker. The series was lauded by the press and public alike for being highly realistic and showing a concern for truth. Apart from a few scenes of medical care - true-to-life but exceptional in emergency rooms - what accounts for the blindness of - both the media and the. TV audience that allows them to ignore the. social, ethnic and- public health problems that abound in emergency rooms? The author, having observed the emergency room in Chicago which is represented in the series, reveals discrimination as the dark side of the decor and discusses the characteristics of the ideology of extolling medical intervention disseminated by this TV drama.■ Jean Peneff : La face cachée d'Urgences, le feuilleton de la télévision Les services d'urgences viennent d'être consacrés thème principal d'une série télévisée à vocation distractive. Ce téléfilm de soixante-dix épisodes diffusés sur trois ans a été réalisé par deux auteurs célèbres, un romancier et un cinéaste, l'un médecin et l'autre metteur en scène éclectique,- des dinosaures, de l'holocauste. Cette série a été saluée: par la presse et le public pour son grand réalisme et son souci de vérité. Quelles sont les conditions pour que, mis à part quelques scènes d'interventions médicales -justes mais exceptionnelles dans les urgences - l'aveuglement des médias et des téléspectateurs évacue les problèmes sociaux, ethniques, de santé publique qui foisonnent aux urgences? L'auteur qui, à Chicago, a observé le service représenté dans la série, révèle l'envers du décors, la discrimination et évoque les caractéristiques de l'idéologie de la célébration médicale diffusée par cette dramatique.Peneff Jean. La face cachée d'Urgences. Le feuilleton de la télévision. In: Genèses, 30, 1998. Emigrés, vagabonds, passeports, sous la direction de Jean Leroy. pp. 122-145