Monitoring Glycan-Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH-π Interactions.

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan-protein interactions that involve N-acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc-containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal (1) H and (19) F atoms that allow both (1) H and (19) F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc- (or GalNAc-) moieties over a range of binding affinities. The CHF2 CONH- moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic-sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF3 CONH- analogue differs more significantly.

Cooperative hydrogen bonding in glyco-oligoamides: DNA minor groove binders in aqueous media.

A strategy to create cooperative hydrogen-bonding centers by using strong and directional intramolecular hydrogen-bonding motifs that can survive in aqueous media is presented. In particular, glyco-oligoamides, a family of DNA minor groove binders, with cooperative and non-cooperative hydrogen-bonding donor centers in the carbohydrate residues have been designed, synthesized, and studied by means of NMR spectroscopy and molecular modeling methods. Indeed, two different sugar moieties, namely, ß-D-Man-Py-γ-Py-Ind (1; Ind=indole, Man=mannose, Py=pyrrole) and ß-D-Tal-Py-γ-Py-Ind (2; Tal=talose), were chosen according to our design. These sugar molecules should present one- or two-directional intramolecular hydrogen bonds. The challenge has been to study the conformation of the glyco-oligoamides at low temperature in physiological media by detecting the exchangeable protons (amide NH and OH resonances) by means of NMR spectroscopic analysis. In addition, two more glyco-oligoamides with non-cooperative hydrogen-bonding centers, that is, ß-D-Glc-Py-γ-Py-Ind (3; Glc=glucose), ß-D-Gal-Py-γ-Py-Ind (4; Gal=galactose), and the model compounds ß-D-Man-Py-NHAc (5) and ß-D-Tal-Py-NHAc (6) were synthesized and studied for comparison. We have demonstrated the existence of directional intramolecular hydrogen bonds in 1 and 2 in aqueous media. The unexpected differences in terms of stabilization of the intramolecular hydrogen bonds in 1 and 2 relative to 5 and 6 promoted us to evaluate the influence of CH-π interactions on the establishment of intramolecular hydrogen bonds by using computational methods. Initial binding studies of 1 and 2 with calf-thymus DNA and poly(dA-dT)2 by NMR spectroscopic analysis and molecular dynamics simulations were also carried out. Both new sugar-oligoamides are bound in the minor groove of DNA, thus keeping a stable hairpin structure, as in the free state, in which both intramolecular hydrogen-bonding and CH-π interactions are present.

The KCNQ1 (Kv7.1) COOH terminus, a multitiered scaffold for subunit assembly and protein interaction.

The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 A crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.

gem-Difluoro-carbasugars, the cases of mannopyranose and galactopyranose.

5a-Difluoro-5a-carbamannopyranose (gem-difluoro-carbamannopyranose) and 5a-difluoro-5a-carbagalactopyranose (gem-difluoro-carbagalactopyranose), close congeners of their respective natural sugars, in which the endocyclic oxygen atom has been replaced by a gem-difluoromethylene group, were synthesized from D-mannose and D-galactose, using a rearrangement strategy.

[Protocol to evaluate the effectiveness of a consciousness-raising and training intervention for primary care professionals, in order to improve detection of domestic violence (ISFVIDAP)]. / Protocolo para la evaluación de la efectividad de una intervención sensibilizadora y formativa en profesionales de atención primaria para la mejora de la detección de la violencia doméstica (ISFVIDAP).

OBJECTIVE: To evaluate the effectiveness of an intervention aimed at primary care physicians and nurses to improve the detection of domestic violence. DESIGN: Community intervention study with control, randomized in clusters, pragmatic, open, and with parallel groups. SETTING: Primary care centres in Spain. POPULATION: Primary care physicians and nurses from the entire country who agree to participate in the study. UNIT OF ANALYSIS: The basic care team (BCT) of doctor and nurse looking after a list is the unit of analysis for evaluating the number of cases detected; and their clinical records are the units of analysis for evaluating recorded cases (suspicion and/or confirmation of mistreatment). SAMPLE SIZE: Sixty eight BCT in each group (136 in the 2 groups) and 1700 clinical records per group (25 per BCT). Altogether, they will cover some 130,000 women of 14 and over. INTERVENTION: A short training programme with homogeneous training contents, aimed at raising the awareness of health professionals and teaching them how to identify risk factors, situations of special vulnerability and alarm signals. The programme also aims to provide health professionals with tools to make the clinical interview easier, when they suspect mistreatment and how to tackle a case once it is detected. MEASUREMENTS: The main measurement will be the mean variation between intervention and control groups in the number of cases of domestic violence detected during the study, through specific recording and mean variation between the initial and final variations in each group. ANALYSIS: A weighted student's t test or, if covariates need to be adjusted, a regression analysis will be used for comparison. All analyses will be based on intention to treat.

Protocolo para la evaluación de la efectividad de una intervención sensibilizadora y formativa en profesionales de atención primaria para la mejora de la detección de la violencia doméstica (ISFVIDAP)

Objetivo. Evaluar la efectividad de una intevención dirigida a médicos y enfermeras de atención primaria, orientada a mejorar la detección de la violencia doméstica. Diseño. Estudio de intervención comunitaria, controlado, aleatorizado en conglomerados, pragmático, abierto y de grupos paralelos. Emplazamientos. Centros de atención primaria de diferentes comunidades autónomas, coordinados a través de la Red de Investigación en Actividades Preventivas y de Promoción de la Salud realizadas en Atención Primaria (red IAPP). Sujetos. Médicos y enfermeras de atención primaria de todo el país que acepten participar en el estudio hasta completar la muestra prevista. Unidad de análisis. Unidad básica asistencial de atención primaria (UBA), formada por un médico y una enfermera que atienden un cupo, mediante registro específico para la evaluación de los casos detectados y mediante auditoría de historias clínicas para los casos registrados (sospecha y/o confirmación de maltrato). Tamaño de la muestra. En total, 136 UBA: 68 Uba experimentales y 68 UBA control, formadas por un médico de familia y una enfermera que atienden un cupo. Se incluyen 1.700 historias clínicas por grupo (25 por UBA) que en conjunto atienden a unas 130.000 mujeres ≥ 14 años. Intervención. Programa de formación breve con contenidos formativos homogéneos y estadarizados, dirigidos a sensibilizar al profesional, enseñar a identificar factores de riesgo y vulnerabilidad, signos y señales de alerta, proporcionar elementos facilitadores de la entrevista clínica ante sospecha de una situación de maltrato y a enseñar el abordaje del problema una vez detectado. Mediciones. La principal medida de resultado será la variación promedio en el número de casos de violencia detectados a lo largo del estudio entre el grupo de intervención y el grupo control, mediante registro específico y variación promdeio de los casos registrados en la historia clínica (a través de una auditoría) entre la evaluación final y la inicial de cada grupo. Análisis La comparación se realizará mediante una prueba de la t de Student ponderara o, en caso de ser necesario para ajustar covariables, un análisis de regresión. Todos los análisis se realizarán por intención de tratar.(AU)

Protocolo para la evaluación de la efectividad de una intervención sensibilizadora y formativa en profesionales de atención primaria para la mejora de la detección de la violencia doméstica (ISFVIDAP) / Protocol to evaluate the effectiveness of a consciousness-raising and training intervention for primary care professionals, in order to improve detection of domestic violence (ISFVIDAP)

Objetivo. Evaluar la efectividad de una intervención dirigida a médicos y enfermeras de atención primaria, orientada a mejorar la detección de la violencia doméstica. Diseño. Estudio de intervención comunitaria, controlado, aleatorizado en conglomerados, pragmático, abierto y de grupos paralelos. Emplazamiento. Centros de atención primaria, de diferentes comunidades autónomas, coordinados a través de la Red de Investigación en Actividades Preventivas y de Promoción de la Salud realizadas en Atención Primaria (redIAPP). Sujetos. Médicos y enfermeras de atención primaria de todo el país que acepten participar en el estudio hasta completar la muestra prevista. Unidad de análisis. Unidad básica asistencial de atención primaria (UBA), formada por un médico y una enfermera que atienden un cupo, mediante registro específico para la evaluación de los casos detectados y mediante auditoría de historias clínicas para los casos registrados (sospecha y/o confirmación de maltrato). Tamaño de la muestra. En total, 136 UBA: 68 UBA experimentales y 68 UBA control, formadas por un médico de familia y una enfermera que atienden un cupo. Se incluyen 1.700 historias clínicas por grupo (25 por UBA) que en conjunto atienden a unas 130.000 mujeres >= 14 años. Intervención. Programa de formación breve con contenidos formativos homogéneos y estandarizados, dirigidos a sensibilizar al profesional, enseñarle a identificar factores de riesgo y vulnerabilidad, signos y señales de alerta, proporcionar elementos facilitadores de la entrevista clínica ante sospecha de una situación de maltrato y a enseñar el abordaje del problema una vez detectado. Mediciones. La principal medida de resultado será la variación promedio en el número de casos de violencia detectados a lo largo del estudio entre el grupo de intervención y el grupo control, mediante registro específico y variación promedio de los casos registrados en la historia clínica (a través de una auditoría) entre la evaluación final y la inicial para cada grupo. Análisis. La comparación se realizará mediante una prueba de la t de Student ponderada o, en caso de ser necesario para ajustar covariables, un análisis de regresión. Todos los análisis se realizarán por intención de tratar

Objective. To evaluate the effectiveness of an intervention aimed at primary care physicians and nurses to improve the detection of domestic violence. Design. Community intervention study with control, randomized in clusters, pragmatic, open, and with parallel groups. Setting. Primary care centres in Spain. Population. Primary care physicians and nurses from the entire country who agree to participate in the study. Unit of analysis. The basic care team (BCT) of doctor and nurse looking after a list is the unit of analysis for evaluating the number of cases detected; and their clinical records are the units of analysis for evaluating recorded cases (suspicion and/or confirmation of mistreatment). Sample size. Sixty eight BCT in each group (136 in the 2 groups) and 1700 clinical records per group (25 per BCT). Altogether, they will cover some 130 000 women of 14 and over. Intervention. A short training programme with homogeneous training contents, aimed at raising the awareness of health professionals and teaching them how to identify risk factors, situations of special vulnerability and alarm signals. The programme also aims to provide health professionals with tools to make the clinical interview easier, when they suspect mistreatment and how to tackle a case once it is detected. Measurements. The main measurement will be the mean variation between intervention and control groups in the number of cases of domestic violence detected during the study, through specific recording and mean variation between the initial and final variations in each group. Analysis. A weighted student's t test or, if covariates need to be adjusted, a regression analysis will be used for comparison. All analyses will be based on intention to treat