Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-B-lactamase

In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear β- lactamase, CphA from Aeromonas hydrophila. According to our results, significant different transition state structures are obtained for the hydrolysis of both antibiotics: while the TS of the CEF is a ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. This feature of the β-lactamase would be in turn, associated to the different mechanisms that the protein employs to hydrolyze the different antibiotics; i.e. the catalytic promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study will be used to shed some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MβL inhibitors against a broad spectrum of bacterial pathogens.We thank FEDER and the Spanish Ministerio de Economía y Competitividad (project CTQ2012-36253-C03), Universitat Jaume I (project P1 1B2011-23) and Generalitat Valenciana (PROMETEOII/2014/022 and ACOMP/2014/277 projects

QM/MM Calculations Suggest a Novel Intermediate Following the Proton Abstraction Catalyzed by Thymidylate Synthase

The cleavage of covalent C–H bonds is one of the most energetically demanding, yet biologically essential, chemical transformations. Two C–H bond cleavages are involved in the reaction catalyzed by thymidylate synthase (TSase), which provides the sole de novo source of thymidylate (i.e., the DNA base T) for most organisms. Our QM/MM free energy calculations show that the C–H → O proton transfer has three transition states that are energetically similar but structurally diverse. These characteristics are different from our previous calculation results on the C–H → C hydride transfer, providing an explanation for differences in temperature dependences of KIEs on these two C–H bond activation steps. The calculations also suggest that the traditionally proposed covalent bond between the protein and substrate (the C6–S bond) is very labile during the multistep catalytic reaction. Collective protein motions not only assist cleavage of the C6–S bond to stabilize the transition state of the proton transfer step but also rearrange the H-bond network at the end of this step to prepare the active site for subsequent chemical steps. These computational results illustrate functionalities of specific protein residues that reconcile many previous experimental observations and provide guidance for future experiments to examine the proposed mechanisms. The synchronized conformational changes in the protein and ligands observed in our simulations demonstrate participation of protein motions in the reaction coordinate of enzymatic reactions. Our computational findings suggest the existence of new reaction intermediates not covalently bound to TSase, which may lead to a new class of drugs targeting DNA biosynthesis

Theoretical site-directed mutagenesis. The Asp168Ala mutant of L-Lactate Dehydrogenase

The reduction of pyruvate to lactate catalyzed by the L-Lactate dehydrogenase has been studied in this paper by means of hybrid Quantum Mechanical / Molecular Mechanical simulations. A very flexible molecular model consisting on the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules has allowed to theoretically mimic site directed mutagenesis studies, most of them previously experimentally performed. The potential energy surfaces obtained for every single mutation, compared with the one corresponding to the native enzyme, have been used to trace the possible reaction pathways and to locate and characterize the structures corresponding to the stationary points. The analysis of the results has been a very powerful tool to conclude about the role of key residues on the vacuole formed in the active site of the enzyme. Our results are in very good agreement with the previous conclusions derived from site directed mutagenesis. This strategy can be extrapolated to other enzyme systems thus opening the door of a very promising methodology that, in combination with the appropriate experimental technique, will enable us to describe enzyme catalysis phenomenon and the particular role of the residues that form the protein. This knowledge placed us in a privileged position to modify the activity of enzymes and to propose efficient inhibitor

An in vitro evaluation of two dentine adhesive systems to seal the pulp chamber using a glucose penetration model

Objectives: To evaluate the sealing capability of Cavit TM G with or without Clearfil TM S3 Bond and Prime & Bond NT placed in the pulp chamber. Study Design: Forty single rooted premolars, extracted for orthodontic and periodontal reasons, with intact coronal surface and mature apices, were standardized to a length of 15 mm. The teeth were instrumented, filled with a gutta-percha master cone and divided into three groups to obturate the pulp chambers: Cavit TM G; Clearfil TM S3 Bond plus Cavit TM G and Prime & Bond® NT plus Cavit TM G. A glucose leakage model was used for evaluating the coronal microleakage. The Mann-Whitney test was used to evaluate the differences in the means of the glucose leakage. Results: An increase in glucose penetration was observed during the first week in groups Cavit TM G and Cavit TM G+PBNT. The glucose penetration values of all groups were similar at 30 and 45 days, and there were no significant differences among them in both time periods (p=0.736 and p=0.581, respectively). Conclusions: The adhesive systems did not improve the capability of Cavit TM G to seal the pulp chamber over time © Medicina Oral S. L

Intraorifice sealing ability of different materials in endodontically treated teeth

Objectives: To evaluate Cavit? G, ProRoot? MTA and Tetric® EvoFlow as intraorifice barriers to prevent coronal microleakage in root canal treatment. Study Design: Forty-two human single rooted teeth were divided randomly in three experimental groups of 10 specimens each and two control groups. The experimental groups were prepared with hand instrumentation and cold lateral condensed technique of the gutta-percha. Four millimetres of coronal gutta-percha were removed and replaced by one of the following filling materials: Cavit? G, Tetric® EvoFlow or ProRoot? MTA. In the experimental groups, leakage was measured by the concentration of leaked glucose in the apical reservoir at 1, 7, 30, and 45 days, using the enzymatic glucose oxidase method. Data were analyzed by means of Mann-Whitney U and Kruskal-Wallis tests at ?=0.05. Results: The glucose penetration results of three experimental groups increased gradually over time. No significant differences were found among groups at 24 hours and 1 week. At thirty and forty-five days, Cavit? and Tetric® EvoFlow values were significantly different (p=0.007 and p=0.023, respectively). Conclusions: The sealing ability of the Cavit? G, ProRoot? MTA and Tetric® EvoFlow used as intraorifice materials tends to be similar over time

Classe invertida (flipped classroom). Experiència en el grau d'Infermeria.

Projecte finalitzat desembre 2015En l’aula invertida (flipped classroom) (FC), els estudiants treballen fora de classe, tasques prèviament preparades amb aquest fi pel professorat. L’objectiu del estudi es motivar als estudiants suplint per FC les classes teòriques magistrals. Superar favorablement FC evita l’examen final (30% de l’avaluació). Context d’aplicació: grup C, 89 estudiants (finalitzen n=81). Programa de Ciències Psicosocials Aplicades a la Salut, assignatura anual, formació bàsica, 9 ECTS, primer curs, Grau d’Infermeria, tarda, segon semestre, curs 2014-15. Metodologia: resposta escrita a preguntes amb suport bibliogràfic i avaluació amb instruments específics estandaritzats. A classe: avaluació per parells i contrastos verbals sobre continguts, tutelats per la professora, que presenta una segona avaluació setmanal (campus virtual). Resultats: Contrast estadístic de notes FC amb examen test, igual al realitzat en grup A (intraclasse). Puntuació (sobre 10) superior a FC (mitja: 7,96 + DE: 0,67) que en l’examen (mitja 5,89 + DE: 1,26), <0.001. També es contrasten resultats d’examen de FC i de grup A (mitja: 6,02 + DE 1,18) (interclasse) i no es troben diferencies estadísticament significatives 0.508 . Degut a que el grup C (FC) no s’havia preparat ni estudiat l’examen, la diferencia de dècimes mostra una adquisició de coneixements favorable per la experiència flipped classroom.2014PID-UB/5

Aula invertida: experiencia en el Grado de Enfermería

En el aula invertida, los estudiantes trabajan autónomamente tareas previamente preparadas con este fin por el profesorado. Objetivo: evaluar el aula invertida como innovación docente y contrastar los resultados con grupo con docencia tradicional. Contexto: primer curso del Grado de Enfermería en la asignatura troncal Ciencias Psicosociales Aplicadas a la Salud (grupo C, n=81). Metodología: cualitativa y cuantitativa con la inclusión de instrumentos estandarizados ad hoc. Destaca la evaluación por pares y contrastes verbales sobre contenidos tutelados por el profesorado. Se realizó evaluación del grupo control y el grupo experimental y se contrastaron los resultados de las cualificaciones obtenidas. Resultados. Puntuación sobre 10 con medias y desviación estándar: Intraclase, superior en el aula invertida (7,96 + 0,67) frente a (5,89 + 1,26) en examen (p-valor < 0,001). Interclase, grupo experimental (5,89 + 1,26) y grupo control (6,02 + 1,16), (p-valor 0,508). Sin hallazgo de diferencias estadísticamente significativas. Conclusión: Sin preparación para el examen y gran esfuerzo adicional, destacó la satisfacción por parte del profesorado y de los estudiantes, el aula invertida se mostró claramente favorable para la adquisición de conocimientos en la asignatura de Enfermería probada

Accuracy of liquid-based brush cytology and HPV detection for the diagnosis and management of patients with oropharyngeal and oral cancer.

Objectives This study aims to evaluate the usefulness of liquid-based brush cytology for malignancy diagnosis and HPV detection in patients with suspected oropharyngeal and oral carcinomas, as well as for the diagnosis of tumoral persistence after treatment. Material and methods Seventy-five patients with suspicion of squamous cell carcinoma of the oropharynx or oral cavity were included. Two different study groups were analyzed according to the date of the sample collection: (1) during the first endoscopy exploration and (2) in the first control endoscopy after treatment for squamous cell carcinoma. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for malignancy diagnosis as well as for HPV-DNA detection on brush cytologies were assessed. Results Before treatment, the brush cytology showed a sensitivity of 88%, specificity of 100%, and accuracy of 88%. After treatment, it showed a sensitivity of 71%, specificity of 77%, and accuracy of 75%. HPV-DNA detection in cytology samples showed a sensitivity of 85%, specificity of 100%, and accuracy of 91% before treatment and an accuracy of 100% after treatment. Conclusions Liquid-based brush cytology showed good accuracy for diagnosis of oropharyngeal and oral squamous cell carcinoma before treatment, but its value decreases after treatment. Nevertheless, it is useful for HPV-DNA detection, as well as to monitor the patients after treatment. Clinical relevance Brush cytology samples are reliable for the detection of HPV-DNA before and after treatment and may be a useful method to incorporate in the HPV testing guidelines

Actigraphic Sleep and Dietary Macronutrient Intake in Children Aged 6-9 Years Old: A Pilot Study

The objective of this study was to examine the relationship between different sleep parameters and energy and macronutrient intake in school-aged children. A total of 203 children 6 to 9 years of age participated in this cross-sectional study. Anthropometric measurements were taken first. Diet was assessed with 3-day food logs and sleep was measured with a questionnaire on sleep quality and a wrist actigraph worn for at least 7 days. A decrease of 165.45 kcal was observed per each additional hour of sleep during the week (β (95% CI) = −165.45 (−274.01, −56.88); p = 0.003). This relationship was also observed for fat (β (95% CI) = −11.14 (−18.44, −3.84); p = 0.003) and protein (β (95% CI) = −13.27 (−22.52, −4.02); p = 0.005). An increase in weekend sleep efficiencies for those under the recommended threshold of 85% also had a similar association with energy (β (95% CI) = −847.43 (−1566.77, 128.09); p = 0.021) and carbohydrate (β (95% CI) = −83.96 (−161.76, −6.15); p = 0.035)) intake. An increase in habitual sleep variability was related with a slight increase in protein intake (β (95% CI) = 0.32 (0.031, 0.62); p = 0.031). Children who slept less had a higher energy intake, especially from fat and protein and those who presented inefficient sleep had a higher carbohydrate intake. Strategies to enhance sleep quality and quantity combined with dietary recommendations could help to improve energy and macronutrient intake levels in children

Aula invertida: experiencia en el Grado de Enfermería

En l’aula inversa, els estudiants treballen autònomament tasques prèviament preparades amb aquest fi pel professorat. Objectiu: avaluar l’aula inversa  com a innovació docent i contrastar els resultats amb el grup amb docència tradicional. Context: primer curs del Grau d’Infermeria en l’assignatura troncal de Ciències Psicosocials Aplicades a la Salut (grup C, n=81). Metodologia: qualitativa i quantitativa amb la inclusió d’instruments estandarditzats ad hoc.  Destaca l’avaluació per parells i contrastos verbals sobre continguts tutelats pel  professorat. Avaluació: Es va realitzar l’avaluació del grup control i del grup experimental i, es van contrastar els resultats de les qualificacions obtingudes.  Resultats. Puntuació sobre 10 amb mitjanes i desviació estàndard: Intraclasse, superior en aula inversa (7,96 + 0,67) al davant (5,89 + 1,26) en examen (p &lt; 0,001). Interclasse, grup experimental (5,89 + 1,26) i grup control (6,02 + 1,16), (p-valor 0,508). No es van trobar diferències estadísticament significatives. Conclusió: Sense preparació per a l’examen i gran esforç addicional, va destacar la satisfacció per part del professorat i dels estudiants. L’aula inversa es va mostrar clarament favorable per a l’adquisició de coneixements en l’assignatura d’Infermeria provada.At flipped classroom, students work autonomously with tasks previously prepared for this purpose by the professorship. Objective: To evaluate the flipped classroom as innovation teaching and contrast group results with traditional teaching. Context: first course of Nursing Degree at Psychosocial Sciences Applied to Health, core subject (Group C, n = 81). Methodology: qualitative and quantitative, including ad hoc standardized instruments and emphasizing the evaluation performed by peers and verbal verification on the content. The whole process was supervised by lecturers. Evaluation: We conducted the evaluation of the control group and the experimental group, and they compare the results of the rating results. Results: Score out of 10 with means and standard deviation: Intraclass, higher flipped classroom (7,96 + 0,67) vs (5,89 + 1,26) in test (p-value &lt;0,001). Interclass, experimental group (5,89 + 1,26) and control group (6,02 + 1,16) (p-value 0,508). Statistically significant differences were not found. Conclusion: Without test preparation and great additional effort, highlighted the satisfaction for lecturers and students, flipped classroom was clearly favorable for the acquisition of knowledge in the subject tested.En el aula invertida, los estudiantes trabajan autónomamente tareas previamente preparadas con este fin por el profesorado. Objetivo: evaluar el aula invertida como innovación docente y contrastar los resultados con grupo con docencia tradicional. Contexto: primer curso del Grado de Enfermería en la asignatura troncal Ciencias Psicosociales Aplicadas a la Salud (grupo C, n=81). Metodología: cualitativa y cuantitativa con la inclusión de instrumentos estandarizados ad hoc. Destaca la evaluación por pares y contrastes verbales sobre contenidos tutelados por el profesorado. Se realizó evaluación del grupo control y el grupo experimental y se contrastaron los resultados de las cualificaciones obtenidas. Resultados. Puntuación sobre 10 con medias y desviación estándar: Intraclase, superior en el aula invertida (7,96 + 0,67) frente a (5,89 + 1,26) en examen (p-valor &lt; 0,001). Interclase, grupo experimental (5,89 + 1,26) y grupo control (6,02 + 1,16), (p-valor 0,508). Sin hallazgo de diferencias estadísticamente significativas. Conclusión: Sin preparación para el examen y gran esfuerzo adicional, destacó la satisfacción por parte del profesorado y de los estudiantes, el aula invertida se mostró claramente favorable para la adquisición de conocimientos en la asignatura de Enfermería probada.