Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis

Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml−1,kcat 1.20 s−1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml−1,kcat 0.23 ml mg−1 s−1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml−1, kcat 0.88 ml mg−1 s−1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.Fil: Calloni, Rodrigo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Muchut, Robertino José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Reconquista; ArgentinaFil: Garay, Alberto Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

Redox Regulation of UDP-glucose Pyrophosphorylase from Entamoeba histolytica.

Amoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo- and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDP-glucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V(max) values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Piattoni, Claudia Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Garay, Alberto. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Rodrigues, Daniel Enrique. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentin

Modelagem molecular como estratégia didática no ensino de química

El Modelado Molecular, a través de la interpretación de los términos físico-matemáticos de un campo de fuerza, podría ser una herramienta útil en la enseñanza de la Química. En este trabajo se presentan los resultados de una actividad de articulación entre docentes y alumnos de la Escuela Industrial Superior y docentes especialistas en Modelado Molecular de la Facultad de Bioquímica y Ciencias Biológicas, ambas instituciones pertenecientes a la Universidad Nacional del Litoral (Santa Fe, Argentina).Fil: Garay, Alberto Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Vignatti, Charito Ivana. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Escuela Industrial Superior; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gutierrez, Carolina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Escuela Industrial Superior; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: García Marín, Nubia. Provincia de Santa Fe. Ministerio de Educacion; Argentin

Causal structure of accelerating black holes

Accelerating black holes are described by the so-called C-metric. In this work, we analyse the causal structure of such black holes by using null geodesics. We construct explicitly the relevant Penrose diagrams. First, we recover well-known results associated with the sub-accelerating black holes. Then, we extend the study to the super-accelerating case, in which an additional essential singularity appears. In addition, we consider accelerating black holes with negative masses. We show that they are equivalent to the geometry described by the black hole metric beyond conformal infinity. We compare our results with the Schwarzschild geometry to facilitate understanding and to highlight the interest of the new features

Productive performance of grasslands with different combinations of orchard grass (Dactylis glomerata L.), perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.)

En la zona central de México existen sistemas de producción animal que tienen como componente importante el uso de praderas puras y asociadas. El objetivo de esta investigación fue evaluar cuatro asociaciones, dos gramíneas y una leguminosa, sembradas en diferentes proporciones. Los tratamientos consistieron de las siguientes asociaciones: 20-40-40, 00-50-50, 40-20-40, y 50-00-50 % de ovillo (Ov), ballico perenne (Ba) y trébol blanco (Tr), respectivamente. Los tratamientos se distribuyeron aleatoriamente en 12 parcelas experimentales de 9 por 8 m, de acuerdo con un diseño de bloques completamente al azar con tres repeticiones. Se evaluó rendimiento de materia seca, tasa de crecimiento, composición botánica y morfológica y altura de la planta. En rendimiento anual las asociaciones que presentaron mayor rendimiento fueron; 40-20-40 y 20-40-40 con 20,182 y 19,146 kg MS ha-1, respectivamente y la menor, la asociación 00-50-50 con 15,896 kg MS ha-1. La asociación; 40-20-40 es la que mayor tasa de crecimiento anual presentó con 56 kg MS ha-1 d-1. Independientemente de la estación, en composición botánica y morfológica las asociaciones; 20-40-40, 40-20-40 y 50-00-50 fue la que mayor hoja de ovillo presentó con un promedio de 50%. La estación de invierno fueron las que mayor porcentaje de hoja de ballico presentó para la asociación 00-50-50 con 32%. Independientemente de la asociación, en la estación de primavera fue donde se obtuvo mayor altura con un promedio de 37 cm y la menor fue en invierno con un promedio de 15 cm. En conclusión, la asociación; 40-20-40 fue mejor en rendimiento anual, tasa de crecimiento y altura y la menor fue la asociación 00-50-50.In central Mexico there animal production systems whose major component using pure and associated grasslands. The objective of this research was to evaluate four associations, two grasses and legume planted in different proportions. Treatments consisted of the following associations: 20-40-40, 00-50-50, 40-20-40, and 50-00-50% of orchard grass (Ov), perennial ryegrass (Ba) and white clover (Tr) respectively. The treatments were randomly distributed in 12 experimental plots of 9 and 8 m, according to a design of a randomized complete block with three replications. Dry matter yield, growth rate, botanical and morphological composition and plant height were evaluated. In the association's annual yield higher performance were presented; 40-20-40 and 20-40-40 with 20.182 and 19.146 kg DM ha-1 respectively and the lowest 00-50-50 association with 15.896 kg DM ha-1. The Association; 40-20-40 is the highest annual growth rate presented with 56 kg DM ha-1 d-1. Regardless of the season, botanical and morphological composition associations; 20-40-40, 40-20-40 and 50-00-50 which was higher clew sheet presented with an average of 50 %, the winter season were the highest percentage of ryegrass sheet presented to the association 00-50-50 to 32%. Regardless of the association in the spring season it was where greater height was obtained with an average of 37 cm and the lowest was in winter with an average of 15 cm. In conclusion the association; 40-20-40 was better in annual yield growth rate and lower height and the association was 00-50-50.Fil: Rojas García, Adelaido Rafael.Fil: Hernández Garay, Alfonso. Colegio de Postgraduados. Campus Montecillo (México)Fil: Ayala, Walter.Fil: Mendoza Pedroza, Sergio Iban. Universidad Autónoma Chapingo (México)Fil: Cancino, Santiago Joaquín. Colegio de Postgraduados. Campus Montecillo (México)Fil: Vaquera Huerata, Humberto. Colegio de Postgraduados. Campus Montecillo (México)Fil: Ortega, Mario Alberto Santiago. Colegio de Postgraduados. Campus Montecillo (México

Comportamiento productivo de praderas con distintas combinaciones de ovillo (Dactylis glomerata L.), ballico perenne (Lolium perenne L.) y trébol blanco (Trifolium repens L.)

In central Mexico there animal production systems whose major component using pure and associated grasslands. The objective of this research was to evaluate four associations, two grasses and legume planted in different proportions. Treatments consisted of the following associations: 20-40-40, 00-50-50, 40-20-40, and 50-00-50% of orchard grass (Ov), perennial ryegrass (Ba) and white clover (Tr) respectively. The treatments were randomly distributed in 12 experimental plots of 9 and 8 m, according to a design of a randomized complete block with three replications. Dry matter yield, growth rate, botanical and morphological composition and plant height were evaluated. In the association's annual yield higher performance were presented; 40-20-40 and 20-40-40 with 20.182 and 19.146 kg DM ha-1 respectively and the lowest 00-50-50 association with 15.896 kg DM ha-1. The Association; 40-20-40 is the highest annual growth rate presented with 56 kg DM ha-1 d-1. Regardless of the season, botanical and morphological composition associations; 20-40-40, 40-20-40 and 50-00-50 which was higher clew sheet presented with an average of 50 %, the winter season were the highest percentage of ryegrass sheet presented to the association 00-50-50 to 32%. Regardless of the association in the spring season it was where greater height was obtained with an average of 37 cm and the lowest was in winter with an average of 15 cm. In conclusion the association; 40-20-40 was better in annual yield growth rate and lower height and the association was 00-50-50.En la zona central de México existen sistemas de producción animal que tienen como componente importante el uso de praderas puras y asociadas. El objetivo de esta investigación fue evaluar cuatro asociaciones, dos gramíneas y una leguminosa, sembradas en diferentes proporciones. Los tratamientos consistieron de las siguientes asociaciones: 20-40-40, 00-50-50, 40-20-40, y 50-00-50 % de ovillo (Ov), ballico perenne (Ba) y trébol blanco (Tr), respectivamente. Los tratamientos se distribuyeron aleatoriamente en 12 parcelas experimentales de 9 por 8 m, de acuerdo con un diseño de bloques completamente al azar con tres repeticiones. Se evaluó rendimiento de materia seca, tasa de crecimiento, composición botánica y morfológica y altura de la planta. En rendimiento anual las asociaciones que presentaron mayor rendimiento fueron; 40-20-40 y 20-40-40 con 20,182 y 19,146 kg MS ha-1, respectivamente y la menor, la asociación 00-50-50 con 15,896 kg MS ha-1. La asociación; 40-20-40 es la que mayor tasa de crecimiento anual presentó con 56 kg MS ha-1 d-1. Independientemente de la estación, en composición botánica y morfológica las asociaciones; 20-40-40, 40-20-40 y 50-00-50 fue la que mayor hoja de ovillo presentó con un promedio de 50%. La estación de invierno fueron las que mayor porcentaje de hoja de ballico presentó para la asociación 00-50-50 con 32%. Independientemente de la asociación, en la estación de primavera fue donde se obtuvo mayor altura con un promedio de 37 cm y la menor fue en invierno con un promedio de 15 cm. En conclusión, la asociación; 40-20-40 fue mejor en rendimiento anual, tasa de crecimiento y altura y la menor fue la asociación 00-50-50

Comparative external validation of the PRECISE-DAPT and PARIS risk scores in 4424 acute coronary syndrome patients treated with prasugrel or ticagrelor

Background: The PRECISE-DAPT and PARIS risk scores (RSs) were recently developed to help clinicians at individualizing the optimal dual antiplatelet therapy duration (DAPT) after percutaneous coronary intervention (PCI). Nevertheless, external validation of these RSs it has not yet been performed in ACS (acute coronary syndrome) patients treated with prasugrel or ticagrelor in a real- world scenario. Methods: 4424 ACS patients who underwent PCI and survived to hospital discharge, from January 2012 to December 2016 at 12 European centers, were included. PRECISE-DAPT and PARIS bleeding RS, as well as PARIS ischemic RS, were computed, and their performance at predicting major bleeding (MB; BARC type 3 or 5) and ischemic events (MI and stent thrombosis) during follow up was compared. Results: After a median follow-up of 14 (interquartile range 12–20.9) months, 83 (1.88%) patients developed MB and 133 (3.0%) suffered an ischemic episode. PRECISE-DAPT performed better than PARIS bleeding RS (c-statistic = 0.653 vs. 0.593; p =.01 for comparison) in predicting MB. The RSs performance for MB prediction remained consistent in STEMI patients (c-statistic = 0.632 vs 0.575) or in those treated with prasugrel (c-statistic = 0.623 vs 0.586). PARIS ischemic RS exhibited superior discrimination in predicting ischemic complications compared to PRECISE-DAPT (c-statistic = 0.604 vs 0.568 p =.05 for comparison). Conclusion: Our data provide support to the use of PRECISE-DAPT in MB risk stratification for patients receiving DAPT in form of aspirin and prasugrel or ticagrelor whereas the PARIS ischemic RS has potential to complement the risk prediction with respect to ischemic events

Average daily ischemic versus bleeding risk in patients with ACS undergoing PCI: Insights from the BleeMACS and RENAMI registries

Background: The risk of recurrent ischemia and bleeding after percutaneous coronary intervention (PCI) for acute coronary syndrome (ACS) may vary during the first year of follow-up according to clinical presentation, and medical and interventional strategies. Methods: BleeMACS and RENAMI are 2 multicenter registries enrolling patients with ACS treated with PCI and clopidogrel, prasugrel, or ticagrelor. The average daily ischemic and bleeding risks (ADIR and ADBR) in the first year after PCI were the primary end points. The difference between ADBR and ADIR was calculated to estimate the potential excess of bleeding/ischemic events in a given period or specific subgroup. Results: A total of 19,826 patients were included. Overall, in the first year after PCI, the ADBR was 0.008085%, whereas ADIR was 0.008017% (P =.886). In the first 2 weeks ADIR was higher than ADBR (P =.013), especially in patients with ST-segment elevation myocardial infarction or incomplete revascularization. ADIR continued to be, albeit non-significantly, greater than ADBR up to the third month, whereas ADBR became higher, although not significantly, afterward. Patients with incomplete revascularization had an excess in ischemic risk (P =.003), whereas non–ST-segment elevation ACS patients and those on ticagrelor had an excess of bleeding (P =.012 and P =.022, respectively). Conclusions: In unselected ACS patients, ADIR and ADBR occurred at similar rates within 1 year after PCI. ADIR was greater than ADBR in the first 2 weeks, especially in ST-segment elevation myocardial infarction patients and those with incomplete revascularization. In the first year, ADIR was higher than ADBR in patients with incomplete revascularization, whereas ADBR was higher in non–ST-segment elevation ACS patients and in those discharged on ticagrelor