Functional studies of p.R132C, p.R149C, p.M283V, p.E431K, and a novel c.652-2A>G mutations of the CYP21A2 gene

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90–95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A.G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A.G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.Fil: Taboas, Melisa Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Gómez Acuña, Luciana Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Scaia, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Bruque, Carlos David. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Buzzalino, Noemí Delia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; ArgentinaFil: Stivel, M.. Gobierno de la Ciudad Autonoma de Buenos Aires. Hospital General de Agudos Carlos Durand.; ArgentinaFil: Ceballos, Nora Raquel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dain, Liliana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentin

Biodiesel production from palm oil in a fixed-bed-reactor with calcium methoxide catalyst

En el presente trabajo se analizó el comportamiento del metóxido de calcio como catalizador para la producción de biodiesel a partir de aceite de palma. Inicialmente se establecieron condiciones de operación en un reactor por lotes, como temperatura de activación CaO (25°C), temperatura de reacción (60°C) y relación molar metanol/aceite (10). Se describió el montaje y puesta en marcha de un reactor de lecho fijo y flujo continuo a escala laboratorio; a las condiciones establecidas se obtuvieron rendimientos a métilesteres del 20% con un tiempo de residencia de 60 min. También se analizó el uso de cosolventes como el terbutanol y el acetato de etilo para eliminar las limitaciones de transferencia de masa entre las fases e incrementar los rendimientos a metil ésteres. Se encontró que el uso de etil acetato como cosolvente incrementó el rendimiento a metil esteres hasta el 31 %. Abstract In this research, the behavior of calcium methoxide as catalyst for the production of biodiesel from palm oil in a continuous system was studied. Initially, operating conditions such as CaO activation temperature (25°C), reaction temperature (60°C) and molar ratio methanol-oil (10)were established for a batch reactor. Then, the schematic diagram of the experimental setup and its implementation were described for a packed-bed laboratory scale reactor with continuous flow; for this system, yields of methyl esters of 20% with a residence time of 60 min were obtained. The use of co-solvent such as tert-Bbutanol and ethyl acetate in the continuous reaction system was also investigated, aiming to eliminate mass transfer artifacts between the phases and eventually increasing yields to methyl esters. The use of ethyl acetate as co-solvent increased methyl esters yields up to 31 %

Surfactant effect in heteroepitaxial growth. The Pb - Co/Cu(111) case

A MonteCarlo simulations study has been performed in order to study the effect of Pb as surfactant on the initial growth stage of Co/Cu(111). The main characteristics of Co growing over Cu(111) face, i.e. the decorated double layer steps, the multiple layer islands and the pools of vacancies, disappear with the pre-evaporation of a Pb monolayer. Through MC simulations, a full picture of these complex processes is obtained. Co quickly diffuses through the Pb monolayer exchanging place with Cu atoms at the substrate. The exchange process diffusion inhibits the formation of pure Co islands, reducing the surface stress and then the formation of multilayer islands and the pools of vacancies. On the other hand, the random exchange also suppress the nucleation preferential sites generated by Co atoms at Cu steps, responsible of the step decoration.Comment: 4 pages, latex, 2 figures embedded in the tex

Movilización de conciencia comunitaria: Una experiencia de Investigación Acción Participativa

17 p.La investigación que se presenta a continuación tuvo dos fases, con sus respectivos objetivos: primero, los objetivos de diagnóstico, orientados a conocer las necesidades, problemáticas y potencialidades de la comunidad de la vereda El Charquito, para realizar junto con ellos el diagnóstico psicosocial; y, segundo, los objetivos de intervención, orientados a fomentar la participación y el compromiso de los actores internos de la vereda El Charquito para transformar de forma positiva las problemáticas identificadas en el diagnóstico de necesidades.Introducción Método Resultados y discusión Construcción de categorías inductivas o emergentes Alcance de la intervención en la comunidad Construcción de conocimiento con la comunidad Ámbitos de transformación en la vereda El Charquito Conclusiones y aportes Referencia

Fasciola hepatica soluble antigens (FhAg) induce ovine PMN innate immune reactions and NET formation in vitro and in vivo

[EN] Fasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.SIOpen Access funding enabled and organized by Projekt DEAL. The study was funded by Agencia Nacional de Investigación y Desarrollo (ANID) Fondecyt Initiation 2020 number 11200103 entitled “Analysis ofFasciola hepatica-induced extracellular traps formation in sheep: implications of this innate immune reaction in the pathogenesis of liver fluke disease” held by TM

Hypogammaglobulinaemia secondary to cow-milk allergy in children under 2 years of age

Symptomatic hypogammaglobulinaemia in children younger than 2 years of age was studied to rule out a primary immunodeficiency. Thirty-four patients were referred to the Immunology Service to study the hypogammaglobulinaemia- associated clinical picture. Food allergy was documented in 10 patients by personal and familial history, presence of specific immunoglobulin E (IgE) and elevated total serum IgE levels. Coeliac disease and human immunodeficiency virus infection were also ruled out. Protein loss through stools was assessed by clearance of α1-antitrypsin (AAT). Serum immunoglobulin levels were determined by nephelometry and functional antibodies were studied by enzyme-linked immunosorbent assay. The cellular immune response was assessed by in vitro lymphocyte proliferation in response to mitogens and cell subsets were analysed by flow cytometry. In five patients of the 10 patients we suspected a protein loss through the mucosa. Four of these five patients showed an increased AAT and the other showed an extensive cutaneous lesion. Immunological studies revealed normal antibody function, in vitro lymphoproliferative responses and cell numbers in four of the 5 patients. One patient showed abnormally low numbers of CD4+ T cells as well as a defective proliferative response to mitogens. After diagnosis of cow milk allergy, milk was replaced with infant milk formula containing hydrolysed proteins. Recovery of immunoglobulin values and clinical resolution were achieved. Hypogammaglobulinaemia during early childhood in some children may be secondary to cow milk allergy, and immunoglobulins and cells may leak through the inflamed mucosa. Resolution of symptoms as well as normalization of immunoglobulin values may be easily achieved by avoidance of the offending allergen.Laboratorio de Investigaciones del Sistema Inmun

Estudios de infección de spongospora subterránea en papa (solanum tuberosum) variedad comercial diacol capiro

Spongospora subterranea (Wallroth) Lagerheim f. sp. subterranea Tomlinson es el agente causal de la roña o sarna polvosa de la papa y, además, es vector del virus potato mop top (PMTV). Es un patógeno protozoario, que causa su infección por medio de zoosporas que se encuentran en quistosoros los cuales son el principal modo de dispersión de la enfermedad. Éstos pueden permanecer en el suelo, raíces y tubérculos como estructuras de resistencia por varios años hasta que las condiciones medio ambientales favorezcan la liberación de zoosporas y se dé una nueva infección en las plantas de papa. En el presente trabajo se evaluó un método de bioensayo con el fin de establecer, bajo condiciones controladas, la infección de Spongospora subterranea en la variedad comercial de papa Diacol Capiro. Para tal fin se realizó un experimento, en el cual se evaluaron los efectos de tres fuentes de inóculo: quistosoros provenientes de suelo, de raíz y de tubérculo, con el fin de determinar la fuente más infectiva y la que provocara el desarrollo de la enfermedad. Las plántulas fueron inoculadas con una solución infectiva con una concentración de 1.6x105 quistososros/ml en condiciones de invernadero. Las evaluaciones fueron realizadas a los 19, 34, 64 y 93 días luego de ser inoculadas. Siguiendo la metodología propuesta y realizado el análisis microscópico respectivo, sólo fue posible encontrar algunas estructuras relacionadas en una observación, lo cual no permite confirmar la presencia general del patógeno luego de la inoculación.   Abstract Spongospora subterranea (Wallroth) Lagerheim f. sp. subterranea Tomlinson causes powdery scab in potato and is also vector to the mop-top virus (PMTV) in potato. Is a plasmodiophorids; infections occur through zoospores which are inside resistance spores called sporeballs or cystosori which are the principal way of the disease dispersion. Sporeballs can remains on soil, roots and tubers as resistance structure, and can still there for many years until the weather conditions being favorable to zoospores release starting a new infections on potato plants. In this study was evaluated a bioassay method with the aim to establish an infection from to Diacol Capiro potato variety on greenhouse conditions. It was carried out an experiment which tried to evaluate three different inoculums sources: cystosori from soil, potato roots and tubers, in order to identify the most infectious source and the one which cause the development of the disease. The plants were inoculated, in greenhouse conditions, by a cystosori infected solutions with a 1.6x105 cystosori/ml concentration. The evaluations were made 19, 34, 64 and 93 days after inoculation. Following this methodology and after all the respective microscopical observations, it was not possible to found many clearly structure related to which does not allowed to confirm the presence of the pathogen after the inoculation.

Relación entre el bienestar y el rendimiento académico en alumnos de primer año de medicina

Background: Stress may affect the sense of wellbeing and academic achievement of university students. Aim: To assess the relationship of academic engagement and burnout with academic achievement among first year medical students. Material and Methods: The Utrecht Work Engagement Scale-Student and Maslach Burnout Inventory Student Survey (MBI-SS) were applied to 277 first year medical students of four universities. Their results were correlated with the grades obtained in the different courses. Results: Moderately high engagement and low burnout levels were detected. There was a high level of satisfaction with studies and a moderate exhaustion level. Academic achievement was associated with the degree of engagement with studies but not with burnout. Conglomerate analysis detected a group of students with high levels of wellbeing, characterized by high levels of academic engagement and low burnout. Other group had moderate levels of engagement and lack of personal fulfilment. Other group, identified as extenuated, had high levels of personal exhaustion and depersonalization. Finally the disassociated group had a low academic engagement, low emotional exhaustion, high levels of depersonalization and lack of personal fulfillment. Conclusions: Academic achievement is associated with the level of engagement with studies but not with burnout

Determination of the aldosterone/plasma renin activity ratio for the screening of primary hyperaldosteronism in essential hypertension: a multicentric study

El hiperaldosteronismo primario (HAP) es una afección caracterizada por la producción inapropiadamente elevada y una relativa autonomía del sistema renina-angiotensina. Estimaciones previas, basadas sólo en la evaluación de hipertensos con hipokalemia, consideraban al HAP como una causa poco frecuente de hipertensión (1%). Sin embargo, estudios actuales fundamentados en el cálculo de la relación aldosterona/ actividad de renina plasmática (RAA) arrojan una incidencia mayor (5-10%), siendo la hipertensión arterial (HTA) normokalémica la presentación más frecuente. Dada la amplitud de los valores de corte de la RAA, el Departamento de Suprarrenal de SAEM diseñó un estudio multicéntrico prospectivo en una población de Argentina con el objetivo de establecer nuestro propio valor y determinar así la prevalencia de HAP. Fueron estudiados 353 individuos de ambos sexos, 104 controles normotensos, sin antecedentes familiares de HTA y 249 pacientes hipertensos. Se indicó dieta normosódica y la suspensión de antihipertensivos que interfieran con el eje mineralocorticoideo. Las determinaciones de la actividad de renina plasmática (ARP), DIA-SorinRIA, y de aldosterona, RIA-DPC, fueron realizadas en un único laboratorio. Se realizó ionograma y se evaluaron parámetros clínicos y bioquímicos de síndrome metabólico. La RAA calculada según el percentilo 95 en los controles, fue establecida en la cifra de 36 como valor de corte para sospechar HAP en los hipertensos, requiriéndose una concentración de aldosterona >15 ng/ml. Con una RAA≥36, se realizaron pruebas confirmatorias de sobrecarga salina o de fludrocortisona. La RAA fue ≥36 en 31/249 pacientes, confirmándose HAP en 8 (7 adenomas y 1 hiperplasia), con una prevalencia del 3.2%. Los restantes no completaron estudios confirmatorios. La presencia de síndrome metabólico fue similar en los hipertensos con y sin HAP. En conclusión, este primer estudio multicéntrico argentino determinó nuestro valor de corte de la RAA en 36. Su aplicación permitió establecer una prevalencia de HAP del 3,2% que, aunque podría estar subestimada, resulta significativamente mayor que la previa histórica y concuerda con la incidencia referida en la bibliografía.Primary hyperaldosteronism (PHA) or Conn's disease was classically suspected in the presence of hypertension (H) and hypokalemia. It was previously considered as a rare cause of H, being reported in only 1% of hypertensive patients. It can be caused by an adrenal adenoma (the former usual presentation) or by adrenal hyperplasia. But since the use of the aldosterone/plasma renin activity ratio (AAR) as the screening method in the last years, it is currently considered as almost the most frequent cause of secondary H., accounting for 5-10% of essential H. Plasma rennin activity (PRA) determination is a laborious procedure with low reproducibility and it directly affects the AAR; thus each laboratory must assess its own cut-off value. Therefore, in the Adrenal Department of the Argentine Society of Endocrinology and Metabolism (SAEM), we performed this multicentric prospective study of a population of Argentina with the aim of assessing our own AAR cut-off level in normotensive controls in order to apply it for PHA screening in essential hypertensive patients. We studied 353 adult subjects: 104 controls, aged 45,18 ± 13,78 years-old ( X±SD), with no history of arterial hypertension in their first-degree relatives and with two separate day-registry of blood pressure≤ 139/85 mmHg and 249 hypertensive patients, aged 51± 13,6 years-old ( X ± SD), with arterial blood pressure≥ 140/90 mmHg in the sitting position. Subjects with cardiac, renal, hepatic and neurological diseases were excluded as well as those with Cushing´s syndrome, hyperthyroidism, untreated hypothyroidism, diabetes mellitus and patients under glucocorticoids, oral contraceptive pills or estrogen therapy. A normal sodium diet was indicated and potassium was supplemented when needed. Blood was withdrawn between 8 and 10:00 a.m. with the subjects in the upright position. Aldosterone (A) was determined by DPC radioimmunoassay (RIA) and PRA, by DIA-Sorin RIA. The A normal levels are 4-30 ng/dl for ambulatory individuals on a normal sodium diet and the PRA normal values are < 3,3 ng/ml/h. In order to avoid false positive results in the hypertensive group, AAR was calculated when A was above 15 ng/dl. We measured the waist circumference and we determined the body mass index. Blood sodium, potassium, calcium, urea, creatinine, cholesterol, HDL-C, LDL-C, triglyceride and liver function tests were performed. Statistical Analysis and Results Since the AAR variable showed a non-normal distribution, the cut-off value was considered as the 95th percentile in the control group, which was calculated as 36. This is also in accordance to the function of the empirical distribution of Collings and Hamilton. In our 249 hypertensive patients, 31 had an AAR ≥ 36. PHA was confirmed in 8: seven has an adrenal adenoma and one had hyperplasia. The prevalence of PHA in our population was 3,2 %, with a 95th confidence interval ranging from 1,4 to 6,2 %. In the remaining 23 patients, confirmatory tests could not be completed. There was no correlation between the severity of the hypertension and the AAR value, with no statistical significant differences between those with or without PHA. Likewise, we found no correlation between PRA and advancing age. In hypertensive patients, metabolic syndrome was more prevalent than in controls, but it was present to the same extent in those with or without PHA. Conclusions To our knowledge, this is the first multicentric study performed in Argentina to determine the aldosterone/ plasma renin activity ratio in our normotensive control population. Our AAR value of 36 agrees with the levels reported in the international literature: thus an AAR ≥ 36 along with an aldosterone ≥ 15 ng/ml in hypertensive patients lead us to suspect PHA and to perform confirmatory tests. Applying these criteria, we found a prevalence of 3,2% of PHA in essential HTA. It is possible that this value may be underestimated due to the fact that confirmatory tests could not be completed in all the hypertensive subjects with an AAR≥ 36. In spite of this, our prevalence is significantly greater than the historical one and it lies in the range reported in the literature.Fil: Pardes, E.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Belli, S.. Hospital Dr. C. Durand; ArgentinaFil: Cornaló, D.. Hospital Rivadavia; ArgentinaFil: Contreras, Liliana Noemi. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; ArgentinaFil: Costa, L.. Hospital Dr. A. Posadas; ArgentinaFil: Chervin. R.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Damilano, S.. Laboratorio de Bioanalítica; ArgentinaFil: Fenili, C.. Laboratorio de Bioanalítica; ArgentinaFil: Gómez, R. M.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Leal Reyna, M.. Complejo Médico Policial "Churruca"; ArgentinaFil: Lupi, S.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Martínez, M.. Hospital Francés; ArgentinaFil: Nofal, M.. Clínica Bazterrica; ArgentinaFil: Ruibal, G.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; Argentin