Sensitive analysis of recombinant human erythropoietin glycopeptides by on-line phenylboronic acid solid-phase extraction capillary electrophoresis-mass spectrometry

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CEMS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N24 and N38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CEMS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest

Characterization of glycoproteins by capillary electrophoresis electrospray mass spectrometry (CE-ESI-MS). Applications to diagnosis in biomedicine

Glycosylation is the most common posttranslational modification in proteins and the carbohydrates participate in many biological processes. The number and type of glycoforms for a certain glycoprotein may change as a consequence of pathological processes. In our work a method for the separation of transferrin sialoforms has been developed, that permits the diagnostic of Congenital Disorders of Glycosylation (CDG) using a polybrene-dextran sulphate coating and CE-ESI-TOF methodologies. In order to improve the sensitivity the use of solid-phase extraction coupled on-line to CE-ESI-MS is studied and the SPE-CE-ESI-MS developed methods are applied for the characterization of rHuEPO glycoforms. The achieved separation and the high mass-resolving power of flight (TOF) mass detection allows to establish the most probable rHuEPO glycoforms

NEU screen shows high accuracy in detecting cognitive impairment in older persons living with HIV

The NEUrocognitive (NEU) Screen is a practical tool proposed to screen for HIV-associated cognitive impairment in the clinical setting. This is a pencil-and-paper method that can be applied rapidly (<=10 minutes for administration) and has no copyright limitations. In this study, we aimed at investigating its diagnostic accuracy in an older population of persons living with HIV (PLWH), with cutoffs set at 30, 40, 50, and 60 years. Data were collected from a sample of 368 PLWH who underwent a comprehensive neuropsychological tests battery (gold standard). Results of statistical tests showed that accuracy of the NEU Screen increased with age of the participants. The highest degree of precision, with a sensitivity of 91% and specificity of 92%, was obtained for people ages 60 years or older (correct classification: 91%). These optimal results point to the great potential of the NEU Screen as a tool for detecting cognitive disorders in older PLWH

Analysis of glycopeptide biomarkers by on-line TiO2 solid-phase extraction capillary electrophoresis-mass spectrometry

In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO2-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O126-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O126 glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides. Under the optimized conditions, the microcartridge lifetime was around 10 analyses and repeatability was acceptable (%RSD values of 9-11% and 6-11% for migration times and peak areas, respectively). The method was linear between 0.5-50 mg·L-1 and 10-50 mg·L-1 for O126 glycoforms containing NeuAc and NeuGc, respectively, and limits of detection (LODs) were up to 100 times lower than by CE-MS. Although optimized for O-glycopeptides, the method proved also successful for preconcentration of N83-glycopeptides, without compromising the separation between glycopeptide glycoforms with different number of sialic acids. Tryptic digests of other glycoproteins (i.e. human apolipoprotein CIII (APO-C3) and bovine alpha-1-acid glycoprotein (bAGP)) were also analyzed, demonstrating the applicability to glycopeptides with different glycan composition and nature

Health decentralization policy and social answer. The Colombian region case

ABSTRACT: During the last twenty years, social participation in Latin America, especially in Colombia has been oriented by a policy of administrative and political decentralization, with consequences in the field of health where both the state and the communities have assumed new spaces and types of relation that impact social organization and justice. Through this framework the project analyses in the Colombian region of the antioqueño southwest, some of the effects health decentralization has had taking into consideration the historic characteristics of the participation process of the population, some central aspects of the legal context of the health and social security system reforms during the last two decades, the perceptions of local actors as regards the process of the implementation of decentralized health, and the actions undertaken by the local state agents and the people in comunitary participation processes for the improvement of life and health conditions.RESUMEN: Durante los últimos veinte años la participación social en América Latina, y particularmente en Colombia, ha estado orientada por la descentralización política y administrativa. Con la aplicación de esta estrategia en el campo de la salud, tanto el Estado como las comunidades han asumido nuevos espacios y formas de relación con impacto en lo social, lo organizativo y lo jurídico. Éste es el marco en el que se inscribe este trabajo, que analiza en la región colombiana del Suroeste antioqueño algunos efectos de la descentralización en salud, teniendo en cuenta las características históricas del proceso de participación de la población en el país; algunos aspectos centrales del contexto legal de las reformas del Sistema de Seguridad Social en Salud en las dos últimas décadas, las percepciones de los actores locales relacionadas con la implementación del proceso descentralizador en salud y las acciones desplegadas por los agentes locales del Estado y por los pobladores en procesos de participación comunitaria para el mejoramiento de las condiciones de vida y salud

On-line solid-phase extraction to enhance sensitivity in peptide biomarker analysis by microseparation techniques coupled to mass spectrometry: capillary liquid chromatography versus capillary electrophoresis

In this study, on-line solid-phase extraction capillary liquid chromatography-mass spectrometry (SPE-CapLC-MS) and on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) were compared for the analysis of the opioid peptide biomarkers dynorphin A (1-7) (DynA), endomorphin 1 (End 1), and methionine-enkephalin (Met). First, a capillary liquid chromatography-mass spectrometry (CapLC-MS) method was established, which allowed limits of detection (LODs) of 0.5 μg/mL for Dyn A and Met, and 0.1 μg/mL for End 1. Then, a column switching setup operated by a 2-position/6-port micro-valve with a C18 enrichment column was assembled for SPE-CapLC-MS. Under optimized conditions, the LODs for the three peptides were lowered up to 1000-fold compared to CapLC-MS, until detecting 0.5 ng/mL concentrations. Repeatability (<0.2 % and <11 % RSD for retention times and peak areas, respectively), linearity (0.5-100 ng/mL), and durability (20 runs) of the enrichment column were appropriate, and the method was applied to analyze human plasma samples. Finally, the established SPE-CapLC-MS method was compared with a valve-free C18-SPE-CE-MS method previously described by our group for the analysis of these opioid peptides, using the same mass spectrometer. Both methods presented an evident difference regarding the need of a valve for the operation and allowed high preconcentration factors and quite similar LODs (until 0.5 and 0.1 ng/mL by SPE-CaLC-MS and SPE-CE-MS, respectively). Some other distinctions related to the instrumental set-up, procedure and method performance were also disclosed and discussed in detail

Preconcentración por extracción en fase sólida en línea a la electroforesis capilar acoplada a la espectrometría de masas

Los factores más importantes a tener en cuenta cuando se desarrollan nuevas técnicas y métodos de separación son la simplificación, rapidez y coste de los análisis, la reducción de la cantidad de reactivos y muestra necesarias, la mejora de la selectividad, eficacia y resolución de las separaciones, la disminución de los límites de detección y la reproducibilidad de los resultados. En este contexto han ido madurando en los últimos tiempos las técnicas microseparativas, implementándose en microchips o dispositivos microfluídicos o en tubos y columnas capilares [1-6]. Estas técnicas de separación en microescala tienen un gran potencial, porque permiten cumplir prácticamente con todos los requisitos anteriormente indicados. Sin embargo, a menudo los límites de detección (LODs) en unidades de concentración no son lo suficientemente bajos debido a los pequeños volúmenes de muestra necesarios para los análisis con estas técnicas [7-11]. Esto, unido a la siempre cuestionada reproducibilidad, han sido los principales impedimentos para se apliquen ampliamente a muestras complejas reales

Glicoproteínas biomarcadoras de cáncer: un reto analítico actual

Las alteraciones en la glicosilación de las proteínas pueden ser la causa de enfermedades graves, como es el caso de los desórdenes congénitos de la glicosilación (CDGs) o pueden aparecer en respuesta a la presencia de un proceso patológico, como ocurre con el cáncer y los procesos inflamatorios. Por ello, las glicoproteínas se utilizan ampliamente en el diagnóstico de diversas patologías [1, 2]. Por ejemplo, el antígeno carbohidratado 19-9 (CA 19-9), el antígeno carcio-embrionario humano (CEA) o el antígeno prostático específico (PSA) se emplean en la actualidad como biomarcadores para el diagnóstico del cáncer de páncreas, colon o próstata, respectivamente. Sin embargo, sólo se mide el aumento de sus niveles por encima de unos valores establecidos, con lo que en muchos casos estos biomarcadores no son totalmente específicos ya que otras enfermedades pueden presentar niveles anormalmente elevados de estas glicoproteínas. Por este motivo, el reto analítico actual consiste en mejorar la selectividad y la sensibilidad de estos biomarcadores, llevando a cabo una caracterización exhaustiva de los puntos de glicosilación y de la composición y estructura de los carbohidratos, comúnmente llamados glicanos

Presence of rare potential pathogenic variants in subjects under 65 years old with very severe or fatal COVID-19

Rare variants affecting host defense against pathogens could be involved in COVID-19 severity and may help explain fatal outcomes in young and middle-aged patients. Our aim was to report the presence of rare genetic variants in certain genes, by using whole exome sequencing, in a selected group of COVID-19 patients under 65 years who required intubation or resulting in death (n = 44). To this end, different etiopathogenic mechanisms were explored using gene prioritization-based analysis in which genes involved in immune response, immunodeficiencies or blood coagulation were studied. We detected 44 different variants of interest, in 29 different patients (66%). Some of these variants were previously described as pathogenic and were located in genes mainly involved in immune response. A network analysis, including the 42 genes with candidate variants, showed three main components, consisting of 25 highly interconnected genes related to immune response and two additional networks composed by genes enriched in carbohydrate metabolism and in DNA metabolism and repair processes. In conclusion, we have detected candidate variants that may potentially influence COVID-19 outcome in our cohort of patients. Further studies are needed to confirm the ultimate role of the genetic variants described in the present study on COVID-19 severityTis work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID19 Research Call, COV20/00181) co-fnanced by European Development Regional Fund (FEDER, A way to achieve Europe) and contributions from Estrella de Levante S.A. and Colabora Mujer Association. CIBERer (Centro de Investigación en Red de Enfermedades Raras) is funded by Instituto de Salud Carlos III.R.L-R.and M.dP.V. are sponsored by the project COV20/00181. M.C., P.M. and B.A. are supported by the Miguel Servet (CP17/00006, CP16/00116) and Juan Rodes (JR17/00020) programs, respectively, of the Instituto de Salud Carlos III, co-fnanced by the European Regional Development Fund (FEDER). R.R. is supported by a postdoctoral fellowship of the Comunidad de Madrid (2019-T2/BMD-13714) and G.N.-M. by a contract of the Comunidad de Madrid (PEJ-2020-AI/BMD-18610

Learning activities in higher education : towards autonomous learning of students?

Una de las acciones que caracteriza a los docentes universitarios es el diseño del aprendizaje de los alumnos. Los docentes continuamente estamos tomando decisiones que, de una u otra forma, afectan a lo que los alumnos aprenden y a la forma en que lo hacen. Generalmente, este proceso se desarrolla en contextos de privacidad. Hasta la entrada en vigor del Espacio Europeo de Educación Superior, la visibilidad del diseño del aprendizaje que el profesorado realizaba era limitada. En esta investigación pretendemos analizar cómo diseñan los profesores universitarios el aprendizaje de sus alumnos. Para ello, nos centramos en describir y analizar las actividades de aprendizaje que el profesorado selecciona para orientar los procesos de adquisición de competencias por parte del alumnado. La recogida de datos en nuestro estudio se ha llevado a cabo mediante entrevistas individuales semiestructuradas con 51 docentes universitarios de las diferentes áreas de conocimiento. Se trata, además, de una selección intencionada de la muestra, ya que hemos entrevistado a docentes con una reconocida trayectoria en proyectos y actividades de innovación. La finalidad de la entrevista consistía en que los participantes describieran una secuencia de aprendizaje que habitualmente planifican e implementan para el desarrollo de la materia que imparten. Las entrevistas se grabaron y analizaron mediante un sistema de categorías que ha permitido clasificar las actividades y tareas que el profesorado utiliza para planificar el aprendizaje de sus alumnos. Hemos agrupado las actividades de aprendizaje en las siguientes categorías: asimilativas, gestión de información, aplicación, comunicativas, productivas, experienciales y evaluativas. Los resultados nos muestran que las actividades asimilativas son las más frecuentes y que no existen en general diferencias entre docentes de diferentes ramas de conocimiento en función de las tareas de aprendizaje que planifican. Estos resultados nos indican que es necesario seguir formando a los profesores para que empleen tareas y actividades más relevantes para un aprendizaje autónomo de los alumnos.One of the activities that characterize teaching is the design of student learning. Teachers are continually making decisions that affect one way or another what students learn and the way they do. Usually this process takes place in contexts of privacy. Until the entry into the European Higher Education Area, the visibility of learning design that teachers performed was limited. In this research we analyze the way the university teachers designed the learning of their students. To do this we focus on describing and analyzing the learning activities that teachers selected to guide the processes of acquisition of competence by students. The collection of data in our study was conducted through semi-structured interviews with 51 university teachers from different areas of knowledge. We have interviewed teachers with a proven track record of innovation projects and activities. The purpose of the interview was that the participants describe a sequence of learning that usually planned and implemented for the development of the subject they teach. The interviews were recorded and analyzed using a category system that has allowed classifying the activities and tasks that teachers used to plan student learning. We have grouped the learning activities in the following categories: assimilative, information management, application, communication, production, experiential and evaluative. The results show that the assimilative activities are the most common and generally there are no differences between teachers from different branches of knowledge in terms of learning tasks that plan. These results call attention to the need to continue to train teachers for the use of tasks and learning activities more relevant to students