Expansion, improvement and application of metabolomic platforms for the determination of vitamin D and its metabolites

Introducción: La principal motivación que dio lugar a esta Memoria de Tesis fue optimizar, validar y aplicar un método para la determinación de vitamina D y sus metabolitos en sangre (suero y plasma). Los analitos a estudiar abarcaron la propia vitamina D (en las formas D2 y D3) y sus principales metabolitos mono y dihidroxilados. Se pretendió soportar el método en: (i) la completa automatización del proceso analítico (para un funcionamiento ininterrumpido y sin atención del operador durante 24 h/día, evitando o minimizando así la sobrecarga de muestras en el laboratorio), además de una adecuada validación. (ii) La multideterminación, que abarcara tantos analitos como fuera posible para obtener información cuantitativa del metabolismo de la vitamina D. (iii) El estudio en profundidad de la información básica sobre las primeras etapas del método para la cuantificación de la vitamina D y sus metabolitos y su impacto en las propiedades analíticas del mismo: la selección del tipo de muestra derivada de la sangre total (suero o plasma) y del contenedor de muestra y, especialmente, el procedimiento óptimo de preparación de la muestra. (iv) La suficiente información sobre la estabilidad de la muestra durante un periodo establecido de dos meses para evaluar el efecto de la temperatura de almacenamiento en las propiedades analíticas del método en función del/los analito/s a determinar. (v) La aplicación masiva a muestras procedentes de diferentes centros de investigación y de pacientes con diferentes enfermedades, para poner así de manifiesto las fortalezas y debilidades del método y ayudar a conocer los cambios que se producen en los metabolitos a causa de enfermedades concretas. Contenido de la investigación: Durante el desarrollo de esta Tesis Doctoral se consiguieron los siguientes objetivos: (i) Automatizar y validar un método basado en el acoplamiento en línea de una estación de extracción en fase sólida (SPE) a un cromatógrafo de líquidos conectado a un espectrómetro de masas en tándem (LC–MS/MS) para la determinación en suero de vitamina D (en las formas D2 y D3) y sus principales metabolitos monohidroxilados (25-hidroxivitamina D2 [25(OH)D2] y 25-hidroxivitamina D3 [25(OH)D3]) y dihidroxilados (1,25-dihidroxivitamina D2 [1,25(OH)2D2], 1,25-dihidroxivitamina D3 [1,25(OH)2D3] y 24,25-dihidroxivitamina D3 [24,25(OH)2D3]) constituyó un objetivo específico clave. El uso de estándares internos marcados isotópicamente (SIL-ISs) y la validación externa de acuerdo con el programa de validación “vitamin D External Quality Assurance Scheme (DEQAS)” contribuyeron a dar un soporte sólido al método. (ii) Ampliar el número de metabolitos determinados fue el objetivo específico del que resultó la discriminación de los epímeros 25(OH)D3, además de mantener el resto de los hidroximetabolitos. La cromatografía de líquidos bidimensional (LC×LC) con una etapa previa de SPE y la subsiguiente detección mediante MS/MS hicieron posible la consecución de este objetivo: un método ampliado y validado mediante aplicación a muestras del programa DEQAS y a un material de referencia certificado (CRM) del NIST. (iii) Conocer la mejor muestra derivada de sangre total (suero o plasma) y el mejor procedimiento de preparación de la muestra fue el tercer objetivo específico. Se utilizaron diferentes tubos de recogida, con y sin recubrimiento interno por gel, para obtener el suero y el plasma. Por otra parte, la información necesaria sobre la etapa de preparación de la muestra se obtuvo comparando cómo afecta a cada uno de los analitos la desproteinización o la SPE previas al análisis mediante LC–MS/MS. (iv) Estudiar la estabilidad de los analitos de las muestras de suero sometidas a diferentes condiciones de conservación durante dos meses fue otro de los objetivos específicos. El estudio estadístico de los datos obtenidos en el seguimiento mediante LC–MS/MS permitió conocer la magnitud de los cambios experimentados por cada uno de los compuestos en estudio, con especial énfasis en los metabolitos. Un logro interesante de este estudio fue la comprobación de que la estabilidad de los analitos se mantiene de forma general cuando las muestras se liofilizan. (v) Validar el excelente comportamiento del método fue el último de los objetivos específicos de la Tesis, al cual el estudiante le dedicó un largo periodo de tiempo. Algunos de los ejemplos de aplicación del método por el doctorando son los siguientes: análisis de 546 muestras de suero de una cohorte de mujeres con cáncer de mama y 558 mujeres control (del proyecto MCC del Instituto de Salud Carlos III, Madrid); 1472 muestras de suero de una cohorte de mujeres pre- y postmenopáusicas (proyecto del mismo instituto); 314 muestras de suero de pacientes con esclerosis múltiple (proyecto sobre vitamina D y esclerosis múltiple en pacientes con síndromes clínicamente aislados, del Hospital San Rafaelle de Milán, Italia). Los datos analíticos han sido y están siendo en la actualidad usados con éxito por los participantes en los proyectos para la obtención de resultados que soporten o destruyan las hipótesis propuestas. Algunos de los datos obtenidos en los análisis realizados se han tratado desde el punto de vista analítico–quimiométrico. 3. Conclusión: Se han desarrollado dos métodos automatizados para el análisis cuantitativo de la vitamina D y sus principales metabolitos proporcionando una rápida separación cromatográfica de las vitaminas D3 y D2 y los metabolitos 1,25(OH)2D3, 24,25(OH)2D3, 1,25(OH)2D2, 25(OH)D3, 25(OH)D2 y 3-epi-C3-25(OH)D3 en muestras de suero (a nivel de nmol/L o pmol/L). Ambas plataformas se han caracterizado y validado mediante programas externos utilizando muestras proporcionadas por DEQAS, para la determinación del total de 25(OH)D y 1,25(OH)2D, y un material de referencia certificado por el NIST para validar la determinación individual de los epímeros del 25(OH)D3. Se han establecido unas recomendaciones en lo que se refiere a la conservación de la muestra (referidas tanto al tiempo de almacenamiento como a los ciclos de congelación/descongelación) deben mantenerse para la medición de todos los metabolitos de la vitamina D3 en estudios clínicos, donde la sensibilidad es un parámetro crítico. Por otro lado, la liofilización y posterior almacenamiento a temperatura ambiente son una estrategia fácil, rápida y robusta para conservar las muestras que se utilizarán en los estudios clínicos. Finalmente, tres centros de investigación nacionales e internacionales han solicitado el análisis de la vitamina D y sus metabolitos en 2890 muestras de suero para determinar el metabolismo de la vitamina D en diferentes estudios clínicos realizados a lo largo del desarrollo de la Tesis

Use of semantic technologies in the development of an ontology for the management of urban projects. the case of the Seville city council

The implementation and start-up of economic activities that generate wealth and employment in any municipality require obtaining municipal licenses and authorizations for which the drafting of a technical activity project is mandatory. All the interested parties benefit from the regulatory compliance with the urban and environmental legal requirements established by the municipalities for this type of projects. However, the numerous non-compliances detected in the "a posteriori" inspections carried out by the municipal technicians are causing that in many Spanish municipalities a high percentage of these projects are not viable and are paralyzed due to non-compliances in aspects such as accessibility for people with disabilities, incorrect location of the machines of the different facilities, smoke and gas outlets, lack of acoustic insulation, etc. The consequence of this situation is the enormous delay in the opening of the establishments, or even that some of the promoters of these projects desist from undertaking the activity. In response to this serious problem, a Knowledge-Based System (KBS) is proposed that would significantly improve the legalization system of economic activity projects processed by a responsible declaration in Spanish municipalities

Improving municipal licenses management forced by the services directive : a case study in the Council of Seville, Spain

Introducción. Se presentan los resultados de una investigación, emprendida para mejorar la eficiencia del sistema de tramitación y autorización administrativa de licencias de actividades en los ayuntamientos españoles, motivada por los cambios en la legislación española debido a la Directiva de Servicios. Material y Métodos. Tras comparar el cumplimiento de la Directiva en una muestra de los diez ayuntamientos con más población de Andalucía, se emplea como caso de estudio el ayuntamiento de Sevilla, analizando sus procedimientos y flujos de información e identificando problemas y carencias. Resultados. Tal y como exige la Directiva, se proponen nuevos procedimientos de administración electrónica, mejoras e innovaciones organizacionales en el sistema de tramitación municipal actual, incluyendo un prototipo de sistema basado en el conocimiento para apoyar las consultas técnicas previas para la implantación de actividades. Discusión. Los resultados son útiles para más de ocho mil ayuntamientos españoles.Introduction. We present the results of an investigation undertaken to improve the efficiency in the administrative permits management system for new economical activities in the Spanish municipalities, caused by the changes in the Spanish law emerged after the Services Directive Material and methods After comparing the Directive fulfilment in a sample of the ten most populated municipalities of Andalusia, the council of Seville is used as study case, analysing its procedures and information flows, and identifying problems and deficiencies. Results Such as is required by the Directive, new e-government procedures, organizational improvements and innovations in the current municipal management system are proposed, including a knowledge based system prototype for supporting technical consultations prior to the implementation of activities. Discussion. The results are useful for more than eight thousand Spanish municipalitie

Neumonía por coinfección de Aspergillus fumigatus y Pneumocystis jirovecii en un paciente no VIH: Reporte de caso

La aspergilosis pulmonar invasiva es una enfermedad presente principalmente en pacientes inmunocomprometidos con alta carga de mortalidad. La neumonía por Pneumocystis jirovecii es una infección oportunista potencialmente mortal que afecta a pacientes inmunocomprometidos por diversas etiologías. La coinfección por estos patógenos en pacientes inmunocompetentes es inusual. Reportamos un caso de un paciente sin las causas tradicionales de inmunocompromiso  en el desarrollo de una  neumonía en  coinfección por Aspergillus fumigatus y Pneumocystis jirovecii.

Serum 25-hydroxyvitamin D and breast cancer risk by pathological subtype (MCC-Spain)

Epidemiologic evidence on the association between vitamin D and breast cancer is still inconclusive. This study analyzes the association between serum 25-hydroxyvitamin D (25(OH)D) and breast cancer risk by pathologic subtype, stage at diagnosis and specific breast cancer risk factors. We conducted a population-based multicase-control study where 546 histologically-confirmed breast cancer cases and 558 population controls, frequently matched by geographic area, age and body mass index, were recruited in 12 Spanish provinces (MCC-Spain). Information was collected by a questionnaire and plasma 25(OH)D was measured by solid-phase extraction on-line coupled to liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS). Odds ratios and 95% confidence intervals were calculated using logistic and multinomial mixed regression models. We found a clear protective effect between 25(OH)D levels and breast cancer risk, with a significant dose-response trend (OR per 10?nmol/L?=?0.88; 95%CI?=?0.82-0.94). While no differences were observed between pre and postmenopausal women, stage at diagnosis, or across strata of the main breast cancer risk factors, the protection was more pronounced for triple negative tumors (OR per 10?nmol/L?=?0.64; p-heterogeneity?=?0.038). Similar results were observed when only cases sampled in the first month after diagnosis were considered. The protective effect of vitamin D on breast cancer risk may be subtype specific, being stronger for more aggressive tumors, which provides a new approach to prevent this disease.The study was funded by Carlos III Institute of Health grants (PI12/00488, PI12/00265, PI12/00715, PI12/01270, PI09/00773 and PI08/1770), by the Spanish Ministry of Health (EC11-273), by the Spanish Ministry of Economy and Competitiveness (IJCI-2014-20900) and by Consejería de Salud de la Junta de Andalucía (PI-0571-2009) competitive calls including peer review for scientific quality. Additional funding was provided by the Spanish Federation of Breast Cancer Patients (FECMA: EPY 1169-10), the Association of Women with Breast Cancer from Elche (AMACMEC: EPY 1394/15), the Marqués de Valdecilla foundation (grant API 10/09), and by Acción Transversal del Cancer, approved by the Spanish Ministry Council on October 11, 2007. None of the funders played any role in conducting research or writing the paper. This article presents independent research. The views expressed are those of the authors and not necessarily those of the Carlos III Institute of Health

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Advances in Computational Social Science and Social Simulation

Aquesta conferència és la celebració conjunta de la "10th Artificial Economics Conference AE", la "10th Conference of the European Social Simulation Association ESSA" i la "1st Simulating the Past to Understand Human History SPUHH".Conferència organitzada pel Laboratory for Socio­-Historical Dynamics Simulation (LSDS-­UAB) de la Universitat Autònoma de Barcelona.Readers will find results of recent research on computational social science and social simulation economics, management, sociology,and history written by leading experts in the field. SOCIAL SIMULATION (former ESSA) conferences constitute annual events which serve as an international platform for the exchange of ideas and discussion of cutting edge research in the field of social simulations, both from the theoretical as well as applied perspective, and the 2014 edition benefits from the cross-fertilization of three different research communities into one single event. The volume consists of 122 articles, corresponding to most of the contributions to the conferences, in three different formats: short abstracts (presentation of work-in-progress research), posters (presentation of models and results), and full papers (presentation of social simulation research including results and discussion). The compilation is completed with indexing lists to help finding articles by title, author and thematic content. We are convinced that this book will serve interested readers as a useful compendium which presents in a nutshell the most recent advances at the frontiers of computational social sciences and social simulation researc

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030