Mecanismo de formación de cilio primario en células epiteliales polarizadas

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-06-2017El cilio primario es una extensión de membrana altamente conservada que protruye de la superficie apical de la mayoría de las células eucariotas. Su estructura consiste en una membrana ciliar que rodea un armazón de microtúbulos, el axonema, que deriva del centriolo más maduro. Aunque su función ha permanecido desconocida durante mucho tiempo, actualmente se sabe que el cilio primario actúa como un biosensor regulando múltiples rutas de señalización y la homeostasis de los tejidos. Durante los últimos años la relevancia clínica y fisiológica del cilio primario se ha hecho evidente debido a que los defectos en su estructura o su disfunción tienen como consecuencia el desarrollo de un grupo de enfermedades genéticas que se agrupan colectivamente con el nombre de ciliopatías. Entre estas enfermedades destacan la poliquistosis renal, la ceguera, la sordera, la obesidad, y los defectos en el desarrollo embrionario. A pesar de que el riñón es el órgano más frecuentemente afectado por las ciliopatías, la biogénesis del cilio primario se ha estudiado mayoritariamente en tipos celulares no polarizados. Hace casi 50 años se propuso que en los epitelios polarizados, como los de los túbulos renales, el proceso de ciliogénesis tiene lugar exclusivamente en la membrana plasmática, mientras que por el contrario, los fibroblastos ensamblan el cilio de manera intracelular. Utilizado el modelo de células epiteliales polarizadas MDCK, he investigado el desconocido proceso de biogénesis del cilio primario en células epiteliales polarizadas. He observado que el cuerpo medio, una estructura basada en microtúbulos localizada en la parte central del puente intercelular formado entre las dos células hijas durante las etapas finales de la división celular, es heredado por una de las dos células en forma de remanente, el cual se posiciona en la periferia de la membrana apical acumulando maquinaria relevante para la ciliogénesis. Posteriormente, este remanente se mueve sobre la membrana apical reuniéndose con el centrosoma. Una vez que se han juntado estos dos orgánulos, el remanente posibilita la formación del cilio primario. Estos hallazgos revelan un mecanismo biológico que conecta funcionalmente el cuerpo medio con el centrosoma y el cilio primario, los otros dos orgánulos celulares basados en microtúbulos. Además, he investigado el papel de la proteína MAL, un componente de la maquinaria general de transporte apical, en el proceso de ciliogénesis. Los resultados obtenidos indican que MAL es necesaria para la correcta condensación de las membranas en la base del cilio, lo que a su vez, es crucial para la elongación eficiente del citado orgánulo. En resumen, este trabajo establece una nueva ruta de ciliogénesis primaria en células epiteliales renales y confiere importancia a las membranas situadas en la base del cilio primario.The primary cilium is a highly conserved membrane extension protruding from the cell surface of most mammalian cells. It consists of a ciliary membrane that surrounds a microtubule-based structure termed the axoneme, which is nucleated from the older of the two centrioles. Although its function has been an enigma for a long time, nowadays it is known to act as a biosensor regulating multiple signaling pathways during development and tissue homeostasis. The physiological and clinical relevance of cilia is evident, since defects in primary cilium function cause a wide spectrum of genetic diseases collectively grouped under the term of ciliopathies. Among the disorders produced by primary cilium dysfunction are cystic kidney disease, blindness, deafness, obesity, and developmental and skeletal abnormalities. The kidney is the organ most frequently affected in ciliopathies. However, despite its importance in the kidney, primary cilium biogenesis has mainly been studied in non-polarized cells. Almost 50 years ago, it was proposed that the process of primary ciliogenesis in polarized epithelia, such as that in kidney tubules, takes place entirely at the plasma membrane, in contrast to fibroblasts that assemble the cilium intracellularly. Using the renal epithelial MDCK cell line, I have investigated the unexplored process of primary cilium biogenesis in polarized epithelial cells. I observed that the midbody, which is a microtubule-based structure that occupies the central part of the intercellular bridge connecting the two sister cells during the final stages of cell division, is inherited by one of the cells as a remnant that localizes at the periphery of the apical membrane, and that accumulates important machinery for cilium biogenesis. The remnant then moves along the apical plasma membrane to a central position to encounter the centrosome. Once the two organelles have met, the remnant enables the centrosome for primary cilium formation. These findings reveal a biological mechanism that functionally links the midbody with the centrosome and the primary cilium, which are the other two main microtubule-based organelles. I have also investigated the role of MAL, a component of the machinery of apical transport, in primary cilium assembly. The results indicate that MAL is required for correct membrane condensation at the ciliary base, which, in turn, is essential for efficient primary cilium elongation. In summary, the work presented establishes a novel pathway of primary ciliogenesis in renal polarized epithelial cells and establishes the importance of the condensation of the membranes at the ciliary base

Análisis de la batería de inicio a la lectura (BIL 3-6) para la predicción de las dificultades del aprendizaje del lenguaje escrito

Esta es la primera parte de un estudio cuyo objetivo es mostrar si la Batería de Inicio a la lectura (BIL 3-6) de Sellés, Martínez, Vidal-Abarca y Gilabert (2008) puede ser considerada como una batería predictiva en lo que se refiere a las dificultades de la adquisición de la lectura. El objetivo presente es conocer la relación de las distintas variables que componen la batería, de manera que se conozca su idoneidad para el fin que se propone. Método: la batería fue aplicada a 87 niños de 2º de Educación Infantil. Resultados: las pruebas que miden los factores Conocimiento Fonológico y Habilidades Lingüísticas correlacionan de forma parcial. Las pruebas que evalúan el Conocimiento Metalingüístico y las pruebas que miden Procesos Cognitivos no presentan correlación que determine la idoneidad de la puntuación global de los factores. La relación entre las pruebas de los factores Conocimiento Fonológico y Habilidades Lingüísticas y Conocimiento Fonológico y Conocimiento Alfabético no presentan correlación de manera absoluta. La puntuación de las profesoras es útil para valorar el aprendizaje de la lectura. Pasados dos años se confirmará si los resultados obtenidos en la batería pueden llegar a predecir las dificultades de aprendizaje de la lectura.This is the first part of a research which purpose is to demonstrate if la Batería de Inicio a la lectura (BIL 3-6) by Sellés, Martínez, Vidal-Abarca y Gilabert (2008) can be considered a predictive test of reading difficulties. The main aim of this study is to evaluate the relationship between the test’s variables to know if it’s suitable for the intended purpose. Method: The test battery was applied to 87 children, students of 2nd Early Childhood Education. Results: The tests that evaluate the factors phonological knowledge and linguistic abilities correlate partially. The tests that evaluate metalinguistic knowledge and the tests that evaluate cognitive processes do not show a correlation that could determine the suitability of the global score of the factors. The tests of the factors phonological 8 knowledge and linguistic abilities and phonological knowledge and literacy skills do not correlate in an absolute way. It was found that the teacher’s evaluation is useful to assess the process of learning to read. After 2 years, it will be confirmed if the obtained result could predict difficulties learning to read

MALL, a membrane-tetra-spanning proteolipid overexpressed in cancer, is present in membraneless nuclear biomolecular condensates

Proteolipids are proteins with unusual lipid-like properties. It has long been established that PLP and plasmolipin, which are two unrelated membrane-tetra-spanning myelin proteolipids, can be converted in vitro into a water-soluble form with a distinct conformation, raising the question of whether these, or other similar proteolipids, can adopt two different conformations in the cell to adapt their structure to distinct environments. Here, we show that MALL, another proteolipid with a membrane-tetra-spanning structure, distributes in membranes outside the nucleus and, within the nucleus, in membrane-less, liquid-like PML body biomolecular condensates. Detection of MALL in one or other environment was strictly dependent on the method of cell fixation used, suggesting that MALL adopts different conformations depending on its physical environment —lipidic or aqueous— in the cell. The acquisition of the condensate-compatible conformation requires PML expression. Excess MALL perturbed the distribution of the inner nuclear membrane proteins emerin and LAP2β, and that of the DNA-binding protein BAF, leading to the formation of aberrant nuclei. This effect, which is consistent with studies identifying overexpressed MALL as an unfavorable prognostic factor in cancer, could contribute to cell malignancy. Our study establishes a link between proteolipids, membranes and biomolecular condensates, with potential biomedical implication

Overall Survival and Biomarker Analysis of Neoadjuvant Nivolumab Plus Chemotherapy in Operable Stage IIIA Non–Small-Cell Lung Cancer (NADIM phase II trial)

PURPOSE Neoadjuvant chemotherapy plus nivolumab has been shown to be effective in resectable non–small cell lung cancer (NSCLC) in the NADIM trial (ClinicalTrials.gov identifier: NCT03081689). The 3-year overall survival (OS) and circulating tumor DNA (ctDNA) analysis have not been reported. METHODS This was an open-label, multicenter, single-arm, phase II trial in which patients with stage IIIA NSCLC, who were deemed to be surgically resectable, were treated with neoadjuvant paclitaxel (200 mg/m2 once a day) and carboplatin (area under curve 6) plus nivolumab (360 mg) once on day 1 of each 21-day cycle, for three cycles, followed by adjuvant nivolumab monotherapy for 1 year (240 mg once every 2 weeks for 4 months, followed by 480 mg once every 4 weeks for 8 months). The 3-year OS and ctDNA analysis were secondary objectives of the trial. RESULTS OS at 36 months was 81.9% (95% CI, 66.8 to 90.6) in the intention-to-treat population, rising to 91.0% (95% CI, 74.2 to 97.0) in the per-protocol population. Neither tumor mutation burden nor programmed cell death ligand-1 staining was predictive of survival. Conversely, low pretreatment levels of ctDNA were significantly associated with improved progression-free survival and OS (hazard ratio [HR], 0.20; 95% CI, 0.06 to 0.63, and HR, 0.07; 95% CI, 0.01 to 0.39, respectively). Clinical responses according to RECIST v1.1 criteria did not predict survival outcomes. However, undetectable ctDNA levels after neoadjuvant treatment were significantly associated with progression-free survival and OS (HR, 0.26; 95% CI, 0.07 to 0.93, and HR, 0.04; 95% CI, 0.00 to 0.55, respectively). The C-index to predict OS for ctDNA levels after neoadjuvant treatment (0.82) was superior to that of RECIST criteria (0.72). CONCLUSION The efficacy of neoadjuvant chemotherapy plus nivolumab in resectable NSCLC is supported by 3- year OS. ctDNA levels were significantly associated with OS and outperformed radiologic assessments in the prediction of survival

PD-(L)1 Inhibitors as Monotherapy for the First-Line Treatment of Non-Small-Cell Lung Cancer Patients with High PD-L1 Expression: A Network Meta-Analysis

Programmed cell death-ligand 1 (PD-L1) has emerged as a potential biomarker for selection of patients more likely to respond to immunotherapy and as a prognostic factor in non-small cell lung cancer (NSCLC). In this network meta-analysis, we aimed to evaluate the efficacy of first-line anti-PD-(L)1 monotherapy in advanced NSCLC patients with high PD-L1 expression (≥50%) compared to platinum-based chemotherapy. We also evaluated efficacy outcomes according to tumor mutational burden (TMB). To that end, we conducted a systematic review. Six clinical trials with 2111 patients were included. In head-to-head comparisons, immunotherapy showed a significant improvement in progression-free survival (PFS: HRpooled = 0.69, 95% CI: 0.52–0.90, p = 0.007), overall survival (OS: HRpooled = 0.69, 95% CI: 0.61–0.78; p < 0.001) and overall response rate (ORR) (Risk ratio (RR)pooled = 1.354, 95% CI: 1.04–1.762, p = 0.024). In the assessment of relative efficacy for PFS through indirect comparisons, pembrolizumab (results from KEYNOTE-024) ranked highest followed by cemiplimab and atezolizumab, with statistical significance determined for some of the drugs. In terms of OS, cemiplimab ranked highest followed by atezolizumab and pembrolizumab, although non-significant OS was determined for these drugs. In conclusion, PD-(L)1 inhibitor monotherapy improves efficacy outcomes in the first line setting of advanced NSCLC patients with high PD-L1 expression. Evaluations with longer follow up are still needed to determine the superiority of any specific drug

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation

The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extensionThis work was supported by the Ministerio de Economıa y Competitividad, Spain [grant numbers BFU2012-32532 and CONSOLIDER COAT CSD2009-00016 to M. A.A.]. G.A. was supported by the Amarouto Program for senior researchers from the Comunidad Autónoma de Madri

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation

The base of the primary cilium contains a zone of condensedmembranes whose importance is not known. Here, we have studiedthe involvement of MAL, a tetraspanning protein that exclusivelypartitions into condensed membrane fractions, in the condensation ofmembranes at the ciliary base and investigated the importance ofthese membranes in primary cilium formation. We show that MALaccumulates at the ciliary base of epithelial MDCK cells. Knockdownof MAL expression resulted in a drastic reduction in the condensationof membranes at the ciliary base, the percentage of ciliated cells andthe length of the cilia, but did not affect the docking of the centrosometo the plasma membrane or produce missorting of proteins to thepericentriolar zone or to the membrane of the remaining cilia. Rab8(for which there are two isoforms, Rab8A and Rab8b), IFT88 andIFT20, which are important components of the machinery of ciliarygrowth, were recruited normally to the ciliary base of MAL-knockdowncells but were unable to elongate the primary cilium correctly. MAL,therefore, is crucial for the proper condensation of membranes at theciliary base, which is required for efficient primary cilium extension

Monitoreo remoto de los parámetros operacionales para el proceso de producción de miel de caña

A local data acquisition, processing and storage system were implemented by means of an open source micro-controlled development card. To continuously measure the variables of the cane honey production process, it was performed using five temperature sensors, pH sensor and a level sensor. Each sensor was associated with a component set with its respective upper and lower reference values, during the processing of a batch of product. The main objective was to determine the effectiveness of using sensors and wireless communication technology to monitor operational parameters, in real time. In general, the recorded temperature, pH and level data obtained from sensors corresponded closely to the changes that occurred in the process, and the wireless communication nodes developed successfully measured and monitored the temperature, pH and level readings in real time. The study also found that the temperature, pH and level readings obtained by the sensors began to standardize more closely within the upper and lower limits. Furthermore, operational parameters could be reasonably predicted by applying a statistical model to measure temperature, pH and level. The present study also found that the operational parameters analyzed showed variability with its consequent effect on the quality of the final product. The findings of this study should serve as the first step towards any future research and development that may take place in the field of agro-industrial process designSe implementó un sistema local de adquisición, procesamiento y almacenamiento de datos mediante una tarjeta microcontrolada de código abierto. Para registrar continuamente las variables del proceso de producción de la miel de caña se utilizó cinco sensores de temperatura, un sensor de pH y un sensor de nivel. Cada sensor se asoció con un conjunto de componentes con sus respectivos valores de referencia superior e inferior durante el procesamiento de un lote de producto. El objetivo principal fue determinar la efectividad del uso de sensores y tecnologías de comunicación inalámbrica para monitorear los parámetros operacionales en tiempo real. En general, los datos de temperatura, pH y nivel registrados por los sensores correspondían estrechamente a los cambios que ocurrieron en el proceso, y los nodos de comunicación inalámbrica desarrollados midieron y monitorearon con éxito las lecturas de temperatura, pH y nivel en tiempo real. El estudio también encontró que las lecturas de temperatura, pH y nivel obtenidas por los sensores comenzaron a estandarizarse dentro de los límites superior e inferior. Además, los parámetros operacionales podrían predecirse razonablemente aplicando un modelo estadístico para medir la temperatura, el pH y el nivel. Los parámetros operacionales analizados mostraron variabilidad con su consecuente efecto sobre la calidad del producto final. Los hallazgos de este estudio deberían servir como el primer paso para el desarrollo futuro en el campo del diseño de procesos agroindustriale

SIMULADOR ESTADISTICO DE CONTAGIOS PARA COVID-19 USANDO 13 VARIABLES DEL SISTEMA DE SALUD (STATISTICAL SIMULATOR OF CONTAGION FOR COVID-19 USING 13 VARIABLES OF THE HEALTH SYSTEM)

Resumen La probabilidad de contagio del COVID-19, ocasionada por el SARS-CoV2 de-pende de varios factores. Por lo cual, organismos internacionales han sugerido distintas medidas de prevención de contagio como son mantener una distancia de al menos dos metros, frecuente lavado de manos y el uso de mascarilla. Por otra parte, a pesar de las medidas establecidas, el COVID-19 puede ser más agresivo con personas de edad avanzada, con padecimientos previos de enfermedades como la diabetes, hipertensión u obesidad. El objetivo de este trabajo es presentar un simulador de contagios para COVID-19 (SM-COVID19) desarrollado en Visual Basic .NET mediante escenarios probabilísticos utilizando datos abiertos de distintos organismos de México para determinar las probabilidades de contagio y del crecimiento de la curva de infectados que son reportados cada día. A partir de estos datos, usando 13 variables del sistema de salud. La propuesta de simulación se enfoca en calcular las probabilidades de contagio en tres distintos escenarios: mejor de los casos, caso promedio y peor de los casos. Estas probabilidades se ajustan a los factores de riesgo que son señalados por la Secretaría de Salud. Con los resultados alcanzados, ha sido posible expresar el crecimiento de la curva de los contagios identificados como positivos al COVID-19 y enfatizar las medias de prevención en la sociedad. Palabra(s) Clave: Contagios, SARS-CoV2, Simulador estadístico. Abstract The probability of contagion of COVID-19, occasioned by SARS-CoV2 depends on several factors. Therefore, international organisms have suggested different measures of prevention of contagion such as to maintain a distance of at least two meters, frequent hand washing, and the use of mask. On the other hand, despite of the established measures, COVID-19 can be more aggressive with elderly people, with previous conditions of diseases like diabetes, hypertension or obesity. The objective of this work is to present a contagion simulator for COVID-19 developed in Visual Basic .NET through probabilistic scenarios using open data of distinct organisms of Mexico to determine the probabilities of contagions and growth of the curve of infected people which are reported every day. From these data, the proposal of simulation focuses on calculating the probabilities of contagions in three different scenarios: best of the cases, average case and worst of the cases. These probabilities adjust to the risk factors that are indicated by the Health Secretary. With the reached results, it has been possible to express the growth of the curve of the identified contagions as positives to COVID-19 and to emphasize the prevention measures in society. Keywords: Contagions, SARS-CoV2, Simulator

Pd-(L)1 inhibitors as monotherapy for the first-line treatment of non-small-cell lung cancer patients with high pd-l1 expression : A network meta-analysis

Altres ajuts: RocheProgrammed cell death-ligand 1 (PD-L1) has emerged as a potential biomarker for selec-tion of patients more likely to respond to immunotherapy and as a prognostic factor in non-small cell lung cancer (NSCLC). In this network meta-analysis, we aimed to evaluate the efficacy of first-line anti-PD-(L)1 monotherapy in advanced NSCLC patients with high PD-L1 expression (≥50%) compared to platinum-based chemotherapy. We also evaluated efficacy outcomes according to tumor mutational burden (TMB). To that end, we conducted a systematic review. Six clinical trials with 2111 patients were included. In head-to-head comparisons, immunotherapy showed a significant improvement in progression-free survival (PFS: HRpooled = 0.69, 95% CI: 0.52-0.90, p = 0.007), overall survival (OS: HRpooled = 0.69, 95% CI: 0.61-0.78; p < 0.001) and overall response rate (ORR) (Risk ratio (RR)pooled = 1.354, 95% CI: 1.04-1.762, p = 0.024). In the assessment of relative efficacy for PFS through indirect comparisons, pembrolizumab (results from KEYNOTE-024) ranked highest followed by cemiplimab and atezolizumab, with statistical significance determined for some of the drugs. In terms of OS, cemiplimab ranked highest followed by atezolizumab and pembrolizumab, although non-significant OS was determined for these drugs. In conclusion, PD-(L)1 inhibitor mon-otherapy improves efficacy outcomes in the first line setting of advanced NSCLC patients with high PD-L1 expression. Evaluations with longer follow up are still needed to determine the superiority of any specific drug