Papel del receptor de dioxina en la regulación de la adhesión y migración celular en fibroblastos: interacciones con Integrina β1 y Caveolina 1

Este trabajo ha sido financiado con proyectos del Ministerio de Economía y Competitividad (SAF2008-00462 y BFU2011-22678), de la Red Temática de Investigación Cooperativa en Cáncer RTICC (RD06/0020/1016) y del Plan de Ayuda a grupos de la Junta de Extremadura (GR10008). Toda la financiación recibida está y co-financiada por el programa FEDER de la Unión Europea. Javier Rey Barroso ha sido beneficiario de mediante una beca de Formación del Profesorado Universitario (FPU, convocatoria de 2008) del Ministerio de Educación y Ciencia, y de una bolsa de viaje para estancias cortas de la RTICC (Convocatoria de 2009).El receptor de dioxina (AhR) es un factor de transcripción con gran peso tanto en toxicología ambiental como en fisiología. Por otra parte, los procesos de adhesión celular y migración han mostrado su importancia tanto en homeostasis como en patología. Este trabajo se centra en estudiar el papel del receptor de dioxina en ambas funciones celulares utilizando fibroblastos de ratón. Éste se ejerce a través del control de la activación de la integrina β1 y la localización y funcionalidad de caveolina 1. En el primer caso, la regulación se produce a través de la combinación del control de la expresión de fibronectina y de la ruta de señalización Cbp-Csk-Src.y tiene consecuencias en la forma celular, la velocidad de migración y la capacidad de contracción e invasión de matriz celular. Por otra parte, la regulación de caveolina 1 por parte de AhR implica cambios en la localización celular de la proteína, así como la sublocalización en dominios específicos de membrana (rafts) y está implicada en la capacidad de reorientación de la célula así como en la de endocitosis vía caveolas. En este caso, el control no se debe a cambios en el estado de fosforilación de caveolina 1 en su residuo Y14 pero sí a cambios en los niveles de colesterol intracelulares, regidos por el receptor de dioxina. Globalmente los resultados ayudan a comprender la importancia de AhR en la fisiología celular, así como las rutas por las cuales ejerce sus funciones.The dioxin receptor (AhR) is a transcription factor with an important role in enviromental toxicology and physiology. Apart from that, cell adhesion and migration processes have shown to be crucial both in homeostasis and pathology. The present work focuses in the study of the role of the dioxin receptor in both cellular functions by using mouse fibroblast. This role is done through the control of integrin β1 activation and the location and functionality of caveolin 1. In the first case, the regulation is driven by the combination of fibronectin expression control and the Cbp-Csk-Src pathway and has consequences in cellular shape, migration speed and contraction and matrix invasion capabilities. On the other hand, the caveolina 1 regulation by AhR involves changes in cellular location of the protein, as well as the sublocation in specific membrane domains (rafts) and is involved in cell re-orientation capability and endocytosis via caveolae. In this case, the control is not caused by phosphorylation state changes in Y14 residue of caveolina 1, but it does it in the case of intracellular cholesterol changes, driven by the dioxin receptor. Globaly, th results help us to understand the importance of AhR in cellular physiology as well as the pathways by AhR execute its functions

Morphological study of skin cancer lesions through a 3D scanner based on fringe projection and machine learning

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Spectroscopic evaluation of red blood cells of thalassemia patients with confocal microscopy: a pilot study

Hemoglobinopathies represent the most common single-gene defects in the world and pose a major public health problem, particularly in tropical countries, where they occur with high frequency. Diagnosing hemoglobinopathies can sometimes be difficult due to the coexistence of different causes of anemia, such as thalassemia and iron deficiency, and blood transfusions, among other factors, and requires expensive and complex molecular tests. This work explores the possibility of using spectral confocal microscopy as a diagnostic tool for thalassemia in pediatric patients, a disease caused by mutations in the globin genes that result in changes of the globin chains that form hemoglobin—in pediatric patients. Red blood cells (RBCs) from patients with different syndromes of alpha-thalassemia and iron deficiency (including anemia) as well as healthy (control) subjects were analyzed under a Leica TCS SP8 confocal microscope following different image acquisition protocols. We found that diseased RBCs exhibited autofluorescence when excited at 405 nm and their emission was collected in the spectral range from 425 nm to 790 nm. Three experimental descriptors calculated from the mean emission intensities at 502 nm, 579 nm, 628 nm, and 649 nm allowed us to discriminate between diseased and healthy cells. According to the results obtained, spectral confocal microscopy could serve as a tool in the diagnosis of thalassemia.This research was funded by Spanish Ministry of Economy and Competitiveness, grant number DPI2017-89414-R. L.R.-B. thanks the Ministry of Science, Innovation and Universities for the PhD (FPI) grant she has received.Peer ReviewedPostprint (published version

Visible and extended near-infrared multispectral imaging for skin cancer diagnosis

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Hyperspectral imaging for skin cancer and blood disorders diagnosis

Hyperspectral imaging is a novel technology for acquiring an image at a large number of wavelengths, thus allowing the study of spectral and spatial details of a sample under analysis. This technology has emerged as a promising imaging modality to be used as a diagnostic tool in several medical applications where spectral information is relevant. In this paper, we outline our most recent achievements in this field. Firstly, hyperspectral imaging systems developed to improve non-invasive diagnosis of skin cancer, consisting of digital silicon and InGaAs cameras and light emitting diodes, are described. Secondly, we present our last advances using hyperspectral technology together with confocal microscopy to improve the diagnosis of blood diseases, in particular, hemoglobinopathies such as thalassemia and cell membrane diseases such as hereditary spherocytosis. Finally, new insights on these topics are discussed.This project has been funded by the Agencia Estatal de Investigación (AEI) (PID2020-112527RB-I00 / AEI / 10.13039/501100011033). L R-B thanks the Ministry of Science, Innovation and Universities for the PhD (FPI) grant she has received.Peer ReviewedPostprint (author's final draft

Membrane protein detection and morphological analysis of red blood cells in hereditary spherocytosis by confocal laser scanning microscopy

In hereditary spherocytosis (HS), genetic mutations in the cell membrane and cytoskeleton proteins cause structural defects in red blood cells (RBCs). As a result, cells are rigid and misshapen, usually with a characteristic spherical form (spherocytes), too stiff to circulate through microcirculation regions, so they are prone to undergo hemolysis and phagocytosis by splenic macrophages. Mild to severe anemia arises in HS, and other derived symptoms like splenomegaly, jaundice, and cholelithiasis. Although abnormally shaped RBCs can be identified under conventional light microscopy, HS diagnosis relies on several clinical factors and sometimes on the results of complex molecular testing. It is specially challenging when other causes of anemia coexist or after recent blood transfusions. We propose two different approaches to characterize RBCs in HS: (i) an immunofluorescence assay targeting protein band 3, which is affected in most HS cases and (ii) a three-dimensional morphology assay, with living cells, staining the membrane with fluorescent dyes. Confocal laser scanning microscopy (CLSM) was used to carry out both assays, and in order to complement the latter, a software was developed for the automated detection of spherocytes in blood samples. CLSM allowed the precise and unambiguous assessment of cell shape and protein expression.This publication is part of the project PID2020-112527RBI00, funded by CIN/AEI/10.13039/501100011033. Laura Rey-Barroso thanks the Ministry of Science, Innovation and Universities for the PhD (FPI) grant she has received (DPI2017-89414-R). The current study has been funded by the Spanish National Agency of Investigation (AEI).Peer ReviewedPostprint (published version

The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol

Background: Adhesion and migration are relevant physiological functions that must be regulated by the cell under both normal and pathological conditions. The dioxin receptor (AhR) has emerged as a transcription factor regulating both processes in mesenchymal, epithelial and endothelial cells. Indirect results suggest that AhR could cooperate not only with additional transcription factors but also with membrane-associated proteins to drive such processes. Results: In this study, we have used immortalized and primary dermal fibroblasts from wild type (AhR+/+) and AhR-null (AhR-/-) mice to show that AhR modulates membrane distribution and mobilization of caveolin-1 (Cav-1) during directional cell migration. AhR co-immunoprecipitated with Cav-1 and a fraction of both proteins co-localized to detergent-resistant membrane microdomains (DRM). Consistent with a role of AhR in the process, AhR-/-cells had a significant reduction in Cav-1 in DRMs. Moreover, high cell density reduced AhR nuclear levels and moved Cav-1 from DRMs to the soluble membrane in AhR+/+ but not in AhR-/-cells. Tyrosine-14 phosphorylation had a complex role in the mechanism since its upregulation reduced Cav-1 in DRMs in both AhR+/+ and AhR-/-cells, despite the lower basal levels of Y-14-Cav-1 in the null cells. Fluorescence recovery after photobleaching revealed that AhR knock-down blocked Cav-1 transport to the plasma membrane, a deficit possibly influencing its depleted levels in DRMs. Membrane distribution of Cav-1 in AhR-null fibroblasts correlated with higher levels of cholesterol and with disrupted membrane microdomains, whereas addition of exogenous cholesterol changed the Cav-1 distribution of AhR+/+ cells to the null phenotype. Consistently, higher cholesterol levels enhanced caveolae-dependent endocytosis in AhR-null cells. Conclusions: These results suggest that AhR modulates Cav-1 distribution in migrating cells through the control of cholesterol-enriched membrane microdomains. Our study also supports the likely possibility of membrane-related, transcription factor independent, functions of AhR.This work was supported by grants to P. M. F-S. from the Spanish Ministry of Science and Innovation (SAF2008-00462 and BFU2011-22678) and from the Junta de Extremadura (GR10008). Research at P. M. F-S laboratory is also funded by the Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health (RD12/0036/0032). J.R.B. was a F.P.U. program fellow from the Spanish Ministry of Education and Sciences. All Spanish funding is co-sponsored by the European Union FEDER program. The support and help of the Servicio de Tecnicas Aplicadas a las Biociencia (STAB) of the Universidad de Extremadura is greatly acknowledged. We are very grateful to Dr. Lisardo Bosca (Instituto de Investigaciones Biomedicas, Madrid, Spain) for providing the Cav-1-GFP and the Cav-1Y14F-GFP expression vectors and to Dr. Miguel A. Alonso Lebrero (Centro de Biologia Molecular Severo Ochoa, Madrid, Spain) for assistance with the sucrose density gradient method. The technical support of Eva Barrasa is greatly appreciated.S

Evaluación de la microscopía confocal como herramienta de diagnóstico en enfermedades de los glóbulos rojos

La esferocitosis hereditaria (EH) provoca mutaciones en las proteínas de la membrana de los glóbulos rojos (GRs) que hacen que las células se deformen y se vuelvan demasiado rígidas para poder viajar a través de los vasos sanguíneos. Estas células anormales se destruyen masivamente en el bazo, lo que provoca anemia grave y esplenomegalia además de ictericia y cálculos biliares. El diagnóstico de la EH requiere la realización de complejas pruebas moleculares en la mayoría de casos. Para evitar la realización de dichas pruebas, la microscopía confocal espectral podría utilizarse en el diagnóstico de estas y otras enfermedades. En este estudio, se tiñó la membrana de los GRs con tintes de color e inmunomarcadores, y, bajo un microscopio Leica TCS8, se analizaron los posibles defectos de membrana expresados como diferencias en color y forma en pacientes con EH

Basedat 2. Gestión de Bases de datos para la docencia

Se presentan un conjunto de 11 guías de usuario y aplicaciones didácticas. Bases de datos SABI, AMADEUS, PITEC, Encuesta Industrial, COMTRADE, OMC, TRADECAN, FDIstat, Contabilidad Nacional de España del INE, Contabilidad Nacional de Eurostat e Indicadores de Desarrollo Mundial

Mutations affecting the actin regulator WD repeat–containing protein 1 lead to aberrant lymphoid immunity

Background: The actin-interacting protein WD repeat–containing protein 1 (WDR1) promotes cofilin-dependent actin filament turnover. Biallelic WDR1 mutations have been identified recently in an immunodeficiency/autoinflammatory syndrome with aberrant morphology and function of myeloid cells. Objective: Given the pleiotropic expression of WDR1, here we investigated to what extent it might control the lymphoid arm of the immune system in human subjects. Methods: Histologic and detailed immunologic analyses were performed to elucidate the role of WDR1 in the development and function of B and T lymphocytes. Results: Here we identified novel homozygous and compound heterozygous WDR1 missense mutations in 6 patients belonging to 3 kindreds who presented with respiratory tract infections, skin ulceration, and stomatitis. In addition to defective adhesion and motility of neutrophils and monocytes, WDR1 deficiency was associated with aberrant T-cell activation and B-cell development. T lymphocytes appeared to develop normally in the patients, except for the follicular helper T-cell subset. However, peripheral T cells from the patients accumulated atypical actin structures at the immunologic synapse and displayed reduced calcium flux and mildly impaired proliferation on T-cell receptor stimulation. WDR1 deficiency was associated with even more severe abnormalities of the B-cell compartment, including peripheral B-cell lymphopenia, paucity of B-cell progenitors in the bone marrow, lack of switched memory B cells, reduced clonal diversity, abnormal B-cell spreading, and increased apoptosis on B-cell receptor/Toll-like receptor stimulation. Conclusion: Our study identifies a novel role for WDR1 in adaptive immunity, highlighting WDR1 as a central regulator of actin turnover during formation of the B-cell and T-cell immunologic synapses