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Este recurso forma parte del Proyecto “Aplicación de realidad virtual sobre el laboratorio de prácticas de Fisiología del Sistema Visual” (“Apoyo al diseño de recursos docentes basados en las nuevas tecnologías” correspondiente a la Línea de actuación Actividades del profesorado para la innovación educativa ref. 222 del IV Plan Propio de Docencia de la Universidad de Sevilla. Este recurso, junto al resto generado con esta ayuda, engloban conceptos teórico-prácticos del laboratorio de prácticas principalmente de Fisiología del Sistema Visual, así como de otras asignaturas como Fisiología Humana I. Estas asignaturas se imparten en el Grado en Óptica y Optometría, Grado en Farmacia, y en el Doble Grado en Farmacia y en Óptica y Optometría. Este recurso de realidad virtual presenta el laboratorio de prácticas del Departamento de Fisiología, con diferente material asociado a las prácticas de Fisiología del Sistema Visual, entre los que se encuentran: el modelo anatómico 3D del globo ocular, escenarios para explorar la retina y la coroides desde el interior del ojo junto a la explicación de profesores del departamento, vídeos docentes de distintas prácticas y un cuestionario tipo test de autoevaluación. Ese recurso de realidad virtual está dirigido a estudiantes de las asignaturas del área de Fisiología de diferentes titulaciones académicas en la que se imparten (principalmente Grado en Farmacia, Óptica y Optometría, Doble grado en Farmacia y Óptica-optometría), principalmente la Fisiología del Sistema Visual, y otras asignaturas con parte de este contenido tales como Fisiología Humana I y Fisiología Humana II. También, será útil para otros estudiantes de grados de ciencias de la salud y ciencias, y para cualquier público interesado en conocer el funcionamiento del sistema visual. Por lo que tiene una función dual: por un lado, mejora el conocimiento por parte de los estudiantes en el campo de la fisiología, y por otro sirve como herramienta de aprendizaje en fisiología para la sociedad.

Este recurso forma parte del Proyecto “Aplicación de realidad virtual sobre el laboratorio de prácticas de Fisiología del Sistema Visual” (“Apoyo al diseño de recursos docentes basados en las nuevas tecnologías” correspondiente a la Línea de actuación Actividades del profesorado para la innovación educativa ref. 222 del IV Plan Propio de Docencia de la Universidad de Sevilla. Este recurso, junto al resto generado con esta ayuda, engloban conceptos teórico-prácticos del laboratorio de prácticas principalmente de Fisiología del Sistema Visual, así como de otras asignaturas como Fisiología Humana I. Estas asignaturas se imparten en el Grado en Óptica y Optometría, Grado en Farmacia, y en el Doble Grado en Farmacia y en Óptica y Optometría. Este recurso de realidad virtual 3D presenta el modelo anatómico del globo ocular, centrándose en las estrcuturas externas asociadas. Además, puede encontrarse una pequeña descripción junto a la localización de cada estructura. Ese recurso de realidad virtual está dirigido a estudiantes de las asignaturas del área de Fisiología de diferentes titulaciones académicas en la que se imparten (principalmente Grado en Farmacia, Óptica y Optometría, Doble grado en Farmacia y Óptica-optometría), principalmente la Fisiología del Sistema Visual, y otras asignaturas con parte de este contenido tales como Fisiología Humana I y Fisiología Humana II. También, será útil para otros estudiantes de grados de ciencias de la salud y ciencias, y para cualquier público interesado en conocer el funcionamiento del sistema visual. Por lo que tiene una función dual: por un lado, mejora el conocimiento por parte de los estudiantes en el campo de la fisiología, y por otro sirve como herramienta de aprendizaje en fisiología para la sociedad.

Este recurso forma parte del Proyecto “Aplicación de realidad virtual sobre el laboratorio de prácticas de Fisiología del Sistema Visual” (“Apoyo al diseño de recursos docentes basados en las nuevas tecnologías” correspondiente a la Línea de actuación Actividades del profesorado para la innovación educativa ref. 222 del IV Plan Propio de Docencia de la Universidad de Sevilla. Este recurso, junto al resto generado con esta ayuda, engloban conceptos teórico-prácticos del laboratorio de prácticas principalmente de Fisiología del Sistema Visual, así como de otras asignaturas como Fisiología Humana I. Estas asignaturas se imparten en el Grado en Óptica y Optometría, Grado en Farmacia, y en el Doble Grado en Farmacia y en Óptica y Optometría. Este recurso de realidad virtual 3D presenta el modelo anatómico del globo ocular, centrándose en todas las estructuras que lo conforman, como pueden ser las distintas capas del globo ocular (esclerótica, coroides y retina) o las diferentes estructuras que atraviesa la luz (córnea, pupila, cristalino), entre otros. Además, puede encontrarse una pequeña descripción junto a la localización de cada estructura. Ese recurso de realidad virtual está dirigido a estudiantes de las asignaturas del área de Fisiología de diferentes titulaciones académicas en la que se imparten (principalmente Grado en Farmacia, Óptica y Optometría, Doble grado en Farmacia y Óptica-optometría), principalmente la Fisiología del Sistema Visual, y otras asignaturas con parte de este contenido tales como Fisiología Humana I y Fisiología Humana II. También, será útil para otros estudiantes de grados de ciencias de la salud y ciencias, y para cualquier público interesado en conocer el funcionamiento del sistema visual. Por lo que tiene una función dual: por un lado, mejora el conocimiento por parte de los estudiantes en el campo de la fisiología, y por otro sirve como herramienta de aprendizaje en fisiología para la sociedad.

Este recurso forma parte del Proyecto “Laboratorio de prácticas de Fisiología del Sistema Visual” del III Plan Propio de Docencia de la Universidad de Sevilla (Ref. 1.2.1 Dotación y mejora de recursos para la docencia (Recursos Audiovisuales), conv. 2021-2022) donde se produce material audiovisual (vídeo didáctico) del laboratorio de prácticas principalmente de Fisiología del Sistema Visual, así como de otras asignaturas como Fisiología Humana I. Estas asignaturas se imparten en el Grado en Óptica y Optometría, Grado en Farmacia, y en el Doble Grado en Farmacia y en Óptica y Optometría. El recurso audiovisual presenta las principales estructuras anatómicas del globo ocular, detallando las capas del globo ocular y las diferentes estructuras que atraviesa la luz. Además, debido a su importancia por ser la capa nerviosa donde comienza el procesamiento de la visión, se hará hincapié en la retina y las diferentes capas de células y conexiones que la forman. Por otro lado, se muestran los músculos extraoculares con sus respectivos movimientos, así como las estructuras y funcionamiento del sistema lagrimal. Por lo tanto, este recurso presenta información detallada de aspectos anatómicos y fisiológicos del globo ocular que favorece la compresión de los conceptos teórico-prácticos impartidos en las asignaturas anteriormente citadas. Ese recurso está dirigido a estudiantes de las asignaturas del área de Fisiología de diferentes titulaciones académicas en la que se imparten (principalmente Grado en Farmacia, Óptica y Optometría, Doble grado en Farmacia y Óptica-optometría), principalmente la Fisiología del Sistema Visual, y otras asignaturas con parte de este contenido tales como Fisiología Humana I y Fisiología Humana II. También, será útil para otros estudiantes de grados de ciencias de la salud y ciencias, y para cualquier público interesado en conocer más sobre cómo funciona el globo ocular. Por lo que tiene una función dual: por un lado, mejora el conocimiento por parte de los estudiantes en el campo de la fisiología, y por otro sirve como herramienta de aprendizaje en fisiología para la sociedad.

Este recurso forma parte del Proyecto “Laboratorio de prácticas de Fisiología del Sistema Visual” del III Plan Propio de Docencia de la Universidad de Sevilla (Ref. 1.2.1 Dotación y mejora de recursos para la docencia (Recursos Audiovisuales), conv. 2021-2022) donde se produce material audiovisual (vídeo didáctico) del laboratorio de prácticas principalmente de Fisiología del Sistema Visual, así como de otras asignaturas como Fisiología Humana I. Estas asignaturas se imparten en el Grado en Óptica y Optometría, Grado en Farmacia, y en el Doble Grado en Farmacia y en Óptica y Optometría. El recurso audiovisual detalla las técnicas para explorar las vías por las cuales ocurren los principales reflejos visuales: de acomodación, fotomotor, corneal, vestíbulo-ocular y optocinético. El estudio de los reflejos visuales es de gran utilidad en la práctica clínica dentro de las diferentes disciplinas médicas y también en la optometría, lo que hace necesaria su correcta interpretación en condiciones fisiológicas. Con este recurso no solo se profundiza en los conceptos teórico-prácticos impartidos en las asignaturas anteriormente citadas, si no que se muestra cómo ponerlos de manifiesto de una manera correcta, por lo que acerca los conceptos fisiológicos aprendidos en las clases teóricas al futuro profesional de los estudiantes. Ese recurso está dirigido a estudiantes de las asignaturas del área de Fisiología de diferentes titulaciones académicas en la que se imparten (principalmente Grado en Farmacia, Óptica y Optometría, Doble grado en Farmacia y Óptica-optometría), principalmente la Fisiología del Sistema Visual, y otras asignaturas con parte de este contenido tales como Fisiología Humana I y Fisiología Humana II. También, será útil para otros estudiantes de grados de ciencias de la salud y ciencias, y para cualquier público interesado en conocer más sobre cómo funciona el sistema visual y su asociación con la exploración neurológica. Por lo que tiene una función dual: por un lado, mejora el conocimiento por parte de los estudiantes en el campo de la fisiología, y por otro sirve como herramienta de aprendizaje en fisiología para la sociedad.

El recurso audiovisual tiene como objetivo profundizar en los conceptos teórico-prácticos de electrofisiología ocular impartidos en las asignaturas mencionadas. En el vídeo se muestra la realización de pruebas electrofisiológicas avanzadas para el estudio del sistema visual, como el registro de potenciales evocados visuales, de los movimientos oculares (electrooculograma), y de la actividad cortical general (electroencefalograma). Ese recurso está dirigido a estudiantes de asignaturas del área de Fisiología (principalmente del Grado en Farmacia, Grado en Óptica y Optometría, y Doble grado en Farmacia y en Óptica y Optometría), abarcando contenido de Fisiología Humana y, especialmente, de Fisiología del Sistema Visual. También será útil para estudiantes de otros grados de Ciencias de la Salud y Ciencias, y para cualquier público interesado en el funcionamiento del cuerpo humano a nivel electrofisiológico. Por lo que tiene una función dual: por un lado, mejora el conocimiento por parte de los estudiantes en el campo de la Fisiología, y por otro sirve como herramienta de divulgación de Fisiología para acercarla a la sociedad. Este recurso favorece la compresión de los conceptos teórico-prácticos de las asignaturas impartidas en el Departamento de Fisiología de la Facultad de Farmacia de la Universidad de Sevilla. Asimismo, el recurso audiovisual suple la escasez de material audiovisual disponible en relación con las diferentes técnicas de electrofisiología que se muestran.

Metabolites produced by an altered gut microbiota might mediate the effects in the brain. Among metabolites, the fecal volatile organic compounds (VOCs) are considered to be potential biomarkers. In this study, we examined both the VOCs and bacterial taxa in the feces from healthy subjects and Alzheimer’s disease (AD) patients at early and middle stages. Remarkably, 29 fecal VOCs and 13 bacterial genera were differentiated from the healthy subjects and the AD patients. In general, higher amounts of acids and esters were found in in the feces of the AD patients and terpenes, sulfur compounds and aldehydes in the healthy subjects. At the early stage of AD, the most relevant VOCs with a higher abundance were short-chain fatty acids and their producing bacteria, Faecalibacterium and Lachnoclostridium. Coinciding with the development of dementia in the AD patients, parallel rises of heptanoic acid and Peptococcus were observed. At a more advanced stage of AD, the microbiota and volatiles shifted towards a profile in the feces with increases in hexanoic acid, Ruminococcus and Blautia. The most remarkable VOCs that were associated with the healthy subjects were 4-ethyl-phenol and dodecanol, together with their possible producers Clostridium and Coprococcus. Our results revealed a VOCs and microbiota crosstalk in AD development and their profiles in the feces were specific depending on the stage of AD. Additionally, some of the most significant fecal VOCs identified in our study could be used as potential biomarkers for the initiation and progression of AD.

According to Braak’s hypothesis, it is plausible that Parkinsońs disease (PD) starts in the enteric nervous system (ENS) to spread the brain via the vagus nerve. Thus, we were wondering whether human inflammatory bowel diseases (IBD) can progress with appearance of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. Analysis of human gastrointestinal tract sections from IBD patients demonstrated the presence of pathogenic phosphorylated α-syn in both myenteric (Auerbach’s) and submucosal (Meissner’s) plexuses. Remarkably, PD subjects exhibit α-syn pathology in identical gastrointestinal locations. Analysis of human midbrain sections from IBD subjects revealed a clear displacement of neuromelanin in some nigral neurons from the ventral mesencephalon, which were inherently associated with presence of α-syn aggregates reminiscent of pale bodies. We also used different dextran sodium sulfate (DSS)-based rat models of gut inflammation (subchronic and chronic) to study the appearance of phosphorylated α-syn inclusions in both Auerbach’s and Meissner’s plexuses (gut), and in dopaminergic neuritic processes (brain) along with degeneration of nigral dopaminergic neurons, which are considered classical hallmarks of PD. Vagotomized DSS-treated animals exhibited pathological α-syn in the gut but failed to show dopaminergic cells degeneration and α-syn aggregation in the ventral mesencephalon. Taken together, these results strongly suggest that Braak’s hypothesis is plausible.

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a −1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a “weighted Goldilocks” scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a −1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a “weighted Goldilocks” scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.

Para nuestro proyecto de “Canal virtual de divulgación sobre Fisiología” se ha utilizado diferentes redes sociales como son Instagram y YouTube, donde se han ido publicando diferentes contenidos sobre Fisiología. Los contenidos subidos a las redes sociales de manera periódica han sido creados por los estudiantes que cursan asignaturas en el Departamento de Fisiología de la Facultad de Farmacia con el objetivo de que accedan de manera atractiva e interactiva al conocimiento básico de nuestras asignaturas, y también adquieran diferentes herramientas de búsqueda y comunicación de información que les será útil como futuros profesionales sanitarios. Este recurso es un Genially organizado en diferentes apartados sobre Fisiología y Fisiopatología de los sistemas a estudiar en las asignaturas del departamento de Fisiología de la Facultad de Farmacia: sistema cardiovascular, la sangre, sistema respiratorio, sistema nervioso, sistema endocrino, aparatos reproductores, sistema digestivo, sistema renal y generalidades. Cada apartado consta con unos vídeos creados por los estudiantes sobre diferentes conceptos estudiados en las asignaturas, con una breve introducción inicial escrita junto al recurso audiovisual. Estos vídeos están directamente enlazados con nuestra cuenta de YouTube, donde quedan todos los vídeos registrados. Además, hay un apartado de “Preguntas” que te dirige a una serie de cuestiones tipo test multirrespuesta que se han ido publicando diariamente sobre los diferentes sistemas fisiológicos en nuestra cuenta de Instagram (@FisioFarma_US), para que los estudiantes puedan repasar conceptos sobre los diferentes temas de las asignaturas.

Aquaporin-4 (AQP4) is the target of the specific immunoglobulin G autoantibody (AQP4-IgG) produced in patients with neuromyelitis optica spectrum disorders (NMOSD). Previous studies demonstrated that AQP4-IgG binding to astrocytic AQP4 leads to cell-destructive lesions. However, the early physiopathological events in Müller cells in the retina are poorly understood. Here, we investigated the consequences of AQP4-IgG binding to AQP4 of Müller cells, previous to the inflammatory response, on two of AQP4’s key functions, cell volume regulation response (RVD) and cell proliferation, a process closely associated with changes in cell volume. Experiments were performed in a human retinal Müller cell line (MIO-M1) exposed to complement-inactivated sera from healthy volunteers or AQP4-IgG positive NMOSD patients. We evaluated AQP4 expression (immunofluorescence and western blot), water permeability coefficient, RVD, intracellular calcium levels and membrane potential changes during hypotonic shock (fluorescence videomicroscopy) and cell proliferation (cell count and BrdU incorporation). Our results showed that AQP4-IgG binding to AQP4 induces its partial internalization, leading to the decrease of the plasma membrane water permeability, a reduction of swelling-induced increase of intracellular calcium levels and the impairment of RVD in Müller cells. The loss of AQP4 from the plasma membrane induced by AQP4-IgG positive sera delayed Müller cells’ proliferation rate. We propose that Müller cell dysfunction after AQP4 removal from the plasma membrane by AQP4-IgG binding could be a non-inflammatory mechanism of retinal injury in vivo, altering cell volume homeostasis and cell proliferation and consequently, contributing to the physiopathology of NMOSD., This study was supported by grants from the University of Buenos Aires (UBA-SECYT, 20020130100682BA, 2018–2021, Argentina) to Claudia Capurro; the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT 2019–01707, Argentina) to Claudia Capurro and the Spanish Ministry of Economy and Competitiveness, co-financed by the Carlos III Health Institute (ISCIII) and European Regional Development Fund (FEDER, PI16/00493) to Miriam Echevarría.

Brain aquaporin 1 (AQP1) and AQP4 are involved in cerebrospinal fluid (CSF) homeostasis and might participate in the origin of hydrocephalus. Studies have shown alterations of perivascular AQP4 expression in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer’s disease (AD). Due to the overlapping of clinical signs between iNPH and certain neurological conditions, mainly AD, specific biomarkers might improve the diagnostic accuracy for iNPH. The goal of the present study was to analyze and quantify the presence of AQP1 and AQP4 in the CSF of patients with iNPH and AD to determine whether these proteins can be used as biomarkers of iNPH. We examined AQP1 and AQP4 protein levels in the CSF of 179 participants (88 women) classified into 5 groups: possible iNPH (81 participants), hydrocephalus associated with other neurological disorders (13 participants), AD (41 participants), non-AD dementia (32 participants) and healthy controls (12 participants). We recorded each participant’s demographic and clinical variables and indicated, when available in the clinical history, the record of cardiovascular and respiratory complications. An ELISA showed virtually no AQP content in the CSF. Information on the vascular risk factors (available for 61 patients) confirmed some type of vascular risk factor in 86% of the patients with possible iNPH and 58% of the patients with AD. In conclusion, the ELISA analysis showed insufficient sensitivity to detect the presence of AQP1 and AQP4 in CSF, ruling out the possible use of these proteins as biomarkers for diagnosing iNPH., This research was funded by grants PI16/00493 and PI19/01096 to M.E. from the Spanish Ministry of Economy and Competitiveness, co-financed by the Carlos III Health Institute (ISCIII) and European Regional Development Fund (FEDER). J.L.T-C. was partially supported by the Regional Government of Andalusia and FEDER funds through a program for recruitment of young researchers.

Parkinson’s disease is a highly prevalent neurological disorder for which there is currently no cure. Therefore, the knowledge of risk factors as well as the development of new putative molecular targets is mandatory. In this sense, peripheral inflammation, especially the originated in the colon, is emerging as a predisposing factor for suffering this disease. We have largely studied the pleiotropic roles of galectin-3 in driving microglia-associated immune responses. However, studies aimed at elucidating the role of galectin-3 in peripheral inflammation in terms of microglia polarization are lacking. To achieve this, we have evaluated the effect of galectin-3 deletion in two different models of acute peripheral inflammation: intraperitoneal injection of lipopolysaccharide or gut inflammation induced by oral administration of dextran sodium sulfate. We found that under peripheral inflammation the number of microglial cells and the expression levels of pro-inflammatory mediators take place specifically in the dopaminergic system, thus supporting causative links between Parkinson’s disease and peripheral inflammation. Absence of galectin-3 highly reduced neuroinflammation in both models, suggesting an important central regulatory role of galectin-3 in driving microglial activation provoked by the peripheral inflammation. Thus, modulation of galectin-3 function emerges as a promising strategy to minimize undesired microglia polarization states., This work was supported by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (RTI 2018-098830-B-I00), from the Consejería de Economía y Conocimiento of Junta de Andalucía (P18-RT-1372 and US-1264806). MJP, MDVC and PGM were supported by a grant from the Junta de Andalucía (CTS 5884) and AEC by an associated post-doctoral grant.

La verdad es un elemento en los procesos de justicia transicional que permite la consecución de otros elementos como son el de justicia, reparación y no repetición. En ese contexto, la presente investigación pretende establecer qué verdad jurídica es la que se aplica, pretendiendo determinar si corresponde a las denominadas verdad real o verdad procesal. Desde otro lado, se busca establecer si la verdad es un instrumento para no olvidar la ocurrencia de hechos que son objeto de justicia transicional o si tiene como fin la determinación del quantum de reparación. Por lo anterior, la pregunta jurídica a resolver es: ¿la verdad en la justicia transicional es de carácter real o procesal? Así las cosas, se trata de determinar qué se entiende por verdad y explorar cómo se ha estudiado y qué modalidades existen. El método de investigación es del orden descriptivo-cualitativo, haciendo énfasis en los conceptos sobre verdad que se soportan en fuentes formales del derecho. La parte descriptiva de esta investigación analiza el funcionamiento de algunas instituciones que hacen parte del proceso de paz colombiano adelantado con las Farc-EP. A manera de ejemplo, las comisiones de la verdad. Truth is an element in the processes of transitional justice that allows the achievement of other elements such as justice, reparation and non-repetition. In this context, the present research seeks to establish what kind of truth is applied legally, trying to determine if it corresponds to the so-called real truth or procedural truth. On the other hand, it tries to establish if the truth is an instrument to not forget the occurrence of facts that are object of transitional justice or has as purpose the determination of the quantum of repair. Therefore, the legal question to be resolved is: Is the truth in transitional justice a real or procedural one? Thus, determining what is understood by truth and exploring how it has been studied and what modalities exist is the main matter of this research. The research methodology revolves around the qualitative approach, emphasizing the concepts about truth that have been elaborated by diverse authors, and from those we will try to determine what truth has been used. The descriptive part of this research also analyzes the functioning of some institutions that are part of the Colombian peace processes advanced with the Farc-EP. By way of example, truth commissions.

We reported that Disabled-2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E-cadherin. Dab2 depletion in Caco-2 cells led to E-cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium-induced colitis increased Dab2/E-cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E-cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E-cadherin delocalization. Epithelial Dab2 decrement preceded that of E-cadherin. We suggest that Dab2, by inhibiting E-cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.

Neuroinflammation underlies neurodegenerative diseases. Herein, we test whether acute colon inflammation activates microglia and astrocytes, induces neuroinflammation, disturbs neuron intrinsic electrical properties in the primary motor cortex, and alters motor behaviors. We used a rat model of acute colon inflammation induced by dextran sulfate sodium. Inflammatory mediators and microglial activation were assessed in the primary motor cortex by PCR and immunofluorescence assays. Electrophysiological properties of the motor cortex neurons were determined by whole-cell patch-clamp recordings. Motor behaviors were examined using open-field and rotarod tests. We show that the primary motor cortex of rats with acute colon inflammation exhibited microglial and astrocyte activation and increased mRNA abundance of interleukin-6, tumor necrosis factor-alpha, and both inducible and neuronal nitric oxide synthases. These changes were accompanied by a reduction in resting membrane potential and rheobase and increased input resistance and action potential frequency, indicating motor neuron hyperexcitability. In addition, locomotion and motor coordination were impaired. In conclusion, acute colon inflammation induces motor cortex microglial and astrocyte activation and inflammation, which led to neurons’ hyperexcitability and reduced motor coordination performance. The described disturbances resembled some of the early features found in amyotrophic lateral sclerosis patients and animal models, suggesting that colon inflammation might be a risk factor for developing this disease. Ministerio de Ciencia e Innovación 18.06.07.3005 PID2019-105632RB-I00, Grant RTI-2018.099908-B-C21 Agencia Estatal de Investigación AEI/10.13039/501100011033

espanolLas Escape Rooms son un tipo de recurso que esta siendo utilizado por muchos educadores, propiciando elementos de colaboracion que ayudan a desarrollar habilidades sociales. En este estudio se ha desarrollado una Escape Room multidisciplinar como herramienta educativa con el fin de mejorar el aprendizaje de los estudiantes y prepararlos para el futuro de la practica profesional. Participaron 145 alumnos y 12 profesores adscritos a 8 areas de conocimiento distintas de la Facultad de Farmacia de la Universidad de Sevilla. Mediante esta innovacion se ha producido una retroalimentacion alumno-profesor, mejorando su relacion y aprendiendo mutuamente. Los resultados obtenidos mediante encuestas de satisfaccion muestran la gran aceptacion que ha tenido la Escape Room entre los alumnos, siendo el estudio valorado con una puntuacion general de 4,83/5. A pesar de que el 93% de los alumnos considero la actividad de alta/muy alta dificultad, el 100% de los mismos destacan que esta herramienta fomenta el trabajo en equipo y el 86,9% considera que refuerza los conocimientos. Este estudio ha despertado en los alumnos la motivacion e interes por las asignaturas implicadas, fortaleciendo el aprendizaje y conocimiento de los conceptos fundamentales adquiridos, y propiciando el trabajo en equipo y la resolucion de problemas. EnglishEscape Rooms are educational resources that are being used by many educators, fostering elements of collaboration that help in social skills development. In this study, a multidisciplinary Escape Room has been developed as an educational tool in order to improve students learning and prepare them for the future of professional practice. 145 students and 12 professors belonging to 8 different areas of knowledge from the Faculty of Pharmacy of the University of Seville participated in this activity. Through this innovation, a student-teacher feedback has been created, improving their relationship and learning from each other. The results obtained through satisfaction surveys show the great acceptance that the escape room had among the students, being the study valued with an overall score of 4.83/5. Although 93% of the students considered the activity of high/very high difficulty, 100% of them emphasize that this tool encourages teamwork and 86.9% consider that it reinforces knowledge. This study has awakened students motivation and interest in the subjects involved, strengthening learning and knowledge of the fundamental concepts acquired, and promoting teamwork and problem solving.

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-β1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers. © 2016 Wiley Periodicals, Inc.

Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCE HIV, like many retroviruses, utilizes a −1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.

The detection of IgG aquaporin-4 antibodies in the serum of patients with Neuromyelitis optica (NMO) has dramatically improved the diagnosis of this disease and its distinction from multiple sclerosis. Recently, a group of patients have been described who have an NMO spectrum disorder (NMOsd) and who are seronegative for AQP4 antibodies but positive for IgG aquaporin-1 (AQP1) or myelin oligodendrocyte glycoprotein (MOG) antibodies. The purpose of this study was to determine whether AQP1 and MOG could be considered new biomarkers of this disease, and if point mutations in the gDNA of AQP4, AQP1 and MOG genes could be associated with the etiology of NMOsd. We evaluated the diagnostic capability of ELISA and cell-based assays (CBA), and analyzed their reliability, specificity, and sensitivity in detecting antibodies against these three proteins. The results showed that both assays can recognize these antigen proteins under appropriate conditions, but only anti-AQP4 antibodies, and not AQP1 or MOG, appears to be a clear biomarker for NMOsd. CBA is the best method for detecting these antibodies, and serum levels of AQP4 antibodies do not correlate with the progression of this disease. So far, the sequencing analysis has not revealed a genetic basis for the etiology of NMOsd, but a more extensive analysis is required before definitive conclusions can be drawn.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus' structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location. Junta de Andalucía CTS 5884 Ministerio de Educación y Ciencia AP2007-04201 European Molecular Biology Organization ASTF45-2012

The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs.

We previously proposed that Dab2 participates in the endocytosis of milk macromolecules in rat small intestine. Here we investigate the receptors that may mediate this endocytosis by studying the effects of age and diet on megalin, VLDLR, and ApoER2 expression, and that of age on the expression of cubilin and amnionless. Of megalin, VLDLR and ApoER2, only the megalin expression pattern resembles that of Dab2 previously reported. Thus the mRNA and protein levels of megalin and Dab2 are high in the intestine of the suckling rat, down-regulated by age and up-regulated by milk diet, mainly in the ileum. Neither age nor diet affect ApoER2 mRNA levels. The effect of age on VLDLR mRNA levels depends on the epithelial cell tested but they are down-regulated by milk diet. In the suckling rat, the intestinal expressions of both cubilin and amnionless are similar to that of megalin and megalin, cubilin, amnionless and Dab2 co-localize at the microvilli and in the apical endocytic apparatus. Co-localization of Dab2 with ApoER2 and VLDLR at the microvilli and in the apical endocytic apparatus is also observed. This is the first report showing intestinal co-localization of: megalin/cubilin/amnionless/Dab2, VLDLR/Dab2 and ApoER2/Dab2. We conclude that the megalin/cubilin/amnionless/Dab2 complex/es participate in intestinal processes, mainly during the lactation period and that Dab2 may act as an adaptor in intestinal processes mediated by ApoER2 and VLDLR.

Background information The myofibroblasts placed underneath the epithelium of the rodent small intestine express reelin, and the reelin absence modifies both the morphology and the cell renewal processes of the crypt–villus unit. In the developing central nervous system, the reelin effects are mediated by the disabled-1 (Dab1) protein. The present work explores whether Dab1 mediates the reelin control of the crypt–villus unit dynamics by examining in the mouse small intestine the consequences of the absence of (i) Dab1 (scrambler mutation) on crypt–villus unit cell renewal processes and morphology and (ii) reelin (reeler mutation) on the intestinal expression of Dab1. Results The effects of the scrambler mutation on the crypt–villus unit renewal processes are remarkably similar to those caused by the lack of reelin. Thus, both mutations significantly reduce epithelial cell proliferation, migration and apoptosis, and the number of Paneth cells; affect the morphology of the villus, and expand the intercellular space of the adherens junctions and desmosomes. The Western blot assays reveal that the Dab1 isoform present in the enterocytes has a molecular weight of ∼63 kDa and that in the brain of ∼82 kDa. They also reveal that the absence of reelin increases Dab1 abundance in both brain and enterocytes. Conclusions All together, the current findings link reelin with Dab1 and suggest that Dab1 functions downstream of reelin action on the homeostasis of the crypt–villus unit.

We previously reported that reelin, an extracellular matrix protein first known for its key role in neuronal migration, reduces the susceptibility to dextran sulphate sodium (DSS)-colitis. The aim of the current study was to determine whether reelin protects from colorectal cancer and how reelin defends from colon pathology. In the colon of wild-type and of mice lacking reelin (reeler mice) we have analysed the: i) epithelium cell renewal processes, ii) morphology, iii) Sox9, Cdx2, Smad5, Cyclin D1, IL-6 and IFNγ mRNA abundance in DSS-treated and untreated mice, and iv) development of azoxymethane/DSS-induced colorectal cancer, using histological and real time-PCR methodologies. The reeler mutation increases colitis-associated tumorigenesis, with increased tumours number and size. It also impairs the intestinal barrier because it reduces cell proliferation, migration, differentiation and apoptosis; decreases the number and maturation of goblet cells, and expands the intercellular space of the desmosomes. The intestinal barrier impairment might explain the increased susceptibility to colon pathology exhibited by the reeler mice and is at least mediated by the down-regulation of Sox9 and Cdx2. In response to DSS-colitis, the reeler colon increases the mRNA abundance of IL-6, Smad5 and Cyclin D1 and decreases that of IFNγ, conditions that might result in the increased colitis-associated tumorigenesis found in the reeler mice. In conclusion, the results highlight a role for reelin in maintaining intestinal epithelial cell homeostasis and providing resistance against colon pathology. España, Junta de Andalucía CTS 5884

Intestinal myofibroblasts secrete substances that control organogenesis and wound repair of the intestine. The myofibroblasts of the rat small intestine express reelin and the present work explores whether reelin regulates crypt-villus unit homeostasis using normal mice and mice with the reelin gene disrupted (reeler). The results reveal that mouse small intestine expresses reelin, its receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VldlR) and the reelin effector protein Disabled-1 (Dab1) and that reelin expression is restricted to myofibroblasts. The absence of reelin significantly reduces epithelial cell proliferation, migration, and apoptosis and the number of Paneth cells. These effects are observed during the suckling, weaning, and adult periods. The number of Goblet cells is increased in the 2-month-old reeler mice. The absence of reelin also expands the extracellular space of the adherens junctions and desmosomes without significantly affecting either the tight-junction structure or the epithelial paracellular permeability. In conclusion, this is the first in vivo work showing that the absence of reelin alters intestinal epithelium homeostasis.

The expression of the phosphoinositides phosphatases Synaptojanins (Synjs) 1 and 2 has been shown in brain and in some peripheral tissues, but their expression in the intestine has not been reported. Herein we show that the small and large intestine express Synj1 and Synj2. Their mRNA levels, measured by RT-PCR, are not affected by development in the small intestine but in the colon they increase with age. Immunostaining assays reveal that both Synjs localize at the apical domain of the epithelial cells and at the lamina propria at sites also expressing the neuron marker calretinin. Synj2 staining at the lamina propria is fainter than that of Synj1. In colonocytes Synjs are at the apical membrane and cytosolic membrane vesicles. Synj2 is also at the mitochondria. Western blots reveal that the intestinal mucosa expresses at least two Synj1 (170- and 139-kDa) and two Synj2 (160- and 148-kDa) isoforms. The observations suggest that Synj1–170, Synj2–160, and Synj2–148 in colonocytes, might participate in processes that take place mainly at the apical domain of the epithelial cells whereas Synj1–139 in those at the enteric nervous system. Experimental colitis augments the mRNA abundance of both Synjs in colon but only Synj2 mRNA levels are increased in colon tumors. In conclusion, as far as we know, this is the first report showing expression, location and isoforms of Synj1 and Synj2 in the small and large intestine and that they might participate in intestinal pathology.

Reelin is an extracellular matrix protein first known for its key role in neuronal migration. Studies in rodent small intestine suggested that reelin protects the organism from intestinal pathology. Here we determined in mice colon, by real time-PCR and immunological assays, the expression of the reelin signalling system; its response to dextran sulphate sodium (DSS) and the response of wild-type and reeler mice to DSS-treatment. DNA methylation was determined by bisulfite modification and sequencing of genomic DNA. In the colon mucosa reelin expression is restricted to the myofibroblasts, whereas both epithelial cells and myofibroblasts express reelin receptors (ApoER2 and VLDLR) and its effector protein Dab1. The muscle layer also expresses reelin. DSS-treatment reduces reelin expression in the muscle but it is activated in the mucosa. Activation of mucosal reelin is greater in magnitude and is delayed until after the activation of the myofibroblasts marker, α-SMA. This indicates that the DSS-induced reelin up-regulation results from changes in the reelin gene expression rather than from myofibroblasts proliferation. DSS-treatment does not modify Sp1 or Tbr1 mRNA abundance, but increases that of TGF-β1 and ApoER2, decreases that of CASK and DNMT1 and it also decreases the reelin promoter methylation. Finally, the reeler mice exhibit higher inflammatory scores than wild-type mice, indicating that the mutation increases the susceptibility to DSS-colitis. In summary, this data are the first to demonstrate that mouse distal colon increases reelin production in response to DSS-colitis via a DNMT1-dependent hypo-methylation of the gene promoter region and that reelin provides protection against colitis España, Junta de Andalucía CTS 5884

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-β1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers. © 2016 Wiley Periodicals, Inc.

In Drosophila, normal and transformed cells compete with each other for survival in a process called cell competition. However, it is not known whether comparable phenomena also occur in mammals. Scribble is a tumor suppressor protein in Drosophila and mammals. In this study we examine the interface between normal and Scribble-knockdown epithelial cells using Madin–Darby Canine Kidney (MDCK) cells expressing Scribble short hairpin RNA (shRNA) in a tetracycline-inducible manner. We observe that Scribble-knockdown cells undergo apoptosis and are apically extruded from the epithelium when surrounded by normal cells. Apoptosis does not occur when Scribble-knockdown cells are cultured alone, suggesting that the presence of surrounding normal cells induces the cell death. We also show that death of Scribble-knockdown cells occurs independently of apical extrusion. Finally, we demonstrate that apoptosis of Scribble-knockdown cells depends on activation of p38 mitogen-activated protein kinase (MAPK). This is the first demonstration that an oncogenic transformation within an epithelium induces cell competition in a mammalian cell culture system.

Expression of reelin, reelin receptors [apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VldlR)] and the Disabled-1 (Dab1) protein was investigated in rat intestinal mucosa. Intestinal reelin and Dab1 mRNA levels were maximal in the early stages of life, reaching adult levels in 1-month-old rats. Expression of reelin mRNA was restricted to fibroblasts, whereas mRNAs of Dab1, ApoER2, VldlR and integrins α3 and β1 were observed in enterocytes, crypts and fibroblasts. Reelin protein was only observed in isolated intestinal fibroblasts and in a cell layer subjacent to the villus epithelium, which seems to be composed of myofibroblasts because it also reacted to α-smooth muscle actin. The Disabled-1 and VldlR protein signals were detected in the crypt and villus cells, and they were particularly abundant in the terminal web domain of the enterocytes. The ApoER2 protein signal was detected in the upper half of the villi but not in the crypts. This is the first report showing that rat intestinal mucosa expresses the reelin–Disabled-1 signalling system.

The kidney synthesizes l -carnitine and reabsorbs it via the Na+/ l -carnitine cotransporter OCTN2. This study investigates the ontogeny of OCTN2, γ-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and γ-butyrobetaine hydroxylase (BBH) in rat kidneys. Foetuses, newborn, suckling, weaning and adult rats were used. The apical membranes of foetal and newborn rat kidneys express OCTN2 transport activity, which is up-regulated by age. Maturation significantly increased the Vmax of this transport system without changing the apparent Kt, which excludes a maturation-related expression of different transporter isoforms. Northern analysis showed a 3.7 kb transcript for OCTN2 in all the ages tested. Northern and RT-PCR assays revealed that maturation increased renal expression of OCTN2 mRNA. Foetuses express TMABA-DH mRNA and this expression increased during postnatal life. BBH mRNA, however, was detected during the suckling period onwards and its abundance was not changed significantly by maturation. This study reports for the first time that, in rat kidneys: (i) an apical OCTN2 transporter is active in rat foetuses, (ii) ontogeny up-regulates OCTN2 activity by increasing the density and/or turnover of the transporters, (iii) the maturation-related changes in OCTN2 are in part mediated by transcriptional mechanism(s) and (iv) the expression of both, TMABA-DH and BBH mRNA is ontogenically regulated. Some of these results were published as an abstract ( Garcia-Delgado et al., 2003 ).

Creatine plays a role in energy storage and transport/shuttle of high-energy phosphate in heart, brain, retina, testis and skeletal muscle. These tissues take creatine from the plasma via a 2Na(+)/1Cl(-)/1creatine cotransporter (CRT). We have previously demonstrated that renal apical membrane presents a 2Na(+)/1Cl(-)/1creatine cotransport activity. The goal of this study was to determine whether this transporter is ontogenically regulated. Na(+)/Cl(-)/creatine transport activity was evaluated by measuring [(14)C]-creatine uptake into renal brush-border membrane vesicles. CRT mRNA expression was measured by Northern and real-time PCR assays. E20 foetuses, newborn, suckling, weaning and adult (2- and 8-month-old) Wistar rats were used. The results revealed that neither the vesicular volume, the binding of creatine to the brush-border membrane vesicles, nor the purity of the brush-border membrane vesicle preparations was affected by maturation. Fetal and neonatal kidneys contained a creatine transporter that was qualitatively indistinguishable from that in the adult: it was concentrative, Na(+)- and Cl(-)-dependent, electrogenic and inhibited by guanidinopropionic acid. Maturation increased this transport activity by increasing the maximal rate of transport (V(max)) without significantly changing the apparent K(m). Northern analysis revealed two transcripts for CRT of 2.7 kb and 4.2 kb in all the ages tested. Northern and real-time PCR assays showed that, as seen with NaCl-dependent creatine transport activity, maturation increased CRT mRNA expression. This study reports for the first time that: (i) an apical renal Na(+)/Cl(-)/creatine cotransporter is already active in rat foetuses and (ii) development regulates Na(+)/Cl(-)/creatine cotransport activity by increasing the density and/or turnover of the transporters.

Oral L-carnitine supplementation is commonly used in sports nutrition and in medicine; however, there is controversy regarding the mechanisms that mediate intestinal L-carnitine transport. We have previously reported that the Na(+)/L-carnitine transporter OCTN2 is present in the small intestinal apical membrane. Herein we aimed to find out if this step of intestinal L-carnitine absorption is ontogenically regulated, and if so, to determine the molecular mechanism(s) involved. L-[(3)H]-Carnitine uptake was measured in the jejunum and ileum of fetuses (E17 and E21), newborn (1 day-old), suckling (15 day-old), weaning (1 month-old) and adult (2 and 6 month-old) Wistar rats. Both, Na(+) -dependent and Na(+) -independent L-carnitine uptake rates, normalized to intestinal weight, significantly increased during the late gestation period, and then declined during the suckling period. After weaning, the rate of Na(+) -dependent L-carnitine uptake is no longer measurable. In E21- fetuses and newborn rats, L-carnitine uptake was higher in the ileum than in the jejunum. The decline in Na(+) -dependent L-carnitine uptake with maturation was mediated via a decrease in the V(max) of the uptake process with no change in its apparent K(m). Semi-quantitative RT-PCR assays showed that OCTN2 mRNA levels were significantly higher in E21-fetuses and newborn rats compared to suckling rats, which were in turn significantly higher than that in adult rats. Neither retardation of weaning nor L-carnitine supplementation prevented the down-regulation of Na(+)/L-carnitine transport activity. The results demonstrate for the first time that intestinal Na(+) -dependent L-carnitine uptake activity is under genetic regulation at the transcriptional level.

We previously proposed that Dab2 participates in the endocytosis of milk macromolecules in rat small intestine. Here we investigate the receptors that may mediate this endocytosis by studying the effects of age and diet on megalin, VLDLR, and ApoER2 expression, and that of age on the expression of cubilin and amnionless. Of megalin, VLDLR and ApoER2, only the megalin expression pattern resembles that of Dab2 previously reported. Thus the mRNA and protein levels of megalin and Dab2 are high in the intestine of the suckling rat, down-regulated by age and up-regulated by milk diet, mainly in the ileum. Neither age nor diet affect ApoER2 mRNA levels. The effect of age on VLDLR mRNA levels depends on the epithelial cell tested but they are down-regulated by milk diet. In the suckling rat, the intestinal expressions of both cubilin and amnionless are similar to that of megalin and megalin, cubilin, amnionless and Dab2 co-localize at the microvilli and in the apical endocytic apparatus. Co-localization of Dab2 with ApoER2 and VLDLR at the microvilli and in the apical endocytic apparatus is also observed. This is the first report showing intestinal co-localization of: megalin/cubilin/amnionless/Dab2, VLDLR/Dab2 and ApoER2/Dab2. We conclude that the megalin/cubilin/amnionless/Dab2 complex/es participate in intestinal processes, mainly during the lactation period and that Dab2 may act as an adaptor in intestinal processes mediated by ApoER2 and VLDLR.

We recently demonstrated that the mucosa of the small intestine of the rat expresses reelin and some components of its signaling system. The current study evaluates whether reelin affects the intestinal gene expression profile using microarray analysis and reeler mice, a natural mutant in which reelin is not expressed. The effect of the mutation on body weight and intestinal morphology is also evaluated. The mutation reduces body and intestinal weight during the first 2 months of age and modifies the morphology of the crypts and villi. For the microarray assays, total RNA was obtained from either isolated epithelial cells or intact small intestine. Of the 45,101 genes present in the microarray the mutation significantly alters the expression of 62 genes in the isolated epithelial cell samples and of 84 in the intact small intestine. The expression of 83% of the genes tested for validation was substantiated by reverse transcriptase polymerase chain reaction. The mutation notably up-regulates genes involved in intestinal metabolism, while it down-regulates genes related with immune response, inflammation, and tumor development. Genes involved in cell proliferation, differentiation, apoptosis, membrane transport and cytoskeleton are also differently expressed in the reeler mice as compared with the control. This is the first report showing that the lack of reelin modifies intestinal morphology and gene expression profile and suggests a role for reelin in intestinal epithelium homeostasis.

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression. J. Cell. Biochem. 112: 354–361, 2011. © 2010 Wiley-Liss, Inc.