Medical physiology 3

Material docente fisiología médica 3 en inglé

Importancia del estrés oxidativo en la diferencia de longevidad entre machos y hembras

RESUMEN Uno de los mayores logros del siglo XX es el aumento de la esperanza de vida de la población humana. En Europa, ésta se ha duplicado entre 1900 y 1992. En todos los casos la esperanza de vida de las mujeres supera a la de los hombres. De hecho, en España en 1900, la esperanza de vida para los hombres era de 33.7 años y para las mujeres de 35.1, es decir, un 3.8% superior en mujeres frente a hombres. En 1992 la esperanza de vida de los hombres era de 73.7 años, mientras que la de las mujeres era 83.8, lo cual supone un 9.9% más en las mujeres que en los hombres. La base de la diferencia de esperanza de vida entre machos y hembras está aún por esclarecer. Este fenómeno no es atribuible a diferencias sociológicas, puesto que también se reproduce en otras especies. En nuestro laboratorio, las ratas Wistar hembras muestran un 16% más de vida media que las ratas Wistar machos. En la presente tesis demostramos que las diferencias de longevidad entre machos hembras tienen una base biológica: los estrógenos suponen una ventaja ante la supervivencia de las hembras, puesto que inducen la expresión de enzimas antioxidantes, y ello las protege frente al estrés oxidativo, y por tanto les confiere una mayor longevidad. Así pues, el estrés oxidativo encontrado en los machos es superior al de las hembras. Se determinó la producción de peróxido de hidrógeno en mitocondrias hepáticas y cerebrales (sinápticas y no sinápticas) aisladas de machos y hembras y observamos que las procedentes de los machos producen una cantidad significativamente superior de peróxido de hidrógeno. Asimismo, los niveles del antioxidante endógeno glutatión reducido son mayores en las mitocondrias procedentes de las hembras en comparación con los machos. A consecuencia del mayor estrés oxidativo en los machos, el DNA mitocondrial de los mismos está hasta cuatro veces más oxidado que el de las hembras. El aumento de este parámetro está asociado a una menor longevidad. Todo ello se puede explicar porque las hembras se comportan como dobles transgénicos, que sobreexpresan las enzimas antioxidantes glutatión peroxidasa y manganeso superóxido dismutasa. Además la expresión del marcador de envejecimiento 16S rRNA está disminuida en los machos en comparación con las hembras, lo cual supone que con una misma edad cronológica, las hembras poseen una edad biológica menos que los machos, es decir son más jóvenes que los machos. Otro parámetro relacionado con la longevidad que determinamos es la actividad telomerasa, la cual además posee un elemento de respuesta a estrógenos, y obtuvimos que las hembras poseen una actividad telomerasa superior en comparación con los machos. Por otro lado, los estudios in vitro realizados en la presente tesis, empleando un cultivo celular de células MCF7, que provienen de carcinoma mamario, indican que el mecanismo por el cual los estrógenos inducen la expresión de enzimas antioxidantes está mediado tanto por los receptores estrogénicos, como por la activación de la vía de señalización de las MAP Kinasas. Además, los efectos antioxidantes del estradiol son mimetizados por la genisteína, el fitoestrógeno más abundante presente en la soja. La importancia de este hecho recae en que en este caso, la genisteína no sólo no posee efectos cancerígenos, sino que previene el desarrollo de diversos cánceres como el mamario o el de próstata. __________________________________________________________________________________________________Females live longer than males in many species, including humans. This can be explained on the basis of the mitochondrial theory of aging. We have investigated the differential mitochondrial oxidative stress between males and females to understand the molecular mechanisms enabling females to live longer than males. Mitochondria are a major source of free radicals in cells. Those from female rats generate half the amount of peroxides than those of males. Mitochondria from females have higher levels of reduced glutathione than those from males. Oxidative damage to mitochondrial DNA in males is four fold higher than that of females. This is due to a higher expression and activities of Mn-superoxide dismutase and of glutathione peroxidase in females, which behave as double transgenics overexpressing superoxide dismutase, and glutathione peroxidase, conferring protection against free radical mediated damage in aging. Moreover, 16S rRNA expression, which decreases significantly with aging, is four times higher in mitochondria from females than in those from males of the same chronological age. Studies in vitro show that oestradiol is an antioxidant, but that estrogenic effects must be transcriptional, by inducing the antioxidant enzyme gene expression. The facts reported here provide molecular evidence to explain the different life span in males and females. The challenge for the future is to find molecules that have the beneficial effects of estradiol, but without its feminizing effects. Phytoestrogens or phytoestrogen-related molecules may be good candidates to meet this challenge

Assessment and risk prediction of frailty using texture-based muscle ultrasound image analysis and machine learning techniques

[EN] The purpose of this study was to evaluate texture-based muscle ultrasound image analysis for the assessment and risk prediction of frailty phenotype. This retrospective study of prospectively acquired data included 101 participants who underwent ultrasound scanning of the anterior thigh. Participants were subdivided according to frailty phenotype and were followed up for two years. Primary and secondary outcome measures were death and comorbidity, respectively. Forty-three texture features were computed from the rectus femoris and the vastus intermedius muscles using statistical methods. Model performance was evaluated by computing the area under the receiver operating characteristic curve (AUC) while outcome prediction was evaluated using regression analysis. Models developed achieved a moderate to good AUC (0.67 <= AUC <= 0.79) for categorizing frailty. The stepwise multiple logistic regression analysis demonstrated that they correctly classified 70-87% of the cases. The models were associated with increased comorbidity (0.01 <= p <= 0.18) and were predictive of death for pre-frail and frail participants (0.001 <= p <= 0.016). In conclusion, texture analysis can be useful to identify frailty and assess risk prediction (i.e. mortality) using texture features extracted from muscle ultrasound images in combination with a machine learning approach.This work was supported by the following grants: Grant PID2020-113839RB-I00 funded by MCIN/AEI/10.13039/501100011033 to C.B. DM acknowledges financial support from the Conselleria d ' Educacio, Investigacio, Cultura i Esport, Generalitat Valenciana (grants AEST/2018/021 and AEST/2019/037) .Mirón-Mombiela, R.; Ruiz-España, S.; Moratal, D.; Borrás, C. (2023). Assessment and risk prediction of frailty using texture-based muscle ultrasound image analysis and machine learning techniques. Mechanisms of Ageing and Development. 215. https://doi.org/10.1016/j.mad.2023.11186021

Segunda campaña de excavación en el asentamiento ibérico final de la Casa de la Cabeza (Requena, València)

Second report about the excavations in Casa de la Cabeza, an iberian rural settlement (IInd century BC)

Tercera campaña de excavación en el asentamiento ibérico final de La Casa de la Cabeza (Requena, València)

Preliminary report

Influence of different types of pulp treatment during isolation in the obtention of human dental pulp stem cells

Background: Different methods have been used in order to isolate dental pulp stem cells. The aim of this study was to study the effect of different types of pulp treatment during isolation, under 3% O 2 conditions, in the time needed and the efficacy for obtaining dental pulp stem cells. Material and Methods: One hundred and twenty dental pulps were used to isolate dental pulp stem cells treating the pulp tissue during isolation using 9 different methods, using digestive, disgregation, or mechanical agents, or combining them. The cells were positive for CD133, Oct4, Nestin, Stro-1, CD34 markers, and negative for the hematopoietic cell marker CD-45, thus confirming the presence of mesenchymal stem cells. The efficacy of dental pulp stem cells obtention and the minimum time needed to obtain such cells comparing the 9 different methods was analyzed. Results: Dental pulp stem cells were obtained from 97 of the 120 pulps used in the study, i.e. 80.8% of the cases. They were obtained with all the methods used except with mechanical fragmentation of the pulp, where no enzymatic digestion was performed. The minimum time needed to isolate dental pulp stem cells was 8 hours, digesting with 2mg/ml EDTA for 10 minutes, 4mg/ml of type I collagenase, 4mg/ml of type II dispase for 40 minutes, 13ng/ ml of thermolysine for 40 minutes and sonicating the culture for one minute. Conclusions: Dental pulp stem cells were obtained in 97 cases from a series of 120 pulps. The time for obtaining dental pulp stem cells was reduced maximally, without compromising the obtention of the cells, by combining digestive, disgregation, and mechanical agent

Application of mesenchymal stem cells in bone regenerative procedures in oral implantology. A literature review

Objective: The aim of this work was to review de literature about the role of mesenchymal stem cells in bone regenerative procedures in oral implantology, specifically, in the time require to promote bone regeneration. Study Desing: A bibliographic search was carried out in PUBMED with a combination of different key words. Animal and human studies that assessed histomorphometrically the influence of mesenchymal stem cells on bone regeneration procedures in oral implantology surgeries were examined. Reults: - Alveolar regeneration: Different controlled histomorphometric animal studies showed that bone regeneration is faster using stem cells seeded in scaffolds than using scaffolds or platelet rich plasma alone. Human studies revealed that stem cells increase bone regeneration. - Maxillary sinus lift: Controlled studies in animals and in humans showed higher bone regeneration applying stem cells compared with controls. - Periimplantary bone regeneration and alveolar distraction: Studies in animals showed higher regeneration when stem cells are used. In humans, no evidence of applying mesenchymal stem cells in these regeneration procedures was found. Conclusion: Stem cells may promote bone regeneration and be useful in bone regenerative procedures in oral implantology, but no firm conclusions can be drawn from the rather limited clinical studies so far performed

Extracellular Vesicles from Healthy Cells Improves Cell Function and Stemness in Premature Senescent Stem Cells by miR-302b and HIF-1α Activation.

Aging is accompanied by the accumulation of senescent cells that alter intercellular communication, thereby impairing tissue homeostasis and reducing organ regenerative potential. Recently, the administration of mesenchymal stem cells (MSC)-derived extracellular vesicles has proven to be more effective and less challenging than current stem cell-based therapies. Extracellular vesicles (EVs) contain a cell-specific cargo of proteins, lipids and nucleic acids that are released and taken up by probably all cell types, thereby inducing functional changes via the horizontal transfer of their cargo. Here, we describe the beneficial properties of extracellular vesicles derived from non-senescent MSC, cultured in a low physiological oxygen tension (3%) microenvironment into prematurely senescent MSC, cultured in a hyperoxic ambient (usual oxygen culture conditions, i.e., 21%). We observed that senescent MCS, treated with EVs from non-senescent MCS, showed reduced SA-β-galactosidase activity levels and pluripotency factor (OCT4, SOX2, KLF4 and cMYC, or OSKM) overexpression and increased glycolysis, as well as reduced oxidative phosphorylation (OXPHOS). Moreover, these EVs' cargo induced the upregulation of miR-302b and HIF-1α levels in the target cells. We propose that miR-302b triggered HIF-1α upregulation, which in turn activated different pathways to delay premature senescence, improve stemness and switch energetic metabolism towards glycolysis. Taken together, we suggest that EVs could be a powerful tool to restore altered intercellular communication and improve stem cell function and stemness, thus delaying stem cell exhaustion in aging

PTEN Mediates the Antioxidant Effect of Resveratrol at Nutritionally Relevant Concentrations

Introduction. Antioxidant properties of resveratrol have been intensively studied for the last years, both in vivo and in vitro. Its bioavailability after an oral dose is very low and therefore it is very important to make sure that plasma concentrations of free resveratrol are sufficient enough to be active as antioxidant. Aims. In the present study, using nutritionally relevant concentrations of resveratrol, we aim to confirm its antioxidant capacity on reducing peroxide levels and look for the molecular pathway involved in this antioxidant effect. Methods. We used mammary gland tumor cells (MCF-7), which were pretreated with different concentrations of resveratrol for 48 h, and/or a PTEN inhibitor (bpV: bipy). Hydrogen peroxide levels were determined by fluorimetry, PTEN levels and Akt phosphorylation by Western Blotting, and mRNA expression of antioxidant genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. Resveratrol treatment for 48 h lowered peroxide levels in MCF-7, even at low nutritional concentrations (1 nM). This effect was mediated by the activation of PTEN/Akt pathway, which resulted in an upregulation of catalase and MnSOD mRNA levels. Conclusion. Resveratrol acts as an antioxidant at nutritionally relevant concentrations by inducing the expression of antioxidant enzymes, through a mechanism involving PTEN/Akt signaling pathway

Cellusim: Un simulador 3D en entorno videojuego para la docencia del laboratorio de cultivos celulares

[EN] Cell cultures allow the maintenance of living cells outside the body. This technique is fundamental to study the biological, biochemical and physiological properties of cells and can be used as an experimental model in vitro in the field of biomedical research. The teaching in the laboratory of this technique presents numerous difficulties at a practical, logistical and economic level. On the other hand, the learning of cell culture protocols is an important part of the student's training in Cell Biology. Based on the routine practices carried out within a cell culture laboratory, we have developed the virtual 3D simulator of a cell culture laboratory "Cellusim". In "Cellusim" you can perform basic tasks of the cell culture of an established cell line of your own (Mel-RC08, Gil-Benso et al., 2012). To make Cellusim more attractive and intuitive to students, it has been developed in the Unity graphic environment, commonly used for the development of video games. The system includes the simulator in Unity, a mysql server with users, passwords and logs and a web of help and explanatory videos. In the simulator the student can virtually execute the essential tasks that are performed in a cell culture laboratory, such as thawing cells, seeding the cells, preparing and changing culture media and making cell subcultures. Cellusim is a training tool that allows users to discover the basics of cell culture techniques in a simple and fast way and without the economic costs or time consumption derived from doing the same work in a real laboratory. In order to evaluate the students' learning, Cellusim can be executed in training mode and in evaluation mode, allowing the student to perform all the processes as many times as necessary to become familiar with the protocols and when be able to execute them in evaluation mode. In evaluation mode, Cellusim records student errors in order to score the acquired training. In this communication we present the Cellusim project and the results of the first experiences with volunteer students of Master's Degrees in the School of Medicine.[ES] Los cultivos celulares permiten el mantenimiento de células vivas fuera del organismo. Esta técnica es fundamental para estudiar las propiedades biológicas, bioquímicas y fisiológicas de las células y puede ser utilizada como modelo experimental in vitro en el ámbito de la investigación biomédica. La enseñanza en el laboratorio de esta técnica presenta numerosas dificultades a nivel práctico, logístico y económico. Por otra parte, el aprendizaje de los protocolos del cultivo celular resultan una parte importante de la formación del alumno de Biología Celular. Basándonos en las prácticas rutinarias llevadas a cabo dentro de un laboratorio de cultivos celulares, hemos desarrollado el simulador virtual 3D de un laboratorio de cultivos celulares “Cellusim”. En “Cellusim” se pueden realizar tareas básicas del cultivo celular de una línea celular establecida propia (Mel‐RC08, Gil‐Benso y cols., 2012). Para hacer Cellusim más atractivo e intuitivo a los alumnos, ha sido desarrollado en el entorno gráfico Unity, utilizado habitualmente para el desarrollo de videojuegos. El sistema incluye el simulador en Unity, un servidor mysql con usuarios, contraseñas y registros y una web de ayuda y videos explicativos. En el simulador el alumno puede ejecutar de modo virtual las tareas esenciales que se realizan en un laboratorio de cultivos celulares, tales como la descongelación de células, el sembrado de las células, la preparación y el cambio de medios de cultivo y el subcultivo celular o técnica de doblaje. Cellusim es una herramienta formativa que permite a los usuarios descubrir los fundamentos de las técnicas básicas de cultivo celular de una manera sencilla, rápida y sin los costes económicos ni el consumo de tiempo derivados de realizar el mismo trabajo en un laboratorio real. Para poder evaluar el aprendizaje de los alumnos, Cellusim puede ser ejecutado en modo entrenamiento y en modo evaluación, permitiendo que el alumno realice todos los procesos las veces que sea necesario para familiarizarse con los protocolos y que cuando esté en condiciones pueda ejecutarlas en modo evaluación. En modo evaluación, Cellusim registra los errores del alumno para poder puntuar la formación adquirida. En esta comunicación presentamos el proyecto Cellusim y los resultados de las primeras experiencias con alumnos voluntarios de Master de la Facultad de Medicina.Monleón, D.; Megías, J.; San Miguel, T.; Borrás, C.; Gil Benso, R.; López Ginés, C. (2018). Cellusim: Un simulador 3D en entorno videojuego para la docencia del laboratorio de cultivos celulares. En IN-RED 2018. IV Congreso Nacional de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 176-183. https://doi.org/10.4995/INRED2018.2018.8714OCS17618