The Impact of Melatonin Supplementation and NLRP3 Inflammasome Deletion on Age-Accompanied Cardiac Damage

This study was partially supported by grants from the Instituto de Salud Carlos III through the projects PI13-981, PI16-00519, PI19-01372, and CB/10/00238 (Co-funded by European Regional Development Fund/European Social Fund "Investing in your future"); the Conserjeria de Economia, Innovacion, Ciencia y Empleo, Junta de Andalucia (CTS-101), Spain, and also by Sohag University, Egypt. M.F.-O and J.F.-M are supported by a FPU fellowship from the Ministerio de Educacion, Spain.To investigate the role of NLRP3 inflammasome in cardiac aging, we evaluate here morphological and ultrastructural age-related changes of cardiac muscles fibers in wild-type and NLRP3-knockout mice, as well as studying the beneficial effect of melatonin therapy. The results clarified the beginning of the cardiac sarcopenia at the age of 12 months, with hypertrophy of cardiac myocytes, increased expression of beta-MHC, appearance of small necrotic fibers, decline of cadiomyocyte number, destruction of mitochondrial cristae, appearance of small-sized residual bodies, and increased apoptotic nuclei ratio. These changes were progressed in the cardiac myocytes of 24 old mice, accompanied by excessive collagen deposition, higher expressions of IL-1 alpha, IL-6, and TNF alpha, complete mitochondrial vacuolation and damage, myofibrils disorganization, multivesicular bodies formation, and nuclear fragmentation. Interestingly, cardiac myocytes of NLRP3(-/-) mice showed less detectable age-related changes compared with WT mice. Oral melatonin therapy preserved the normal cardiomyocytes structure, restored cardiomyocytes number, and reduced beta-MHC expression of cardiac hypertrophy. In addition, melatonin recovered mitochondrial architecture, reduced apoptosis and multivesicular bodies' formation, and decreased expressions of beta-MHC, IL-1 alpha, and IL-6. Fewer cardiac sarcopenic changes and highly remarkable protective effects of melatonin treatment detected in aged cardiomyocytes of NLRP3(-/-) mice compared with aged WT animals, confirming implication of the NLRP3 inflammasome in cardiac aging. Thus, NLRP3 suppression and melatonin therapy may be therapeutic approaches for age-related cardiac sarcopenia.Instituto de Salud Carlos III (European Regional Development Fund/European Social Fund "Investing in your future") PI13-981 PI16-00519 PI19-01372 CB/10/00238Junta de Andalucia CTS-101Sohag UniversityGerman Research Foundation (DFG

Age and Chronodisruption in Mouse Heart: Effect of the NLRP3 Inflammasome and Melatonin Therapy

Age and age-dependent inflammation are two main risk factors for cardiovascular diseases. Aging can also affect clock gene-related impairments such as chronodisruption and has been linked to a decline in melatonin synthesis and aggravation of the NF- B/NLRP3 innate immune response known as inflammaging. The molecular drivers of these mechanisms remain unknown. This study investigated the impact of aging and NLRP3 expression on the cardiac circadian system, and the actions of melatonin as a potential therapy to restore daily rhythms by mitigating inflammaging. We analyzed the circadian expression and rhythmicity of clock genes in heart tissue of wild-type and NLRP3-knockout mice at 3, 12, and 24 months of age, with and without melatonin treatment. Our results support that aging, NLRP3 inflammasome, and melatonin affected the cardiac clock genes expression, except for Rev-erba, which was not influenced by genotype. Aging caused small phase changes in Clock, loss of rhythmicity in Per2 and Rora, and mesor dampening of Clock, Bmal1, and Per2. NLRP3 inflammasome influenced the acrophase of Clock, Per2, and Rora. Melatonin restored the acrophase and the rhythm of clock genes affected by age or NLRP3 activation. The administration of melatonin re-established murine cardiac homeostasis by reversing age-associated chronodisruption. Altogether, these results highlight new findings about the effects aging and NLRP3 inflammasome have on clock genes in cardiac tissue, pointing to continuous melatonin as a promising therapy to placate inflammaging and restore circadian rhythm in heart muscle. Additionally, light microscopy analysis showed age-related morphological impairments in cardiomyocytes, which were less severe in mice lacking NLRP3. Melatonin supplementation preserved the structure of cardiac muscle fibers in all experimental groups.Instituto de Salud Carlos III (Ministerio de Economia y Competitividad, Spain) (European Regional Development Fund/European Social Fund "Investing in your future") PI13-981 PI16-00519 PI19-01372 CB16-10-00238 CB16/10/00239Junta de Andalucia CTS-101Spanish Governmen

Analysis of Plasma MicroRNAs as Predictors and Biomarkers of Aging and Frailty in Humans

Although circulating microRNAs (miRNAs) can modulate gene expression and affect immune system response, little is known about their participation in age-associated frailty syndrome and sarcopenia. The aim of this study was to determine miRNAs as possible biomarkers of age and frailty and their correlation with oxidative and inflammatory state in human blood. Three inflammation-related miRNAs (miR-21, miR-146a, and miR-223) and one miRNA related with the control of melatonin synthesis (miR-483) were analyzed. Twenty-two healthy adults, 34 aged robust, and 40 aged fragile patients were selected for this study. The expression of plasma miRNAs was assessed by RT-qPCR; plasma cytokines (IL-6, IL-8, IL-10, and TNFα) were analyzed by commercial kits, and plasma advanced oxidation protein products (AOPP) and lipid oxidation (LPO) were spectrophotometrically measured. Fragile subjects had higher miR-21 levels than control subjects, whereas miR-223 and miR- 483 levels increased at a similar extend in both aged groups. All cytokines measured increased in aged groups compared with controls, without differences between robust and fragile subjects. The fragile group had a TNFα/IL-10 ratio significantly higher than robust and control groups. Aged groups also had higher AOPP and LPO levels than controls. Women presented higher AOPP and LPO levels and increased expression of miR-483 compared with men. Positive correlations between miR-21 and AOPP and between miR-483 and IL-8 were detected. The expression of miR-21 and the TNFα/IL-10 ratio were correlated positively with the presence of frailty, which suggests that these markers can be considered as possible biomarkers for age-related frailty.This work was partially supported by grants from the Ministerio de Economía, Industria y Competitividad y por el Fondo de Desarrollo Regional Feder, Spain nos. RD12/ 0043/0005, PI13-00981, and CB16-10-00238 and from the Universidad de Granada, Spain no. CEI2014-MPBS3

Cuentos de nunca acabar. Aproximaciones desde la interculturalidad

Cuentos de nunca acabar. Aproximaciones desde la interculturalidad, surge después de la pandemia y su imposibilidad de socializar “en persona” con los compañeros de eventuales encuentros, porque la Comprensión Lectora tenía que reinventarse para su nueva reflexión cognitiva, adaptación contextual y reconstrucción del conocimiento. Este renovado enfoque de la realidad postpandemia, concebido en el marco de la educación intercultural comunitaria, busca potencializar los entornos naturales, sociales y culturales como recursos de aprendizaje multidisciplinario a través del lenguaje animado de los cuentos. En este marco, había que dinamizar la asignatura de Comunicación Oral y Escrita, que se dicta en los Primeros Niveles de los Centros de Apoyo de Otavalo, Cayambe, Latacunga y Riobamba, mediante un eje transversal donde los estudiantes escriban fundamentados en valores de la cosmovisión andina, considerando que provienen de varios lugares de la sierra y amazonía ecuatoriana. Todo surgió del encuentro presencial de un sábado cualquiera donde los estudiantes realizaban ejercicios narrativos, logrando una apreciable respuesta de imaginación, más emotiva que la clásica tarea de las Unidades, tanto así que, pasados unos días, seguían llegando sus escritos a mi correo. Entonces nos pusimos manos a la obra, cada estudiante tendría dos opciones como Actividad Integradora, la primera consistía en escribir un cuento de su propia inspiración, y la segunda analizar un clásico para comentar sus valores y antivalores. La mayor parte de estudiantes decidió escribir su propio cuento, de donde se escogieron algunas participaciones que podrían considerarse originales, para una edición que, respetando la transcripción de la tradición oral que prima en los sectores comunitarios, nos concretamos en revisar la puntuación y ortografía para publicarlos. Con esto buscamos innovar la Actividad Integradora, por algo más práctico y operativo para configurar los Objetos de Aprendizaje que buscamos. Así nació, en medio del camino, este libro de Cuentos de nunca acabar. Aproximaciones desde la interculturalidad, que ponemos en sus manos. Hernán Hermosa Mantilla Quito, junio de 202

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

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

Chronodisruption: melatonin deficiency and innate immunity. Connection with mitochondrial dysfunction during aging

Cardiovascular diseases are the leading cause of death in the world, with aging being the main risk factor associated with these pathologies (Global Health and Aging, 2019). The world’s population is aging at an unprecedented rate, and study of the mechanisms underlying this process is vitally important from a health, economic and social point of view. Aging is characterized by a deregulation of the immune system resulting in a subclinical, sterile, asymptomatic and chronic pro-inflammatory state known as inflammaging (Franceschi and Campisi, 2014). This inflammatory condition, coupled with oxidative stress, leads to mitochondrial dysfunction and subsequent apoptosis, facilitating the release of reactive species, ATP, and mtDNA. These hazard signals are recognized by the NLRP3 inflammasome, a multiprotein complex responsible for the maturation of pro-inflammatory cytokines dependent on NF-κB, including IL-1β. In this way, this process perpetuates a vicious cycle that results in systemic inflammation that is accompanied by symptoms of immunosenescence and activation of the innate immune pathway. Alterations in the regulation of mitochondrial homeostasis, including mechanisms of mitochondrial dynamics, autophagy, apoptosis, as well as decreased antioxidant defense that occurs with aging, such as the Nrf2-dependent pathway, may be necessary for activation of NLRP3 inflammasome. In addition, the effect this inflammasome may have on mitochondrial function or on the antioxidant pathway of Nrf2 during cardiac aging remains unknown. Interestingly, numerous scientific studies relate inflammaging to the disruption of circadian rhythms, which allow the organism to adapt and anticipate environmental changes to ensure optimal physiological performance (Acuña-Castroviejo et al., 2017; Acuña-Fernández et al., 2020; Volt et al., 2016). In mammals, circadian rhythms are regulated by a central clock, located in the suprachiasmatic nucleus, and by peripheral clocks, located in virtually all tissues, including the heart. While there seems to be a connection between aging, clock genes and innate immune response, the molecular mechanisms that link these processes remain a mystery. To date, the influence of NF-κB on the disruption of circadian rhythms during aging has been demonstrated. However, little is known about NLRP3's involvement in aging-associated chronodisruption. It should be noted that aging manifests the progressive loss of strength and muscle mass. This process is defined as sarcopenia and is considered one of the main causes of reduced physical performance and impaired cardiorespiratory function in patients with heart failure (Curcio et al., 2020). Numerous clinical and experimental studies have shown that aging is associated with histological, structural, and functional changes in cardiac tissue (Lakatta, 2002). Our group demonstrated that the absence of the NLRP3 inflammasome reduced sarcopenia in skeletal muscle (Sayed et al., 2019). Given these results, we consider it of interest to analyze the involvement of NLRP3 inflammasome activation in structural alterations in the aging heart. Melatonin is a hormone synthesized by the pineal gland, as well as by most organs and tissues, including the heart. Pineal melatonin has chronobiotic actions and its production decreases with age. This decline has been linked to changes in circadian rhythms, increased inflammation, and development of cardiac pathologies (Hardeland, 2012). Extrapineal melatonin has antioxidant and anti-inflammatory properties (Hawthorn et al., 2012). In experimental models that include chronic and acute inflammation as well as aging in the mouse heart, melatonin decreased innate immune response, counteracted oxidative stress, and improved the activity of cardiac mitochondria (García et al., 2015; Rodríguez et al., 2007). In addition, melatonin administration has been shown to improve muscle function and reduce inflammation in athletes (Leonardo-Mendonca et al., 2017). Considering this theoretical framework, our work focused on the study of the causal relationship between chronodisruption, melatonin deficiency, and innate immunity, as well as the involvement of the NLRP3 inflammasome-mediated immune response during cardiac aging. To accomplish this, the aims were to evaluate cardiac tissue of wild type C57/Bl6 mice and mice with a C57/Bl6 background knocked-out for NLRP3 inflammasome (NLRP3-/-) of 3, 12 and 24 months of age in the following parameters: 1.- Mitochondrial pathway: mitochondrial dynamics, autophagy, apoptosis and mitochondria ultrastructure. 2.- Antioxidant pathway dependent on Nrf2. 3.- Biological clocking: expression of clock genes, rhythmicity, acrophase, amplitude and mesor. 4.- Histological study and MRI of heart muscle. 5.- Effects of melatonin treatment on the parameters mentioned above. The results of this doctoral thesis show the deleterious effect NLRP3 inflammasome has on mitochondrial function during aging, as its absence prevented damage to mitochondrial dynamics and structure. Melatonin treatment also reestablished mitochondrial dynamics, had an anti-apoptotic action, restored the Nrf2 dependent antioxidant pathway, and preserved mitochondrial structure during aging. With reference to the biological clock pathway, it could be found that aging, melatonin, and presence of the NLRP3 inflammasome had significant effects on expression observed in the clock genes, except for the Rev-erbα gene, which was not affected by the mouse genotype. Small phase changes were observed in the Clock gene, loss of rhythmicity in Per2 and Rorα and a tendency for mesor to decrease with aging. The NLRP3 inflammasome influenced the acrophase of Clock, Per2 and Rorα, suggesting some negative impact on the function of the myocardium. Melatonin restored rhythms and acrophases in cardiac tissue, highlighting its clinical potential in the prevention and treatment of chronodisruption. Besides these changes, the results indicate that the local chronobiotic system of the heart is highly protected against aging. Finally, it was concluded that NLRP3 is involved in cardiac sarcopenia, as 24-month-old mutant mice had less thickening of the ventricular wall, less fibrosis, lower expression of inflammatory cytokines, and lower mitochondrial damage compared to wild type mice. Again, we observed a prophylactic effect of melatonin in preserving the structure and number of cardiomyocytes and reducing pro-inflammatory and hypertrophic markers as well as apoptosis. It is therefore inferred that suppression of the NLRP3 inflammasome and implementation of melatonin therapy may be beneficial therapeutic approaches to ameliorate cardiac aging and sarcopenia.Tesis Univ. Granada.Formación de Profesorado Universitario (FPU) - Spanish Ministry of Science, Innovation and UniversitiesInternational Mobility Scholarship for PhD StudentsISCIIIJunta de Andalucía - P18-RT-69

Alternaivas de aprendizaje para la educación física

The investigation arises for the necessity of facilitating to the professors in alternative formation of learning with an integrative, personalized and systemic focus that contains to the necessary elements for the attention to school with special educational necessities, as way of contributing to its integral preparation according to the development areas in the physical, social, conductual and talkative. The use of the empiric methods will allow to determine the main difficulties in the teaching of the Physical Educationin school with special educational necessities and the deficiencies in the integral formation, the strategy is structured starting from the modulation of the different elements that compose it after having determined the necessities and the students' interests andthe objective institutionalism. Se it sustains in philosophical, educational and didactic postures that are adjusted to the educational politics in the superior education and it possesses general characteristics that they distinguish it, premises that guaranteed their efficiency, the contents and the roads for their implementation, evaluation. Making emphasis in the use of the texts martianos like one of the alternatives (interpretation, comment, significance, valuation, present time) it is without place to doubts, a process guided to the formation and education (in their widest and comprehensive meanings) that allows to improve the communication, the language, the oral expression, to promote national and universal culture, value political and ideological of the students with special educational necessities.La investigación surge por la necesidad de facilitar a los profesores en formación alternativas de aprendizaje con un enfoque integrador, personalizado y sistémico que agrupa a los elementos necesarios para la atención a escolares con necesidades educativas especiales, como modo de contribuir a su preparación integral de acuerdo a las áreas de desarrollo en lo físico, social, conductual y comunicativo. La utilización delos métodos empíricos permitirá determinar las principales dificultades en la enseñanza de la Educación Física en escolares con necesidades educativas especiales y las deficiencias en la formación integral, la estrategia se estructura a partir de la modelación de los diferentes elementos que la componen luego de haber determinado las necesidades e intereses de los estudiantes y los objetivos institucionales. Se sustenta en posturas filosóficas, educativas y didácticas que se ajustan a la política educativa en la educación superior y posee características generales que la distinguen, premisas que garantizaran su eficiencia, los contenidos y las vías para su implementación, evaluación. Hace énfasis en la utilización de los textos martianos como una de las alternativas (interpretación, comentario, significación, valoración, actualidad) es sin lugar a dudas, un proceso orientado a la formación y educación, en sus más amplias y abarcadoras acepciones, que permite mejorar la comunicación, el lenguaje, la expresión oral, promover cultura nacional y universal

Melatonin/Nrf2/NLRP3 Connection in Mouse Heart Mitochondria during Aging

Aging is a major risk for cardiovascular diseases (CVD). Age-related disorders include oxidative stress, mitochondria dysfunction, and exacerbation of the NF- B/NLRP3 innate immune response pathways. Some of the molecular mechanisms underlying these processes, however, remain unclear. This study tested the hypothesis that NLRP3 inflammasome plays a role in cardiac aging and melatonin is able to counteract its e ects. With the aim of investigating the impact of NLRP3 inflammasome and the actions and target of melatonin in aged myocardium, we analyzed the expression of proteins implied in mitochondria dynamics, autophagy, apoptosis, Nrf2-dependent antioxidant response and mitochondria ultrastructure in heart of wild-type and NLRP3-knockout mice of 3, 12, and 24 months-old, with and without melatonin treatment. Our results showed that the absence of NLRP3 prevented age-related mitochondrial dynamic alterations in cardiac muscle with minimal e ects in cardiac autophagy during aging. The deficiency of the inflammasome a ected Bax/Bcl2 ratio, but not p53 or caspase 9. The Nrf2-antioxidant pathway was also una ected by the absence of NLRP3. Furthermore, NLRP3-deficiency prevented the drop in autophagy and mice showed less mitochondrial damage than wild-type animals. Interestingly, melatonin treatment recovered mitochondrial dynamics altered by aging and had few e ects on cardiac autophagy. Melatonin supplementation also had an anti-apoptotic action in addition to restoring Nrf2-antioxidant capacity and improving mitochondria ultrastructure altered by aging.Instituto de Salud Carlos III (Ministerio de Economia y Competitividad, Spain) PI16-00519 PI19-01372 CB16-10-00238European Regional Development Fund/European Social Fund "Investing in your future"Junta de Andalucía CTS-101German Research Foundation (DFG

The Impact of Melatonin and NLRP3 Inflammasome on the Expression of microRNAs in Aged Muscle

Muscular aging is a complex process and underlying physiological mechanisms are not fully clear. In recent years, the participation of the NF-kB pathway and the NLRP3 inflammasome in the chronic inflammation process that accompanies the skeletal muscle’s aging has been confirmed. microRNAs (miRs) form part of a gene regulatory machinery, and they control numerous biological processes including inflammatory pathways. In this work, we studied the expression of four miRs; three of them are considered as inflammatory-related miRs (miR-21, miR-146a, and miR-223), and miR-483, which is related to the regulation of melatonin synthesis, among other targets. To investigate the changes of miRs expression in muscle along aging, the impact of inflammation, and the role of melatonin in aged skeletal muscle, we used the gastrocnemius muscle of wild type (WT) and NLRP3- knockout (NLRP3−) mice of 3, 12, and 24 months-old, with and without melatonin supplementation. The expression of miRs and pro-caspase-1, caspase-3, pro-IL-1β, bax, bcl-2, and p53, was investigated by qRT-PCR analysis. Histological examination of the gastrocnemius muscle was also done. The results showed that age increased the expression of miR-21 (p < 0.01), miR-146a, and miR-223 (p < 0.05, for both miRs) in WT mice, whereas the 24-months-old mutant mice revealed decline of miR-21 and miR-223 (p < 0.05), compared to WT age. The lack of NLRP3 inflammasome also improved the skeletal muscle fibers arrangement and reduced the collagen deposits compared with WT muscle during aging. For the first time, we showed that melatonin significantly reduced the expression of miR-21, miR-146a, and miR-223 (p < 0.05 for all ones, and p < 0.01 for miR-21 at 24 months old) in aged WT mice, increased miR-223 in NLRP3− mice (p < 0.05), and induced miR-483 expression in both mice strains, this increase being significant at 24 months of age.Instituto de Salud Carlos III (Ministerio de Economía y Competitividad, Spain)European Regional Development Fund/European Social FundConsejería de Innovación, Ciencia y Empresa, Junta de Andalucía (CTS-101)FPU fellowships from the Ministerio de Educació