Abstracts of the XL Congress of the Spanish Society of Physiological Sciences (SECF)

Joint meeting between Spanish and Portuguese physiologists

19-22 September 2022, Badajoz, Spain

1. Welcome letter

Dear friends and physiologist colleagues,

In the last congress held in Cádiz (September 2018), the Spanish Society of Physiological Sciences trusted on us to organize the next SECF Congress (XL Congress of the Spanish Society of Physiological Sciences: joint meeting between Spanish and Portuguese physiologists) in Extremadura, in the city of Badajoz, originally scheduled for September 2020. Unfortunately, it could not be held due to the COVID-19 pandemic.

It is now when, together with the SECF, we resume the celebration of the congress that will take place in Badajoz from September 19 to 22, 2022.

I do not want to miss this opportunity without remembering all those anonymous people and relatives who left us because of Covid-19, and especially those physiologist colleagues and friends that we have lost.

Now more than ever, we must get back to this type of scientific meeting that will undoubtedly help us to gradually redirect our lives from both a professional and a personal point of view.

Thus, this XL Congress of the Spanish Society of Physiological Sciences: joint meeting between Spanish and Portuguese physiologists is not just any other congress. It is the Congress, the one that will lead us to the normalization of our personal and professional lives, meet again with physiologist friends, and connect science with celebrations in harmony.

Spanish-Portuguese physiologists have the challenge of holding a congress that is big in every aspect, special, the first after a pandemic that will always remain in our memory.

Although it came by surprise, we assumed the assignment to organize the congress with enthusiasm. It is a challenge and an honour to host the SECF congress 23 years after the last one held in Extremadura in 1999. It must also be emphasized that, after 39 editions of the SECF congress (since its constitution in 1952 and the first congress held in Madrid in 1953), it will be the first time that it is chaired by a woman.

We will try to do our best to maintain the high scientific and sociocultural level achieved in previous SECF meetings. From September 19 to 22, 2022, in Badajoz, we will try to combine Science and Spanish-Portuguese fraternization, as well as good environment and fun. As we organize the congress with our Portuguese neighbours, we intend that this allows us to establish collaborative ties between groups of physiologists of both nationalities and that this enriches the congress not only from a scientific point of view but also from a human point of view. We hope to make a congress that is unforgettable in all aspects and for this we must count on the most important thing: all of you, the attendants. We await you with open arms. Welcome.

To accomplish our aims, we request your help in developing the scientific program that will consist of plenary lectures, symposia, and communications (oral and poster). We also want to encourage undergraduate and master students to present their final degree projects, either poster or oral communication, in a dedicated session. We have also planned a novel symposium dedicated to young pre- and post-doctoral researchers, and another one on animal experimentation that will be valid and certified as a training activity for the maintenance of the corresponding accreditation. All together, we intend to achieve the most suitable environment to discuss from the simplest physiological processes to the most integrative ones, promoting the translation of basic findings to society's problems.

The scientific program will be complemented with social events in the evenings which will include tourism and Spanish-Portuguese gastronomy. All attendants can participate in these activities that are included in registration fee. We will also celebrate the traditional gala dinner and will try to get an optimal price that allows the assistance of anyone who wishes.

Badajoz is one of the least explored places of Spain, but it has a relevant touristic attraction that should be taken more seriously and not only like a crossing place. There are some amazing places to visit, as you will find out. For example, the beautiful Plaza Alta where the markets were held under its arches during the Middle Ages. Also, the Alcazaba, a fortification made by Muslims, which means citadel, is said to be the largest in Europe and one of the largest in the world, considering the perimeter of its walls. The bastioned wall that we can see in Badajoz is a military fortification formed by different defensive elements such as forts, towers, moats, etc. We can also highlight the gorgeous Metropolitan Cathedral Church, next to the City Hall in Plaza de España, the Plaza de la Soledad with amazing buildings and the Hermitage of the Virgen de la Soledad, the Patron Saint of the city. Badajoz is also the “bridges city”, with its 4 bridges over the Guadiana River, which has fantastic gardens where you can walk and do sports. Badajoz is located in a strategic crossing, very close to the border with Portugal, and between the roads that link Castilla with Andalucía. It is also an eminently commercial city. In fact, the commerce in our city surprises those who visit it.

We hope we have convinced you to come to Badajoz to participate in the XL SECF Congress from September 19 to 22, 2022.

We are awaiting you in Badajoz. We are sure that with your presence it will be an exceptional congress. See you later!

Ana Beatriz Rodríguez Moratinos

President of the Organizing Committee

XL Congress of the Spanish Society of Physiological Sciences

(Joint Meeting between Spanish and Portuguese physiologists)

Badajoz, September 19-22, 2022.

2. Summary of the Scientific Program

XL CONGRESS OF THE SPANISH SOCIETY OF PHYSIOLOGICAL SCIENCES

(Joint meeting between Spanish and Portuguese physiologists), 19-22 September 2022

MONDAY, 19th SEPTEMBER

TUESDAY, 20th SEPTEMBER

WEDNESDAY, 21st SEPTEMBER

THURSDAY, 22nd SEPTEMBER

3. Committees

ORGANIZING

Meritxell López Gallardo (Universidad Complutense de Madrid)

Jorge Juan García Seoane (Universidad Complutense de Madrid)

Vicente Martínez Perea (Universidad Autónoma de Barcelona)

Eva Mª Marco López (Universidad Complutense de Madrid)

Antonio González Mateos (Universidad de Extremadura)

Jesús Francisco Rodríguez Huertas (Universidad de Granada)

Mª Inmaculada García Fernández (Universidad de Málaga)

Ana Beatriz Rodríguez Moratinos (Universidad de Extremadura)

José Antonio Pariente Llanos (Universidad de Extremadura)

LOCAL

Ana Beatriz Rodríguez Moratinos (Universidad de Extremadura)

José Antonio Pariente Llanos (Universidad de Extremadura)

Javier Espino Palma (Universidad de Extremadura)

María Garrido Álvarez (Universidad de Extremadura)

Cristina Carrasco Romero (Universidad de Extremadura)

Mª Ángeles Gómez Zubeldia (Universidad de Extremadura)

Mª Pilar Terrón Sánchez (Universidad de Extremadura)

Elena Fernández Delgado (Universidad de Extremadura)

Samuel Estirado Rivera (Universidad de Extremadura)

Patricia Cosme Canito (Universidad de Extremadura)

Lourdes Franco Hernández (Universidad de Extremadura)

SCIENTIFIC

Cristina Camello Almaraz (Universidad de Extremadura)

Pedro Javier Camello Almaraz (Universidad de Extremadura)

Mamede de Carvalho (Universidade de Lisboa)

Antonio González Mateos (Universidad de Extremadura)

Adelino Leite Moreira (Universidade do Porto)

Silvia Conde (Universidade Nova de Lisboa)

Marcos Maynar Mariño (Universidad de Extremadura)

Eduardo Ortega Rincón (Universidad de Extremadura)

Pedro Cosme Redondo Liberal (Universidad de Extremadura)

Isabel Rocha (Universidade de Lisboa)

Juan Antonio Rosado Dionisio (Universidad de Extremadura)

Paulo Correia De Sa (Instituto de Ciências Biomédicas de Abel Salazar)

Ginés María Salido Ruíz (Universidad de Extremadura)

Raquel Seiça (Universidade de Coimbra)

Vera Geraldes (Universidade de Lisboa)

José Antonio Tapia García (Universidad de Extremadura)

Raquel Tarazona Lafarga (Universidad de Extremadura)

Juan Manuel Moreno Vazquez (Universidad de Extremadura)

Francisco Luna Giles (Universidad de Extremadura)

Emilio Viñuelas Zahinos (Universidad de Extremadura)

Pablo Carús (Universidade de Evora)

Jonathan Delgado Adámez (Instituto Tecnológico Agroalimentario de Extremadura, CICYTEX)

Javier Rocha Pimienta (Instituto Tecnológico Agroalimentario de Extremadura, CICYTEX)

COLLABORATORS

Esther de Jesús Almeida García (University of Extremadura)

Ángel Verdasco Espada (University of Extremadura)

María Cantero Calleja (University of Extremadura)

Ángela Sánchez Fernández (University of Extremadura)

Nerea Ye Keituqwa Rebollo (University of Extremadura)

María Xiang Pozo Moreno (University of Extremadura)

Enrique Hernández Mayordomo (University of Extremadura)

4. Collaborating institutions

5. Sponsors

6. Abstract book cover

7. Table of contents

LECTURES

Opening Lecture

Antonio Negrín Award Lecture

SYMPOSIA

Symposium 1: Physiological determinants of food choices and consumption

Symposium 2: Immunosenescence and cancer immunotherapy

Symposium 3: Autonomic nervous system in disease: From sensors, to circuits, to reflex

Symposium 4: Physiopathological role of intestinal microbiota: A new player in health and disease

Symposium 5: Molecular pathophysiology of cancer

Symposium 6: Cardiovascular physiology: Pulmonary hypertension - the right ventricle under pressure

Symposium 7: Multi-organ damage: Role of inflammasome

Symposium 8: Circadian rhythms: Clock, sleep and metabolism

Symposium 9: Animal research in the 21st century

Symposium 10: Teaching in Physiology

Symposium 11: Environmental physiology

Symposium 12: Physiological aspects of assisted reproduction

ORAL COMMUNICATIONS

Oral session 1: Neurophysiology

Oral session 2: Cellular physiology

Oral session 3: Cardiovascular physiology

Oral session 4: Endocrinology and Reproduction

Oral session 5: Metabolism and Nutrition

Oral session 6: Final Degree/Master Projects & Teaching in Physiology

Oral session 7: Animal research

Oral session 8: Neurophysiology & Cellular physiology II

Oral session 9: Miscellaneous

POSTERS

Poster session 1

Poster session 2

Poster session 3

LECTURES

PL-01 (Opening Lecture / Plenary Lecture 1)

Aging is physiological: Reflections of an optimistic gerontologist

^*Jose Viña

Freshage Research Group, Department of Physiology, Faculty of Medicine, University of Valencia

Aging is one of the greatest challenges of today's society, both on a personal and human level as well as on an economic level. Life expectancy at birth has grown more in the 20th century than in all recorded history. Currently, Spain is the fourth oldest country in the world (after Japan, Singapore and Switzerland). The maximum life expectancy today is around 110 years. The voices that indicate that we can live many more years, and even be immortal, have no scientific basis in light of current knowledge. This presentation will emphasize that aging is a normal, physiological process, and not a disease. Naturally, there are diseases associated with aging, but this does not indicate that aging itself is not a process that falls within the field of physiology. The mechanisms of aging are not completely understood, although there is a great deal of information about them and the cellular signaling pathways that lead to the loss of resilience associated with aging. It is of great interest to emphasize healthy physiological aging. In medical terms, unhealthy aging leads to frailty which leads to disability. The mechanisms of frailty will be analyzed, the possible interventions that lead to a considerable delay in the transition to disability as well as possible physiological interventions to delay unhealthy aging at the cognitive level. Finally, and as an epilogue, we will analyze what are the general ideas, based on scientific facts, to promote a physiological, healthy and productive aging.

Keywords: Aging, frailty, resilience, cognition

Corresponding author: Jose Viña, https://orcid.org/0000-0001-9709-0089

PL-02 (Antonio Negrín Award Lecture / Plenary Lecture 2)

Selective autophagy in the physiology of healthy aging

¹Ana M Cuervo

¹ Institute for Aging Studies, Albert Einstein College of Medicine, New York, NY 10461 USA.

All the cells of the organism contain mechanisms dedicated exclusively to ensuring quality control of proteins and organelles. The loss of function of these control systems is a feature common to all aged organisms, and it has also been shown that it contributes to aggravating the symptoms of a wide group of age-related diseases. In our group, we are interested in understanding the mechanisms and physiological relevance of one of these cellular recycling systems, specifically autophagy, with the aim of better understanding the consequences of its functional failure with age. In this talk, some of our most recent advances in the molecular dissection of a selective form of autophagy known as chaperone-mediated autophagy (CMA) will be described. To better understand the physiological relevance of this type of autophagy, we have generated mouse models where the CMA process can be directly measured in multiple organs and reduce or increase its activity. It will be presented some results obtained in these models in which the importance of CMA has been demonstrated in a variety of functions that include the regulation of the metabolism of sugars and lipids, the activation of stem cells, the maintenance of circadian rhythms and response to stressful situations, among others. Finally, some of the efforts made to pharmacologically modulate this type of autophagy in the context of diseases associated with aging will be also described.

Keywords: autophagy, chaperones, aging, cellular recycling.

Corresponding author: Ana Maria Cuervo, https://orcid.org/0000-0002-0771-700X

SYMPOSIA

Symposium 1: Physiological determinants of food choices and consumption

S1-01

Food-saliva interactions: Mechanisms and implications

^*1Elsa Lamy

¹ MED – Mediterranean Institute for Agriculture, Environment and Development; IIFA – Instituto de Investigação e Formação Avançada; University of Evora.

Saliva is the fluid that lies in the oral cavity and that bathes oral structures. Besides having a recognized role in oral protection and health, saliva plays a role in food intake. This role can be seen in different ways. At one hand, saliva is mixed with food and interacts with its components; this can interfere the physical-chemical nature of these molecules and/or the way they reach the sites of sensory reception (taste, aroma, etc.). Moreover, the chemical composition of saliva can affect the receptors bathed by this fluid. For example, higher levels of Na⁺ results in continuous higher stimulation of salty taste receptors, affecting the detection thresholds. Other inorganic and/organic saliva compounds can have similar effect. All this, together, show that saliva can affect the way food is perceived in the mouth. So, saliva may help to explain food choices. At the other hand, saliva is a fluid whose composition changes with many factors, many of which are not completely understood until the moment. The type of diet induces changes in saliva proteome, as it has been seen in animal models and humans. So, we can hypothesize that salivary proteome may be useful as a source of biomarkers of dietary habits, although this needs to be further explored. This dynamic nature of a fluid that interferes in oral food perception needs to be further explored and can be of major interest in the context of nutrition. These different aspects will be presented and discussed in this presentation.

Keywords: Food choices, ingestive behavior, oral food perception, saliva

Corresponding author: Elsa Lamy (ecsl@uevora.pt)

S1-02

Health eating: The importance of the first 8000 days of life

¹Pedro Moreira

¹ Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto

Decisions about what, how much and when to eat are complex processes influenced by many factors. Appetite traits are the drivers of these processes, reflecting a complex combination of internal hunger and/or satiety cues, with environmental cues such as attitudes and psychosocial factors related to food selection and dietary choices. In this communication, we will consider some constructs and measures to assess pregnant, parental, and children´s eating behaviors, and their potential role in shaping, children's self-regulation, eating habits and weight status, during infancy, childhood, and adolescence. In this context, data will be presented to stress the importance of investing as early as possible - since preconception, and during the critical periods from conception until the first 1000 days of life, and during the times of growth and development over the next 7000 days - involving health professionals, parents, and educators, to establish adequate eating habits for a lifetime.

Keywords: appetite; intake; infancy; childhood.

Corresponding author: Pedro Moreira, https://orcid.org/0000-0002-7035-7799

S1-03

Mechanisms involved in the control of feeding behavior and food intake in relation to food flavor

^*Emilio Martinez de Victoria Muñoz

Department of Physiology. University of Granada. Granada. Spain

Control of eating behavior involves complex mechanisms including homeostatic, hedonic, and cognitive. Homeostatic mechanisms are related to nerve and humoral signals that inform about the general state of the organism regarding gastrointestinal function, energy needs and energy stores. These homeostatic mechanisms affect the orosensory properties of the food consumed, especially taste and smell that determine, along with other sensory modalities, the flavor of a food and therefore affects it the hedonic aspects. Within the hedonic aspects we will focus on those related to changes in food intake throughout a meal episode and that affect the pleasure of eating a food based on its palatability (hunger, satiation, satiety, preferences, aversions, reward systems). Finally, we will address the factors related to food choice and intake in relation to the perception of flavor (taste, smell, texture) and its role in the processes of satiation and satiety and therefore in the total intake of energy. The latter is important to prevent the development of overweight and obesity that affect a significant part of the world's population and are related to other morbidities.

Keywords: Food behaviors, flavor. orosensorial, obesity.

Corresponding author: Emilio Martinez de Victoria, https://orcid.org/0000-0002-2507-6541

Symposium 2: Immunosenescence and cancer immunotherapy

S2-01

Immunosenescence and cancer immunology: study in acute myeloid leukaemia patients

^*1Raquel Tarazona, ¹I Valhondo, ¹B Sánchez Correa, ¹N López-Sejas, ²F Hassouneh, ³A Pera, ¹JG Casado, ^2,3 R Solana

¹ Immunology Unit, Department of Physiology, University of Extremadura, Cáceres, Spain ² Maimonides Biomedical Research institute of Cordoba (IMIBIC). Reina Sofía University Hospital, Córdoba, Spain

³ Immunology Unit, Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain

The incidence of cancer increases with age and is therefore generally considered a disease of old age. Different mechanisms have been proposed to explain the increased incidence of cancer with age, including age-associated decline in innate and adaptive immunity, a process termed immunosenescence. The involvement of immunosenescence in the increased risk of cancer in the elderly has been widely debated and it has been proposed that alterations in immunosurveillance against cancer could, at least in part, contribute to this process. Age-related alterations in natural killer (NK) cells include changes in the redistribution of NK cell subsets and downregulation of activating receptors and cytotoxic proteins. In T cells, immunosenescence also alters T cell compartment, phenotype, and function. The impact of latent cytomegalovirus (CMV) and other chronic infections on the immune system has also been recognized as a major force contributing to NK and T cell senescence. In addition, cancer can also induce immune cell senescence in a process called premature senescence or cancer-induced senescence, further contributing to immune system impairment in older patients with cancer. Acute myeloid leukaemia is primarily a disease of older adults, and treatment options are often limited due to its toxicity and other comorbidities that often occur in older patients. Cancer immunotherapy has opened new opportunities in the fight against cancer. In this context, an additional effort should be made to optimize immunotherapy strategies against cancer in the elderly patient.

Supported by Junta de Extremadura, grant numbers IB16164, IB20132 and GR21178 (to RT); Ministry of Science and Innovation of Spain grant number SAF2017-87538-R (to RT); Spanish Instituto de Salud Carlos III, Ministry of Health, grant number PI21/01125 (to RS) and Secretaría General de Investigación, Desarrollo e Innovación en Salud de la Junta de Andalucía, grant number PECART-0060-2020 (to R.S.); co-financed by the European Union (FEDER).

Keywords: Acute myeloid leukaemia, cancer, cytomegalovirus, immunosenescence.

Corresponding author: Raquel Tarazona.

S2-02

Regulatory T cells, immunosenescence and cancer. Prospects for vaccine development

^*Alexander Batista-Duharte

GC01-Immunology and Allergy Group, Maimonides Institute for Biomedical Research of Cordoba (IMIBIC), 14004 Cordoba, Spain.

Regulatory T cells (Tregs) are CD4(+)CD25(+)Foxp3(+) lymphocytes that play a central role in the maintenance of self‐tolerance avoiding the development of autoimmune, inflammatory, and allergic diseases. However, their hyperstimulation is involved in the immunosenescence process and contributes to increased susceptibility to diseases associated with aging, including infections, cancer, and reduced vaccine efficacy by suppressing T-cell responses. The cancer research field has made considerable advances in dissecting the role of Tregs in cancer progression and suppressing immune responses to vaccines. Transient depletion of Tregs in murine models has been shown to enhance immune responses to different anti-tumor and anti-infectious vaccines. Similar results have been observed in humans by using certain anti-tumor therapy targeting Tregs, including immune checkpoint inhibitors. Herein, we discuss novel insights into the role of Tregs in immunosenescence and cancer, and current strategies under research to develop Tregs-targeted molecular adjuvants to improve vaccine efficacy. These strategies include monoclonal antibodies, antisense oligonucleotides, and peptides against immune checkpoints, Foxp3, and other Tregs pathways. Safety aspects and other challenges of these immunomodulation strategies will also be addressed.

Supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 847468. IMIBIC Fellowship Programme for Personalised and Precision Medicine (IMIBIC-P2Med).

Keywords: Regulatory T cells, immunosenescence, cancer, vaccines.

Corresponding author: Alexander Batista-Duharte, https://orcid.org/0000-0002-1875-0518.

S2-03

Monitoring immunodynamics in cancer immunotherapy

^*1,2,3Paulo Rodrigues-Santos

¹ Institute of Immunology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

² Immunology and Oncology Laboratory, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

³ Centre for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal

Although immune checkpoint inhibitor therapy became a routine for a growing number of cancers, only a few clinical trials consider immune endpoints and immunodynamic monitoring in their design. For several cancers, tumour grade remains as the most important prognostic factor. The race for biomarker discovery often do not consider the changing immune landscape context in a specific patient and cancer type. There is also a need for non-invasive approaches, like the analysis of peripheral immune profile, using immunological assays and gene analysis methods. Our group have been studying the immune profiles of patients with different types of cancers (chronic myeloid leukaemia, non-small cell lung cancer, metastatic melanoma, soft tissue sarcoma, and metastatic urothelial carcinoma) trying to obtain information that contributes to the immune characterization and prognostic evaluation on these patients. Monitoring of major effector and suppressor immune factors demonstrated the potential contribution for clinical management of cancer patients. Disease and therapeutic impact on the immune response against tumours has been further analysed using deep immunophenotyping and immune-related soluble proteomics and transcriptomics. Using clustering analysis we identified constellations of immune cells, cytokines and gene signatures correlated with overall survival. Recently, the description of new methods that permit single-cell analysis of immunometabolism using flow cytometry encouraged us to design immuno-oncology research projects to add functional profile energy metabolism to our core immune monitoring laboratory portfolio.

Supported by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Program and through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia, under the projects POCI-01-0145-FEDER-007440, UIDB/04539/2020 and UIDP/04539/2020.

Keywords: cancer immunotherapy, monitoring, immune checkpoints, immunodynamics.

Corresponding author: Paulo Rodrigues-Santos, https://orcid.org/0000-0001-7519-1620.

S2-04

Methods for the optimization of new chimeric antigen receptors

Pablo Gonzalez-Garcia, Juan P Muñoz Miranda, Lucia Olvera, Ricardo Fernandez-Cisnal, Antonio Gabucio, Cecilia Fernandez-Ponce, ^*Francisco Garcia-Cozar

Departamento de Biomedicina, Biotecnología y Salud Pública, Universidad de Cádiz.

Chimeric Antigen Receptor (CAR)-T cell therapy, constituted a breakthrough in immune therapy, achieving complete remission in more than 80% of patients with advanced hematological malignancies refractory to other treatments. Notwithstanding the good results obtained by CAR-T cell therapies in Chronic lymphocytic leukemia (CLL), attaining a similar success in other malignancies is still lacking. While applications in other areas such as infectious diseases are still in its infancy. The rate of improvement of CAR constructs is always hindered by difficulties originating from primary cell transduction and thus a reliable alternative that allows for rapid testing of optimized CARs need to be devised.

Keywords: chimeric antigen receptor (CAR), gene immunotherapy

Corresponding author: Francisco Garcia-Cozar, https://orcid.org/0000-0003-3720-259X

S2-05

Role of gene therapy in cancer inmunotherapy

^*1,2Francisco Martin

¹ Departamento de Bioquimica y Biología Molecular 3 e inmunología. Facultad de Medicina. Universidad de Granada. Avda. de la Investigacion 11, 18071, Granada, Spain.

² Department of Genomic Medicine, Pfizer-University of Granada-Andalusian Regional Government Centre for Genomics and Oncological Research (GENYO), PTS, Avda. de la Ilustración 114, 18016 Granada, Spain.

Gene therapy (GT) is a field that applies gene transfer technologies with the aim of treating diseases. Recent advances in the delivery methods, genetic modification strategies as well as in cell culture have led to the first approvals of gene therapy medicaments and opened new opportunities for safer and more efficient therapies for several diseases without treatment. Cancer immunotherapy (CI) has been for long an important target for gene therapy by expressing different transgenes on tumor cells and/or immune cells. In fact, the developments of new gene therapy-based immunotherapies have led to impressive successes in the treatment of B-cell malignances with 6 advance therapy medicinal products (ATMPs) approved by the FDA and/or EMA. This ATMPs, named CAR-T cells, are generated by genetically modify patient´s T cells to express a protein (chimeric antigen receptor-CAR) that instruct the T cells to destroy the tumor cells. However, these therapeutic benefits do not apply equally to solid tumors due to the absence of an ideal tumor specific antigen (TSA) and to the physical and functional barriers that the CAR-T cells encounter at the tumor microenvironment (TME). We and others are using all the Gene Therapy tools to re-engineer T cells to overcome these barriers and destroy the tumor cells without cause severe side effects to the patients. In this presentation I will summarize the different approaches that our group is working in this direction: from genome editing to generate universal CAR-T cells to regulated lentiviral vectors to externally control the potency of CAR-T cells.

Keywords: gene therapy, cancer immunotherapy, tumor specific antigen, CAR-T cells.

Corresponding author: Francisco Martin

Symposium 3: Autonomic nervous system in disease: From sensors, to circuits, to reflex

S3-01

Leptin: Beyond the hunger paradigm

^*Candelaria Caballero Eraso

Unidad Médico-Quirúrgica de Enfermedades Respiratorias, Instituto de Biomedicina de Sevilla (IBiS), Virgen del Rocio Hospital. Sevilla

Leptin is a circulating hormone that was identified in 1994. It´s mainly produce in the white adipose and play an important role in the metabolic regulation, being a master mediator of food intake and body energy balance. Obese subject present high leptin levels, however, they usually have leptin resistance which is defined as a failure of high-circulating levels of leptin to decrease hunger. Leptin acts by binding to leptin receptor that has several isoforms- The ObRb isoform is found in almost all tissues and can activate the intracellular leptin signaling cascade. This receptor is expressed at high levels in the brain and other peripheral organs such as carotid body. Apart from being involved in feeding behavior, leptin play a role in the glucose metabolism and insulin secretion, in the modulation of immune and inflammatory response and is related with oxidative. Moreover, in the last years it has been described that leptin plays an important role in the control of breathing by signaling either centrally or peripherally and has been related with some diseases such as obstructive sleep apnea (OSA) and hypertension induction acting through carotid body. OSA is a major health problem, is characterized by recurrent collapse of the upper airway during sleep provoking intermittent inspiratory flow limitation and cessation of airflow, leading to intermittent hypoxia (IH) and hypercapnia during sleep. Is associated with obesity and with cardiovascular morbidity and mortality. OSA and IH produce oxidative stress and increase peripheral leptin levels and leptin resistance, being leptin resistance implicated in the pathogenesis of OSA.

Keywords: Leptin, ventilatory response, obstructive sleep apnea, hypertension.

Corresponding author: Candelaria Caballero Eraso.

S3-02

Viscerosensorial pathways in nutrient postingestive signalling

^*1,2Ana B Fernandes, ^1,2Joaquim Alves da Silva, ^1,3Rui M Costa, ^1,2Albino Oliveira-Maia

¹ Champalimaud Research and Clincial Centre, Champalimaud Foundation, Lisbon, Portugal

² Nova Medical School; Lisbon Portugal

³ Zuckerman Institute, New York, USA

Internal sensory systems relay vital information to the brain about physiological states, including energy and nutritional balance. Rapid sensing of gut nutrients by the brain is mediated by communication through peripheral sensory neurons. Vagal afferent signals have been extensively studied in this context, and have critical impacts on food intake. Infusion of nutrients into the gut is also associated to striatal dopamine release and robust conditioned food preferences, suggesting that viscerosensory neurons may also generate signals that compute reward value. Despite evidence of a gut-brain circuit for nutrient sensing a direct causal link from the periphery to the activity of brain reward areas has not been established. We investigated how postingestive nutrient signals modulate dopamine neuron activity to control food-seeking behavior and how these signals are transmitted to the brain to produce such effects. Our results show that postingestive sucrose signals lead to modulation of VTA dopaminergic activity, which is required for postingestive modulation of food seeking. Also, deletion of the NMDA receptor in these neurons, which affects bursting and plasticity, abolishes lever pressing for postingestive sucrose delivery. Furthermore, lesions of the hepatic branch of the vagus nerve significantly impair postingestive-dependent VTA dopamine neuron activity and food seeking, whereas optogenetic stimulation of left vagus nerve neurons significantly increases VTA dopamine neuron activity. These data established a necessary role of vagus-mediated dopamine neuron activity in postingestive-dependent food seeking, which is independent of taste signaling, providing critical contributions to understand central and peripheral mechanisms underlying food-seeking behavior.

Keywords: Viscerosensorial pathways, postingestive nutrient signals.

Corresponding author: Ana B Fernandes.

S3-03

Critical role of the interaction gut microbiota - sympathetic nervous system in the regulation of blood pressure in rat

^1†Marta Toral, ^1†Iñaki Robles-Vera, ¹Néstor de la Visitación, ^1,2Miguel Romero, ³Tao Yang, ^1,2Manuel Sánchez, ¹Manuel Gómez-Guzmán, ^1,2,4Rosario Jiménez, ³Mohan K Raizada, ^*1,2,4Juan Duarte

¹ Department of Pharmacology, School of Pharmacy, Centro de Investigación Biomédica, University of Granada, Granada, Spain, ² Instituto de Investigación Biosanitaria de Granada, ibs.GRANADA, Granada, Spain, ³ Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL, United States, ⁴ CIBERCV, University of Granada, Granada, Spain

†These authors have contributed equally to this work as first authors

Association between gut dysbiosis and neurogenic diseases, such as hypertension, has been described. The aim of this study was to investigate whether changes in the gut microbiota alter gut-brain interactions inducing changes in blood pressure (BP). Recipient normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were orally gavaged with donor fecal contents from SHR and WKY. We divided the animals into four groups: WKY transplanted with WKY microbiota (W-W), SHR with SHR (S-S), WKY with SHR (W-S) and SHR with WKY (S-W). Basal systolic BP (SBP) and diastolic BP (DBP) were reduced with no change in heart rate as a result of fecal microbiota transplantation (FMT) from WKY rats to SHR. Similarly, FMT from SHR to WKY increased basal SBP and DBP. Increases in both NADPH oxidase-driven reactive oxygen species production and proinflammatory cytokines in brain paraventricular nucleus linked to higher BP drop with pentolinium and plasmatic noradrenaline (NA) levels were found in the S-S group as compared to the W-W group. These parameters were reduced by FMT from WKY to SHR. Increased levels of pro-inflammatory cytokines, tyrosine hydroxylase mRNA levels and NA content in the proximal colon, whereas reduced mRNA levels of gap junction proteins, were found in the S-S group as compared to the W-W group. These changes were inhibited by FMT from WKY to SHR. According to our correlation analyses, the abundance of Blautia and Odoribacter showed a negative correlation with high SBP. In conclusion, in SHR gut microbiota is an important factor involved in BP control, at least in part, as consequence of its effect on neuroinflammation and the sympathetic nervous system activity.

Funded by: Comisión Interministerial de Ciencia y Tecnología, Ministerio de Economía y Competitividad (MINECO) (SAF2017-8489-R, AGL2015-67995-C3-3-R, and SAF2014-55523-R), Junta de Andalucía (Proyecto de Excelencia P12-CTS-2722 and CTS-164) with support from the European Union, and Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (CIBER-CV, CIBER-EHD).

Keywords: gut dysbiosis, hypertension, neuroinflammation, sympathetic nervous system.

Corresponding author: Juan Duarte, jmduarte@ugr.es

S3-04

Carotid body: coupling oxygen sensing with energy homeostasis

^*Silvia V Conde

NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Metabolic diseases such as obesity and type 2 diabetes (T2D) are major public health problems, defined by WHO as worldwide epidemics. Deregulation of the sympathetic nervous system (SNS) is key to the generation of these diseases. Altered thermogenesis, lipolysis, insulin secretion, pathways known to be regulated by SNS, are present in animal models and humans with metabolic diseases, however, poor attention has been given to the afferent pathways and the stimuli that trigger this sympathetic activation. In the last decades it was postulated that the carotid bodies (CB), small peripheral chemoreceptors classically defined as O₂ sensors, could be the missing link between sympathetic overactivation and metabolic diseases. Extensive literature has been published in these last years related with the metabolic sensing properties of the CB and the contribution of its dysfunction to the genesis of metabolic diseases placing this organ in the spotlight as new therapeutic target for the treatment of these pathologies. In the present talk I will review the role of the CB in the control of glucose and lipid homeostasis, exploring in deep the mechanisms promoting CB overactivation in metabolic diseases – insulin, leptin, incretins, inflammatory cytokines-, the CB-associated chemoreflexes and neural pathways/circuitries involved in glucose and lipid regulation as well as the role of the CB in the link neural-immuno-metabolism. I will also debate strategies to adjust CB activity and its reflex responses, which can be used for prevention and treatment of metabolic diseases, envisioning new therapeutic horizons.

Funding: Portuguese Foundation for Science and Technology Research Grant EXPL/MED-NEU/0733/2021.

Keywords: carotid bodies, oxygen sensing, metabolic diseases.

Corresponding author: Silvia V Conde, https://orcid.org/0000-0002-5920-5700

Symposium 4: Physiopathological role of intestinal microbiota: A new player in health and disease

S4-01

Influence of lifestyle on gut microbiota: how can help each other?

^*1Mar Larrosa, ²Rocío González

¹Department of Nutrition and Food Science, Faculty of Pharmacy, Complutense University of Madrid, Spain, ²Universidad Europea de Madrid, Spain.

The composition of the gut microbiota is influenced by intrinsic host factors (age, sex, genetics, etc) and external factors such as stress, drug intake, physical exercise, diet, etc. It is estimated that about 20% of the factors that influence the microbiota are known, while the remaining 80% of the factors that have an impact in its composition are still unknown. One of the known factors that most determine the composition of the gut microbiota is diet, and this is not unexpected since the microorganisms that are in our intestine ultimately feed on what we eat. A substantial modification in our diet causes relevant changes in the gut microbiota in a matter of 4-6 days, however, the gut microbiota remains stable throughout our life, unless we make drastic changes in our lifestyle and remain constant over time. The gut microbiota has the ability to be resilient to most changes, but when the microbiota loses this ability and becomes unbalanced the microbiota enters in dysbiosis. Physical exercise is another factor that modifies the gut microbiota, therefore, a healthy lifestyle, understood as a healthy diet and the practice at least of the minimum dose of physical exercise recommended by the World Health Organization, results in a healthy microbiota. Our studies indicate that there is a high correlation between the practice of exercise and a healthy diet, and that people with an active lifestyle compared to those with a sedentary lifestyle have a microbiota characterized by a high species diversity, a greater number of "healthy" bacterial species, with redundant metabolic functions and a greater capacity for resilience to changes and therefore to dysbiosis.

Keywords: microbiota, diet, physical exercise, lifestyle.

Corresponding author: Mar Larrosa, https://orcid.org/0000-0002-8863-4686

S4-02

Modulation of intestinal microbiota in the development of non-alcoholic fatty liver disease (NAFLD)

^*1,2Sonia Sánchez-Campos

¹ Institute of Biomedicine (IBIOMED), University of León, León Spain.

² Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Carlos III Institute of Health, Madrid, Spain.

In the last years, gut microbiota has been identified as an essential factor in obesity and non-alcoholic fatty liver disease (NAFLD/MAFLD) development. For that reason, different therapeutic alternatives based on gut microbiota modulation have been proposed, such as probiotics, prebiotics or symbiotics, lifestyle interventions as physical exercise, and microbiota transplantation. We have investigated the potential benefit of quercetin administration combined with intestinal microbiota transplantation on obesity-associated NAFLD development in an experimental model (high-fat diet, HFD-fed mice). HFD and quercetin administration result in specific microbiota profiles associated with protected or non-protected metabolic phenotypes against NAFLD, which are transferable by means of gut microbiota transplantation to germ-free mice. Protected phenotype associates with increased Oscillospira and Desulfovibrio genera and reduction of Bacteroides and Oribacterium genera, enhanced specific secondary bile acids, which probably inhibit hepatic lipogenesis, and augmented bile flow. This gut-liver cross-talk, mediated by plasma bile acids, may have clinical and nutritional relevance against NAFLD. Nowadays, the protective role of Akkermansia muciniphila is proposed against obesity and NAFLD. In this regard, we have evaluated the potential therapeutic effects of quercetin and A. muciniphila synbiotic supplementation along with a nutritional intervention on the development of early obesity and NAFLD in rats. The beneficial effects observed with the synbiotic are mediated by the modulation of gut microbiota composition and its functionality, leading to a specific microbiota profile, the increase of primary and hydrophilic bile acid species and the induction of gut-liver bile flow. In conclusion, the symbiotic administration of A. muciniphila and quercetin is a feasible strategy to face juvenile obesity and NAFLD development, in a mechanism that also involves the modulation of bile acid signaling, the liver inflammatory status, the process of lipogenesis and the gut microbiota composition.

Supported by BFU2017-87960-R, PID2020-120363RB-I0, GRS2126/A/2020, LE017-P20. CIBERehd is funded by Carlos III Institute of Health. All procedures were performed in accordance with the European Research Council guidelines for animal care and use and under the approval by the local animal Ethics Committees.

Keywords: Akkermansia muciniphila, gut microbiota, non-alcoholic fatty liver disease, quercetin.

Corresponding author: Sonia Sánchez-Campos, https://orcid.org/0000-0003-2672-734X.

S4-03

Importance of gut microbiota in obesity

^*Francisco J Tinahones

Department of Endocrinology and Nutrition, Virgen de la Victoria University Hospital, Instituto de Investigación Biomédica de Málaga (IBIMA), Faculty of Medicine, University of Málaga, Málaga, Spain.

The obesity epidemic has increased significantly in recent decades. Traditional theories arguing that this weight gain is an Imbalance between energy expenditure and intake have been superseded and at this time is considered too simplistic. New environmental factors have been proposed to explain this increased prevalence. The gut microbiota plays an important role both locally and systemically. There is a symbiotic relationship between the microbiota and its host. Several studies have linked the development of highly prevalent disorders, such as type 2 diabetes and obesity, to the gut microbiota. The intestinal microbiota has changed drastically in recent decades and these changes have been linked to pathologies that have increased significantly in this century. Obesity itself has been associated with certain gut microbiota profiles. The composition of the microbiota of obese subjects differs from that of lean individuals and after a weight loss there are important changes in the microbiota that are dependent on the weight change strategy that we have used. Numerous studies have related the etiopathogenesis of obesity to changes in the microbiota through different mechanisms such as the overgrowth of microorganisms that obtain energy more efficiently from the diet, changes in endotoxemia, intestinal permeability, insulin resistance, hormonal environment, expression of genes that regulate lipogenesis, interaction with bile acids, as well as changes in the proportion of brown adipose tissue. Currently, the use of prebiotics and probiotics and other innovative techniques such as antibiotic therapy or intestinal microbiota transplantation have been proposed as possible tools to control the development of metabolic diseases such as obesity.

Keywords: microbiota, obesity.

Correspondingauthor: Francisco José Tinahones, fjtinahones@uma.es, https://orcid.org/0000-0001-6871-4403

S4-04

Role of microbiota in the control of arterial hypertension and its modulation by probiotics

^*Miguel Romero Pérez

Department of Pharmacology, Center for Biomedical Research (CIBM), University of Granada. Granada, Spain.

There are numerous studies in animal models and in humans that indicate a direct association between hypertension and the gut microbiota. In this study, we focus on the analysis of intestinal dysbiosis associated with hypertension and discuss the current knowledge of the mechanisms and bacterial mediators involved in the regulation of blood pressure (BP) and the use of probiotic bacteria in the treatment of the hypertension. The profile of the ecological parameters and the bacterial genera composition of intestinal dysbiosis in hypertension varies according to the experimental model of hypertension. In hypertension associated with activation of the renin-angiotensin system, an increase in the Firmicutes/Bacteroidetes (F/B) ratio has been observed, as well as a deficit of bacteria that produce short-chain fatty acids (SCFAs), acetate and butyrate. In renin-independent models, no changes were observed in F/B, nor in the proportion of SCFA-producing bacteria. Intestinal dysbiosis in hypertension seems to be a consequence of the alteration of the integrity of the intestinal epithelium, derived from the excessive sympathetic discharge in the intestine, which favors a less hypoxic environment and the growth of aerobic bacteria. Recent evidence supports that the gut microbiota may protect or promote the development of hypertension by interacting with the secondary lymphatic organs of the gut and altering the polarization of T helper (Th)17/T regulatory (Treg) cells, inducing changes in the infiltration of T cells in vascular tissues. This regulation of lymphocyte polarization seems to be related to the colonic content of SCFAs and to an enrichment of genes for the biosynthesis and export of lipopolysaccharides (LPS) in the hypertensive microbiota. In general, the gut microbiota is now recognized as a target for prebiotic, probiotic, and postbiotic dietary interventions aimed at lowering BP. Chronic consumption of probiotic bacteria, such as Lactobacillus ssp, and Bifidobacterium ssp, reduces BP in renin-dependent and renin-independent animal models. Additionally, a meta-analysis of human studies supports that probiotic supplementation slightly lowers BP. The mechanism of the antihypertensive effect of probiotics is related to the improvement of intestinal dysbiosis, the increase in butyrate-producing bacteria, the improvement of intestinal integrity, the decrease in the Th17/Treg ratio in the mesenteric lymph nodes and the lower Th17 infiltration in the vascular wall. However, the position of probiotics within antihypertensive therapy has not been finalized yet.

Keywords: Hypertension, gut microbiota, probiotic, immune response.

Corresponding author: Miguel Romero Pérez, https://orcid.org/0000-0003-0578-1099

Symposium 5: Molecular pathophysiology of cancer

S5-01

Essential role of Orai1 in Notch1 activation in angiogenesis

^*Tarik Smani

Group of Cardiovascular Pathophysiology, Institute of Biomedicine of Seville, University Hospital of Virgen del Rocío, University of Seville, CSIC, Seville, Spain; Department of Medical Physiology and Biophysics, Faculty of Medicine, University of Seville, Seville, Spain

Angiogenesis plays important roles in physiological and pathological settings such as tissue repair, embryo development, wound healing, or cancer tumor growth. Angiogenesis is, in fact, a hallmark of cancer, being necessary for both cancer growth/progression and metastasis. The process is initiated when endothelial cells are rapidly activated by pro-angiogenic molecules that induce endothelial cell sprouting, migration, and proliferation, vascular tube formation, and anastomosis of the newly formed tubes, which will ensure blood flow to tumor tissues. Increasing evidence suggest a role of Orai1, the pore forming subunit of store-operated Ca²⁺ channels, in the stimulation of endothelial cell and angiogenesis by pro-angiogenic growth factors. Here, we found that vascular endothelial growth factor (VEGF) induced a significant increase in the intracellular Ca²⁺ concentration in Human Umbilical Vein Endothelial Cells (HUVEC), which was significantly inhibited by silencing the expression of Orai1 by siRNA. In addition, Orai1 downregulation prevented HUVEC proliferation and migration, as well as tube formation in cells seeded on matrigel. Similarly, silencing of Orai1 also attenuated endothelial cells sprouting assessed in 3D Organ-on-a-chip culture. Furthermore, we investigated the effect of blood serum collected from patients with breast cancer on angiogenesis. First, we found an increased amount of VEGFA in patients compared to control. Second, the incubation of HUVEC with patients’ serum stimulated their migration and tube formation, correlating with the upregulation of Orai1 and notch1. Interestingly, silencing of Orai1 prevents the expression of notch1 and its target genes Hes1, Hey1, and VEGF-receptor, suggesting a role of Orai1 in the transcription of Notch1 and related genes. Therefore, herein we demonstrate the relevant role of Orai1 in activating notch1 signaling pathway and angiogenesis.

Keywords: angiogenesis, breast cancer, Orai1, Notch1

Corresponding author: Tarik Smani

S5-02

Molecular basis of Ca ²⁺ remodeling in colorectal cancer and its reversal by polyamine depletion

^1,2 Enrique Pérez Riesgo, ^1,2Elena Hernándo-Pérez, ^1,2Lucía Núñez, ^*1Carlos Villalobos

¹ Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain.

² Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain.

Colorectal cancer (CRC) is the third most common cancer worldwide and the second most deadly form of cancer with even worst figures in Spain. Alterations in the APC/Wnt/β-catenin pathway that result in c-myc activation and excess of polyamine biosynthesis are the most frequent cause of CRC development. In addition, data suggest that CRC is also associated to the remodeling of intracellular Ca²⁺ homeostasis including changes in resting levels of intracellular Ca²⁺, enhanced store-operated Ca²⁺ entry (SOCE) and changes in Ca²⁺ store content, Ca²⁺ release from intracellular stores and mitochondrial Ca²⁺ uptake. We have used cell models, paired tumor samples from CRC patients and statistical analysis from data repositories to characterize transcriptomic remodeling of genes involved in intracellular Ca²⁺ transport systems in CRC. We found upregulation and downregulation of genes coding for voltage-gated Ca²⁺ channels, TRP channels, molecular players involved in SOCE, Ca²⁺ release channels, Ca²⁺ pumps and exchangers and Ca²⁺ transport in mitochondria. Significant differences were found among the several CRC models used for analysis. Polyamine depletion in cell models treated with difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase, the limiting step in polyamine synthesis, reversed Ca²⁺ and transcriptomic remodeling in CRC. Effects of DFMO were only observed in cancer cells but not in normal cells. These data indicate that excess polyamine synthesis may contribute to Ca²⁺ remodeling in CRC and polyamine depletion may help to reverse the tumoral phenotype to prevent CRC.

Supported by Ministerio de Ciencia e Innovación (RTI2018-099298-B-100), Junta de Castilla y León (CCVC8485) and Asociación Española Contra el Cáncer (AECC).

Keywords: intracellular calcium, colorectal cancer, transcriptomics, polyamines

*Corresponding author: Carlos Villalobos, ORCID: 0000-0002-0429-3846

S5-03

Stress kinases in liver cancer

¹ María Crespo, ¹ Magdalena Leiva, ¹ Antonia Tomás-Loba, ¹ Elisa Manieri, ¹ Ivana Nikolic, ¹ Alfonso Mora, ¹ Bárbara Gonzalez-Terán, ² Miguel Marcos, * ¹ Guadalupe Sabio

¹ Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

² Department of Internal Medicine, University Hospital of Salamanca-IBSAL, Department of Medicine, University of Salamanca, Salamanca, Spain

Obesity, a worldwide pandemic health condition, is involved in the development and progression of diabetes, cancer and heart failure. One important common factor in obesity is the activation of stress kinases, p38 and JNK, in several organs, including adipose tissue, liver, muscle, immune and central nervous system. The correct activation in time, intensity and duration is crucial because it contributes to the metabolic cellular adaptation, while uncontrolled activation, like in obesity, has been suggested to contribute to obesity burden. In consequence, these pathways have been proposed as potential targets for the development of novel therapeutic approaches for the treatment of the metabolic syndrome. In the last years, the scientific community has made major efforts to understand how these kinases are regulated in a cell specific manner and what is their contribution to obesity development, insulin resistance and liver cancer development. Recently, several studies have uncovered new and unexpected functions of the less classical family members of both pathways including an important function regulating liver circadian clock. Here, we will provide an overview of the role of the stress kinases in the metabolic control and discuss how alteration of these kinases in hepatocytes trigger chronic perturbation of circadian rhythms and liver cancer.

Financial support: M.C. was fellow of FPI-MINECO (BES-2017–079711). M.L. was supported by Spanish grant MINECO-FEDER SAF2015-74112-JIN and Fundación AECC: INVES20026LEIV. I.N. was funded by EFSD/Lilly grants (2017 and 2019), the CNIC IPP FP7 Marie Curie Programme (PCOFUND-2012-600396), an EFSD Rising Star award (2019) and grant MINECO IJC2018-035390-I. G.S. received funding from the following programmes and organizations: European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number ERC 260464; EFSD/Lilly European Diabetes Research Programme; Fundación AECC PROYE19047SABI; BBVA Foundation Leonardo Grants program for Researchers and Cultural Creators (Investigadores-BBVA-2017) IN[17]_BBM_BAS_0066; MINECO-FEDER SAF2016-79126-R and PID2019-104399RB-I00; and the Comunidad de Madrid (IMMUNOTHERCAN-CM S2010/BMD-2326 and B2017/BMD-3733). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015–0505). M. M. was supported by ISCIII and FEDER PI16/01548 and PI20/00743 and Junta de Castilla y León GRS 2291/A/2020.

Keywords: stress kinases, obesity, liver cancer.

Corresponding author: Guadalupe Sabio, ORCID 0000-0002-2822-0625.

S5-04

Orai1α modulates TRPC1 channel function

^*1Juan A. Rosado, ¹Jose Sanchez-Collado, ¹Jose J Lopez, ¹Isaac Jardin, ¹Alejandro Berna-Erro, ¹Pedro J Camello, ¹Carlos Cantonero, ²Tarik Smani, ¹Gines M Salido

¹ Department of Physiology (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain. ² Department of Medical Physiology and Biophysics, University of Seville, Seville, Spain.

Store-operated Ca²⁺ channels (SOCs) mediate Ca²⁺ influx in response to depletion of the intracellular Ca²⁺ stores. Prototypical SOCs consist of a hexameric assembly of Orai subunits with a predominant role for Orai1 over its paralogs Orai2 and Orai3. Two Orai1 variants are present in mammalian cells: Orai1α, the full-length Orai1, containing 301 amino acids, and Orai1β, generated by a process of alternative translation initiation from a methionine at position 64 in the Orai1α variant. TRPC1 channels are activated by multiple pathways. Store-dependent activation TRPC1 depends on Orai1-mediated Ca²⁺ influx which triggers translocation of TRPC1 to the plasma membrane to mediate Ca²⁺ influx. By using APEX2 proximity labelling assay, co-immunoprecipitation and the analysis of the fluorescence of G-GECO1.2 fused to Orai1α or a dominant-negative (dn) Orai1α mutant we have found that agonist stimulation and Ca²⁺ store depletion enhance the interaction of TRPC1 with Orai1α but not with Orai1β in HeLa cells. Analysis of Mn²⁺ influx as a surrogate of Ca²⁺ demonstrated that expression of a dnOrai1α mutant significantly attenuated divalent cation entry through TRPC1 and impaired TRPC1 translocation to the plasma membrane in HeLa cells. By contrast, both Orai1 variants seem to be equally relevant for TRPC1-mediated cation entry and I_SOC current in HEK-293 cells. This talk highlights the main experimental advances in the understanding of the store-dependent regulation of TRPC1 channel function by Orai1.

Supported by Grants PID2019-104084GB-C21 and PID2019-104084GB-C22 funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe, and Junta de Extremadura-cofinanciado por la Unión Europea (Grants IB20007, IB18025 and GR21008).

Keywords: Orai1α, Orai1β, TRPC1, Ca²⁺ influx.

Corresponding author: Juan A. Rosado, https://orcid.org/0000-0002-9749-2325.

Symposium 6: Cardiovascular physiology: Pulmonary hypertension - the right ventricle under pressure

S6-01

Experimental models of right ventricular dysfunction: a window for investigating translation in pulmonary hypertension

¹Rui Adão

¹ UnIC@RISE, Department of Surgery and Physiology, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal

Right ventricular (RV) heart failure may be the ultimate cause of death in patients with acute or chronic pulmonary hypertension (PH). Despite major pharmacological advances since the last decade, PH remains a deadly disease and new therapies continue to be focused on pulmonary artery vasodilation, leaving aside the RV function, while side effects of some PH-specific drugs might even include deterioration of RV function. Thus, there is an increased need to develop RV-specific therapies and to understand RV adaptation to overload and progression to RV maladaptation and failure, although the RV's complex anatomy, physiology, and deterioration in response to pressure overload could represent a greater difficulty in achieving this goal. To understand the underlying pathophysiology of right heart failure and to aid in the development of new treatments we need solid animal models that mimic the pathophysiology of human disease. Several experimental models of RV dysfunction have been developed over the past three decades providing a particular insight in RV pathophysiology. They range from flow induced to pressure overload induced right heart failure and have been introduced in both small and large animals. In general, large animals are good candidates for surgical models, while murine models have the advantage of selective gene knockout or knockin allowing the investigation of specific molecular pathways involved in RV remodeling. The insult causing RV dysfunction varies between models and can occur secondary to ischemic, hypoxic, toxic, or mechanical (pressure or volume overload) stressor to the RV. Considering experimental models of PH, a gradual development of pressure overload is more representative of PH pathophysiology. Another essential concept in analyzing different models of RV pressure overload is whether the model produces adaptive or maladaptive remodeling of the RV. When initiating new pre-clinical or basic research studies it is key to choose the right animal model to ensure successful translation to the clinical setting. We do, however, still need to refine and develop animal models of RV failure to create more precise pathophysiologic and biologic modeling to ensure the best possible pre-clinical research and successful clinical translation.

Keywords: right ventricle, pulmonary hypertension, animal model, pre-clinical research.

Corresponding author: Rui Adão, https://orcid.org/0000-0003-2203-436X

S6-02

Is exercise good for the right ventricle in pulmonary hypertension: what the animal studies show?

^*1,2Daniel Moreira-Gonçalves

¹ Centro de Investigação em Atividade Física, Saúde e LAzer (CIAFEL), Faculdade de Desporto da Universidade do Porto, Porto, Portugal

² Departamento de Cirurgia e Fisiologia, Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Exercise training (ET) is widely recognized by its preventive and therapeutic effects in several chronic diseases but only recently it started to be recognized as safe and beneficial in pulmonary arterial hypertension (PAH). In fact, guidelines for PAH treatment use to recommended that any physical activity should be limited as it could aggravate the disease progression and increase the risk of sudden cardiac death. However, accumulating evidence is showing a positive effect of supervised ET in functional capacity and quality of life (on top of the best standard care with approved medications), paralleled by a reduced rate of major adverse events, such right heart failure, mortality and worsening of the disease. Current guidelines now recommend that PAH patients should be encouraged to be active within symptom limits and, when physically deconditioned, they should undertake supervised ET under medical therapy. Despite consensus in the literature regarding the favorable effects of ET in PAH, the mechanisms underlying these clinical improvements remain to be defined. Existing evidence points for adaptations at the level of skeletal muscle but the impact of ET on right ventricular (RV) function and remodeling cannot be discarded. This would be of great clinical importance as survival is closely related to RV performance. Clinical data is very scarce but there is preliminary evidence supporting that ET may indeed improve RV function. Several pre-clinical studies corroborate this impact of ET on cardiac function and show additional improvements in markers of cardiac remodeling, neurohumoral activation, metabolism, oxidative stress and inflammation. While it is tempting to suggest that ET may have a favorable hemodynamic effect in PAH, we still need large multicentre trials, specifically designed to evaluate cardiac function and remodeling, before any final conclusion can be made.

Keywords: Physical exercise, right ventricle, pulmonary hypertension

Corresponding author: Daniel Moreira-Gonçalves

S6-03

Novel mechanisms in pulmonary hypertension: microRNAs and right ventricular dysfunction

Carla I Marques

Faculty of Medicine, Coimbra Institute for Clinical and Biomedical Research (iCBR), Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Clinical Academic Centre of Coimbra (CACC), Coimbra, Portugal.

Pulmonary hypertension (PH) is a severe progressive cardiopulmonary disorder that carries a high mortality rate, for which there is no cure at this time. This disease characterized by increased pulmonary vascular resistance and pulmonary arterial pressure, leading to right heart failure. Despite improvements in treatment, the overall prognosis for PH remains poor. In most right heart failure patients, the disease is not detected in an early phase and, consequently, profound structural alterations in the right ventricle (RV) are established. Initially the right ventricle responds with compensated hypertrophy, which is often rapidly followed by decompensated hypertrophy changes leading to right heart decompensation and failure, which is the ultimate cause of death. However, the molecular mechanisms of what drives the switch from compensated to decompensated hypertrophy remain poorly understood. MicroRNAs have been identified as key regulators of biological processes that drive the progression of several diseases. Although several studies have suggested that changes in miRs profile are implicated in PH, the underlying molecular mechanisms, including the identification of targets, remain largely elusive. There are differences in the miRNA expression profile between RV hypertrophy and RV failure, which suggests specific changes in the signature of miRNAs during PH progression. In our work, we investigated whether circulating miR-424 can be used as biomarkers in PH, as well as the signaling pathways modulated by this miRNA, such SMURF1 levels. The results shown that the levels of circulating miR-424 are increased in PH patients when compared with healthy subjects, and correlated with decreased cardiac output. Therefore, miR-424 levels could be used has diagnostic and prognostic value in PH patients, correlating with markers of disease severity. In conclusion, there is little doubt that miRNAs yield tremendous potential to become clinically useful diagnostic and prognostic biomarkers, as well as novel therapeutic targets.

Keywords: MicroRNAs; Pulmonary hypertension; Right ventricular dysfunction.

Corresponding author: Carla I Marques, https://orcid.org/0000-0002-1817-4813

S6-04

How to preserve the right ventricle in pulmonary hypertension: insights from animal studies

^*1Carmen Brás-Silva

¹ UnIC@RISE, Department of Surgery and Physiology, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive obstruction of small calibre pulmonary arteries caused by sustained vasoconstriction and extensive vascular remodelling. The narrowing of pulmonary arteries is accompanied by a gradual increase in pulmonary vascular resistance and mean pulmonary artery pressure, pushing the right ventricle (RV) to undergo structural and functional changes. Eventually, the RV is not able to cope with the increase in load and heart failure (HF) develops. Currently available treatments for PAH were developed to restore an imbalance of vasoactive factors, arising because of endothelial and smooth muscle cell dysfunction, and primarily function by inducing vasodilation. These traditional medications include the prostacyclin analogs and receptor agonists, phosphodiesterase 5 inhibitors, endothelin-receptor antagonists, and cGMP activators. These medications prolong life, but mortality remains unacceptably high. The major determinant of symptoms and outcome in patients with PAH is RV function, and a challenge for the field is identifying novel drug targets that can reverse the vascular structural changes and improve RV failure. Animal models that mimic the pathophysiology of human disease help to understand the underlying pathophysiology of right heart failure and to develop new to develop RV-specific therapies. In the last years, a line of research, ongoing in our laboratory, devoted to studying the role of several pharmacological agents in PAH and RV HF implemented several experimental models of PAH and RV overload. We have shown that neuregulin-1 and urocortin-2 ameliorate PAH, decreasing RV hypertrophy and pulmonary vascular remodelling. The role of these agents in the RV was established by the pulmonary artery banding model which isolates the pathology to the RV: identifying changes in isolated and skinned cardiomyocytes clearly shows there is a direct impact on cardiomyocyte biology. A separate role in the pulmonary vasculature was identified by studying vascular remodelling, and the tone of isolated pulmonary artery rings. Another major strength of these studies is the use of multiple endpoints to assess efficacy, including mortality, exercise capacity, pulmonary and RV remodelling, and RV function.

Keywords: pulmonary hypertension, right ventricle, therapy, pre-clinical research.

Corresponding author: Carmen Brás-Silva, https://orcid.org/0000-0003-1527-3776

Symposium 7: Multi-organ damage: Role of inflammasome

S7-01

Inflammasome and renal damage

^*Alberto Lázaro Fernández

Laboratorio de Fisiopatología Renal, Instituto de Investigación Sanitaria Gregorio Marañón, Hospital General Universitario Gregorio Marañón, Madrid, España

Departamento de Fisiología, Facultad de Medicina, Grupo UCM 951579, Universidad Complutense de Madrid, España

Renal failure is a global public health problem affecting more than 750 million people worldwide in which the kidneys are no longer able to function effectively. When renal failure occurs suddenly (within hours or days), it is called acute kidney injury (AKI) in contrast to the condition characterized by a gradual loss of kidney function over time representing chronic renal failure (CKD). In both cases the process may end in end-stage kidney disease where the only possibilities are renal replacement therapies or transplantation. Inflammation has been considered to play a decisive role in renal failure contributing to the development of renal tissue damage and amplifying kidney injury. Currently, it is known that inflammasomes (multi-protein complexes that result in the activation of procaspase-1 to caspase-1, with the consequent maturation and release of the pro-inflammatory cytokines IL-18 and IL-1β and TNFα) participate in the inflammatory reactions in kidneys, leading to pathological lesions and kidney injury by inducing pyroptosis, apoptosis and inflammatory/fibrotic pathways among others. Several studies have examined and defined the role of inflammasomes and, in particular, the NLRP3 to a wide range of CKD and AKI in animal models including ischemia-reperfusion injury, sepsis, toxics or rhabdomyolysis-induced AKI and fibrotic unilateral ureteral obstruction. Although renal failure has no treatment, the inflammasome also represents a novel therapeutic target. Thus, IL-1β-targeting therapies and caspase-1 and NLRP3 specific inhibitors have shown beneficial effects in cellular and animal models of renal damage, but the transition to the human clinic will have to be studied and validated in future clinical trials. In our research group, we have found that cilastatin, a specific inhibitor of the renal dehydropeptidase I designed to prevent hydrolysis, proximal tubule uptake and cell toxicity of imipenem, is able to protect the kidney against AKI of any etiology by suppressing apoptosis, oxidative stress and inflammation. Part of the observed protection is mediated by a decrease in the expression and activation of inflammasome components, thus preventing lesion exacerbation. Our results highlight the role of the renal inflammasome as a valid and promising therapeutic target for the prevention and treatment of renal failure.

Keywords: kidney Injury, inflammasome, interleukins, cilastatin.

Corresponding author: Alberto Lázaro Fernández

S7-02

Inflammasome and lung damage

^*Luis Puente-Maestu

Hospital General Gregorio Marañón-Universidad Complutense Madrid (Spain)

The primary function of human lungs —passively exchange oxygen and carbon dioxide—need an alveolar surface between 80 m² to 120 m², matched by pulmonary capillaries. Such huge area is constantly exposed to an assortment of environmental and systemic aggressions of diverse kind, mechanical, chemical, infectious and inflammatory. To keep their structure functional, the respiratory system is endowed whit a remarkably effective set of defensive mechanism, of which innate and acquired immunity are the ultimate barrier. The Inflammatory response of the lungs is complex and involves a variety of defense and repair mechanisms. Numerous types of cells are involved including epithelial, neuroendocrine, mesenchymal and activated immunity cells. Each releases cytokines and mediators to modify activities of other inflammatory, mucosal and mesenchymal cells. Orchestration of these cells and molecules leads to progression of inflammation. Clinically, acute inflammation is seen after the exposure to tobacco smoke or other irritants, after mechanical injury such as mechanical ventilation, in pneumonia and acute respiratory distress syndrome (ARDS) among other processes, whereas chronic inflammation is represented by asthma, chronic obstructive pulmonary disease (COPD) or interstitial diseases. The alveolar-capillary membrane is a very thin structure with a geometric mean thickness of 0,67 μm, which function is easily impaired by excessive inflammation and its consequences such as increased capillary permeability can be life threatening or neumocytes injury. Because the lungs are constantly exposed to harmful pathogens, inflammation has to be delicately balanced by a range of anti-inflammatory responses, which are essential for the health of the lungs. A full understanding of the underlying mechanisms is vital in the treatment of patients with lung inflammation that is leading to the development of powerful anti-inflammatory medicines, that is radically changing the natural history of a wide range of different pulmonary conditions.

Keywords: inflammasome, lung damage, chronic obstructive pulmonary disease.

Corresponding author: Luis Puente-Maestu.

S7-03

Inflammasome and cardiac damage

^*Javier Díez Martínez

Centro de Investigación Médica (CIMA), Universidad de Navarra

The inflammasome is a multimeric protein complex that plays an essential role in the innate immune response by triggering the cleavage and activation of the proinflammatory cytokines interleukins (IL)-1β and IL-18. Inflammasome activation in the heart is responsible for myocardial remodelling (i.e., hypertrophy, fibrosis, and pyroptosis). Besides inflammatory cells, the inflammasome in other cardiac cells initiates local inflammation through intercellular communication. The cardiac inflammasome is activated under various pathologic conditions that contribute to the progression of hart failure (HF), including pressure overload, acute or chronic overactivation of the sympathetic system, myocardial infarction, and diabetic cardiomyopathy. IL-1β has been shown to promote myocardial remodelling in experimental models, and the presence of elevated circulating concentrations of this cytokine has been reported in patients with chronic HF. Additionally, our group has demonstrated that hat the excess of circulating IL-1β cytokine has prognostic implications for acute decompensated HF (J Am Coll Cardiol 2019;73:1016-1025). Furthermore, blocking IL-1β with the monoclonal antibody canakinumab reduced hospitalizations and mortality in HF patients, suggesting that the inflammasome is involved in HF development and/or decompensation. The current research on the role of the inflammasome in HF is focused on the NLRP3 inflammasome. The functions of other types of inflammasome in HF -for example, of AIM2 and NLRC4,- need to be clarified. The different functions of inflammasomes in different types of HF (i.e., with reduced, midly reduced and preserved ejection fraction) also merit further investigation. Finally, some other inflammasome inhibitors different from canakinumab are currently being investigated in clinical trials in patients with HF suggesting that it is a promising therapeutic target for this syndrome.

Keywords: inflammasome, cardiac damage, heart failure.

Corresponding author: Javier Díez Martínez

S7-04

Inflammasome and neuronal damage

¹Diego San Felipe, ^1,2Beatriz Martín-Sánchez, ¹Miguel A Martínez-López, ¹Sara Rubio-Casado, ¹Ricardo Llorente, ^1,2María A González-Nicolás, ³Eva M Marco, ^1,2Alberto Lázaro, ^*1Meritxell López-Gallardo

¹ Department of Physiology, Faculty of Medicine, Universidad Complutense de Madrid, Madrid, Spain.

² Laboratory of Renal Physiopathology, Department of Nephrology, Instituto de Investigación Sanitaria Gregorio Marañón, Hospital General Universitario Gregorio Marañón, Madrid, Spain.

³ Department of Genetics, Physiology and Microbiology, Faculty of Biological Sciences, Universidad Complutense de Madrid, Madrid, Spain.

Sepsis is a life-threatening organ dysfunction caused by the host uncontrolled inflammatory response to an infection. This multiorgan failure can result in brain impairments affecting different areas of the Central Nervous System (CNS), including the retina. Herein, by using the systemic administration of lipopolysaccharide (LPS) as an animal model for sepsis known to induce systemic inflammasome, we aimed to analyze whether LPS may provoke retinal damage; and if DF401, a compound that effectively reversed the LPS-induced damage in other organs, may also be effective in the management of LPS-induced retinal injury. Therefore, in the present study we have concomitantly administered 28 male Wistar rats with LPS (10 mg/kg, i.p.), or its vehicle (VH); and DF401 (150 mg/kg, i.p.), or its VH, and animals were sacrificed 24 hrs. post-LPS administration. Then, body weight was registered, and eyes harvested for immunohistochemical analysis in retinal sections where Brn3, GFAP and Iba1 were employed to immunostain Retinal Ganglion Cells (RGCs) and macroglia and microglial cells, respectively. LPS induced body weight loss 24 hours post injection. LPS induced a subtle reduction in the number of Brn3+ cells in the whole retina, with effects being more marked in the nasal inferior retina, where the DF401 administration reverted or prevented such decrease. The analysis of glial markers demonstrated that LPS provoked a generalized increase in GFAP expression, together with an increase of Iba1+ cells number. LPS promoted the activation of microglia towards a migratory morphotype that was no longer present in case of DF401 administration. In addition, the combination of LPS and DF401 prevented or reverted the increase in Iba1+ cells within the nasal superior and inferior subregions. Present results provide evidence for neuronal damage and glia activation in the retina of male Wistar rats following systemic LPS administration. In addition, DF401 has demonstrated a potential protective role in the LPS-induced retinal inflammation although further research is still needed to elucidate the target and pharmacological mechanisms of this drug.

Keywords: LPS-induced sepsis, ganglionar and glial retinal cells, DF401, inflammation.

Corresponding author: Meritxell López-Gallardo, https://orcid.org/0000-0002-9291-623X

Symposium 8: Circadian rhythms: Clock, sleep and metabolism

S8-01

Circadian timing system: from physiology to clinic

^*Miguel Meira e Cruz

Sleep Unit, Cardiovascular Center of University of Lisbon, Lisbon School of Medicine, Portugal

Circadian Timing System (CTS) is a complex biomolecular machinery from which derives a genetic controlled temporal dictation synchronizing functions to optimize life and its environmental interaction. Sleep is the most conspicuous circadian rhythm taking part of about 1/3 of human’s life. Circadian clocks are also important in both the aetiology and treatment of several cardiovascular and metabolic disorders which are highly impacted by sleep and sleep disorders. In the modern world several factors may implicate with a circadian misalignment and with sleep deficits which may contribute to an increase in cardiometabolic morbidity and mortality. Therefore, researchers and clinicians should be aware of the influence that CTS and its desynchrony have on heath and disease so they can optimize diagnostic and therapeutic tools based on a temporally driven contribute to the concept of personalized medicine.

Keywords: circadian timing system, circadian clock, sleep disorders

Corresponding author: Miguel Meira e Cruz, mcruz@medicina.ulisboa.pt

S8-02

Melatonin and circadian rhythms: Cardio-metabolic impact

*Darío Acuña-Castroviejo

Centro de Investigación Biomédica, Parque Tecnológico de Ciencias de la Salud, Universidad de Granada, Avda. del Conocimiento s/n, 18016 Granada (Spain)

Cardiovascular diseases (CVD) constitute the leading cause of death in the world, and aging is by far the major risk factor for cardiac dysfunction. Aging involves subclinical inflammatory activation, with NF-kb and NLRP3 inflammasome as the main components of such response. But inflammation also induces a pro-oxidative response, responsible for the oxidative damage to the cell and mitochondria, leading to a bioenergetic failure. In turn, melatonin exerts profound anti-inflammatory and antioxidant properties, with the mitochondria as the main intracellular target. Here, we analyzed the impact of the NRLP3 inflammasome in the myocardial clock during aging and the role of melatonin in preventing it. For this study, we used wild-type and NLRP3-deficient mice of 3, 12, and 124 months of age, with and without melatonin treatment. The absence of NLRP3 prevented the age-dependent myocardial failure and mitochondrial impairment. Our results support that aging, NLRP3 inflammasome, and melatonin affected the cardiac clock genes expression, including Bmal1, Clock, Per2, Cry1 and RORα, but not Rev-erbα, which was not influenced by genotype. Aging caused phase changes in Clock, loss of rhythmicity in Per2 and Rorα, and mesor alteration of Clock, Bmal1 and Per2. The acrophases of Clock, Per2 and Rorα, were affected by the NLRP3 inflammasome, which affect myocardial function. Treatment with melatonin restored acrophases and rhythms of clock genes affected by age or NLRP3. The administration of melatonin recovered cardiac homeostasis by reversing the age-associated chronodisruption. Together, our results underline new outcomes related to the effects of age and NLRP3 inflammasome on clock genes in cardiac tissue, and support melatonin therapy as a promising therapy to counteract inflammaging and restoring circadian rhythm in heart muscle.

Keywords: heart, melatonin, inflammasome, clock genes

Corresponding author: Dario Acuña-Castroviejo, dacuna@ugr.es

S8-03

The clock in extreme environments: Lessons from physiology to the clinic

^*Michele dos Santos Gomes da Rosa

Cardiovascular Centre, Faculty of Medicine, University of Lisbon, Lisbon, Portugal

Humanity is on the verge of a new era in space exploration. Exposure to space radiation and microgravity are the main risks to human health, especially on long-term missions. Fatigue can also become a problem in the enclosed environment of the spacecraft due to a constant noise of onboard equipment, irregular light patterns, and the absence of a normal circadian rhythm (similar to earth's), all of which can alter the sleep-wake cycle and result in reduced sleeping time and/or poor-quality sleep. As they orbit the Earth, astronauts witness 16 sunrises and sunsets every 24 hours. While seeing the sunrise every 90 minutes may seem like an incredible experience, it can also make it difficult for astronauts to have a regular sleep pattern. Despite pre-mission training and preparation, living aboard a space station can be disorienting to some body functions. The sleep and circadian rhythm disorders that appear in manned spaceflight are also characteristics found in advancing age, such as sleep fragmentation, reduced nocturnal sleep tendency and sleep efficiency, reduced daytime alertness, and increased daytime nap are common to these two conditions. In the face of all these issues, aerospace medical research can be seen as a resource to improve ground medical care and patient management on Earth. In this seminar we will address some similarities between the health care used in manned space missions and terrestrial health care to identify possible approaches used in space that could become solutions to challenges encountered in the terrestrial environment.

Keywords: circadian rhythms, physiology space, extreme environments

Corresponding author: Michele Gomes da Rosa, michelerosa@medicina.ulisboa.pt

S8-04

Chronodisruption in cancer

*César Rodríguez, Germaine Escames

Centro de Investigación Biomédica, Parque Tecnológico de Ciencias de la Salud, Universidad de Granada, Avda. del Conocimiento s/n, 18016 Granada (Spain)

Tumor cells are characterized by uncontrolled cell proliferation resulting in abnormal and accelerated tissue growth. In contrast, “healthy” cells often proliferate with a division rate of ~24 h. This is due to the direct control of cell cycle checkpoints by the intracellular circadian clock machinery. At the molecular level, these clocks are based on clock genes, which participate in auto-regulatory feedback loops. In the core loop, the transcription factors CLOCK and BMAL1 activate the expression of Per and Cry genes, whose protein products negatively feedback on their own expression. Several additional feedback loops contribute to this canonical mechanism, including the orphan nuclear receptors REV-ERBα and RORα. Accumulating epidemiological and genetic evidence indicates that circadian rhythms’ disruption may be linked to cancer. Moreover, circadian clock alteration due to mutations of single clock genes, such as Per2 or Bmal1, accelerates tumor growth or the whole carcinogenesis process. This is putatively due to an increase in proliferation rate upon circadian rhythm disruption. Intriguingly, cancer prognosis and survival has been associated with the level of circadian disruption in patient tumor tissues. It is well known that the broken clock is recovered by melatonin administration, that is accompanied by the normalization of the innate immunity and mitochondrial homeostasis. In our group we have shown that melatonin induces an increase in apoptosis and a decrease in proliferation in head and neck cancer cells, and these effects correlate with a synchronization of Bmal1 as well as Sirt1, clearly related to mitochondrial function.

Keywords: chronodisruption, cancer, clock genes

Corresponding author: Germaine Escames, gescames@ugr.es

Symposium 9: Animal research in the 21st century

S9-01

The legal requirements in research with animals

*María Reyes

Animalary and Research Animal Section, University of Extremadura, Badajoz/Cáceres, Spain

The use of animal in research has always indued an ethical debate both within and out of the research community. At the very first beginning of animal experimentation, there were no established rules or legislation. There was even a period in which it was assumed that animals did not feel nor suffer like human beings do. Afterwards, a trend emerged against this practice, and it was considered that the use of live animals should follow a series of ethical rules in order to carry a proper control of their use. Avoiding suffering of animals and to promote a significant reduction in the number of animals used were then major aims, in addition to a refinement of the procedures that would allow obtaining more and better results with a minimum use of animals. The whole of biomedical research carried out does not need to use animals. Alternative methods exist, which can provide the same results. However, in some procedures it is unavoidable to count with a living being to obtain realistic and reliable results. In order to limit animal experimentation to ​​what is considered strictly essential for the obtention of important and justified benefits, a series of regulations and laws have been set in Europe, which must be thoroughly followed. These regulations require the specific accreditation of researchers with specific skills and make it mandatory to request a positive evaluation and prior authorization of the procedures to be applied in research with animals. Being aware of all the necessary procedures to work on research with animals will avoid undesirable delays in the development of certain projects, which may affect the research team involved, the facilities, or even the budget. All the bureaucracy involved may discourage the researcher at the very first beginning. Even more, it could be considered an excessive control from the Government’s´ side. Nevertheless, getting familiar with the forms and the necessity of this legislative framework will end up with self-consciousness of researchers, in the sense that everything is designed with the aim to achieve animal welfare and to assure reliable experimental results.

Keywords: responsible animal experimentation, experimental animal legislation, RD rule 53/2013.

Corresponding author: María Reyes.

S9-02

Studies about oral physiology and ingestive behaviour using animal models

¹Elsa Lamy, ^1,2Fernando Capela-Silva F

¹ MED – Mediterranean Institute for Agriculture, Environment and Development; IIFA – Instituto de Investigação e Formação Avançada; University of Evora.

² Biology Department; School of Sciences and Technology, University of Evora.

Ingestive behavior requires complex regulation, that makes its study challenging. Among the diversity of factors influencing it, the way food is perceived inside the mouth is relevant for acceptance and future choices. Different classes of sensory signals (e.g., taste, retronasal aroma and texture) are detected in the mouth, which interact with each other to modulate the final perception. If, on one hand humans have the capacity of report what they are sensing, allowing to relate sensory perception and acceptance, on the other hand, more invasive procedures, such as the induction of metabolic or hormonal changes, for example, can require animal models. Moreover, in animals we have the possibility of study innate and spontaneous behavior, not so easy to access in humans. At the same time, to used animals for these purposes can be challenging. For example, for assessing sensory perception. In this case, special technology, able to measure particular types of behavior, integrated with the interpretation of such behavior, is needed. One of the particular aspects of oral physiology, to which we have been dedicated for more than 15 years, is saliva composition, related with ingestion and metabolism. In this case, challenges in working with animals should also be considered. Simple things like the acquirement of samples can be difficult. Whereas in humans we can consider saliva collection as non-invasive to the individual, in animals this varies among species. For example, in large domestic ruminants, familiar with human management (e.g., dairy cows) we can access high amounts of this fluid, whereas in small breed dogs, not used with the procedure, this can be a difficult task. In this presentation we will present examples of work done relating oral physiology with food acceptance and choices and will highlight the major advantages and limitations.

Keywords: Animal models, ingestive behavior, food sensory acceptance, saliva

Corresponding author: E. Lamy

S9-03

Animal models in biomedicine, a key piece with countless possibilities

Patricia Santofimia Castaño

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France

Cancer remains one of the world's leading causes of mortality. According to the latest available data, the number of diagnosed tumors in the world population continues to grow and the number of new cases is expected to increase, reaching 29.5 million in 2040. Fortunately, cancer survival has risen sharply in recent decades. However, this decrease in mortality is not uniform across all tumors.

Further research must be done in order to find new therapeutic targets and to improve the effectiveness of chemotherapy, mostly, in aggressive tumors. Thus, in this regard, there is an urgent demand to innovate new preclinical models that recapitulate the tumor-microenvironment interface, the pressure of the immune system, and molecular and morphological characteristics of the tumor.

The 2D cell line cultures are the most commonly used in vitro model. However, they have a low fidelity to mimic the architectural and functional complexity of the tumor. 3D culture methods are very promising tools to better mimic the tumor biology found in vivo, offering many advantages over monolayer culture.

However, in vivo models are much more clinically relevant, addressing many of the drawbacks of in vitro models. Genetically modified mice are a widely used tool for studying cancer; by activating oncogenes and/or inactivating tumor suppressor genes. These models can be implemented with the combination of constitutive or conditional knock-out mice in the whole organism or the organ of interest. On the other hand, xenotransplantation or allotransplantation are other interesting strategies to investigate the tumor response. In this regard, the patient-derived tumor xenografts offer a lot of advantages because they retain the morphological characteristics of the primary tumor as well as its metastatic potential, reflecting patient’s response to chemotherapy.

Recently, several in ovo models have been developed, which consist of the implantation of tumor cells either in the chorioallantoic membrane of a chicken egg or different organs in the chicken embryo. In addition, innovative advancements, such as 3D Bio-printing, organs-on-a-Chip, or swine or fish models are increasing the number of choices of our preclinical models, improving our investigations, and ensuring the future benefit of the patients.

Keywords: Animal models, pancreatic cancer, Transgenic models, knockout models

Corresponding author: Patricia Santomifia.

S9-04

Gnotobiotic Animals Facility of the Center for Scientific Instrumentation of the University of Granada. How to work with these animals and what to expect from them

^*1Ana I Nieto, ²Gustavo Ortíz-Ferrón, ¹María J García-Chicano, ¹Mariano Mañas

¹ Unidad de Gnotobióticos. Centro de Instrumentación Científica. Universidad de Granada.

² Unidad de Citometría. Centro de Instrumentación Científica. Universidad de Granada.

The germ-free (GF) mouse exists in a sterile environment completely devoid of microorganisms. Moreover furthermore, gnotobiotic mice can be modified by mono or poly-inoculation with known microbial organisms (gnotobiote, from the Greek words “gnostos”, for known, and “bios”, for life). Both have proven to be an important tool in understanding the complex relationship between a host and its microbial residents. Gnotobiotic animals have been used in a wide variety of models demonstrating the critical importance of various microbiota partners and their interaction with the mammalian host such as, cancers, metabolic, autoimmune and central nervous system diseases. However, the maintenance and ultimately the monitoring of gnotobiotic rodents is a critical, labor-intensive, and costly process (e.g., sterility, not absence of specific pathogens, must be demonstrated in germfree animals). Gnotobiotic Unit from University of Granada (UGR) is one of the few facilities in Europe houses so-called gnotobiotic mice. These mice are born in germ-free conditions, and investigators control the microbiota by inoculating the animals with specific microorganisms. The facility is setup with 4 rigid methacrylate isolators that can contain 12 cages each; each of the cage is capable of housing 7-10 mouse or to dedicate to breeding. In addition, the Gnotobiotic facility count with a rack of isopositive cages (Isocages) from Tecniplast^©. In this system, each cage is hooked up to an air supply that is filtered twice through a HEPA filter before it gets into the individual cage. Animal were transferred from the germ-free isolators into Isocages in a biosafety cabinet prepared by thorough spraying with Virkon. The inoculation was realized also in the cabinet All the material is sterilized through autoclavable stainless steel DPTE® containers. Furthermore, our Flow Cytometry Unit can be leveraged for robust microbiome research using gnotobiotic mice with defined microbiota. Flow cytometry and cell sorting are becoming particularly powerful in microbiome research. Our mission is to be leaders in microbiome germ free and gnotobiotic research through providing expertise, resources and service. Our germ-free facility enables internal and external researchers, as well as industry partners to perform state-of-the-art in vivo microbiome research using experimental conditions with precise microbial status.

Keywords: germ-free, gnotobiotic, microbiota, cytometry.

Corresponding author: Ana I. Nieto, https://orcid.org/0000-0001-9698-3686

S9-05

Animal research in the 21st century: the challenge of including sex as a biological variable

^*1Eva M Marco

¹ Department of Genetics, Physiology, and Microbiology, Faculty of Biological Sciences, Complutense University of Madrid, Madrid, Spain.

Females have long been neglected in animal research. The National Institutes of Health (NIH) included the enrollment of women in clinical research in 1993, but it was not until 2015 that female animals were included as mandatory in the preclinical research in the United States of America. In Spain, the situation is far from ideal, although nowadays most national calls for proposals requires an explanation if both sexes are not included in the research project. Although in the last decades male bias in human studies has notably decreased, it has not been the case for non-human studies. Accounting for sex as a biological variable in preclinical research should be mandatory, and it should be considered at every step of the research project: from the initial development of research questions and study design to the final reporting of findings. Considering sex as a biological variable may be critical to the interpretation, validation, and generalizability of research findings. Appropriate analysis and transparent reporting of data by sex may also enhance the reproducibility and translational value of preclinical biomedical research. Moreover, animal welfare must also be considered when referring to sex bias in research since remarkable sex differences have been reported regarding stress response and pain perception. Considering sex as a biological variable should be our challenge in animal research for the next decades, and both researchers and the general population should join forces to convince institutions and national committees to require a balance in male and female animals in preclinical investigations to achieve more relevant knowledge that could be applied to the clinic.

Supported by Complutense University of Madrid, UCM Research Group: 951579.

Keywords: animal models, sex bias, animal welfare, 3Rs.

Corresponding author: Eva M Marco, https://orcid.org/ 0000-0002-2457-475X

Symposium 10: Teaching in Physiology

S10-01

Scape Room as a tool to improve motivation in science-based subjects

¹Silvia M. Arribas, ²Juan Arredondo Lamas, ²Alejandro Samhan Arias, ²Ana I. Rojo, ²Gemma Domínguez Muñoz, ¹David Ramiro-Cortijo, ^3,4Jesús Arribas Blázquez

¹ Departmnent of Physiology; ² Department of Biochemistry, Faculty of Medicine, Universidad Autónoma de Madrid; ³ Scape Diem, Murcia, Spain; ⁴ Colegio Miralmonte, Murcia, Spain.

Motivation is one of the key elements in the quality learning, and it has been demonstrated that it leads to better learning strategies and performance. Although the motivation to learn is the responsibility of the student, the teachers should help to boost it, using tools that awaken extrinsic motivation, which, in turn, can trigger students’ intrinsic motivation. The support of the teacher may be particularly relevant in subjects with a high science load, which often require high levels of student motivation to achieve optimal learning outcomes. For this propose, gamification is becoming more and more popular as a learning tool. Scape Room, one of the learning techniques based on gamification, is a format well received by students. We have used this technique in two education contexts: 1) High school, to encourage interest in scientific careers (Escape Room with Science, ERS), and 2) Nursing degree, to increase the interest in Biochemistry, which is a subject that students perceive unrelated to their professional practice. ERS is a project integrated by 25 researchers from the Universidad Autónoma de Madrid and Instituto de Salud Carlos III. Its objective is to boost interest in science-based degrees, eliminating taboos and gender biases. We have developed 4 online Escape Rooms, designed by an expert through Genially platform. They are based on our scientific material and include a 1 min video of our research activity and literacy assessment questionnaire, currently under analysis. We expect that the challenge to solve a problem related to science, being the main actors will be a source of motivation for the students to choose a scientific career. Regarding nursing students, we have implemented two Escape Rooms intended to help students review the course contents in a playful manner and favor their engagement and motivation, employing OneNote software. Besides, we have set up an Escape Room as an optional part of the final evaluation to be solved in teams. Opinions collected through a survey show that Escape Rooms promoted learning and were useful as a self-evaluation and review tool.

Supported by Fundación Española para la Ciencia y la Tecnología (FECYT; FCT-19-14506).

Keywords: Scape room, science-based subjects, gamification, motivation.

Corresponding author: S.M. Arribas, https://orcid.org/0000-0001-7103-6105

S10-02

Development of an inter-university competition (FisioCómic) as a tool to promote creative learning in Physiology

^*1Beatriz Gal, ²Carlos Romá, ³Silvia Arribas

¹ Universidad Camilo José Cela

² Universitat de València

³ Universidad Autónoma de Madrid

Physiology is a basic subject in Health Sciences Degrees, which provides the fundamentals for understanding body function as a whole. It is also an integrating subject with other basic disciplines such as Anatomy, Histology, etc. However, first year students tend to feel it as a barrier in their academic path towards most clinically relevant subjects and fail to develop the necessary critical thinking required to reach a holistic physiological understanding. Developing ludic-competitive activities included into a gamification strategy may help to improve their motivation for learning integrative physiological concepts. The objective of this work was to promote creative thinking and motivation through gamification for learning Physiology in Health Sciences degrees. For this purpose, an extra-curricular teaching action (FisioCómic) has been developed in a national inter-university environment for Health Sciences degrees students. The participants required having studied any aspect of Physiology, whether cellular or human. Each university could present up to a maximum of 4 teams (made up of 2 to 5 students from similar or different degrees). Each team had the support of a teacher who act as a tutor and who was in charge of supervising the students' work throughout the preparatory phase. For the selection of the best proposals, a first eliminatory phase was carried out. Each university, through its coordinator, vote for the 4 best comics selected according to a rubric, excluding those presented by their own institution. In a second phase, the 10 best rated comics were evaluated by a jury made up of comic designers and scientific communication professionals. This first edition, which was organized by the Autonomous University of Madrid, the European University and the University of Valencia, involved 20 teams from different Spanish universities participated. Analysis of the first three prizes reveals the ability of the activity to promote integrative knowledge of complex physiological concepts, such as phototransduction, plasticity in the form of long-term potentiation and the reward route. In conclusion, learning Physiology through this type of initiative has been a very positive experience and highly motivating for students, who have developed skills such as creative thinking. By opening this initiative to other aspects of Physiology and related disciplines, both in the human and animal physiological domains, can help to integrate concepts across basic disciplines and to promote critical thinking.

Keywords: Physiology, creative thinking, graphic medicine, gamification

Corresponding author: Beatriz Gal

S10-03

Use of Twitter to promote the critical analysis of scientific knowledge among undergraduate students of Medicine

^*1Gregorio Segovia, ¹Julian Bustamante, ¹Victoria Cachofeiro, ¹Natalia De las Heras, ¹Jesús A Fernández-Tresguerres, ¹José A García-Baró, ¹Ricardo Gredilla, ¹Asunción López-Calderón, ¹Ana I Martín, ¹Sergio Paredes, ¹Miguel A Pozo, ¹Teresa Priego, ¹María A Vicente

¹ Department of Physiology, Faculty of Medicine, University Complutense of Madrid, Madrid, Spain

The aim of the teaching innovation project presented here was to improve the capacity for critical analysis of scientific knowledge among students of the Degree in Medicine through the use of Twitter. Students of the Human Physiology course (2nd year) organised themselves into teams ("Scientific news agencies") to share scientific studies related to the concepts included in the syllabus of the course via Twitter, using the hashtag #FisioHumUCM. Subsequently, the teaching staff made a selection of the shared news to create an online newspaper ("Human Physiology News Bulletin"), which was organised into thematic blocks (Physiological Systems). Finally, each team presented one of the news items in the newspaper as part of the continuous assessment of each thematic block. 23 working teams of 10 members each were created and posted a total of 3837 tweets. Most of the tweets consisted of a link to a scientific news item with or without a short commentary. Some teams also made threads of tweets commenting on the news item and relating it to the subject matter. At the end of the course, an online survey (Google Forms) was conducted to evaluate the project using Likert scales (scale from 1 to 10). A total of 129 students (56% of the participants) answered the survey. Of the students who took the survey, 31.8% used Twitter frequently, 29.5% used it occasionally and 38.8% did not have a Twitter account. The overall rating of the project was 8 (ds=1.55). The students responded that they liked the activity of sharing news on Twitter (7.98; ds=1.73) and that they found the news presentations interesting (8.09; ds=1.76). They also considered that the project had increased their ability to critically analyse biomedical research and its communication (7.65; ds=1.71) and that the methodology had increased their interest and curiosity in the subject (8.05; ds=1.69). We believe that the use of Twitter combined with the presentation of scientific news is a useful tool to improve the ability to critically analyse biomedical scientific evidence, a basic skill that students of the Degree in Medicine should acquire.

Supported by University Complutense of Madrid (INNOVA-DOCENCIA 2020-2021).

Keywords: Twitter, project-based learning, critical analysis, research.

Corresponding author: Gregorio Segovia, https://orcid.org/0000-0001-5669-0889

S10-04

Biomedical research divulgation as a learning hub in Physiology

Teresa Fernández Agulló, María J Alonso Gordo, Custodia García Jiménez

Department of Basic Health Sciences, Faculty of Health Sciences, Rey Juan Carlos University, Alcorcón

Teaching the subject “Human Physiology” for medical students at University Rey Juan Carlos includes a research activity related to hot topics with a physiological basis. Subgroups of students work under the supervision of a tutor throughout the term to elaborate a written report that is defended through oral presentations. With the pandemic situation, this activity was moved to a remote format. This prompted us to consider other alternatives to evaluate the communication skills in physiology acquired by our students. We have proposed to the students the choice between a classical oral presentation (8 min) or the elaboration of a divulgative video (3 min) on the subject chosen. Throughout this activity, students read scientific papers, which allows them to appreciate the value of research while working on transversal skills such as Capacity for analysis and synthesis, Capacity for organization and planning, Oral and written communication, information management, decision making, teamwork, ethical commitment, autonomous learning and creativity. The activity is evaluated through a written report (similar for all) plus the ability to defend their work by oral presentation or videos followed in both cases by public discussion. The aim of this activity is to make students more involved in order to explore if the skills for oral defense of a scientific work are better acquired through more attractive formats. As secondary objectives, it introduces students in the research carried out in their environment and how knowledge is generated based on evidence in Health Sciences.

Keywords: divulgation, video, creativity, communication.

Corresponding author: Teresa Fernández, ORCID

Symposium 11: Environmental physiology

S11-01

The role of the microbiome – Retrospective and future perspectives in a One Health model

^*1Pedro Brogueira, ¹Manuela Vilhena

¹ Master in One Health: Human and Animal Public Health, University of Évora, Évora, Portugal

Microbiota refers to a community of microorganisms in a specific environment. When considering the role of microorganisms, the interactions between the various species and their function, we are defining microbiome. We can define the microbiome from two perspectives: ecological, describing the community of commensal and symbiotic species that share our body space; or in terms of genomics and interrelations, the microbiome being the entire collection of microorganisms and their genomes, considering their function and the interactions between them. The human being must be perceived as a holobiont, a mutualistic ecosystem composed of the three domains of life – Bacteria, Archae and Eukaryota. In general, human organs and systems depend on this interaction to be considered biologically and functionally complete. In this regard, we can understand the microbiome in its role as a facilitator in our evolution as a superorganism, an assembly of multiple organs and systems in which vital functions are performed in order to keep homeostasis and assure survival. This concept of coevolution enlightens the need of a holistic understanding of the microbiota. As an evolutionary process, it is modulated by the environment, largely influenced by the surrounding biodiversity, namely plants and animals. Such understanding would be vital for one to predict the consequences of the development of specific microbiomes, as a result of increasing domestication and breeding activities, crop management, overcleaning of home environments and other anthropogenic shifts that shape microbiota diversity and drive host-microbiome interactions over time, sometimes leading to a state of dysbiosis. By changing environmental conditions in a way that microbiomes also shift their structure and function from dysbiosis into a healthy state, a synergy is visible across humans, animals and cropping systems. Understanding each particular microbiome assembly and functional capacities in terms of host-microbe, environment-microbe and microbe-microbe interactions can pave the way to tailor made interventions, like the so-called personalized medicine or precision agriculture. This One Health approach can bring considerable gains in terms of enhancing productivity and sustainability of diverse systems. The main goal is to improve health of humans, animals, plants and whole ecosystems, connecting all the fields and producing innovative evidence.

Keywords: Microbiome, Coevolution, Sustainability, One Health

Corresponding author: Pedro Brogueira, pbrogueira@gmail.com

S11-02

Physiological ramifications of human exposure to the micro-nano-plastics invasion

^*M Ramiro Pastorinho

Department of Medical and Health Sciences, School of Health and Human Development, and CHRC (Comprehensive Health Research Centre), University of Évora, Portugal.

The post II World War industrial development witnessed a large-scale production of plastics. This manufacture has steadily grown annually, increasing 22-fold to the present 364 million tons, with the production forecasted to triple by 2050. The scientific community has been devoting growing attention to microplastics (MPs<5 mm) and nanoplastics (NPs<0.1 μm). During manufacture chemical additives such as plasticizers, BFRs, antioxidants, pigments, fillers, etc. are incorporated into plastics, and leach over time as they are not chemically bound to the polymer matrix. In addition, transport throughout the environment causes MPs and NPs to come across multiple pollutants and due to plastics’ hydrophobic surface, what has been called the “chemical sponge” effect occurs. M-N-Ps have been detected throughout the marine, freshwater and terrestrial environments, as well as in items commonly consumed by/in contact with humans. Upon exposure, NPs exhibit strong biological penetration capability, being able to reach and penetrate organs, including placenta and brain. Recent studies have detected these materials in human samples. However, critical data are lacking to accurately estimate NPs’ human exposure and effects: they are seldom measured (some sources being ignored); methods for identifying and measuring particles are highly variable between studies rendering comparisons difficult and leading to under-or-overestimations; most studies do not identify the polymers in the analyzed samples, a problem since each polymer has different additive chemicals and affinities for contaminants; sample analysis results seldom include mass measurements; studies of human and animal health effects are too few and too limited, and test for unrealistic doses; most human exposure media are not sampled, while others are over studied (i.e., seafood ingestion). In face of this, despite the acceleration in scientific research, no broad conclusions toward human health can, at this point, be drawn, since exposure-dose has yet to be established, being the impacts on biological functions loaded with uncertainty. In this presentation we detail micro and nanoplastic exposure pathways, highlight the most important additives and absorbed leachable contaminants and summarize their potential risk to human health under current knowledge of cellular uptake routes, intracellular fate, toxic effects and toxic mechanisms.

Keywords: additives, contaminants, pollutants, “chemical sponge effect”.

Corresponding author: Ramiro Pastorinho, https://orcid.org/0000-0001-5205-4973

S11-03

How human physiology reacts to a long-term microgravity

¹Michele dos Santos Gomes da Rosa

¹ Cardiovascular Centre, Faculty of Medicine, University of Lisbon, Lisbon, Portugal

Targets the next years will see tourism and commercial space activity increase. EMEA, Americas, and Asia Pacific Nations are Driving R&D into space tourism at an accelerated rate to offer services and choice for citizens to journey 100 Km above the Earth’s surface. But this is just the beginning. Greater safety, better vehicle capacity and varying options of comfort might result in more people travel beyond the “karmaran line”. The human factors of such travel are still being studied, not only for space tourism but for moon, Mars and other deep-space missions. Similarly, space technology itself has advanced and matured enough for Humanity to resume space exploration beyond the near-earth orbit. But a long duration inter-planetary space flights will increase the “degree of difficulty” associated with health monitoring of space travellers. While the fidelity of data being telemetered back to earth is not likely to be an issue, the transmission delay may become an issue worth thinking about. More importantly, I believe that the bigger challenge is going to be in ensuring the physiological well-being of space crews assigned to human settlements on the Moon. Later, longer transit times to Mars, will add another challenging dimension requiring to evolution of interventional physiological strategies- both passive and active – to ensure the health of long duration space crews. These fundamental aspects to space travel and space living preparedness are the premise and expertise that will be showed in this seminar. We will put humans into space is safe.

Keywords: microgravity; physiology space; environmental space

Corresponding author: Michele Gomes da Rosa, michelerosa@medicina.ulisboa.pt

S11-04

Physiopathological effects upon heavy metals exposure: focus in environmental lead exposure

^*1,2,3Liana Shvachiy

¹ Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany

² Cardiovascular Centre of the University of Lisbon, Portugal

³ Institute of Physiology, Faculty of Medicine of the University of Lisbon, Portugal

Heavy metal exposure toxicity has skyrocketed as a result of the twentieth century's industrial activity. The most frequent heavy metals that cause human poisoning are mercury, lead, chromium, cadmium, and arsenic. The bioaccumulation of these heavy metals causes a wide range of harmful consequences in various human tissues and organs. Heavy metals interfere with biological functions such as growth, proliferation, differentiation, damage repair, and apoptosis. The mechanisms of action for these metals to cause toxicity are similar, including the formation of reactive oxygen species (ROS), the weakening of antioxidant defences, enzyme inactivation, and oxidative stress. One of the most common heavy metals that have been occurring in the environment and studied for its systematic toxicity is lead. Exposures to lead during developmental phases can alter the normal course of development, with lifelong health consequences. Permanent Pb exposure leads to behavioural changes, cognitive impairment, sympathoexcitation, tachycardia, hypertension, and autonomic dysfunction. However, the effects of intermittent Pb exposure, which has become more common in recent years, have not been investigated. We wanted to see how lead affected people's health at different levels of exposure, including a new animal model of intermittent low-level lead exposure. In our project we focus on different environmental lead exposure profiles and their long-lasting physiological effects. As the main route of lead exposure is ingestion, we replaced the tap water by a low-level lead acetate solution. We applied an interdisciplinary, longitudinal, and comparative approach in an animal model of Wistar rats from foetal period to adulthood. We characterize the new, intermittent low-level lead exposure profile and compare it to the other lead profiles that have been studied before, through time, showing the differences between the various time-points through the lifetime of the animal. We demonstrated new insights on the effects of environmental lead exposure on nervous and cardiovascular systems during developmental phases, which can help create new public policy strategies to prevent and control further adverse health effects.

Funding: LS is supported by Foundation for Science and Technology (FCT) - SFRH/BD/143286/2019

Keywords: heavy metals, pathophysiology, environmental lead exposure, cardiorespiratory impairment.

Corresponding author: Liana Shvachiy, shvachiy.liana@gmail.com, https://orcid.org/0000-0002-4576-0017

Symposium 12: Physiological aspects of assisted reproduction

S12-01

Surplus embryos from IVF cycles. Unprecedented research possibilities

^*1José L Cortés

¹ Eppendorf Ibérica SLU, Spain

In Spain, there is an estimation of 500.000 surplus embryos from IVF cycles. This country currently represents a permissive legal and ethical environment for the use of excess embryos for research, thanks to Spanish Reproduction Law 14/2006. Although IVF cycles must not be planned specifically for the generation of human embryos to be used for research, cryopreserved embryos fewer than 14 days past fertilization may be used for that purpose, regardless of the length of time that they have been kept frozen. The only requirement for the use of these embryos is that the procedures must be

linked to a specific research project. During the last 20 years, many countries, including Spain, have set up stem cell banks to provide researchers with human embryonic stem (hES) cell lines and facilitate hES cell research, about all in the optimization of the methodology. In our case, in the Andalusian Stem Cell Bank, we proposed the use of the laser for the isolation of the inner cell mass, and the use of mesenchymal stem cells as growth surface for this aim. The arrival of the induced pluripotent cells stopped the derivation of new hES cell lines. So, new research projects had to be found for these surplus embryos. Some examples: Long Interspersed Element-1 (LINE-1 or L1) is a family of active non-LTR retrotransposons that comprise around a fifth of our genome and can lead to human genetic disease. It is not known when exactly LINE-1 retrotransposition begins to re-model the human genome. The group where I collaborate report endogenous L1 expression and mobilization during early stages of human embryonic development. We found that L1 mRNA and the L1-encoded ORF1 protein are expressed at multiple stages of human embryogenesis (2-cell to blastocyst stage). In base to this study, is possible to develop a single-cell high- throughput sequencing methodology to characterize endogenous de novo full-length L1 retrotransposition events in human embryos. Also, we could discover a novel phylogenetically- restricted cell type consistent with the human early embryo being a clonal selection arena, and an emergence and antiviral activity of a human placental protein of retroviral origin.

Keywords: embryonic stem cells, Long Interspersed Element-1, retrotransposition, surplus embryos

Corresponding author: José L Cortés

S12-02

Studies on gametes and embryos with implantation failure

^*Susana Cortés Gallego

Directora Laboratorio de Reproducción Asistida de la Clínica Tambre, Madrid, España

Recurrent implantation failure (RIF) is the term used to refer cases in which women have had at least three failed in vitro fertilization (IVF) transfer attempts with good quality embryos. Embryo implantation failure can be a consequence of multiple factors like abnormal uterine anatomical conditions, sperm/oocyte or embryos factors, maternal age or the specific type of IVF protocol. Furthermore, RIF has multiple known causes, but many are not routinely considered as part of the posttreatment analysis, such as those associated with lifestyle (smoking status of both parents, elevated body mass index or stress levels) and other causes related to pre-existing conditions that have only weak or no apparent connection to fertility. Immunological factors such as cytokine levels or the presence of specific autoantibodies as well as any infectious organisms in the uterus leading to chronic endometritis may be related with RIF. Sperm analysis (sperm DNA damage, FISH, chromosperm, …), pre-implantation genetic screening, thrombophilia study, immunological factors and endometrial receptivity should be considered and evaluated in these patients. Inadequate culture conditions, and suboptimal embryo development play also a significant role in the ethiology of recurrent implantation failure. A good IVF Quality Control program is fundamental. Preimplantation genetic screening does not increase implantation or live birth rates, but it rules out failures derived from the transfer of embryos with chromosomal abnormalities. Assisted hatching to overcome zona hardening could be useful in selected cases. Blastocyst transfer could contribute to improve implantation and pregnancy rates. Novel embryo assessment (time-lapse imaging and metabolomics) and sperm selection procedures (Chip-fertile; MACs), may help in better evaluation of embryo quality and viability and help in selecting embryos with the highest implantation potential. Treatment approaches should be individualised for each particular case. There is not just one treatment option, in fact there are many depending on the ethiology and the severity of the problem.

Keywords: implantation failure, embryo factors; immunological factors¸ endometrial receptivity.

Corresponding author: Susana Cortés Gallego

S12-03

Importance of the culture medium and its influence on the state of blastocyst

^*Yosu Franco

Hospital Ruber Internacional

Establishing an optimal culture system is critical to the proper development of human embryos and the success of a breeding program. The improvement of culture media has led to the development of a new generation of incubators, causing policies on the number of embryos to be transferred have changed favorably. When embryos are cultured in vitro, about 50% will stop developing during the first days of culture, due to different causes, including chromosomal abnormalities, suboptimal culture conditions or inadequate oocyte maturation, among others. It is well-known that the embryo is naturally exposed to nutrient gradients within the female reproductive tract and that, depending on its development, it exhibits changes in the use of these nutrients. Determining the nature of each gradient has facilitated the formulation of state-specific culture media design to facilitate embryo development through the preimplantation period. The use of these media when transferring embryos at D5 vs. D3 has shown a significant increase in the implantation rate per blastocyst of 50.5% vs. 30.1%. Even certain medium components can increase blastocyst euploidy.

Keywords: culture medium, blastocyst, embryo transfer

Corresponding author: Yosu Franco.

S12-04

Influence of vitamin D levels in assisted reproduction treatments

¹Juana Mª Molina, ¹Julio A Gobernado, ¹Isabel Molpeceres, ¹Luis Rodríguez-Tabernero, ²Sara Martínez

¹ Clinical Universitary Hospital of Valladolid, Spain, and ² Serrania of Ronda Hospital, Spain

Vitamin D is essential for the proper functioning of the human body. Its fundamental functions in terms of calcium metabolism are known, but in recent years its possible association with the female reproductive system and its possible influence on human reproduction treatments have been postulated. This prospective observational study was designed with the objective of knowing the prevalence of Vitamin D deficiency in patients undergoing in vitro fertilization (IVF) treatments, its seasonal variation, and the relationship between these levels with the results of the treatments. 8.5% of the patients presented severe deficit, 71.2% moderate deficit, presenting normal values only 20.3% of the studied population. The seasonal variations of the levels of Vitamin D showed statistical significance, finding higher values in summer and autumn where 30-40% of the patients reached normal levels, while in spring and winter it was less than 10%. The antimullerian hormone showed a lineal relationship between its levels and those of Vitamin D, being these results statistically significant. On the other hand, when grouping the patients according to the deficit, the differences observed did not present statistical significance. Greater FSH requirements were observed during treatment for patients with greater Vitamin D deficiency, without these results being statistically significant. No significant differences were found between vitamin D levels and the number of follicles larger than 14 mm observed in transvaginal ultrasound prior to oocyte retrieval, the total number of oocytes obtained, or the number of metaphase II oocytes. Fertilization and blastulation rates were higher in patients with severe Vitamin D deficiency, without being these results statistically significant. The number of biochemical and evolutionary pregnancies was analyzed, and when these data were related to Vitamin D levels, higher pregnancy rates were observed in the group with severe deficiency, without the results being statistically significant. The different published studies, like our study, provide contradictory data, so more studies and a larger sample size are necessary to be able to reach conclusions and thus establish evidence-based recommendations.

Keywords: Vitamin D, in vitro fertilization, antimullerian hormone, pregnancy rates.

Corresponding author: Juana María Molina

ORAL COMMUNICATIONS

Oral session 1: Neurophysiology

O1-01

Serum myeloperoxidase, but not the cerebrospinal-fluid enzyme, is closely linked to neuronal loss in the substantia nigra and motor severity degree in Parkinson´s disease

Emilio Fernández Espejo

Reial Acadèmia de Medicina de Catalunya, 08001 Barcelona & Real Colegio Oficial de Médicos de Sevilla, 41013 Sevilla.

Myeloperoxidase (MPO) has been implicated in the development of Parkinson´s disease. To date, no study has examined how MPO level in biofluids relates to motor features of idiopathic PD. The objective of this study was to look at the relationship of MPO in serum and cerebrospinal fluid (CSF) with clinical variables of the disease. MPO concentration and activity were measured in patients and controls, and degree of nigrostriatal dopaminergic cell loss was evaluated with single-photon emission computed tomography (SPECT) with ¹²³I-Ioflupane, specific radioligand of the dopamine transporter (DAT). The results revealed that MPO activity and concentration in blood serum, not in the CSF, were significantly higher in the patients vs. controls (p<0.0001). Significant correlation was found between serum MPO concentration and rating scales of motor severity (Hoehn-Yahr staging; MDS-UPDRS part III), as well as percentage reduction of DAT binding on basal ganglia (p<0.0001), adjusted for age and gender. To sum up, content and activity of serum myeloperoxidase, not CSF MPO, are enhanced in PD patients relative to healthy individuals. Serum MPO level correlates with motor severity degree and reduction of dopamine-transporter binding on basal ganglia, an indirect measure of neuronal loss in the substantia nigra. These results would open new avenues for treatment and could help explain PD pathogenesis.

Supported by Sociedad Andaluza de Neurología (SUBAIA2015/006), and RETICS Red de Trastornos Adictivos, Instituto de Salud Carlos III (RD06/001/002).

Keywords: myeloperoxidase, serum, cerebrospinal fluid, Parkinson´s disease.

Corresponding author: https://orcid.org/0000-0003-0724-6049

O1-02

Changes in chemokines and their receptors involved in phagocytosis, demyelination, chemotaxis, and coagulation contribute to explain the differences between APP/PS1 and wild type mice

^*1Soraya L. Valles, ^1,2Adrián Jordá, ¹Ignacio Campo-Palacios

¹ Department of Physiology, School of Medicine, University of Valencia, Valencia, Spain, and

² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

In Alzheimer’s disease (AD) increase in inflammation is distinctive. The amyloid precursor protein plus presenilin-1 (APP/PS1 mice) is a model for AD. Chemokines secreted by Central Nervous System (CNS) cells could play important roles in AD. Data looking for the chemokines involved in inflammatory mechanisms are not further understood. Changes occurred in the inflammation process in AD, could be necessary to improve strategies to act in specific inflammatory targets. Chemokines and their receptors involved in phagocytosis, demyelination, chemotaxis, and coagulation were the objective of our study. Female APPswe/PS1 double-transgenic mice (B6C3-Tg) were used and cortex brain of 20-22 months of age were obtained and used to quantify chemokines and chemokine receptors expression using RT-PCR and Western-blot technique. Significant inflammatory changes were detected in APP/PS1 compared to Wild type mice. CCR1, CCR3, CCR4, and CCR9 were elevated and CCR2 were decreased compared with Wild type mice. Their ligands CCL7, CCL11, CCL17, CCL22, CCL25, and CXCL4 showed an increase expression, however, changes were not observed in CCL2 in APP/PS1 compared to Wild type mice. Changes in protein expression could contribute to explain the differences between Alzheimer patients and elderly people without AD. This would provide a new strategy for the treatment of AD, with the possibility to act in specific inflammatory targets.

Supported by Oskar Fisher Foundation, USA.

Keywords: inflammation, chemokines, APP/SP1, Alzheimer’s disease

Corresponding author: Soraya L. Valles, https://orcid.org/0000-0003-4861-4266

O1-03

Ablation of carotid body activity improves cognitive function and attenuates the levels of neurodegenerative markers in prediabetic rats

Adriana M Capucho, ^*Ana Chegão, ^*Bernardete F Melo, Fátima O Martins, Natália Madeira, Joana F Sacramento, Rosalina Fonseca, ^#Hugo Vicente Miranda, ^#Sílvia V Conde

¹ NOVA Medical School, NMS, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056, Lisboa, Portugal

^* Equal contribution; ^# Co-senior authors

Type 2 diabetes (T2D) is an important risk factor for neurodegenerative disorders. Importantly, hyperactivation of the carotid bodies (CBs) was identified in T2D and the silencing of these organs prevents and reverses systemic pathological features of T2D. We hypothesize that brain dysmetabolism aggravates neurodegenerative processes, and that the modulation of CBs activity will balance brain metabolism and prevent neurodegeneration. Herein we investigated if resection of Carotid Sinus Nerve (CSN), the CBs sensitive nerve, prevents the impact of metabolic deregulation on the neurodegenerative process. Male Wistar rats with 10 weeks of age were fed a high fat-high sucrose (HFHSu) diet (60% lipid-rich diet plus 35% sucrose in drinking water), or normal chow diet (NC). Fourteen weeks post-diet, animals were randomly divided and submitted to CSN resection or sham surgery and followed for more 7 weeks. Insulin sensitivity and glucose tolerance were evaluated together with behavioural tests: open field, y maze and block test throughout diet protocol. After sacrifice, the hippocampus and prefrontal cortex were collected for analysis of synaptic and neurodegenerative markers, and insulin pathway-related proteins. HFHSu animals exhibited insulin resistance and glucose intolerance, and CSN resection reversed these phenotypes. Additionally, HFHSu animals spent 62% less time in the novel arm in the y-maze test vs the NC group (p<0.05). CSN denervation in HFHSu animals increases the time animals spent in the novel arm by 128% (p<0.05). In the block test HFHSu animals sniff less time the novel block by 52% vs NC animals (p<0.05), and CSN resection did not modify this effect. HFHSu animals do not shown differences in advanced glycated end-products (AGEs) levels at the prefrontal cortex vs NC animals, however AGEs levels were increased by 32.7% (p<0.05) at the hippocampus. Prediabetes animals’ vs NC animals also exhibited at the: 1) prefrontal cortex increased levels of alpha-synuclein (20.1%, p<0.05), amyloid precursor protein (APP, 29.8%, p<0.05) and Tau (24.9%, p<0.05); 2) hippocampus increased levels of (alpha-synuclein (23.9%, p<0.05), APP (30.3%, p<0.05) and Tau (13.2%). These effects were prevented by CSN resection. The ablation of CBs activity may prevent dysmetabolism-induced neurodegenerative processes through the improvement in peripheral metabolic function.

Supported by: Portuguese Foundation for Science and Technology BFM: PD/BD/128336/2017; FOM: CEECIND/ 04266/ 2017; JFS: CEEC IND/02428/2018; no conflicts of interest to declare.

Keywords: Type 2 diabetes, carotid body, neurodegenerative markers

O1-04

Repaglinide as a potential treatment against motor and cognitive decline associated with neurodegeneration in C. elegans and Mus musculus

^*1,2Inés Sánchez Romero, ¹Juan A Fernández Cabrera, ²Mercedes M Pérez-Jiménez, ²Manuel J. Muñoz Ruiz, ¹Ángel M Carrión Rodríguez.

¹ Universidad Pablo de Olavide, Department of Anatomy, Physiology and Cell Biology, Seville, Spain. ² Centro Andaluz de Biología del Desarrollo (CABD)—Universidad Pablo de Olavide (UPO), Department of Molecular Biology and Biochemical Engineering, UPO/ CSIC/JA, Sevilla, Spain.

The aging process is associated with cognitive and motor decline and an increased risk of suffering neurodegenerative diseases. One of the common hallmarks of these neurodegenerative diseases is the deregulation of intracellular Ca²⁺ levels and the loss of proteostasis, which leads to the accumulation of phatogenic protein aggregates in the brain. Repaglinide (RPG) is an antidiabetic drug used clinically that works as an inhibitor of DREAM, a calcium sensing protein, whose blockade delays the onset of cognitive decline associated with age in mice. To determine if RPG treatment improves the symptoms associated with neurodegeneration by protein aggregation, pharmacological and behavioral experiments were carried out in C. elegans and Mus musculus models of neuroaggregopathies. Our results show that chronic treatment with RPG can reduce motor symptoms in models of protein aggregation in C. elegans, as well as reverse cognitive deficits in aged mice injected with amyloid oligomers in the hippocampus. Experiments in course will determinate the effect of the RPG treatment on protein aggregation and on the neuroinflammatory aspect of the pathological nervous system. Taken together, our preliminar results suggest that RPG could be an anti-aging drug and a potential treatment for neurodegenerative diseases in humans.

Keywords: DREAM, repaglinide, neurodegeneration, aging.

Corresponding author: Inés Sánchez Romero, https://orcid.org/0000-0002-6137-7528

O1-05

Rat duodenal nerve plexuses degeneration in physiological aging

¹Laura López-Pingarrón; ² Henrique Almeida; ¹María Soledad Soria-Aznar; ³Ahmad Agil; ³Vanessa Blanca; ¹José Joaquín García-García

¹ Department of Pharmacology, Physiology, Legal and Forensic Medicine, Faculty of Medicine, University of Zaragoza, Spain

² Department of Biomedicine, Faculty of Medicine, University of Porto, Portugal

³ Department of Pharmacology, Faculty of Medicine, University of Granada, Spain

The digestive tract owns an intrinsic inervation called enteric nervous system (ENS), formed by two plexus: myenteric (Auerbach´s), and submucosus (Meissner´s), establishing a vast number of connections to each other. Both are enriched in enteric glial cells with involvement in neuron support. The aim is to quantify effects of aging in the rat duodenum ENS, addressing the structure of both plexus and examining quantitative changes, such as their areas they are occupied and the density of cells there occurring. 12 Wistar male rats were arranged into three groups: young (3 months aged); adult (one year old); and old (two years old). After anesthesized, the abdominal viscera were exposed and pieces of duodenum were collected, fixed in buffered formalin and embedded in paraffin. Sections were processed for immunohistochemical study employing antibodies anti- S-100 (1:200) for the study of the glial cells, and anti- caspase-7 (1:100) to analyze cells undergoing apoptosis. S-100 positive area of plexuses in relation to the area of the submucosal and muscular wall, respectively was established. The area occupied by the submucosal did not show significant quantitative changes along ageing; however, there was an age-related decrement in myenteric plexus, which was very significant (p≤0,01 vs 3 months: 3,25%±0,33) and significant (p≤0,05 vs 12 months: 2,83%±0,34) when compared to the old rats (1,97%±0,16).

Also, a study of S-100 areas comparing the gray intensity was made, whereby increased in gray intensity scale in the S-100 labelling was considered inversely proportional to the presence of glial cells. S-100 myenteric plexus labelling declines in aging, or increase in intensity scale, (p≤0,01 vs 3 months: 66,18±1,6) in adult rats (79,91±2,0) and decrease (p≤0,05 vs 3 months) in old rats (71,70±1,0). The results of cell apoptosis density in myenteric plexus, number of caspase-7+ cells per mm² of muscular layer, reveals a significant increase (p≤0,05 vs 3 months: 1,07±0,23) in adult rats (2,47±0,44).

Our findings suggest that the glial cells of the myenteric plexus decrease in physiological aging, mainly in the oldest group; whereas apoptosis in the middle age is a likely cause that may be influenced by other factors.

Supported by Aragon Govern (B37_17D) and collaboration of Tissue Engineering Research group of Granada.

Keywords: enteric nervous system (ENS), apoptosis, glial cells, duodenal aging

Corresponding author: Laura López-Pingarrón, https://orcid.org/0000-0002-2455-680X

O1-06

Cellular proliferation of the rat duodenal nervous plexus in physiological aging

¹Laura López-Pingarrón; ² Henrique Almeida; ¹María Soledad Soria-Aznar; ¹Marcos César Reyes-Gonzales; ³Antonio Muñoz-Hoyos; ¹José Joaquín García-García

¹ Department of Pharmacology, Physiology, Legal and Forensic Medicine, Faculty of Medicine, University of Zaragoza, Spain

² Department of Biomedicine, Faculty of Medicine, University of Porto, Portugal

³ Department of Pediatrics Faculty of Medicine, University of Granada, Spain

The neurons and supporting glial cells of the submucosal and myenteric nervous plexuses are grouped in the enteric ganglia, as small clusters which are interconnected by nerve fiber bundles. These nerve cells connect by their processes with other neurons, in order to provide a network to innervate the muscular layer. Our aim was to analyse the effect of aging on the neuronal cell population of the duodenal nervous plexuses. 12 Wistar male rats were divided into three groups of four animals as follows: young (3 months); adult (one year old); and old (two years old). Upon anesthesia and laparotomy, pieces of duodenum were collected, fixed in buffered formalin, embedded in paraffin and sectioned. Sections were processed for immunohistochemical study, employing NSE (neuronal specific enolase) (1:1000), for studying the neurons of the duodenal plexuses; and Ki-67 (1:200) to analyze proliferating cells. For each marker at submucosal and myenteric plexuses, positive cells per mm² of surface area of the submucosal and muscular walls were counted, respectively. By the quantify of neurons cells NSE+, there weren´t any differences between younger an older groups. However, the gray scale intensity for NSE+, at the inner muscular layer, revealed a significant decrease of marking, or increase in gray scale intensity (p≤0,05 vs 3 months: 192,20±2,8) in adult (210,54±2,2) and older rats (209,90±1,9). Cellular proliferation density, in relation to the submucosal and muscular layers areas, for Ki-67 antibody, was increased in both duodenal nerve plexuses: submucosal (p≤0,05 vs 3 months: 2,03±0,30 and 12 months: 2,59±0,47) and myenteric (p≤0,05 vs 3 months: 0,27±0,14 and 12 months: 0,27±0,16) in older rats (6,37±1,29; 1,27±0,24) for these plexuses, respectively. The changes observed in the duodenal plexuses propound a more important degeneration of the neuronal processes ending at the inner muscular layer than of their bodies located in plexuses, in relation to physiological aging. Cellular proliferation is increased in the oldest group, suggesting a new form of regeneration at the level of both plexuses, possibly to compensate for lost cells.

Supported by Aragon Govern (B37_17D) and colaboration of Tissue Engineering Research group of Granada

Keywords: myenteric plexus, cellular proliferation, neuronal axons, aging of neuronal bodies

Corresponding author: Laura López-Pingarrón, https://orcid.org/0000-0002-2455-680X

Oral session 2: Cellular physiology

O2-01

Role of STIMATE and STIMATE-MUSTN1 during SOCE activation in HEK293

cells

¹Alejandro Berna-Erro, ²Pedro Camello, ¹Juan Antonio Rosado, ^*1Pedro Cosme Redondo.

¹ Department of Physiology (Phicell group), Faculty of Veterinary, University of Extremadura, Cáceres, Spain, and ² Department of Physiology, Faculty of Nursing and Occupational Therapy, University of Extremadura, Cáceres, Spain

STIMATE has been defined as a scaffolding protein that promotes the contact of the membranes that belong to the endoplasmic reticulum to plasma membrane. As a result, STIMATE facilitates the interaction between STIM1 and Orai1, which would lead to enhanced store operated calcium entry (SOCE). Interestingly, the translational machinery sometimes ignores the stop codon of STIMATE resulting in a naturally occurring read-through mRNA transcription with the adjacent downstream transcription factor gene MUSTN1 (STIMATE-MUSTN1) and, this fusion protein role has been poorly investigated up today, despite being identified in several cell types. In STIM1 and STIM2 double knock-out HEK293 cells, we have overexpressed different fluorescent tagged STIM1 and Orai1 proteins, as well as RFP-STIMATE and untagged-STIMATE-MUSTN1 to increase our knowledge regarding the role of these two proteins in SOCE. Single-cell experiments using cells overexpressing either STIMATE or STIMATE-MUSTN1, demonstrated a reduction of the TG-evoked SOCE, with a greater SOCE inhibition in cells overexpressing STIMATE. Confocal fluorescence colocalization analysis revealed that STIMATE overexpression interferes in the coupling between STIM1 and Orai1 probably due to increased STIM1/STIMATE colocalization. Meanwhile, STIMATE-MUST1 overexpression had no significant effect in the association of STIM1 and Orai1. Altogether, our data suggest that despite STIMATE could be required for SOCE activation, cells overexpressing STIMATE can present reduced TG-evoked SOCE. The fact that cells overexpressing STIMATE-MUSTN1 exhibit normal SOCE as compared to those overexpressing the empty vector, and those overexpressing STIMATE-MUSTN1 could be explained as a modulatory mechanism to avoid alteration in STIMATE during SOCE activation.

Supported by Junta de Extremadura (IB18020, GR21008) and Grant PID2019-104084GB-C21 funded by MCIN/AEI/ 10.13039/501100011033.

Keywords: SOCE, STIMATE, STIMATE-MUSTN1, STIM1

*Corresponding author: Pedro C Redondo, https://orcid.org/0000-0002-2067-2627

O2-02

TRPM2 channel mediated JNK/mTOR signaling pathway regulates autophagy in off springs’ heart tissue of high dose alcohol exposure during pregnancy: Impact of gender difference

¹A Cihangir Uğuz, ² Aslı Okan Oflamaz, ³ Seher Yilmaz, ⁴ Evrim Suna Arikan Söylemez, ² Züleyha Doğanyiğit

¹ Department of Biophysics, Faculty of Medicine, Karamanoğlu Mehmetbey University, Karaman, Turkey

² Department of Histology and Embryology, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey

³ Department of Anatomy, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey

⁴ Department of Medical Biology, Faculty of Medicine, Afyonkarahisar Health Sciences University, Afyonkarahisar, Turkey

Alcohol consumption during pregnancy may cause some harmful consequences on the fetus. Its excessive intake triggers both structural and molecular changes. Increased alcohol exposure in the embryonic period may result growth retardation, neurodevelopmental abnormalities, and morphological changes, including a series of characteristic differentiations. These abnormalities are called as fetal alcohol syndrome (FAS). As a member of Transient Receptor Potential super family, TRP Melastatin 2 cation channels were determined to be crucial for autophagy regulation in variety of diseases’ molecular signaling cascades. The mammalian target of rapamycin (mTOR) and c-Jun N-terminal kinase (JNK) pathways control different functions including proliferation and cell death during development. Hence, we ought to determine the JNK/mTOR pathway and possible involvement of TRPM2 channel expression levels in heart tissue during embryonic period. The main objective of the current study is to compare obtained results according to gender differences as above mentioned parameters. Moreover, we determined MDA, LDH, TAS and TOS levels as an oxidative stress indicator. The mRNA levels of the TRPM2 gene were found to be downregulated in hearts of male and female rats’ compared to the related control group. This downregulation was determined to be more significant in female off springs. MDA, VEGF, LDH and TOS levels were altered and TAS levels were decreased in alcohol exposure group. Female off springs had increased MDA, LDH and TOS levels but decreased TAS levels compared to males. The rate of cardiovascular damage was more intense in both male and female heart tissues exposed to alcohol on histological evaluation. JNK and mTOR expression levels were significantly increased in off springs of alcohol exposure. However, there were no significant difference between female and male rats. Although, further studies are needed to understand the underlying mechanisms, the present findings suggest that TRPM2 cation channels have crucial role in regulation of JNK/mTOR signaling cascade.

The current study was supported by the Scientific Research Unit of Yozgat Bozok University with a grant number 6602c TF/21-471

Keywords: alcohol consumption, pregnancy, JNK/mTOR pathway, TRPM2 channels.

Corresponding Author: A. Cihangir Uğuz, https://orcid.org/0000-0002-5778-581X.

O2-03

Functional consequences of the Kv1.3-KCNE4 complex in leukocyte physiology

Magalí Colomer-Molera, Daniel Sastre, María Navarro-Pérez, Anna Benavente-García, Jesusa Capera, ^*Antonio Felipe

Molecular Physiology Laboratory, Departament de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, Spain.

The voltage-gated potassium channel Kv1.3 plays a crucial role during the immune response. The regulatory subunit KCNE4 acts as an ancillary peptide of Kv1.3, modulating K⁺ currents, controlling channel abundance at the cell surface and enhancing the C-type inactivation of the channel. KCNE4-dependent regulation of the oligomeric complex fine-tunes the physiological role of Kv1.3. In light of the therapeutic relevance of Kv1.3, KCNE4 emerges as a modulator of the immune system physiology. For a better understanding of the role of KCNE4 in the regulation of the immune system, we manipulated KCNE4 expression in various leukocyte cell lines. While the KCNE4 expression in Jurkat T lymphocytes is limited, CY15 dendritic cells, a model of professional antigen-presenting cells, robustly express the ancillary subunit. Overexpression of KCNE4 affects Kv1.3-dependent physiology of T lymphocytes, including the channel translocation at the immunological synapse (IS), proliferation, and IL-2 production. Contrarily, the increased Kv1.3/KCNE4 ratio and free Kv1.3 upon LPS-dependent activation in CY15 correlates with an increase in current density. The architecture of the complex relies on the differential expression of Kv1.3 and KCNE4, determining the stoichiometry of the Kv1.3/KCNE4 complex. Single fluorescence bleaching steps demonstrated that oligomers present an open Kv1.3/KCNE4 stoichiometry, with up to four KCNE4 peptides per channel. Moreover, an increasing number of KCNE4 subunits in the Kv1.3 complex correlates with a decrease in current density as a consequence of the impaired membrane trafficking of the channel. However, the acceleration of the C-type inactivation of the channel is independent of the number of KCNE4 present in the complex. Altogether, we demonstrated the role of the KCNE4- in the immune system by modulation of Kv1.3. Additionally, the Kv1.3/KCNE4 channelosome composition determines the amount of channel’s activity, depending on KCNE4 availability. Therefore, KCNE4 expression remodelling in the immune system has functional consequences for Kv1.3-dependent physiological functions.

Supported by the Ministerio de Ciencia e Innovación (MICINN/AEI), Spain (PID2020-112647RB-I00 and 10.13039/501100011033) and European Regional Development Fund (FEDER).

Keywords: potassium channels, leukocytes, immune response, functional complex.

Corresponding author: Antonio Felipe, https://orcid.org/0000-0002-7294-6431.

O2-04

The spatial organization and membrane targeting of the regulatory Kvβ2.1 subunit is mediated by S‑palmitoylation

Silvia Cassinelli, Sara R Roig, María Navarro‑Pérez, Carla Viñola-Renart, Irene Estadella, Jesusa Capera, ^*Antonio Felipe

Molecular Physiology Laboratory, Departament de Bioquimica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, Spain.

The voltage-dependent potassium (Kv) channel Kvβ family was the first cloned group regulating Kv channels. Unlike the Kvβs modulation on Kv channels, the physiological role of these proteins is far from being determined. Similar to Kv α subunits, Kvβs are expressed in excitable and non-excitable cells and they are strongly regulated in leukocytes. Although they exhibit cytosolic distribution, Kvβs may traffic to the cell surface in close contact with plasma membrane Kv channels, and this is crucial for a proper immune response. Indeed, Kvβ2.1 locates to adjacent Kv1.3 channels at the cell membrane within the immunological synapse during lymphocyte activation. The aim of this study was to decipher the structural elements that participate in the cellular distribution of Kvβs. Surprisingly, Kvβ peptides targeted the cell surface in the absence of pore-forming α-subunits. Furthermore, Kvβ2.1, but not Kvβ1.1, targeted lipid raft microdomains, undergoing S-palmitoylation, and concomitantly with a localization within the immunological synapse. Distal cysteines C301 and C311 were mostly responsible for the specific acylation of Kvβ2.1. In addition, several insults altered Kvβ2.1 membrane localization. Proliferation enhanced the protein surface targeting, whereas PKC activation impaired lipid raft localization. However, PSD95 stabilized Kvβ2.1 in these domains. Our data provides essential information about molecular mechanisms by which Kvβ2 clusters into immunological synapses during leukocyte activation.

Supported by the Ministerio de Ciencia e Innovación (MICINN/AEI), Spain (PID2020-112647RB-I00 and 10.13039/501100011033) and European Regional Development Fund (FEDER).

Keywords: potassium channels, leukocytes, immune response, regulatory subunits.

Corresponding author: Antonio Felipe, https://orcid.org/0000-0002-7294-6431.

O2-05

Comparison between proapoptotic effects of Pt(II) and Pd(II) complexes with thiazoline or thiazine-based ligands in tumour cell lines

*¹Elena Fernández-Delgado, ¹Samuel Estirado, ²Francisco Luna-Giles, ¹Ana B. Rodríguez, ¹José A. Pariente, ¹Javier Espino, ²Emilio Viñuelas-Zahínos.

¹ Department of Physiology (Neuroimmunophysiology and Chrononutrition Research Group), Faculty of Science, University of Extremadura

² Department of Organic and Inorganic Chemistry (Chemistry of Coordination Group), Faculty of Science, University of Extremadura

Since Rosenberg discovered cisplatin (CisPt) back in 1965, an enormous attention has been paid to platinum-based drugs, being used as treatment for a wide range of tumours. However, to overcome their disadvantages (acquired resistance, severe side effects, etc.), new compounds should be developed, being the coordination of bioactive ligands to transition metals the widest used strategy. Ligands with heterocycles bearing donor atoms such as thiazoline or pyrazole have showed apoptotic properties. Other transition metals with a similar coordination chemistry to Pt(II) such as Pd(II) has been studied as a substitute of Pt(II). Therefore, in this work it has been carried out the synthesis and structural characterization of eight complexes of Pt(II) and Pd(II) coordinated with 2-(1-pyrazolyl)-2-thiazoline (PzTn), 2-(1-pyrazolyl)-1,3-thiazine (PzTz), 2-(3,5-diphenyl-1-pyrazolyl)-2-thiazoline (DPhPzTn) and 2-(3,5-diphenyl-1-pyrazolyl)-1,3-thiazine (DPhPzTz). The goal was evaluating their potential anticarcinogenic capacity in three tumoral cell lines (HeLa, HL-60 and U-937) and checking if the metal ion and the structural factors of the ligand may influence their biological activity. The cytotoxic potential was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, challenging the tumoral cell lines with increasing concentrations (0–100 μM) of the compounds for 24 h. Our findings proved that those compounds that included aromatic rings in their structure showed lower IC₅₀ values, for both Pt(II) and Pd(II) complexes in all three tumoral cell lines. Moreover, Pt(II) complexes showed much lower IC₅₀ values that the Pd(II) complexes in all cases, even lower than CisPt. The accumulation of Pt(II) and Pd(II) was also tested in HeLa cells by inductively coupled plasma mass spectrometry (ICP-MS). Cells were incubated for 5 h with IC₅₀ of all compounds (including CisPt) and it was observed that PtDPhPzTz and PtDPhPzTn accumulated into cells much more efficiently than their Pt(II) analogues, the Pd(II) complexes and CisPt, thereby indicating that the enhanced lipophilicity of this two compounds boosted their cellular uptake and accumulation. Altogether, our results indicated that the incorporation of aromatic groups to the ligands increased biological activity and, for these complexes, Pt(II) is more cytotoxic as metal centre than Pd(II).

This work was supported by Junta de Extremadura grants (ref. GR21042, GR21075, IB18013, PD18020 and TA18002).

Keywords: Apoptosis, Cancer, Pt(II) complexes, Pd(II) complexes

Corresponding author: Elena Fernández-Delgado, https://orcid.org/0000-0001-6987-6586

O2-06

Melatonin-conjugated formulations of metallodrug-functionalized mesoporous silica drug delivery systems produce cytotoxicity and apoptosis in HeLa cells

^#1Samuel Estirado, ¹Elena Fernández-Delgado, ²Emilio Viñuelas-Zahínos, ³Santiago Gómez-Ruiz, ³Diana Díaz-García, ²Francisco Luna-Giles, ¹José A Pariente, ^*1Javier Espino.

¹ Department of Physiology (Neuroimmunophysiology and Chrononutrition Research Group), and ² Departament of Organic and Inorganic Chemistry (Chemistry of Coordination Research Group), Universidad de Extremadura, Badajoz; ³ Department of Biology, Physical Geology and Inorganic Chemistry (Comet-Nano Group), Universidad Rey Juan Carlos, Madrid, Spain.

^# Presenting author

Metal-based antitumor therapies, such as cisplatin, have been used successfully for more than 40 years. However, the use of metal drugs presents problems associated with their low stability and their low degree of internalization in the target cells. The recent development of nanotechnology has revealed that nanoparticles can be very useful as drug carriers, as they can easily cross the cell membrane. These nanoparticles can be coupled to anticancer agents, thus leading the effects of chemotherapeutic drugs to cancer cells. Herein, we designed effective drug delivery systems based on the synthesis of novel Pt(II) or Pd(II) complexes containing a thiazoline derivative ligand, TdTn (2-(3,4-dichlorophenyl)imino-N-(2-thiazolin-2-yl)thiazolidine). Thus, both complexes (PtTdTn and PdTdTn) and the ligand (TdTn) were encapsulated together with a melatonin derivative (5-methoxytryptamine, 5-MT) on functionalized mesoporous silica nanomaterials (MSN). The morphology and size of the resulting silica-based systems were analysed by transmission and scanning microscopy. Interestingly, the particles have a hexagonal quasi-spherical shape and show high dispersibility, not forming large aggregates, which is beneficial for their subsequent use in biological studies. To evaluate the potential antitumoral capacity of the MSN particles, the cytotoxicity of all materials against human cervical adenocarcinoma HeLa cells was first determined. The IC₅₀ values for all series of materials were obtained and compared with those of the free compounds, so that the IC₅₀ values in quantity of either metal or ligand showed far lower values for the MSN-based materials than for the free compounds. The most cytotoxic system was MSN-PdTdTn and, when including 5-MT, all three materials (MSN-PtTdTn, MSN-PdTdTn, and MSN-TdTn) were even more cytotoxic than those without the melatonin derivative. Finally, the induction of apoptosis was studied by challenging double-stained (Annexin V-FITC and Hoechst 33258) HeLa cells with the IC₂₅ dose of every material, which produced a significant increase in the populations of late apoptotic and secondary necrotic cells in all cases. Altogether, these materials represent a promising therapeutic tool for future studies in this field, with special emphasis on the highly cytotoxic nature of 5-MT-functionalized MSN particles.

This work was supported by Junta de Extremadura (IB18013, GR21042 and GR21075 grants; PD18020 and TA18002 fellowships).

Keywords: Melatonin, cancer, metallodrugs, mesoporous silica nanoparticles.

^*Corresponding author: Javier Espino, https://orcid.org/0000-0002-8549-9343.

O2-07

Demyelinating disease: Focus on oligodendrocytes and TRPA1 channel

^*1,2Bilal Cig, ¹Nicola Hamilton

¹ Wolfson Centre for Age-Related Diseases, Institute of Psychiatry, Psychology and Neuroscience, Guy's Campus, King's College London, London SE1 1UL, UK.

² Kirsehir Ahi Evran University Medicine Faculty Department of Physiology Kirsehir, 40100, Turkey

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) characterized by inflammatory and neurodegenerative processes. The myelin sheath, which is wrapped around axons by oligodendrocytes and is very important for brain function, is damaged in a calcium (Ca²⁺) dependent manner, thereby eliminating action potential propagation. Demyelination is a heterogeneous condition that occurs against the background of an acute or chronic inflammatory process. The activity of the disease process is reflected by the presence of lesions with ongoing destruction of myelin. Axonal and neuronal destruction in lesions is an important substrate for permanent neurological deficit. Although demyelination is a hallmark of the disease, its relationship with neurodegeneration is still not fully resolved and research is still ongoing. Current therapies target pathological immune responses to counter inflammatory processes. Given the efficacy and safety limitations of current treatments, it appears that new treatments are needed to prevent demyelination or promote repair mechanisms. Demyelination is characterized by disruption of Ca²⁺ homeostasis. TRP ankyrin 1 (TRPA1), known as a member of the relatively calcium-high permeable transient receptor potential (TRP) family, is the only member of the ankyrin subfamily that has so far been identified in mammals and is widely expressed in cells and tissues. In resident tissue cells, inflammatory and immune cells, TRPA1 regulates a number of cellular processes such as cytokine production, cell differentiation and cytotoxicity. Therefore, TRPA1 activation has been proposed as a protective mechanism to detect harmful agents and mediate inflammatory responses in a variety of pathological conditions, including various inflammatory diseases. Special attention has been given to the contribution of TRPA1 to the transition of inflammatory and immune responses from an early defensive response to a chronic pathological state. Although our understanding of the roles of TRP channels in glia is still in its infancy, recent studies have reported that TRPA1 deficiency significantly reduces cuprizone-induced demyelination by reducing apoptosis of mature oligodendrocytes. It may be a promising therapeutic target to limit damage. Here we will address the potential protective roles of TRPA1 in demyelination.

Keywords: Multiple sclerosis, demyelinating disease, oligodendrocytes, TRPA1 channel

Corresponding author: Bilal Cig

O2-08

Regulation of potassium ion channels by 17β-estradiol and the endocrine disruptor Bisphenol-A in the human beta cell line EndoC-βH1

¹Roberto Sempere-Navarro, ^1,2Regla M Medina-Gali, ^1,2Esther Fuentes, ^1,3Juan Martinez-Pinna, ^*1,2Angel Nadal

¹ Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universitas Miguel Hernández, Elche, Spain.

² Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Spain.

³ Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante,

Estrogens, including 17β-estradiol (E2), increase glucose stimulated insulin secretion, insulin biosynthesis and protect pancreatic β-cells from apoptosis via activation of estrogen receptors (ERs). In contrast, the plastic-derived endocrine disruptor Bisphenol-A (BPA), which exerts multiple effects in this cell type via ERs, induces insulin resistance and is associated with a high risk of developing Type 2 Diabetes. Pancreatic β-cells express several voltage-activated K⁺ ion channels, including K_v2.1/2.2 and big-conductance Ca²⁺-dependent K⁺ channel (K_Ca1.1) which are involved in the regulation of cell cycle progression, proliferation, and cell function. We sought to investigate how ER ligands regulate K⁺ channels. For this purpose, we measured voltage-activated K⁺ currents by whole-cell patch-clamp recordings in the human beta cell line EndoC-βH1. E2 (1nM) had no effect on voltage-activated K⁺ currents. Conversely, 1nM BPA partially inhibited voltage-activated K⁺ currents. To determine which K⁺ channel subtypes were altered by BPA exposure, we measured K⁺ currents in the absence or presence of iberiotoxin (IbTx), a specific blocker of K_Ca1.1 channels, or stromatoxin-1 (ScTx1), a selective inhibitor of K_v2.1/2.2 channels. In EndoC-βH1, 1nM BPA decreased the peak K⁺ current density in the presence of 100nM IbTx, but it did not alter K⁺ currents in the presence of 100nM ScTx1, indicating that BPA reduces K_v2.1/2.2 but not K_Ca1.1 currents. Additionally, we measured EndoC-βH1 K_v2.1, K_v2.2 and K_Ca1.1 channel expression, after 48h of BPA or E2 (1pM, 1nM, 1μM) exposure compared to vehicle by qRT-PCR, observing a decrease in the expression of the genes codyfying for K_v channels. Moreover, to investigate which ERs of the EndoC-βH1 cell line might contribute to K⁺ current decrease, we measured K⁺ currents in the presence of 1nM DPN (ERβ agonist), 100nM G1 (GPER agonist), or 1nM PPT (ERα agonist). Neither G1 nor PPT decreased K⁺ currents but 1nM DPN decreased EndoC-βH1 peak K⁺ current density. Ultimately, we measured K⁺ currents of cells treated with 1nM BPA with 1μM PHTPP (ERβ antagonist). PHTPP abolished the effects produced by BPA. Our findings suggest that inhibition of K_v2.1/2.2 channels might contribute to BPA induced β-cell dysfunction, and that ERβ could have a role in this effect.

Funded by the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI), PID2020-117294RB-I00 (AN, JM-P) and Generalitat Valenciana, PROMETEOII/2015/016 (A.N.). CIBERDEM is an initiative of the Instituto de Salud Carlos III.

Keywords: estrogens, endocrine disruptor bisphenol-A, potassium ion channels, diabetes

Corresponding author: Angel Nadal, https://orcid.org/0000-0003-4178-2152

Oral session 3: Cardiovascular physiology

O3-01

Effects of the inhibitor of Ca ²⁺ /calmodulin-dependent protein kinase II, KN-93, on intrinsic ventricular electrophysiological modifications produced by local stretch

¹Germán Parra, ^1,4Irene del Canto, ^2,4Manuel Zarzoso, *^2,4Luis Such-Miquel, ^1,4Patricia Genovés, ^1,4Óscar J Arias, ¹Johan E Ortiz, ¹María J Cardells, ^3,4Francisco J Chorro, ^1,4Luis Such, and ^1,4Antonio M Alberola.

¹ Department of Physiology, Faculty of Medicine, Universitat de València, Valencia, Spain,

² Department of Physiotherapy, Faculty of Physiotherapy, Universitat de València, Valencia, Spain,

³ Department of Medicine, Faculty of Medicine, Universitat de València, Valencia, Spain and

⁴ INCLIVA, Valencia, Spain and CIBERCV, Valencia, Spain.

It is currently accepted that alterations in cardiac Ca2+ homeostasis, as it occurs in myocardial stretch, is directly related to arrhythmia triggering, although as it is well known, the mechanisms underlying arrhythmia are complex in nature. For this reason, to maintain the homeostatic factors of the calcium cycle at their physiological level, has been established as a therapeutic maneuver. KN-93 is a methoxybenzenesulfonamide compound and is an extensively used inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) when abnormally active, contributing to diastolic cytosolic Ca++ release and arrhythmogenesis. We have studied the effects of KN93 (1 μM) on the modifications of ventricular intrinsic electrophysiological properties (related with refractoriness and heterogeneity) produced by acute local stretch. Materials and Methods: Twenty adult male White New Zealand rabbits were heparinized and euthanized by sodium thiopental injection (European Ethic Guidelines). Their hearts were excised, isolated, perfused and fibrillated (without to interrupt the perfusion), in a Langendorff system. A pacing electrode and a recording multielectrode were placed on the left ventricle epicardium submitted to stretch. The stretching was induced by an intraventricular device. Ventricular fibrillation (VF) was triggered by pacing and spectral VF analysis was performed to determine VF dominant frequency (DF) and VF spectral concentration (SpC), as refractoriness and heterogeneity indexes. Determinations were performed, previously, during and after stretch in both control and KN-93 groups. An ANOVA test was used for comparisons. Significance when p<0.05. Results: DF increased after three minutes of stretch respect to pre (18,4 ± 3,8; n=11, vs. 14,3 ± 2,97; n=11, Hz) and three minutes post stretch (18,4 ± 3,8; n=11, vs. 13,7 ± 2,15; n=11, Hz) in the control group. SpC decreased after three minutes of stretch respect to pre stretch (17,4 ± 1,66 vs. 26,9 ± 4,45; n=9, percentage) and decreased after three minutes post stretch respect to stretch (17,4 ± 1,66; n=9, vs. 27,8 ±3,89; n=11, percentage) in the control group. No differences in DF and SpC were found in treated group, although basal values were significantly higher and lower respectively than control group. Conclusions: KN93 prevents ventricular refractoriness and heterogeneity modifications produced by local stretch.

Supported by Instituto de Salud Carlos III - CIBERCV (CB16/11/00486).

Keywords: isolated rabbit heart, cardiac electrophysiology, myocardial stretch.

Corresponding autor: Luis Such-Miquel,https://orcid.org/0000-0001-8499-7183

O3-02

Nitric oxide inhibits the calcium entry through T-type voltage gated-calcium channels in response to phenylephrine in the renal artery from healthy rabbits

*¹Andrea Suarez, ¹Begoña Belmonte, ¹Solanye Guerra-Ojeda, ¹Patricia Genovés, ¹Martín Aldasoro, ¹David Verdú, ¹Eva Serna, and ¹María Dolores Mauricio.

¹Department of Physiology, University of Valencia ^.

Voltage-gated calcium channels (VGCCs) are involved in blood vessel contraction. L-, P/Q- and T-type channels are differentially expressed in human intrarenal arteries and are of functional relevance for depolarisation-induced vasoconstriction. For instances, L-type VGCCs have been described in afferent arteriole while T-type VGCCs are located in both afferent and efferent arteriole, suggesting that T-type VGCCs could play an important role in the regulation of renal haemodynamic. On the other hand, calcium influx is necessary for the synthesis of nitric oxide (NO), a potent endothelium-derived relaxing factor, that is reduced in endothelial dysfunction. The aim of the present study was to analyse if T-type VGCCs may increase their function in the absence of NO, aggravating the tendency to vasoconstriction in the endothelial dysfunction. For experiments, healthy rabbit renal rings were mounted for isometric tension recording in organ baths chambers containing Krebs-Henseleit solution. Concentration-response curves to phenylephrine (Phe) (10⁻⁹ - 3x10⁻⁵M) were obtained in the absence and presence of L-NAME (10⁻⁴M), an inhibitor of NO synthase, Nickel (5x10⁻⁵M), a blocker of T-type VGCCs, and the combination of both. The results showed that L-NAME left-shifted the concentration-response curve to Phe (pD₂ = 6.34 ± 0.08 for control vs 6.64 ± 0.06 for L-NAME, P < 0.05), indicating that reduced contraction is associated with the release of NO. Nickel right-shifted the concentration-response curve to Phe (pD₂ = 6.34 ± 0.08 for control vs 5.56 ± 0.14 for Nickel, P < 0.05), suggesting the involvement of T-type VGCCs in Phe-induced vasoconstriction. Moreover, the blocking effect of Nickel was further increased in the presence of L-NAME: area under curve (AUC) values were 131.00 ± 30.56 AU for control, and 278.30 ± 33.80 AU for L-NAME, P < 0.05. According to these findings, the inhibition of NO causes T-type VGCCs become more active in the renal artery from healthy rabbits, thus increasing calcium influx and contraction. As a consequence, blood flow within the renal circulation could be compromised, potentially contributing to hypertensive state in cardiovascular disease. Therefore, the blockage of T-type VGCCs could be a therapeutic target for the treatment of excessive vasoconstriction.

Supported by Universitat de Valencia. Convocatòria d’Accions Especials, expedient UV‐INV‐AE‐1544052.

Keywords: vascular reactivity, hypertension, voltage-gated calcium channels, nitric oxide.

Corresponding author: Andrea Suárez https://orcid.org/0000-0003-0932-7870

Autorización procedimiento experimentación animal: 2015/VSC/PAE/00233

O3-03

Effects of KN93 on the modifications of the functional refractory period and the refractoriness dispersion produced by ventricular local stretch

*^1,4Luis Such-Miquel, ^1,4Manuel Zarzoso, ²Germán Parra, ^2,4Patricia Genovés, ^2,4Óscar J Arias, ^2,4Irene del Canto, ²Johan E Ortiz, ²María J Cardells, ^2,4Antonio M Alberola, ^2,4Luis Such, ^3,4Francisco J Chorro.

¹ Department of Physiotherapy, Faculty of Physiotherapy, Universitat de València, Valencia,

Spain,

² Department of Physiology, Faculty of Medicine, Universitat de València, Valencia, Spain,

³ Department of Medicine, Faculty of Medicine, Universitat de València, Valencia, Spain and

⁴ INCLIVA, Valencia, Spain and CIBERCV, Valencia, Spain.

KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) is widely used to inhibit CaMKII in circumstances in which this enzyme becomes abnormally active causing hyperphosphorylation of the ryanodine receptor and diastolic release of calcium from the sarcoplasmic reticulum, which in turn causes arrhythmias. We have studied the effects of KN93 (1μM) on refractoriness and its dispersion during ventricular local stretch. Materials and Methods: Twenty-two adult male New Zealand White rabbits were heparinized and euthanized by sodium thiopental injection (European Ethic Guidelines). The hearts were excised, isolated, perfused and fibrillated (without to interrupt the perfusion), in a Langendorff system. A pacing electrode and a recording multielectrode (121) were placed on the left ventricle epicardium submitted to stretch. An intraventricular device induced the stretching. Ventricular fibrillation (VF) was triggered by pacing and it was analyzed in the time domain to calculate the 5th percentile (5p) of the fibrillatory intervals (VV), which is an estimation of refractory functional period during the arrhythmia, and VV dispersion (coefficient of variation, CV, of the VV). VV are related with refractoriness. Determinations were performed, previously, during and after stretch in both control and KN-93 groups. An ANOVA test was used for comparisons. Significance when p<0.05. Results: 5p decreased after three minutes of stretch respect to pre stretch (38 ± 4 vs. 52 ± 10; n=10) and increased after three minutes post stretch respect to stretch (54 ± 8 vs. 38 ± 4; n=10) in the control group. No differences were found in treated group, although basal, stretch and post stretch values of 5p were significantly higher in treated than in control group. There were no differences in CV, neither in the control group nor in the treated group between pre stretch, stretch and post it. Conclusions: KN93 prevents against ventricular functional refractory period modifications produced by local stretching.

Supported by Instituto de Salud Carlos III - CIBERCV (CB16/11/00486).

Keywords: isolated rabbit heart, cardiac electrophysiology, myocardial stretch.

Corresponding autor: Luis Such-Miquel, https://orcid.org/0000-0001-8499-7183

O3-04

Effectiveness of an R-based software to detect closed-loop cardiovascular interactions and baroreflex impairment in human subjects from the EUROBAVAR data set

^*1,2Alvaro Chao-Ecija, ^1,2Daniel Carrasco-Gomez, ^1,2Marc Stefan Dawid-Milner

¹ Department of Physiology, Faculty of Medicine, University of Malaga, Malaga, Spain, and

² Autonomic Nervous System Unit, CIMES, IBIMA, University of Malaga, Malaga, Spain

We recently developed an R-based software that could model closed-loop cardiovascular interactions. In this study, we applied this tool on the EUROBAVAR data set to further test its applicability. This set consists of 21 human beat-to-beat blood pressure recordings with their corresponding inter-beat intervals (IBI) obtained during supine rest and standing positions. Of these subjects, two are known to have a baroreflex impairment. The aim of this work is to test the effectiveness of our software to identify cardiovascular regulatory mechanisms and baroreflex impairment from these data. After downloading the recordings, we first uploaded each recording into our software to model the interactions present between systolic blood pressure (SBP) and IBI by employing a wavelet detrending and multivariate autoregressive modeling algorithm. Then, our software estimated causal coherence and Gaussian-weighted baroreflex sensitivity (BRS) indexes from each model at the low frequency (LF, 0.04-0.15 Hz, sympathetic) and high frequency (HF, 0.15-0.4 Hz, parasympathetic) bands. Immediate variability transfer from SBP to IBI was also computed for each subject. Estimates were compared using the Wilcoxon test for paired samples. Our results showed that, when standing, the estimates of only two subjects, B005 and B010, were below percentile P10 of the BRS distribution (0.719 ms/mmHg at HF, 1.678 ms/mmHg at LF) at both bands. A literature review indicated that these two subjects had a baroreflex impairment. In non-baroreflex-impaired subjects, causal coherence from IBI to SBP at LF was significantly predominant at rest when compared with the coupling from SBP to IBI (p < 0.001). This predominance disappeared during standing due to changes in the couplings, suggesting a baroreflex interaction. Closed-loop BRS supine-to-standing ratios in these subjects were 1.69 ± 0.93 (LF band) and 3.1 ± 1.32 (HF band), showing a significantly decreased BRS during standing position (p < 0.01, LF; p < 0.001, HF). Immediate transfer also decreased during standing (p < 0.001). In conclusion, our software managed to evaluate causal closed-loop interactions between cardiovascular variables from the data set, evidencing a baroreflex coupling, and was able to identify baroreflex impairment. Thus, this allows it to be a useful tool for baroreceptor evaluations.

Keywords: cardiovascular regulation, baroreflex impairment, spectral analysis, autoregressive models.

Corresponding author: Alvaro Chao-Ecija, https://orcid.org/0000-0002-2691-6936

O3-05

Role of carotid body in the control of adipose tissue catecholamine resistance in obesity

^*1Fátima O Martins, ¹Bernardete F Melo, ¹Joana F Sacramento, ^2,3Daniela Rosendo-Silva, ⁴Elena Olea, ^2,3,5Paulo Matafome, ¹Silvia V Conde

¹ NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Portugal

² Institute of Clinical and Biomedical Research (iCBR), Faculty of Medicine and CIBB, University of Coimbra, Portugal

³ Clinical-Academic Center of Coimbra, Coimbra, Portugal

⁴ Universidad de Valladolid, Valladolid, Spain

⁵ Instituto Politécnico de Coimbra, Coimbra Health School, Coimbra, Portugal

Obesity is an epidemic worldwide. Adrenergic signaling in white (WAT) and brown (BAT) adipose tissues promotes lipolysis and glucose uptake and activates BAT thermogenesis. Dopamine signaling also contributes to these effects. However, during obesity the effect of catecholamines on adipose tissue is blunted. The carotid bodies (CB) are metabolic sensors involved in energy homeostasis with key function in dysmetabolism. We found that the CB regulates both WAT and BAT through the modulation of catecholaminergic activity. We tested if obesity is associated with decreased catecholaminergic signaling in the WAT and BAT and investigated the effects of CB modulation. Male Wistar rats were assigned to a HF group (60% fat) or a control group fed with a standard diet (NC). After 10 weeks, half the groups were submitted to bilateral carotid sinus nerve (CSN) resection or to a sham procedure. 9 weeks after, WAT and BAT depots were collected and kept at -80ºC for western blot analysis of proteins from catecholaminergic system, inflammatory markers and to measure catecholamines by HPLC. Experiments followed the 2010/63/EU European Union Directive and were approved by NMS Ethics Committee and Portuguese Authority for Animal Health. Differences between means were calculated using One-Way ANOVA and considered significantly for p<0.05. HF diet decreased significantly norepinephrine (NE), epinephrine (Epi) and dopamine (DA) in WAT. In WAT, HF diet did not change D2R or b3R but decreased D1R levels, an effect not changed by CSN resection, and increased b2R, an effect abolished in CSN-resected animals. In BAT, HF diet or CSN resection did not change D1R and D2R levels, but HF diet decreased b3R levels, an effect abolished in CSN-resected animals. HF diet or CSN resection did not alter IL1bR and IL6R levels in BAT, but in WAT HF diet increased IL1bR levels, an effect attenuated in HF-CSN resected animals. HF diet differentially altered catecholaminergic signaling in WAT and BAT, with major roles for D1R and b2R on WAT and b3R on BAT. CSN resection restored adrenergic signaling in both tissues and attenuated inflammation, unraveling a new mechanism to overcome catecholamines resistance in obesity.

Supported by FCT (CEECIND/04266/2017, CEEC IND/02428/2018, EXPL/MED-NEU/0733/2021).

Keywords: carotid body, adipose tissue, catecholamines, obesity.

Corresponding author: Fátima O. Martins, https://orcid.org/0000-0003-2161-459X.

O3-06

MPO as a surrogate inflammation biomarker for physiological changes induced by doxorubicin in rats

Ana I. Afonso², Ângela Amaro-Leal^1,2; Filipa Machado², Isabel Rocha^1,2, Vera Geraldes^1,2

¹ Faculdade de Medicina da Universidade de Lisboa

² Centro Cardiovascular da Universidade de Lisboa

Myeloperoxidase (MPO) is a recognized biomarker of inflammation released in high quantities by immune cells. The association of MPO with cardiovascular disease is extensively known. Anthracyclines, such as doxorubicin (DOX) are the most prescribed antineoplastic drugs, although they have various adverse effects, particularly on the heart, leading to progressive cardiomyopathy and heart failure. In the present work, we sought to investigate the effect of a crescent dose of DOX on physiological homeostasis and the role of MPO as surrogate marker of cardiac dysfunction induced by DOX.

For that, female Wistar rats, with 12 weeks of age, were DOX-treated for 4 weeks: DOX16 (n=6), a cumulative dose of 16 mg/kg (i.p. 4 mg/kg/week), DOX20 (n=6), a cumulative dose of 20 mg/kg (i.p. 5 mg/kg/week). A control group, CTR (n=6) (i.p. saline). At the end of the protocol, animals were anaesthetized and blood pressure (BP), electrocardiogram, heart rate (HR) and respiratory frequency (RF) were recorded. Unpaired t-test were used (signif. p<0.05).

Our results showed that both cumulative DOX doses triggered hypotension when compared with CTR group: a significant decrease in mean BP (DOX16: 90.38 ± 10.08; DOX 20: 75.85 ± 12.03; CTL: 129 ± 3.5 mmHg, p=0.0047 and p=0,0017 respectively), systolic BP (DOX16: 103.1 ± 10.19; DOX20: 94.91 ± 12.61; CTL: 150.1 ± 5.34 mmHg, p=0.0022 and p=0,0024 respectively) and diastolic BP (DOX16: 79.6 ± 9.9; DOX20: 61.22 ± 10.98; CTL: 111.6 ±3.93 mmHg, p=0.0133 and p=0.0015 respectively).

MPO serum concentrations measurements revealed that DOX treatment tends to increase MPO serum levels. In fact, DOX20 group MPO levels were significantly higher than the other groups (DOX20: 238.9 ± 41.1; CTL: 31.9 ± 1.67 pg/ml, p=0.0018) and (DOX20: 238.9 ± 41.1; DOX16: 43.79 ± 9.25 pg/ml, p=0.0003).

These findings suggest that the DOX effects on physiological parameters are different according to the cumulative dosage, with the highest dosage causing an increase in MPO levels and a more pronounced hypotensive effect. Although complementary data is still needed, our data suggest that MPO serum elevation reflects physiological dysfunction caused by DOX.

Keywords: Cardiovascular function, Doxorrubicin, Inflammation, Myeloperoxidase

Corresponding author: Vera Geraldes, https://orcid.org/0000-0003-1275-3459

O3-07

Effect of CaMKII inhibition by KN-93 on intrinsic myocardial ventricular refractoriness and sinus chronotropism in an isolated and perfused rabbit heart model

*¹Ortiz, J.E., ²Genovés, P., ²Arias, O., ³Such-Miquel, L., ³Zarzoso, M., ¹Parra, G., ¹Cardells, M.J., ⁴del Canto, I., ¹Alberola., A.M., ⁵Chorro, F.J., ¹Such, L.

¹ Department of Physiology, Universitat de València, 46010 Valencia, Spain.

² CIBERCV, Carlos III Health Institute, 28029 Madrid, Spain.

³ Department of Physiotherapy, Universitat de València, 46010 Valencia, Spain.

⁴ Department of Electronic Engineering, Universitat de València, 46010 Valencia, Spain.

⁵ Department of Medicine, Universitat de València, 46010 Valencia, Spain.

The overactivation of Ca²⁺/calmodulin-dependent kinase II (CaMKII), that causes certain pathophysiological circumstances, is involved in the hyperphosphorylation of ryanodine receptors, which produces Ca²⁺ diastolic release, thus favoring the triggering of ventricular arrhythmias. So, the effect of differents CaMKII inhibitors in the mentioned pathophysiological circumstances has been subject of several investigations. In this work, the electrophysiological consequences of CaMKII inhibition by the administration of KN-93 on intrinsic myocardial ventricular refractoriness and the effects on intrinsic sinus chronotropism, have been investigated. We have analyzed in 19 New Zealand male rabbits, the effect of KN-93 infusion at two concentrations (0.1μM and 1μM) in an isolated and perfused heart model. The animals were heparinized and euthanized by sodium thiopental intravenous injection (European Ethic Guidelines). The hearts were excised, isolated and perfused, in a Langendorff system. A pacing electrode and a recording multielectrode (121) were placed on the left ventricle epicardium. For the analysis of refractoriness, the effective and functional ventricular refractory periods (VERP and VFRP respectively) were determined, applying the ventricular extrastimulus test (VEET) with a pacing cycle length of 250ms (VEET-250) and 150ms (VEET-150) and sinus cycle length (SCL) was determined as a measure of the chronotropism. VERP and VFRP, as well as the SCL, were determined before and after KN-93 infusion at 2 mentioned concentrations. Statistical analysis was performed using the t-Student test for paired samples and statistical significance was considered when p<0.05. VFRP decreased after KN-93 infusion compared to baseline, for VEET-250 at the concentration of 0.1μM (119.6 ±12.5ms vs. 128.1 ±9.7ms, respectively; n=9) and at 1μM for VEET-150 (112.1 ±13.8ms vs. 123.4 ±6.4ms, respectively; n=8). A decreasing trend was observed for VERP and VFRP in VEET-250 (p=0.09 and p=0.07, respectively), and for VERP in VEET-150 (p=0.09) at 1μM. Regarding SCL, an increase was found after the infusion of KN-93 compared to the baseline value at 1μM concentration (330.5 ±61.5ms vs. 319.2 ±58.9ms; n=10). The 0.1μM concentration did not cause significant changes in the SCL. The results allow us to conclude that the inhibition of CaMKII with KN-93 (1μM) depresses intrinsic sinus chronotropism and decreases intrinsic ventricular refractoriness.

Keywords: Ventricular refractoriness, sinus chronotropism, CaMKII inhibition, cardiac electrophysiology.

Corresponding author: Johan Ortiz Guzmán https://orcid.org/0000-0003-0068-7749

Oral session 4: Endocrinology and Reproduction

O4-01

Interaction between oxidative stress and NLRP3 inflammasome pathway after Bisphenol F treatment in the liver of Long Evans lactating dams and its perinatal effect on postnatal day 6 offspring

^*1Beatriz Linillos-Pradillo, ²Sergio D. Paredes, ¹Julio García, ³Margret Schlumpf, ³Walter Lichtensteiger, ²J.A.F. Tresguerres ,¹Elena Vara and ¹Lisa Rancan.

¹ Department of Biochemistry and Molecular Biology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

² Department of Physiology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

³ GREEN Tox and Institute of Veterinary Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

Bisphenol F (BPF) is now replacing Bisphenol A (BPA) in the manufacturing of a broad range of products containing polycarbonates and epoxy resins due to an increased concern over BPA endocrine-disrupting effects. Nevertheless, free monomers of this bisphenol can be released into the environment and enter the food chain, highly likely resulting in human exposure to low doses of BPF. The aim of this study was to investigate the effect on the liver of different doses of BPF in lactating dams and also in their offspring. 36 female Long Evans rats were randomly distributed according to oral treatment into three groups: Control, BPF 0.036 mg/kg b.w./day; and BPF 3.6 mg/kg b.w./day. The levels of antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH/GSSG) and lipid damage markers (MDA, LPO) were measured using colorimetric methods. Inducers of oxidative stress (Ho1d, iNOS, eNOS), inflammation (IL-1β, IL-18, IFN-γ and TNF-α) and components of the NLRP3-inflammasome pathway (NLRP3, CASP-1 and Pycard) were measured by qRT-PCR and Western blotting in liver of lactating dams and PND6 offspring. Mean values were analyzed using Prism-6. Hematoxylin and eosin staining were used for histological studies. Treatment with the low dose of BPF (BPF 0.036 mg/kg b.w./day) showed an increase in oxidative stress and a reduction in antioxidant defense. Activation of the NLRP3- inflammasome pathways was also observed, with inflammation and activation of CASP-1 in the liver of lactating dams. The same occurred in both male and female PND6 offspring. Similar effects without significant differences were observed at higher doses of BPF. BPF treatment triggers an inflammatory response through upregulation of NLRP3-inflammasome pathway and apoptosis in the liver of lactating dams and PND6 offspring following a non-monotonic dose-response curve.

Supported by the European Union’s Horizon 2020 Research and Innovation Programme (project ENDpoiNTs; grant number: 825759).

Keywords: Bisphenol F, NLRP3, apoptosis, oxidative stress.

Corresponding author: Beatriz Linillos-Pradillo, https://orcid.org/0000-0003-0344-9877

O4-02

Effect of developmental exposure to Bisphenol F (BPF) on behavior of adult Long Evans rat’s offspring

^*1Beatriz Linillos-Pradillo, ¹Lisa Rancan, ²Lara Miguélez, ³Margret Schlumpf, ¹Elena Vara ³Walter Lichtensteiger, ²J.A.F. Tresguerres and ²Sergio D. Paredes.

¹ Department of Biochemistry and Molecular Biology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

² Department of Physiology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

³ GREEN Tox and Institute of Veterinary Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

The recent restriction of the use of Bisphenol A (BPA), due to its effects on the brain and behavior, has led to the entry into the market of analogues of this compound, such as BPF. BPF is increasingly used due to its low viscosity and better solvent resistance than BPA. It has been found to be used in food packaging, pipe coating, dental sealants, lacquers, plastics and adhesives, as well as in various consumer products. This compound is absorbed orally and distributed throughout the body, even crossing the placental barrier. BPF has an endocrine disrupting potential as active as BPA, acting as an estrogenic, progesterone and anti-androgen agent. This study aimed to investigate the effects of parental exposure to BPF on offspring behavior. The offspring of 2.5 month-old Long Evans rats were distributed according to the diet treatment received and sex: Control (n=6 females and n=6 males) or BPF 0.036 mg/kg b.w./day (n=9 females and n=9 males). The behavioral tests performed were object recognition (short- and long-term memory and learning) and elevated plus maze (anxiety-like responses). Regarding the elevated plus maze, BPF females remained a shorter amount of time in the central platform and in the open arms, while they remained longer in the closed arms than control females. BPF males remained a shorter amount of time in open arms compared to control males. In short-term memory, BPF females spent a similar amount of time with the familiar object and the new object. However, control females spent longer with the new object than with the familiar object. In long-term memory, the same occurred in both sexes, BPF males and females spending a similar amount of time with the new object as with the familiar one. However, control group spent more time with the new object and less time with the familiar one. Parental exposure to BPF appears to alter short-term memory in female offspring and alter long-term memory and increase anxiety behaviors in both sexes of offspring.

Supported by the European Union’s Horizon 2020 Research and Innovation Programme (project ENDpoiNTs; grant number: 825759).

Keywords: Bisphenol F, anxiety, memory, offspring.

Corresponding author: Beatriz Linillos-Pradillo, https://orcid.org/0000-0003-0344-9877

O4-03

Effect of permethrin (PMT) on the liver of Long Evans lactating dams and their offspring related to inflammation, glutathione system, and insulin signaling pathways

*¹Beatriz Linillos-Pradillo, ¹Lisa Rancan, ²María Ortiz, ³Margret Schlumpf, ³Walter Lichtensteiger, ²J.A.F. Tresguerres ,¹Elena Vara and ²Sergio D. Paredes.

¹ Department of Biochemistry and Molecular Biology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

² Department of Physiology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

³ GREEN Tox and Institute of Veterinary Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

Previous studies have shown that permethrin (PMT), a commonly used pesticide, induces cellular damage through mechanisms such as oxidative stress and inflammation, and may also have some interaction with insulin. In relation to the liver, one of the main organs of lipid and glucose metabolism, it has been observed that permethrin increases lipogenesis and decreases fatty acid oxidation. The aim of this work was to investigate the effects caused by treatment at two different doses of PMT on the liver of lactating dams and their offspring. Female Long Evans rats were randomly distributed according to oral treatment into three groups: Control, PMT 0.36 mg/kg b.w./day, and PMT 3.6 mg/kg b.w/day. After 60 days of treatment, lactating females and their offspring were sacrificed at post-natal day 6, and livers were collected. mRNA and protein expression of pro-inflammatory molecules (IL-1β, TNF-α), oxidative stress markers (GPx, GST, γGCS), molecules involved in glutathione and insulin metabolism (INS-R, IRS-1) and the main insulin-activated signaling pathways (PI3K/AKT/mTOR and MAPKs) were measured by RT-qPCR and Western blotting. Mean values were analyzed using Prism-6. Hematoxylin and eosin staining were used for histological studies. In lactating dams treated with 3.6 mg/kg PMT, increased inflammation (IL-1β) and oxidative stress (decreased GST and GPx) were observed. In the offspring, the most important alterations were found in the insulin signaling pathway, generally due to a decrease in its activity. Treatment with high dose PMT triggers an inflammatory and potentially cytotoxic response in adult mothers. It may also act in the offspring by generally down-regulating the levels of proteins involved in its signaling by its receptor-bound pathways (PI3K/AKT/mTOR and MAPKs). Exposure to low dose PMT causes less alterations, mainly affecting pathway intermediary molecules such as AKT and RAF. PMT could act as an endocrine disruptor in the liver by modifying mRNA and protein expressions of several molecules related to oxidative stress, inflammation and insulin metabolism.

Supported by the European Union’s Horizon 2020 Research and Innovation Programme (project ENDpoiNTs; grant number: 825759).

Keywords: PMT, Inflammation, oxidative stress, PI3K/AKT/mTOR.

Corresponding author: Beatriz Linillos-Pradillo, https://orcid.org/0000-0003-0344-9877

O4-04

Low doses of Bisphenol A induce hepatotoxicity through oxidative stress, inflammation and apoptosis in Long Evans lactating rats and its perinatal effect on female postnatal day 6 offspring

^*1Beatriz Linillos-Pradillo, ²Sergio D. Paredes, ²Lara Miguélez, ²Hassana Idrissi, ³Margret Schlumpf, ³Walter Lichtensteiger, ²J.A.F. Tresguerres, ¹Elena Vara and ¹Lisa Rancan.

¹ Department of Biochemistry and Molecular Biology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

² Department of Physiology, School of Medicine, Complutense University of Madrid, Madrid, Spain.

³ GREEN Tox and Institute of Veterinary Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

Bisphenol A (BPA) is a phenolic compound widely used in industry especially for the elaboration of plastics that on the other hand could be used for food protection or packaging. Monomers of BPA can be released and enter the food chain, resulting in continuous and ubiquitous low-dose human exposure. This exposure during prenatal development is especially critical, since the fetus is extremely sensitive to chemical exposure that could lead to alterations in the ontogeny of several tissues so to increase the risk of developing diseases later in adulthood. The aim of this study was to evaluate whether BPA administration to pregnant rats could induce hepatotoxicity by generating liver oxidative stress, inflammation and apoptosis, and whether these effects may be also observed in female postnatal day 6 (PND6) offspring. 27 Long Evans rats were randomly distributed according to oral treatment into three groups: Control, BPA 0.036 mg/kg b.w./day; and BPA 3.42 mg/kg b.w/day. The levels of antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH/GSSG) and lipid and DNA damage markers (MDA, LPO, NO, 8-OHdG) were measured using colorimetric methods. Inducers of oxidative stress (Ho1d, iNOS, eNOS), inflammation (IL-1β) and apoptosis (AIF, BAX, Bcl-2 and BCL-XL) were measured by qRT-PCR and Western blotting in liver of lactating dams and female PND6 offspring. Animals treated with the low dose of BPA 0.036 mg/kg showed a significant reduction in the activity of antioxidant enzymes and increased lipid and DNA damage both in lactating dams and PND6 offspring together with an increase in markers of oxidative stress, inflammation and proapoptotic factors. Higher dose of BPA showed similar effects without significant differences with the low dose. Low dose of BPA caused hepatotoxicity in lactating dams and had a perinatal effect in female PND6 offspring by increasing oxidative stress levels and triggering an inflammatory response and apoptosis pathways in the liver, the organ responsible for detoxification of this endocrine disruptor.

Supported by the European Union’s Horizon 2020 Research and Innovation Programme (project ENDpoiNTs; grant number: 825759).

Keywords: Bisphenol A, oxidative stress, inflammation, apoptosis.

Corresponding author: Beatriz Linillos-Pradillo, https://orcid.org/0000-0003-0344-9877

O4-05

Morphometric study of the umbilical cord in in-vitro- derived pigs

^*1Ester Párraga-Ros, ² Úrsula Álvarez-Martín, ^2,3 Pilar Coy, ¹Juan Seva, ^2,3 Raquel Romar

¹ Department of Anatomy and Comparative Pathology, Faculty of Veterinary Medicine, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain.

² Department of Physiology, Faculty of Veterinary Medicine, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain.

³ Institute for Biomedical Research of Murcia (IMIB), Murcia, Spain.

The umbilical cord is the vital fetus-placenta connection and represents one of the greatest sources of precursor cells. Histologically it is a single amniotic epithelium that encloses a mucoid connective tissue, and a vein and two arteries lacking tunica adventitia. Instead, there is a special mucoid connective tissue named Wharton’s Jelly (WJ) that is divided into a perivascular and an intermediate zone, the first being the most abundant in mesenchymal stromal cells. Morphological characteristics of the umbilical cord and its components have been related to fetal malformations, preterm birth and low birth weight. The objective of this study was to compare the WJ and the vascular area in the umbilical cord of pigs born from in-vitro- and in-vivo-produced embryos (the latter born by artificial insemination of sows; AI group). In-vitro embryos (IVP) were produced after insemination in-vitro of matured oocytes and further in-vitro culture up to blastocyst stage in media supplemented with (RF-IVP group) or without (C-IVP group) reproductive fluids (1% porcine oviductal fluid and 1% uterine fluid). Blastocysts produced were surgically transferred at day 7 post-in-vitro fertilization. After birth, umbilical cord samples of 15 animals (5 per group) were collected and productive parameters recorded. Samples were fixed (10% buffered formaldehyde solution) and paraffin-embedded. Complete sections of 5 μm thickness were stained (hematoxylin-eosin) and digitized with a Histech MIDI II 3D scanner at 0.172 pixels/μm. The virtual microscope SlideViewer 2.5 3D Histech was used for the digital analysis of the total umbilical area, the thickness of both WJ’s zones, and each vessel’s area. Data were analyzed by one-way ANOVA (SPSS Statistics 28) and the differences were compared by Tukey’s test (P<0.05). The thickness of the WJ-perivascular zone was significantly higher in the C-IVP group (AI: 572,4 ± 39,3 μm; C-IVP: 662,2 ± 42,3 μm; RF-IVP: 501,1 ± 35,7 μm), and was significantly correlated with piglet birth weight, placental weight and placental efficiency (Pearson <0.05). No significant differences in the WJ-intermediate zone thickness, vascular and total umbilical areas were found. These results might be related to the subsequent development of pathophysiological changes in IVP animals during their growth.

This study is part of project PID2020-113366RB-I00 funded by MCIN/AEI/10.13039/501100011033/ and “FEDER Una manera de hacer Europa”.

Keywords: Umbilical cord, Wharton’s Jelly, in-vitro fertilization, pig

Corresponding author: Ester Párraga-Ros (ester.parraga@um.es), https://orcid.org/0000-0001-7704-2549

O4-06

Deepening into the early onset pathogenic mechanisms of preeclampsia: NADPH oxidase isoforms and oxidative disbalance in a rodent model with excess sFlt-1

^*1,2Álvaro Santana-Garrido, ^1,2Claudia Reyes-Goya, ¹Pablo Espinosa-Martín, ^2,3Luis M. Beltrán-Romero, ^1,2Carmen M. Vázquez, ^1,2Alfonso Mate

¹ Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, España; ² Epidemiología Clínica y Riesgo Cardiovascular, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/Consejo Superior de Investigaciones Científicas/Universidad de Sevilla, 41013 Sevilla, España; and ³ Departamento de Medicina, Facultad de Medicina, Universidad de Sevilla, 41009 Sevilla, España.

Preeclampsia (PE) is a gestational hypertensive syndrome that affects around 3-10% of pregnancies worldwide. This syndrome is well-described by new-onset hypertension in the second half of pregnancy that might be accompanied by proteinuria and/or organ damage. PE can be subdivided into early-onset and late-onset PE depending on the development of the disease, although both forms are associated with an increase in the anti-angiogenic marker, sFlt-1 (soluble fms-like tyrosine kinase-1). The precise mechanisms that trigger this disorder remain unknown at present. In this sense, oxidative stress has been postulated as one of the key factors responsible for the observed manifestations. The aim of this study was to shed light on the role of oxidative stress in a rat model of early-onset PE following continuous sFlt-1 infusion. PE was mimicked using osmotic minipumps loaded with sFlt-1 and implanted intraperitoneally from gestational day 7 (GD7) until GD19. To confirm successful induction of a preeclampsia-like disorder, blood pressure, proteinuria, and sFlt-1/PLGF ratio were routinely measured throughout the experiment. Oxidative stress-related parameters, including evaluation of reactive oxygen species (ROS), nitrosylation protein levels and NADPH oxidase activity/protein/mRNA expression (isoforms NOX1, NOX2, NOX4), as well as the activity and expression of antioxidant enzymes (namely, glutathione peroxidase, GSH-Px; glutathione reductase, GSH-Red; and superoxide dismutase, SOD), were measured in placenta and kidney homogenates obtained from normal pregnant (NP) and preeclamptic (PE) rats. Nitric oxide (NO) levels and the expression of eNOS and arginase isoforms were also analyzed in both groups of animals. Our results showed increased ROS levels and NADPH oxidase activity/gene expression in tissues of preeclamptic rats. Preeclampsia also presented with alterations in NO metabolism and in the activity/expression of antioxidant enzymes. Noteworthy, combined results on ROS sources and the expression of SOD2 isoform suggest the presence of mitochondrial dysfunction in our model of PE.

Supported by Consejería de Salud, Junta de Andalucía (PI-0456-2018). Álvaro Santana-Garrido is a recipient of an FPU predoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FPU17/03465).

Keywords: NADPH oxidase, oxidative stress, preeclampsia, sFlt-1.

Corresponding author: Alfonso Mate, https://orcid.org/0000-0002-2719-8825

O4-07

Reproductive fluids reduce the rate of telomere shortening during the first year of life in in-vitro derived-pigs and cattle

¹Claudia García Cobarro, ^1,2 Raquel Romar, ^1,3 Guillermo Ramis Vidal, ^1,2Sebastián Cánovas, ⁴Alfonso Gutiérrez-Adán, ^1,2Pilar Coy

¹ Department of Physiology, Faculty of Veterinary Medicine, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain; ² Institute for Biomedical Research of Murcia (IMIB), Murcia, Spain; ³ Department of Animal Production, Faculty of Veterinary Medicine, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain; and ⁴ Animal Reproduction Department, INIA-CSIC, Madrid, Spain.

The shortening rate of telomere length in blood cells is a potential predictive tool for health and lifespan. It is unknown whether the differences in telomere shortening observed between individuals are genetically determined or whether early environmental factors produce such differences. In this study, we analyzed the relative length of leukocyte telomeres throughout the first year of life in two animal colonies of the bovine and porcine species. The animals were generated by artificial insemination (AI) (control group) or by in vitro production (IVP) and transfer of embryos, either using conventional culture media (C-IVP, with bovine serum albumin as the only protein source) or enriched culture media (ET-IVP, with reproductive fluids as an extra source of proteins). Blood samples obtained 3 days, one month (day 28-30), 4 months (day 120), and one year (day 365) after birth were used. Cellular DNA was extracted, purified, and quantified following standardized protocols. Subsequently, the telomere length was determined by real-time quantitative PCR, normalizing the telomere amplification values with respect to the amount of the constitutive gene Rn18S (relative quantification). Statistical analysis (one way ANOVA and Bonferroni in cattle, two-sample t-test for inhomogeneous variances in pig, Pearson correlation and regression analysis) of the relative telomere size with respect to age and animal group allowed us to observe the dynamics of shortening that occurs in both species. The results showed a progressive shortening in telomere length with age, with the greatest shortening occurring in cattle at 30 days and in pigs at 120 days. As for the speed of shortening, in cattle, it was observed that the C-IVP group suffered a greater shortening between days 30 and 120 compared to the AI and ET-IVP groups. In pigs, a greater reduction was also observed in the C-IVP group than in the ET-IVP group. The results in both species suggest that supplementation with reproductive fluids during IVP reduces the rate of shortening of telomere length during the early development of individuals.

Supported by PID2020-113366RB-I00 funded by MCIN/AEI/10.13039/501100011033/ and “FEDER Una manera de hacer Europa”.

Keywords: Telomere length, pig, cow, reproductive fluids.

Corresponding author: Pilar Coy Fuster, https://orcid.org/0000-0002-3943-1890

O4-08

Statistical analysis of the self-reported persistence of long-COVID19 symptoms in naturally cycling women depending on the phase of the menstrual cycle in which they tested positive

¹Ainhoa Rodriguez Muguruza, ²Cristina Carrasco Romero, ²Ana B Rodriguez Moratinos, ¹Arantza Etxeberria Agiriano

¹ Philosophy Department, University of the Basque Country, Donostia-San Sebastian, Guipúzcoa, Spain; and ² Department of Physiology, University of Extremadura, Badajoz, Extremadura, Spain

Self-reported symptoms and surveys during the COVID-19 pandemic have alerted on the effects of infection in different collectives. They indicate a causal relation between the COVID-19 infection and alterations in the menstrual cycle of naturally cycling women (NCW). NCW go through significant metabolic and endocrinological changes during their menstrual cycle, due to variations in the levels of the sexual hormones involved. Fluctuations in oestrogen, progesterone, and testosterone prompt changes related to the ovarian and endometrial cycles, and also affect the immune system. We present an interdisciplinary attempt on explanation, involving both physiology and philosophy of science, to explore the interactions between the immune and the reproductive systems occurring as effects of COVID-19 effects in menstruating women. The University of Extremadura conducted a survey with over 17,000 participants and gathered data on hospitalisations, alterations in length and due date of the first post-COVID-19 menstruation, and duration of post-COVID-19 symptoms. The study sampled n=1922 individuals with a regular menstrual cycle and taking no contraception, diagnosed with COVID-19. Results suggest that the immune and menstrual cycle fluctuations may be matched, related to the effects of oestrogen and progesterone, a hormone having lengthily documented immunosuppressive effects. Inferring the phase of the menstrual cycle in which the respondents to the survey tested positive, our statistical analysis can identify variations in the likelihood for NCW to experience menstrual alterations and to develop long-term post-COVID-19 symptoms depending on the phase of the menstrual cycle in which they tested positive. From this analysis, our results showed that NCW that tested positive for COVID-19 during their ovulatory phase were significantly less likely to experience menstrual alterations, whilst those testing positive during their follicular phase were significantly the most likely to experience them. Moreover, NCW that tested positive for the SARS-CoV-2 virus during their ovulatory phase were also less likely to experience post-COVID-19 symptoms whilst those that tested positive during their luteal phase were the most likely to experience them; these findings, however, were not statistically significant and will require further studies.

Keywords: COVID-19, immune system, menstrual cycle, self-reported survey

Corresponding author: Ainhoa Rodríguez, ORCID: 0000-0001-5748-3494

Oral session 5: Metabolism and Nutrition

O5-01

Enteral feeding of human milk MCP-1, EPA and DHA alter the risk of BPD in preterm infants

^1,2David Ramiro-Cortijo, ²Pratibha Singh, ³William Yakah, ²Joanne Brown, ²Esli Medina-Morales, ³Kristin L. Santoro, ^*3Camilia R. Martin.¹Department of Physiology, Faculty of Medicine, Universidad Autónoma de Madrid, Madrid, Spain; ²Division of Gastroenterology and ³Department of Neonatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Bronchopulmonary dysplasia (BPD) is one of the most common inflammation-related neonatal diseases of prematurity affecting approximately 50% of extremely preterm infants. High levels of pro-inflammatory cytokines in breast milk (BM) may predispose the offspring to inflammation through immunological modulation. In addition, omega(n)-6 and n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) play a critical role in lung development. We aimed to test the role of BM cytokines, and n-6 and n-3 LC-PUFAs in the development of BPD in preterm infants in the first month of life. Fifty-two infants born less than 28 weeks from the Nutrition and Infant Health program were enrolled at Beth Israel Deaconess Medical Center (Boston, MA, USA). Inclusion criteria were infant on full enteral feedings at day 14, receiving greater than 70% of mother’s BM, and feeding samples available at day 14 and day 28. BPD was defined as a need for supplemental oxygen at 36 weeks postmenstrual age (62.3% in the cohort). In feeding samples, IL-6, IL-8, and MCP-1 levels were determined by semiquantitative enzyme-linked Immuno-Assay and arachidonic (ARA), α-linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) were quantified by GC/MS. The association between cytokines and LC-PUFAs with BPD was tested by multivariate logistic regression models using a stepwise time-oriented approach, adjusted for maternal/neonatal variables. In feeding samples at day 14, ARA, ALA, EPA, and DHA levels, but not ARA:DHA or n-3:n-6 ratios, were significantly lower in BPD infants compared to non-BPD. At day 28, in feeding samples the level of MCP-1 was significantly higher in BPD than non-BPD infants. Adjusted models at day 14 corroborated the protective association between EPA (aOR=0.65 [0.41; 0.90]; P=0.027) and DHA (aOR=0.51 [0.25; 0.91]; P=0.039) with BPD. In the first month of life, low BM n-3 LC-PUFAs, and potentially later an increase in MCP-1, were biomarkers of BPD development. Lipids and cytokines are part of a network of metabolites that needs to be explored in depth. Additionally, the exposure to bioactive compounds should be explored.

Supported by Charles H. and Judy Hood Family Infant Health Research Program and National Institute of Diabetes and Digestive and Kidney Diseases (NIH R01 DK104346).

Keywords: Bronchopulmonary dysplasia, extremely premature, cytokines, LC-PUFAs.

Corresponding author: Camilia R. Martin, https://orcid.org/0000-0003-2783-6126

O5-02

Maternal nutritional pattern and gestational age is associated with secretion of cytokines in breastmilk

^1,2David Ramiro-Cortijo, ¹Santiago Ruvira, ¹Andrea Gila-Diaz, ³Gloria Herranz Carrillo, ¹Pilar Rodriguez-Rodriguez, ⁴Camilia R. Martin, ¹Silvia M. Arribas.

¹ Department of Physiology, Faculty of Medicine, Universidad Autonoma de Madrid, Madrid, Spain; ² Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; ³ Division of Neonatology, Hospital Clínico San Carlos, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain; and ⁴ Department of Neonatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Breast milk (BM) macronutrients are modulated by gestational age, lactation period, and maternal diet. However, the influence of these factors on bioactive BM compounds, such as cytokines, is less explored. Cytokines play a key role in early neonatal development, particularly in preterm infants since inflammation is an important modulator of perinatal diseases. We aimed to explore the contribution of maternal nutritional pattern, lactation period, and gestational age on BM cytokine pro- and anti-inflammatory profile. Forty-three mothers were retrospectively enrolled at Hospital Universitario Clínico San Carlos (Madrid, Spain). Mothers provided a BM sample at days 7 and 28, filled a 72h-recall dietary form (72hRD), and their anthropometry was evaluated by bioimpedance. From 72hRD, nutritional dimension I (NDI) was calculated by an equation based on energy, proteins, carbohydrates, different types of fats, cholesterol, calcium, sodium, and water intakes. In addition, net endogenous acid production (NEAP) was calculated based on protein and potassium intakes. BM IL-10, IL-13, IL-8, MCP-1, and TNFα levels were determined by a Meso Scale Discovery U-PLEX panel. The association between cytokines, lactation period and gestational age was tested by linear regression models. No significant differences in anthropometry were detected from day 7 to 28. NDI and NEAP were positively correlated (rho=0.35; P=0.007); these parameters did not correlate with lactation period or gestational age. From day 7 to 28 the anti-inflammatory cytokine IL-13 increased, while the pro-inflammatory IL-8, MCP-1 and TNFα decreased. Inverse associations close to significance were found at day 7 between NDI and MCP-1 (β=-518.6±292.9; P=0.08), and at day 28 between IL-8 and gestational age (β=-17.5±9.83; P=0.09). We conclude that along the first month of lactation, BM cytokines progress towards an anti-inflammatory pattern; 2) maternal nutrition influences MCP-1 BM content, which is a pro-inflammatory cytokine; 3) maternal nutrition does not influence IL-8 BM content. However, this cytokine is influenced by prematurity, being higher in lower gestational ages probably related to mammary gland development.

Supported by crowdfunding project PRECIPITA “Composicion de la leche maternal prematuridad y parametros maternos” (13 September 2020; FECYT, Spain), and Nutriben-AlterPharma^®.

Keywords: Maternal dietary pattern, breast milk, cytokines, prematurity.

Corresponding author: David Ramiro-Cortijo, https://orcid.org/0000-0001-7442-1586

O5-03

Unveiling the preventive role of wild olive oil (acebuche) in ocular hypertension and fibrosis in L-NAME-induced hypertension

^*1,2,3Álvaro Santana-Garrido, ^1,2Claudia Reyes-Goya, ³Helder André, ^1,2,3Carmen M. Vázquez, ^1,2Alfonso Mate

¹ Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, España; ² Epidemiología Clínica y Riesgo Cardiovascular, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/Consejo Superior de Investigaciones Científicas/Universidad de Sevilla, 41013 Sevilla, España; and ³ Department of Clinical Neuroscience, St. Erik Eye Hospital, Karolinska Institutet, 11282 Stockholm, Sweden.

Arterial hypertension contributes to enhanced production of reactive oxygen species (ROS) that favors hypertensive organ-damage via different mechanisms, including inflammatory and profibrotic processes. Fibrosis plays a crucial role in the development/progression of ocular diseases, including vascular retinopathies such as diabetic retinopathy (DR) and age-related-macular degeneration (AMD). Although the beneficial effect of extra virgin olive oil (EVOO) in fibrotic diseases has been previously reported, the potential therapeutic effects of the wild olive tree (also known in Spain as acebuche, ACE) are largely unknown. The aim of this study was to evaluate fibrotic processes in the retina of hypertensive mice and to assess the beneficial effects of an ACE-supplemented diet in this regard. To this purpose, C57B/6J mice were rendered hypertensive by administration of NG-nitro-L-arginine-methyl-ester (L-NAME), and simultaneously subjected to dietary supplementation with either ACE oil or EVOO for comparison purposes. Retinal vasculature and layers were analyzed by fluorescein angiography (FFA) and optical coherence tomography (OCT), respectively, and intraocular pressure (IOP) was measured by rebound tonometry. Different retina samples were obtained for Sirius Red staining and to estimate the protein/mRNA expression of fibrosis-related biomarkers: i) collagen isoforms (COL1a1-2, COL3a1, COL4a1-2); ii) MMPs isoforms and their inhibitors, TIMPs; iii) CTGF and TFGb isoforms (TFGb-1, TFGb-2, TFGb-3) and receptors (TFGb-R1, TFGb-R2). The co-localization of COL4a1 and TFGb-Rs in the retinal endothelial cells was assayed by immunofluorescence with CD-31 endothelial marker. Our results showed an increase in IOP values in L-NAME hypertensive animals, which was prevented by ACE diet. Furthermore, ACE diet prevented the dysregulation of retinal vasculature and thickness found in this hypertensive model. L-NAME-induced hypertension resulted in modified expressions of collagen/TFGb isoforms and MMP/TIMP ratios, these alterations being reversed by ACE supplementation. ACE diet also reduced the expression of COL4a1 and TFGb-Rs in retinal endothelial cells. In general, ACE diet showed better outcomes compared to EVOO. Our results support the use of ACE oil-enriched diets as a potential nutraceutical tool to lower IOP values and to counteract hypertension-related retinal fibrotic damage.

Supported by Grant 18.06.07.3004 PID2019-109002RB-I00 funded by MCIN/AEI/10.13039/501100011033. A patent application (P202030625) was filed with the Oficina Española de Patentes y Marcas (OEPM). Á. S.-G. is recipient of an FPU predoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FPU17/03465). Part of these results were obtained during an FPU mobility grant (EST19/00305).

Keywords: Arterial hypertension, acebuche oil, fibrosis, ocular hypertension.

Corresponding author: Alfonso Mate, https://orcid.org/0000-0002-2719-8825

O5-04

Effects of exposure to high amounts of carrageenan on the nervous and immune systems and redox state of young mice

*^1,2Judith Félix, ³Teresa Requena, ^1,2Mónica De La Fuente

¹ Department of Genetics, Physiology and Microbiology (Animal Physiology Unit). Faculty of Biology. Complutense University of Madrid. Spain.

² Research Institute of the Hospital 12 de Octubre, Madrid. Spain.

³ Department of Food Biotechnology and Microbiology, Institute of Food Science Research, CIAL (CSIC). Madrid, Spain.

The kind of diet in the childhood affects the development and functions of the homeostatic systems such as the nervous and immune systems and, consequently, the health state at adult age and the rate of aging in each individual. Currently, there are many additives that are added to all kinds of foods, such thickeners. One of them is carrageenan (E 407), which is obtained from different families of red seaweeds and can be divided into groups according to the number of sulfate groups in each polysaccharide repeating unit. Although the maximum allowed levels of carrageenan intake are revised from time to time, we are currently exposed to carrageenan in amounts above the optimal ones, especially during childhood, and the effects that this high exposure may have on our health are unknown. Therefore, the aim of the present study is to determine the effects of carrageenan intake on the nervous and immune systems and on the redox state in 8-week-old mice. For this purpose, mice of the ICR-CD1 strain of 8±1 week old were used and 4 experimental groups were formed: control females (n=10), carrageenan females (n=10), control males (n=10) and carrageenan males (n=10), which received 540 mg/kg/day of κ-carrageenan (sulfate group in C4 of β- D-galactose) diluted in 200μl of PBS and the control groups received 200μl of PBS for 15 days. After this time, a battery of behavioral tests was performed, and peritoneal leukocytes were extracted to evaluate different immune functions and their redox state. The results show that animals receiving carrageenan show, in comparison to controls, higher levels of anxiety and nervousness, as well as an impaired immune function and a redox imbalance in favor of oxidative components. Therefore, it can be concluded that exposure to high amounts of carrageenan during childhood generates a deterioration of health that could lead to the appearance of different oxidative-inflammatory diseases throughout life, as well as an accelerated aging.

Keywords: Behavior, carrageenan, immunity, redox state.

Corresponding author: Mónica De La Fuente, https://orcid.org/0000-0002-5969-097X

O5-05

Associations between adherence to unhealthy dietary patterns with body composition, liver, metabolic and inflammatory biomarkers in the UK Biobank study

^1,2Carmen Piernas, ³Anna K Sweetman, ²Min Gao, ^3,4Jennifer Carter, ^2,3,5Aurora Perez-Cornago, ^2,4Susan A Jebb

¹ Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Spain

² Nuffield Department of Primary Care Health Sciences, University of Oxford, UK

³ Nuffield Department of Population Health, University of Oxford, UK

⁴ NIHR Oxford Biomedical Research Centre, University of Oxford, UK.

⁵ Cancer Epidemiology Unit, Nuffield Department of Population Health, University of Oxford, UK.

Unhealthy dietary patterns (DP) previously identified in the UK Biobank population were characterized by high energy density, saturated fat, free sugars and low intake of dietary fibre. This DP showed strong associations with incident type 2 diabetes, cardiovascular disease (CVD) and all-cause mortality in this population. There is limited evidence on the associations between DPs with several biomarkers and cardiometabolic risk factors, some of which may be potential mediators of the reported associations with longer term health outcomes. We aimed to investigate cross-sectional and prospective associations between DP and body composition (fat mass [FM] and appendicular skeletal muscle mass [aSMM]), adiposity measures (body mass index [BMI], waist circumference [WC], as well as liver, metabolic and inflammatory biomarkers collected at baseline. We also investigated changes from baseline to follow up in a subsample of participants with available data. The main sample included 101,905 individuals from the UK Biobank, who provided detailed dietary information on two or more occasions with available blood biochemistry analyses and bio-impedance measurements. A subsample (n=26,751) had repeated measurements at follow up. Sex-stratified multivariable linear regression models were used with DP modelled in categories of adherence as well as continuously (per SD). We found that higher DP adherence was significantly linearly associated with FM, BMI and WC, but did not show a linear association with aSMM. Among participants with prospective follow-up body composition data (an average of 8.1 years after baseline), those adhering the most to the DP gained on average: FM (1.26kg in men, 1.26kg in women), and those adhering the least lost FM (0.09kg in men, 0.26kg in women). We also found significant positive associations between DP adherence and other biomarkers such as low-density and high-density lipoprotein cholesterol, triglycerides, apolipoprotein B, gamma glutamyl transferase, alanine aminotransferase, diastolic blood pressure and C-reactive protein. In conclusion, adherence to this unhealthy DP is positively associated with a wide range of cardiometabolic risk factors simultaneously, including general and central adiposity measures as well as blood lipids and other metabolic and inflammatory markers. This underscores the importance of looking at the diverse physiological effects of diet rather than single surrogate outcomes.

Supported by the National Institute for Health Research – Applied Research Collaborations Oxford and the Ramon y Cajal Fellowship, Spanish State Plan for Scientific and Technical Research and Innovation 2017-2020 (RYC2020-028818-I).

Keywords: dietary patterns, cardiometabolic health, body composition, cohort study

Corresponding author: Carmen Piernas, https://orcid.org/0000-0002-7536-922X

O5-06

Binge drinking reduces adolescent Wistar rats’ adipose tissue mass contributing to insulin resistance

^*1Inés Romero-Herrera, ¹María del Carmen Gallego-López, ¹Fátima Nogales, ²Karick Jotty, ¹M. Luisa Ojeda, ¹Olimpia Carreras

¹ Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain; and ² Natural and Exact Sciences Faculty, University of Cartagena, Cartagena, Colombia

Chronic alcoholism leads to a lipid imbalance at the adipose-liver axis that reduces lipid storage in the white adipose tissue (WAT) mass and increases lipid deposition in liver, not only resulting in alcoholic fatty liver disease, but also contributing to develop insulin resistance (IR). However, little is known about these effects after acute binge drinking (BD), which is the consumption pattern of greatest concern among teenagers, being the adolescence a period of continuous growth and metabolic changes. Therefore, the objective of this study was to analyse the macroscopic changes in WAT, the lipid oxidation and serum profile, and the SIRT-1 and IRS-1 expressions in the WAT of adolescent male Wistar rats under BD conditions. To this end, two groups of rats were tested during three weeks: control (standard base diet) and BD (standard base diet and i.p. alcohol 20%). BD rats presented lower body weight and body mass index, although the abdominal circumference remained almost unchanged. Serum triglycerides and HDL were increased, whereas cholesterol and LDL were decreased in the BD group, being this lipid profile also consistent with the adipose-liver axis disruption. The lipid peroxidation was determined by the malondialdehyde (MDA) measurement, which resulted increased in the BD animals, confirming the oxidative stress present. Besides, SIRT-1 was highly overexpressed in the WAT of the BD rats, indicating a lipolytic state. The IRS-1 expression was reduced in the WAT of the BD group, showing an affectation in the insulin signalling pathway and lipolysis. This situation may lead to IR. Although further studies are still required, these findings suggest that BD significantly decreases the WAT mass by the generation of a high lipolysis and lipid dyshomeostasis, which could contribute to future liver steatosis and IR. Deep understanding of the underlying mechanisms could be useful in the prevention of the worrying increase in IR during adolescence and of future cardiovascular complications during adulthood.

Supported by Junta de Andalucía (CTS-193). Inés Romero-Herrera is funded by a predoctoral Junta de Andalucía contract of research and teaching personnel number USE-22212-V.

Keywords: adipose tissue, binge drinking, lipolysis, insulin resistance.

Corresponding author: Inés Romero-Herrera, https://orcid.org/0000-0001-5394-1849

O5-07

Folic acid by modulating antioxidant enzyme balance avoids binge drinking-oxidative damage in liver, kidney and heart of adolescent rats

^*1María del Carmen Gallego-López, ¹Inés Romero-Herrera, ¹M Luisa Ojeda, ¹Olimpia Carreras, ²Rui Rua, ¹Fátima Nogales

¹ Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain; and ² Department of Dental Medicine, Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal

Binge drinking (BD) is an acute pattern of alcohol consumption mainly widespread among teenagers. It leads to important health consequences, both in short and long terms, such as cardiometabolic damage. BD is extremely pro-oxidant, promoting biomolecules’ oxidation and tissues’ damage. Folic acid (FA) is an antioxidant which is sharply decreased in chronic alcoholics. In fact, the supplementation with this vitamin clearly improves the oxidative profile of these patients. However, there are few studies linking BD and FA. Recently, it has been demonstrated that FA homeostasis in adolescent rats' tissues is disrupted after BD exposition. The objective of this study was to compare how and if FA supplementation to adolescent rats exposed to BD improves the oxidative balance in liver, kidney and heart. For this, four groups of adolescent male Wistar rats were treated for three weeks: control (base diet: 2 ppm FA), control supplemented with FA (8 ppm FA in food), BD (base diet and i.p. alcohol 20%) and BD with FA (supplemented diet and i.p. alcohol 20%). The oxidative stress markers in the tissues (lipid and protein oxidation) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) were determined by spectrophotometry. FA supplementation restored the antioxidant enzyme balance disrupted by BD, avoiding oxidative stress, but it acted differently on each tissue. In the liver, FA decreased SOD activity; in the kidneys, it increased GPx and GR activities; and in the heart, it reduced the four enzymes’ activities equalling the values to those of control. FA can act as an antioxidant by different mechanisms; therefore, in this study, in each of the tissues analysed FA seems to act through different routes. In conclusion, more studies are required to know the exact antioxidant mechanism of FA in these tissues. However, it is clear that FA is implicated in the oxidative stress which appears after BD exposition, and therefore, FA supplementation could be a safe and cheap therapy to prevent BD-oxidative damage during adolescence.

Supported by Junta de Andalucía (CTS-193). María del Carmen Gallego-López (this author is funded by a predoctoral University of Seville contract of research and teaching personnel VI-PPITUS).

Keywords: folic acid, binge drinking, adolescent rats, oxidative stress.

Corresponding author: María del Carmen Gallego-López, https://orcid.org/0000-0003-2272-5603.

O5-08

Alterations in NO metabolism do not explain hypertension induced by fetal undernutrition in rats but associate with nitrosative damage

¹Pilar Rodríguez-Rodríguez, ²Anuson Poasakate, ¹Santiago Ruvira, ³Perla Y Gutierrez-Arzapalo, ⁴Rainer Böger, ⁴Juliane Hannemann, ⁴Nicole Lüneburg, ¹Silvia M Arribas

¹ Departmnent of Physiology, Faculty of Medicine, Universidad Autónoma de Madrid, Spain; ² Department of Physiology, Khon Kaen University, Thailand; ³ Centro de Investigación y Docencia en Ciencias de la Salud, Universidad Autónoma de Sinaloa, Mexico; ⁴ Department of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Germany.

Exposure to insufficient nutrition in fetal life programs hypertension in male offspring. Nitric oxide (NO) is a key factor in blood pressure control, maintaining normal vascular tone and structure. We aimed to investigate the role of NO in fetal programing of hypertension. Male and female offspring from rats exposed to undernutrition during the second half of gestation (MUN) were studied at 21 days (normotensive) and 6 months of age (hypertension developed only in males). In aorta we analyzed total and phosphorylated endothelial NO synthase (eNOS, p-eNOS) and 3-nitrotyrosine (Western blot). In plasma we assessed L-arginine, asymmetric and symmetric dimethylarginine (ADMA, SDMA; LC–MS/MS), nitrates (NOx, Griess reaction), carbonyl and lipid peroxidation (spectrophotometry). 21-day-old MUN males showed significantly higher plasma L-arginine, ADMA, SDMA and carbonyls than controls, with no difference in L-arginine/ADMA ratio; MUN females only showed higher plasma L-arginine and SDMA compared to controls. 6-month-old MUN males showed increased aortic expression of p-eNOS/eNOS and 3-nytrotirosine, and elevated plasma L-arginine/ADMA, NOx and carbonyl levels; MUN females only showed higher plasma NOx than controls. We conclude that hypertension induced by fetal undernutrition in male adult offspring is not linked to low NO availability; in fact, NO is raised related to the higher eNOS expression and lower ADMA inhibition, possible compensatory mechanisms in response to high blood pressure. The higher nitrosative damage in MUN males, related to the pro-oxidant environment, can contribute to cardiovascular damage induced by fetal programming.

Supported by the COCARDIOLAC project from the Spanish Ministry of Science and Innovation (RTI2018-097504-B-I00).

Keywords: Fetal programing of hypertension, ADMA, nitrosative stress, sex.

Corresponding author: Silvia M. Arribas Rodríguez, https://orcid.org/0000-0001-7103-6105

Oral session 6: Final Degree/Master Projects & Teaching in Physiology

O6-01

Adjuvant effect of melatonin and a Pt(II) thiazoline complex in HeLa cells. Results of the use of mesoporous silica nanoparticles as drug delivery system on the cytotoxic activity of both compounds

¹Ángel Verdasco, ¹Samuel Estirado, ¹Elena Fernández-Delgado, ³Santiago Gómez-Ruiz, ³Diana Díaz-García, ²Emilio Viñuelas-Zahínos, ²Francisco Luna-Giles, ¹Javier Espino, ^*1José A Pariente.

¹ Department of Physiology (Neuroimmunophysiology and Chrononutrition Research Group), and ² Departament of Organic and Inorganic Chemistry (Chemistry of Coordination Research Group), Universidad de Extremadura, Badajoz; ³ Department of Biology, Physical Geology and Inorganic Chemistry (Comet-Nano Group), Universidad Rey Juan Carlos, Madrid, Spain.

Many studies have shown that co-administration of melatonin with conventional chemotherapeutic improves the sensitivity of cancers to induction of cell death. Interestingly, melatonin incorporated into nanosized materials exhibits superior effectiveness than free melatonin. In fact, melatonin-loaded nanoparticles showed superior antioxidant, anti-inflammatory, and antitumor properties in distinct cell types and biological tissues, both in vivo and in vitro experimental conditions, compared to melatonin in its free form. Herein, we reported the potentiating effects of a melatonin derivative (5-methoxytryptamine, 5-MT) with a Pt(II) thiazoline complex (PtTdTn) on cytotoxicity activity in epithelial cervix carcinoma HeLa cells. Moreover, we studied if mesoporous silica nanoparticles loaded with melatonin derivative and/or Pt(II) complex increases the cytotoxic activity of the free compounds. Our results showed both 5-MT (100 μM-5 mM) and PtTdTn (1 μM- 100 μM) produced a dose-dependent decrease in cell viability (IC₅₀ (5-MT) = 0.99 mM; IC₅₀ (PtTdTn) = 51.75 μM). Co-incubation of the IC₅₀ dose of both 5-MT and PtTdTn potentiated the cytotoxic effects on HeLa cells compared to the effects displayed by compounds alone. Additionally, the administration of mesoporous silica nanoparticles loaded with 5-MT and/or PtTdTn significantly increased cellular cytotoxicity compared to that displayed by the free compounds. In summary, our findings provided evidence that melatonin and the complex PtTdTn could be potentially applied to human cervix carcinoma treatment as powerful synergistic agents, which can be improved with the use of mesoporous silica nanoparticles.

This work was supported by Junta de Extremadura (IB18013, GR21042 and GR21075). EF and JE hold research pre-doctoral (jointly financed by European Social Fund, ref. PD18020) and post-doctoral (ref. TA18002) fellowship respectively, both from Junta de Extremadura.

Keywords: Melatonin, thiazoline, Pt(II) complex, mesoporous silica nanoparticles.

Corresponding author: José A Pariente, ORCID: 0000-0002-9094-9943

O6-02

Influence of lifestyle habits on university academic performance

^*1Celia Esteban, ¹Ana B. Rodríguez, ¹Cristina Carrasco.

¹ Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain.

Academic performance measures the level of knowledge demonstrated after a learning process and is affected by a multitude of internal and external factors. Since it is an important concept in education, in this way it is possible promote the implementation of mechanisms that stimulate students, allowing them to efficiently take advantage of their potential and increasing their performance. Thus, it is very interesting to determine the lifestyle habits that improve academic performance. For this purpose, 24 university students (12 men and 12 women) enrolled in Grade’s and Master’s degree courses at the University of Extremadura (Spain) were selected to participate in the study. Sociodemographic characteristics, dietary intake, physical activity, anthropometry, mood, sleep quality, use of new technologies and academic performance were collected at three time points within one academic year. Students were classified according to their academic performance (medium-low and medium-high). Statistical comparison between the two groups revealed significant differences in age, parent’s level of education, mood, sleep quality, use of new technologies and number of meals. Further analysis using binary logistic regression indicated that only age proved to be the most influential factor in the rating of university students. Thus, younger students clearly showed better academic performance. The rest of the variables were not significant when constructing the model. However, it can be deduced from the results obtained that not working, living at the family home, having more highly trained parents and a good sleep hygiene, using less the new technologies, being less anxious and eating more meals per day are aspects that may facilitate the learning processes and academic performance, having a beneficial influence on learning processes and improving academic performance.

Supported by Junta de Extremadura-FEDER (BB021; GR21042).

Keywords: academic performance, learning processes, lifestyle habits, university students.

Corresponding autor: Celia Esteban, ORCID: 0000-0002-5974-6101.

O6-03

Application of edible coatings with functional properties to improve the preservation of foodstuffs

¹Isabel P Fuentes, ¹Javier Rocha-Pimienta, ²María Garrido, ¹Jonathan Delgado-Adámez

¹ Department of Biotechnology and Sustainability, Technological Agri-Food Institute (INTAEX), Center for Scientific and Technological Research of Extremadura (CICYTEX), Badajoz, Spain

² Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain

Market demand for less perishable food, which allow reducing food waste, has increased the research of new techniques to prolong the shelf-life of foodstuffs. One strategy is the use of edible coatings and films, which maintains the quality and safety of food and has been of great interest in recent years. The objective of this work was based on the study of the application of edible coatings with biobased biopolymers to improve food preservation. Different chitosan-based biopolymers were prepared, using lactic acid and glycerol as plasticizers, gelatin as a solidifying agent, and cherry phenolic extract and rosemary essential oil with antimicrobial and antioxidant properties. The methodology used was as follows: solid-liquid extraction to obtain the phenolic extract and hydrodistillation method to obtain the essential oils. The phenolic profile of cherry extract and the volatile compound profile of rosemary essential oil were determined by liquid and gas chromatography, respectively. The antimicrobial activity was performed with the micro and macro dilution methods and the disk diffusion method to measure its bacteriostatic and bactericidal capacity, respectively. The antioxidant activity was determined by the ABTS method. Our results showed the following: regarding the aromatic composition of rosemary essential oil, the main component is eucalyptus with 20.77%. In addition, the phenolic profile showed that the anthocyanin with the greatest presence is cyanidin-3-rutinoside. These phytochemicals have antimicrobial and antioxidant properties that can palliate the microbiological deterioration of food and oxidative processes. The antimicrobial activity in microdilution showed a higher percentage of inhibition in Escherichia coli than in Listeria innocua for the different dilutions (1:1, 1:10 and 1:100). Therefore, edible coatings and films are very promising techniques to improve food quality, preservation and packaging, as their functional properties can ensure sustainable development and provide safe foodstuffs to consumers.

M.G. holds a post-doctoral fellowship (ref. TA18029) from Junta de Extremadura. J.R.-P. thanks to Junta de Extremadura for the predoctoral formation contract (ref. PD18018). Authors acknowledge to Agrupación de Cooperativas Valle del Jerte for the supply of the raw material to develop the study.

Keywords: cherry phenolic extract, rosemary essential oil, edible coatings, food packaging.

Corresponding author: Isabel Paula Fuentes.

O6-04

Relationship between dioxine receptor (Ahr) expression and pluripotency markers in human hepatocellular carcinoma patients

^*1Sergio Calderón, ^1,2Claudia M Rejano-Gordillo, ^1,2Francisco J González-Rico, ^1,2Ana Ordiales-Talavero, ^1,2Beatriz Díaz-Marín, ^1,2Jaime M Merino, ^2,3Noelia de Armas, ^2,3Gerardo Blanco-Fernández, ^1,2Pedro Fernández-Salguero

¹ Departmento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain, ² Instituto Universitario de Investigación Biosanitaria de Extremadura (INUBE), Badajoz, Spain and ³ Cirugía HBP y Trasplante Hepático, Hospital Universitario de Badajoz, Badajoz, Spain

Human hepatocellular carcinoma (HCC) is the fourth tumour with the highest mortality rate and the sixth most diagnosed. It is characterized by its classic chemotherapy drugs resistance and by its difficulty in having early diagnosis, hence the necessity of studying different genetic markers that could serve as therapeutic or diagnostic targets. On the other hand, it is known that the aryl hydrocarbon receptor (Ahr) has great implication in liver tumours and it has been observed that it can act as a tumour suppressor or as an oncogene. The goal of this study is to examine the relationship between Ahr expression and other liver tumour markers’ expression (OCT4, NANOG, Sox2, LGR5 and Cyp1a1) in human HCC patients. To achieve this goal, the patients were classified into high Ahr expression and low Ahr expression groups and compared their protein expression through Western-Blotting with their transcriptional expression through RT-qPCR. It was observed that there were any differences between the protein expression in patients with high and low Ahr levels. On the other hand, it was observed a higher mRNA expression in NANOG and lower in Sox2 and Cyp1a1 in those patients with high Ahr expression. While studying patients with low Ahr levels it was observed a higher transcriptional expression of OCT4, NANOG and Sox2. With these results it can be concluded that the presence of Ahr in HCC patients has an impact on the transcriptional levels of OCT4, Sox2 and Cyp1a1, but not on NANOG’s. It did not show any impact in the protein level of any of these markers. This suggests the existence of a translational regulation system between these markers and Ahr, which plays an important role.

The study using human samples has been carried out under the approval of the ethical committee of the University of Extremadura and Hospital Universitario de Badajoz.

Keywords: Human hepatocarcinoma, dioxin receptor, pluripotency, liver cancer.

Corresponding author: Sergio Calderón-Vicente, https://orcid.org/0000-0003-1328-2847

O6-05

Virus and autoimmunity

¹Ismael Rodríguez, ¹Ana Beatriz Rodríguez, ¹Cristina Carrasco.

¹ Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain.

Autoimmune diseases are pathologies with a very complex diagnosis and treatment. They are also considered as the final consequence of a series of immunological events that occur in a genetically susceptible person. Unlike other conditions such as infectious diseases, autoimmune diseases are not transmitted between individuals, because there are caused by isolated genes or groups of predisposing genes. Regarding their treatment, as there is no specific, there are several medicines focused on reducing the activity of the immune system. However, very few therapies can slow down the disease and regenerate damaged functions, tissues, and organs. In terms of origin of this kind of diseases, environmental, immunological factors and loss of self-tolerance may be involved. In recent years, it has been suggested that infectious agents such as viruses could also cause them, including enteroviruses and type I diabetes mellitus or Graves' disease, endogenous retroviruses and systemic lupus erythematosus, and Chikungunya virus and rheumatoid arthritis, among others. In vivo studies have indicated two possible mechanisms of action: the cross-reactivity between the infectious agent and the host antigens and the immunoreactivity disorders derived from the infection. Additionally, there would be a third pathway consisting in a nonspecific activation, where pathogens may weaken the self-tolerance through cell death or promoting an imbalance between cytokines. This Final Master Project offers a bibliographical review on the current evidence of the relationship between viruses and autoimmune diseases, as well as the available preventive and/or therapeutic options and possible future treatments. New and supplementary studies are needed to deep into the mechanisms underlying the development of autoimmune diseases. By this way, effective and safety treatments could be developed such as the use of plant virus nanoparticles, which may help in reducing symptoms and organ damage, as well as slowing down the progression of the disease.

Keywords: autoimmune disease, virus, treatment, prevention.

Corresponding author: Ismael Rodríguez.

O6-06

A simulation laboratory for the study of countercurrent oxygen transport across fish gills. Insights on pysiological variables and environmental factors

*Mario Díaz

Department of Physics, School of Sciences, Universidad de La Laguna, 38206 Tenerife, Spain

Gas exchange across fish gills is a paramount example of adaptive physiology. Thanks to the appearance of countercurrent systems, where water and blood follow opposite directions, the efficiency of oxygen transport is largely improved in marine and freshwater fish. However, laboratory experiments aimed to illustrate how this particular topology functions is enormously complex and require sophisticated technologies and setups. In NEMO 3.0, we have intended to develop a simulation environment to illustrate how the countercurrent system works at the branquial level in marine and freshwater teleost fish, at the same time that different physiological parameters may be manipulated to assess the contribution of different variables, including those derived from Fick’s Law. The simulation environment also allows experimental appraisals of modifications in venous oxygen concentration, blood pH, Bohr Effect, haemoglobin concentrations, fractional perfusion, effective gill area, as well as ventilation/perfusion relationships. As oxygen availability depends on environmental pO₂ and O₂ solubility, which, in turn is affected by temperature, salinity and pH, we have incorporated these variables in a dedicated section of the software. Further, based on real data taken from FishBase database, we have implemented allometric relationships of representative sedentary and active fish which may be used for comparative purposes and also as model species in the simulations. NEMO 3.0 is based on a system of ‘2n’ differential equations which were solved using Mathematica and implemented in a graphical interface unit using Matlab. The software, accessory files and documentation are available in Spanish and English languages, and are currently accessible for free distribution amongst academic and university communities.

Keywords: Computer-assisted learning, fish gills, countercurrent exchange, oxygen transport

Corresponding author: Mario Díaz

O6-07

Peer-to-peer medical student education: design, experience and evaluation of the impact in learning of 2 ^nd year medical students from the University of Malaga

¹Daniel Carrasco-Gomez, ¹Alvaro Chao-Ecija, ¹Marc Stefan Dawid-Milner

¹ Faculty of Medicine, University of Malaga. Autonomic Nervous System Unit, Department of Physiology, CIMES, Malaga

It is well-known that gamification is a rising educational technique, implemented in many settings by teachers, especially in primary education. Escape rooms have been trending in the past years among young adults, as teamwork, ingenuity and knowledge merge in a both distended but competitive environment. Inspired by it, a physiology escape room was designed for second year medical students of the Faculty of Medicine in Malaga on a peer-to-peer teaching basis. Indeed, physiology intern students from higher courses structured a practical escape room that covered the topics of the first human physiology course, focusing on cardiovascular and respiratory physiology. As a result, these students replaced regular teachers in this activity, taking control of the design, promotion, preparation, execution, evaluation and follow-up of the event. The escape room was done in mid-December, when all practical and theoretical content of the subject had already been delivered. It was divided in four stages, two for each topic, adding a final case, connected to the beginning of the activity. Participants were asked to team up freely in groups of 4-6 students. This activity was done during three years, readapting the structure to evaluate impact on students' learning and exam results. Our statistical analysis showed an increase in final exam grade of students that participated in escape room with a magnitude of 1.03 ± 0.65 points (p < 0.005) compared with those who did not, as well as an increase in global final grade with a magnitude of 1.34 ± 0.83 points (p < 0.005). Additionally, the students that participated showed a significantly higher performance in the final exam regarding cardiovascular (p < 10^-4) and respiratory-related questions (p < 0.005). Furthermore, positive feedback was given by participants on how they found the activity, independently on the impact in grade (as this was done prior to the exam). We conclude this teaching tool should be furtherly assessed in following years in order to implement a peer-to-peer teaching structure that can maximize its utility to medical students.

Keywords: teaching, escape room, peer-to-peer education, physiology

Corresponding author: Daniel Carrasco-Gomez

O6-08

An educational project for teaching Physiology along with science university degrees

^*Isabel Sánchez-Vera, Juan Francisco Aranda, Mª José Borrego, Esther Escudero, Josué García-Yagüe, Lucía Guerra, José Luis Lavandera, María del Nogal, Úrsula Muñoz, Cruz Sádaba, Antonio Sobrado, Hortensia Torres.

Departamento de Ciencias Médicas Básicas, Facultad de Medicina, Universidad CEU-San Pablo,CEU Universities, Madrid, Spain

Physiology subjects are studied in all science degrees, such as medicine, pharmacy, optics, nutrition, dentistry, physiotherapy, nursing, biomedical engineering, and genetics, among others. The aim of this study is to continue to educate our students in Physiology knowledge throughout their university career once they have passed the subject. For this proposal, we organized three different activities for them, since the 2019 academic year. 96 students and all the physiological area teachers participated in this project. Students participate on a voluntary basis. First, we organize teaching sessions about physiological topics once a month. Thus, students can remember concepts studied and relate them to other biomedical subjects. Likewise, teachers are used to updating, unifying criteria, and sharing content and teaching tools, while sharing with students the desire to continue learning and training. Second, we organize scientific sessions where teachers explain their experimental investigations or revise different research projects once a month too. In this way, students have more information about the teacher's investigation and can participate in it. They are encouraged to carry out Final Degree Projects or Doctoral Theses and are motivated to pursue scientific careers. As a result, more students applied for an undergraduate scholarship with one of the Physiology professors, and we now have more internal students than in previous years. Third, the students can participate in the cultural classroom. This consists of a biweekly newsletter where students receive recent news about scientific research, conferences, meetings, workshops, congresses, and whatever else is related to scientific and physiological science. Besides, teachers publish content related to the subject on the Instagram social network. In addition, some stories uploaded to Instagram consist of material prepared by the students themselves, which gives them visibility and motivates them. Currently, 114 publications and numerous stories have been uploaded. Some of them are featured. The engagement rate reached 1053 followers at this moment.

Keywords: Teaching method innovations, Physiology teaching, newsletter, Instagram

Corresponding author: Isabel Sánchez-Vera, https://orcid.org/0000-0003-1278-5338

Oral session 7: Animal research

O7-01

Cannibalism/infanticide and maternal aggression towards pups in laboratory animals: a bibliometric approach

^1,2José C. Bravo, ³Lierni Ugartemendia, ^*,2Ana B. Rodríguez, ²José A Pariente^{, 3}Rafael Bravo

^{1Animal Facility of University of Extremadura and 2} Neuroimmunophysiology and Chrononutrition Research Group. Faculty of Science. University of Extremadura. Badajoz, Spain. ³ Institute of Molecular Medicine, McGovern Medical School at the University of Texas Health Science Center, Houston, TX 77030, USA

Animal welfare has evolved during the past decades to improve not only the quality of life of laboratory animals, but even the quality and reproducibility of scientific investigations. Bibliometric analyses are defined as a method in which scientific literature is evaluated through computer-assisted techniques to obtain new information from bibliographic data. This kind of techniques are on the rise in scientific research - including animal welfare - to complete current knowledge with academic databases. Our aim was to elucidate whether scientific research about cannibalism/infanticide relates to maternal aggression towards the offspring in laboratory rodents. To perform our research, we carried out a specific search for published articles about each concept in the Web of Science (Clarivate Analytics PLC^®). Results were analyzed in the open-source environment RStudio with the package Bibliometrix. Firstly, we obtained 228 articles for cannibalism/infanticide and, secondly, for maternal aggression towards the pups 134 published articles. We noticed that the interest in infanticide cannibalism started during 1950s, while researchers showed some interest in maternal aggression towards the pups during 1980s. Our results showed that maternal aggression had better citations in scientific literature. Furthermore, we noticed some concepts appeared in both searches (e.g.: oxytocin or medial preoptic area in brain) and even we observed a gap between cannibalism/infanticide and maternal aggression towards the pups with only 18 published articles in common for both searches. Therefore, our investigation indicates researchers to combine both concepts in further research in the context of cannibalism for a better dissemination and higher impact in laboratory animal welfare research.

Supported by Junta de Extremadura (FEDER - GR21042).

Keywords: cannibalism, infanticide, maternal aggression, bibliometric analysis

Corresponding author: Ana B. Rodríguez, https://orcid.org/0000-0001-6063-0504

O7-02

The protective effect of dexpanthenol and tocopherol in an experimental model of liver toxicity induced by paracetamol

A Karanfil, *F Şirinyıldız, R Özmerdivenli

Department of Physiology (Medicine), Aydin Adnan Menderes University, Aydin, Turkey

It has been determined that hepatotoxicity is increasing due to the increasing use of drugs, and paracetamol is the focal point due to its easy supply. It has been shown that oxidative stress plays a role in the formation of many diseases, including hepatotoxicity. Disruptions in the balance between oxidative damage and the antioxidant system cause the emergence and progression of diseases. Dexpanthenol, a B5 provitamin, is the alcohol form of pantothenic acid, and it is known that dexpanthenol has a healing effect on wound healing and epithelialization. Tocopherol is taken as fat-soluble in the diet, has an antioxidant effect due to the aromatic ring with the phenolic hydroxyl group in its structure, and is one of the most important antioxidants responsible for maintaining the integrity of the cell membrane. In this study, the protective effects of dexpanthenol and tocopherol in an experimental liver toxicity model induced with paracetamol were investigated. In our study, Wistar albino rats were divided into control group, toxicity group, dexpanthenol treatment group, tocopherol treatment group, and dexpanthenol+tocopherol combined treatment groups. Toxicity was administered by intragastric administration of high-dose paracetamol, and rats were sacrificed 24 hours after this administration. While dexpanthenol applications were administered intramuscularly at the literature dose until toxicity was established, tocopherol applications were administered intraperitoneally at the literature dose until toxicity was established. Histological and biochemical analyzes were performed on blood and liver tissue samples taken from sacrificed rats. As a result of the tests, it was determined that the liver tissue MDA, MPO, and GSHPx parameters changed significantly in the toxicity group compared to the control group, and these values ​​in the treatment groups were close to the results of the control group. Histopathological evaluations in liver tissue also supported the tissue biochemistry results. AST, ALT, and LDH results from serum also showed that dexpanthenol and tocopherol applied as tissue samples reduced the effects of toxicity. As a result; It has been observed that Dexpanthenol and Tocopherol have protective effects against liver damage caused by paracetamol. It is very important to evaluate the results of clinical practice for the next step.

Keywords: Dexpanthenol, hepatotoxicity, paracetamol, tocopherol

Corresponding author: Ferhat Sirinyildiz, ORCID: 0000-0001-8800-978.

O7-03

A review on the analysis of six different experimental anemia models

^*1F Şirinyıldız, ¹D İşler, ²S Ünay

¹ Department of Physiology (Medicine), Aydin Adnan Menderes University, Aydin, Turkey, ² Department of Biophysics (Medicine), Lokman Hekim University, Ankara, Turkey

While a large number of anemia developing due to different etiological causes has been defined in the community, the representation of these, anemia in experimental studies and animal experiments shows a wide variation at this rate. When the pathophysiological mechanisms of the experimental study and the expectations from the therapeutic approaches are considered together, the suitability of the chosen model gains importance. Choosing the right animal, experimental time, time and cost savings, ethical compliance, limited or extensive opportunities, high representativeness of the applied pathophysiological mechanisms, biosafety level and easy reproducibility are of great importance in the model to be applied. In this study, models of anemia such as adenine-induced, turpentine-induced, phlebotomy, phenyl hydrazine-induced, 5/6 nephrectomy and benzene-induced are discussed. Adenine-induced anemia model: In this model, especially applied in male rodents, intragastrically administered adenine is metabolized. 2,8-dihydroxyadenine forms crystals in the proximal tubular epithelium, leading to inflammation and subsequently to tubulointerstitial fibrosis and anemia. Turpentine-induced anemia model: It has been shown that 24 hours after a single turpentine injection, serum iron levels drop below 40% of baseline levels and markedly increase IL-6 levels after turpentine injection. Phlebotomy anemia model: In this model, the blood of the animals is taken repeatedly until anemia is created by reducing the hematocrit value to a certain level and the hRBC count is reduced. Phenylhydrazine-induced anemia model: Phenylhydrazine, which causes damage to the red blood cell, shows secondary effects in the spleen and liver. It induces reticulocytosis, increased osmotic resistance. 5/6 nephrectomy anemia model: This model can be achieved either by ligation of renal vessels feeding the renal poles or by surgical removal of both kidney poles followed by contralateral nephrectomy. Benzene-induced anemia model: Chronic exposure to benzene leads to progressive bone marrow failure, and this pattern is acquired aplastic anemia characterized by severe anemia, neutropenia, and thrombocytopenia caused by immunotoxicity and oxidative stress. As a result, the different anemia models that have been developed are of great importance in terms of developing correct approaches to clinical anemia with their advantages and disadvantages. The increase in these experimental studies will lead to clinically compatible results.

Keywords: Anemia, animal models, experimental.

Corresponding author: Ferhat Şirinyıldız, ORCID: 0000-0001-8800-978

O7-04

Modulatory effects of rifaximin on inflammation and gut commensal microbiota during dextran sulfate sodium (DSS)-induced colitis in mice

^1,2Marina Ferrer, ^*1,2,3 Vicente Martínez

¹ Department of Cell Biology, Physiology and Immunology, Universidad Autónoma de Barcelona. Barcelona, Spain.

² Neuroscience Institute, Universidad Autónoma de Barcelona. Barcelona, Spain.

³ Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd). Instituto de Salud Carlos III, Madrid, Spain.

Rifaximin is a wide-spectrum antibiotic that ameliorates intestinal inflammation in inflammatory bowel disease (IBD), although the mechanism of action remains to be fully elucidated. The objective of this study was to characterize the effects of rifaximin in gut commensal microbiota (GCM) and innate immune parameters related to host-bacterial interactions, namely Toll-like receptors (TLRs) and antimicrobial peptides, in a murine model of colitis. To this aim, C57BL/6NCrl female mice received 3% DSS in the drinking water (5 days). Animals were treated, in a preventive manner, with rifaximin (50 or 150 mg/kg/day, 8 days, PO) or doxycycline (30 mg/kg/day, 8 days, PO). Daily clinical signs, histological colonic inflammation, GCM (16S rRNA gene sequencing), local expression (RT-qPCR) of Toll-like receptors (TLRs) (2, 3, 4, 5 and 7), antimicrobial peptides (defensin α24 -DEFα24-, resistin-like molecule β -RELMβ- and regenerating islet-derived protein IIIɣ -RegIIIɣ-), inflammatory markers (IL-6, INFɣ, TNFα, IL-1β, RANTES, IL-10) and pregnane X receptors (PXR) were assessed. All animal procedures were approved by the Ethical Committee of the Universidad Autònoma de Barcelona (protocol 1906). Animals receiving DSS showed clinical signs indicative of the development of colitis. Regardless the dose tested, rifaximin did not affect the clinical course of colitis, while doxycycline, attenuated clinical signs. Similarly, colitis-associated up-regulation of inflammatory markers was not affected by rifaximin, while doxycycline completely prevented this response. Colitis up-regulated RegIIIɣ, while other antimicrobial peptides were unaffected. Rifaximin did not affect colitis-associated RegIIIɣ up-regulation, while doxycycline completely normalized its expression. PXR was down-regulated during colitis, a change not affected by rifaximin but prevented by doxycycline. Regardless the treatment, minor changes in TLRs expression were observed. DSS-induced colitis was associated to a dysbiotic state. Rifaximin–treated animals, with or without colitis, showed similar bacterial composition. Doxycycline-treated-animals showed extensive changes in the microbiota, with similarities between inflamed and non-inflamed conditions. In the DSS-induced colitis model rifaximin does not show neither clear anti-inflammatory nor antimicrobial activities. The mechanism mediating the beneficial effects of rifaximin in IBD remains elusive.

Keywords: inflammatory bowel disease, gut commensal microbiota, host-bacterial interactions, rifaximin.

Corresponding author: Vicente Martínez, https://orcid.org/0000-0001-5193-715X

O7-05

Characterization of parameters that influence acceptance of a new food in rats

¹Santiago Ruvira, ¹Pilar Rodríguez-Rodriguez, ^2,3Silvia Cañas, ¹Ignacio Monedero-Cobeta, ¹David Ramiro-Cortijo, ⁴David Muñoz-Valverde, ^2,3Yolanda Aguilera, ¹Silvia M. Arribas.

¹ Department of Physiology, Faculty of Medicine, ² Department of Agricultural Chemistry & Bromatology, Faculty of Science, Universidad Autónoma de Madrid, Madrid, Spain; ³ Institute of Food Science Research (CIAL), Madrid, Spain; ⁴ Gabinete Veterinario, Universidad Autónoma de Madrid, Madrid, Spain.

Oral gavage is commonly used to test compounds in experimental animals. Possible problems are stress and inflammatory response if repetitive use, being also inadequate in pregnancy and lactation. Test compounds can also be given as a supplement in a gelatin but requires animal training to accept a new food. Our aim was to study in rats the degree of neophobia, some parameters that may influence acceptance (age, sex and fasting), and to test a protocol in lactating rats. 1-month-old (9 females; 14 males) and 5-month-old (31 females; 16 males) Sprague-Dawley rats, with or without 4 hours of fasting, were placed in an empty cage with a cube of neutral gelatin (size according to rat weight) for 2h, assessing if the cube was eaten. The procedure was repeated daily for up to 7 days (excluding weekends). The day the whole cube was eaten was considered the acceptance day. Rats that did not eat the cube by day 7 were considered neophobic. A group of adult females (n=10) was trained prior to mating and the acceptance of a gelatin containing 250 mg/kg cocoa shell extract tested during lactation. Mann Whitney U test was used for statistical analysis. In young rats neophobia was similar in both sexes (11,1% females; 7,1% males; P=0.640). In adult rats a significantly higher number of male rats were neophobic (16.1% females; 43.8 males; P=0.040). In non-neophobic rats the cube was eaten in a median of 2 days (minimum 1; maximum 6 days), without statistical differences between young and adult rats (P=0.657) or between male and female rats (P=0.189). Fasted rats ate the gelatin in shorter time, with a median of 1 day (P=0.007). Rats trained prior to gestation and tested during lactation ate daily the gelatin containing the test compound immediately after it was offered within 1-3 minutes. The use of gelatin as vehicle for supplementation in rats is feasible and requires minimum training, being facilitated by fasting. However, adult males exhibit a high degree of neophobia, and it would be important to detect neophobic animals prior to the study.

Supported by the COCARDIOLAC project from the Spanish Ministry of Science and Innovation (RTI2018-097504-B-I00).

Keywords: New food administration, neophobic, non-invasive techniques, sex.

Corresponding author: Silvia M. Arribas Rodríguez, https://orcid.org/0000-0001-7103-6105

O7-06

Cystine and glutathione (GSH) promote metastatic dissemination of human melanoma xenografts in mice

^1,2Elena Obrador, ¹Rosario Salvador-Palmer, ¹Rafael López-Blanch, ¹María Oriol-Caballo, ²Paz Moreno-Murciano, and ^1,2José M. Estrela

¹ Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, Valencia 46010, Spain

² Scientia BioTech S.L., 46002 Valencia, Spain

N-acetylcysteine (NAC) is a direct Cys donor and a promoter of glutathione (GSH) synthesis. GSH regulates melanoma growth and NAC has been suggested to increase melanoma metastases in mice. We found that high therapeutic doses of NAC do not increase the growth of melanoma xenografts but can cause metastatic spread and distant metastases. Nevertheless, this is not due to an antioxidant effect since NAC, in fact, increases the generation of reactive oxygen species in the growing metastatic melanoma. Trolox, an antioxidant vitamin E derivative, administered in vivo, decreased metastatic growth. Metastatic cells isolated from NAC-treated mice showed an increase in the nuclear translocation of Nrf2, as compared to controls. Nrf2, a master regulator of the antioxidant response, control the expression of different antioxidant enzymes and of the γ-glutamylcysteine ligase (the rate-limiting step in GSH synthesis). Cystine uptake through the xCT cystine-glutamate antiporter (generating intracellular Cys) and the γ-glutamylcysteine ligase activity are key to control metastatic growth. This associated to an increase in the utilization of L-Gln by the metastatic cells, another metastases promoter. Our results demonstrate the potential of NAC as an inducer of melanoma metastases spread and suggest that cautions should be taken when administering GSH promoters to cancer patients.

Supported by Scientia BioTech S.L. (Valencia, Spain), grant number SBT-001.

Keywords: cystine, glutathione, melanoma, metastases.

Corresponding author: José M. Estrela, https://orcid.org/ 0000-0002-2540-9190

O7-07

Long-lasting visceral hypersensitivity in a model of dextran sulfate sodium (DSS)-induced colitis in rats

^1,2Sergio López-Estévez, ¹Josep Manuel López-Torrellardona, ¹Marc Parera, *^1,2,3 Vicente Martínez

¹ Department of Cell Biology, Physiology and Immunology, Universidad Autónoma de Barcelona. Barcelona, Spain.

² Neuroscience Institute, Universidad Autónoma de Barcelona. Barcelona, Spain.

³ Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd). Instituto de Salud Carlos III, Madrid, Spain.

Persistent visceral hypersensitivity is a key component of functional and inflammatory gastrointestinal diseases. Current animal models fail to fully reproduce the characteristics of visceral pain in humans, particularly as it relates to persistent hypersensitivity. This work explores the validity of dextran sulfate sodium (DSS)-induced colitis in rats as a model to mimic chronic intestinal hypersensitivity. Exposure to DSS (5% for 7 days) was used to induce colitis in rats. Thereafter, changes in viscerosensitivity (visceromotor responses to colorectal distension -CRD-), the presence of somatic referred pain (mechanosensitivity of the hind paws, von Frey test) and the expression (qRT-PCR) of sensory-related markers (colon, lumbosacral DRGs and lumbosacral spinal cord) were assessed at different time points during the 35-day period after colitis induction. All animal procedures were approved by the Ethical Committee of the Universidad Autónoma de Barcelona (protocols 3958 and 3961). Following colitis, a sustained increase in visceromotor responses to CRD, lasting up to day 35 post-colitis, were observed, indicative of the presence of persistent visceral hypersensitivity. Responses in animals without colitis remained stable over time. In colitic animals, somatic referred hypersensitivity was also detected, although the responses observed were inconsistent. DSS-induced colitis was associated to a differential expression of sensory-related markers (with both pro- and anti-nociceptive action) in the colon, lumbosacral DRGs and lumbosacral spinal cord; thus, indicating the presence of peripheral and central sensitization. DSS-induced colitis in rats is associated to the generation of a long-lasting state of visceral (colonic) hypersensitivity. This model reproduces the changes in intestinal sensitivity characteristics of inflammatory and functional gastrointestinal disorders in humans and can be used in the characterization of new pharmacological treatments against visceral pain.

Keywords: hypersensitivity, intestinal inflammation, pain sensitization, visceral pain

Corresponding author: Vicente Martínez, https://orcid.org/0000-0001-5193-715X

Oral session 8: Neurophysiology & Cellular physiology II

O8-01

Role of quercetin and low-level laser therapy in chemotherapy-induced peripheral neuropathy

^*1Unay Simge, ^1,2Bilgin Mehmet Dincer

¹ Aydin Adnan Menderes University, Healthy Science Institute, Department of Biophysics, Aydin, Turkey

² Aydin Adnan Menderes University, School of Medicine, Department of Biophysics, Aydin, Turkey

Chemotherapy-induced peripheral neuropathy (CIPN) is caused by the side effects of chemotherapeutic drugs. Pharmacological agents and photobiostimulators are used in the treatment of chemotherapy-induced peripheral neuropathy to minimize neuronal loss resulting from cisplatin. However, these methods' mechanisms of action and efficacy are not clear and are being investigated. We investigated the effects of quercetin and low-level laser therapy (LLLT) in the PC12 cell line that created cisplatin-induced peripheral neuropathy. PC12 cell line was used. Cisplatin, quercetin, and low-level laser therapy were treated with PC12 cells. Assays on cell viability, induction of apoptosis, alterations in mitopotential, LDH, and RT-PCR analysis were performed. Our results indicated that cisplatin caused an increase in apoptosis, mitochondrial membrane potential, LDH activity, and a decrease in cell viability, expressions of GAP-43, and synapsin I gene in PC12 cells. Apoptosis, mitochondrial membrane potential, and LDH activity decreased when cisplatin with quercetin and cisplatin with low-level laser therapy was applied in PC12 cells. In this study, the appropriate doses of quercetin and low-level laser therapy may be used for the treatment of cisplatin-induced peripheral neuropathy through quercetin and low-level laser therapy affected cells. The results revealed the efficacy of applications of quercetin and low-level laser therapy in vitro cisplatin-induced peripheral neuropathy model.

The study was supported by the scientific and technological research council of Turkey (TUBITAK), No: 220S547, and Aydin Adnan Menderes University Scientific Research Projects Unit, No: TPF-21002. S. Unay thanks the Higher Education Council of Turkey (YOK) for 100/2000 Ph.D. Scholarship.

Keywords: Chemotherapy-induced peripheral neuropathy, cisplatin, low-level laser therapy, quercetin

Corresponding author: Unay Simge

O8-02

DREAM protein inhibition as a potential treatment against NAFLD and metabolic syndrome and its associated neurologic signs

¹José M Hernández Curiel, ¹Rocío Manzano González, ¹Ángel Carrión Rodríguez

¹ Universidad Pablo de Olavide, Physiology, Anatomy and Molecular Biology Department, Seville, Spain.

High fat diet (HFD) chronic intake induces metabolic syndrome in mice, characterized by a body weight increase and insulin resistance (IR). Furthermore, non-alcoholic fatty liver disease (NAFLD) is strongly linked to metabolic syndrome appearance. Obesity is a risk factor in the development of neurodegenerative diseases, neuropsychiatric disorders and is associated with cognitive decline. DREAM/kchip3/calsenilin (DREAM) is a multifunctional protein that belongs to the neuronal Ca2+ sensor family. Previous studies have demonstrated the relevance of DREAM in nociception and in learning and memory processes. In the present study we characterize the consequences of DREAM, genetic or pharmacologic, inhibition in the molecular (qPCR analysis), cellular (H-E histological assays), metabolic and behavioural alterations caused by HFD intake in mice. Our results showed that chronic pharmacological and genetic DREAM inhibition block the metabolic syndrome and NAFLD development, and both of its neurologic comorbidity symptoms: the anxiety-related behaviour, and the cognition deficiency. Therefore, in this study we demonstrate: DREAM inhibition may be a potential treatment to restore neurological symptoms related with metabolic syndrome; and to block metabolic syndrome and NAFLD induced by HFD intake.

Keywords: non-alcoholic fatty liver disease, high fat diet, DREAM.

Corresponding author: José Manuel Hernández Curiel

O8-03

The effects of curcumin on peripheral nerves in the statin-administered rats

^*1Fatih Sırıken, ¹Cengiz Ünsal

¹ Department of Physiology, Faculty of Veterinary Medicine, University of Aydın Adnan Menderes, Aydın, Turkey.

Cardiovascular diseases (CVDs) are the leading cause of mortality and about 1/3 of deaths worldwide occur due to CVDs. Statins are a class of cholesterol lowering drugs, commonly being used for the prevention or treatment of CVDs. Safe and tolerable long-term use of statins is important for chronic hypercholesterolemia. This study aimed to investigate the possible protective effects of curcumin in polyneuropathy, which is expected to be induced by the statin administration in rats. For this purpose, four groups were designed as statin group (40 mg/kg atorvastatin); statin (40 mg/kg atorvastatin) + curcumin (400mg/kg) group; curcumin (400 mg/kg) group; and control group (vehicle). Atorvastatin and curcumin were given orally for 30 days. At the end of the experiment, nerve conduction velocity (NCV), Na-K ATPase activity, total oxidant and antioxidant capacity, levels of TNF-α and IL-6 were measured. Atorvastatin led to a reduction in NCV, compared with the other groups (P<0.001). In addition, Na-K ATPase activity was lower in statin group than that of the control and curcumin groups (P=0.002). Atorvastatin increased TNF-α levels (P=0.014) but did not have effect on IL-6 levels (P=0.239). While the total oxidant capacity was not different between groups, the total antioxidant capacity increased in the atorvastatin+curcumin group (P=0.002) compared to the other groups. These findings suggest that statins may have the potential to induce polyneuropathy by decreasing Na-K ATPase activity. Importantly, curcumin administration ameliorates the reduced NCV and Na-K ATPase activity. Collectively, curcumin intake may have protective effects on peripheral neuropathy that might occur during statin treatment.

This study was supported by Aydın Adnan Menderes University Scientific Research Projects Unit (Project no: VTF 21020).

Keywords: Atorvastatin, curcumin, rat, nerve conduction velocity.

Corresponding author: Fatih Sırıken, ORCİD:0000-0001-5119-8772

O8-04

The steroid sulfatase inhibitor STX64 improves mice aging-associated deficiencies in cognition

^*1Juan A Fernández-Cabrera, ¹Inés Sánchez-Romero, ¹Ángel M Carrión-Rodríguez, ²Mercedes M Pérez-Jiménez, ¹Sara Esteban-García

¹ Department of Physiology, Anatomy and Cellular Biology, University Pablo de Olavide, Seville, Spain, and ² Department of Molecular Biology and Biochemical Engineering, University Pablo de Olavide, Centro Andaluz de Biología del Desarrollo (CABD), Seville, Spain

Aging produces in brain a progressive oxidative process and neuroinflammation that deteriorates cognition. These brain dysfunctions provoke high economic and social costs to developed societies. For this reason, the search for treatments to prevent cognitive deficits associated with aging is a priority. Previous results suggest that STX64, a steroid sulfatase (STS) inhibitor, improves cognition in neurodegenerative diseases. Here we have designed cellular (immunohistochemistry), molecular (qPCRs with primers of inflammatory markers and cellular populations) and behaviour (mainly, object recognition and passive avoidance for the study of learning and memory) assays to know if subchronic (6 months) oral treatment with STX64 (2mg/L in water from a concentrated stock of 5mg/mL in DMSO) rescues cognitive dysfunction present in aging mice. The mouse strain used in our studies was C57. Our behavioural results show that STX64 treatment improved cognition in aging mice, reflected as an improvement in object recognition and in passive avoidance tests. At cellular level, aging mice treated with STX64 showed an increase in microglial response and in hippocampal adult neurogenesis. By last, qPCR assays indicated that hippocampus from aging mice treated with STX64 express lower levels of inflammatory factors mRNA compared with untreated aging mice, though new studies are in progress to distinguish the microglial phenotypes of these animals using phenotype-specific primers. Then, our results indicates that STX64 may be a drug with potential therapeutic interest for the treatment of cognitive deficits associated to aging.

Key words: STX64, aging, hippocampus, neurogenesis.

Corresponding author: Juan A Fernández Cabrera, https://orcid.org/0000-0003-4133-4325

O8-05

Galanin and neuropeptide Y interaction enhances proliferation of granule precursor cells and expression of neuroprotective factors in the rat hippocampus: role in depression and memory

¹Marina Mirchandani-Duque, ¹Miguel A Barbancho, ¹Alexander López-Salas, ¹Jose E Alvarez-Contino, ¹Natalia García-Casares, ²Kjell Fuxe, ^1,2,3Dasiel O Borroto-Escuela, ^*1,2Manuel Narváez

¹ Instituto de Investigación Biomédica de Málaga, Facultad de Medicina, Universidad de Málaga, 29071 Malaga, Spain

² Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden

³ Department of Biomolecular Science, Section of Physiology, University of Urbino, 61029 Urbino, Italy

Neuropeptide Y(NPY) Y1 receptor (Y1R) and galanin (GAL) receptor 2 (GALR2) interact in brain regions responsible for mood control and learning and memory processes, emphasizing the hippocampus. The current study assesses the sustained memory performance and antidepressive-like effects induced by GALR2 and NPYY1R agonists coadministration and their neurochemical hippocampal correlates. Object-in-place task and forced swimming test were conducted together with in situ proximity ligation assay (PLA) to manifest the formation of GALR2/Y1R heteroreceptor complexes. We evaluated cell proliferation through a 5-Bromo-2’-deoxyuridine (BrdU) and PCNA expression study within the hippocampus. The GalR2 agonist M1145 and GAL were demonstrated to act with the Y1R agonist to improve memory retrieval and antidepressive-like actions at 24 hours in both tasks, enhancing the cell proliferation in the DG of the hippocampus through BrdU and PCNA expression and the GALR2/Y1R heteroreceptor complexes upon agonist coactivation. Our results may provide the basis for developing heterobivalent agonist pharmacophores targeting Y1R-GALR2 heterocomplexes. It involves especially the neuronal precursor cells of the dentate gyrus in the hippocampus for the novel treatment of Alzheimer’s disease or depression.

Supported by the UMA18-FEDERJA-100 and Proyecto Jovenes Investigadores (B1-2019_04) and Proyecto Puente (B4-2021) UMA, Spain to MN.

Keywords: Neuropeptide Y1 receptor, galanin 2 receptor, receptor–receptor interaction, reurogenesis.

Corresponding author: Manuel Narváez

O8-06

Role of insulin-growth factor II on mitochondrial recovery in a cellular model of Parkinson's disease

¹Nadia Valverde, ²Silvana Y Romero-Zerbo, ²Estrella Lara, ²Silvia Claros, ¹José Pavía, ¹Elisa Martín-Montañez, ^*2María García-Fernández

¹ Department of Pharmacology and Pediatrics, Faculty of Medicine, University of Málaga, Málaga, Spain, and

² Department of Human Phisiology, Faculty of Medicine, University of Málaga, Málaga, Spain

Insulin-growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Mitochondrial-oxidative damage along with accumulation and aggregation of abnormal proteins, have a critical role in pathoetiology of Parkinson´s disease (PD). ROS cause damage to cellular macromolecules such as DNA, lipids and proteins, affecting several cellular processes resulting in necrosis and apoptotic cell death. Our objective was to assess the IGF-II role in the recovery of the oxidative damage produced on mitochondrial structure and function in a cellular model of PD. SN4741 cell line was treated as follows: MPP⁺ alone, in presence of IGF-II or co-incubated with IGF-II and BMS (Ins/IGF-I receptors antagonist) or AB (anti-IGF-II-receptor). To assess the effect of IGF-II in the recovery of MPP⁺ damage, this treatment was removed after 2 h and replaced during another 2 h by medium, IGF-II or IGF-II + BMS or IGF-II + AB. Mitochondrial structure, localization and morphology was studied by western blot/ immunochemistry of Mitofilin and electron microscopy (EM); functionality by Mitotracker-Deep-Red^TM (MTR) and oxygen consumption rate (OCR) (Seahorse-analyzer). Cell death was analysed through annexin-V by confocal microscopy. In morphological and structural studies, MPP⁺ treated cells showed swollen mitochondria with partial/total loss of cristae, and electron-lucent matrix, inducing a mitochondrial number reduction. IGF-II retrieved normal-shaped mitochondria with intact cristae and outer/inner membranes. Moreover, treatment with MPP⁺ significantly reduced the expression levels of Mitofilin compared to the control group (Wb). This expression was found in areas where mitochondria did not exist or had a very weak mark (MTR), indicating mitochondrial destruction or dysfunction. IGF-II in coadministration, recovered the expression of Mitofilin (Wb), remaining associated with normal mitochondrial function (MTR). Additionally, the decrease in OCR levels after MPP⁺ administration (80%), was recovered in presence of IGF-II. IGF-II prevented MPP⁺ cell-death. In all the processes studied, IGF-II did not show significant differences vs control. The BMS-receptor blockage did not modify the IGF-II responses; and AB limited its effect. In conclusion IGF-II recovers mitochondrial structure and function due to MPP⁺ injuries. This improvement is carried out through the specific IGF-II receptor.

Supported by M.G-F. & L.J.S. Proyectos I+D+I-Programa Operativo-FEDER Andalucía 2014-2020 (UMA18-FEDERJA-004) Junta de Andalucía.

Keywords: Insulin-growth factor II, mitochondria, Parkinson disease, oxidative damage.

Corresponding author: María I. García Fernández, https://orcid.org/0000-0002-6436-1361.

O8-07

Hypoxia induces the activation of pancreatic stellate cells through modulation of antioxidant response and glycolytic metabolic shift

¹Matías Estarás, ¹Cándido Ortiz-Placin, ²Patricia Santofimia-Castaño, ¹Antonio González

¹ Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain; and ² Centre de Recherche en Cancérologie de Marseille, INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France.

Pancreatic stellate cells (PSCs) play a pivotal role in the fibrosis that develops in pancreatic diseases such as chronic pancreatitis or pancreatic cancer. In the tumour microenvironment, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O₂) on PSCs physiology was investigated. Oxidative stress markers were studied by fluorescence and spectrophotometric techniques and by high-performance liquid chromatography. The expression of antioxidant and metabolic enzymes was analysed by quantitative reverse transcription polymerase chain reaction and Western blot. For the studies of Oxidative Phosphorylation (OxPhos) and glycolytic activities, SeaHorse analysis was performed. Cell viability was studied by crystal violet. Cell migration was studied employing the wound healing assay. The expression of smooth muscle α-actin (α-sma) and collagen I was assayed by western blot and confocal analysis respectively. Lipid and protein oxidation was increased under low oxygen availability. In this pro-oxidant environment, PSCs exhibited activation of different elements of the antioxidant response, such as an increase of the antioxidant enzymes expression or the consumption of glutathione. Regarding energy metabolism adaptation, PSCs underwent a shift from mitochondrial to glycolytic metabolism. Under hypoxia, PSCs increased the proliferation, the migration, the expression of α-sma and collagen-I deposition. In conclusion, under hypoxia, PSCs undergo several adaptations of the antioxidant response and energy metabolism. These adaptations allow to PSCs to increase their activation state and promote the development of pathological fibrotic tissue.

Suported by Junta de Extremadura-FEDER (GR21037), Ministerio de Ciencia e Innovación (EQC2018-004646-P) and Valhondo Calaff Foundation.

Keywords: pancreatic stellate cells, hypoxia, fibrosis, pancreas

Corresponding author: Matías Estarás (meh@unex.es), https://orcid.org/0000-0003-3462-7904

O8-08

G protein-coupled estrogen receptor activation by Bisphenol-A disrupts protection from apoptosis conferred by estrogen receptors ERα and ERβ in pancreatic beta cells

^1,2Ignacio Babiloni-Chust, ^1,2Reinaldo S dos Santos, ^1,2Regla M Medina-Gali, ^1,2Atenea A Perez-Serna, ¹José Antonio Encinar, ^1,3Juan Martinez-Pinna, ^4,5Jan-Ake Gustafsson, ^1,2Laura Marroqui, ^*1,2Angel Nadal

¹ Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universitas Miguel Hernández, Elche, Spain; ² Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Spain; ³ Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain; ⁴ Department of Cell Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA; and ⁵ Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden

17β-estradiol protects pancreatic beta cells from apoptosis via the estrogen receptor α (ERα) and β (ERβ), and the G protein-coupled estrogen receptor (GPER/GPR30). Conversely, the endocrine disruptor Bisphenol-A (BPA), which exerts multiple effects in beta cells via the same estrogen receptors, increased basal apoptosis. We investigated the molecular initiated events underlying these opposite actions. Apoptosis was determined by nuclear dyes, flow cytometry and luminescence. Estrogen receptors were inhibited by small interfering RNAs. ERα and ERβ heterodimers were studied by molecular dynamics simulations, proximity ligand assay and co-immunoprecipitation. All experiments were performed in rat INS-1E and human EndoC-βH1 cells and dispersed islet cells from wild-type and ERβ knockout mice. BPA exposure induced reactive oxygen species production and induced apoptosis. GPER agonist G1 also induced apoptosis, but to a less extent than BPA. Either gene downregulation or pharmacological inhibition of each receptor resulted in augmented beta cell apoptosis in the absence of external stimuli. BPA- and G1-induced apoptosis was abolished in ERα-, ERβ- and GPER-silenced cells as well as in dispersed islet cells from ERβ knockout mice. Moreover, pharmacological blockade of each receptor also abrogated BPA- and G1-induced apoptosis. ERα and ERβ pharmacological agonists had no effect on beta cell viability. BPA interaction with ERα and ERβ as well as GPER activation by G1 or BPA decreased ERαβ heterodimerization. Our data suggest that ERα, ERβ and GPER play a key role in beta cell survival, where BPA-induced changes in GPER activation as well as in ERαβ heterodimer formation may increase beta cell apoptosis.

Experimental procedures were performed according to the Spanish Royal Decree 1201/2005 and the European Community Council directive 2010/63/EU. The ethical committee of Miguel Hernandez University reviewed and approved the methods used herein (approvals ID: UMH-IB-AN-01–14 and UMH-IB-AN-02-14).

Supported by: Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) PID2020-117294RB-I00, Generalitat Valenciana PROMETEO II/2020/006 and European Union’s Horizon 2020 research and innovation programme under grant agreement GOLIATH No. 82548.

Keywords: Apoptosis, beta-cells, estrogen receptors, endocrine disruptors

Corresponding author: Angel Nadal, nadal@umh.es, ORCID: 0000-0003-4178-2152

O8-09

Role of ND-13, a DJ-1/Nrf2 pathway activator, in the prevention of inflammasome activation in diabetic nephropathy

¹Virginia Celdran, ¹Maksym Kupchyk, ¹Adrian Luna-Garcia, ²Inmaculada Ros, ²Florentina Rosique, ²Laura Martinez-Alarcon, ²Leonor Andujar, ¹Cristina Molina, ¹Laura Hurtado, ^*1Santiago Cuevas

¹ BioMedical Research Institute of Murcia (IMIB-Arrixaca); and ² Clinical University Hospital Virgen de la Arrixaca, 30120, Murcia, Spain

Inflammasome is a crucial regulator of renal inflammation and a key factor in the pathogenesis of renal diseases. DJ-1 is a renal protein with antioxidant and anti-inflammatory properties with the capacity to prevent Nrf2 degradation. ND-13 is a peptide consisting of 13 highly conserved aminoacids from the DJ-1 sequence. In this study, we determined the capacity of ND-13/Nrf2 pathway to attenuate inflammasome activation. Mouse bone marrow macrophages (BMM) were treated with Bardoxolone, A Nrf2 inducer, and ND-13. Diabetes was induced in C57Bl/6 mice via injection of STZ and treated with ND-13. PBMCs were isolated from the blood of patients with diabetic nephropathy and controls and were plated and stimulated with LPS/ATP and treated with ND-13 and MCC950. The IL-1β concentration in the medium of BMM increased by NLRP3 inflammasome stimulation by LPS/ATP, and decreased in macrophages pre-treated with Bardoxolone (65.07±26%, n=4, P<0.05) but not pre-treated with ND-13, however, in presence of H₂O₂ (100nM), ND-13 significantly decreased IL-1β release after NLRP3 activation (88.6±1.2%, n=4, P<0.05). These data were confirmed by Bardoxolone and ND-13 concentration-response curve. Peritoneal macrophages from diabetic mice were obtained and plated. STZ treatment increased the IL-1β production compared with the control, suggesting that inflammasome may be activated in diabetes and ND-13 treatment normalized its activity. PBMCs isolated from the blood of patients were plate and stimulated with LPS/ATP, and patients with diabetic nephropathy presented a trend to increase IL-1β release compared to controls and diabetic individuals and ND-13 could have a role in the prevention of inflammasome activation. All these data point out that DJ-1/Nrf2 pathway stimulation is a promising approach to decreasing immune cells inflammasome activation, and ND-13 could be a new approach to attenuate inflammation in renal diseases.

Keywords: diabetic nephropathy, DJ-1, Nfr2, ND-13

Corresponding author: Santiago Cuevas, santiago.cuevas@imib.es

O8-10

Immunomodulation of pancreatic cells/macrophage interaction and its effect in a diabetes mellitus type 1 progression in a BB rat model.

^*1,2Mª Carmen González-Montelongo, ^1,2Francisco Martín-Loro, ^1,2Fátima Cano-Cano, ^1,2,4Laura Gómez-Jaramillo, ^1,3Belén Cuevas, ³Eva Zubía, ³Mª Jesús Ortega, ^1,2†Manuel Aguilar-Diosdado, ^*1,2†Ana I Arroba

¹ Research Unit, Instituto de Investigación e Innovación en Ciencias Biomédicas de la Provincia de Cádiz (INiBICA), University Hospital “Puerta del Mar”, Cádiz, Spain; ² Department of Endocrinology and Metabolism, Instituto de Investigación e Innovación en Ciencias Biomédicas de la Provincia de Cádiz (INiBICA), University Hospital “Puerta del Mar”, Cádiz, Spain; and ³ Departamento de Química Orgánica, Facultad de Química, Universidad de Cádiz, Cádiz, Spain.

^† These authors have contributed equally to this work.

Diabetes mellitus type 1 (DM1) is a chronic autoimmune disease characterized by the absence of endogenous insulin production and development of hyperglycemia. It is known to be caused by a combination of environmental and genetic factors that trigger the β-cell autoimmunity. The inflammatory context in which the disease and its complications develop is decisive for the progress of DM1. Previously, our group has shown the ability of macrophages to change polarization (phenotype) due to the action of various natural compounds. Bioactive extracts from algae, such as 3-arilftalidas have shown properties against diabetic retinopathy with an inflammatory component. Immunomodulatory effects of the 3-arilftalidas bioactive compound were evaluated on b-pancreatic cells (INS-1), macrophages cells (Raw264.7) and pancreatic islets from BB rats (autoimmune Diabetes Type 1 animal model). The aim of this work was to investigate the effects of the 3-arilftalidas on inflammation associated with early prediabetes stages by immunomodulation of specific immune systems and the possible role of systemic immune cells in DM1 progression. INS-1 and Raw264.7 cells were stimulated with cytokines or lipopolysaccharide (LPS), respectively, as an inflammatory stimulus and/or arilftalidas as anti-inflammatory inductor. The effects of conditioned medium (CM) from Raw264.7 on INS-1 cells were analysed to determine the immune system's contribution to DM1 progression and immuno-modulation. The pancreatic islet from BB rat, in a prediabetes status (normoglycaemia), at 7 weeks was cultured in the presence or absence of 3-arilftalidas, and with both CM from Raw264.7 cells. Pro/anti-inflammatory and signaling pathways were analyzed by qPCR and Western blotting. In the DM1 animal model, we found pancreatic inflammatory events that go before hyperglycemic detection. Raw264.7 under a diabetic environment and pancreatic islets from BB rats treated with 3-arilftalidas reduce the pro-inflammatory markers, such as Nos2, Il1b, Il6 and Tnfa expression. CM induced an immune differential response on INS-1 cells and pancreatic islet from wild type rats. The inflammatory processes that precede DM1 are able to be modulated by 3-arilftalidas, which exerts an anti-inflammatory effect on pancreatic islets and systemic immune cells that could be an effective alternative for DM1 treatment and/or prevention.

Supported by ITI-0012-2019; PI18-01287; ITI-0029-2017

Keywords: diabetes mellitus type 1, INS-1 cells, Raw264.7 cells, BB rat model

^*Corresponding authors: María del Carmen González-Montelongo, https://orcid.org/0000-0003-2332-6153; and Ana I. Arroba, https://orcid.org/0000-0002-5136-1725.

Oral session 9: Miscellaneous

O9-01

Chrono saliva: circadian changes in salivary protein profile

¹Elsa Lamy, ¹Carla Simões, ¹Laura Carreira, ¹Inês Caeiro, ^1,2 Fernando Capela e Silva

¹ MED – Mediterranean Institute for Agriculture, Environment and Development; IIFA – Instituto de Investigação e Formação Avançada; University of Evora, Evora, Portugal

² Medical and Health Sciences Department, School of Health and Human Development; University of Evora, Evora, Portugal

Saliva has different functions that go from the protection of the oral cavity to food-related functions, namely participating in ingestion and digestion and in the oral food perception. Besides this, saliva has gained popularity for its potential as a non-invasive source of biomarkers. However, as a biological fluid, its secretion and composition are also influenced by biologic clocks. This is particularly important when searching for salivary biomarkers, whose levels may not represent the same, at different hours of the day, but also highly important for the different roles of saliva, previously reported. Despite some reports about variations in some salivary molecules, through the day, the circadian variation of salivary proteome has been few studied. The present work aimed to assess the variations in salivary protein profile during individuals wake time. Six different individuals collected unstimulated saliva at 10 time points, from 7a.m. to 12p.m. This procedure was repeated, for each individual, in 3 different days, to assess intra-individual variation according to the day. SDS-PAGE was used to separate proteins according to molecular mass. Additionally, salivary amylase enzymatic activity and leptin levels were determined. The variations, through the day, in the proportion of the different salivary proteins is not the same. For example, salivary amylase variation seems to be dependent on intake episodes, but it increases after waking and returns to lower values during the evening. Other proteins, such as cystatins have their higher values at night. Despite these circadian variations, that will be detailed in the presentation, it is interesting to see that it still exists inter-individual variations and, for the same individual, variations from one day to another, reinforcing that different factors affect saliva proteome and highlighting the need to deeply study this body fluid.

Key words – Alpha-amylase, Circadian rhythm, Salivary proteome, Leptin

Corresponding author: Elsa Lamy, https://orcid.org/0000-0002-9370-1337

O9-02

Differences in extracellular and intracellular iron concentrations between athletic and physically inactive young people

¹Marcos Maynar-Mariño, ¹Diego Muñoz, ¹María C Robles-Gil, ^1,2Francisco J Grijota, ^1,3,4Ignacio Bartolomé, ^1,5Jesús Siquier-Coll, ¹Ángel García, ¹Sara Clemente, ^*1Víctor Toro-Román

¹ Exercise Physiology Lab, Faculty of Sports Sciences, University of Extremadura, Cáceres, Spain.

² Faculty of Life and Natural Sciences, University of Nebrija, Campus La Berzosa, Hoyo de Manzanares, Spain.

³ Faculty of Law and Social Science, Isabel I University, Burgos, Spain

⁴ Faculty of Law and Social Science, Avila Catholic University, Avila, Spain

⁵ SER Research Group. Center of Higher Education Alberta Giménez, affiliated to Comillas Pontificial University, Palma Mallorca, Spain.

Iron (Fe) is one of the most widely studied trace mineral elements. Fe metabolism and homeostasis could be altered by exercise and nutrition. Physical training could impair Fe status through several mechanisms, such as inflammation, hemolysis, or losses through sweat or urine. This study aimed to analyze the influence of physical training on serum, plasma, urine, erythrocyte, and platelet Fe concentrations. Forty men divided into a training group (TG; n=20; 18.15 ± 0.27 years) and a control group (CG; n = 20; 19.25 ± 0.39 years) participated in this study. The Biomedical Ethics Committee approved the protocol of the University of Extremadura (Cáceres, Spain) following the guidelines of the Helsinki ethical declaration, updated at the World Medical Assembly in Fortaleza (2013), for research involving human subjects (13/2021). The TG was composed of soccer players of the highest youth category who performed 10 hours of regular training sessions per week. The CG consisted of young people who did not follow any training routine and had not practiced any sport for at least the previous six months. The participants' anthropometry, body composition, nutritional intake, and cardiorespiratory capacity were assessed. Plasma, serum, urine, erythrocyte, and platelet samples of Fe were obtained and analyzed by inductively coupled plasma mass spectrometry. Samples were frozen at -80 °C until analysis. Intracellular values were expressed as absolute values and relative to the number of erythrocytes/platelets. The TG showed higher plasma and serum Fe concentrations (p<0.05). However, TG obtained lower concentrations, both in absolute and relative values, in erythrocytes and platelets compared to the CG (p<0.01). Regular physical training could influence Fe concentrations in extracellular and intracellular compartments.

This study has been partially subsidized by the Aid for Research Groups (GR21003) from the Regional Government of Extremadura (Department of Employment, Companies and Innovation), with a contribution from the European Union from the European Funds for Regional Development.

Keywords: erythrocytes, platelets, exercise, iron.

Corresponding author: Víctor Toro-Román, https://orcid.org/0000-0001-9607-1759

O9-03

Acute effect of exposure to high temperature on performance in the Wingate test

^*1Víctor Toro-Román, ¹Diego Muñoz, ¹María C Robles-Gil, ^1,2Francisco J Grijota, ^1,3,4Ignacio Bartolomé, ^1,5Jesús Siquier-Coll, ¹Ángel García, ¹Sara Clemente, ¹Marcos Maynar-Mariño

¹ Exercise Physiology Lab, Faculty of Sports Sciences, University of Extremadura, Cáceres, Spain.

² Faculty of Life and Natural Sciences, University of Nebrija, Campus La Berzosa, Hoyo de Manzanares, Spain.

³ Faculty of Law and Social Science, Isabel I University, Burgos, Spain

⁴ Faculty of Law and Social Science, Avila Catholic University, Avila, Spain

⁵ SER Research Group. Center of Higher Education Alberta Giménez, affiliated to Comillas Pontificial University, Palma Mallorca, Spain.

Warming up improves exercise performance due to possible biochemical or contractile adaptations of the muscle. Studies that have analyzed the acute effects of high temperature on performance have used maximum temperatures of 45ºC. However, the possible effects of extreme temperatures have not been investigated. Therefore, the study aimed to analyze the effect of high temperatures (100±2°C) on performance in a lactic anaerobic power test (Wingate test). A total of 21 subjects (19.76±1.22 years; 1.69±0.12 m; 67.89±11.78 kg) participated in the present study. The Biomedical Ethics Committee approved the protocol of the University of Extremadura (Cáceres, Spain) following the guidelines of the Helsinki ethical declaration, updated at the World Medical Assembly in Fortaleza (2013), for research involving human subjects. The study was divided into three days; a familiarization period where the participants performed the Wingate test and were introduced into the sauna at 80ºC monitored by electrocardiogram; a second day where the subjects performed the Wingate test in normothermia (20±2ºC), and a third day where they performed the Wingate test after a 15-minute exposure to high temperatures (100±2ºC) in sauna (Harvia C105S Logix Combi Control; 3-15 W; Finland) and relative humidity of 20%. Between days there was a difference of 48 hours. Heart rate, body temperature, subjective perception of exertion (RPE), revolutions per minute, and power were assessed. Regarding the differences between groups, after exposure to high temperatures, higher power values were obtained at 5" and 10" compared to the values obtained in normothermia (p<0.05). As for the differences throughout the test, there were differences concerning 5", 10", and 15" (p<0.05). Power losses were greater in the hyperthermia group (p<0.05). A 15 min exposure to high temperatures (100±2°C) prior to a lactic anaerobic power test improves performance during the first 10". However, it produces lower power values in the final stretch of the test.

This study has been partially subsidized by the Aid for Research Groups (GR21003) from the Regional Government of Extremadura (Department of Employment, Companies and Innovation), with a contribution from the European Union from the European Funds for Regional Development.

Keywords: Hyperthermia, anaerobic power, sauna.

Corresponding author: Víctor Toro-Román, https://orcid.org/0000-0001-9607-1759

O9-04

Effects of seawater consumption on IL-6 release after a triathlon event

^*1,2Jerónimo Aragón-Vela, ²Marta Palma Morales, ^1,2Jesus F Rodríguez Huertas, ²Rafael A Casuso

¹ Department of Physiology, Faculty of Sport Sciences, University of Granada, Granada, Spain; and ² Institute of Nutrition and Food Technology “José Mataix”, Biomedical Research Centre.

Triathlon is an endurance event in which athletes have limited access to fluid intake, as fluid intake is restricted at different stages. For this reason, an efficient hydration strategy must be established to avoid a significant negative impact on both aerobic performance and athlete health. Seawater intake now appears to be a suitable hydration alternative for this type of endurance event. Indeed, there seems to be a relationship between seawater consumption and IL-6 release, which could maintain energy status during exercise and improve post-exercise recovery. Therefore, the aim of this study was to evaluate the efficacy of hydration of seawater during a triathlon training under thermoneutral environmental conditions and its relationship with IL-6 response. Fifteen trained male triathletes (age = 38.8 ± 5.62 years old; BMI = 22.58 ± 2.51 kg/m²) randomly performed 3 triathlons, one of them consuming seawater containing 198 mg of chloride and 12 mg of magnesium per 10 ml (QUINTON HYPERTONIC 30 AB, Laboratories Quinton, France), the other one consuming tap water-hydration at libitum and the last one with isocaloric placebo. A triathlon training session consisting of an 800 m swim, 90 km bike ride and 10 km run was carried out. Blood samples were taken at rest- and post-exercise, where markers of inflammation were assessed. The most prominent results showed that post-exercise plasma IL-6 levels were significantly increased in the experimental group (p=<0.0001) compared to the control group. While the placebo group and the control group did not show any post-exercise increase. In conclusion, the release of IL-6 post-exercise by the consumption of seawater opens a new field of research. On the one hand, it should be tested whether chronic ingestion of seawater can be ergogenic through the improvement of the oxidative capacity of skeletal muscle and post-exercise recovery. On the other hand, it should also be tested whether subjects suffering from metabolic diseases could benefit from the anti-inflammatory effects of seawater.

This research was funded by Quinton (QUINTON HYPERTONIC 30 AB, Laboratories Quinton, France).

Keywords: Exercise physiology, endurance exercise, seawater, hydration.

Corresponding author: Jesus F. Rodriguez Huertas, https://orcid.org/0000-0002-7446-0857

O9-05

An interesting clinical case of a patient with autoimmune polyglandular syndrome type II (Schmidt’s syndrome)

¹Juan M Pacheco Bolaños, ²Elisa García Calvo, ³María García Duque, ¹Rodrigo Santos Santamarta, ⁴Ricardo I Cereceda García, ¹Yolanda Calvo Martin.

¹ Parque Alameda – Covaresa Health Center, Río Hortega University Hospital, Valladolid, Spain.

² Pintor Oliva Health Center, Río Carrión Hospital, Palencia, Spain.

³ Endocrinology Service, Río Hortega University Hospital, Valladolid, Spain.

⁴ Parquesol Health Center, Río Hortega University Hospital, Valladolid, Spain.

Autoimmune polyglandular syndrome type II (Schmidt’s syndrome) consists of Addison’s disease (primary autoimmune adrenal insufficiency), autoimmune thyroid disease (usually chronic autoimmune thyroiditis but occasionally Graves' disease) and type I diabetes mellitus. Approximately one-half of cases are familial and several modes of inheritance (autosomal recessive, autosomal dominant, and polygenic) have been reported. This syndrome is very rare and may occur in both sexes at any age of their lifetime, but it commonly affects middle-aged females. The prevalence is 1-2 per 100000 population. We are reporting a case of a twenty-five-year-old lady who was diagnosed with type I diabetes after developing diabetic ketoacidosis, furthermore she was diagnosed with autoimmune hypothyroidism with positive anti-TPO antibodies and anti-thyroglobulin antibodies. After that, she started treatment with insulin and levothyroxine with a good clinical response. Seven years later, at the age of thirty-two, she debuted with symptoms of asthenia, hypotension, skin hyperpigmentation and glycemic imbalances. Laboratory tests revealed a very low basal cortisol (<3 μg/dL) and a very high ACTH hormone (>1250 pg/ml). Antibodies against 21-alpha-hydroxylase may be elevated. Primary adrenal insufficiency was diagnosed in the context of type I diabetes and autoimmune hypothyroidism; all of this constitutes Schmidt’s syndrome. A great improvement was evidenced with treatment with glucocorticoids and mineralocorticoids (fludrocortisone and hydrocortisone). Adrenal insufficiency is the initial manifestation in about 50 percent of patients, it occurs simultaneously with autoimmune thyroid disease or diabetes mellitus in about 20 percent and follows them in about 30 percent. Primary hypogonadism can occur first, and ovarian failure is more frequent than testicular failure. Hypopituitarism due to autoimmune hypophysitis, preferentially causing ACTH deficiency. Other nonendocrine autoimmune disorders, such as vitiligo, myasthenia gravis, thrombocytopenic purpura, Sjögren's syndrome, rheumatoid arthritis, and primary antiphospholipid syndrome, occur occasionally. Hypoparathyroidism does not occur in this disorder. The objective of the work is to know this disease as well as to understand the physiology that determines its pathology. The patient is currently well controlled with the treatment and after an abortion, she has given birth to a healthy son, with the challenge that this represents in the context of this disease.

Keywords: autoimmune polyglandular syndrome type II, primary autoimmune adrenal insufficiency, autoimmune hypothyroidism, type I diabetes mellitus.

Corresponding author: Juan Miguel Pacheco Bolaños, ORCID: 0000-0002-2683-2855.

O9-06

Immunology response to lipids and the lipidic profile are prognosis biomarkers in COVID-19 patients

¹Isabel Sánchez-Vera, ¹Esther Escudero, ¹Úrsula Muñoz, ¹Ignacio Piédrola, ²Sara Martínez, ²Ana Sánchez, ²Ana Gradillas, ²Coral Barbas, ³Jose Felipe Varona, ³Andrés Ruiz, ³Jose María Castellano, ¹María Cruz Sádaba.

¹ Instituto de Medicina Molecular Aplicada (IMMA), Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities. Urbanización Montepríncipe, 28660 Boadilla del Monte, Madrid, Spain.

² Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte, Madrid, Spain.

³ Hospital Universitario HM Madrid. Urbanización Montepríncipe, Madrid, Spain.

Covid-19 pandemic is a major health crisis worldwide. The progression of the disease is heterogenous, some of the patients are asymptomatic, others develop mild or severe forms. The latter is related with lipid metabolic disorder and disseminated intravascular coagulopathy, associated with the presence of antibodies to lipids (aPLs). We aimed to stablish the relation between the lipid profile and the presence of aPL and the progression of the disease. Serum samples from COVID-19 patients with good (n=40) and poor (n=40) evolution and control group (n=40) were included. Lipid profile was analyzed using UHPLC-ESI-QTOF-MS. More than 200 phospholipids and glucolipids were identified. A high-sensitive ELISA assay developed in our lab was used to analyze IgG and IgM to phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol, phosphatidylserine, sphingomyelin, sulfatides, cardiolipin. We observed that COVID-19 patients showed increased levels of lysophosphatidylcholine (22:5/0:0) compared with control group. On the contrary, COVID-19 patients with a poor evolution showed decreased levels of phosphatidylinositol (16:2/24:2 and 16:2/24:1) than those with good evolution. In this line, patients with COVID-19 of good (0.440±0.038) and poor evolution (0.420±0.034; p<0.0001) COVID-19 patients showed higher IgM to phosphatidylcholine than control group (0.218±0.029; p<0.0001). Moreover, COVID-19 of good evolution showed higher levels of serum IgM to phosphatidylinositol (0.233±0.026; p<0.0001) than patients of poor evolution (0.128±0.026) and control group (0.103±0.028; p<0.0001). The humoral immune response is related with the lipidic profile in COVID-19. IgM to phosphatidylcholine is a main immune response to SARS-CoV-2 and IgM to phosphatidylinositol is essential in the defense to this virus. Further studies will carry out to define the role of them in the physiopathology of the disease and/or as biomarkers.

Funding by grant MCOV20V2 from Fundación Universitaria San Pablo CEU and NAWA project Poland. S.M. thanks CEINDO for a predoctoral fellowship grant FPI19/06206.

Keywords: COVID-19, SARS-CoV-2, lipidic profile

Corresponding author: Isabel Sánchez-Vera

O9-07

Increased glycosaminoglycans content induces changes in fibroblasts cell physiology in mucopolysaccharidosis patients.

^*1Alba Diaz-Pizarro, ¹Angel Carlos Roman, ¹Sonia Mulero-Navarro

¹ Department of Biochemistry, Molecular Biology and Genetics, Faculty of Science, University of Extremadura, Badajoz, Spain

Mucopolysaccharidoses are rare diseases in which there is an accumulation of glycosaminoglycans (GAGs) due to different defects in the lysosomal degradation of these molecules. GAGs are a class of polysaccharides that are present in all mammalian cells, and they are usually covalently attached to proteins, forming the proteoglycans. These molecules are present not only on the cellular membrane, but also in the intracellular milieu and extracellular matrix. In the last few years, it has been shown how GAGs can modify the extracellular matrix mechanical and molecular structure, having a profound role in multiple physiological and pathological processes. The objective of this study was to test if there were differences between the extracellular matrix secreted by human dermal fibroblasts from healthy people (control) and from patients suffering distinct types of mucopolysaccharidosis. Analysing the results, we showed that the changes caused by the accumulation of different GAGs led in all cases to a decrease of the cell adhesion and an increase of the proliferation, invasion and the detachment. These changes were concomitant to molecular alterations in the transcriptomic profiles of the cells derived from patients. Even though further studies are needed in which we will explore the molecular mechanisms underlying these changes, these results suggest that human dermal fibroblasts from patients that are suffering different mucopolysaccharidoses have altered properties regarding to important physiological processes for the cell biology.

Keywords: Mucopolysaccharidoses, glycosaminoglycans, fibroblasts

Corresponding author: Alba Diaz-Pizarro

O9-08

Non-invasive induction and electrographic detection of cortical spreading depolarization (CSD) using graphene micro-transistor arrays to investigate CSD induced inflammation

^*1,2Kağan Ağan, ^*1Rob C Wykes

• ¹Department of Clinical and Experimental Epilepsy, Queen Square Institute of Neurophysiology, University College of London, London, United Kingdom

² Experimental Animals Application and Research Centre, Düzce University, Düzce, Turkey

Spreading Depolarization (SD) is the largest disruption of brain homeostasis possible in living neural tissue. It is a slowly propagating wave of neuronal depolarization often associated with activity suppression of neuronal activity across the cortex at a velocity of 2–9 mm/min. Cortical spreading depolarization (CSD) is known to induces pro-inflammatory gene expression in brain tissue. However, many studies investigating this have used invasive mechanisms to either induce or electrographically record the CSDs, and or use anaesthesia, which can have indirect effects on inflammation. A recent paper demonstrated the ability to non-invasively induce CSD to more accurately determine the inflammatory response using optogenetics. We have previously shown that it is possible to both non-invasively optogenetically induce CSD and electrographically detect CSD through the skull using innovative graphene transistor arrays. Importantly this was performed in awake mice. We have an experimental platform available to directly correlate CSD frequency with the early inflammatory response in wild-type mice, and also in mouse models of familial hemiplegic migraine and ischemic stroke. For each model we have two groups, low CSD frequency, (2 optogenetially evoked CSDs in 2 Hrs), and high frequency (10 CSDs induced in 2hrs). 4 hours after induction of the 1st CSD, animals are sacrificed and their brains removed and perfusion fixed. Post-hoc immunohistochemistry is performed to determine expression levels of the following markers (MAP-2, IBA-1, Cox-2 and IL1β). We aim to determine in awake brain, without any confounders due to invasive induction or recording, the impact CSD frequency has on the early inflammatory response in 3 experimental groups. Results from this study will be presented and discussed.

Keywords: graphene transistor, in vivo, optogenetic

Corresponding authors: Kağan Ağan https://orcid.org/0000-0003-1472-570X; Rob C. Wykes, https://orcid.org/0000-0002-6141-6822

O9-09

Determination of antibacterial activity of propolis extract obtained from Amasya province of Turkey

Ceren Başkan¹, Belgin Sırıken^*2, Çetin Kılıç³, Fatih Sırıken⁴, Ayhan Güler⁵

¹ Amasya University, Sabuncuoğlu Şerefeddin Health Services Vocational School, Amasya, Türkiye.

² Ondokuz Mayis University, Faculty of Veterinary Medicine, Department of Aquatic Animal Diseases, Samsun,Türkiye.

³ Amasya University, Amasya Sabuncuoglu Serefeddin Education and Research Hospital, Amasya, Türkiye.

⁴ Aydın Adnan Menderes University Research Hospital, Clinic of Nutrition and Dietetics, Aydın, Türkiye.

⁵ Hakkari University, Faculty of Education, Department of Physical Education and Sports, Center, Hakkari, Türkiye.

Propolis is a mixture of substances used by bees to defend the hive. It exhibits a broad spectrum of biological and pharmacological properties such as antimicrobial, antioxidant, anti-inflammatory, immunomodulatory, antitumor, anticancer, antiulcer, hepatoprotective, cardioprotective and neuroprotective actions. Nosocomial infections caused by Staphylococcus aureus (S. aureus) are one of the important infections worldwide. In particular, antibiotic resistant S. aureus treatment is difficult, and it causes high infections with high mortality. Therefore, researchers have searched different alternative agents like bioactive compounds contained in natural products such as propolis for the treatment of the S. aureus and other microbial infections. Therefore, this study aimed to determine the antibacterial activity of propolis collected from Amasya province against clinical S. aureus isolates. For this aim, total of 30 clinical S. aureus isolates, which were previously isolated, were used in this study. Antibacterial activity of propolis was investigated using the disc diffusion method. Staphylococcus aureus ATCC 25923 was used as a reference strain. Gentamycin and DMSO were used as a negative control. As a result, propolis extract showed antibacterial activity against 29 of 30 (96.7%) isolates. In addition, it was observed that propolis extract had a higher inhibitory effect than the antibiotic used as the control group, especially against 5 isolates (16.66%). According to our findings, propolis extracts can be considered as an alternative antibacterial agent due to its potential to inhibit S. aureus clinical isolates.

Keywords: Antibacterial activity, disc diffusion, propolis, Staphylococcus aureus

Corresponding author: Belgin Sırıken, ORCID: 0000-0002-5793-1792

O9-10

Nutritional supplementation in premenstrual síndrome

¹Andrea Herrera, ²Miriam Al Adib, ¹Ana Beatriz Rodríguez, ¹Cristina Carrasco

¹ Neuroimmunophysiology and Chrononutrition Research Group. Faculty of Science. University of Extremadura. Badajoz, Spain

² Clínicas Miriam Gine. Obstetrics and gynecology clinic, Almendralejo. Badajoz, Spain.

Premenstrual syndrome (PMS) is a common disorder among women of reproductive age, being considered by the World Health Organisation as a public health problem in modern societies. It is characterised by the appearance of a series of symptoms that develop during the luteal phase of the menstrual cycle, disappearing at the onset of menstruation. To date, although its exact aetiology is unknown, it is considered to be a multifactorial disorder, influenced by different biological, psychological and social factors. There are various approaches to mitigating the symptoms, ranging from lifestyle corrective measures to pharmacological treatments − which are not completely effectives, expensive and have considerable side effects −. For this reason, more and more women with PMS are considering the food supplements as a therapeutic alternative, due to the scientific evidence of their beneficial effect. Therefore, the aim of this study was to validate the potential therapeutic use of the food supplement formulation for PMS PREMENCALM® (Naturlider) in women of reproductive age, analysing its impact on the characteristic symptoms and other aspects of the pathophysiology. Participants (N=39) were randomly divided into placebo group and PREMENCALM® group. During 3 complete menstrual cycles, participants took the assigned product once a day, about 30-60 minutes before bedtime. Baseline and final values were obtained before and after the treatment period, respectively. Blood and urine samples were obtained to assess levels of total antioxidant status, the serotonin metabolite 5-hydroxyindolacetic acid, C-reactive protein and IL-10. Other study variables included body composition, quality of life, subjective sleep quality, trait-state anxiety and symptomatology. The results showed that taking PREMENCALM® for 3 months resulted in a significant increase in antioxidant status and a significant decrease in mood-anxiety in the PREMENCALM® group compared to the placebo group. There was also a slight improvement in subjective sleep quality, quality of life and affective symptoms. To our knowledge, this is the first pilot study to demonstrate the efficacy of a food supplement formulated with a cocktail of active ingredients in the treatment of PMS. Based on the results obtained, it is thought that PREMENCALM® could be useful in the treatment of this gynaecological disorder.

Supported by Junta de Extremadura-FEDER (BBB021; GR21042) and financed by the Agreement UEx-Naturlider Import Export S.L.: Ref. 100/21.

Key words: nutritional supplementation, premenstrual syndrome, antioxidant status, quality of life.

Corresponding author: Andrea Herrera, https://orcid.org/0000-0001-7858-6847

POSTERS

Poster session 1

P1-01

Adrenergic effects of ranolazine in isolated rabbit aorta

¹Constanza Aldasoro, ¹Andrea Suarez, ¹Begoña Belmonte, ¹Solanye Guerra-Ojeda, ¹Patricia Marchio, ^1,2Adrian Jorda, ^*1Ignacio Campo-Palacio, ¹Antonio Iradi, ¹Soraya L Valles, ^#1Martin Aldasoro.

¹ Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain ² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

^* Presenter Author

Ranolazine is effective on heart failure and heart rhythm disorders. Its cardioprotective effects are mainly mediated by the inhibition of late sodium current, stabilizing cardiac electrical activity, and improving coronary blood flow. Ranolazine also reduces ischemia by improving endothelial function at therapeutic concentrations. Also, improves vascular function in coronary and rat renal arteries. The mechanisms involved are the enhancement of endothelium-dependent and independent vasodilation, the decrease in adrenergic tone and the inhibition of L-type Ca²⁺ channels. Rings of aorta isolated from 36 New Zealand rabbits were mounted in organ baths. Concentration-response curves to ranolazine were constructed in rings precontracted with noradrenaline, vasopressin and KCl. In rings precontracted with noradrenaline, relaxation to ranolazine was tested in the absence and presence of endothelial factors inhibitors, K⁺ channel blockers and Ca²⁺ channels. The effects of ranolazine on frequency-response electrical stimulation and on concentration-response curves to noradrenaline were performed in the absence and presence of endothelial factors inhibitors and K⁺ channel blockers. Endothelial nitric oxide synthase, α1, α2, β1, β2 and, β3 adrenergic receptors and large conductance Ca²⁺-activated K⁺ channel protein expressions were measured by Western blot technique. Ranolazine (10^-6-10^-5M) induced concentration-dependent relaxation only in rings precontracted with noradrenaline that was inhibited by, NG-nitro-l-arginine methyl ester (10^-4 M), charybdotoxin (10^-7 M) and verapamil (10^-6 M). Ranolazine diminished adrenergic contractions induced by electrical field stimulation (2-4 Hz) and nor-adrenaline (10^-9-10^-5 M) that were prevented by tetraethylammonium (10^-3 M) and charybdotoxin (10^-7 M). Ranolazine significantly increased β2 and β3, and decreased α1 and α2 adrenergic receptor expression. Ranolazine diminishes the adrenergic vasoconstriction, acting as α1 antagonist, and by increasing large conductance Ca^2+-activated K⁺ channel involvement. The relaxant effects of ranolazine are partially mediated by endothelial nitric oxide, endothelial PGI_2, large conductance Ca^2+-activated K⁺ channels and the blockade of voltage-dependent Ca²⁺ channels.

Keywords: α1 adrenergic receptor, β2 adrenergic receptor, ranolazine, sympathetic nervous stimulation.

Corresponding author: Ignacio Campo-Palacio, https://orcid.org/0000-0001-6630-0493

P1-02

Analysis of pro-inflammatory monocyte subsets and their inflammatory intermediates in an acute and ischemia/reperfusion myocardial infarction rat model

^#2Elisa Bevilacqua, ^#1,2Débora Falcon, ²Antonio Ordóñez-Fernández, ^1,2Tarik Smani, ^*1,2Raquel del Toro

¹ Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla. Seville. Spain.

² Cardiovascular Pathophysiology Group, Institute of Biomedicine of Seville- IBiS, University of Seville /HUVR/CSIC, Seville, Spain

^# These authors contributed equally.

Primary percutaneous transluminal coronary angioplasty (PTCA) is nowadays considered the best therapy to improve survival of patients after an acute myocardial infarction (AMI). Nevertheless, despite successful revascularization procedure, molecular alterations have been observed in the myocardium which trigger ventricular adverse remodeling. This remodeling involves inflammatory cell infiltration, cardiomyocytes apoptosis and interstitial fibrosis. We used two different surgical procedures, PTCA (ischemia-reperfusion, I/R) and AMI in adult rats, to study inflammatory cell infiltration and secretion of inflammatory intermediates within the myocardium after the ischemic event. Moreover, we analyzed whether these molecules change the phenotype of the resident cells upon an injury in the heart. For that purpose, we performed FACS analysis of neutrophils and monocytes in peripheral blood, bone marrow and spleen. Subsequently, we performed IHC (CD68) to detect pro-inflammatory M1 macrophages in the rat myocardium. Finally, we used an in-vitro model to study how inflammatory intermediates influences myocardium cell populations transcriptomics. We analyzed 3 inflammatory cell populations including neutrophils and two monocyte subsets based on the cell surface expression of CD43 and His48. CD43^lowHis48^high (classical monocytes) the most prevalent subset and CD43^highHis48^low (non-classical monocytes) which are the most pro-inflammatory subset. FACS analysis of neutrophils and monocytes showed an increased level of these cells in both injury models until 2 weeks post-ischemic event. First there are a high increase of neutrophils followed by an increase of the two monocytes subsets. We also studied the extra-hematopoiesis of these populations in the spleen, beyond the bone marrow. We observed a significant increase of CD43^lowHis48^high population in the spleen one week after the ischemic event. In parallel the infiltration of M1 macrophages was increased in the myocardium until 4 weeks, indicating a long extravasation of inflammatory cells after the ischemic event. On the other hand, fibroblast and cardiomyocytes co-culture with inflammatory intermediates increase the expression of fibrotic and apoptotic genes. In conclusion, the level of inflammatory cell populations is increased in IAM and I/R rat models. Moreover, these cells infiltrate in the myocardium and change the transcriptome of the resident cells towards a pro-apoptotic and pro-fibrotic phenotype.

Keywords: Inflammatory cells, myocardial infarction, ventricular adverse remodeling, ischemia/ reperfusion injury.

Corresponding author: Raquel del Toro, https://orcid.org/0000-0002-1379-7990

P1-03

Cuneiform nucleus stimulation modifies laryngeal activity and subglottic pressure in rats

^1,3L Carrillo-Franco, ^1,2,3MV Lopez-Gonzalez, ^1,2,3M Gonzalez-Garcia, ¹C Casermeiro-Garcia, ^1,2,3A Díaz-Casares, ^1,2,3MS Dawid-Milner

¹ Department of Human Physiology, Faculty of Medicine, University of Malaga, Malaga (Spain)

² Unit of Neurophysiology of the Autonomic Nervous System (CIMES), University of Malaga, Málaga (Spain)

³ Biomedical Research Institute of Malaga (IBIMA)

The Cuneiform nucleus (CnF) of the mesencephalon has afferent and efferent connections with different regions of the CNS involved in cardiorespiratory control, i.e. dorsolateral part of the Periaqueductal Gray matter (dlPAG) and Parabrachial complex (PBc). In recent studies, we have characterized functional interactions between all these hypothalamic (DMH-PeF), mesencephalic (dlPAG) and pontine regions (PBc, A5 region) that are also involved in changes of laryngeal caliber, due to the abduction and adduction of the vocal folds controlled by motoneurons located in the caudal portion of the nucleus Ambiguus (Amb). The aim of this study was to test the possible role of the CnF in laryngeal control and its effect on vocalization, and to characterize the electrophysiological relationships between the CnF and those pontine-medullary neuronal circuits involved in cardiorespiratory control and in changes of laryngeal caliber. Experimental studies were carried out with non-inbred male rats (n=14), SPF, Sprague-Dawley (250-300 g) housed under standard conditions. Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/kg, i.v., as necessary). A double up and down tracheal cannulation was performed to measure subglottic pressure and airflow. Subglottic pressure was recorded with an aneroid transducer (Hugo Sachs Elektronik D-7801, ±0,1 psi) by passing a stream of humidified warm medical air upwards with a thermal mass digital air flow meter controller (Bronkhorst Hi-Tec F-201CV-AGD-22-V). Bilateral parietostomy allowed access to CnF. Microinjections of PBS-Evans Blue (250 nl, pH 7.4 ± 0.1, 5-s duration) or glutamate (0,25M, 250 nl) were performed. Respiratory flow, pleural pressure, blood pressure and heart rate were also recorded. PBS-Evans Blue microinjections within CnF did not produce any significant changes in any of the cardiorespiratory variables recorded. However, glutamate microinjections within the CnF evoked a decrease of laryngeal resistance (subglottal pressure) (p<0,001) accompanied with an inspiratory facilitatory response consisted of an increase in respiratory rate (p<0,001), together with a pressor (p<0,01) and tachycardic response (p<0,001). The results of our study contribute with new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.

Supported by Junta de Andalucía (CTS-156).

Keywords: Subglottic pressure, laryngeal motoneurons, CnF, nucleus ambiguus

Corresponding author: L. Carrillo-Franco (laura_carrillo@uma.es)

P1-04

Renal vascular disfunction induced by endoplasmic reticulum stress

^*Cristina Contreras, Alfonso Gómez del Val, Mercedes Muñoz, Claudia Rodríguez, Ana Sánchez, Marta Guixe, Vitor Samuel L Fernandes, Sara Benedito, Luis Rivera, ^*Dolores Prieto

Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense de Madrid, Spain

The endoplasmic reticulum (ER) is wherein protein synthesis, maturation, and folding take place, as well as lipid synthesis and calcium reservoir. Any condition that alters ER homeostasis leads to lose functionality, accumulating misfolded proteins, which triggers signalling pathways to solve this stress situation (Yoshida, FEBS J, 2007). ER stress (ERS) has been related to metabolic disorders such as obesity and diabetes (Kaur, Cardiol Res Pract, 2014), affecting vascular function through reduced NO bioavailability, inflammation and reactive oxygen species (ROS) production leading to endothelial dysfunction (Kwasniewska, Diabetol Metab Syndrom, 2015). We have demonstrated the differential implication of NADPH oxidases (Nox) as sources of ROS production in renal arteries. While both Nox2 and mitochondrial Nox4 at the endothelium generate vasodilator hydrogen peroxide (H₂O₂) under physiological conditions, Nox1 and Nox2 are up-regulated and responsible for oxidative stress in metabolic syndrome (Muñoz, Redox Biol, 2020). The aim of this study was to assess the implication of ERS in vascular dysfunction of renal preglomerular arteries and the molecular mechanism involved. To induce ERS, male Wistar rats were treated with vehicle or 0,30 mg tunycamicin/Kg i.p. 3 times each 3 days before the sacrifice. Renal arteries were dissected and mounted in microvascular myographs to assess vascular reactivity in response to ERS. We observed that ERS impaired NO-mediated relaxant response to acetylcholine (Ach), while nitric oxide (NO)- and prostanoid-independent EDH-type relaxations were preserved or augmented. Furthermore, vasoconstrictor responses to phenylephrine were significantly reduced in ERS. About the role of oxidative stress in the impaired endothelial relaxations of renal arteries from ERS rats, relaxant responses to Ach were blunted by the mitochondria-targeted superoxide (O₂^.-) scavenger mito-TEMPO (0,1 mM) in control, but not in ERS arteries. Treatment with the selective NADPH oxidase inhibitor apocynin (30 mM) did not change renal Ach relaxations neither in controls nor in the ERS model. Basal H₂O₂ levels were reduced in ERS renal arteries and cortex, while Nox-derived O₂^.- was inhibited by apocynin in control but not in ERS arteries. These results demonstrate endothelial dysfunction associated to ERS in renal arteries by impaired ROS metabolism, reduced vasodilator H₂O₂, that blunts endothelium dependent vasodilatation.

Keywords: Endoplasmic reticulum stress (ERS), oxidative stress, renal arteries.

*Corresponding authors: Dolores Prieto, http://orcid.org/0000-0001-7049-5991; Cristina Contreras, http://orcid.org/0000-0002-7015-7922.

P1-05

Locally advanced non-small cell lung cancer and cardiovascular events

¹Carmen Corral Fernández; ²José M Gimeno Montes, ¹María González de Dueñas; ¹Beatriz Baños Pérez; ¹Esther Agudo Rey; ¹Victoria Vera Barragán; ¹Yesika Ríos Kavadoy; ¹Joaquín J Cabrera Rodríguez; ¹Juan Quirós Rivero; ¹María F Ropero Carmona; ¹Julia L Muñoz García.

¹ Service of Radiation Oncology, Universitary Hospital of Badajoz, Spain, and

² Service of Cardiology, Universitary Hospital of Badajoz, Spain.

Worldwide, lung cancer is the leading cancer in incidence and mortality. The objective of this study was to prove if the incidence of cardiovascular events (CVE) and oncological results in patients with locally advanced non-small cell lung cancer (NSCLC) treated with chemotherapy and radiotherapy was related with the dose of radiotherapy received by the heart and cardiovascular risk factors. For this study, we examined a retrospective analysis of 160 patients with locally advanced NSCLC treated, in Radiation Oncology Service Badajoz, between 2009 and 2017 with 3D-radiotherapy (total dose: 60-74Gy, dose per fraction: 2Gy) and chemotherapy. The mean age was 67 years (40-84). Male sex 93.5%. Squamous cell carcinoma was the most frequent histology (50%). The cardiac dosimetric parameters (V5C, V30C), pulmonary (mean dose and V20P); the cardiovascular risk factors, included SCORE and cardiovascular events was recorded in each patient. Statistical analysis was actuarial survival and multivariate analysis of the relationship between the variables under study and CVE and survival rates. The results of the most outstanding analysis were: 3 years Overall Survival (OS) and Disease-Free Survival (DFS) were 19.5% and 15.3% respectively. The median V20 pulmonary was 22.75, mean dose pulmonary 14.58, V30C 9.5 and V5C 44.75, and the most frequent CVE were arrhythmia (10.6%) and acute coronary syndrome (7.5%). The multivariate analyses between cardiac dosimetric parameters and the toxicity observed, shown statistical relationship between V30C and CVE (p 0,013). 10.75% was the limit of V30C from which the risk of CVE. According with the results of other publications where is confirmed the relationship between cardiac toxicity and 3D-RTE, our results shown a statistical relationship between V30C>10,75% and the the risk CVE. So we believe that V30C should be a dosimetric limit to be taken into account to reduce the cardiac toxicity.

Keywords: Non-small cell lung cancer, cardiovascular events, radiotherapy, dosimetric limit.

Corresponding author: Carmen Corral Fernández.

P1-06

Supplementation with two new extracts of white tea or black and green tea improves endothelial function and prevents the development of hypertension in mice with metabolic syndrome

^*1M De la Fuente-Muñoz, ¹M de la Fuente-Fernández, ¹M Román-Carmena, ¹D Sanz-Fuentes, ¹A Núñez-Manzano, ²AM Inarejos-García, ¹MC Iglesias-de la Cruz, ¹AL García-Villalón, ¹M Granado

¹ Department of Physiology, Faculty of Medicine, Universidad Autónoma de Madrid

² Department of Functional Extracts, Health & Wellness, ADM® Valencia

Metabolic syndrome is highly prevalent in the population and is one of the main risk factors for the development of metabolic and cardiovascular diseases, which has prompted the search for new preventive and therapeutic strategies, ideally of natural origin and with less side effects than conventional pharmacological treatments. Among these, tea consumption has shown beneficial health effects due to its composition of bioactive substances, mainly flavan-3-ols and xanthines. Thus, the aim of this study was to analyze the possible beneficial effects of two new tea extracts, one of white tea (ADM® White Tea Powdered Flavanols - Xanthines: WT) and the other composed by a proprietary blend of black and green tea (ADM® Tea Complex; TC), on metabolic syndrome-induced alterations in vascular function and hypertension developement. For this purpose, C57BL/6J mice were fed for 20 weeks either with a standard diet (Controls) or with a diet rich in fat and sugars (HFHS), supplemented or not with 1.6% of white tea extract (HFHS+WT) or 1.6% Tea Complex (HFHS+TC). During the treatment period blood pressure was measured and after the sacrifice, vascular reactivity experiments were performed on aortic segments using the organ bath technique. The gene expression of the main markers of inflammation and oxidative stress was analyzed by real-time PCR. Finally, to determine the basal production of vascular superoxide anion radical we used dihydroethidium staining. The results showed that supplementation with both extracts reduced the development of hypertension in obese mice and improved endothelial dysfunction in aortic segments, showing an increased vasodilator response to acetylcholine, increased eNOS expression, decreased expression of pro-inflammatory cytokines such as IL-6, IL-1β, TNF-α and MCP-1, and increased expression of antioxidant enzymes such as SOD-1, GPX3 and GSR, together with a reduction in vascular superoxide anion radical production. In conclusion, the supplementation of obese mice with White Tea Powdered Flavanols - Xanthines or Tea Complex improves endothelial function and prevents the development of hypertension in obese mice possible through their anti-inflammatory and antioxidant effects.

Keywords: metabolic syndrome, tea extracts, endothelial dysfunction, hypertension.

Corresponding author: M. De la Fuente-Muñoz.

P1-07

Takotsubo syndrome or stress cardiomyopathy in cancer disease

^*1José M Gimeno Montes, ²Carmen Corral Fernández, ¹Miguel Sánchez Sánchez, ¹Javier Corral Macias, ¹Rosa Navarro Romero, ¹Natalia Torrijos López, ¹Ramón E Rubí Matamoros¹, ¹José M Rojo Pérez, ¹Clara Nuevo Gallardo, ¹Javier Pérez Cervera, ¹Estrella Suárez Corchuelo, ¹Juan M Nogales Asensio, ¹María R González Fernández, ¹José R López Minguez

¹ Service of Cardiology, Universitary Hospital of Badajoz, Spain

² Service of Radiation Oncology, Universitary Hospital of Badajoz, Spain.

Stress cardiomyopathy is characterized by acute left ventricular dysfunction characterized by apical hypokinesia frecuently reversible. It was called Takotsubo because that is the name of a convex vessel with a narrow neck, traditionally used to fish for octopus. A triggering event, either emotional or physical, is usually identified that causes an excess of catecholamines. Although the precise pathophysiological mechanisms are incompletely understood, is postulated that the catecholamines and endothelin exert their vasoconstrictor effects primarily in the coronary microvasculature where α₁-receptors and endothelin receptor type A predominate, and also, catecholamine toxicity on cardiomyocytes due to catecholamines can decrease myocyte viability through cyclic adenosine monophosphate (cAMP) mediated Ca²⁺ overload. This syndrome is important because it is characterized by being a great simulator of others pathologies cardiacs. We propose a clinical case of a patient who, a few days to being informed that she had breast cancer, started with chest pain for which she consulted in the emergency service. The following alterations were found in the complementary tests: on the electrocardiogram, we looked sinus rhythm with negatives T waves in precordial leads; on the echocardiogram preserved left ventricular ejection fraction but hypokinesia of the apex; and elevation of myocardial damage enzymes on the analytics. With all that, the principal suspicion was acute coronary syndrome due to critical obstruction of the left anterior descending artery. So that was started treatment with antiplatelet, anticoagulant, and antianginal agents, and finally was performed an cardiac catheterization, that no showing lesions coronary. Next, the ventriculography helped to confirm the diagnosis, visualizing typical morphology Takotsubo. Subsequently, prior medication was discontinued and beta-blockers were introduced. She presented favorable evolution with disappeared all the previous alterations, presenting normality of the complementary cardiological tests and being asymptomatic without new episodes of chest pain.

Keywords: Takotsubo, stress cardiomyopathy, catecholamines, acute coronary syndrome.

Corresponding author: José María Gimeno Montes.

P1-08

Cardiovascular toxicity associated with ibrutinib

^*1José M Gimeno Montes, ²Carmen Corral Fernández, ¹Miguel Sánchez Sánchez, ¹Javier Corral Macias, ¹Rosa Navarro Romero, ¹Natalia Torrijos López, ¹Ramón E Rubí Matamoros¹, ¹José M Rojo Pérez, ¹Clara Nuevo Gallardo, ¹Javier Pérez Cervera, ¹Estrella Suárez Corchuelo, ¹Juan M Nogales Asensio, ¹María R González Fernández, ¹José R López Minguez

¹ Service of Cardiology, Universitary Hospital of Badajoz, Spain

² Service of Radiation Oncology, Universitary Hospital of Badajoz, Spain.

Ibrutinib is a Bruton's tyrosine kinase inhibitor drug that has shown great efficacy against chronic lymphatic leukemia and other B-cell tumors. But the recent appearance in the literature of cardiovascular toxicity associated, so that the objective of this study was to verify the incidence of cardiovascular events due to ibrutinib in the Universitary Hospital of Badajoz. For this, we developed a retrospective review of 41 patients who have received treatment with ibrutinib. We got these of their digital clinical history, and we picked up the baseline characteristics of the patients (mainly the cardiovascular risk factors) and the appearance of cardiovascular toxicity described in the literature (atrial fibrillation, major or minor bleeding, hypertension, heart failure, ventricular tachycardia, atrioventricular block and death). With a mean age of 68.4 years, with a predominance of males (58%). The appearance of a total of 8 cardiovascular events is observed, being four cases (9.75%) of atrial fibrillation in an average of 3.2 months, three cases (7.32%) of arterial hypertension in 2,7 months on average and one case (2.4%) of sudden death after 17 days. Overall, the mean survival in patients who have suffered cardiovascular toxicity (5.6 months) is lower compared to those who have not (16 months), with a p value of 0.125. In addition, we did not get an increase in bleeding events in patients with ibrutinib who developed atrial fibrillation. This is important because this drug has an antiplatelet effect, that together with the need for anticoagulation this pathology, there was fear of major bleeding events. This result is probably due to the better safety profile of the new direct oral anticoagulants (DOACs). We can conclude that the appearance of cardiovascular events was not infrequent in the population that received ibrutinib in our hospital. Only one serious event (sudden death) was observed, the rest being easy to control by the clinician, but assuming the appearance of these, a reduction in survival was not statistically significant, probably due to the small sample size.

Keywords: Ibrutinib, cardiovascular events, atrial fibrillation, arterial hypertension.

Corresponding author: José María Gimeno Montes.

P1-09

Endoplasmic reticulum stress (ERS)-induced endothelial dysfunction in penile arteries

Alfonso Gómez del Val, Cristina Contreras, Marta Guixé, Mercedes Muñoz, ^*Ana Sánchez, ^*Dolores Prieto

Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense de Madrid, Spain

Endoplasmic reticulum (ER) is involved in Ca²⁺ storage, lipid and glucose metabolism, and signal transduction, and reacts to biological stresses via an ER stress (ERS) response consisting of increased production of unfolded proteins (UPR). Metabolic syndrome, a constellation of metabolic abnormalities including central obesity, hyperglycemia, dyslipidemia, and hypertension, increases the risk of cardiovascular diseases, and has been linked to vascular endothelial dysfunction and recently to the development of ERS. Erectile function is primarily a vascular event that relies on vasodilation largely due to both nerve- and endothelium-derived nitric oxide (NO), these mechanisms being impaired in insulin resistant obese rats. We believe that ER stress is a key factor in the endothelium dysfunction of penile arteries. The aim of this study was to elucidate the mechanisms by which oxidative stress and ERS impair penile vascular function in a rat model of tunicamycin-induced ERS. Endothelial function was assessed in vitro in isolated rat dorsal penile arteries mounted in microvascular myographs and the effects of the mitochondrial antioxidant mitoTEMPO (30 nM) and the NADPH oxidase (Nox) inhibitor apocynin (10 μM) were investigated on the endothelium-dependent relaxations to acetylcholine (Ach). Extracellular acidification rate (ECAR) was quantified in isolated penile arteries by using a SeaHorse XF extracellular flux analyser. Vasodilation induced by Ach in penile arteries was significantly reduced in tunicamycin-treated rats compared to controls indicating endothelial dysfunction. Ex vivo treatment with apocynin or mitoTEMPO ameliorated endothelial dysfunction and restored endothelium-dependent relaxations in penile arteries from ERS rats. Plasma lactate levels were elevated and ECAR measurements in intact penile arteries showed a significant increase in glycolytic acidification in rats with ERS induced by tunicamycin. Our results demonstrate that chemically-induced ERS leads to Nox- and mitochondria- derived oxidative stress causing endothelial dysfunction associated to metabolic impairment of penile arteries. Inhibition of ERS could be a therapeutic target to ameliorate penile endothelial dysfunction and thus erectile dysfunction in metabolic disease.

Supported by PID2019-105689RB-I00.

Keywords: Endoplasmic reticulum stress (ERS), endothelium, oxidative stress, penile arteries

^*Corresponding authors: Dolores Prieto, https://orcid.org/0000-0001-7049-5991; Ana A. Sánchez Pina, https://orcid.org/0000-0002-8432-8702

P1-10

Impact of dorsolateral periaqueductal grey matter stimulation in laryngeal activity and subglottic pressure in rats

^1,2,3M Gonzalez-Garcia, ^*1,2,3MV Lopez-Gonzalez, ^1,3L Carrillo-Franco^1,3, ¹C Casermeiro-Garcia¹, ^1,2,3A Diaz-Casares, ^1,2,3MS Dawid-Milner

¹ Department of Human Physiology, Faculty of Medicine, University of Malaga, Malaga (Spain)

² Unit of Neurophysiology of the Autonomic Nervous System (CIMES), University of Malaga, Málaga (Spain)

³ Biomedical Research Institute of Malaga (IBIMA)

The stimulation of the dorsolateral Periaqueductal Gray matter (dlPAG) produces an increase in sympathetic tone that includes an increase in cardiorespiratory parameters (blood pressure, heart rate and respiratory frequency). PAG and nucleus retroambiguus (nRA) are necessary to produce vocalization. The nRA modifies the activity of laryngeal motoneurons located in the nucleus ambiguus. Rostral and ventral pontine structures involved in cardiorespiratory control, Parabrachial complex (PBc) and A5 Region (A5) are also involved in changes of laryngeal caliber. A high expression of FOXP2 protein, a transcription factor closely related to vocalization, at the level of the PAG, PBc and A5 has been shown. The aim of this study was to characterize the relations between mesencephalic regions (dlPAG) involved in cardiorespiratory control and their possible role in modulating laryngeal activity. Experimental studies were carried out with non-inbred male rats (n=14), SPF, Sprague-Dawley (250-300 g) housed under standard conditions. Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/kg, i.v., as necessary). A double tracheal cannulation for the recording of respiratory airflow and subglottic pressure was carried out. Subglottic pressure was recorded with an aneroid transducer (Hugo Sachs Elektronik D-7801, ± 0,1 psi) by passing a stream of humidified warm medical air upwards through the larynx at a constant rate of 30-70 ml/min with a thermal mass digital air flow meter controller (Bronkhorst Hi-Tec F-201CV-AGD-22-V). Thus, at constant air flow, changes in pressure indicate changes in laryngeal resistance. Microinjections of PBS-Evans Blue (250 nl, pH 7.4 ± 0.1, 5-s duration) or glutamate (0,25M, 250 nl) were performed within dlPAG. Respiratory flow, pleural pressure, blood pressure, heart rate and ECG activity were also recorded. Microinjection of PBS-Evans Blue within dlPAG did not produce any significant cardiorespiratory changes. However, glutamate microinjections within the dlPAG evoked a decrease of laryngeal resistance (p<0,001) accompanied with an increase in respiratory rate (p<0,001) together with a pressor (p<0,001) and tachycardic response (p<0,001). The results of our study contribute with new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.

Supported by Junta de Andalucía (CTS-156)

Keywords: Subglottic pressure, laryngeal motoneurons, dlPAG, nucleus ambiguus

Corresponding author: MV López-González, https://orcid.org/0000-0001-9023-3710

P1-11

A nutraceutical product based on a mixture of algae and extra virgin olive oils and olive leaf extract attenuates sepsis-induced cardiovascular and muscle alterations in rats

^1,3Daniel González-Hedström, ²Álvaro Moreno-Rupérez, ¹María de la Fuente-Fernández, ¹Mario de la Fuente-Muñoz, ¹Marta Román-Carmena, ¹Sara Amor, ¹Angel L García-Villalón, ²Asunción López-Calderón, ²Ana I Martín, ²Teresa Priego, ^*1,4Miriam Granado

¹ Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain.

² Departamento de Fisiología, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.

³ R&D Department, Pharmactive Biotech Products S.L.U. Parque Científico de Madrid. Avenida del Doctor Severo Ochoa, 37 Local 4J, 28108 Alcobendas, Madrid

⁴ CIBER Fisiopatología de la Obesidad y Nutrición. Instituto de Salud Carlos III, Madrid.

Nutraceuticals are products of natural origin widely used for the treatment and/or prevention of highly prevalent chronic diseases. However, its possible use in the prevention of acute life-threatening diseases has been poorly studied. Sepsis causes cardiovascular and skeletal muscle damage due to a systemic inflammatory state. Its incidence has increased in recent years and is associated with a moderate mortality rate, which makes necessary the search of preventive strategies that help to reduce its impact on public health. The aim of this work was to evaluate the possible beneficial effect of a new nutraceutical based on a mixture of algae oil (AO) and extra virgin olive oil (EVOO) supplemented with an olive leaf extract (OLE) in the prevention of cardiovascular and skeletal muscle alterations induced by sepsis in rats. For this purpose, male Wistar rats were treated with the nutraceutical or with water p.o. for 3 weeks and after the treatment they were injected with 1mg/kg LPS twice (12 and 4 hours before sacrifice). Pretreatment with the nutraceutical prevented the LPS-induced decrease in cardiac contractility before and after the hearts were subjected to ischemia-reperfusion. At the vascular level, supplementation with the nutraceutical did not prevent hypotension in septic animals, but it attenuated endothelial dysfunction and the increased response of aortic rings to the vasoconstrictors norepinephrine and angiotensin-II induced by LPS. The beneficial effects on cardiovascular function were associated with an increased expression of the antioxidant enzymes SOD-1 and GSR in cardiac tissue and SOD-1 and Alox-5 in arterial tissue. In skeletal muscle, nutraceutical pretreatment prevented LPS-induced muscle proteolysis and autophagy and significantly increased protein synthesis as demonstrated by decreased expression of MURF-1, atrogin-1, LC3b and increased MCH-I and MCH -IIa in gastrocnemius muscle. These effects were associated with a decrease in the expression of TNFα, HDAC4 and myogenin. In conclusion, treatment with a new nutraceutical based on a mixture of AO and EVOO supplemented with OLE is useful to prevent cardiovascular and muscular changes induced by sepsis in rats. Thus, supplementation with this nutraceutical may constitute an interesting strategy to reduce the severity and mortality risk in septic patients.

Supported by Comunidad de Madrid, Doctorados Industriales 2017 (IND2017/BIO7701).

Keywords: Sepsis, nutraceutical, algae and extra virgin olive oils, olive leaf extract

Corresponding author: Miriam Granado, https://orcid.org/0000-0001-9178-8822

P1-12

Vascular effects of insulin and ranolazine mediated by eNOS

¹Solanye Guerra-Ojeda, ¹Andrea Suarez, ¹Begoña Belmonte, ¹María D Mauricio, ¹Kenia Alvarez-Gamez, ¹Constanza Aldasoro, ^1,2Adrian Jorda, ¹Ignacio Campo-Palacio, ¹Jose Mª Vila, ^*1Martin Aldasoro

¹ Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain an ² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

Insulin induces vasodilator effects that are largely dependent on endothelial nitric oxide (NO) release. Known signaling for this effect is initiated by phosphatidylinositol 3-kinase (PI3K), generating PIP3 that activates PDK and AKT, two kinase enzymes that mediate most of the metabolic effects of insulin as well as the phosphorylation of eNOS on Ser1177 and its subsequent activation. Multicenter clinical studies have shown different antidiabetic effects of ranolazine, a protective drug in coronary ischemia, although they are not conclusive about its possible intervention in the sense of facilitating the peripheral effects of insulin by reducing tissue resistance to the hormone. The hypothesis is that ranolazine would facilitates the vascular effects of insulin by increasing endothelial and smooth muscle sensitivity to the hormone. The study was carried out on rabbit aorta using the isometric contraction technique in segments with or without the endothelial cell layer. The concentrations used, both insulin and ranolazine, correspond to the physiological ones in the case of insulin (10^-8-10^-9 M) and to those obtained in patients treated with the drug in the case of ranolazine (10^-6--10^-5 M) Using Western Blot, the expression of AKT, pAKT, eNOS and p-eNOS was analyzed in the presence of insulin, ranolazine and insulin + ranolazine. Insulin causes relaxation of the rabbit abdominal aortic segments. This effect is mediated by the insulin receptor and inhibited by L-NAME, indicating that NO is the final mediator of vasodilation. Insulin increases the vasodilation induced by sodium nitroprusside (NO donor drug) in the aortic segments and inhibits the contractile effects of endothelin 1 and TXA₂. We have shown that insulin promotes the expression of p-eNOS and pAKT. All the vascular effects of insulin were facilitated in the presence of ranolazine. Therefore, our results suggest that ranolazine is a therapeutic possibility in type 2 diabetes, caused by cellular resistance to insulin.

Financial support from the University of Valencia. (Specific Key 20180268).

Keywords: Insulin, ranolazine, vascular function, insulin resistance.

Corresponding author: Martin Aldasoro, http://orcid.org/0000-0001-6630-0493

P1-13

Cytochrome P450 derivatives are involved in renal endothelial dysfunction and vascular oxidative stress in obesity

¹Mercedes Muñoz, ¹María E López-Oliva, ¹Estéfano Pinilla, ¹Claudia Rodríguez, ²María del Pilar Martínez, ³Javier Sáenz-Medina, ¹Cristina Contreras, ¹Ana Sánchez, ¹Alfonso Gómez del Val, ¹Luis Rivera, ^*1Dolores Prieto

¹ Department of Physiology and ² Department of Anatomy and Embryology, Universidad Complutense de Madrid, Spain and ³ Department of Urology, Hospital Universitario Puerta de Hierro-Majadahonda, Madrid, Spain

Oxidative stress derived from Nox, COX-2 and xanthin oxidase is involved in kidney endothelial dysfunction in obesity and insulin-resistant states (Dey et al., Obes Res 12:1278, 2004; Muñoz et al., Free Rad. Biol. Med. 84:77, 2015; Muñoz et al., Redox Biol. 28:101330, 2020). On the other hand, increased activity of soluble epoxide hydrolase (sEH), the enzyme hydrolyzing epoxyeicosatrienoic acids (EETs), decreased levels of EETs and increased circulating and adipose levels of 20-hidroxyeicosatetranoic acid (20-HETE) have been involved in the pathogenesis of metabolic disease vascular complications (Elmarakby et al., Am J Physiol 2011; 301, R1307; Gilani et al., Am J Physiol 2011; 315, R934). The present study aimed to assess whether obesity may induce changes in CYP enzymes that may contribute to oxidative stress and renal endothelial dysfunction. Endothelial function and oxidative stress were assessed in preglomerular arteries of obese Zucker rats (OZR) and their lean counterparts lean Zucker rats (LZR) and the effects of CYP2C and CYP4A inhibitors sulfaphenazole and HET0016, respectively, were examined on the endothelium-dependent relaxations and O₂^.- and H₂O₂ production in intact arteries. Non-nitric oxide (NO) non-prostanoid endothelium-derived hyperpolarization (EDH)-type responses were preserved but resistant to CYP2C9 blockade in OZR in contrast to those in LZR. Sulfaphenazole did not further inhibit reduced arterial H₂O₂ levels and CYP2C11 and CYP2C23 enzymes were downregulated in intrarenal arteries from obese rats. CYP4A blockade restored impaired NO-mediated dilatation and reduced augmented O₂^.- production in kidney arteries from OZR. The current data demonstrate that both decreased endothelial CYP2C11/CYP2C23-derived vasodilator H₂O₂ and augmented CYP4A-derived 20-HETE contribute to endothelial dysfunction and vascular oxidative stress in obesity. CYP4A inhibitors ameliorate arterial oxidative stress and restore endothelial function which suggests its therapeutic potential for the vascular complications of obesity-associated kidney injury.

This work was supported by PID2019-105689RB-I00 Spain.

Keywords: reactive oxygen species, CYPC epoxygenases, CYP4 hydroxylase, obesity

Corresponding author: Dolores Prieto, http://orcid.org/0000-0001-7049-5991

P1-14

T-type voltage gated-calcium channels participate in the contractile response to phenylephrine without affecting vasodilation in rabbit aorta

^*1Andrea Suarez, ¹Begoña Belmonte, ¹Solanye Guerra-Ojeda, ¹José M Vila, ¹Óscar J Arias, ¹Alicia Valls, ¹Eva Serna, ¹María D Mauricio

¹ Department of Physiology, University of Valencia.

Among calcium channels presents in vascular cells, L-type voltage-gated calcium channels (VGCCs) have been the most described. However, recent studies have shown that T-type VGCCs also play a key role in the control of vascular tone. For instance, it has been shown that T-VGCCs are implicated in endothelial NO release. Therefore, an increased understanding of the role of T-type VGCCs may shed light on the regulation of Ca²⁺ signaling pathways at the vascular level and could provide a therapeutic target for patients with cardiovascular disease. The aim of the present study was two-fold: first, to analyse the participation of T-type VGCCs in response to the vasoconstrictor phenylephrine (Phe), and secondly, to determine their participation in response to the endothelium-dependent and endothelium-independent vasodilators, acetylcholine (Ach) and sodium nitroprusside (SNP), respectively. For experiments, isolated aorta rings (3mm long) from seventeen healthy rabbits were mounted for isometric tension recording in organ baths chambers containing Krebs-Henseleit solution. Concentration-response curves to Phe (10^-9- 3x10^-5M), Ach (10^-9- 3x10^-5M) and SNP (10^-9- 10^-6M) were obtained in the absence and presence of Nickel (5x10^-5M), a T-Type VGCC blocker, to assess whether T-type VGCCs could influence the vasodilator and vasocontractile response. Data showed that Phe-induced vasoconstriction was right-shifted in the presence of Nickel (pD_{2 =} 6.55 ± 0.08 for control vs 6.20 ± 0.07 for Nickel; P < 0.05). Conversely, Nickel did not modify vasodilation to Ach and SNP, a nitric oxide donor. Although further studies are needed to understand the complex mechanism of the calcium channels, the present results suggest that activation of T-type VGCCs promotes calcium influx causing vasoconstriction in response to phenylephrine in the rabbit aorta, without affecting NO-mediated vasodilation.

Supported by Universitat de Valencia. Programa propi d’Investigació del Vicerectorat d’Investigació de la UV, convocatòria d’Accions Especials, expedient UV‐INV‐AE‐1544052.

Autorización procedimiento experimentación animal: 2015/VSC/PAE/00233

Keywords: T-type voltage-gated calcium channels, vascular tone, aorta, organ bath.

Corresponding author: Andrea Suárez, https://orcid.org/0000-0003-0932-7870

P1-15

Tempol, a SOD mimetic, enhances vasodilation in response to exogenous NO in human saphenous vein

¹Andrea Suarez, ¹Begoña Belmonte, ¹Solanye Guerra-Ojeda, ²Cristina Rueda, ²Marina Juez, ³Iván Martín, ³José A Rincón, ³Claudia M Aguirre, ¹José M Vila, ^*1María D Mauricio

¹ Department of Physiology, University of Valencia, Spain.

² Servicio de Cirugía Cardiovascular. Hospital General Universitari de Valencia, Spain.

³ Servicio de Cirugía Cardiovascular. Hospital Universitari i Politècnic La Fe, Valencia, Spain

Vascular oxidative stress plays a fundamental role in the initiation and progression of endothelial dysfunction, a hallmark of cardiovascular disease. Specifically, the increase in the production of superoxide anion (O₂^-), rapidly reacts with nitric oxide (NO) to form peroxynitrites (ONOO^-), leading to a reduction in NO bioavailability. On the other hand, the vascular wall has antioxidant defence mechanisms that eliminate O₂^-, such as superoxide dismutases (SOD) to form hydrogen peroxide (H₂O₂). Our aim was to analyse whether O₂^- is released in response to angiotensin II-induced contraction and in response to bradykinin and sodium nitroprusside-induced vasodilation. In this regard, we use tempol, an exogenous SOD mimetic, that might improve NO bioavailability. Accordingly, 3-mm-long human saphenous vein segments from patients undergoing coronary bypass graft surgery were isolated and mounted in an organ bath containing Krebs-Henseleit solution to record isometric tension. Concentration-response curves to angiotensin II (10^‑11-3x10^-6 M), bradykinin (10^-9-3x10^-5 M), and sodium nitroprusside (10^-10-3x10^-6 M) were obtained in the absence and presence of tempol (10^-3 M). Our results showed that tempol did not modify the response to angiotensin II and bradykinin, indicating the O₂^- is not released by these agonists. However, endothelium independent vasodilatation to sodium nitroprusside was potentiated in the presence of tempol, (pD = 7.9 ± 0.1 for control and 8.7 ± 0.1 for tempol, P<0.05) suggesting that the removal of O₂^-increases the NO bioavailability. These data provide evidence that antioxidant treatment could improve NO-mediated vasodilation in the human saphenous vein undergoing coronary surgery.

Supported by Universitat de Valencia. Programa propi d’Investigació del Vicerectorat d’Investigació de la UV, convocatòria d’Accions Especials, expedient UV‐INV‐AE‐1544052.

Informe favorable Comité de Ética Hospital Universitario y Politécnico La Fe, Valencia. Nº de registro 2020-675-1.

Keywords: Oxidative stress, vascular reactivity, nitric oxide, human saphenous vein.

Corresponding author: María Dolores Mauricio, https://orcid.org/0000-0002-7695-2898

P1-16

Electrophysiological effects of Rimacalib (SMP 114), inhibitor of Ca ²⁺ /calmodulin-dependent protein kinase II, on left ventricle submitted to local stretch in an isolated heart model

^*1,4Luis Such-Miquel, ^2,4Óscar J Arias-Mutis, ²Johan E Ortiz, ^2,4Patricia Genovés, ²Germán Parra, ²María J Cardells, ^1,4Manuel Zarzoso, ^2,4Antonio M Alberola, ^2,4Luis Such, ^3,4Francisco J Chorro

¹ Department of Physiotherapy, Faculty of Physiotherapy, Universitat de València, Valencia, Spain,

² Department of Physiology, Faculty of Medicine, Universitat de València, Valencia, Spain,

³ Department of Medicine, Faculty of Medicine, Universitat de València, Valencia, Spain and

⁴ INCLIVA, Valencia, Spain and CIBERCV, Valencia, Spain.

Alterations in cardiac Ca²⁺ homeostasis, as occurs in myocardial stretching during some pathologies, are directly related to arrhythmia triggering, although as it is well-known the particular mechanisms underlying arrhythmia are unidentified. For this reason, maintaining the homeostatic factors of the calcium pathway at their physiological levels, has been established as a therapeutic manoeuvre. Rimacalib (SMP 114) is a Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor, widely used as an inhibitor of CaMKII, and when the latter is abnormally active, contributes to diastolic cytosolic Ca²⁺ release and arrhythmogenesis. We have studied the effects of SMP 114 (0.1 and 1 μM) on several intrinsic electrophysiological properties in ventricle (isolated rabbit heart) submitted to acute local stretch in order to investigate its possible preventing electrophysiological consequences of the mentioned manipulation. Eleven adult male White New Zealand rabbits were heparinized and euthanized by sodium thiopental injection (European Ethic Guidelines). The hearts were excised, isolated, perfused and fibrillated (without interrupting the perfusion), in a Langendorff setup. A pacing electrode and a recording multielectrode (121 equi-spaced electrodes) were placed on the left ventricle epicardium submitted to stretch, induced by an intraventricular device. SMP 114 was injected through the aortic root at two different concentrations after basal determinations. Ventricular fibrillation (VF) was triggered by pacing and VF spectral analysis was performed to determine VF dominant frequency (DF) and VF spectral concentration (SpC), as refractoriness and heterogeneity indexes respectively. Determinations were performed, previously and during 3^rd minute of stretch in both basal and SMP 114 situation. An ANOVA test was used for comparisons. Significance when p<0.05. Initial DF was different than stretch DF (16.5±3.7 vs 20.1±3.7Hz) and so did in SpC (23.6±5.6 vs 17.7±3.5). No further differences were observed at SMP 114 0.1 μM nor 1 μM in both unstretched and stretched ventricle. In conclusion, SMP 114 prevents the intrinsic electrophysiological changes produced by acute local stretch.

Supported by Instituto de Salud Carlos III - CIBERCV (CB16/11/00486).

Keywords: isolated rabbit heart, cardiac electrophysiology, myocardial stretch.

Corresponding author: Luis Such-Miquel, https://orcid.org/0000-0001-8499-7183

P1-17

Aging and the effect of nifedipine in Ca ²⁺ signals in human skeletal muscle cultures

^1,2B Calle-Ciborro, ^1,2N Díaz Rodriguez, ³MT Espín Jaime, ⁴FJ Santos, ^1,2MJ Pozo, ^1,2 PJ Camello, ^*1,2 C Camello Almaraz

¹ Department of Physiology, Faculty of Nursing and Occupational Therapy, University of Extremadura, Cáceres, Spain

² Instituto de Biomarcadores Patológicos Moleculares y Metabólicos, University of Extremadura, Spain

³ Faculty of Medicine, Hospital Universitario, University of Extremadura, Badajoz, Spain

⁴ Hospital Universitario, Cáceres, Spain

Cultured skeletal muscle cells are a key model to study metabolic and developmental aspects of skeletal muscle physiology. In addition, several groups have shown that cultures retain diverse features of the donor across multiple passages. Ca²⁺ signals in response to several agonists play an important role in the regulation of muscle metabolism and differentiation. Given that aging alters some aspects of Ca²⁺ signalling, we have studied the effects of age in Ca²⁺ signals in cultures prepared from donors above (aged) and below (young) 60 years old. Ca²⁺ signals were studied using fura-2 and fluorescence microscopy. ATP-induced signals were studied using a two pulses protocol with an intermediate 10 min period allowing recovery or treatment with pharmacological inhibitors. The ATP peak response in cultures from young donors was in part due to influx of extracellular calcium, because it was clearly inhibited, but not suppressed, in low Ca²⁺ medium. The route for this entry seems to be different to capacitative calcium entry (CCE), as indicated by the lack of effect of specific CCE inhibitors (GSK7975 and Synta66). The same was true for L-type Ca²⁺ channels, because nifedipine only induced a slight decrease in the response. In cultures from aged individuals the second pulse of ATP showed a significant decrease, and in contrast to cultures from young donors was insensitive to removal of extracellular Ca²⁺. In a series of experiments with thapsigargin to activate CCE, this influx pathway was inhibited not only by its specific inhibitors GSK7975 and Synta66, but also by nifedipine, indicating that depletion of internal stores coactivates voltage operated calcium channels. In cultures from aged donors CCE was slightly inhibited and was insensitive to nifedipine, similar to the behaviour of the response to ATP. In conclusion, aging induces a conserved detrimental effect on the ATP- and CCE-evoked Ca²⁺ signals, in both cases associated to a likely deregulation of voltage operated Ca²⁺ channels.

Supported by Junta de Extremadura (IB18025, GR21095 y GR18119).

Keywords: skeletal muscle, Ca²⁺ signals, aging, capacitative calcium entry

Corresponding author: Cristina Camello Almaraz, ORCID: 0000-0003-0882-8497

P1-18

Role of Ca ²⁺ in IL6 secretion in human skeletal muscle cultures

^1,2B Calle-Ciborro, ³MT Espín Jaime, ⁴FJ Santos, ^1,2MJ Pozo, ^1,2C Camello Almaraz, ^*1,2PJ Camello

¹ Department of Physiology, Faculty of Nursing and Occupational Therapy, University of Extremadura, Cáceres, Spain

² Instituto de Biomarcadores Patológicos Moleculares y Metabólicos, University of Extremadura, Spain

³ Faculty of Medicine, Hospital Universitario, University of Extremadura, Badajoz, Spain

⁴ Hospital Universitario, Cáceres, Spain

Scientific evidence of the last two decades reveals that, in addition to contraction and metabolism, skeletal muscle is an important endocrine tissue, delivering both local and classic, long-range hormones collectively termed myokines. IL-6 was the first myokine reported to be released into circulation upon contraction, and plays a key role in inflammation, metabolism and cell fate. IL-6 secretion from muscle has been reported to rely on Stat3 pathway, but there is scarce information on the role of Ca²⁺ signalling elements in this process. We have studied the role of Ca²⁺ in IL-6 secretion in human cultured skeletal muscle cells obtained from adult donors under the guidelines and approval of Bioethic Committes of Hospital Universitario and University of Extremadura. IL-6 was assayed in the cultures supernatant using ELISA. IL-6 secretion in response to ATP, a known stimulus for cultured and developing muscle, was dependent on Ca²⁺ signals, given the inhibitory effect of the calcium chelator BAPTA. The secretion was also impaired by removal of extracellular Ca²⁺, indicating a role for influx of extracellular calcium. The Ca²⁺ influx route involved in the IL-6 secretion seems to be voltage-operated Ca²⁺ channels, because nifedipine, verapamil and NNC decreased the secretion, while Synta66, a specific blocker of store operated Orai channels, was without effect. Release of calcium from intracellular stores also participates in IL-6 release, as shown by impairment with ryanodine and the IP3 receptor inhibitor 2-APB. Regarding the downstream pathways activated by Ca²⁺, it is likely that calcineurin is not involved, given the lack of effect of rapamycin and FK506, while the calmodulin inhibitor calmidazolium decreased the IL-6 secretion. As a whole, our results suggest that the release of IL-6 from skeletal muscle cells in response to ATP is mediated by voltage-operated Ca²⁺ channels, Ca²⁺ release from intracellular stores and calmodulin-dependent pathways different to calcineurin.

Supported by Junta de Extremadura (IB-18025, GR21095 y GR18119).

Keywords: skeletal muscle, Ca²⁺ signals, IL-6, primary culture

Corresponding author: Pedro J Camello, https://orcid.org/0000-0001-5026-7166

P1-19

Transfer of mitochondria from normal colon cells to cancer colon cells, reverses remodelling of store-operated channels in colon cancer cells

^*1,2Verónica Feijóo, ^1,2Sendoa Tajada, ^1,2Lucía Núñez, ¹Carlos Villalobos

¹ Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain. ² Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid. Valladolid, Spain.

During cancer process there is a metabolic reprograming which provides survival advantages to tumor cells. The cornerstone of this reprograming is the Warburg effect consisting in the rewiring of aerobic metabolism to glycolisis due to defective mitochondrial synthesis of ATP. Due to this effect, most tumor cells display enhanced mitochondrial potential (∆Ψ), the driving force for mitochondrial calcium uptake. Mitochondria are critical players in intracellular calcium homeostasis. For instance, they control the calcium-dependent inactivation of store-operated channels involved in cell proliferation and other cancer hallmarks. In addition to metabolic reprogramming, cancer cells undergo a deep remodeling of intracellular calcium homeostasis. To learn about the contribution of cancer mitochondria to this remodeling we asked whether transfer of mitochondria from normal cells may influence calcium remodeling in cancer cells. For this end we isolated mitochondria from normal, human colonic NCM460 cells and labelled them with a fluorescent marker. Then we adapted a protocol of mitoception and transfer of exogenous, normal mitochondria to human colon cancer HT29 cells before investigating intracellular calcium homeostasis in mitocepted cells. Our preliminary results showed that HT29 cells with normal mitochondria (HT29 mitocepted) show a lower store-operated calcium entry (SOCE) than control HT29 cells. In contrast, when colon cancer cells are self-mitocepted with mitochondria isolated from colon cancer cells, SOCE is enhanced. These results suggest that transformed mitochondria may modulate dramatically CRAC channels involved in store-operated calcium entry likely acting on the slow calcium-dependent inactivation of these channels.

Keywords: store-operated calcium entry (SOCE), mitochondrial transfer, mitoception

Corresponding author: Verónica Feijóo

P1-20

Relevant role of Orai1 variants in the formation of spheroids and the self-renew process in cancer stem cells derived from the triple negative breast cancer cell line MDA-MB-231

^*Isaac Jardin, Joel Nieto-Felipe, Sandra Alvarado, José Sanchez-Collado, José J López, Ginés M Salido, Juan A Rosado

Department of Physiology (Phycell group), Institute of Molecular Pathology Biomarkers, University of Extremadura, Cáceres, Spain.

During the last decade, evidence favors the hypothesis that exclusively a sub-population of cancer cells within a tumor, termed cancer stem cells (CSC) have the ability for tumor initiation. Breast CSC, with low proliferative rates, an asymmetric division and self-renewing capacity, form discrete cluster of cells denominated mammospheres. Aberrant Ca²⁺ homeostasis has been proposed as a hallmark for cancer initiation and progression. Store operated calcium entry (SOCE) is the main mechanism for Ca²⁺ influx from the extracellular medium in not excitable cells. SOCE is mediated by members of the STIM, Orai and TRPC families, whose expression is altered in several cancers, including breast cancer. By combining molecular biology, biochemistry, and microscopy imaging approaches, we have analyzed the implications of Orai1 in the capability for CSC to form spheroids and self-renew. Our results show that Orai1 is over-expressed in mammospheres derived from MDA-MB-231, both at the mRNA and proteins levels. MDA-MB-231-derived CSC where Orai1 was knocked-down by a shRNA showed a significant impairment in sphere forming efficiency. Moreover, the self-renewal ability of those cells was inhibited. Similar results were obtained in CSC derived from MDA-MB-231 cells with CRISPR-mediated knockout of Orai1 (Orai1-KO). Furthermore, over-expression of Orai1α and Orai1β in CSC derived from MDA-MB-231 Orai1-KO significantly restored their ability to form spheroids and self-renew, with similar efficiency. Summarizing, our results support an important role of Orai1α and Orai1β in the ability of CSC derived from the triple negative MDA-MB-231 cell line to form spheroids, hence in their capability for tumor initiation, marking Orai1 proteins as appropriate candidates for new therapies and treatment against breast cancer metastasis.

Supported by PID2019-104084GB-C21 funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe, and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grants IB20007 and GR21008) to J.A.R., J.J.L. and I.J. are supported by contracts from Junta de Extremadura (TA18011 and TA18054, respectively). J.N.-F., S.A. and J.S.-C. are supported by contracts from Ministry of Science, Innovation, and Universities, Spain.

Keywords: Breast cancer, cancer stem cells, Orai1, SOCE.

Corresponding author: Isaac Jardin (ijp@unex.es), https://orcid.org/0000-0003-4575-8264.

P1-21

Saraf implication in the vascular remodeling

^*1,2Marta Martín-Bórnez, ³José Sánchez-Collado, ¹Antonio Ordoñez-Fernández, ³Juan A Rosado, ^1,2Tarik Smani

¹ Group of Cardiovascular Pathophysiology, Institute of Biomedicine of Seville, University Hospital of Virgen del Rocío/University of Seville/CSIC, Seville, Spain; ² Department of Medical Physiology and Biophysics of the University of Seville; and ³ Department of Physiology, Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain

Orai1 and STIM1, molecular components of Store-Operated Calcium Entry (SOCE), have been associated with vascular smooth muscle cells (VSMCs) proliferation in vascular remodeling. Nevertheless, the role of SARAF (SOCE Associated Regulatory Factor), a regulatory protein involved in STIM1 inhibition, has not been firmly established in the vascular remodeling. The objective of this study was to examine the role of SARAF and Orai1 in VSMCs proliferation and neointima formation after balloon injury of rat carotid arteries. To aim this, experiments were conducted in an animal model of rat carotid angioplasty to characterize neointima formation. VSMC isolated from rat coronary artery was also used to examine cell proliferation. The formation of neointima after balloon injury of rat carotid arteries was confirmed by haematoxylin and eosin staining of tissue sections up to 3 weeks after surgery. Injured arteries showed significant higher expression of SARAF, STIM1 and Orai1 compared to control tissues, corroborating the presence of these regulatory proteins in the neointima layer. Proximity ligation and co-immunoprecipitation assays revealed that SARAF interacts with Orai1 in the neointima. Furthermore, selective silencing of SARAF and Orai1 by small interfering RNA (siRNA) inhibited VSMC proliferation. In conclusion, our data suggest that SARAF is involved in VSMC proliferation and neointima formation after vascular injury.

This research was funded by Agencia Estatal de Investigación [PID2019-104084GB-C22/AEI/10.13039/501100011033].

Keywords: SARAF, Orai1, store-operated calcium entry, vascular remodeling.

Corresponding autor: Tarik Smani, tasmani@us.es https://orcid.org/0000-0002-1877-7438

P1-22

Sphingomyelinases and mitochondria‐associated endoplasmic reticulum membranes are involved in ethanol-induced extracellular vesicle secretion by altering lipid metabolism

^1,2Susana Mellado, ²Francesc Ibáñez, ¹Soraya Vallés, ²Consuelo Guerri, ^*1,2María Pascual

¹ Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

² Department of Molecular and Cellular Pathology of Alcohol, Príncipe Felipe Research Center, Valencia, Spain

Recent evidence demonstrate the involvement of the extracellular vesicles (EVs) as key players in the intercellular communication in physiopathological conditions. We previously showed that ethanol treatment increases the number of secreted EVs astroglial cells and their content in inflammatory molecules, spreading and amplifying the neuroinflammatory response. Considering that cholesterol/sphingomyelin are important for EVs biology, and the importance of mitochondria-associated endoplasmic reticulum membranes (MAMs) in their homeostasis, we have evaluated if MAMs and sphingomyelinases (SMases) could participate in ethanol-induced EVs release. We used BV2 microglial cell line and brains from C57/BL6 wild-type mice. EVs were isolated from the extracellular medium of BV2 cultures cells treated with or without ethanol at 10, 50 and 100 mM. Radioactive metabolic tracers and thin layer chromatography identification were used as a quantitative method to assay phospholipid transfer, sphingomyelinase activity and cholesterol uptake/esterification. Inhibitors of SMases (desipramine and GW4869) and MAMs (cyclosporin A) were also used to analyze the role of lipid metabolism in ethanol-induced alterations of EVs secretion. We demonstrated that ethanol increased EVs secretion and altered their content in BV2 microglial cells. Ethanol also activated MAMs, in a dose-dependent manner, in crude membrane fractions from murine brain and BV2 cells. Ethanol also alters the lipid metabolism by increasing cholesterol uptake and esterification, and SMase activity in BV2 microglia cells. Notably, the ethanol-induced increase in EVs secretion was abolished by using SMases or MAMs inhibitors in BV2 cells, which suggests the role of SMases and MAMs in ethanol-induced EVs secretion. Collectively, these results strongly support a lipid‐driven mechanism, specifically via SMases and MAM, to explain the effect of ethanol on EVs secretion in glial cells.

Supported by the Spanish Ministry of Health‐PNSD (2019‐I039).

Keywords: extracellular vesicles, lipid metabolism, alcohol, neuroinflammation.

Corresponding author: María Pascual, https://orcid.org/0000-0003-1420-631X.

P1-23

Imaging flow cytometry is useful for studying key characteristics of blood circulating exosomes in patients diagnosed with pancreatic cancer

Cándido Ortíz^1*, Matías Estarás¹, Isabel Jaén², Adela Rojas², Diego López-Guerra², Gerardo Blanco-Fernández², Noelia De Armas², José M. Mateos-Rodríguez³, Miguel Fernández-Bermejo³, Carmen Galán⁴, Pedro L. Fernández-Cordero³, Sandra Chamizo⁴, Daniel Vara³, Antonio González¹, José A. Tapia¹.

¹ Department of Physiology, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.

² Hepatobiliary-Pancreatic Surgery and Liver Transplant Unit, Hospital Universitario de Badajoz, Badajoz, Spain.

³ Department of Gastroenterology, Hospital Universitario de Cáceres, Cáceres, Spain.

⁴ Hematology and Hemotherapy Service, Hospital San Pedro de Alcántara, Cáceres, Spain.

Pancreatic cancer is a highly malignant and incurable disease. It represents the fourth leading cause of cancer-related death worldwide. Some of the characteristics of pancreatic cancer, such as metastasis or growth, rely on the interchange of exosomes between pancreatic tumor cells and surrounding or distant cells. Such exosomes are a heterogenous mixture of small vesicles (<150 nm) expelled by both normal and transformed cells that are involved in cell communication and regulation. Recently it has been proposed that growth factor receptors EGFR and cMet, and AXL tyrosine kinase could be related with pancreatic cancer growth and detection. Aiming the study of putative biomarkers for human pancreatic cancer cells, we isolated exosomes from blood samples of healthy donors (n=10) and patients diagnosed with pancreatic cancer (n=8). Exosomes were then permeabilized and labeled with antibodies against cMet-FITC, EGFR-FITC and AXL-PE and subsequently analyzed by imaging flow cytometry. Results showed that the exosomes bearing cMET in the membrane were virtually identical between control and patient samples (respectively 4.4±0.3 vs 4.3±0.1 million/ml). Similarly, the concentration of exosomes positives to EGF were statistically identical, although it tended to be somewhat higher in controls compared to patients (5.6±0.2 vs 3.4±0.2 million/ml, respectively). However, the concentration of exosomes bearing AXL were statistically higher (p<0.05, Student’s t-test) in patients diagnosed with pancreatic cancer (15.3±5.6 million/ml) compared to healthy donors (5.1±0.5 million/ml). Interestingly these differences were even higher in EVS with a diameter above 1 μm. For example, in patient samples were detected 4.4±2.3 million/ml of AXL-bearing EVs with a diameter between 2 and 4 μm vs 0.8±0.3 million/ml of such structures detected in control (p<0.001). In summary, we have applied imaging flow cytometry to investigate circulating human exosomes. We found significant and reliable differences among patient and donors in the concentration of AXL-positive exosomes. Further studies are clearly required to determine the usefulness of these findings in the diagnosis or prognosis of pancreatic cancer. However, we can conclude that imaging flow cytometry coupled with a subset of specific markers could be useful to find biomarkers for the (early?) detection of human pancreatic cancer.

Supported by EQC2019-006152-P, EQC2019-006153-P, GR21037

Keywords: Exosomes, Imaging flow cytometry, Pancreatic cancer, AXL

*Corresponding author: Cándido Ortíz https://orcid.org/0000-0003-0529-4047

P1-24

Differential plasma membrane recycling of Orai1α and Orai1β

Pedro C Redondo, Sandra Alvarado, Isaac Jardin, Joel Nieto-Felipe, Jose J Lopez, Gines M Salido, ^*Juan A Rosado

Department of Physiology (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, Universidad de Extremadura, Caceres, Spain.

Orai1 is the major component of the CRAC (Ca²⁺ release-activated Ca²⁺) channels that conduct store-operated Ca²⁺ entry upon intracellular Ca²⁺ stores depletion. The identification of two Orai1 variants in mammalian cells: Orai1α, the full-length Orai1, and Orai1β, generated by a process of alternative translation initiation from a methionine at position 64 in the Orai1α variant, has raised the question whether both variants are redundant or, in contrast, exhibit different properties and cellular functions. Here we have investigated the ability of both Orai1 variants to recycle in the plasma membrane upon agonist stimulation. By using biotinylation and confocal microscopy in Orai1 knockout HEK-293 cells expressing either Orai1α or Orai1β we have found that both Orai1variants are expressed in the plasma membrane in resting conditions and agonist stimulation enhances the surface expression of Orai1α but was unable to enhance plasma membrane expression of Orai1β. Interestingly, in cells co-expressing Orai1α and Orai1β agonist stimulation enhanced the surface expression of both variants. Orai1 variants plasma membrane recycling was independent of changes in cytosolic Ca²⁺ concentration but requires functional Arf6. These findings provide evidence of different plasma membrane recycling ability of Orai1α and Orai1β and suggest that Orai1α might support agonist-stimulated plasma membrane translocation of Orai1β.

Supported by Grants PID2019-104084GB-C21 funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe, and Junta de Extremadura-cofinanciado por la Unión Europea (Grants IB20007 and GR21008).

Keywords: Orai1α, Orai1β, Ca²⁺ influx, plasma membrane recycling.

Corresponding author: Juan A Rosado, https://orcid.org/0000-0002-9749-2325.

P1-25

Role of NMDARs in spike timing-dependent plasticity at layer 2/3-2/3 synapses of the mouse barrel cortex

^*Joaquín Sánchez-Gómez, Antonio Rodríguez-Moreno.

Laboratorio de Neurociencia Celular y Plasticidad (Edificio 21). Departamento de Fisiología, Anatomía y Biología Celular. Universidad Pablo de Olavide, Sevilla, Spain

Spike timing-dependent plasticity (STDP) is an attractive candidate to mediate the synaptic changes that support circuit plasticity in sensory cortices during development. In horizontal layer 2/3-2/3 synapses of the somatosensory cortex, synaptic plasticity on excitatory connections has been proposed to mediate the integration of sensorial information. It has been reported that NMDA-type glutamate receptors (NMDARs) are necessary for the induction of STDP in diverse excitatory synapses along the central nervous system. The goal of this work was to determine the involvement of NMDARs in STDP in L2/3-2/3 synapses of juvenile mice (P13-21). For this we performed electrophysiological recordings from the cortical layer 2/3 of slices prepared from P13-P21 mice using the whole-cell configuration of the patch-clamp technique. To induce t-LTD or t-LTP, post-pre or pre-post pairing protocols respectively, were applied after a stable EPSP baseline period of 10 min. To determine the involvement of NMDARs we applied the mentioned pairing protocols under specific pharmacological blockade of NMDARs with DAP5 and MK801 and with antagonists of NMDARs containing specific subunits (Glun2A, GluN2B, and Glun2C/D with Zn²⁺, Ro256981 and PPDA respectively). Our results indicate that t-LTP requires NMDARs that contains the GluN2A and GluN2B subunits and t-LTD requires NMDARs-containing GluN2B and GluN2C subunits. We concluded that both t-LTD and t-LTP are present in these synapses at the postnatal age studied with a postsynaptic locus of expression. However, the t-LTP and t-LTD observed are mediated by different NMDARs.

Keywords: spike-timing dependent plasticity, barrel cortex, NMDAR.

Corresponding author: Joaquín Sánchez-Gómez.

P1-26

Cytotoxic activity of polyphenol-rich sweet cherry extracts optimized by response surface methodology

¹Patricia Cosme, ²Sara Martillanes, ²Jonathan Delgado-Adámez, ²Javier Rocha-Pimienta, ¹Javier Espino, ^*1Maria Garrido

¹ Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain

² Department of Biotechnology and Sustainability, Technological Agri-Food Institute (INTAEX), Center for Scientific and Technological Research of Extremadura (CICYTEX), Badajoz, Spain

Natural by-products of the agri-food industry constitute an underexploited source of bioactive molecules with potential health benefits. The aim of this study was to optimize the extraction process for the recovery of cherry polyphenols using a response surface methodology and evaluate the potential cytotoxic effect of the cherry extracts obtained. Lapins, Van and Ambrunes cherry cultivars from Jerte Valley (Spain) were utilized as raw material. A central composite design and response surface methodology were applied to optimize the cherry extraction, where concentration of EtOH, time and temperature were utilized as experimental factors. The combination of EtOH with water (52.2:47.8 v/v) at 40.5 ºC during 70.3 min in agitation (avoiding light) turned out to be the optimal conditions to obtain the extracts with the highest concentration of polyphenols. The profile of phenolic content was analyzed before carrying out the cytotoxic evaluation in human cervical cancer HeLa cell line. Our findings showed that all the extracts assayed possess a relevant phenolic content, being especially high in the extract elaborated with Lapins cultivar. The cytotoxic studies revealed that all three cherry extracts assayed produced a dose-dependent reduction in cell viability in HeLa cells, the most remarkable effect being obtained with the extract elaborated with Lapins cultivars. In conclusion, non-commercial cherry fruit from Jerte Valley can provide pharmacological benefits beyond its nutritional value.

J.E. and M.G. hold post-doctoral fellowships (ref. TA18002 and TA18029, respectively) from Junta de Extremadura. J.R.-P. thanks to Junta de Extremadura for the predoctoral formation contract (ref. PD18018). Authors acknowledge to Agrupación de Cooperativas Valle del Jerte for the supply of the raw material to develop the study.

Keywords: sweet cherry, by-product, antioxidant, cancer.

Corresponding author: María Garrido, https://orcid.org/0000-0002-6987-1950

P1-27

Association between hours of sleep and body mass index in university students from Extremadura

¹Sandra Dávila, ¹Cristina Carrasco, ¹Ana B Rodríguez, ^*2María del Pilar Terrón

¹ Department of Physiology, Faculty of Sciences, University of Extremadura, Badajoz, Spain. ² Department of Physiology, Faculty of Medicine and Health Sciences, University of Extremadura, Badajoz, Spain.

The prevalence of overweight and obesity has increased dramatically in the population in recent decades, becoming a major public health epidemic worldwide. These are fundamentally due to an imbalance between the calories ingested and the calories used. However, in this relationship there are many other factors that influence from genetics, to social and environmental. Thus, one of the factors that has been associated with obesity in recent years is insufficient sleep. Recent data suggests that poor sleep habits may contribute to obesity risk, opening a new avenue for possible intervention. Therefore, the objective of this study was to analyze the effect of sleep quality on body mass index (BMI) and fat percentage, in a group of master's students from the University of Extremadura. To do this, 24 volunteers, aged between 20 and 35 years, participated in the study, who filled out a questionnaire that was made up of two sections: study of anthropometric parameters and study of sleep quality. Thanks to the data provided by the BMI and body fat percentage measurements, it was possible to establish two groups: volunteers with normal weight and volunteers with overweight. It was seen how overweight students had a tendency to wake up earlier and go to bed later than students with normal weight. In addition, in general, the overweight group presented greater sleep disturbance and a greater tendency to suffer from daytime dysfunction. Therefore, it can be concluded that although both groups analyzed showed quite similar sleep quality and no significant differences were observed, whether a relationship was observed between sleep duration and BMI value although more studies with a larger number of participants would be necessary.

Supported by Junta de Extremadura (GR21042).

Keywords: sleep duration, BMI, overweight/obesity, university students

Corresponding author: Mª del Pilar Terrón, https://orcid.org/0000-0002-1060-8277

P1-28

Supplementation with a carob extract (CSAT+ ^® ) enhances body weight loss and favours the recuperation of metabolic health in mice with metabolic syndrome

¹M de la Fuente-Fernández, ¹M de la Fuente-Muñoz, ² D González-Hedström, ²A Espinel, ¹S Amor, ¹M Román-Carmena, ¹AL García Villalón, ¹M Granado

¹ Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid

² Departamento de I+D, Pharmactive Biotech Products S.L.U.

Metabolic syndrome (MetS) is a multifactorial disease characterized by dyslipidaemia, abdominal obesity, insulin resistance and hypertension. Among the pathophysiological mechanisms involved, a state of chronic low-grade inflammation and increased oxidative stress stand out. The high prevalence and incidence of MetS in the population makes necessary the search of new preventive and therapeutic strategies, if possible, with fewer side effects than conventional pharmacological treatments. Among them, carob is a promising candidate due to its antidiabetic, anti-inflammatory and antioxidant effects. The aim of this study was to analyze the possible beneficial effects of a carob extract branded under the name CSAT^+® (Pharmactive Biotech Products, S.L.U., Spain) in the recovery of metabolic health in mice with MetS. For this purpose mice were fed with a high fat (60% kcal from fat)/high sugar diet for 23 weeks to induce MetS. During the last two weeks, 4 groups of mice were switched to a less caloric diet (25% kcal from fat) supplemented or not with CSAT+^® 4.8% and/or subjected to aerobic training. Both caloric reduction and aerobic training improved the lipid profile and attenuated MetS-induced insulin resistance measured as HOMA-IR. However, only supplementation with CSAT^+® enhanced weight loss and significantly lowered plasma levels of IL-6. Moreover, CSAT+^® supplementation was the most effective strategy to improve glucose metabolism in the liver and in skeletal muscle, recovering glycogen synthase enzyme levels and improving insulin sensitivity. In the liver the reduction of insulin resistance was mediated by a decrease in the gene expression of pro-inflammatory markers such as MCP-1 and TNFα, as well as by an upregulation in the mRNA levels of antioxidant enzymes such as GSR and SOD-1. However, in skeletal muscle the improved insulin sensitivity was mostly due to reduction of oxidative stress. In conclusion, supplementation with CSAT+^® improves insulin sensitivity and lipid profile in mice with MetS through its anti-inflammatory and antioxidant properties. For this reason, it could be used as a supplement in dietary regimens to enhance body weight loss and to favour the recuperation of metabolic health.

Keywords: Metabolic syndrome, carob, inflammation, antioxidant.

Corresponding author: M de la Fuente-Fernández.

P1-29

Selenium nanoparticles and selenite supplementation impact white adipose tissue insulin receptor expression and adipogenesis differently during adolescence

^*1María del Carmen Gallego-López, ¹Adrián Martínez, ¹Fátima Nogales, ¹Olimpia Carreras, ¹Inés Romero-Herrera, ²Ana Alcudia, ³Eloísa Pajuelo, ¹M Luisa Ojeda

¹ Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain; ² Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seville, Seville, Spain; and ³ Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain

Adolescence is a period of intense growth and endocrine changes where obesity and insulin resistance processes are increasing. Selenium (Se) homeostasis is tightly related to lipid metabolism, since it interferes with insulin signaling in adipocytes modulating adipogenesis, among other mechanisms. Se has important antioxidant properties through the selenoprotein Glutathione Peroxidase (GPx) enzyme. However, these effects are Se form and dose-dependent. Nanoparticles offer interesting chemical properties which could enhance photoelectric and biological properties of Se. The objective of this study was to test the actions of low-dose selenite and nanoparticles of Se (NPSe) on white (WAT) and brown adipose (BAT) tissue deposition, insulin secretion and GPx-1 and IRS-1 expression in WAT of adolescent rats. To this aim, three groups of adolescent male Wistar rats, control (C), selenite supplemented (S) and NPSe supplemented (NS), were treated for three weeks. Selenite and NPSe supplementation was received orally by water intake; receiving two-fold more Se than C. NPSe were obtained by reducing selenium tetrachloride in the presence of ascorbic acid. S rats had significantly higher longitude and abdominal circumference, but proportioned, together to higher BAT mass and BAT/WAT ratio than C rats. In WAT they presented higher GPx-1 and IRS-1expression, and higher insulin serum levels. NS rats had higher longitude but lower abdominal circumference-longitude ratio than C rats. Moreover, they presented a significant decrease in WAT mass and higher BAT/WAT ratio. They had similar GPx-1 expression in WAT, but significantly higher IRS-1 levels than S rats. NS animals had normal levels of insulin in serum. Thus, selenite supplementation increased insulin secretion and IRS-1 expression in WAT, leading to a balanced overall anabolic body insulin response. By contrast, NPS supplementation did not affect insulin secretion but significantly increased IRS-1 expression in WAT; nonetheless, it was not enough to increase adipogenesis. These substantially different effects on WAT are not mediated by GPx-1. However, it seems that during adolescence low Se supplementation could be considered as a therapeutic approach to avoid obesity in form of NPSe, or to avoid poor growth state or cachexia in form of oral selenite.

Supported by Junta de Andalucía (CTS-193). María del Carmen Gallego-López (this author is funded by a predoctoral University of Seville contract of research and teaching personnel VI-PPITUS).

Keywords: selenite, nanoparticles, adipose tissue, insulin.

Corresponding author: María del Carmen Gallego-López, https://orcid.org/0000-0003-2272-5603

P1-30

Assessment of halal meat quality and its physiological effects in non-Muslim consumers

^*1María de los Ángeles Gómez, ²Sergio Gómez, ³Miguel Fajardo, ⁴Luis M Luengo, ⁵ María T Pérez, ⁶Cristina Carrasco, ⁶Ana B Rodríguez

¹ Department of Physiology, Faculty of Medicine, University of Extremadura, Badajoz, Spain; ² Service of Clinical Analysis, University Hospital of Badajoz, Spain; ³ Service of Microbiology and Parasitology, University Hospital of Badajoz, Spain; ⁴ Service of Endocrinology and Nutrition, University Hospital of Badajoz, Spain; ⁵ Meat and Meat Products Institute, Faculty of Veterinary, University of Extremadura, Cáceres, Spain; and ⁶ Department of Physiology, Faculty of Sciences, University of Extremadura, Badajoz, Spain.

The differential production of halal meat − in terms of feeding, sanitary treatment and animal welfare − suggests the higher quality of the final product; consequently, it is expected that its consumption could have greater health benefits than conventional meat. However, to date there are no scientific studies verifying this hypothesis. Therefore, the study objective is to analyze the nutritional profile of the halal meat and evaluate the effect of its consumption on physiological parameters of great importance for health. For this purpose, a comparative analysis between halal and non-halal meat was performed, including the fatty acids profile, percentage of moisture, proteins, and intramuscular fat, pH, and water activity. Later, an interventional pilot study was carried out, which consisted in the exclusive consumption of halal meat (lamb and beef) for a month in healthy individuals (35-65 years) of both sexes. To study the effects of the consumption of halal meat on participant´s body composition, gut microbiota, biomarkers and antioxidant status, specific analyses were performed before and after the study period. Compared to conventional meat, the results showed that halal meat presented significant differences in the composition of fatty acids, especially in the content of polyunsaturated fatty acids of the beef sample. On the other hand, the consumption of halal meat for a month produced a non-significant improvement in the body composition and antioxidant status of the participants, without changing the biomarkers compared to baseline values. Likewise, the intestinal microbiota was not modified, with a non-significant decrease in antibiotic resistance in the colonies of E. coli. Therefore, it can be concluded that, although a positive trend is observed in relation to the nutritional profile of halal meat and the physiological biomarkers, more studies are needed to confirm the possible healthy effect of its consumption.

Supported by Junta de Extremadura-FEDER (BB021; GR21042) and Financed by the Agreement UEx-Golden Worldwide Trade S.L.: Ref. 141/18

Keywords: halal meat, body composition, intestinal microbiota, antioxidant status.

Corresponding author: Mª Ángeles Gómez, ORCID: 0000-0003-1902-6051.

P1-31

Halal meat: amino acid content, sleep-wake cycle and mood state profile after consumption in non-Muslim consumers

^*1María de los Ángeles Gómez, ²Antonio González, ³Cristina Carrasco, ³Ana B Rodríguez.

¹ Department of Physiology, Faculty of Medicine, University of Extremadura, Badajoz, Spain

² Department of Physiology, Faculty of Veterinary, University of Extremadura, Cáceres, Spain ³ Department of Physiology, Faculty of Sciences, University of Extremadura, Badajoz, Spain.

The growing interest of consumers for quality meat products and production that is more respectful of the animal and the environment, makes halal meat a gastronomic trend worldwide today. In recent years, the halal industry, and especially the meat sector, has become a global market with great projection. Since diet, particularly high-protein diet, plays an essential role in regulating the sleep-wake cycle, it is suspected that the consumption of this kind of meat could report positive effects on different physiological variables, including sleep and mood. However, there are no scientific studies that verify this hypothesis. Therefore, the objective has been to analyze the protein and amino acid contents of halal meat and its relationship with the sleep-wake cycle and the mood state profile of the general population. For this, the protein and amino acid contents of halal and non-halal meat was analyzed in a comparative manner. Subsequently, a pilot study was carried out with a nutritional intervention consisting in the exclusive consumption of halal meat for one month in 25 healthy individuals of both sexes. Variables analyzed before and after the experimental period included activity during wakefulness, objective and subjective quality of sleep, mood state profile and levels of the urinary metabolites of serotonin and melatonin. Compared to conventional meat, halal beef presented significantly higher levels of the amino acids involved in the sleep-wake cycle histidine, tyrosine and tryptophan. On the other hand, the consumption of halal meat during a month caused a slight improvement in men´s wake activity and mood state profile, as well as in women´s subjective sleep quality. Significantly higher urine levels of serotonin metabolite were also reported, particularly in men. Therefore, it can be concluded that the differential content of high-quality proteins and certain amino acids in halal meat could have a positive effect on the sleep-wake cycle and mood state profile of non-Muslim consumers.

Supported by Junta de Extremadura-FEDER (BB021; GR21042; GR21037) and Ministry of Economy and Competiveness (BFU2016-79259-R). Financed by the Agreement UEx-Golden Worldwide Trade S.L.: Ref.141/18

Keywords: halal meat, amino acids, sleep-wake cycle, mood-state profile.

Corresponding author: Mª Ángeles Gómez, ORCID: 0000-0003-1902-6051.

P1-32

Sex differences in mesenteric adipose tissue inflammatory response in diet-induced obese mice

¹A Idoate-Bayón, ¹N Sáinz, ^2,3JA Martínez-Climent, ^1,3,4MP Lostao, ^1,3,4MJ Moreno-Aliaga.

¹ University of Navarra, Center for Nutrition Research and Dept. of Nutrition, Food Sciences

and Physiology. School of Pharmacy and Nutrition. Pamplona, Spain.

² Center for Applied Medical Research University of Navarra, Pamplona, Spain. CIBERONC, ISCIII

³ Navarra's Health Research Institute (IdiSNA).

⁴ CIBEROBN, ISCIII

Several studies have suggested relevant sex differences in adipose tissue metabolism and inflammatory status in different animal models. It has been suggested that the mesenteric adipose tissue could play a critical role in the development of insulin resistance in comparison to other intra-abdominal adipose depots. The aim of the current study was to characterize potential differences in whole-body adiposity and insulin resistance as well as on mesenteric adipose tissue between male and female Prdm1^flox/flox mice in response to a high fat diet. Two-month-old Prdm1^flox/flox male and female mice were fed ad libitum with a control diet (CD; 13% lipids) or a high-fat diet (HFD; 60% lipids) for 3 months. After this period, body weight gain, body fat mass (magnetic resonance), and glucose and insulin tolerance tests (GTT, ITT) were performed. Mesenteric adipose tissue was obtained, weighed and used for histological analysis (Hematoxylin-Eosin) and for gene expression assays by RT-PCR. All experimental procedures were approved by the Ethics Committee for Animal Experimentation of the University of Navarra. Body weight gain and whole-body fat mass were significantly higher in male than in female mice both under CD and HFD. In parallel, male mice were more glucose intolerant and insulin resistance than female mice even when fed a CD. Sex differences were observed also on the weight and adipocyte size of the mesenteric fat. Indeed, female mice displayed smaller adipocyte area as compared to male mice, especially when fed HFD, suggesting that female are more protected to the diet-induced adipocyte hypertrophy. Interestingly, the significant upregulation of proinflammatory genes (F4/80, Cd11c, Ccl2) that accompanied adipocyte hypertrophy in mesenteric fat of HFD-male mice was not observed in HFD-female mice. Il-6 mRNA levels were reduced in mesenteric fat of female mice independently of the type of diet. In summary, our results suggest that male Prdm1^flox/flox mice are more prone to develop obesity, glucose intolerance and mesenteric adipose tissue inflammation than female mice. These data suggest that sex-dependent differential physiological responses should be considered when designing experimental protocols for metabolic studies.

Funding: PID2019-106982RB-I00 (MICINN/AEI/FEDER), CB12/03/30002 (CIBERobn, ISCIII). A. Idoate-Bayón was supported by a fellowship: Spanish GovernmentFPI PRE2020-094419.

Keywords: mesenteric adipose tissue, inflammatory, obesity, sex.

Corresponding autor: María P. Lostao, https://orcid.org/0000-0002-7319-3451.

P1-33

Overweight in older adults of Extremadura: sociodemographic factors, diseases, and medication

^*1Rocío Jerez, ²Ana B Rodríguez, ³María Ángeles Gómez, ²Cristina Carrasco

¹ Department of Nursing, University Center of Mérida, University of Extremadura, Mérida, Spain

² Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain

³ Department of Physiology, Faculty of Medicine, University of Extremadura, Badajoz, Spain

Aging can be defined as the set of morphological and physiological modifications that appear because of the action of time on living beings. In the aging process, there are two simultaneous phenomena, thus is, a normal physiological decline, and an increase in the prevalence of different diseases. In recent years, there has been an increased prevalence of overweight and obesity, which has been observed more urgently in older people of developed populations. Therefore, the problem of overweight and its associated comorbidities has become increasingly important. The objective of this study was to analyze the factors associated with overweight in the population over 65 years of Extremadura. For this purpose, a cross-sectional study was carried out using a questionnaire specifically designed for it, with which data has been collected in sociodemographic terms (age, sex, marital status, cohabitation, income levels and studies, retirement age and place of residence) and physical health (diagnosed diseases, medication, anthropometry, and levels of physical activity). A total of 115 participants (49 men and 69 women) from both rural and urban areas responded to the questionnaire and were classified according to the presence or not of overweight. The results showed that low education and income levels, polypharmacy and the consumption of diuretics were significantly associated with the presence of overweight in the study population. However, no significant differences in weekly physical activity levels were detected between subgroups. These findings may contribute to a better understanding of the factors influencing overweight in older population of Extremadura.

Supported by Junta de Extremadura-FEDER (BB021; GR21042).

Keywords: older adults, overweight, polypharmacy, physical activity.

Corresponding author: Rocio Jerez, ORCID: 0000-0002-0261-5709

P1-34

Evaluation of the antioxidant and antiproliferative potential properties of phytocomplexes extracted from Mediterranean area fruits

^*1Pasquale Marino, ¹Michele Manfra, ²Giacomo Pepe, ²Manuela G Basilicata, ²Stefania Marzocco, ¹Alfredo Procino, ²Pietro Campiglia, ³Isabel Gomez-Monterrey, ⁴Javier Espino, ⁴Maria Garrido, ⁴José A Pariente

¹ Department of Science, University of Basilicata, Potenza, Italy; ² Department of Pharmacy, University of Salerno, Fisciano (SA), Italy; ³ Department of Pharmacy, University of Naples Federico II, Naples, Italy; ⁴ Department of Physiology (Neuroimmunophysiology and Chrononutrition Research Group), Faculty of Science, University of Extremadura, Badajoz, Spain.

Nowadays, high attention has been paid to natural compounds in fruits and vegetables with potential nutraceutical properties. In this regard, dietary polyphenols have been widely demonstrated to be able to not only reduce oxidative and inflammatory stress, but also decrease proliferation of cancer cells. However, the biological activity of various food plants has not yet been studied. This work aims to characterize the nutraceutical potential of four fruits such as, Malpighia emarginata, Arbutus unedo, Goji berries, Annona cherimola. In detail, the bioactive compounds were isolated from these fruits using food grade solvents. The antioxidant potential of polyphenol extracts was evaluated by ABTS radical scavenging assay and in H₂O₂-treated intestinal epithelial cells (IEC-6). In in vitro cell-based assay, ROS intracellular production was evaluated by the probe 2’,7’-dichlorofluorescein-diacetate (H₂DCF-DA). Our results showed that the extracts (12.5-100 μg/mL) significantly inhibited ROS production in IEC-6 cells, with the greatest capacity shown by Annona cherimola extract. The ABTS assay also highlighted an interesting antioxidant activity of the extracts with particular reference to the Malpighia emarginata extract. Additionally, we evaluated the antiproliferative activity of extracts using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay on human cervical cancer (HeLa) and breast cancer (MCF-7) cell lines. Our data showed that the extracts exhibit a dose-dependent effect on cell viability of HeLa and MCF-7 cell lines, with the greatest reduction obtained from Goji berries extract. In conclusion, the results obtained suggest a potential use of extracts tested in the onconutraceutical field.

J.E. and M.G. hold post-doctoral fellowships (ref. TA18002 and TA18029, respectively) from Junta de Extremadura.

Keywords: nutraceutical, antioxidant, antiproliferative, cancer.

Corresponding author: Pasquale Marino

P1-35

Comparative evaluation study of the nutritional composition and ingredients of interest in health and food safety, in private label foods and specific brands of high consumption consumed in Granada

^*1,2Mª Alba Martínez Burgos, ²Marco Santana Jiménez, ^1,2Mª Dolores Yago Torregrosa, ^1,2Mariano Mañas Almendros, ^1,2Emilio Martínez-Victoria Muñoz

¹ Department of Physiology. University of Granada.

² Institute of Nutrition and Food Technology “José Mataix” (INYTA), Center for Biomedical Research (CIBM). University of Granada.

The appearance of new diseases related to food has aroused scientific interest in the study of the effects caused by the consumption of critical nutrients and ingredients of interest, such as food additives. The current diet, followed by the Spanish population, turns towards unhealthy trends, in which the consumption of processed foods is on the rise. At the same time, food composition databases are fundamental tools for their study and their applications in health and in the food industry. To carry out a comparative nutritional study of processed foods, private labels and specific commercial brands, and inform the consumer of their repercussions on health, through the creation of a nutritional traffic light. The methodology used has been: Food choice protocol, Database to compile data, NOVA system, WHO nutritional profile, Nutritional traffic light, Nutri-Score type and Final data template for inclusion in BEDCA. A total of 120 processed foods have been analyzed, according to the designed protocol, from the most consumed food groups, from white brands and specific brands, distributed in the main supermarkets in Granada (Mercadona, Corte Inglés, Carrefour, Alcampo, Lidl). Subsequently, we have carried out a comparative study of processed foods, private labels and commercial brands, profiling differences and similarities of nutrients, additives, and critical ingredients. We have made classifications according to the NOVA international system (determines degree of processing) and the nutritional profile of the WHO. Finally, we have implemented a nutritional traffic light, type Nutri-Score, as an informative signal to the consumer, in relation to the greater or lesser health risk of said foods, according to the components evaluated in them. All this will be included in BEDCA. For the first time, we have carried out a comparative study in Granada, through which we can inform consumers of the similarities and differences in the nutritional composition of private label foods and specific brands, widely consumed in our city. Likewise, we have classified the degree of processing of highly consumed foods in Granada. In addition, BEDCA will be used as an essential tool, which informs the user of their diet and its impact on health.

Keywords: Processed foods, white and specific brands, health, BEDCA.

Corresponding autor: Mª Alba Martínez-Burgos, http://orcid/0000-0002-3095-6543

P1-36

Sugar intake in children of primary education in Extremadura

¹Juan A Pérez, ¹Cristina Carrasco, ¹Ana B Rodríguez, ^*2María del Pilar Terrón

¹ Department of Physiology, Faculty of Sciences, University of Extremadura, Badajoz, Spain. ² Department of Physiology, Faculty of Medicine and Health Sciences, University of Extremadura, Badajoz, Spain.

The term total sugar refers to both sugar naturally present in foods and free sugar. Sugar is found naturally in fruits, vegetables, and some grains, as well as in the form of lactose in milk and milk products. It is important to note that free sugars are linked to several adverse health conditions, both in the short and long term. Consuming foods and beverages high in added sugars during childhood is linked to the development of risk factors for heart disease, including an increased risk of obesity and elevated blood pressure. Today's food environment is characterized by a cheap and plentiful supply of sugar, and a continuing increase in its consumption. In recent decades, the consumption of sugary drinks and industrial pastries has increased dramatically, especially in children. Therefore, the objective of this study was to examine the differences by school (urban and rural) and sex of healthful versus less healthful food group intake in a diverse sample of schoolchildren. 59 urban schoolchildren and 52 rural schoolchildren (age 6 to 11 years, from 1st, 3rd and 5th grades of primary education) participated in the study. Intake of healthful (fruits, vegetables, unsweetened beverages) and less healthful (sweet and salty snacks, sugar-sweetened beverages) food groups was determined via a food frequency questionnaire. In 1st grade, the results showed that there was a significantly lower consumption of water by male children in rural areas compared to urban ones, at recess. In the 3rd year of Primary, the sandwich and commercial juice are the most consumed among male students in urban areas, while the drink of choice in rural areas is water. In the 5th year of Primary School, yogurt was more consumed in the urban group and fruit in rural students as for dessert. Perhaps because the sample was not large enough, this study provided a slight association between dietary intake and educational level although future work is necessary to corroborate these results.

Supported by Junta de Extremadura (GR21042)

Keywords: sugar intake, diet, body weight, children.

Corresponding author: María del Pilar Terrón, https://orcid.org/0000-0002-1060-8277

P1-37

Study of the combined effect of genetic variants and dietary exposure to obesogenic disruptors in altered body mass index

^1,6Viviana G Ramírez-López, ^1,3Yolanda Gálvez-Ontiveros, ^3,4,5Inmaculada Salcedo-Bellido, ^3,6,7Mª Jesús Álvarez-Cubero, ^2,8Mª Dolores Yago-Torregrosa, ^*2,8Mª Alba Martínez-Burgos, ^1,3,8Ana Mª Rivas-Velasco

¹ Department of Nutrition and Bromatology. University of Granada; ² Department of Physiology. University of Granada; ³ Biosafety Research Institute. Ibs-Granada; ⁴ Department of Preventive Medicine and Public Health. University of Granada; ⁵ Biomedical Research Consortium in Epidemiology and Public Health (CIBERESP); ⁶ Genyo. Pfizer-University of Granada-Andalusian Government Center for Genomics and Cancer Research; ⁷ Department of Biochemistry and Molecular Biology III. University of Granada; and ⁸ Institute of Nutrition and Food Technology “José Mataix” (INYTA), Center for Biomedical Research (CIBM). University of Granada.

Obesity is one of the most serious health problems that exist worldwide, with an increasing incidence in the last 20 years. Genetic and environmental factors are involved in this disease, which is characterized by a high genetic predisposition. Thus, alterations in body mass index (BMI) have been associated with a large number of single nucleotide polymorphisms (SNPs). A strong influence of exposure to endocrine disruptors considered obesogenic (bisphenols and parabens), has been shown in the epidemic growth of obesity. Therefore, the study of the interaction between genetic and environmental factors could elucidate, in part, the lack of heritability in obesity. To study the role of polymorphisms rs9939609 of the FTO gene (fatmass andobeses-associated gene) and rs9436303 of the leptin receptor (LEPR) in BMI variation, and its relationship with exposure to bisphenols and parabens, at through diet. This study was carried out in a cohort of 101 Spanish individuals (16-24 years old), from whom saliva samples were taken with buccal swabs. Once the genomic DNA was extracted, the polymorphisms of interest were genotyped by means of quantitative PCR (qPCR), with Taqman probes. Estimation of dietary exposure to parabens and bisphenols if done through food frequency questionnaires (FFQs); the analytical determination of these compounds in each food is carried out using the UHPLC-MS/MS system. Finally, they applied linear regression analysis to associate genetic variants and BMI alterations, according to low and high exposure to parabens and bisphenols. The risk allele G of the LEPR variant rs9436303 is significantly associated with increased BMI (exp (β) = 1.20, 95% CI: 1.04 - 1.38, p = 0.011). It says significant permanence association with high exposure to bisphenols (exp (β) = 1.27, 95% CI: 1.03 - 1.57, p = 0.024). This trend was found to be more relevant in individuals with high exposure to parabens (exp (β) = 1.33, 95% CI: 1.08 - 1.63, p = 0.009). This work shows, for the first time, that the interaction between dietary exposure to obesogens and alterations in the leptin receptor could negatively affect its signaling pathway, favoring an increase in BMI, and a consequent development of obesity.

Keywords: Obesity, endocrine disruptors, diet, polymorphisms.

Corresponding autor: Mª Alba Martínez-Burgos, http://orcid/0000-0002-3095-6543

P1-38

SIRT-1 increases beta-cell function in adolescent binge drinking rats despite the fact that oxidative damage occurs

^*1Inés Romero-Herrera, ¹María Ramblado, ¹María del Carmen Gallego-López, ¹Fátima Nogales, ¹Olimpia Carreras, ¹M Luisa Ojeda

¹ Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain

Binge drinking (BD) is adolescents' most common alcohol consumption model. This alcohol consumption during adolescence, which is characterized by being highly pro-oxidant, has been widely associated with damage to the nervous system. However, it has recently been shown that BD during adolescence also affects different tissues, such as the liver, kidney, and heart. This causes cardiovascular damage and alters hepatic energy metabolism, developing steatosis and insulin resistance (IR), which can end up producing metabolic diseases in later life. The pancreas is an important organ, implicated in the energetic metabolism balance by the secretion of the hormone insulin. This tissue, especially the endocrine part, is very sensitive to oxidative stress, since it has a low proportion of antioxidant enzymes, such as glutathione peroxidase (GPx). Thus, the objective of this study was to analyze if BD exposition in adolescent rats produces oxidative damage in the pancreas, affecting its functionality. For this, two groups of adolescent male Wistar rats, exposed or not to BD, were used. Serum insulin and glucose levels as well as pancreatic oxidative balance, β-cells functionality and SIRT-1 expression, one protein implicated in the energetic balance that protects the endocrine pancreas, were determined. Although BD decreased weight gain at the end of the experimental period, relative weight and total proteins were increased in pancreas. This tissue presented oxidative disbalance in BD rats, since the activity of the antioxidant enzymes was altered producing lipid oxidation. BD exposition significantly increased serum insulin and glucose levels with respect to the control group; thus, IR was occurring. However, β-cells functionality was not altered. Finally, SIRT-1 expression was increased in BD adolescent rats. Based on this, it can be concluded that, although BD produces oxidative stress in the pancreas, it does not affect the β-cells function, since due to the protection that SIRT-1 provides, there is a high secretion of insulin. Consequently, the IR observed after BD exposure is not related to the insulin synthesis, but it is most probably linked to the action of this hormone in target tissues.

Supported by Junta de Andalucía (CTS-193). Inés Romero-Herrera (this author is funded by a predoctoral Junta de Andalucía contract of research and teaching personnel number USE-22212-V).

Keywords: binge drinking, pancreas, β-cells, SIRT-1.

Corresponding author: Inés Romero-Herrera, https://orcid.org/0000-0001-5394-1849

P1-39

Epistasis between SLC6A4 and BDNF: Improving mood and social cognition through a tryptophan-enriched diet in adults aged 50 and older

¹Lierni Ugartemendia, ¹Rafael Bravo, ²Martin Reuter, ¹M Yolanda Castaño, ³Vera Zamoscik, ²Thomas Plieger, ³Peter Kirsch, ^*1Ana B Rodríguez

¹ Department of Physiology. Faculty of Science. University of Extremadura, Badajoz, Spain

² Biological & Personality Psychology, Laboratory of Neurogenetics, Department of Psychology, University of Bonn, Bonn, Germany

³ Department of Clinical Psychology, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

Aging is a multifactorial process which leads to an increased incidence of cognitive decline and mood disorders. Both serotonin transporter (SLC6A4) and Brain-Derived Neurotrophic Factor (BDNF) genes are involved in different signaling pathways. However, there is evidence that they interact to modulate neurogenesis and neural plasticity in emotion processing circuits. Therefore, our purpose was to elucidate if the daily administration of 500mg tryptophan to a cohort of people older than 50 years results in an enhancement of serotonin metabolism modulated by the BDNF gene. We recruited 63 healthy adults who were randomly assigned to a placebo or a tryptophan-treated group. Participants from both groups followed the same protocol. At baseline, blood samples were taken to genotype for serotonin-transporter-linked polymorphic region (5-HTTLPR) including rs25531 polymorphism and for rs6265 polymorphism on the BDNF gene. In addition, before (T₀) and after completing the study (T₁) urine samples were collected to measure 5-hydroxindolacetic acid (5-HIAA) and BDNF levels, while psychological questionnaires (to measure mood and social cognition), and a one-week dietary record (to evaluate the adherence to MIND diet) were assessed. Our results, while consistent with our previous investigations, indicated an interaction between functional variants of SLC6A4 and BDNF when a tryptophan-supplemented diet is administered. In fact, people prone to develop depression regarding SLC6A4 and BDNF polymorphisms (with at least one S’ or Met allele) were those with a greatest benefit from the tryptophan treatment.

Supported by the Joint Programming Initiative “Healthy Diet for a Healthy Life”, MINECO (PCIN-2015-228), Junta de Extremadura (GR21042), YSONUT Laboratories, and BMBF (01EA1606, 01EA1606).

Keywords: mood, social cognition, tryptophan, precision nutrition

Corresponding author: Ana B. Rodríguez, https://orcid.org/0000-0001-6063-0504

P1-40

Nutritional and nutricosmetic approach in skin cancer patients

^*1Maria G Valle, ¹Maria L Lorenzo, ²Virginia Ortega

¹ Department of Nutrition and Bromatology, Faculty of Pharmacy, University of Granada, Granada, Spain

² Department of Dermopharmacy, Official College of Pharmacist of Granada, Granada, Spain

The anticancer therapy and associated symptoms, increase the risk of malnutrition in oncologic patients, leading to decreased tolerance to treatment, affecting life quality and increased complications. To follow a diet rich in antioxidants, essential fatty acids and/or supplemented with nutricosmetics, are contributing factors in prevention, development and treatment of the disease, and also reduces the risk of relapse of a second neoplasia. The object of the following study is to show findings which with suggest the importance of the Mediterranean Diet (MD), in the relationship of risks of suffering skin cancer in the general public, in particular melanoma and basal cell carcinomas. We propose personal modifications in each patient, which are associated to the supplementation with nutricosmetics. Nutritional recommendations should be personalized, changing some aspects, and associating to the MD: organics oil, essential fatty acids w3, w6 and w9, lycopene and “Polypodium spp.” extract as oral photoprotection. The oral supplementation with nutricosmetics as photoprotectors shows protectors effects against photoaging and phototoxicity and helps to protect the skin integrity from damaged produced by ultraviolet radiation. Vitamin E and Zinc supplements should also be included to maintain skin integrity. Substances like olive oil, present in MD, which contains all phenolic compounds and alpha tocopherol, present strong antioxidant and anti-inflammatory properties, influence in cell proliferation, cell cycle progression and apoptosis, so it´s very interesting in these patients. There is also evidence of anticancer effect of substances like resveratrol and nicotinamide. Nicotinamide, vitamin B3 and NAD+ precursor, have been shown to reduce the rate of non-melanoma skin cancer and actinic keratosis in high-risk patients. Recent studies show that it may be the cause of the decrease of NAD+ levels with age and/or after ultraviolet radiation can affect processes involved in genomic stability. Therefore, it is interesting to develop a nutritional proposal or protocol, complementary recommendations that help and improve the life quality of these patients during all the therapeutic process.

Keywords: mediterranean diet, skin cancer, nicotinamide, nutricosmetics

Corresponding author: Maria Guadalupe Valle Pastelero (mvallepastelero@gmail.com)

P1-41

Evaluation of metabolic function and oxidative stress in high fat guinea pig animal models

^*1,3Esther Valverde-Pérez, ^2,3Elena Olea ¹Marta I Pablos ^1,3Ana Obeso, ⁴Joana L Fernandes, ⁴Silvia V Conde, ^1,3Asunción Rocher, ^1,3Jesús Prieto-Lloret

¹ Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain.

² Departamento de Enfermería, Grupo de Investigación en Cuidados Enfermeros (GICE), Universidad de Valladolid, 47005 Valladolid, Spain.

³ Unidad de Excelencia, Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid-CSIC, 47005 Valladolid, Spain.

⁴ CEDOC, NOVA Medical School, Faculdade de Ciências Médicas, Universidad de Lisboa, Portugal.

Metabolic diseases, such as obesity and type 2 diabetes, linked mainly to hypercaloric diets, are a growing issue in developed countries. Data from experimental models in human and animals have shown the relationship between the carotid body (CB) induced sympathetic overactivation and metabolic diseases. Our previous work using hypercaloric diets in rat animal models have extensively supported this evidence. To test the effects of two different hypercaloric diets on the metabolic function and redox status in the guinea pig (GP), an animal model characterised by their hypo-functional CBs. Four-months-old male Harley GPs were divided into three groups (n=6 each) and fed with a control (C), High Fat (HF: 20% fat) or High Fat + High Sucrose (HFS: 20% fat 10% sucrose) diet for 14 weeks. Several parameters were analyzed in samples of adipose tissue, liver and plasma. We evaluated the oxidative status in the liver through the aconitase/fumarase. Plasma samples were used to measure the insulin and leptin levels using ELISA kits. The significance of the differences between the mean values was calculated by one way ANOVA with Bonferroni multiple comparison test. Differences were considered significant at p < 0.05. Despite what happens in rat animal models subjected to hypercaloric diets, GP with similar diets didn´t increase the body weight, even adipose tissue weight diminished (C 12.3±1.2g; HF 5.8±0.8g; HFS 4.9±1.8g; **p<0.01). Meanwhile the triglycerides levels were unchanged (C 165.8±11.01mg/dl; HF 233.2±56.8mg/dl; HFS 201.5±23.3mg/dl; p>0.05). In the liver, we observed significant differences in lipid depositions between the experimental groups and controls. When measuring the aconitase/fumarase levels we observed that in the experimental groups these values were reduced by half, this difference being significant in both groups (C 1.07±0.06; HF 0.57±0.05; HFS 0.61±0.06; ***p<0.001). We found histological differences in the liver of HF and HFS animal groups, accompanied by redox alterations suggesting hepatic alteration of the GP subjected to hypercaloric diets. Further studies will be needed to clarify this issue.

Supported by Unidad de excelencia IBGM Universidad de Valladolid-CSIC (Ref. CCVC8485), Consejería de Educación, Junta de Castilla y León (Spain) and Portuguese Foundation for Science and technology (Ref CEECIND/04266/2017 to FO Martins and PD/BD/128336/2017 to BF Melo). UVa PROYEMER-2021-57 (E. Olea).

Keywords: guinea pig, metabolism, high fat diets, oxidative stress

Corresponding author: Esther Valverde Pérez, https://orcid.org/0000-0002-1822-5713

P1-42

In response to a punctual stress male and female tyrosine hydroxylase haploinsuficient mice show deteriorate immunity and redox state

^1,2Judith Félix, ^1,2Antonio Garrido, ³Eduardo Ortega, ^*1,2Mónica De la Fuente

¹ Department of Genetics, Physiology and Microbiology (Animal Physiology Unit). Faculty of Biology. Complutense University of Madrid. Spain; ² Research Institute of the Hospital 12 de Octubre, Madrid. Spain; and ³ Department of Physiology. Faculty of sciences. University of Extremadura, Badajoz, Spain.

The maintenance of health depends on the correct function of the homeostatic systems (nervous, endocrine, and immune systems) and the adequate communication between them. An inadequate stress response leads to impaired neuroimmunoendocrine communication, increasing morbidity and mortality. Accordingly, catecholamines (CA), which are the end products of the sympathetic-adreno-medullary axis (SAM), constitute one of the main acute stress response pathways involved in that neuroimmunoendocrine crosstalk. Since the main limiting enzyme in CA synthesis is tyrosine hydroxylase (TH), we have reported that female mice with an haploinsufficiency of the Th gene (TH-HZ), which show low plasmatic and endogenous leukocyte CA amounts, exhibit an impairment of homeostatic systems. Indeed, they show premature immunosenescence and oxidative stress triggering in an accelerated aging. Therefore, the aim of this study was to determine the effect of an acute stress (10 minutes of restriction by a clamp) on several parameters of immune function and redox state in TH-HZ mice in comparison with WT, considering the sex-related differences that could exist. WT (n=12) and TH-HZ (n=12) virgin female and male mice of ICR-CD1 strain at the late adult age (9 ± 1 month) were restrained for 10 minutes with a clamp. Then, peritoneal leukocytes were extracted for immune function and oxidative state assays. The results show that this punctual stress in female WT improved their innate immunity but in male WT it was slightly impaired. However, in TH-HZ, regardless of sex, their innate immunity was impaired. The adaptive immunity, in male and female WT mice was not affected, whereas in TH-HZ it was impaired. Moreover, while the WT managed to regulate their redox state after this stress, the TH-HZ did not, seeing an increase in oxidant compounds. It can conclude that after a punctual stress, the control WT may even be beneficial in their innate immunity (only females) and antioxidant defenses, the TH-HZ mice of both sexes showed deterioration of all immune functions and redox state. This confirms the need of adequate CA synthesis to deal with any punctual stress, as well as that the TH-HZ mice are a good model of premature aging.

Keywords: Punctual stress, restrain, sex, tyrosine hydroxylase.

Corresponding author: Mónica De la Fuente del Rey, https://orcid.org/0000-0002-5969-097X

P1-43

Utility of imaging flow cytometry to study the concentration and characteristics of circulating human extracellular vesicles (EVs) in plasma and serum samples

Cándido Ortíz¹*, Matías Estarás¹, José M. Mateos-Rodríguez², Miguel Fernández-Bermejo², Carmen Galán³, Pedro L. Fernández-Cordero², Sandra Chamizo³, Daniel Vara², Isabel Jaén⁴, Adela Rojas⁴, Diego López-Guerra⁴, Gerardo Blanco-Fernández⁴, Noelia De Armas⁴, Antonio González¹, José A. Tapia¹.

¹ Department of Physiology, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.

² Department of Gastroenterology, Hospital Universitario de Cáceres, Cáceres, Spain.

³ Hematology and Hemotherapy Service, Hospital San Pedro de Alcántara, Cáceres, Spain.

⁴ Hepatobiliary-Pancreatic Surgery and Liver Transplant Unit, Hospital Universitario de Badajoz, Badajoz, Spain.

The extracellular vesicles (EVs) are small membrane-comprised structures that play important roles in cellular communication and regulation. The ubiquity of the EVs, particularly the exosomes (<150 nm), and the noninvasive methods that can be used for their collection, make them excellent biomarkers. In spite of this potential the knowledge on EVs concentration and characteristics is very limited, mainly due to the intrinsic limitations of the methods applied for their characterization. Recently has emerged the imaging flow cytometry as a useful tool to detect and characterize EVs. Consequently, we have studied by imaging flow cytometry (ImageStreamX, Amnis) circulating human EVs in serum and plasma samples from healthy donors (n=10) and patients diagnosed with pancreatic cancer (n=8). EVs were isolated by using kits for plasma (Invitrogen, 4484450) or serum samples (Invitrogen, 4478360), and by ultracentrifugation. After isolation exosomes were labeled with CD81-PE, a universal EV marker, and analyzed to determine their concentration and characteristics. Results showed a high concentration of EVs in serum samples from donors and patients (respectively 41.2±5.1 and 38.9±10.5 million/ml). EVs detected in serum after ultracentrifugation were 29.9±5.4 from donors and 28.4±4.3 million/ml from patients. Similarly, with plasma isolating kits the samples rendered 39.2±9.5 and 22.5±0.4 million/ml in donors and patients, whereas ultracentrifugation rendered only 28.9±6.1 and 14.2±1.4 million/ml. Therefore, EVs concentration by ultracentrifugation were systematically lower than those obtained with the kits, although significant differences only were found when comparing plasma samples (p<0.05, Student’s t-test). EVs comprises different vesicle types classified by their sizes. In samples from serum more that 85% of events displayed sizes compatible with exosomes (<150 nm). This percent was lower in samples from plasma, where 25% of EVs displayed a size compatible with exosomes. Finally, the percent of events analyzed in the cytometer as single events were consistently higher than 80% and the average processing time of the samples was less than 2 min. All these results confirm the ability of the imaging flow cytometry to analyze EVs with velocity and precision, making feasible a multiparametric evaluation of the EVs in blood looking for their potential use as biomarkers.

Supported by EQC2019-006152-P, EQC2019-006153-P, GR21037

Keywords: Exosomes, Imaging flow cytometry, Pancreatic cancer, Blood samples

*Corresponding author: Cándido Ortíz https://orcid.org/0000-0003-0529-4047

P1-44

miRNomic profile discriminates between very low birth-weight infants with late-onset Gram-positive sepsis from non-septic controls

^*1David Verdú, ¹Alicia Valls, ¹Andrea Suárez,²María Cernada, ²Alejandro Pinilla, ²Anna Parra-Llorca, ¹María D Mauricio, ¹Eva Serna

¹ Department of Physiology, University of Valencia.

² Heath Research Institute Hospital La Fe, Valencia, Spain.

Neonatal sepsis is a major health concern among neonates with higher morbidity and mortality rate and responsible for up to 20% of all deaths in very low birth weight (VLBW) infants. The gold standard for sepsis diagnosis is a positive blood culture which sometimes yields false-negative results either due to small sample volume and/or the administration of antibiotics to mother prior to delivery. Moreover, results are often unavailable before 48-72h, thus delaying a therapeutic decision. The present study aims to identify differentially expressed miRNA pattern in neonates with late-onset Gram-positive sepsis (LOS) would help for an earlier diagnosis and therapy. This is a prospective, observational, case-control study performed in the Division of Neonatology of the University and Polytechnic Hospital La Fe (Valencia, Spain). The study was approved by the Scientific and Ethics Committee for Biomedical Research (2019/0195). Venous blood (0.5 mL) was used to determine small non-coding RNA expression profiling using the GeneChip miRNA 4.0 Array. The results indicated a tridimensional PCA which discriminates miRNome between Gram-positive sepsis and controls samples. The 1-way ANOVA was performed on Gram-positive septic neonates and controls identifying 217 differentially expressed miRNAs (p-value <0.01); 168 miRNAs were overexpressed (77,42 %) and 49 underexpressed (22,58 %) in septic group vs control group. We identified the transcriptomic profile and, we distinguished 4,297 differential expression genes (FDR<0,05). We combined 217 differentially expressed miRNAs with 4,297 differentially expressed genes to evaluate microRNA–mRNA regulation/interaction. The result was 33 potential miRNAs whereby relevant biological processes are associated with the immune system and the inflammatory response. The most relevant subnetworks were protein targets of proteasome endopeptidase complex, TNF, TGFB1, SP1, NF-kB family, mitogen-activated protein kinase, TP53, ubiquitin, INS and MAPK1. Our conclusions propose that specific miRNAs discriminate between VLBW infants with LOS from non-septic controls and may be potential therapeutic targets for infections caused by Gram-positive bacteria through the modulation of the neonatal immune system.

This research was funded by University of Valencia (Spain), UV-INV-AE18-775130 and “Instituto de Salud Carlos III”, PI18/01292.

Keywords: Sepsis neonatal, miRNomic signature, very low birth-weight neonates, Gram-positive bacteria.

Corresponding author: David Verdú, https://orcid.org/0000-0003-0790-2501

P1-45

Optimization of human "memory-like" NK cell culture techniques: phenotypic and functional analysis

^*1Eva Sánchez-Hernández, ¹Marina González, ¹Sofía Carreira Santos, ¹Raquel Tarazona, ¹Javier G Casado, ¹Nelson López-Sejas

¹ Immunology Unit, Department of Physiology, University of Extremadura, 10003 Cáceres, Spain

Natural killer cells belong to the lymphoid lineage of the innate immune response. These cells are responsible for the elimination of virus-infected cells or tumor cells. Their activation involves a balance between activating and inhibitory receptors expressed on their surface. Their antitumor activity makes them a valuable tool for immunotherapy against cancer, however, it is necessary to optimize in vitro expansion protocols to increase their number and cytotoxic potential. Memory-like NK cells were recently identified and characterized as a promising therapeutic option because of their enhanced cytotoxic capacity against tumor cells in vitro. Currently, it is possible to induce the phenotype of memory-like NK cells through several approaches, such as incubation with cytokine cocktails, fusion proteins, or by co-culture with tumor cells. The overall objective of this work was to analyze the effect of IL-12/15/18 pre-activation on NK cells to induce the in vitro expansion of memory-like NK cells. The methodology of this work was based on cell culture techniques using peripheral blood-derived NK cells and multiparametric flow cytometry. The study has been carried out under the approval of the Ethics Committee of the University of Extremadura (Nº: 118/2020). Our results allowed us to identify phenotypic and functional differences between IL-12/15/18-stimulated NK cells (hereinafter defined as cytokine-induced memory-like NK cells), and control NK cells. Moreover, it is interesting to note that cytokine-induced memory-like NK cells exhibited potent cytotoxic activity against tumor cell lines under in vitro conditions. Nevertheless, the functional response decreases under long term culture conditions. Here we hypothesize that functional variations are caused by transcriptional and transductional modifications in activating/inhibitory receptors expressed on their cell surface. In conclusion, we consider that our preliminary results represent an advance in the optimization of in vitro culture conditions for the expansion of memory-like NK cells. In terms of clinical relevance, this study may represent a step forward to enable the application of adoptive cell transfer of memory-like NK cells.

Supported by Junta de Extremadura (IB20132 and GR21178) and the Ministry of Science and Innovation of Spain (SAF2017-87538-R), co-financed by the European Union (FEDER).

Keywords: cancer immunotherapy, cytokine-induced memory-like NK cells, NK cell cytotoxicity, phenotypical characterization.

Corresponding author: Eva Sánchez-Hernández.

P1-46

Evolution of the duodenal lymphogram in adult celiac patients after the withdrawal of gluten from the diet

^*1Roberto Pariente Rodríguez, ¹Carlota García-Hoz Jiménez, ¹Miren G Roy Ariño

¹ Department of Immunology, Ramón y Cajal University Hospital, Madrid.

Celiac disease (CD) is characterized by changes in the subpopulations of intraepithelial lymphocytes (IEL), and its determination by flow cytometry (FCM) is the duodenal lymphogram. Our group has established the cut-off points that define a celiac lymphogram in adults: percentage of TCRγδ+ IELs >14% and percentage of CD3 surface negative (sCD3^-) IELs <4%. Duodenal lymphogram adds specificity to the histological findings and enable diagnosis even in unconventional presentations of CD. The aim of this study was to know how the lymphogram evolves in adult patients with active CD when starting a gluten-free diet (GFD). This is a retrospective study on a cohort of 59 active CD patients. We have analysed the results of the histological lesions and lymphograms before and after the withdrawal of gluten from the diet, and the levels of serum anti-transglutaminase antibodies (AcTG2). As a control group, 410 patients were included in whom CD was ruled out. As for results, on a gluten-free diet, 90% of patients maintain a %IEL TCRγδ+ >14% and 56% maintain a %IEL sCD3^- lower than 4%, without being able to establish a clear relationship with the degree of villous atrophy. In this sense, 52.4% of patients maintained a complete celiac lymphogram and only 6.8% had a non-celiac lymphogram after GFD (median time of GFD 24 months). The remaining 40.6% of the celiac patients on a GFD had a partial celiac lymphogram (predominantly patients with increased TCRγδ subset but recovery of sCD3^- subpopulation above 4%). Most patients maintain abnormalities in the lymphogram despite a correct adherence to GFD, with negative serology (AcTG2) and recovery of mucosal integrity. The celiac lymphogram is a marker of CD, both in the inflammatory stage and after treatment when the inflammation and injury have been reversed. IEL sCD3^- subpopulation could represent a useful marker to monitor GFD compliance.

Keywords: celiac disease, duodenal lymphogram, intraepithelial lymphocytes.

Corresponding author: Roberto Pariente Rodríguez.

P1-47

Activation of mitochondrial K _ATP channels relax penile arteries via activation of the endothelial NO pathway

Alfonso Gómez del Val, Cristina Contreras, ^*Dolores Prieto, ^*Ana Sánchez Pina

Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense de Madrid, Spain

Besides its essential role in energy production, mitochondria are involved in adaptive functions such as regulation of vascular tone. In cerebral arteries, mitochondrial depolarization that follows mitoK_ATP channel activation at the endothelium evokes a nitric oxide (NO)-mediated vasodilator response via activation of the PI3K-Akt-eNOS pathway. Normal erectile function is primarily a vascular event that relies on vasodilation, which is largely due to both nerve- and endothelium-derived nitric oxide (NO). The purpose of this study was to assess whether mitochondrial K_ATP (mitoK_ATP) channels are involved in vasodilatation of penile arteries. The effects of the mitoK_AT channel activators BMS-191095 and diazoxide were investigated in the absence and presence of endothelium and of blockers of NO synthesis (L-NOARG 100) or K⁺ channels, in intact small penile arteries from Wistar rats mounted in microvascular myographs. The effect of mitoK_ATP channel activation was also investigated in penile arteries from genetically obese Zucker rats (OZR). BMS-191095 and diazoxide (0.1-10 µM) evoked concentration-dependent relaxations of penile arteries, BMS -191095 being about 2-orders of magnitude more potent than diazoxide. Relaxant responses to BMS-191095 were largely reduced by mechanical endothelium removal and by blockade of the NO synthase (NOS) with L-NOARG. The selective inhibitors of K_ATP channels (glibenclamide) and of large conductance Ca²⁺-activated K⁺ (BK_Ca) channels (iberiotoxin) also inhibited the vasodilator response to BMS-191095. Moreover, relaxations induced by mitoK_ATP channel activation were impaired in penile arteries from obese rats compared to controls. The present study demonstrates a vasodilator response in penile arteries following activation of mitoK_ATP channels at the endothelium, involving NO release and activation of BK_Ca channels in VSM. Impaired relaxant response to mitoK_ATP channel activation in arteries from obese rats suggest that mitochondrial dysfunction may underlie penile endothelial dysfunction in insulin resistant states.

Supported by PID2019-105689RB-I00

Keywords:  Mitochondrial K_ATP channel, endothelium, nitric oxide, penile arteries

^*Corresponding authors: Dolores Prieto, https://orcid.org/0000-0001-7049-5991; Ana A. Sánchez Pina, https://orcid.org/0000-0002-8432-8702

Poster session 2

P2-01

Sodium valproate treatment prevents endothelial dysfunction in rabbit aorta with acute myocardial infarction

¹B Belmonte, ¹A Suarez, ¹S Guerra-Ojeda, ¹JM Vila, ¹M Aldasoro, ¹G Parra, ¹E Serna and *¹MD Mauricio.

¹ Department of Physiology, University of Valencia

Acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide that involve epigenetic modifications, such as histone deacetylation. Sodium valproate (VPA) inhibits histone deacetylases, making it a promising drug for the treatment of AMI. However, the effects of VPA at the vascular level are poorly understood. Therefore, the aim of this work was to assess whether AMI causes vascular dysfunction in the rabbit aorta, and whether VPA can reverse it. White New Zealand rabbits were divided into three groups: sham (n = 9), AMI (n = 15) and AMI + VPA (n = 14). To study vascular reactivity, 3 mm long abdominal aorta segments were isolated and mounted in an organ bath containing Krebs-Henseleit solution and isometric tension was recorded. Concentration-response curves to acetylcholine (Ach) and sodium nitroprusside (SNP) (10^-9-3x10^-5 M) were performed to determine endothelium-dependent and independent relaxation respectively. Acetylcholine curves previously incubated with L‑NAME (10^-4 M) to inhibit nitric oxide (NO) synthesis were also performed. Ach-mediated relaxation was significantly reduced in the AMI group (pD₂ = 7.18 ± 0.02 for AMI vs 7.49 ± 0.09 for sham, P=0.01) indicating endothelial dysfunction. VPA reversed this effect (pD2 = 7.45 ± 0.09 for AMI + VPA vs 7.18 ± 0.02 for AMI, P=0.02). Furthermore, ACh-mediated relaxation was decreased in the presence of L- NAME in the three groups, with the higher blockage in the AMI + VPA group. The production of NO was calculated by area under curve (AUC) and the data were 71 ± 12 AU for sham, 80.2 ± 29 AU for IAM, and 150 ± 15 AU for IAM + VPA, P < 0.05 for IAM + VPA compared to the other groups. These results indicate that VPA increases NO production. In contrast, SNP-mediated relaxation was unchanged in the three groups. In conclusion, our data suggest that AMI causes endothelial dysfunction in rabbit aorta, and the treatment with VPA reverses this response by increasing the NO production.

Supported by Universitat de Valencia. Programa Propi d’Investigació del Vicerectorat d’Investigació de la UV, convocatòria d’Accions Especials, expedient UV‐INV‐AE‐1544052. Autorización procedimiento experimentación animal: 2015/VSC/PAE/00233

Keywords: Endothelial dysfunction, Acute myocardial infarction, Sodium valproate, Nitric oxide.

Corresponding author: María Dolores Mauricio https://orcid.org/0000-0002-7695-2898

P2-02

Keeping animal competences in habilitated staff for animal experimentation

^*1,2José C. Bravo, ¹María Reyes

¹ Animal facility of University of Extremadura. Badajoz, Spain

² Neuroimmunophysiology and Chrononutrition Research Group. Faculty of Science. University of Extremadura. Badajoz, Spain.

Competences for animal experimentation involve that knowledge and skills required to perform those functions allowed to certified breeders suppliers and users in animal experimentation. Keeping these habilitations are essential to staff in animal research but also for faculty when teaching in high education with laboratory animals. Currently, in Spain, maintenance, implementation and further development is reflected in Ministerial Order ECC/566/2015. Additionally, a continuous training is a requirement to keep these competences as indicated by this regulation, but also its maintenance and further verification by competent authorities. Then, animal competences has to be requested every 8 years with minimum specific training time for each particular function. Furthermore, the mentioned training has to ensure an adequate professional retraining through updating knowledge or through educational activities. The indicated educational activities might be training courses, workshops, or seminars. However, other options are also available like new skills in work centre, project evaluation, scientific publications, technical and scientific reports or committee memberships, among others. To conclude, once the competence has been renewed, each individual person needs to keep the competence updated, at least, every 8 years through the indicated activities and in agreement with Ministerial Order ECC/566/2015.

Keywords: animal competences, animal experimentation, Ministerial Order ECC/566/2015.

Corresponding author: José Carlos Bravo (jcbravo@unex.es), https://orcid.org/0000-0001-5400-0952

P2-03

Effects of PEG-coated magnetite nanoparticles on molecular and biochemical parameters in the heart and liver of normotensive and hypertensive rats

¹Andrea Micurova, ¹Michal Kluknavsky, ²Martin Skratek, ¹Peter Balis, ³Monika Okuliarova, ²Jan Manka,¹Iveta Bernatova

¹ Institute of Normal and Pathological Physiology, Centre of Experimental Medicine, Slovak Academy of Sciences, Bratislava, Slovak Republic;

² Institute of Measurement Science, Slovak Academy of Sciences, Bratislava, Slovak Republic;

³ Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic

This study investigated the biological effects of polyethylene glycol (PEG)-coated magnetite nanoparticles (~30 nm core size, ~51 nm hydrodynamic size, 1 mg Fe/kg/day, administered intravenously) in the left heart ventricle (LHV) and liver of Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHR) 90 min post infusion. Iron oxide nanoparticles (IONs) had no effect on the blood pressure and heart rate of ION-treated rats, however there was significant decrease of plasma corticosterone in ION-treated SHR (SHR_ION ) vs. control SHR (SHR_C) and ION-treated WKY (WKY_ION). Nitric oxide synthase activity was unaltered in the LHV, but significantly decreased in the liver of SHR_ION vs. SHR_C. For superoxide productions, there were significantly higher levels found in WKY_ION compared to WKY_C in both the LHV and liver. No such changes in superoxide production were found in SHR. Saturation magnetization (a parameter associated with content of natural iron-containing compounds) of the liver of SHR_C was significantly higher than in WKY_C, however, in LHV we found the reduction in saturation magnetization of SHR_C compared to WKY_C. ION-originated iron content was significantly lower in the liver of SHR_ION vs. WKY_ION. In the LHV we found no significant change, however there was a decreasing tendency in nanoparticles-derived iron content in SHR_ION compared to WKY_ION. There were no ION-dependent changes in iNOS, eNOS, NRF2 and PPARg gene expressions vs. the respective control group in both tissues investigated. In the liver, IONs significantly reduced the expressions of SOD1, SOD2 and FTH1 in SHR_ION vs. SHR_C. In the LHV, IONs elevated SOD1 gene expression in WKY and TFR1 in SHR vs. the respective controls. In conclusion, the results showed reduced incorporation of IONs and lower superoxide production in the liver and LHV of SHR compared to WKY suggesting considerable influence of high blood pressure on pharmacodynamics and kinetics of PEG-coated iron oxide nanoparticles.

This study was supported by the grants Nos. APVV-16-0263, VEGA-2/0160/17 and 2/0157/21.

Keywords: iron oxide nanoparticles, iron metabolism, antioxidant defense.

Corresponding author: Iveta Nernátová, http:// orcid.org/0000-0002-6120-706X

P2-04

Environmental lead exposure effects in animal behaviour and physiology: a comparative study

^*Liana Shvachiy

Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany

Cardiovascular Centre of the University of Lisbon, Portugal

Institute of Physiology, Faculty of Medicine of the University of Lisbon, Portugal

Lead (Pb) is a heavy metal whose widespread use has resulted in environmental contamination and serious health issues, especially when it is exposed during the developmental stages. Behavioural alterations, inflammation, hypertension, and autonomic dysfunction have all been linked to chronic lead exposure. However, numerous environmental lead exposure scenarios exist, and the effects of them have not been compared in a large-scale investigation. The effects of developmental, intermittent, and permanent exposure to Pb on glial activation, behavioural, physiological, and autonomic parameters in rats of both sexes exposed to water containing lead acetate from the foetal period were investigated in the current study. Developmental Pb-exposed pups were given lead until they were 12 weeks old, intermittent Pb-exposed pups were given tap water until they were 20 weeks old, then leaded water for the second time from 20 to 28 weeks old, and permanent Pb-exposed pups were given lead until they were 28 weeks old. At 28 weeks of age, behavioural assessments for anxiety, locomotor activity, and memory assessment were done following the exposure procedures. Because the PerPb group had more obvious effects and a substantial rise in LF and HF bands as well as anxiety levels, the data suggest that the length of Pb exposure is more important than the timing of exposure. In summary, this is the first study to characterize and compare physiological, autonomic, behavioural, and molecular changes in diverse environmental low-level lead exposures from the foetus to adulthood, with the duration of exposure being the key predictor for larger deleterious effects. These kinds of studies are of great importance, hence showing the importance of the surrounding environment in health from childhood until adulthood, leading to the creation of new policies for toxicant usage control.

Keywords: environmental lead exposure; cognitive impairment; hypertension; cardiorespiratory impairment

Corresponding author: Liana Shvachiy

P2-05

Pomegranate extract reverses loss of motor coordination in aging frail mice. Functional and molecular studies.

^*1David Verdú, ¹Alicia Valls, ¹Aitor Carretero, ²Clara Bueno-Fernandez, ¹Esther García-Domínguez, ¹José Viña, ¹Eva Serna.

¹ Department of Physiology, University of Valencia, Valencia, Spain.

² Central Unit for Research in Medicine (UCIM), University of Valencia, Valencia, Spain.

Aging is defined as the biological process that consists of the progressive deterioration of the organism in apparently healthy individuals. The changes that occur during aging make the individual frailer. Frailty is an age-associated biological syndrome characterized by decreased biological reserves leading to a decline and deterioration of functional properties at the cellular, tissue and organ level. The cerebellum is responsible for the complex motor functions like maintaining balance and posture, coordination of voluntary movements, motor learning and cognitive tasks. During aging most of these functions are deteriorated which results in falls and accidents. The aim of this work was to elucidate the effect of pomegranate extract (PE) during 4 months of supplementation in elderly mice to prevent the frailty. The animal study was approved by the University of Valencia Ethics Committee for Research and Animal Welfare (License reference: A20201110122413). Male C57Bl/6J eighteen-month mice were assigned to one of two groups: control group or PE supplementation (150mg/kg/day) group. Mice were evaluated for the “Valencia Score” for frailty at pre-supplementation and post-supplementation times using rotarod performance test (motor coordination), ladder climbing test (neuromuscular function), grip strength (muscular force), treadmill (endurance) and monthly involuntary weight loss were conducted. Finally, animals were sacrificed to obtain biological samples to determinate lipid peroxidation as an oxidative stress parameter in cerebellum and cortex. Our results showed that 16 weeks of PE supplementation improved motor skills of C57Bl/6J aged mice in motor coordination, neuromuscular function and monthly weight loss but no changes in grip strength and endurance were found. Furthermore, PE seemed to decrease malondialdehyde, a lipid peroxidation marker, after supplementation in cerebellum homogenates, but no changes in cerebral cortex were found. We report an improvement of motor skills, less weight loss and cerebellum oxidative state in a PE nutritional supplementation model for elderly frail mice.

This work was supported by the following grants: Instituto de Salud Carlos III CB16/10/00435 (CIBERFES), (PID2019-110906RB-I00/ AEI / 10.13039/501100011033) from the Spanish Ministry of Innovation and Science.

Keywords: Frailty, pomegranate extract, motor coordination, cerebellum.

Corresponding author: David Verdú, https://orcid.org/0000-0003-0790-2501

P2-06

Comparative analysis of the quality of sleep in relation to the time of physical exercise

Alejandro Corbacho, ^*Lourdes Franco

Department of Physiology, Faculty of Medicine and Health Science, University of Extremadura, Badajoz, Spain

Sleep is a necessary reversible phase of decreased responsiveness, motor activity and metabolism. At the same time, it is an important factor to take into account when carrying out any daily activity, since lack of sleep causes daytime disturbances, affecting alertness, attention, and daytime cognitive functions both in adolescents and adults. In the case of athletes, sleep has been considered a key factor in their relation to performance, relating a low quality of sleep with a decrease in performance in competitive sports. In addition, there are also studies that relate physical exercise with an improvement in the quality of deep sleep. In other words, in general, those who exercise are more likely to achieve deep sleep than those who don't. One of the most common alterations among athletes is the alteration or poor quality of sleep, as well as the training schedule. The aim of this study was to assess how exercise time affects sleep quality. For this purpose, 124 volunteers (73 men and 51 women) practicing sports (age 30.01±13.15 years) were selected, who completed the Pittsburgh Sleep Quality Questionnaire (PSQI) and sociodemographic questions. In this study it was concluded that men have a better quality of sleep than women, and women spend more time in bed despite having greater daytime sleepiness. In addition, the group of "good sleepers" performs physical exercise first thing in the morning, despite how much time they spend in that exercise.

Supported by Junta de Extremadura (GR21042).

Keywords: athletes, sleep, Pittsburgh Sleep Quality, sport

Correspondingauthor: Lourdes Franco, https://orcid.org/0000-0002-2695-5660

P2-07

Physiological impact of seasonal time changes on Spanish youth and adult population

¹Rocío Lozano, ¹Ana B. Rodríguez, ^*1Cristina Carrasco

¹ Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain.

Seasonal time change is a method that has been implanted for years in most of the countries. By using this resource, the clocks are put forward or back for one hour to achieve energy saving and to provide a better quality of life for the population. However, these purposes have been questioned in recent years by many publications that report the increased occurrence of certain diseases and other health disorders after the seasonal time changes. Due to the current controversy, more research is needed to a better understanding of the health consequences. For this reason, the objective of this cross-sectional study has been analysed the influence of seasonal time changes on the sleep quality, mood, and food consumption patterns of Spanish youth and adult population. A cross-sectional study was performed in 40 participants divided in two age groups (20-35 and 50-65 years). Data collection was carried out in the weeks before the winter and summer time changes, as well as one month after these time points. Results showed that both seasonal time changes negatively affected the quality of sleep and the mood of the participants. Moreover, differences were observed according to sex and age range. Regarding the food consumption patterns, the summer time change produced an increase in the consumption of foods such as cereals, sausages, eggs, fish and seafood, and beer. Therefore, the current evidence for the impact of these biannual time changes on sleep quality and mood strengths the scientific and social debate on the need to eliminate these procedures for the risk they may pose to public health.

Supported by Junta de Extremadura-FEDER (BB021; GR21042).

Keywords: seasonal time change, sleep quality, mood, food consumption pattern.

Corresponding autor: Cristina Carrasco, ORCID: 0000-0003-2763-4903.

P2-08

Comparative analysis of sleep quality in students under stress

Clara María Ventura, Lourdes Franco

Department of Physiology, Faculty of Medicine and Health Sciences, University of Extremadura, Badajoz, Spain

Sleep is an integral part of life, a biological necessity that allows the restoration of physical and psychological functions essential for living. On the other hand, anxiety is a natural adaptive mechanism that allows us to become alert, being the physiological response to stress. At times, the anxiety response system is overwhelmed and functions incorrectly, being anxiety disproportionate to the situation, and generating a deterioration in the psychosocial and physiological functioning of the individual. Anxiety disorders are, collectively, the most common psychiatric illness. Currently, more and more young students suffer from this disorder. Based on this premise, a study has been carried out to verify the relationship between the quality of sleep and the appearance of stress and anxiety. For this, an online survey has been carried out with students of legal age from different parts of Spain and who are studying at the university. The survey consisted in two parts, the first with questions related to sociodemographic variables that we consider could be related to the variables to be analysed, and the second with questions from the Pittbourgh subjective quality of sleep questionnaire and the State-Trait Anxiety questionnaire (STAI). A total of 291 students, 150 women and 141 men between 18-41 years old, has participated in this study. The conclusion reached was that women suffer from greater anxiety and have poorer quality of sleep than men. Contrary to expectations, social sciences students and law students suffer from the highest levels of anxiety but have the best quality of sleep. The fact of being away from the family nucleus, a bad study environment, living in a student residence, combining work with studies or having a dependent person in charge are factors that negatively influence anxiety and sleep; however, playing sports or having a pet are protective factors.

Supported by Junta de Extremadura (GR21042).

Keywords: anxiety, sleep quality, Pittsburgh, STAI

Correspondingauthor: Lourdes Franco, https://orcid.org/0000-0002-2695-5660

P2-09

Menstrual cycle disturbances following COVID-19 vaccination: a cross-sectional study in Spanish women with amenorrhea

¹María González, ²Miriam Al-Adib, ¹Ana B Rodríguez, ^1*Cristina Carrasco

¹ Neuroimmunophysiology and Chrononutrition Research Group. Faculty of Science. University of Extremadura. Badajoz, Spain

² Clínicas Miriam Gine. Obstetrics and Gynecology clinic, Almendralejo. Badajoz, Spain.

The COVID-19 pandemic has caused high infection and mortality rates in all countries of the world. Among preventive measurements, a vaccine has been developed in record time to halt the spread of the pandemic. However, the influence of the COVID-19 vaccine on women´s health has not been studied in clinical assays, particularly in women with amenorrhea. For this purpose, the main objective of this study was to determine the occurrence of menstrual cycle alterations in this population following vaccination. An anonymous online survey was completed by a total of 548 women with amenorrhea (18-69 years). Collected data included COVID-19 vaccination characteristics (vaccine brand name, date and number of doses) and menstrual cycle disturbances following COVID-19 vaccination (occurrence of menstruation, spotting, other menstrual bleeding, breast soreness, hot flashes, premenstrual cycle, and duration of the changes). McNemar mid-p test was used to compared the occurrence of disturbances between doses 1 and 2. After receiving the second dose of the vaccine, a significantly higher percentage of women experienced menstrual cycle disturbances (38.5% Dose 1 vs. 44.6% Dose 2; McNemar = 9.15; p = 0.002) and the concurrence of two or more symptoms (11.2 % Dose 1 vs. 15.3% Dose 2; McNemar = 15.53; p = 0.044) in comparison to the first one. Among the main disturbances, significant differences were detected between doses for the variables spotting (9.8% Dose 1 vs. 12.4% Dose 2; McNemar = 4.83; p = 0.054), breast soreness (6.5% Dose 1 vs. 10.2% Dose 2; McNemar = 4.83; p = 0.054) and abnormal menstrual bleeding (10.6% Dose 1 vs. 14.9% Dose 2; McNemar = 6.78, p = 0.010). It should be noted that experiencing hot flashes was more frequent after the first dose than the second one, although the statistical significance was marginal (8.6% Dose 1 vs. 6.5% Dose 2; McNemar = 3.57; p = 0.061). Therefore, further studies are needed to determine the possible relationship between vaccines and alterations in the menstrual cycle of women in general, and particularly in women with amenorrhea.

Supported by Junta de Extremadura-FEDER (BBB021; GR21042).

Keywords: women´s health, menstrual cycle, amenorrhea, COVID-19 vaccine.

Corresponding author: Cristina Carrasco, ORCID: 0000-0003-2763-4903.

P2-10

Influence of life habits on the menstrual cycle symptomatology of young women

¹Rosalía Rangel, ¹Ana B. Rodríguez, ^*1Cristina Carrasco

¹ Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain.

The occurrence of different symptoms derived from the menstrual cycle seriously affects the daily lives of many women in reproductive age. Adopting healthy lifestyle may help to improve the quality of life at different phases of the menstrual cycle. For this purpose, the main objective of this study was to determine the influence of life habits (physical activity and diet) and sleep quality on the symptoms of the menstrual cycle in young women. A total of 19 women (18-26 years) were enrolled in this cross-sectional study. Participants were evaluated during the three phases of three complete menstrual cycles according to symptomatology (using an android mobile application "My Calendar"), physical activity, diet, and sleep quality. At follicular phase, results showed that the intensity of most of the studied symptoms was significantly higher compared to luteal and ovulatory phases of the menstrual cycle; for this reason, a bivariate logistic regression analysis was performed focusing on the factors influencing symptomatology at this phase. Neither the life habits (physical activity and diet) nor the quality of sleep significantly influenced the menstrual symptoms in women with high symptomatology at the follicular phase. Additionally, the mood of the participants in terms of anxiety was compared between phases. Lower levels of anxiety were significantly registered at the ovulatory phase compared to the rest of the phases of the menstrual cycle. The lack of conclusive scientific evidence makes it necessary to carry out more studies to determine the lifestyle habits that may influence menstrual symptomatology in young women.

Supported by Junta de Extremadura-FEDER (BB021; GR21042).

Keywords: menstrual cycle, symptomatology, life habits, anxiety.

Corresponding autor: Cristina Carrasco, ORCID: 0000-0003-2763-4903.

P2-11

Renal physiology practical during COVID-19 pandemic: experimentation at home

^*1Margarita González-Vallinas, ¹Verónica García, ¹Jesús Fernández, ¹Elena Hernando-Pérez, ¹Enrique Pérez-Riesgo, ¹Ana Sánchez, ¹Javier García-Sancho, ¹Asunción Rocher, ¹Lucía Núñez

¹ Department of Biochemistry and Molecular Biology and Physiology, Faculty of Medicine, University of Valladolid, Spain.

Laboratory practical classes in health sciences represent a key teaching resource to promote student motivation and learning. However, the situation of the COVID-19 pandemic has complicated in-person activities, thus making necessary to propose alternative strategies that meet the same teaching objectives. In this work, we have modified the renal physiology practical of the second year of Medicine Degree, historically carried out in the laboratory, to make it suitable for students to perform at home. This practice aims to highlight the renal mechanisms that regulate volume, pH and osmolarity of body fluids, by analysing urine flow, pH and ion concentration after student ingestion of different solutions (water, isotonic sodium chloride solution or isotonic sodium bicarbonate solution). The data obtained from the new renal physiology practical (at home) adequately reflect the renal mechanisms of interest, being similar to those obtained from the practical performed in previous years (at the laboratory). This indicates that the students have correctly executed the practical at home, following the protocol and the materials provided. Moreover, the new version has been better accepted by the students since the participation was notably higher (94%) than in the laboratory version during the past three years (61-73%). In addition, adequate understanding by the students of the renal mechanisms analysed was illustrated by the high scores obtained in an online questionnaire developed for this purpose, in which a large majority of the students scored above 9. The new protocol designed to carry out the renal physiology practical at home has been proved to be useful for self-learning under similar conditions to the in-person laboratory version and received a much higher acceptance by the students. This “home” version of the practical could be useful for all studies that include renal physiology subjects, especially those taught by online learning modalities.

Supported by University of Valladolid (PID 123, 2020-21).

Keywords: renal physiology, teaching, medicine, practical.

*Corresponding author: Margarita González-Vallinas, https://orcid.org/0000-0003-4968-6675

P2-12

Combination of active methodologies as an effective way of teaching Physiology

*¹Alicia Valls, ¹Andrea Suárez, ¹David Verdú, ¹Solanye Guerra-Ojeda, ²Marta Serna-García, ¹María Dolores Mauricio, ¹Eva Serna.

¹Departamento de Fisiología,, Universidad de Valencia.

² Departamento de Odontología, Facultad de Ciencias de la Salud, Universidad Europea de Valencia.

The teaching of Human Physiology applied in the Degree in Physiotherapy at the Universitat de València is based on master lectures, practical classes, review sessions, seminars and multimedia material developed by the teaching team. The review sessions are carried out with gamification in the form of individual competition, while in the seminars, we apply an Escape Room in groups of 4 or 5 students. All these methodological strategies aim to be used to develop and assess critical thinking, problem-solving and self-assessment, demonstrate understanding of subject knowledge, and enable students to relate concepts from the different blocks of the course to one another. To assess the impact and suitability of these strategies on our students, we collected their opinion using a survey that was analysed afterwards. Students were asked to rate the different methodologies used during the course through a questionnaire consisting of 5-point Likert scale questions ranging from negative to positive scales (strongly disagree/disagree/neutral/agree/strongly agree) and open-ended questions. On the other hand, they could also evaluate each methodology by a 1 - 10 qualification. A sum of forty-one students participated in the survey. Results showed that their grade in the subject was moderately high. In detail, the marks they gave for each item were 7.66 for the master lectures, 8.63 for the practical classes, 8.12 for review sessions, 7.80 for seminars, and 8.20 for the multimedia material. Our results indicated that the favourite methodology for 46% of the students was the practical class, followed by the seminars (23%). They remarked that both methodologies were interactive and participative. In addition, the survey revealed that the master lectures were essential for transmitting knowledge quickly and efficiently. In conclusion, all the methodologies used are valid and complementary, helping the students to relate concepts and internalise them. The students consider the combination of these methodologies as a whole to be a useful and effective way of studying physiology and do not want to suppress any of them.

Keywords: teaching Physiology, active methodologies, gamification.

Corresponding author: Alicia Valls, https://orcid.org/0000-0001-5653-9409

P2-13

Current strategies to develop antitumor Chimeric Antigen Receptor (CAR)-T cell therapies from donor T cells: a systematic review

^1,2Carlos Acebal, ^*1,2Margarita González-Vallinas

¹ Unit of Excellence Institute of Biology and Molecular Genetics (IBGM), University of Valladolid (UVa)-CSIC, Valladolid, Spain.

² Department of Biochemistry and Molecular Biology and Physiology, Faculty of Medicine, University of Valladolid, Spain.

CAR-T cells are novel antitumor therapies consisting of T cells that express a chimeric antigen receptor (CAR), which enables the recognition of specific tumor antigens. Until now, CAR-T cell therapies approved for commercialization use the patient's own T cells as starting material (autologous use). Although these therapies have demonstrated impressive clinical results in cancer treatment, they have some limitations, such as high production costs and time, difficulties in standardizing the manufacturing process, and production failures due to patient T cell dysfunction. These drawbacks could be overcome by producing CAR-T cell therapies from healthy donor T cells (allogeneic use); however, this entails the risk of graft-versus-host disease (GvHD) and therapy rejection, two challenges that must be handled in the design of the manufacturing procedure. In this work, we present a systematic review of the different strategies studied for the development of allogeneic CAR-T cell therapies, most of them reported during the last five years. We found that all the strategies identified could be divided into two main groups: those that include genetic modifications (in addition to CAR integration) and those based on the use of alternative sources or subpopulations of T cells, such as umbilical cord blood-derived T cells or virus-specific T cells, among others. Moreover, some studies combined both T cell source/subpopulation selection and genetic modification to further improve allogeneic CAR-T cell therapies. Additional preclinical and clinical data will help to determine the most convenient strategy to develop allogeneic CAR-T cell therapies, which will make this promising antitumor treatment available to a larger number of patients.

This work has been supported by Beca Consejo Social (academic year 2021-22), University of Valladolid (to C.A.).

Keywords: CAR-T cells, allogeneic, cancer treatment, systematic review

^*Corresponding author: Margarita González-Vallinas, https://orcid.org/0000-0003-4968-6675

P2-14

Effects of testosterone on the seminal vesicles in rats

^*G Blanco, M López, N Pintado, ML Villarino

Physiology Department, Science Faculty, Universidad de Extremadura (Badajoz)

Testosterone is a male sex hormone with anabolic properties and one of s target organs is the seminal vesicles. The objective of this study is to analyse the effect of testosterone on the functionality of the seminal vesicle of male rats. A number of 54 male Wistar rats were used, of which 26 were used as control group (O) and 28 were given testosterone (O+T). Firstly, a surgical intervention was carried out in which the testicles were removed from all animals, then those to be treated were injected with a solution of testosterone in sunflower oil (0.2 mg/day) for seven days. A second surgical intervention was performed in which the seminal vesicles were removed. It should be noted that during the surgical interventions, rats were anesthetized with isoflurane Finally, those are weighed on a scale, along with the corresponding animal weighed in kilograms. Our results show that administration of testosterone (O+T group) have a mean weight of 0.22 ± 0.02 kg which then, related to the mean weight of the vesicles in this group (0.81 ± 0.51 g), presented a mean relative weight of 3.13 ±1.02 g/kg body weight. Meanwhile, the animals belonging to the control group (O) presented a mean rat weight of 0.23 ± 0.04 kg and a mean vesicle weight of 0.08 ± 0.02 g, concluding in a mean relative weight of 0.34 ± 0.08 g/kg body weight. Therefore, we can conclude that testosterone produces an increase in the weight of the seminal vesicles (causing hypertrophy) since this hormone stimulates the secretion of the products of the seminal vesicles. So, those animals that have not been treated with this hormone present vesicles considerably smaller in size than those that have been treated.

Keywords: testosterone, orchidectomy, seminal vesicle, male rat.

Corresponding author: G Blanco

P2-15

Mobile phone use and its effects on anxiety levels and sleep quality in young people (18-25 years old)

^*1Elena Fernández-Delgado, ¹Ana B. Rodríguez.

¹ Department of Physiology (Neuroimmunophysiology and Chrononutrition Research Group), Faculty of Science, University of Extremadura, Badajoz, Spain.

Since the director of Motorola, Martin Cooper, made that first call from a mobile phone back in 1973, the evolution of this device has been astonishing in last decades, ceasing to be just an easy way to make calls to a little computer of the size of your hand. Its use and availability have increased too, going from being a luxury to an essential. Furthermore, young people of 18 to 30 years are one of the groups where its use has increased quicker. Although it is important to remind that the age of acquisition of a mobile phone is getting smaller, even reaching the 9-10 years. On the other hand, main threaten at these ages use to be more focus on psychological disorders than on physical ones, being anxiety and depression the most common ones, which can often be associated with a bad sleep quality. Therefore, the objective of this study is to evaluate how the use of mobile phones affects young people, determining their anxiety levels, mobile addiction, and sleep quality. Moreover, we also wanted to check if any difference between sexes were found. To do that, a representative population of 195 people from 18 to 25 years was studied. They filled out an anxiety questionnaire, STAI (State - Trait Anxiety Inventory), a mobile phone addiction test and Pittsburg sleep quality index. It could be observed significative differences when compare women (N=121) versus men (N=74) for trait-anxiety levels (general anxiety) and quality of sleep, being both worst in women. On the other hand, it wasn’t observed significant differences for state-anxiety (anxiety in a particular moment) and for mobile phone addiction. Nevertheless, it is important to highlight that for both women and men, a high dependence to the mobile phone was founded, being more than 20 points up to the Spanish mean. In conclusion, young people have a strong addiction to mobile phone regardless of their sex, but higher anxiety levels and worst quality of sleep was found for women in comparison with men.

This work was supported by Junta de Extremadura grants (ref. GR21042, PD18020).

Keywords: Mobile phone, anxiety, sleep quality, students

Corresponding author: Elena Fernández-Delgado, https://orcid.org/0000-0001-6987-6586

P2-16

Effects of dietary supplements in assisted reproduction

^#Sara Matamoros, ^*Javier Espino, Ana B Rodríguez

Department of Physiology (Neuroimmunophysiology and Chrononutrition Research Group), Faculty of Science, University of Extremadura, Badajoz, Spain

^# Presenting author

Fertility rates in Spain have been progressively decreasing over the last 10 years. It is estimated that currently 15-17% of the population of childbearing age suffers from some type of infertility. This generates socioeconomic problems at the population level and psychological problems at the individual level, which is the cause of a current increasing demand for assisted reproductive techniques (ART), including artificial insemination (AI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Although ART have been developed and improved over the years, their success rates are not very high. Nutrition is an essential factor in fertility, in both the male sex and the female sex, and therefore the use of certain dietary supplements in addition to ART can improve the outcomes of ART. The most relevant supplements in this regard are omega-3 fatty acids, folic acid, vitamins B12, D, C and E, zinc, selenium, melatonin, myo-inositol and coenzyme Q₁₀. The present study addresses the main beneficial effects of supplementation with these micronutrients on fertility in men and women undergoing ART.

This work was supported by Junta de Extremadura (GR21042). J.E. holds a post-doctoral fellowship from Junta de Extremadura (TA18002).

Keywords: assisted reproductive techniques (ART), dietary supplements, female infertility, male infertility.

^*Corresponding author: Javier Espino, https://orcid.org/0000-0002-8549-9343

P2-17

Use of adenosine deaminase (ADA) as a biomarker for lameness and growth speed in pigs

¹Lisette Ramírez, ^1,2Raquel Romar, ^1,2Jon Romero-Aguirregomezcorta, ^1,2Sebastián Cánovas, ³Alba Ortín, ^1,2Pilar Coy

¹ Department of Physiology, Faculty of Veterinary Medicine, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain.

² Institute for Biomedical Research of Murcia (IMIB), Murcia, Spain.

³ Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain.

Adenosine deaminase 2 (ADA) is an enzyme considered a biomarker of the immune system, which is found in serum, saliva, and lymphoid tissue. A direct relationship between ADA in saliva and the immune status of the animal has been shown, but there are few studies on the use of ADA as a biomarker for growth rate or lameness. Here, a colony of pigs (N=27, 22 females and 5 males, Large-White x Landrace living in semi-free conditions in a sanctuary located at the Teaching Farm of the University of Murcia (Spain) was used. From birth to the present, their individualized growth curves were recorded. At 3.5-year age, saliva samples were taken to detect ADA, and the animals suffering from lameness, one of the most frequent pathologies in the pig sector, were identified. ADA measurement was performed using a commercial automated spectrophotometric assay (Diazyme Laboratories, Poway, CA, USA) adapted to the Olympus AU600 analyzer, a validated method for pig saliva. Statistical analysis was performed in ``R´´ version 4.0.3 (R Core Team 2020). The variables used for the analysis were ADA2 values in saliva and presence/absence of lameness at the age of 3.5 years, and average daily weight gain (ADG) () at different age points. Spearman's correlation coefficient (s) was used to measure the correlations between the variables. The results showed a negative correlation (s=-0.4) between ADA values and ADG in the period between days 45 and 90. These ADA values could indicate a greater predisposition to diseases in animals with low ADG at that time due to a worse immune status. No correlation with the ADA values was found for the incidence of lameness (s=-0.19). However, a strong relationship was observed between ADG in 90-135 days and the incidence of lameness at 4 years of age (s=0.52). Likewise, positive correlations were observed between ADG at 0-45 days (s=0.45), 45-135 (s=0.46) and 0-135 days (s=0.47) and the incidence of lameness at 4 years. These data indicate that rapid growth rate during the first 6 months of life predisposes animals to lameness in adulthood.

Supported by PID2020-113366RB-I00 funded by MCIN/AEI/10.13039/501100011033/ and “FEDER Una manera de hacer Europa”.

Keywords: Adenosine deaminase (ADA), pig, growth, lameness

Corresponding author: Pilar Coy Fuster, https://orcid.org/0000-0002-3943-1890

P2-18

New generation of cosmetics components

¹Carmen Ramiro, ¹Ana B Rodríguez, ²Lourdes Franco

¹ Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain and ² Department of Physiology, Faculty of Medicine and Health Science, University of Extremadura, Badajoz, Spain.

Skin is the largest organ in the human body and along with the skin appendages, performs important functions of barrier and protection against pathogenic agents, reception of stimulus and maintenance of homeostasis in the organism. The growing interest of today’s society in the use of cosmetic products made from natural components, along with concern about personal appearance and skin aging, has increased the number of investigations that try to find aternatives for synthetic chemical compounds, widely included in cosmetics that can be harmful to consumer’s skin. As a result of this bibliographic review, a great diversity of research has been found on natural components derived from algae, marine sponges, plants, foods, fungi and bacteria with the potential to be added to cosmetic formulations due to their anti-aging, antioxidant and anti-pigmenting properties. This project aims to offer an updated review of the most innovative components used in the cosmetic industry and the scientific evidence that supports the benefits derived from those components.

Supported by Junta de Extremadura (IB18013).

Keywords: skin aging, photoaging, natural cosmetics, nutricosmetics

Corresponding author: Carmen Ramiro

P2-19

Effects of physical exercise on the cholinergic system in the 3xTg-AD model for Alzheimer's disease

¹Jesús Andrade, ¹Nayeli Barrón, ²Luis Oskar Soto-Rojas, ¹Erika Orta, ^*1Sofía Diaz Cintra

¹ Instituto de Neurobiología, UNAM Campus Juriquilla, Querétaro, México

² Facultad de Estudios Superiores de Iztacala, UNAM, Estado de México, México

Alzheimer's disease (AD) is the most common dementia and neurodegenerative disease in the world and is characterized by a cognitive impairment with symptoms like memory loss, disorientation, and learning problems, affecting the quality of life of those who suffer from it. In addition, to its two main histopathological characteristics: neuritic plaques and neurofibrillary tangles, the cholinergic system is also affected. This system is widely related to cognitive processes therefore its neurodegeneration during the pathology arises as a possible hypothesis for the development of the disease. Among the non-pharmacological treatments is physical exercise, which is a type of physical activity characterized by being planned, structured and repetitive, proving to be safe as part of the treatment of the disease, however, some of the underlying mechanisms still are in the study. One of the most widely used animal models to study the mechanisms of the disease is the triple transgenic 3xTg-AD mouse which overexpresses three human proteins (PS1M146V, APPSWE, and tauP301L) involved in the disease. The aim of this study was to analyse the effect of physical exercise on the cholinergic system in the 3xTg-AD model for AD. A total of 40 10-month-old male and female mice, 3xTg-AD (n=20) and NoTg (n=20) equally divided into exercise and sedentary groups were used. An intervention of voluntary physical exercise was carried out for 2 months with a frequency of 5 times a week. Subsequently, working memory was evaluated using T Maze test and the gait with the footprint measures. The anatomical analysis of the cholinergic system was made for the number of the somas localized immunohistochemically in two cholinergic nuclei: Meynert's basalis and medial septum. Our results prove an improvement in cognitive capacity, as well as preservation of the neurons of the cholinergic system, without changes in motor capacities. Intervention with voluntary physical exercise favors cognitive abilities, through preservation of the cholinergic system associated with AD in symptomatic stages of the disease.

Keywords: Alzheimer, exercise, acetylcholine, cognition.

Corresponding author: Sofía Y Diaz (yoldi@unam.mx), ORCID: 0000-0002-3354-2717

P2-20

Electrolysis technique on the muscle damage induced by notexin in rats

^1,2Juan Campos-Campos, ^1,2Adrian Jorda, ¹Carlos Colmena, ¹Ignacio Campo-Palacio, ¹Kenia Alvarez-Gamez, ^*1Soraya L Valles

¹ Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

Muscle injury caused by exercise generates an inflammatory process in the muscle. This inflammation can be controlled with the application of EPI (percutaneous inter-tissue electrolysis) technique that will trigger an anti-inflammatory process which will favor the recovery period. This technique is used in the different fields of health sciences, applied by physiotherapists, podiatrists and rehabilitators. This study was carried out using female Wistar rats distributed in three different groups: control (C), notexin (NOT) and notexin with PIE (NOT+PIE6). We used PIE technique, based on electrical stimulation with a continuous current of 4 pulses at an intensity of 6 mA for 5 seconds, and applied to the quadriceps of a rear leg. We analysis motor functions and our tests detected an increase in time and foot faults when crossing a beam in the NOT group compared to C group. Furthermore, a significant decrease in both variables was detected in NOT+PIE6 compared to NOT group. Muscle power was measured with a grip strength test, obtaining far better performances in NOT+PIE6 rats when compared to NOT rats. Moreover, the PIE technique prevented the increase of pro-inflammatory proteins, IL-6 and chemokines CCL3, CCL4, and CCL5 with their receptor CCR5, induced by notexin in the quadriceps. At the same time, the study evidenced a decrease in both CCR8 and NF-κB expressions after PIE treatment. On the other hand, we obtained evidence that demonstrated anti-inflammatory properties of PIE technique, thus being the increase in IL-10 and IL-13 in NOT+PIE6 group compared to NOT group. In conclusion, these results support the idea that PIE technique improves muscle recovery after toxic damages, which would justify its employment.

Financial support from the University of Valencia. (Specific Key 20180268).

Keywords: percutaneous inter-tissue electrolysis, motor function and balance, inflammation, muscle recovery.

Corresponding author: Soraya L. Valles, https://orcid.org/0000-0003-4861-4266

P2-21

Rho-GTPases in chick inner ear development

¹Sergio Villa-Carballar, ¹Antuca Callejas Marin, ²Joaquín Rodríguez-León, ^3,4José L Ferrán, ¹Luis O Sánchez-Guardado, *¹Matías Hidalgo-Sánchez

¹ Department of Cell Biology, Faculty of Science, University of Extremadura, Badajoz, Spain; ² Department of Human Anatomy and Embriology, Faculty of Medicine, University of Extremadura, Badajoz, Spain; ³ Department of Human Anatomy and Psychobiology, School of Medicine, University of Murcia, Murcia, Spain; and ⁴ Institute of Biomedical Research of Murcia-IMIB, Virgen de la Arrixaca University Hospital, Murcia, Spain

The Guanosine Triphosphate (GTP)-binding Proteins of the Rho family (Rho-GTPases) regulate numerous processes in all eukaryotic cells. Members of the Rho-GTPase family are key regulators of signals that determine changes in the actin cytoskeleton required for adhesion, motility, cytokinesis and membrane trafficking, as well as signaling that regulates the growth of the neural axon cone or any cell extension. They function as molecular switches by alternating between an inactive state when bound to GDP and an active state when associated to GTP. Our work focuses on the study of the possible role of some Rho-GTPases (RhoA, RhoB, and RhoU) in the avian inner ear development. The inner ear is a complex three-dimensional sensory structure with auditory and vestibular functions. It originates from the otic placode, a thickened transitional portion of the cephalic ectoderm on both side of the developing hindbrain. The otic placode invaginates and subsequently detaches from the ectoderm to generate the otocyst, an ovoid vesicular structure physically independent of the ectoderm from which it originates. The otic epithelium will give rise to all sensory and non-sensory elements of the adult membranous labyrinth, as well as otic neurons of the vestibular and auditory ganglia. Our studies of the expression patterns of the RhoA, RhoB and RhoU genes show that these genes would be involved in the specification of the sensory and non-sensory elements of the avian inner ear, in addition to regulating otic neurogenesis. In particular, RhoA expression delimits the presumptive territory of almost all developing sensory elements, whereas RhoB expression stains some non-sensory elements such as the developing endolymphatic apparatus and some aspects of the growing semicircular canals. The RhoU expression is very similar to that described for RhoA gene, except for relevant low levels of expression in the developing macula sacculi and macula neglecta. The expression patterns of RhoA, RhoB, and RhoU genes strongly suggest that they could also be involved in the formation of the vestibular and auditory ganglia. These studies allow experimental works to be carried out, both in ovo and in vitro, using drugs that block or activate the function of these Rho-GTPases.

Funding: (1) Junta de Extremadura, GR21167; (2) Junta de Extremadura, Fondo Europeo de Desarrollo Regional, “Una manera de hacer Europa”, IB18046; (3) PGC2018-098229-B-100: and (4) Séneca Foundation (19904/GERM/15).

Keywords: Otic epithelial specification, otic neurogenesis, otic vesicle, sensory elements.

Corresponding author: *Matías Hidalgo-Sánchez, mhidalgo@unex.es, https://orcid.org/0000-0001-8573-8829.

P2-22

Influence of 12-week concurrent training on exosome cargo and its correlation with cardiometabolic health parameters in men with obesity: A pilot study

^*1Brisamar Estébanez, ²Francisco Amaro-Gahete, ³Cristina Gil-González, ¹Marta Rivera-Viloria, ¹Javier González-Gallego, ³David Jiménez-Pavón, ¹María J Cuevas

¹ Institute of Biomedicine (IBIOMED), University of León, León, Spain. ² Department of Physical Education and Sports, Faculty of Sport Sciences, University of Granada, Granada, Spain. ³ MOVE-IT Research Group, Department of Physical Education, Faculty of Education Sciences University of Cádiz, Cádiz, Spain.

Obesity has become the biggest pandemic in today's societies, with the increasing risk to develop obesity-associated diseases, such as type 2 diabetes or cardiovascular diseases. Exosome release and cargo varies depending on the physiological state of the cell, so that they could play a fundamental role in obesity. The beneficial effects that exercise has both on weight loss and cardiovascular health parameters in obese is well known, may occur through intercellular communication mediated by exosomes released in response to exercise. This study was to investigate the role of a 12-week concurrent training (CT) in modifying the levels of plasmatic exosome markers and proteins in obese patients and explore their relationship with the exercise-promoted cardiometabolic changes. Nine adult male obese patients were randomly divided into a control group (n=4) and a training group (n=5), which completed 36 sessions of aerobic and strength exercises. Before and after the training period, cardiometabolic health parameters were evaluated and blood samples were drawn. Exosomes were isolated from plasma by ultracentrifugation plus 0,2 μm filtering and exosome markers (CD9, CD63, CD81, Flot-1, GAPDH and LAMP2) and carried proteins (b-actin, CD14, HSP60, HSP70, LAMP-1, VDAC1) were evaluated by Western blot. No significant changes were observed in the levels of any of exosomal markers and proteins, nor in the cardiometabolic parameters analyzed in response to CT. However, associations in CT-induced changes between CD81 and both fat mass and weight, Flot-1 and VO2 max, HSP70 and both CRP and left ventricle diastolic diameter, or CD14 and leptin were found. Although the current CT was no able to modify the exosome cargo or cardiometabolic parameters, associations between the CT-promoted changes in cardiometabolic parameters and exosome-carried proteins could indicate a relationship to be considered for future treatments in patients with obesity. However, further investigation is needed to broaden the knowledge about the modifications in exosome cargo and release in response to different exercise modalities in the obese population.

Keywords: aerobic and resistance training, cardiometabolic health, extracellular vesicles, obesity.

^*Corresponding author: Brisamar Estébanez, https://orcid.org/0000-0001-5034-9508.

P2-23

Modifications in circulating cfDNA, TLR4- and SOD1-related inflammatory and oxidative responses in young subjects after 10-week eccentric training

^*1Brisamar Estébanez, ²Sergio Maroto-Izquierdo, ¹Marta Rivera-Viloria, ¹José A de Paz, ¹Javier González-Gallego, ¹María J Cuevas

¹ Institute of Biomedicine, University of León, León, Spain.

² Department of Health Sciences, European University Miguel de Cervantes, Valladolid, Spain.

Unaccustomed eccentric exercise, especially overload protocols, has been documented to produced muscle damage. This acute response results in the release of specific muscle proteins into the circulation that could induce inflammatory responses, stimulating leukocytes, macrophages, muscle cells or fibroblasts, along with increased antioxidant capacity to counteracts exercise-evoked circulatory oxidative stress. However, excessive or prolonged response could lead to muscle fibrosis by the impediment of satellite cells migration and fusion to form the regenerated muscle fiber as a consequence of the impairment in the connective tissue production and deposition. This study was to evaluate the inflammatory and oxidative stress responses to a middle-term eccentric training in the peripheral blood mononuclear cells (PBMCs) of young subjects. Twenty-eight physically active young males (20.25±0.423 years) were randomly distributed into a control group (CG; n=10), a training group (TG; n=9), at submaximal load (90% one-repetition maximum (1RM) for the eccentric phase), or an overtraining group (OG; n=9), at supramaximal load (120% 1RM), to complete 20 sessions of unilateral leg press over 10 weeks for the dominant leg. Blood samples were obtained the week prior and after the training protocol and PBMCs, plasma, serum and plasmatic exosomes were isolated for Western blot (TLR4, NF-kB, NRF2 and SOD1) and cfDNA analysis. Results exposed a time by group effect in TLR4 expression (p=0.040), showing an increase in the OG after training (p=0.015), along with a time effect in SOD1 (p=0.041) and exosomal cfDNA levels (p=0.017). Post-exercise increases were found in the CG for exosomal and serum cfDNA (p=0.019 and p=0.027, respectively), but not in the exercised groups. No significant time by group effect or post-training modifications were observed in NF-kB, NRF2 and plasma cfDNA. Our findings indicate that the current eccentric training modulates the inflammatory and antioxidant responses in circulatory immune cells. The increases in exosomal and serum cfDNA in CG, together with the non-significant decrease in the trained groups, could imply a decrease in the damage-related biomolecules release as a physiological adaptation in the damage-inflammation-regeneration/remodeling process. Given the lack of significant results in the NF-kB and NRF2 expression, it is necessary to further investigate their nuclear translocation.

Keywords: cfDNA, eccentric exercise, inflammation, oxidative status.

^*Corresponding author: Brisamar Estébanez, https://orcid.org/0000-0001-5034-9508.

P2-24

Cosmetics and sustainability in skin health

^*1Irene García, ¹Ana Beatriz Rodríguez, ²Lourdes Franco

Department of Physiology, Faculty of Medicine and Health Science, University of Extremadura, Badajoz, Spain; and ² Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain

Over the past 50 years, the reach of cosmetic companies has become global, and the understanding of its underlying scientific principles has sharpened and expanded. The largest challenge in cosmetic products nowadays is the sustainability of obtaining the materials that compose it. When idealizing and developing a new cosmetic product, two contradicting challenges must be considered: the increased material demand and the limited supply of resources. The growing economic aspect of the cosmetic industry over the last few decades has thus fueled the need for harmless and efficient natural raw ingredients for these products. One field that has large potential for future development in terms of cosmetic science is that of marine bioresources. Algal species are now actively and proficiently being incorporated in diverse cosmetics as a reliable organic ingredient, also adding value to these products. Due to their abundance in beneficial metabolites, unicellular microalgae, such as Chlorella vulgaris, have gained special attention, due to their richness in compounds such as polysaccharides and pigments, which have the ability to enhance the health of the skin by acting as anti-ageing, antioxidant, anti-inflammatory and collagen-boosting agents Due to their beneficial potential for the skin, microalgae are therefore a source of raw material for one of the most promising and lucrative sectors of the biotechnological industry (cosmetics industry), suitable for all skin types and sustainable.

Supported by Junta de Extremadura (GR21042).

Keywords: Marine bioresources, Chlorella vulgaris, cosmetic, sustainability

Corresponding author: Irene García, https://orcid.org/0000-0002-2695-5660

P2-25

Long-term exposure to isoflavones compromise sperm quality in adult male Wistar rats

¹Sara Cáceres, ¹Belen Crespo, ¹Gema Silván, ¹Maria Josefina Illera, ²Angela Alonso-Diez, ²Paloma Jimena de Andrés, *¹Juan Carlos Illera.

¹ Department of Physiology, Faculty of Veterinary Medicine, University Complutense of Madrid, Spain, and

² Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University Complutense of Madrid, Spain.

Isoflavones are compounds present in vegetables such as soybeans. They have a similar structure to endogenous estrogens, giving them the ability to bind with high affinity to the estrogen receptor affecting hormonal modulation, so they are considered endocrine disruptors. Due to soy consumption is increasing in Western society, the present study aims to observe the effects of prolonged exposure to isoflavones in adult male rats. To this purpose, fifty male rats of 90 days were exposed to a mixture of isoflavones in low doses (17 mg/kg/day genistein + 12 mg/kg/day daidzein) and high doses (170 mg/kg/day genistein + 120 mg/kg/day daidzein) dissolved in drinking water for 6 months. A control group of twenty-five animals was used. During the experiment, blood samples were collected from the dorsal aorta. Each month, 5 rats from each group were humanely euthanized. Immediately after, at necropsy, the epididymis was collected to perform sperm quality tests (sperm count, sperm motility, sperm viability and membrane integrity), in addition to the testicles for homogenization. Testosterone, dihydrotestosterone, 17β-estradiol, and estrone sulphate levels were analyzed in blood samples and testicular homogenates using enzyme-immunoassay techniques. The experimental protocols adhered to the guidelines of the Council of European Union and were approved by the Institutional Animal Care and Use Committee of the University Complutense of Madrid, and Animal Protection Area of the Community of Madrid, Spain (Ref: PROEX 175/19). The results showed that in low and high doses groups, motility and sperm count significantly decrease compared to control group, while sperm viability and membrane integrity are also compromised at low doses and significantly decreased at high doses. Hormonal results revealed decreased testosterone and dihydrotestosterone levels in both serum and tumor homogenates, while 17β-estradiol and estrone sulphate levels were augmented. These results demonstrate that the consumption of isoflavones at low and high doses over a long period of time increased estrogen levels in males compromising sperm quality by decreasing the concentrations of circulating and testicular androgens.

Keywords: Isoflavones, Androgens, Estrogens, Sperm Quality.

Corresponding author: Juan C Illera. https://orcid.org/0000-0002-9178-3121

P2-26

Melatonin counteracts hypoxia-induced activation of pancreatic stellate cells resolving oxidative stress

¹Matías Estarás, ¹Cándido Ortiz-Placin, ¹Antonio González

¹ Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain.

Pancreatic stellate cells (PSCs) are major players in the fibrosis that develops in pancreatic cancer. Due to the rapid growth of the tumoral tissue, a hypoxia condition is established, under which cells included in the mass can proliferate. Additionally, melatonin has gained attention as an agent with therapeutic potential against pancreatic cancer. The anti-inflammatory and anticancer activity of melatonin have been well documented, but the antifibrotic action of this indolamine was less known. In this study we have investigated the effect of hypoxia (1% O₂) on PSCs physiology and whether melatonin exerts any modulatory action. PSCs cultures were subject to hypoxia and melatonin treatment (1 mM-1 μM). PSCs proliferation was evaluated by crystal violet assay. The expression of smooth muscle α-actin (α-sma) and collagen I deposition were studied by western blot and confocal analysis respectively. Oxidative stress markers were studied by fluorescence and spectrophotometric techniques and HPLC. The status of the enzymes studied was analysed by RT-qPCR. Melatonin treatment reduced the proliferation of PSCs and the increase of the expression of α-sma and collagen I evoked by hypoxia. Moreover, lipid peroxidation and protein oxidation were diminished by melatonin treatment under hypoxia. The compound increased the expression of several antioxidant enzymes. We conclude that the prooxidant environment evoked by hypoxia acts like an activator for PSCs. Melatonin, a powerful antioxidant, resolves this pro-oxidant condition and reduces the activation of PSCs. Therefore, melatonin could be taken into consideration as potential therapeutic agent for pancreatic fibrosis.

Suported by Junta de Extremadura-FEDER (GR21037), Ministerio de Ciencia e Innovación (EQC2018-004646-P; EQC2019-005660-P) and Valhondo Calaff Foundation.

Keywords: pancreatic stellate cells, melatonin, fibrosis, oxidative stress

Corresponding author: Matías Estarás (meh@unex.es), https://orcid.org/0000-0003-3462-7904

P2-27

Identification of neuropilin-1 as a novel biomarker on prognosis and invasive-related features in colorectal and liver cancer patients

^*1,2Paula Fernández-Palanca, ^1,2Tania Payo-Serafín, ^1,2Flavia Fondevila, ^1,2Carolina Méndez-Blanco, ^1,2Beatriz San-Miguel, ^2,3Marta R Romero, ^1,2María J Tuñón, ^2,3Jose JG Marin, ^1,2Javier González-Gallego, ^1,2José L Mauriz

¹ Institute of Biomedicine (IBIOMED), University of León, León, Spain

² Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institute of Health Carlos III, Madrid, Spain

³ Experimental Hepatology and Drug Targeting (HEVEPHARM), Salamanca Biomedical Research Institute (IBSAL), University of Salamanca, 37007 Salamanca, Spain

Neuropilin-1 (NRP1) is a transmembrane protein closely related to tumor progression and poor prognosis through modulating cellular processes including cell survival, angiogenesis and migration. Colorectal cancer (CRC) and liver cancer, mainly constituted by hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), are the second and third causes of cancer-related death, respectively, and, despite recent advances in the therapeutic landscape, high mortality and recurrence rates are major challenges that need to be addressed. Here, we evaluated the prognostic, diagnostic and clinicopathological value of NRP1 in both CRC and liver cancer. A systematic review and meta-analysis were conducted to determine the association of NRP1 expression with survival parameters, tumor development and invasion-related clinicopathological features in human patients with CRC or liver cancer. The study protocol was registered in PROSPERO (CRD42022307062). We included articles from five different databases published from inception to January 31^st, 2022. To evaluate the overall effect size, hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were extracted or estimated by Parmar method and pooled. The chi-square-based Q test and I² statistic were used to detect the presence of heterogeneity among studies, identifying its source by meta-regression and subgroup analysis. Publication bias was analyzed by funnel plot asymmetry and Egger’s test. Statistical meta-analysis was performed with STATA 16 software. After literature search, 15 potential studies, comprising 3407 patients, were identified and included in the meta-analysis. A significant correlation was found between NRP1 overexpression and lower survival in liver cancer patients, observing also a strong association with tumor development in HCC patients. Moreover, high levels of NRP1 were significantly correlated with vascular invasion in liver cancer and with metastasis in both CRC and liver cancer patients. Altogether, these results report that NRP1 overexpression seems to be strongly associated to shorter patient survival, tumor pathogenesis and increased risk of vascular invasion and metastasis in CRC and liver cancer patients, which supports the potential role of NRP1 as prognostic and diagnostic biomarker in both tumor types, CRC and liver cancer.

Supported by the project PID2020-119164RB-I00 (MCIN/AEI/10.13039/501100011033).

Keywords: colorectal cancer, liver cancer, neuropilin-1, prognosis

Corresponding author: José L. Mauriz, https://orcid.org/0000-0003-3160-8599

P2-28

Role of the hypoxia-derived response and autophagy interplay in the loss of lenvatinib sensitivity: Neuropilin-1 as a potential therapeutic target in human hepatocellular carcinoma

^*1,2Paula Fernández-Palanca, ^1,2Tania Payo-Serafín, ^1,2Beatriz San-Miguel, ^1,2María J Tuñón, ^1,2Javier González-Gallego, ^1,2José L Mauriz

¹ Institute of Biomedicine (IBIOMED), University of León, León, Spain

² Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institute of Health Carlos III, Madrid, Spain

Hepatocellular carcinoma (HCC) constitutes one of the most frequent and deadly cancer worldwide. Despite recent advances in the treatment landscape, tumor cells often develop chemoresistance against targeted drugs such as lenvatinib, where the double-edged process of autophagy and the hypoxia-derived response have been strongly associated to the loss of therapeutic effectiveness. Neuropilin-1 (NRP1) is a co-receptor of proteins highly involved in tumor progression, emerging as an interesting target in several tumor types. We aimed at assessing the role of NRP1 on the mechanisms underlying lenvatinib efficacy under hypoxia in an in vitro model of human HCC. We employed two human HCC cell lines Huh-7 and Hep3B, CoCl₂ as hypoximimetic, lenvatinib for cell treatment, and bafilomycin A1 (Baf) as specific autophagy inhibitor. Protein expression was assessed using Western blot and immunocytochemistry, and cell viability by MTT, nuclear Ki67 immunofluorescence and colony formation assay. For gene silencing, a pool of NRP1 or HIF1A siRNA was employed for cell transfection, and autophagy was evaluated by acridine orange staining. GraphPad Prism 8 was used for statistical analysis, considering significance when p<0.05. Lenvatinib showed a dose-dependent efficacy represented by lower cell viability of Huh-7 and Hep3B cells along with a decreased NRP1 protein expression. To determine the underlying mechanism of this NRP1 downregulation, we combined lenvatinib and Baf. Lenvatinib promoted an autophagy-dependent degradation of NRP1 and its blockage prevented it and diminished lenvatinib effectiveness. Interestingly, hypoxia induction also reduced NRP1 levels and induced autophagy, being again prevented by Baf treatment. Hypoxia-inducible factor 1α (HIF-1α) silencing led to NRP1 downregulation and prevented the recovery of NRP1 expression when autophagy was blocked. Additionally, although autophagy abrogation partially restored cell viability, HIF-1α silencing avoided this effect. Finally, although autophagy blockade restored NRP1 expression and cell viability even in presence of lenvatinib and hypoxia induction, HIF-1α silencing successfully prevented these effects. Altogether, these results suggest that NRP1 exerts a key role in the loss of therapeutic efficacy of lenvatinib associated to the autophagy modulation by the HIF-1α-derived hypoxia response, highlighting the potential use of NRP1 as therapeutic target in human HCC.

Supported by the project PID2020-119164RB-I00 (MCIN/AEI/10.13039/501100011033).

Keywords: hepatocellular carcinoma, hypoxia, lenvatinib, neuropilin-1

Corresponding author: José L. Mauriz, https://orcid.org/0000-0003-3160-8599

P2-29

Forkhead box O3 upregulation accounts for cytoprotective autophagy activation and sorafenib resistance acquisition in human hepatocellular carcinoma

^1,2Flavia Fondevila, ^1,2Carolina Méndez-Blanco, ^1,2Paula Fernández-Palanca, ^*1,2Tania Payo-Serafín, ³Jos van Pelt, ³Chris Verslype, ^1,2Javier González-Gallego, ^1,2José L Mauriz

¹ Institute of Biomedicine (IBIOMED), University of León, León, Spain

² Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institute of Health Carlos III, Madrid, Spain

³ Laboratory of Clinical Digestive Oncology, Department of Oncology, Leuven Cancer Institute (LKI), KU Leuven and University Hospitals Leuven, Leuven, Belgium

Sorafenib resistance is responsible for the poor prognosis of hepatocellular carcinoma (HCC), and autophagy has been shown to promote or counteract chemosensitivity. The transcription factor forkhead box O3 (FOXO3) has been indicated to regulate autophagy in several cancer types, being also related to the modulation of cell sensitivity to antitumor drugs. Despite the potential linkage between FOXO3, autophagy and sorafenib resistance in HCC, this interesting crosstalk remains understood. Therefore, our objective was to unravel the involvement of autophagy and its potential modulation by FOXO3 in the acquisition of sorafenib resistance in HCC. We employed two sorafenib-resistant HCC in vitro models (HepG2S1 and HepG2S3), independently generated from the non-resistant HepG2. UALCAN platform was used for FOXO3 evaluation in HCC patient samples. Autophagolysosomes were visualized with acridine orange staining. LC3-LAMP2 colocalization, FOXO3 nuclear translocation and Ki67 levels were assessed by ICC/IF. FOXO3 silencing was performed through siRNAs transfection. Expression was analyzed by WB and qRT-PCR; ROS production by DCFDA assay; cell viability by MTT or CellTiter-Glo; and apoptotic subG1 population by flow cytometry. Bafilomycin A1, chloroquine, rapamycin and regorafenib treatments were also used. Differences were considered statistically significant when p<0.05. Sorafenib-resistant cell lines, which demonstrated to be autophagy-competent, displayed an enhanced basal autophagic flux compared to parental HepG2 cells, showing increased autophagolysosome content, LC3-LAMP2 colocalization and autophagy markers expression. Autophagy suppression with bafilomycin A1 or chloroquine abolished the improved autophagy and caused sorafenib-resistant cell death, indicating that such overinduced autophagy is essential for chemoresistant cell survival. Interestingly, enhanced FOXO3 expression in HCC samples, which was linked to poor patients’ survival, positively correlated with autophagy markers. In vitro, sorafenib-resistant HCC cells displayed FOXO3 upregulation, related to the oxidative stress. FOXO3 knockdown impaired the increased autophagy of sorafenib-resistant cells, reduced its proliferation and viability, and augmented its apoptotic rate. These findings suggest that sorafenib resistance acquisition is also mediated by FOXO3 upregulation and subsequent activation of cytoprotective autophagy, a novel pro-survival mechanism that the second-line drug regorafenib inhibits. Thus, FOXO3/autophagy axis emerges as a promising therapeutic target whose inhibition could allow sorafenib resistance overcoming in HCC.

Supported by the project PID2020-119164RB-I00 (MCIN/AEI/10.13039/501100011033).

Keywords: autophagy, chemoresistance, forkhead box O3, hepatocellular carcinoma.

Corresponding author: José L. Mauriz, https://orcid.org/0000-0003-3160-8599

P2-30

Hypoxia-inducible factor 1α-dependent response to hypoxia through apoptosis and autophagy modulation promotes cell survival in hepatocellular carcinoma

^*1,2Tania Payo-Serafín, ^1,2Paula Fernández-Palanca, ^1,2Carolina Méndez-Blanco, ^1,2Flavia Fondevila, ^1,2Beatriz San-Miguel, ³Andrés García-Palomo, ⁴Juan J Ortiz de Urbina, ^1,2Javier González-Gallego, ^1,2María J Tuñón, ^1,2José L Mauriz

¹ Institute of Biomedicine (IBIOMED), University of León, León, Spain

² Centro de investigación biomédica en red de enfermedades hepáticas y digestivas (CIBERehd), Institute of Health Carlos III, Madrid, Spain

³ Service of Medical Oncology, Complejo Asistencial Universitario de León (CAULE), Hospital of León, León, Spain

⁴ Pharmacy Service, Complejo Asistencial Universitario de León (CAULE), Hospital of León, León, Spain

Hypoxia, a common feature in advanced solid tumors, is known to play a crucial role in tumorigenesis and the loss of sensitivity to chemotherapy in hepatocellular carcinoma (HCC). The adaptive cell response to this hypoxic microenvironment is mainly mediated by the hypoxia-inducible factor 1 and 2 alpha (HIF-1α and HIF-2α), which can induce the expression of many genes involved in cell survival and resistance to chemotherapy, modulating an autophagy and/or apoptosis-mediated cell death. Our aim was to elucidate the role of HIF-1α in the survival of HCC cells by regulating autophagy and/or apoptosis. For this, we used several human HCC cell lines (HepG2, Hep3B, Huh-7, PLC/PRF/5, SNU-387, SNU-423, SNU-449 and SNU-475) to perform a screening of HIF-1α overexpression under hypoxia. We treated HCC cells with 100 μM CoCl₂ to mimic hypoxia. Protein expression was assessed by Western blot and immunofluorescence and laser confocal imaging; and gene expression was analyzed by qRT-PCR. HIF-1α gene silencing was carried out using siRNA transfection. Cell viability was evaluated by MTT assay and proliferation by Ki67 levels assessment. Autophagolysosomes visualization was assessed by acridine orange staining and fluorescence microscopy. GraphPad Prism 8 software was employed to perform statistical analysis and differences were considered significant when p<0.05. We selected those HCC cell lines that showed an increased expression of HIF-1α under hypoxic conditions. After HIF-1α gene silencing, a decreased level of Ki67 expression as well as viability percentage was detected, revealing a possible relationship between HIF-1α expression and a pro-survival capacity of liver tumor cell lines. Pro-apoptotic proteins levels were significantly higher in HIF-1α knockdown HCC cells under a hypoxic microenvironment, suggesting that HIF-1α promotes cell survival by avoiding apoptosis. The evaluation of the autophagy flux displayed a reduction in the amount of autophagolysosomes when HIF-1α was silenced, also observing a modulation in several autophagy markers. These findings indicate that HIF-1α expression and stabilization might contribute to cell survival in solid tumors under hypoxia through apoptosis evasion and autophagy regulation. Therefore, HIF-1α activity blockage may constitute a promising therapeutic strategy to overcome cell survival under hypoxic microenvironment in HCC.

Supported by the project PID2020-119164RB-I00 (MCIN/AEI/10.13039/501100011033).

Keywords: apoptosis, autophagy, hepatocellular carcinoma, hypoxia.

Corresponding author: José L. Mauriz, https://orcid.org/0000-0003-3160-8599

P2-31

Modulatory effect of melatonin and Akkermansia muciniphila on gut microbiota composition in a mice model of early liver fibrosis

¹Sara Román-Sagüillo, ^1,2María Juárez-Fernández, ¹Beatriz San-Miguel, ¹Susana Martínez-Flórez, ¹Alicia Andrés-Amo, ^1,2Paula Fernández-Palanca, ^1,2José L Mauriz, ^1,2María V García-Mediavilla, ^1,2Esther Nistal, ^1,2María J Tuñón, ^*1,2Sonia Sánchez-Campos, ^1,2Javier González-Gallego^1,2

¹ Institute of Biomedicine (IBIOMED), University of León, León, Spain.

² Network Biomedical Research Center for Liver and Digestive Diseases (CIBERehd), Carlos III Health Institute, Madrid, Spain.

Early liver fibrosis has demonstrated to be a reversible disease, although the involved mechanisms are unknown and there are not available effective treatments. Melatonin has been proposed as a therapeutic alternative due to its antioxidant, anti-inflammatory and antifibrogenic properties, while the bacterium Akkermansia muciniphila has been identified as a possible probiotic with a protective effect in metabolic diseases. The objective is to evaluate the effect of melatonin and/or A. muciniphila supplementation on gut microbiota composition in an in vivo model of early liver fibrosis. C57BL/6J mice were divided in two groups: group fed with control diet (C group) and group fed with Western Diet supplemented with fructose in drinking water and injected with CCl₄ subcutaneously (WD group) for 8 weeks. Subsequently, they were divided into subgroups according to diet and daily treatment received: melatonin and/or A. muciniphila, for 4 weeks. After sacrifice, parameters related to liver fibrosis, gut-liver axis and gut microbiota composition were analysed. The WD group developed intestinal dysbiosis associated with the early liver fibrosis status. Phyla such as Desulfobacterota and Cyanobacteria were increased with Western diet, while Bacteroidota showed an opposite pattern. At genus level, an alteration was also observed in WD group, in which genera such as Alistipes, Blautia, Lactobacillus, Lactococcus, Muribaculum and Ruminococcus were modified due to the diet. Moreover, regarding α-diversity, a significant decrease in Shannon index was observed also due to Western Diet. However, the treatment with melatonin and A. muciniphila tended to reverse these changes. Thus, Principal Coordinate Analysis at genus level showed not only a clear separation of bacterial communities between control and WD groups according to the first axis, but also a significative influence of the different treatments in WD groups in bacterial communities clustering. Finally, relative abundance of Akkermansia genus was increased in the supplemented groups, highlighting that melatonin also increased the presence of this microorganism. In conclusion, the combination of melatonin and A. muciniphila has a modulatory effect on gut microbiota composition that could be linked with the beneficial effects of the intervention.

Funding: PID2020-120363RB-I00, GRS2126/A/2020, LE017-P20. CIBERehd funded by ISCIII.

Keywords: Akkermansia muciniphila, early liver fibrosis, gut microbiota, melatonin.

Corresponding author: Sonia Sánchez-Campos, https://orcid.org/0000-0003-2672-734X

P2-32

Creatine supplementation promotes recovery from chronic colon inflammation induced in rat

Alejandro Ruiz-Calero, Gema Sotelo-Parrilla, María D Vázquez-Carretero, María L Calonge, Pablo García-Miranda, María J Peral

Department of Physiology, Faculty of Pharmacy, University of Sevilla, Spain

Ulcerative colitis is a chronic pathology characterized by relapse and remission periods of intestinal inflammation. Creatine is a dietary supplement with pleiotropic beneficial effects. It has been suggested the use of creatine as adjuvant treatment for ulcerative colitis, since it may improve the cellular bioenergetics, modulate immune system, and exhibit anti-inflammatory effects. Creatine kinase (CK) plays a key role for cells with high energy requirements as colonic epithelial cells, and we have previously described that dietary creatine is taken up by these cells through a specific creatine transporter (CrT1). The goal of the study was to investigate whether creatine prevents or reduces the severity of chronic colitis and/or helps restore colon health. For this purpose, 45-day-old male rats were used. Chronic colitis was induced by cyclical administration of 5% sodium dextran sulphate (DSS) in drinking water to mimic inflammation recurrent periods as those of ulcerative colitis in humans. Control rats group drank normal water. Each of these groups were divided into either creatine supplemented group or non-creatine supplemented group. Procedures concerning the protection of experimental animals were in accordance with European (2010/63/UE) and Spanish (BOE 34/1337, 2013) normative, and protocols approved (15/01/2020/001) by the Animal Ethics Committee. Clinical symptoms and, macroscopic and histological alterations of the colon were evaluated to assess colitis severity. Inflammatory parameters mRNA relative abundance was measured by PCR, and colon epithelial integrity by immunofluorescence assays. ANOVA followed by Tukey´s test was used (p<0.05) (n=3-6 animals per group). We found that in DSS-treated rats, when the colitis became chronic and during the recovery period, creatine supplementation decreased clinical symptoms, colon shortening, histological score and proinflammatory cytokines IL-6 and IL-1β mRNA abundance and increased that of the anti-inflammatory IL-10. Immunofluorescence assays revealed that creatine contributed to maintain epithelial integrity in the colon mucosa of DSS-treated rats as E-cadherin expression was increased. In addition, creatine also increased colonic CrT1 and cytosolic CK expression. In conclusion, these observations suggest that creatine attenuates the severity of chronic colitis and promotes the recovery by stimulating creatine metabolism and epithelial restauration.

Supported by Grant 18.06.07.3005 PID2019-105632RB-I00, funded by MCIN/AEI/ 10.13039/501100011033.

Keywords: creatine, colon inflammation, epithelial integrity, recovery

Corresponding author: Pablo García-Miranda, https://orcid.org/0000-0003-2588-6904

P2-33

Creatine supplementation improves motor activity and short-term memory impaired during chronic colitis development in rat

Gema Sotelo-Parrilla, Alejandro Ruiz-Calero, Pablo García-Miranda, María L Calonge, María D Vázquez-Carretero, María J Peral

Department of Physiology, Faculty of Pharmacy, University of Sevilla, Spain

Creatine is a dietary supplement best known for enhancing muscular performance, but it is also suggested to be beneficial in several neurological and non- neurological diseases. Ulcerative colitis is a chronic pathology characterized by recurrent periods of intestinal inflammation. Alterations in behaviour have been also found in patients and animal models of this disease. We have previously reported that motor activity and short-term memory were impaired along chronic colon inflammation development in rats. Our aim was to study whether creatine supplementation has positive effects on these behaviour alterations. For that, we used 45-day-old male rats. Chronic colitis was induced by cyclical administration of 5% sodium dextran sulphate (DSS) in drinking water. Control rats group drank normal water. Each of these groups were divided into either creatine supplemented group or non-creatine supplemented group. The procedures concerning the protection of experimental animals were in accordance with European (2010/63/UE) and Spanish (BOE 34/1337, 2013) normative, and protocols approved (15/01/2020/001) by the Animal Ethics Committee. Clinical alterations were evaluated to assess colitis severity by using the disease activity index (DAI) that evaluates body weight gain, stool consistency, and presence of blood in faeces. Behavioural analysis of the animals was carried out using the “open field test” to evaluate motor activity and the “novel object recognition test” (time for test:10 minutes) to assess the short-term memory. ANOVA followed by Tukey´s test was used (p<0.05) (n=8-15 animals per group). Our results reveal that creatine supplementation improved horizontal and vertical motor activity, impaired in the DSS-treated rats, by decreasing the immobility time and increasing the number of rearings. In addition, creatine increased the discrimination index, indicating an improvement in the short-term memory, also decreased in the chronic colitis. We conclude that creatine exerts beneficial effects since it recovers motor activity and memory reduced in rats due to chronic colon inflammation.

Supported by Grant 18.06.07.3005 PID2019-105632RB-I00, funded by MCIN/AEI/ 10.13039/501100011033.

Keywords: creatine, colitis, motor activity, memory

Corresponding author: María J Peral, https://orcid.org/0000-0003-0343-7550

P2-34

Squamous cell carcinoma, complication as a consequence of hidradenitis suppurative and effects of radiotheraphy

^*1Carmen Corral Fernández, ²José M Gimeno Montes, ¹María González de Dueñas, ¹Beatriz Baños Pérez, ¹Esther Agudo Rey, ¹Victoria Vera Barragán, ¹Yesika Ríos Kavadoy, ¹Joaquín J Cabrera Rodríguez, ¹Juan Quirós Rivero, ¹María F Ropero Carmona, ¹Julia L Muñoz García

¹ Service of Radiation Oncology, Universitary Hospital of Badajoz, Spain, and

² Service of Cardiology, Universitary Hospital of Badajoz, Spain.

Hidradenitis suppurative (HS), also known as Verneuil's disease or inverse acne, is a chronic and recurrent inflammatory disease of the apocrine glands and hair follicles of the skin. Clinically, the disease presents as lesions and fistulous tracts that suppurate chronically, according to the Hurley classification. Malignant degeneration of HS to squamous cell carcinoma (SC) is unusual, 3.2%, most frequently located in the perianal region. This type of SC has a high metastatic capacity (30%) compared to the classic cutaneous SC (5%). Treatment consists of resection of the affected area of ​​skin with wide margins. In addition, complementary radiotherapy (RT) is an effective treatment in the management of cutaneous SC with response rates greater than 90%. Our patient was a 53-year-old man follow-up by the Dermatology Service for type III HS of several months' evolution with failure of topical and systemic treatments. On January 5, 2021, the patient is evaluated by Surgery Service for a large excrescent and painful lesion, of 20 cm in size in the sacrococcygeal region with extension intergluteal area, that prevented him from carrying out his daily activities. Drainage and biopsies were performed, with pathological results compatible with SC. The patient was presented to a multidisciplinary committee, due to the large size of the lesion, surgery was ruled out and radiotherapy was chosen. Between January 20 and February 19, 2021, treatment was completed with 3D external radiotherapy, with 6 MV photons, at a dose per fraction of 3 Gy in 20 sessions, administering a total dose of 60 Gy. After radiotherapy treatment, the patient presented a favourable clinical evolution with a little doubtful tumour remnant on physical examination, but not visualized in the imaging test. Revaluated by the multidisciplinary committee, salvage surgical was adopted. The intervention consisted in resection with wide margin, with pathological results of complete response. Surgery is the treatment of choice in this type of tumour, but due to the volume of some lesions, many of them are unresectable. Radiotherapy is an effective alternative option in patients who are not candidates for surgery.

Keywords: Hidradenitis suppurative, squamous cell carcinoma, radiotherapy.

Corresponding author: Carmen Corral Fernández.

P2-35

Unravelling the inflammatory processes in early stages of diabetic nephropathy in rat models and the potential effect of (Ss)-DS-ONJ

^*1,2Laura Gómez-Jaramillo, ^1,2Fátima Cano-Cano, ³Elena M Sánchez-Fernández, ³Carmen Ortiz Mellet, ⁴José M García-Fernández, ¹Marta Iturregui, ^*1,2‡Ana I Arroba, ^1,2‡Manuel Aguilar-Diosdado

¹ Unidad de Investigación, Instituto de Investigación e Innovación en Ciencias Biomédicas de la Provincia de Cádiz (INiBICA), Cádiz, Spain; ² Departamento de Endocrinología y Nutrición, Hospital Universitario Puerta del Mar. Universidad de Cádiz, Cádiz, Spain; ³ Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Sevilla, Spain; and ⁴ Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Sevilla, Spain,

^‡ These authors contributed equally to this work

Type 1 diabetes mellitus (T1DM) is a chronic disease, which is associated with the development of complications including nephropathy. DN is a multifactorial and complex complication that originates from the damage or danger signals released by injured renal cells, which can trigger remodeling processes by stimulating renal cells and activating immune cells of both the innate and adaptive systems. Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) at early stages of the disease. In the early inflammatory phase of renal fibrosis, cytokines produced by infiltrating and local cells play a decisive role in initiating the phenotypic change of epithelial-to-mesenchymal transition (EMT). (S_S-DS-ONJ) belongs to a family of compounds called sulfur-linked sp²-iminosugar glycolipids (sp²-IGLs). Increasing evidence suggested that sp²-IGLs may offer anti-inflammatory protection against diabetic complications. The aim in this study was to decipher the inflammatory processes in the context of the DN and the potential therapeutic effect of the (S_S)-DS-ONJ compound as a modulator in each specific process involved in DN process. In vitro assays were carried out on mouse cortical tubular epithelial cell line (MCTs) that reproduces the inflammatory features of kidney failure when they were cultured in a pro-inflammatory environment using a cocktail of cytokines. Ex vivo assays were performed with insight the potent anti-inflammatory effect of (S_S)-DS-ONJ on adult kidney (AK) explants from Wistar rat stimulated with CKs or BB rat that develops diabetes spontaneously at 11 old-weeks, however showing an inflammatory progress to overt since 7 weeks of age. We demonstrate that the glycolipid mimetic (S_s)-DS-ONJ is able to abolish inflammation by induction of autophagy flux and provokes inhibition of inflammasome complex in in vitro and ex vivo model, using AK explants from BB rats. The contribution of (S_s)-DS-ONJ in reducing inflammatory events are mediated by inhibition of classical stress kinase pathways and blocking inflammasome complex activation. The (S_s)-DS-ONJ treatment is able to block the epithelial-to-mesenchymal transition (EMT) progression, but only when the IL18 levels are reduced by the treatment. These findings suggest that (S_s)-DS-ONJ could be a promising novel and multifactorial treatment for DN.

Supported by ITI-0012-2019; PI18-01287; ITI-0029-2017.

Keywords: Inflammation, epithelial mesenchymal transition, autophagy, (S_s)-DS-ONJ.

^*Corresponding authors: Ana I. Arroba, ORCID: 0000-0002-5136-1725; Laura Gómez-Jaramillo, ORCID: 0000-0001-5104-1425

P2-36

DHP-I inhibitor ameliorates sepsis-induced acute renal failure by blocking apoptosis in male Sprague-Dawley rats

^*1María A González-Nicolás, ¹Blanca Humanes, ^1,2Alberto Lázaro

¹ Renal Physiopathology Laboratory, Department of Nephrology, Instituto de Investigación Sanitaria Gregorio Marañón, Hospital General Universitario Gregorio Marañón, Madrid, Spain.

² Department of Physiology, Faculty of Medicine, Universidad Complutense de Madrid, Spain

Sepsis is a life-threatening organ dysfunction caused by dysregulated host response against infection, and the acute renal failure (ARF) is currently recognized as one of the most severe complications related to sepsis. In fact, the mortality of septic patients with ARF is about 75%, while those without ARF ranges between 27 and 32%. Apoptosis has been recognized as an important factor in the physiopathology of septic complications, enhancing ARF progress. Cilastatin, a renal dehydropeptidase I inhibitor located in the apical membrane of proximal tubule cells, has shown its usefulness in the protection of ARF induced by nephrotoxic drugs. Here, we evaluated the utility of cilastatin as a protector against renal damage induced by sepsis, focusing on its protective role against cell death by apoptosis. Sepsis was developed on male Sprague-Dawley rats by cecal ligation puncture model (CLP). The animals were divided into 4 groups: control, CLP, control + cilastatin and CLP + cilastatin. Cilastatin (150 mg/kg, bw i.p.) was administered immediately and at 24h after induction of sepsis. Renal damage was evaluated 48h after surgery by measuring serum creatinine, blood urea nitrogen (BUN), glomerular filtration rate (GFR), proteinuria and renal morphology, as well as apoptotic mediators such as caspases, DNA fragmentation and pro/anti apoptotic molecules. Sepsis increased serum creatinine, BUN and protein excretion levels and decreased the GFR in comparison with the control group. These renal effects were confirmed by the presence of severe morphological changes. Treatment with cilastatin improved renal function and tissue morphology after damage. In addition, cilastatin also reduced cell death by decreasing the activation of caspases, Bax and TUNEL-positive cells, increasing antiapoptotic factors such as BCL2. This study provides evidence that cilastatin reduces in vivo ARF-induced by sepsis by preventing apoptosis and might represent a novel therapeutic opportunity for septic patients susceptible to renal damage in clinical practice.

Funding: ISCIII (PI14/01195); co-financed with FEDER funds of UE and RETIC REDinREN/RD16/0009/0026.

Keywords: DHP-I, apoptosis, sepsis, kidney.

Corresponding author: Alberto Lázaro, https://orcid.org/0000-0002-1095-679X.

P2-37

The effects of bisphenol A exposure during early life on urine biomarkers after furosemide treatment in Wistar rats

Paula Nuñez, Juan Arguelles, Carmen Perillan

Department of Functional Biology, Faculty of Medicine and Health Sciences, University of Oviedo, Oviedo, Spain

Bisphenol A (BPA) is an endocrine disrupting compound of plastics that has been ubiquitously detected in human biological samples. Data have demonstrated that early exposure to BPA results in adverse outcomes in adult life, including cardiovascular diseases. The kidney participates in blood pressure homeostasis regulating electrolyte concentrations and extracellular fluid volume. Changes in urine function parameters are associated with variations in renal function. The aim of this work was to study the effects of two low BPA doses in the dam, during pregnancy and lactation, in the normal and stimulated renal function of their adult offspring. The University of Oviedo's Experimental Animal Use Committee approved all experimental procedures (PROE 15/2016). Each Wistar dam was assigned to one of three different treatment groups: Control, BPA5 or BPA10. Control rats had access to drinking water, whereas the BPA groups received drinking water containing BPA (5 mg/L or 10 mg/L; BPA Sigma-Aldrich, Saint Louis) to mimic the most likely route of human exposure. The experiment was performed in offspring when they were adult, at 3 months of age. Pre-test urine samples were obtained after 24h in metabolism cages. In the test, furosemide (Sanofi-Aventis Laboratories, Barcelona) was injected (10 ml/kg bw sc) to induce natriuresis and diuresis. Rats were placed in metabolism cages, and urine was collected after the 1st hour after furosemide injection. On the next morning, urine was also collected for the remainder of 19h after furosemide injection. Reagent strips for urinalysis (Multistix strips, Bayer HealthCare Inc., Elkhart) was also used to test 10 parameters (glucose, bilirubin, ketones, SG, blood, pH, protein, urobilinogen, nitrite and leukocytes). In pre-test samples, biomarkers (creatinine, osmolality, total proteins) were significant higher in control group compared to BPA groups, but values are normal for all groups. In post-test samples, biomarkers were similar in all groups at each time. The strips results were negative to glucose, bilirubin, ketones, blood, urobilinogen, nitrite and leukocites. Urine showed normal pH values (6.5 to 7.5). We can conclude that normal and stimulated renal function was not affected by these BPA exposures during early life.

Supported by University of Oviedo (PAPI-18-EMERG-32, PAPI-20-PUENTE-12, PAPI-19-PUENTE-15)

Keywords: bisphenol A, kidney, rat, fetal programming

Corresponding author: Paula Nuñez, https://orcid.org/0000-0002-7877-9515.

P2-38

Use of urine biomarkers to study the normal and stimulated renal function in Wistar rats exposed to chronic low doses of bisphenol A

Paula Nuñez, Juan Arguelles, Carmen Perillan

Department of Functional Biology, Faculty of Medicine and Health Sciences, University of Oviedo, Oviedo, Spain

Bisphenol A (BPA) is one of the main volume chemicals produced worldwide. Nephrons which reabsorb many filtered molecules, are the primary site of kidney injury associated with nephrotoxicity. Renal diseases usually produce a decreased ability to concentrate urine and to conserve large molecules, as for example, proteins or glucose. The aim of the present work was to determine the effect of long-term and low-dose BPA treatment in normal and stimulated renal function in male Wistar rats. The University of Oviedo's Experimental Animal Use Committee approved all experimental procedures (PROE 15/2016). During 3 months, the control group had access to drinking water (vehicle), whereas the BPA group received drinking water containing BPA (5 mg/L; Sigma-Aldrich, Saint Louis) to mimic the most likely route of human exposure. Pre-test urine samples were obtained after 24h in metabolism cages. In the test, furosemide (Sanofi-Aventis Laboratories, Barcelona) was injected (10 ml/kg bw sc) to induce natriuresis and diuresis. Rats were placed in standard metabolism cages, and urine was collected 1 hour after furosemide injection. On the next morning, urine was also collected for the remainder of 19h after furosemide injection. Samples were refrigerated for later analysis. Rats received two such tests separated by 8 days to study kidney sensitization to the furosemide stimulus. Reagent strips for urinalysis (Multistix strips, Bayer HealthCare Inc., Elkhart) was also used to test 10 parameters (glucose, bilirubin, ketones, SG, blood, pH, protein, urobilinogen, nitrite and leukocytes). Urine osmolality and total proteins were similar in both groups, and equivalent between test 1 and test 2 at each time. The strips results were negative to glucose, bilirubin, ketones, blood, urobilinogen, nitrite and leukocites. The urine samples showed normal pH values, range from 6.5 to 8.0. As expected, urine density decreased 1h post-test and reached normal values 19h post-test. We can conclude that normal and stimulated renal function was not affected by this BPA exposure in adult male rats.

Supported by University of Oviedo (PAPI-18-EMERG-32, PAPI-20-PUENTE-12, PAPI-19-PUENTE-15).

Keywords: bisphenol A, kidney, rat, urine biomarkers

Corresponding author: Paula Nuñez, https://orcid.org/0000-0002-7877-9515

P2-39

Group dynamics of survival in the desert to apply the knowledge of renal physiology

Isabel Sánchez-Vera, María del Nogal, Úrsula Muñoz

Departamento de Ciencias Médicas Básicas, Facultad de Medicina, Universidad CEU-San Pablo,CEU Universities, Madrid, Spain

One of the skills medical students need to acquire while studying the subject of Physiology is the application of their knowledge into their future work and problems solving within their area of study. In addition, the ability of team working is also very important in their career, since as physicians they will have to work integrated into multidisciplinary teams formed by different professionals. The aim of this study was to put into practice the knowledge learned in renal physiology classes about the role of kidney in body fluids homeostasis, simulating a situation that could happen in real life and solving it through a group dynamic, using teams of six students formed randomly. Students were given a simulated situation where they were lost in the desert and need to survive in that situation. To be able to do it, they had to correctly solve a series of questions and problems on topics related to renal physiology. For each question correctly answered, they were rewarded with an object chosen from a list of 18. Some of them were beneficial for survival, such as a bottle of water, but others were better avoided because of their negative effects on maintenance of plasma osmolarity, such as a bag of chips. Each object was previously scored by the teachers, but the students were blind about that information during the group dynamic. Once the activity finished, the points of each group were counted to obtain a single winning group, which was the one that had answered the largest number of questions and had obtained the highest score by choosing better objects to survive in the desert. With this group dynamic, students learned how to work in a team and the practical application of the theoretical knowledge acquired in class. In the survey that the students filled out at the end of the activity, their perception about this experience was very positive.

Keywords: Competency based teaching, Group dynamics, Decision making, Teaching method innovations.

Corresponding author: Sánchez-Vera, Isabel; https://orcid.org/0000-0003-1278-5338

P2-40

The use of digital devices and its effects on behavior and cognitive capacities

^*Patricia M Serrano Pastor, Primitivo Mena Arias, Lourdes Franco

Department of Physiology, Faculty of Medicine and Health Science, University of Extremadura, Badajoz, Spain

For a few decades, technologies have been part of our day-to-day. It is not surprising, therefore, that so much research has been done on its effects on people's behavior and abilities. In order to find out its consequences on attention, language, reading, learning, interpersonal relationships, empathy and psychological health, a review has been carried out with a total of 44 articles from the last ten years, including both original studies and bibliographic reviews found between the PubMed and Elsevier platforms. As results we obtained: 1. There was a positive confirmation between attention dispersion or ADHD symptoms and the use of technologies. 2. Language learning was diminished in relation to technologies. 3. Reading in digital format involved a lower understanding of the text than on paper. 4. ICT in the academic field lead to a greater distraction on the part of the students and greater difficulties in integrating knowledge. 5. Both the presence of technologies and the use of them during a conversation entail lower scores of intimacy and satisfaction with the personal relationship than in the absence of these devices. 6. Empathy is reduced with violent video games and technology abuse, but its levels are higher in cooperative video games and in online activities that lead to real face-to-face encounters, so more research is needed in this regard. 7. The vast majority of studies showed a disturbance between the use of technologies and depression, OCD, anxiety and its manifestations, social isolation and behavioral problems.

Supported by Junta de Extremadura (GR21042).

Keywords: technologies, attention, language, psychological aspects.

Corresponding author: Patricia María Serrano Pastor, https://orcid.org/0000-0002-2695-5660

P2-41

Distribution of antioxidants intake and its effects in European population

¹Miguel Romero, ²Ilda de Jesús Casimiro, ³Rafael Bravo Santos, ^*2David González-Flores

¹ Final Degree Student, University of Extremadura, Badajoz, Spain

² Department of Anatomy, Cell Biology and Zoology, University of Extremadura, Badajoz, Spain

³ Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, United States.

Due to the contemporary growth of the elderly global population and the resulting incidence increase of chronic disorders derived from senescence, there exists a necessity for establishing an understanding regarding the mechanisms involved in the process of aging and its consequences for human health. This study focuses on the search for relevancy regarding one of the most discussed elements on this matter, the effects of oxidative stress, through a visualization of the distribution of antioxidant intake throughout occidental European territory and their possible effect on the incidence of cancer, one of the most societally impactful senescence related diseases. This process consisted firstly of a bibliographic review with the objective of collecting dietary intake data belonging to western European countries, regarding four model antioxidants: vitamins C and E, selenium and copper. The collected data was processed through the programming language R, resulting in mean dietary intakes for each country that were then represented on a geographic map. The result was a heterogenous distribution of intakes, with countries belonging to central and Southern-Mediterranean regions showing higher levels of antioxidant consumption compared to those situated in Atlantic and Nordic regions. The higher levels were occupied by Austria and Germany, countries representing central Europe, while Denmark was consistently shown to result in the lowest values for all four antioxidant intakes. Once the visualization of the distribution was over, the student proceeded with the search for possible relations between the intake data and the incidence of several varieties of cancer on the represented countries through a Pearson’s correlation test. Within the 40 resulting coefficients, 29 were negative, with 6 of them presenting values below r = -0.7. This majority conformed by negative cases suggest a possible suppressing effect of antioxidant intake over the incidence of cancer. While the lack of data in this study pertaining to other possible etiological causes for cancer makes it un-viable to establish firm conclusions, the results are in agreement with the redox control theory and are inviting towards the realization of further studies on the present matter.

Keywords: antioxidants, health, computational biology, aging.

Corresponding author: David González-Flores, https://orcid.org/0000-0002-4543-8810.

P2-42

Sperm selection methods for male infertility

¹Cristina Rodríguez, ²Margarita Fernández, ^*2David González-Flores

¹ Final Degree Student, University of Extremadura, Badajoz, Spain

² Department of Anatomy, Cell Biology and Zoology, University of Extremadura, Badajoz, Spain

Currently, infertility problems are a great obstacle in order to have offspring; many of these infertility problems in the couple have their origin in the male. The sperm selection techniques that are commonly used in assisted reproduction clinics and that are more established are swim-up and selection gradients. These methods recover the spermatozoa with better mobility and morphology but do not differentiate between those that are genetically normal and those with a good capacity to fertilize the ovule, whereas the most advanced techniques such as MACS, microfluidic chambers, PICSI, IMSI, among others, can select the sperm with the set of exceptional characteristics so that fertilization is reached, resulting in a healthy baby. When a couple undergoes assisted reproductive technology treatment, selecting the best sperm is an essential element. Both the conventional and the most advanced techniques solve the problems of male infertility, favoring full-term pregnancy with completely healthy babies.

Keywords: spermatozoa, sterility, selection gradients, assisted reproductive technology.

Corresponding author: David González-Flores, https://orcid.org/0000-0002-4543-8810

Poster session 3

P3-01

Protective effects of foam rolling against inflammation and notexin induced muscle damage in rat

^#1Carlos Colmena, ^1,2Juan Campos-Campos, ^1,2Adrian Jorda, ¹Ignacio Campo-Palacio, ¹Constanza Aldasoro, ^*1Soraya L Valles

¹ Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

^#Presenter Author

Foam rolling technique is used by physical therapists and fitness professionals. It is known that high-intensity exercise can cause inflammation and damage in muscle tissue. Despite the lack of basic science studies to support or refute the efficacy of foam rolling, the technique is very widely used in the sports world. Here, we investigated whether foam rolling could attenuate muscle damage and inflammation. Female Wistar rats were assigned to control (C), foam rolling (FR), notexin without foam rolling (N) and notexin with foam rolling (NFR) groups. A 4.5 x 2 cm foam roller was used to massage their hind legs (two 60-second repetitions twice a day for 3 days). Motor function tests (Balance Beam Test and Grip strength) were used. We detected an increase in time and foot faults when crossing a beam in the N group compared to C and FR rats. In contrast, a significant decrease was detected in both tests in NFR compared to N rats. Muscle power was measured with a grip strength test and better performance was detected in NFR rats compared to N rats. Furthermore, an increase of pro-inflammatory proteins was noted in the N group, while there was a decrease in the NFR group. Moreover, an increase in PPAR-γ (anti-inflammatory protein) in the NFR group compared to the N group demonstrates the anti-inflammatory properties of the foam rolling technique. In summary, applying foam rolling after damage has benefits such as a reduction of pro-inflammatory proteins and increase in anti-inflammatory proteins, resulting in muscle recovery and better performance.

Financial support from the University of Valencia. (Specific Key 20180268).

Keywords: Foam Rolling, motor function tests, inflammation, muscle recovery.

Corresponding Author: Soraya L. Valles

P3-02

Influence of codiagnosis of chronic fatigue syndrome on the perceived and physiological stress of fibromyalgia patients

^1,2María Dolores Hinchado, ¹Eduardo Otero, ¹María Carmen Navarro, ^1,3Leticia Martín-Cordero, ^1,4Isabel Gálvez, ^*1,2Eduardo Ortega

¹ Immunophyisiology Research Group, Instituto Universitario de Investigación Biosanitaria de Extremadura (INUBE), 06006 Badajoz, Extremadura, Spain; ² Immunophysiology Research Group, Physiology Department, Faculty of Sciences, University of Extremadura, 06071 Badajoz, Extremadura, Spain; ³ Immunophysiology Research Group, Nursing Department, University Center of Plasencia, University of Extremadura, 10600 Plasencia, Extremadura, Spain; and ⁴ Immunophysiology Research Group, Nursing Department, Faculty of Medicine and Health Sciences, University of Extremadura, 06006 Badajoz, Extremadura, Spain.

Fibromyalgia (FM) and chronic fatigue syndrome (CFS) are two diseases that are frequently codiagnosed. Both present similar pathophysiology, including a potential dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which could be related to high levels of pain, stress, and anxiety in these patients. The absence of specific objective biomarkers to confirm this hypothesis and the great controversy regarding the influence of the codiagnosis of CFS in patients with FM led us to propose the following objectives: 1) to determine whether the codiagnosis of CFS has a negative influence on the perceived and physiological stress of patients with FM and 2) to examine whether the perceived stress levels correspond to a dysregulation of the HPA axis by determining the systemic concentration of cortisol and dehydroepiandrosterone (DHEA), since the balance between the two ensures the proper functioning of the HPA. For this purpose, we performed an initial assessment of subjective stress using the Perceived Stress Scale (PSS) in three experimental groups: an age-matched reference group of “healthy women” without FM (RG, n=70), women with only FM (FM-only, n=35) and women with FM and associated CFS (FM+CFS, n=35). Following this assessment, systemic cortisol and DHEA concentrations were determined in a representative subgroup (n=15) of each experimental group. Codiagnosis of CFS did not negatively affect perceived stress in FM patients, although both experimental groups reported higher perceived stress than the RG. However, the FM+CFS group had a lower cortisol concentration than the FM-only group, closer to the values of the RG. There were no significant differences in systemic DHEA concentration, with very close values to those of the reference group. The cortisol/DHEA ratio mitigated the differences found in cortisol between the FM-only and FM+CFS groups, with significantly higher values for both experimental groups compared to the RG of women not diagnosed with FM. Codiagnosis with CFS does not influence the elevated levels of perceived stress in FM patients. Furthermore, there is a clear dysregulation of the HPA axis manifested by an altered cortisol/DHEA ratio, which could be a potential objective biomarker of perceived stress in FM patients, without a clear influence of CFS.

Supported by Junta de Extremadura (GR21079). Clinical trial registry number: NCT05323838 (ClinicalTrials.gov).

Keywords: Fibromyalgia, chronic fatigue syndrome, stress.

^*Correspondig author: Eduardo Ortega, https://orcid.org/0000-0002-7007-7615

P3-03

Effect of weight cycling (yo-yo dieting) on sensorimotor and stress/anxiety responses in C57BL/6J mice

^1,2Leticia Martín-Cordero, ¹María Carmen Navarro, ^1,3María Dolores Hinchado, ¹Eduardo Otero, ^1,4Silvia Torres-Piles, ⁵Mónica de la Fuente, ^1,6Isabel Gálvez, ^*1,3Eduardo Ortega.

¹ Immunophyisiology Research Group, Instituto Universitario de Investigación Biosanitaria de Extremadura (INUBE), 06006 Badajoz, Extremadura, Spain; ² Immunophysiology Research Group, Nursing Department, University Center of Plasencia, University of Extremadura, 10600 Plasencia, Extremadura, Spain; ³ Immunophysiology Research Group, Physiology Department, Faculty of Sciences, University of Extremadura, 06071 Badajoz, Extremadura, Spain; ⁴ Immunophysiology Research Group, Department of Medical-Surgical Therapy, Faculty of Medicine and Health Sciences, University of Extremadura, 06006 Badajoz, Extremadura, Spain; ⁵ Department of Genetics, Physiology and Microbiology (Animal Physiology Unit), Faculty of Biology, Complutense University of Madrid (UCM), Madrid, Spain; Institute of Investigation Hospital 12 Octubre, Madrid, Spain; and ⁶ Immunophysiology Research Group, Nursing Department, Faculty of Medicine and Health Sciences, University of Extremadura, 06006 Badajoz, Extremadura, Spain.

Obesity is associated with neuroendocrine dysregulation and altered metabolic homeostasis, together with impaired neural pathways and a higher prevalence of anxiety/depressive symptoms. Although diet-induced weight loss is a common strategy for obese individuals, it is still not clear whether some of the alterations linked to obesity persist despite weight normalization. Moreover, poor adherence to dietary strategies can lead to yo-yo diets, resulting in several cycles of weight loss and weight regain, also known as weight cycling. The impact of weight cycling on health is still not fully understood, but it can be implicated in many potential deleterious health consequences. The aim of this study was to evaluate the effect of weight cycling caused by yo-yo dieting on the sensorimotor response and stress/anxiety response in two groups of C57BL/6J mice. The control lean group fed a standard diet. The other group was subjected to a weight cycling protocol consisting in undergoing a yo-yo diet: 1) high-fat diet-induced weight gain period (8-26 weeks of age); 2) standard diet-induced weight loss period (26-48 weeks of age); 3) high-fat diet-induced weight regain period (48-66 weeks of age); 4) standard diet-induced weight loss period (66-80 weeks of age). At the end of each weight gain cycle animals were obese, and at the end of each weight loss cycle animals presented similar weight to that of control lean animals. At 80 weeks of age, weight, fasting glucose concentrations and lipid profile were assessed, as well as sensorimotor and exploratory activity, and stress/anxiety responses using standard behavioural tests for rodents. Results showed that, at the end of the weight cycling protocol, animals presented higher total cholesterol concentration than control lean animals, but fasting blood glucose and HDL cholesterol concentrations were similar to those of lean animals. Balance and motor coordination as well as exploratory activity were poorer, and stress/anxiety levels were higher in the yo-yo diet group at the end of the protocol, irrespective of aging. Therefore, weight cycling caused by yo-yo dieting over a lifetime can impair sensorimotor capacity and increase anxiety and stress, even when normal weight and glucose levels have been reached.

Supported by Junta de Extremadura (IB18011; GR21079).

Keywords: Obesity, behaviour, weight cycling, yo-yo diet.

^*Corresponding author: Eduardo Ortega, https://orcid.org/0000-0002-7007-7615

P3-04

Facilitation of insulin effects by ranolazine in astrocytes in primary culture

¹Kenia Alvarez-Gamez, ¹Ignacio Campo-Palacio, ^1,2Adrian Jorda, ^1,2Juan Campos-Campos, ¹Carlos Colmena, ¹Constanza Aldasoro, ¹Martin Aldasoro, ¹Soraya L Valles

¹ Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

Ranolazine (Rn) is a drug used in chronic coronary ischemia. It has proven to possess, also, beneficial effects on the central nervous system, as well as an antidiabetic influence, by reducing blood glucose levels and hemoglobin A1c (HbA1c). However, no effects of Rn on cellular sensitivity to insulin (Ins) have been demonstrated yet. The present study aimed to investigate the permissive effects of Rn on the actions of Ins in astrocytes in primary culture. Ins (10^-8 M), Rn (10^{-6 M}) or Ins+Rn (10^-8 M and 10⁻⁶ M respectively) were added to astrocytes during 24 h. Rn and/or Ins produced increase in cell viability or proliferation compared to control cells. Ins increased the protein expression of p-AKT, p-ERK, p-eNOS, Mn-SOD, COX-2 and the anti-inflammatory protein PPAR-γ. On the contrary, no significant changes were found in the protein expression of Cu/Zn-SOD, NF-κB and IκB. The presence of Rn produced increase in p-ERK and p-PPAR-γ and a significant decrease in COX-2 protein expression. Furthermore, Rn significantly increased the effects of Ins on the expression of p-AKT, p-eNOS, p-ERK, Mn-SOD and PPAR-γ. On the other hand, Rn+Ins produced a significant decrease in COX-2 expression. In conclusion, Rn facilitated the effects of insulin on the p-AKT, p-eNOS, p-ERK, Mn-SOD and PPAR-γ, signaling pathways, as well as on the anti-inflammatory and antioxidant effects of the hormone.

Financial support from the University of Valencia. (Specific Key 20180268).

Keywords: Ranolazine, Insulin, astrocytes, inflammation, antioxidants.

Corresponding author: Soraya L. Valles, https://orcid.org/0000-0003-4861-4266

P3-05

Inflammation in APP/SP1 mice, a model of Alzheimer'disease

Ignacio Campo Palacios, Adrian Jorda, Soraya L Valles

Department of Physiology, University of Valencia, Valencia, Spain

Alzheimer's disease (AD) is a degenerative illness highly study, but with poor results in investigation and recovery patients. Research scientific community have accepted the theory of amyloid as the most important. Actually, a new drug based in diminish amyloid plates, Aduconubab, is giving to patients with poor results. Our group and others, are looking for another cause to produce AD. Inflammation is characteristic process of the illness and to investigate it, we analyses the changes in occurred in APP/PS1, a mice with high production of amyloid. Changes in cytokines and chemokines are detected in APP/SP1 compared with Wild Type mice. Increases in pro-inflammatory proteins are noted and theses are associated with quimiotaxis of chemokines and cytokines to the brain across blood brain barrier (BBB). Furthermore, glial fibrillary acidic protein (GFAP), a characteristic protein of astrocytes, is increase in APP/PS1 compared with Wilde Type mice, demonstrating an astrocytes defense in front of AD at young mice (3 mouths age). On the contrary, an increase in this protein and microglia proteins are highly expressed in APP/PS1 compared with Wild Type mice at 28 days age, showing an exacerbation present of these cells with astrogliosis mechanism too. Our results, demonstrate a presence increase of inflammation, astrogliosis, glial cell presence and neurodegeneration in APP/PS1 brain mice. There are other solutions to diminish AD neurodegeneration and progression of the illness.

Supported by Oskar Phisher Foundation, USA.

Keywords: Alzheimer's disease, inflammation, astrocytes, microglia

Corresponding Author: Soraya L. Valles, https://orcid.org/0000-0003-4861-4266

P3-06

Mild cognitive impairment improvement with transcranial laser stimulation

^*1Ignacio Campo-Palacio, ^1,2Adrian Jorda, ^1,2Juan Campos-Campos, ¹Constanza Aldasoro, ¹Carlos Colmena, ¹Kenia Alvarez-Gamez, ¹Martin Aldasoro, ¹Soraya L. Valles, ^#1Antonio Iradi

¹ Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

^* Presenter Author

Mild cognitive impairment (MCI) is an alteration in cognitive function, considered a precursor to Alzheimer's disease and related to vascular dysfunction. Despite the existing therapeutic alternatives to delay the development of cognitive impairment, none of them has shown benefit in the patient, so new treatments are required to meet the needs of this population. Studies, carried out by our group, point to the possible benefits of using transcranial laser stimulation (TLS) in patients with MCI, since it stimulates cellular processes at the brain level that improve vascular irrigation. Mitochondria are the energy producers (ATP) in the neural cell (neurons, astrocytes, oligodendroglia and microglia). In Alzheimer's disease, a decrease in the production of energy by the mitochondria has been detected, with an increase in oxidative stress and an increase in chronic inflammation (astrogliosis and occasionally unrestrained activation of microglia). The application of the transcranial laser increases the amount of ATP produced by the mitochondria. This effect could favor the delay of deterioration; however, more studies are needed to evaluate the efficacy of this technique on the function and possible neurocognitive improvement of amnesic MCI patients.

Financial support from the University of Valencia. (Specific Key 20180268).

Keywords: Mild cognition impairment, Alzheimer’disease, ATP, transcranial laser stimulation.

^#Corresponding author: Antonio Iradi

P3-07

Inflammation, chemotaxis and coagulation changes in APP/PS1 compared to wild type mice

^*1Juan Campos-Campos, ¹Ignacio Campo-Palacio, ^1,2Adrián Jordá, ¹Kenia Alvarez-Gamez, ¹Constanza Aldasoro, ¹Sol Guerra-Ojeda, ¹Andrea Suarez, ^#1Soraya L. Vallés

¹ Department of Physiology, School of Medicine, University of Valencia, Valencia, Spain, and

² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

^* Presenter Author

Alzheimer’s disease (AD) had not been yet understood. Changes in inflammation could be necessary to improve strategies to act in AD. The amyloid precursor protein plus presenilin-1 (APP/PS1 mice) is a model for AD. Chemokines secreted by Central Nervous System (CNS) cells could play important roles in AD. Chemokines and their receptors involved in inflammation were the objective of this study. Female APPswe/PS1 double-transgenic mice (B6C3-Tg) were used. Cortex brain of 20-22 months of age were obtained and used to quantify chemokines and chemokine receptors expression using RT-PCR and Western-blot technique. Significant increase in GFAP protein expression was detected showing an elevation in number of reactive astrocytes in transgenic compared to wild type mice. IL-3 and ABDF1 (intervenes in the transport across cell membrane) were up-regulated in APP/PS1. On the contrary, CCR5 expression was downregulated in transgenic mice but, their both chemokines, CCL3 and CCL4 were highly expressed indicating a possible gliosis and probably an increase in chemotaxis from lymphocytes and T cell generation. Moreover, CCR8 increase expression with diminution of its CCL1 chemokine, both normally involved in protection from bacterial infection and demyelination. Control of inflammatory proteins will be the next step in understanding the progression of AD and in determining the mechanisms that can develop in this disease.

Supported by Oskar Fisher Foundation. USA.

Keywords: inflammation, chemokines, APP/SP1, Alzheimer’s disease

^#Corresponding author: Soraya L. Valles, https://orcid.org/0000-0003-4861-4266

P3-08

Changes in chemokines and chemokines receptors involved in phagocytosis, demyelination and chemotaxis in APP/PS1 mice

^*1Carlos Colmena, ^1,2Adrián Jorda, ¹Ignacio Campo-Palacio, ¹Kenia Alvarez-Gamez, ¹Juan Campos-Campos, ¹Sol Guerra-Ojeda, ¹Constanza Aldasoro, ^#1Soraya L. Valles

¹ Department of Physiology, School of Medicine, University of Valencia, Valencia, Spain, and

² Department of Nursing, Faculty of Nursing and Podiatry, University of Valencia, Valencia, Spain

^* Presenter Author

Alzheimer’s disease (AD) had not been yet understood and it is known that Inflammation is distinctive. The amyloid precursor protein plus presenilin-1 (APP/PS1 mice) is a model for AD. Chemokines secreted by Central Nervous System (CNS) cells could play important roles in AD. Data looking for the chemokines involved in inflammatory mechanisms are not further understood. Chemokines and their receptors involved in phagocytosis, demyelination, chemotaxis, and coagulation were the objective of our study. Female APPswe/PS1 double-transgenic mice (B6C3-Tg) were used and cortex brain of 20-22 months of age were obtained and used to quantify chemokines and chemokine receptors expression using RT-PCR and Western-blot technique. Significant inflammatory changes were detected in APP/PS1 compared to Wild type mice. CCR1, CCR3, CCR4, and CCR9 were elevated and CCR2 were decreased compared with Wild type mice. Their ligands CCL7, CCL11, CCL17, CCL22, CCL25, and CXCL4 showed an increase expression, however, changes were not observed in CCL2 in APP/PS1 compared to Wild type mice. Changes in protein expression could contribute to explain the differences between Alzheimer patients and elderly people without AD. This would provide a new strategy for the treatment of AD, with the possibility to act in specific inflammatory targets.

Supported by Oskar Fisher Foundation, USA.

Keywords: inflammation, chemokines, APP/SP1, Alzheimer’s disease

Corresponding author: Soraya L. Valles, https://orcid.org/0000-0003-4861-4266

P3-09

Role of transient receptor potential channels in epilepsy: Current treatment approaches

¹Nigar Ebrar Coskun, ¹Özkan Yesiltas, ²Abdülhadi Cihangir Uğuz

¹ Faculty of Medicine, Karamanoglu Mehmetbey University, Karaman, Turkey

² Department of Biophysics, School of Medicine, Karamanoglu Mehmetbey University, Karaman, Turkey

Epilepsy is a combination of the neurological disorders. It is one of the most common brain disorders, affect nearly 1% of the world population. It occurs as a result of abnormal and excessive electrical discharge in cortical neurons which cause seizures. However, the exact reasons of the underlying mechanisms have not been identified yet. Although epilepsy can be treated with antiepileptic drugs, 30% of the patients suffers from drug-resistant seizures. Therefore, newly designed next generation antiepileptic drugs are needed for an effective treatment of epilepsy. Calcium ions [Ca²⁺]_i have a crucial role in neuronal signaling. Transient Receptor superfamily has 28 members which functions as a cation permeable channel in mammals. The development and identification of the TRP channel agonists or inhibitors are promising targets for new therapeutic drug design for treatment of epilepsy. Identification of these ion channels will provide new clues to a better understand of the pathophysiology of epilepsy. Increasing amount of newly published articles highlights the role of TRP channels in epileptogenesis. Neuronal hyperexcitability originate from ion channel mediated neuronal firing and the abnormality in neuronal network caused uncontrolled synchronization of a group of neurons are the main reasons of the epilepsy. Altered [Ca²⁺]_i levels have long been well known as a major contributor of epilepsy. Ankyrin transient receptor potential channels (TRPA) was found to be upregulated during epilepsy. Canonical transient receptor potential channels (TRPC) contributes to epilepsy above mentioned two steps. Vanilloid transient receptor potential channels (TRPV) is one of the mediator of the epilepsy. Melastatin transient receptor potential channels (TRPM) family was determined to be involved in pathogenesis of epilepsy. Finally, the implication of these channels’ agonist or blocker mechanisms on seizure generation and propagation will be discussed in the presentation. We will figure out the latest findings in epilepsy treatment by focusing on ion channels gating manners and newly discovered drugs. As indicated, TRP channels play a key role in the pathogenesis of epilepsy. Their modulation is crucial in disease progression. Further studies are needed for discovering new molecular pathways and drug design for an effective treatment.

Keywords: Epilepsy, transient receptor potential channels

Corresponding author: A. Cihangir Uğuz, https://orcid.org/0000-0002-5778-581X

P3-10

Fornix demyelination in the early onset of Alzheimer’s disease

^*1Artemis Ftara, ²Rut Campos, ³José L León, ⁴María-Ángeles Lloret, ³Natalia Castillo, ⁵Begoña López, ²Ana Cervera-Ferri, ¹Ana Lloret

¹ CIBERFES, Institute of Health Research-INCLIVA, Department of Physiology, Faculty of Medicine, University of Valencia, Avda. Blasco Ibañez 17, 46010, Valencia, Spain

² Department of Human Anatomy and Embryology, Faculty of Medicine, University of Valencia, Avda. Blasco Ibañez 17, 46010, Valencia, Spain

³ Department of Neuroradiology, Ascires Biomedical Group, Calle Menéndez y Pelayo 25, 46010, Valencia, Spain

⁴ Department of Clinical Neurophysiology, University Clinic Hospital of Valencia, Avda. Blasco Ibañez 19, 46010, Valencia, Spain

⁵ Department of Neurology, University Clinic Hospital of Valencia, Avda. Blasco Ibañez 19, 46010, Valencia, Spain

In Alzheimer’s disease (AD) the process of demyelination has been demonstrated and indeed it is an early event that occurs before atrophy of grey matter and may even precede Aβ and tau pathologies. The degree of myelin loss is also an indicator of progression from Mild Cognitive Impairment (MCI) to AD and correlates with accelerated memory decline. Demyelination has also been identified at medial temporal lobes which includes the fornix. Moreover, it has been proposed that detection of fornix myelin loss is more sensitive than volume measures and maybe a potential early marker for loss of medial temporal lobe connectivity. We propose that this demyelination occurs specifically at the hippocampal commissure, the connection between both hippocampi. We have assessed myelin content by immunohistochemistry in 10 young APP/PS1 mice (4-5 months) as model of AD and 10 young wildtype mice as controls from the same colony. We have also recruited 10 non-demented controls, 10 MCI and 10 moderate AD patients, to whom we have performed DTI at the hippocampal commissure and volumetric studies by MRI. Also, electroencephalography in the resting state with closed eyes by EEG recordings was performed in all subjects. In AD mice, we observed a significant demyelination in the hippocampal commissure compared to controls. Our preliminary results indicate that the fractional anisotropy (FA) decreases in MCI and AD patients compared to controls and the apparent diffusion coefficient (ADC) increases in patients. Our results are compatible with a demyelination process in the hippocampal commissure. Moreover, our first EEG recordings’ analyses show diminished interhemispheric phase coherence between the temporal electrodes in the 8-13 Hz frequency band in the resting state with closed eyes.

Keywords: Demyelination, inter-hippocampal commissure, Alzheimer’s disease

Corresponding author: Artemis Ftara

P3-11

Kainate receptor modulation of glutamatergic synaptic transmission in the CA2 region of the hippocampus in mice

^*1Aníbal Gallardo-Recio, ¹Rafael Falcón-Moya, ¹Irene Martínez-Gallego, ¹Antonio Rodríguez-Moreno

¹ Cellular Neuroscience Laboratory and Plasticity, Department of Physiology, Anatomy and Cell Biology, University Pablo de Olavide, Seville, Spain

Kainate (KA) receptors (KARs) are important modulators of synaptic transmission. We studied here the role of KARs on glutamatergic synaptic transmission in the CA2 region of the hippocampus using whole-cell patch-clamp recordings from CA2 pyramidal neurons. We observed that KA depresses glutamatergic synaptic transmission at Schaffer collateral-CA2 synapses; an effect that was antagonized by NBQX (a KA/AMPA receptors antagonist) under condition where AMPA receptors were previously blocked. The study of paired-pulse facilitation ratio indicated a presynaptic locus of action for KAR. Additionally, we determined the action mechanism for this depression of glutamate release mediated by the activation of KARs. We found that inhibition of protein kinase A suppressed the effect of KAR activation on evoked excitatory post-synaptic current, an effect that was not suppressed by protein kinase C inhibitors. Furthermore, in the presence of Pertussis toxin, the depression of glutamate release mediated by KAR activation was not present, invoking the participation of a G_i/o protein in this modulation. Finally, the KAR-mediated depression of glutamate release was not suppressed in dn-SNARE mice.

Keywords: CA2, kainate receptor, glutamate, dn-SNARE.

Corresponding author: Aníbal Gallardo Recio (email: agalrec@alu.upo.es)

P3-12

Role of LPA ₁ in use-dependent short-term synaptic plasticity in rats

^*Ángela Gento-Caro, Esther Vilches-Herrando, David González-Forero, Bernardo Moreno-López.

Área de Fisiología, Facultad de Medicina, Universidad de Cádiz, Cádiz, Spain

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, acts as a local messenger involved in synaptic neuromodulation. In this context, LPA mainly acting on receptor LPA₁ engages two intracellular signaling cascades regulating the strength of excitatory and inhibitory synaptic transmission. The aim of this work was to investigate whether LPA-LPA₁ signaling is involved in use-dependent short-term plasticity at excitatory synapses. In brainstem slices from rat pup (P5-P9), a high-frequency stimulation (HFS, @20-Hz, 60-s) protocol was applied to the ventrolateral reticular formation and excitatory postsynaptic currents (EPSCs) evoked in hypoglossal motoneurons (HMNs) were analyzed during and after (@0.2-Hz) HFS. Exogenous application of LPA (1uM) to the bath solution altered both, synchronic and asynchronic neurotransmitter release during HFS. It is well established that the asynchronic component increases during the train by accumulation of presynaptic [Ca²⁺]i. In addition, LPA delayed the kinetic of EPSC recovery after HFS. Addition to the bath solution of the LPA₁ inhibitor AM095 (10 uM) also affected synchronic and asynchronic components but, as expected, in a direction opposite to that of LPA. Furthermore, AM095 sped up EPSC recovery after HFS. Finally, the addition of a non-permeable Ca²⁺ chelator, EGTA (11 mM), to the internal solution of the recording pipette differentially affected AM095-induced alterations in EPSCs during and after HFS. Taken together, these results indicate that LPA-LPA₁ signaling mediates short-term depression and recovery kinetics during and after HFS, respectively. The mechanism of action of endogenously synthesized LPA appears to be dependent on postsynaptic [Ca²⁺]i.

Supported by MINECO/FEDER (BFU2015-71422-R; PID2019-110960GB-I00); 2014-2020 ERDF Operational Programme, Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia (FEDER-UCA18-108475).

Keywords: Motoneurons, lysophosphatidic acid, short-term synaptic plasticity.

Corresponding author: Ángela Gento-Caro

P3-13

Study of the cisplatin-induced retinal damage in male Wistar rats: looking for possible therapeutic agents

^*@1,2Beatriz Martín-Sánchez, ^@1Sara Rubio-Casado, ¹Diego San Felipe, ¹Miguel A Martínez-López, ¹Ricardo Llorente,²María A González-Nicolás, ³Eva M Marco, ¹Meritxell López-Gallardo,^1,2Alberto Lázaro

¹ Department of Physiology, Faculty of Medicine, Universidad Complutense de Madrid, Madrid, Spain.

² Laboratory of Renal Physiopathology, Department of Nephrology, Instituto de Investigación Sanitaria Gregorio Marañón, Hospital General Universitario Gregorio Marañón, Madrid, Spain.

³ Department of Genetics, Physiology and Microbiology, Faculty of Biological Sciences, Universidad Complutense de Madrid, Madrid, Spain.

^@ These authors contributed equally to this work.

Cisplatin is a widely used antineoplastic agent employed in the treatment of many forms of cancer. Nephrotoxicity has been reported as the main secondary adverse effect resulting from cisplatin administration, although neurotoxicity, including ototoxicity and visual deterioration, has also been described. In the present study, we aimed to investigate the effects of cisplatin administration in the retina of male Wistar rats and to evaluate whether DF401 will be able to prevent or reverse the expected cisplatin-induced retinal damage. For that purpose, 12 animals were employed for a preliminary analysis: half received a single dose of cisplatin (CISP, 5 mg/kg, i.p.) and the other half received a vehicle (VH, saline) injection; from that day on, DF401 (150 mg/kg, i.p.) or its vehicle (VH, saline) was administered for 5 consecutive days until sacrifice, when retinas were obtained for the forthcoming immunohistochemical analysis. Retinal Ganglion Cells (RGCs) and macroglial and microglial cells were determined based on their expression of Brn3, GFAP and Iba1, respectively. Preliminary results indicate that CISP may induce a generalised reduction in the studied retinal cell types, although a statistically significant reduction was only observed for GFAP+ cells within the temporal inferior retina, and for Iba1+ cells within the same retinal subregion as well as for the nasal central and inferior subregions. A more in detail analysis showed that DF401 co-administered with CISP increased GFAP+ cells in the nasal central and inferior regions, whereas their combination diminished Iba1+ cells within the nasal-superior retina. In general, considering data from the whole retina, the combination of CISP and DF401 maintained the number of Brn3+ cells as well as GFAP expression similar to control (VH+VH) levels, although this observation was not so clear for the number of Iba1+ cells. Present results suggest that the nasal retina could be particularly sensitive to the CISP-induced effects; and DF401 seems to interact specifically regarding macroglial and microglial expression in specific retina subregions. Further analyses are still required to confirm the reported effects, and to better understand the neurobiological mechanisms involved.

Keywords: Ganglionar and glial retinal cells, neurotoxicity, antineoplastic agent, DF-401.

Corresponding author: Alberto Lázaro Fernández, https://orcid.org/0000-0002-1095-679X

P3-14

Endogenous pH 6.0 β-galactosidase activity in developing sensory systems of vertebrates

¹José A de Mera-Rodríguez, ¹Guadalupe Álvarez-Hernán, ¹Alejandro Arias-Montecino, ¹Andrea Ruiz-Salguero, ¹Cristina Pérez-Mora, ²Violeta Calle-Guisado, ^*1Gervasio Martín-Partido, ²Joaquín Rodríguez-León, ¹Javier Francisco-Morcillo.

¹ Department of Anatomy, Cell Biology and Zoology, Faculty of Science, University of Extremadura, Badajoz, Spain, and ² Department Anatomy, Cell Biology and Zoology, Faculty of Medicine and Health Sciences, University of Extremadura, Badajoz, Spain.

The histochemical detection of β-galactosidase enzymatic activity at pH 6.0 (β-gal-pH6) has become one of the most commonly used markers of cell-aging. Additionally, there are multiple examples of β-gal-pH6 expression during specific time windows in embryonic senescent cells located in transient structures that degenerate during tissue development. However, it has also been described that β-gal-pH6 labelling is also present in non-senescent cells, indicating that this histochemical method may not be a marker of senescence in all contexts. In the case of the central nervous system, strong β-gal-pH6 signal is detected in differentiating neurons at early stages of embryo development. Similar results have been described in neurons during development of sensory systems. It has been described that lysosomal β-gal is the origin of the β-gal-pH6 activity detected in subpopulations of early differentiating and mature retinal neurons. Furthermore, these recently differentiated β-gal-pH6-positive retinal cells also express p21, a typical senescence marker during embryonic development. In the case of the developing olfactory epithelium, β-gal-pH6 staining is restricted to mature OMP-immunoreactive neurons but it is absent from GAP43-positive immature olfactory neurons. Therefore, it is important to discriminate between senescent cells and post-mitotic cells in studies about aging of the central nervous system because some accepted markers of senescence are less specific than originally was expected.

Supported by Junta de Extremadura, Fondo Europeo de Desarrollo Regional, “Una manera de hacer Europa” (GR15158, GR18114, IB18113).

Keywords: senescence, cell differentiation, histochemistry, immunohistochemistry.

Corresponding author: Gervasio Martín-Partido (gmartin@unex.es), https://orcid.org/0000-0002-8208-744X

P3-15

Queen bee acid upregulate the expression of autophagy proteins through SIRT1/FOXO axis

^*1,2Marta Paredes-Barquero, ^1,2,3Guadalupe Martínez-Chacón, ^1,2Eva Alegre-Cortés, ^1,2Saray Canales-Cortés, ^1,2Alberto Gimenez-Bejarano, ^1,2,3Elisabet Uribe-Carretero, ¹Laura Sánchez-Molinero, ¹Nerea Dominguez-Rojo, ^1,2Patricia Gómez-Suaga, ^2,3Sokhna MS Yakhine-Diop, ^1,2,3Rosa A González-Polo, ^1,2,3José M Fuentes, ^1,2,3Mireia Niso-Santano

¹ Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Cáceres, Spain.

² Instituto de Investigación Biosanitaria de Extremadura (INUBE), Cáceres, Spain.

³ Centro de Investigación Biomédica en Red de Enfermedades (CIBERNED), Madrid, Spain.

Autophagy is a highly conserved intracellular catabolic pathway that removes toxic protein aggregates and cellular organelles to maintain neuronal homeostasis. The impaired regulation of autophagy leads to the accumulation of protein aggregates and thereby, neurodegeneration. Therefore, the activation of autophagy may become a potential target against neurodegeneration through the prevention or degradation of the protein aggregates. Queen bee acid (QBA, 10-hydroxy-2-decenoic acid) is the major fatty acid component of, and found exclusively in, royal jelly, which has anti-tumor, anti-inflammatory and antibacterial activities and promotes neurogenesis and neuronal health. We have previously demonstrated that autophagy induction plays a critical role in the beneficial health effects of QBA. The present study investigates the mechanism by which QBA induces autophagy due to the fact that, so far, it has not yet been elucidated. Our results showed that QBA upregulates the expression of several autophagy proteins throughout the activation of Sirtuin 1 and forkhead box transcription factor (FOXO). QBA potentiates the expression of Sirtuin1 and increases it deacetylase activity. Hence, Sirtuin 1 may play a key role in the autophagy induced by QBA.

This research was supported by a Grant (IB18048) from Junta de Extremadura, Spain and a Grant (RTI2018-099259-A-I00) from Ministerio de Ciencia e Innovación, Spain. This work was also partially supported by “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. G.M-C is supported by Fundación ONCE. E.A-C is supported by a pre-doctoral fellowship of Valhondo Calaff Foundation. S.C-C and E.U-C were supported by a FPU fellowship [FPU19/04435 and FPU16/00684, respectively] from the Ministerio de Ciencia, Innovación y Universidades, Spain. M. P-B and A.G-B received fellowships from the “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (Universidad de Extremadura). M.N-S was supported by the “Ramon y Cajal” Program [RYC-2016–20883], and P.G-S, was funded by “Juan de la Cierva Incorporación” Program [IJC2019-039229-I], Spain. S.M.S.Y-D was supported by Isabel Gemio Foundation and CIBERNED [CB06/05/0041]. JMF received research support from Isabel Gemio Foundation and the “Instituto de Salud Carlos” III, CIBERNED [CB06/05/0041].

Keywords: Autophagy, QBA, neurodegeneration, SIRT1

Corresponding author: Marta Paredes-Barquero, https://orcid.org/0000-0002-5698-8180

P3-16

Pharmacological activation of dopamine D4 receptor prevents morphine-induced tolerance at the rat dorsal horn level

¹Marina Ponce-Velasco, ¹María A Real, ¹Alicia Rivera, ^*2Belén Gago

¹ Department of Cell Biology, University of Málaga, Málaga, Spain

² Department of Human Physiology, University of Málaga, Málaga, Spain

Morphine is one of the most effective drugs used for pain management. However, prolonged exposition to morphine produces a wide variety of side effects such as tolerance to analgesia, hyperalgesia and addiction. Downregulation of the mu opioid receptor (MOR) and its uncoupling to G-proteins in the dorsal horn are likely to contribute to the development of morphine tolerance. Previous studies demonstrated that dopamine D4 receptor (D4R) activation prevents morphine addiction by modulating dopamine signaling from nigral dopamine cells. This effect seems to be the result of an antagonistic receptor-receptor interaction involving a D4R-MOR heteroreceptor which could exist in the dorsal striatum. As D4R is expressed in dorsal horn neurons, we hypothesize that D4R could interfere in the development of morphine-induced tolerance to its analgesic effects. Here, using a chronic paradigm of combined treatment of morphine with the D4R agonist PD168.077, we investigated the nociceptive response to three noxious stimuli: thermal (tail-flick test), mechanical (von Frey test) and chemical (formalin test). Moreover, using immunohistochemistry, western blot and qRT-PCR, we have evaluated alterations in the primary circuitry of pain and the balance between glutamate and GABA within dorsal horn. Results from the evaluation of analgesic activity of chronic combined treatment of morphine with PD168,077 showed that D4R prevents the development of morphine-induced analgesic tolerance. The present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interaction in the dorsal horn that could help to the development of a new pharmacology strategy in the treatment of pain.

Supported by CTS161 and UMA20-FEDERJA-122 (Junta de Andalucía, Spain)

Keywords: morphine, D4 receptor, tolerance, noxious stimuli

Corresponding author: Belén Gago

P3-17

AP2α immunoreactivity in the developing avian retina

¹Andrea Ruiz-Salguero, ¹José A de Mera-Rodríguez, ¹Guadalupe Álvarez-Hernán, ¹Alejandro Arias-Montecino, ¹Cristina Pérez-Mora, ²Violeta Calle-Guisado, ^*1Gervasio Martín-Partido, ²Joaquín Rodríguez-León, ¹Javier Francisco-Morcillo

¹ Department of Anatomy, Cell Biology and Zoology, Faculty of Science, University of Extremadura, Badajoz, Spain, and ² Department Anatomy, Cell Biology and Zoology, Faculty of Medicine and Health Sciences, University of Extremadura, Badajoz, Spain

The spatial and temporal expression profiles of the activating protein 2 alpha (AP2α) during embryogenesis suggest that it is important for normal tissue development. Indeed, severe malformations of the face and skull, as well as abnormalities of sensory organs, result from inactivation of the AP2α gene in mammals. It has been shown that AP2α gene is expressed in the optic lobes in Drosophila during development and in different populations of retinal cells in vertebrates, suggesting a role in the differentiation of the visual system. In the present study we analyzed the spatial and temporal distribution of the AP2α transcription factor in the developing retina of two precocial (Gallus gallus and Coturnix coturnix) and an altricial (Taeniopygia guttata) bird species. The expression pattern of AP2α during avian retinal development was highly conserved. In the early differentiating retina, immunoreactivity was mainly found in the vitreal region of the presumptive neural retina, but also in sparse ovoid nuclei dispersed throughout the neuroblastic layer, with the major axis oriented vitreo-sclerally. In the laminated retina, AP2α immunoreactivity was detected as a thick band of nuclei located in the amacrine cell layer. Subpopulations of these AP2α-immunopositive cells also expressed typical markers of amacrine cells such as calretinin, calbindin, doublecortin, or parvalbumin. Therefore, the early and cell-specific expression of AP2α in the developing bird avian retina suggest a role for this transcription factor in the early steps of amacrine cell differentiation, but also in the maintenance of the amacrine cell phenotype.

Supported by Junta de Extremadura, Fondo Europeo de Desarrollo Regional, “Una manera de hacer Europa” (GR15158, GR18114, IB18113).

Keywords: cell differentiation, development, retina, immunohistochemistry.

Corresponding author: Gervasio Martín-Partido (gmartin@unex.es), https://orcid.org/0000-0002-8208-744X

P3-18

Impaired rate-dependent depression of the hoffman reflex in subjects with overwise and obesity

Jaime Pérez-Huitrón¹ Marcelino Rodriguez-Aguila¹ Francisco Alcántara-Reyes¹ Luisa Salinas-Hernández¹ Daniela Torres-Loera² Carlos Cuellar-Ramos³ Oscar Garcia⁴ Eduardo Trujillo-Condes¹

¹ Faculty of Medicine, Universidad Autónoma del Estado de México

² Faculty of Sciences, Universidad Autónoma del Estado de México

³ School of Sport Sciences, Universidad Anáhuac México

⁴ Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado

According to the National Center for the Prevention of Chronic Diseases (CDC) of México, the prediabetes is a condition in which blood glucose levels are higher than normal, but without presenting criteria for the diagnosis of type-2 diabetes (CDC, 2022).The criteria to determine a prediabetic subject are: 8-hour fasting glucose of 100 to 125 mg/dl; oral glucose tolerance test with a 75-gram load of 140-199mg/dl and glycosylated hemoglobin (Hb1Ac) 5.7 to 6.4%. Meanwhile, it is not associated with a specific symptomatology, but it is associated a body mass index (BMI) greater than 25 kg/m2, that is, overweight and obesity in their different degrees (ADA, 2022).

Overweight and obesity have been considered a proinflammatory state that conditions multi-organ disorders, ranging from fatty liver to peripheral nervous system disorders such as neuropathy (ADA, 2022).

It was recently described that the phenomenon of stimulation Frequency Dependent Depression in the Hoffman reflex (RDDH) is affected in a murine model of obesity induced by high caloric intake (Nguyen et al., 2017). In humans with type 1 and 2 diabetes and peripheral neuropathy, it has been described that RDDH could be a biomarker of nerve damage, associated with the loss of the spinal inhibition mechanism (Calcutt, 2014).

Currently there are no tests that evaluate nerve fiber damage in prediabetic subjects, then with this project to describe the findings found in frequency-dependent depression of the Hoffman reflex (RDDH) in prediabetic and healthy subjects with overweight and obesity.

The problem group was formed by 10 subjects with a BMI >25kg/m2 (Range 25-78 years, 3 men/ 7 women) vs 10 subjects with a BMI of 18.5-24.9 kg/m2 (Range 25-78 years, 4 men/6 women); who underwent the RDDH test in the lower limb at different frequencies (0.2, 1.0, 2.0, 5.0 and 10.0 Hz) with a total of 10 pulses for each one. The Shapiro-Wilk test was performed and if passed p>0.05, the T-Student test was used to compare the two groups; otherwise The Mann-Whitney was used. P values ​​<0.05 were considered significant. Significant depression of RDDH was observed in subjects with prediabetes at 1 Hz (p < 0.01), 5 Hz (p < 0.01), and 10 Hz (p 0.1), but not at 0.2 Hz and 2 Hz (p > 0.5). Therefore, the RDDH test can be used for the early evaluation of neuropathy in subjects with alterations in glycemia coupled with clinical diagnosis.

Keywords: Hoffman reflex, prediabetes, neuropathy, overweight, obesity, rate-dependent depression

P3-19

Molecular network in neurodegenerative diseases

¹S Unay

¹ Aydin Adnan Menderes University, Healthy Science Institute, Department of Biophysics, Aydin, Turkey

The aim of this study is to highlight the mechanisms and the role of network biology in causing neurodegenerative diseases such as Alzheimer’s (AH), Parkinson’s (PD), and Huntington’s (HD). Alzheimer's (AD), Parkinson's (PD), and Huntington's (HD) diseases are among the most common neurodegenerative diseases. In addition to a mutation in a gene, genetic, epigenetic, and environmental factors cause neurodegenerative diseases. Technologies such as RNA sequencing are preferred in the molecular explanation of neurodegenerative diseases. However, it is very difficult to interpret extensive data with these methods and it is a classical analysis method. Healthy and diseased molecular networks are extensively explored and extensive data integrated because of the so-called network biology platform. In the clinic, network biology is very successful in describing the link between neurodegenerative diseases and other diseases. This platform is especially preferred for examining the mechanisms of gene structure that trigger neurodegenerative diseases and identifying diagnostic biomarkers and therapeutic targets. With this approach, links between neurodegenerative diseases and diseases such as cancer and diabetes have emerged. In conclusion, network biology is considered to be a practical platform for the clinical diagnosis of neurodegenerative diseases and the identification of potential therapeutic targets. In addition, considering the results of studies conducted in recent years, it is predicted that the network biology platform will accelerate the discovery of new therapeutic targets and significantly improve the clinical management of neurodegenerative diseases.

Keywords: Neurodegenerative diseases, network biology

Corresponding author: S. Unay

P3-20

Evidence for a role of chaperone p11 in neurotransmission and use-dependent synaptic plasticity in rats

^*Esther Vilches-Herrando, Ángela Gento-Caro, Isis Gastaldo-Jordán, David González-Forero, Bernardo Moreno-López

Physiology Area, University of Cádiz, Cádiz, Spain

Because synaptic communication is key for information processing in the brain, deciphering molecular signals involved in synaptic dynamic is the subject of intense investigation. p11 (S100A10) generally acts as an adaptor protein that regulates intracellular trafficking and plasma membrane expression of specific receptors and ion channels. On this background, we recently reported that p11 sets up motoneuron (MN) intrinsic excitability. Although several evidences support the hypothesis that this chaperone could be pivotal in the control of synaptic function and plasticity, this issue has not been addressed yet. In brainstem slices from rat pups (P7-P9), different frequency stimulation (@10, @20, @40, @60-Hz, 10-s) protocols were applied to the ventrolateral reticular formation. Evoked excitatory postsynaptic currents (EPSC) in hypoglossal MNs (HMNs) were recorded and analyzed off-line. As expected, the magnitude of synaptic depression was stronger after application of the highest frequency stimulation (HFS, @60-Hz, 10-s) protocol. In addition, spontaneously occurring AMPAergic synaptic currents (sEPSC_AMPA) and AMPAergic miniature EPSCs (mEPSC_AMPA) were recorded. A small-interfering RNA against p11 (siRNA_p11; 5 μg/5 μl) was injected into the fourth ventricle at P5, taken administration of a non-interfering siRNA (cRNA) as control. siRNA_p11 reduced (-46.4 ± 1.7%) mRNAp11 expression in the brainstem of P7-P9 rats. p11 knockdown was accompanied by a reduction in the amplitude of recorded EPSCs (cRNA: 0.467 ± 0.056 nA; siRNA_p11: 0.338 ± 0.048 nA). siRNA_p11 altered both, synchronic and asynchronic neurotransmitter release during HFS. Particularly, total charge transfer for the asynchronic component of the synaptic response across the train was strongly attenuated by this treatment (cRNA: 1.58 ± 0.372 nC; siRNA_p11: 0.66 ± 0.119 nC). Since the asynchronic component increases during train by accumulation of presynaptic [Ca²⁺]i, siRNA_p11-induced reduction in this factor supports a pre-synaptic site of action of endogenous p11. Kinetic of EPSC recovery after HFS was also altered in siRNA_p11 relative to cRNA-treated pups. Finally, sEPSC_AMPA and mEPSC_AMPA showed a reduction in frequency and amplitude under siRNA_p11 treatment. Altogether, these outcomes support a pivotal role of p11 in excitatory neurotransmission and use-dependent plasticity by, at least, a pre-synaptic mechanism of action.

Supported by MINECO/FEDER (BFU2015-71422-R; PID2019-110960GB-I00); 2014-2020 ERDF Operational Programme, Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia (FEDER-UCA18-108475).

Keywords: p11, siRNA_p11, plasticity, EPSC.

Corresponding author: Esther Vilches-Herrando

P3-21

Protective effect of acorn cupule bioactive compound in neurodegeneration. Involvement of autophagy/mitophagy

^*1,2Saray Canales-Cortés, ³Guadalupe Lavado, ^1,2Alberto Gimenez-Bejarano, ^1,2,4Elisabet Uribe-Carretero, ¹Gema Duque González, ^1,2Marta Paredes-Barquero, ^1,2Eva Alegre-Cortés, ⁵Vicente Climent, ⁶Pedro J Camello, ¹Laura Sánchez-Molinero, ¹Nerea Domínguez-Rojo, ¹Mercedes Blanco-Benítez, ^4,7,10Ana Aiastui, ^{4,7,8,9,10,11}Adolfo López de Munain, ^1,2,3Guadalupe Martínez-Chacón, ^1,2,3Patricia Gómez Suaga, ^1,2,3Mireia Niso-Santano, ^1,2,3Sokhna MS Yakhine-Diop, ³Ramón Cava, ^1,2,3José M Fuentes, ^1,2,3Rosa A González-Polo

¹ Universidad de Extremadura, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Enfermería y Terapia Ocupacional, Cáceres, Spain; 2 Instituto de Investigación Biosanitaria de Extremadura (INUBE), Cáceres, Spain; ³ Universidad de Extremadura, INBio G-C, Departamento Facultad de Veterinaria, Cáceres, Spain; ⁴ Centro de Univestigación Biomédica en Red de Enfermedades (CIBERNED), Madrid, Spain; ⁵ Universidad de Extremadura, Departmento de Anatomía y Embriología Humana, Facultad de Medicina, Badajoz, Spain; ⁶ Universidad de Extremadura, Departamento de Fisiología, Facultad de Enfermería y Terapia Ocupacional, Cáceres, Spain; ⁷ Cell Culture Plataform, Donostia University Hospital, San Sebastián, Spain; ⁸ Neuroscience Area of Biodonostia Health Research Institute, Donostia University Hospital, San Sebastián, Spain; ⁹ Donostia University Hospital, Department of Neurology, San Sebastian, Spain; ¹⁰ Ilundain Foundation, San Sebastian, Spain; ¹¹ Department of Neurosciences, University of the Basque Country UPV-EHU, San Sebastián, Spain.

Autophagy is a highly conserved catabolic process that plays a key role in the maintenance of cellular and organismal homeostasis by facilitating the turnover of cytoplasmic structures, allowing cells to adapt to changing and stressful conditions. Impaired autophagy is directly involved in all major age-related human diseases including neurodegenerative diorders such as Parkinson's disease (PD). PD is the second most common neurodegenerative disease in worldwide. Its etiological mechanisms are not yet known in a precise way, because it is a complex disease of multifactorial origin. In PD, cellular degradation process is deregulated, inducing aggregates of several proteins, affecting the formation of autophagosomes or lysosomal biogenesis. Therefore, a dysregulation of autophagy aggravates the pathogenesis of PD. The acorn has been an ancient staple food, an important source of a natural compound known as ellagitannins, whose metabolism through the intestinal microbiota originates substances known as urolithins. The urolithins have been reported to have significant antiproliferative, anticancer, and antiaging beneficial effects. In addition, it has been described that urolithin A induces autophagy and mitophagy, both in vivo and in vitro. In this work, we have analyzed the possible beneficial effects that different bioactive compounds of acorn cupule (a by-product of agrifood industry, used as a possible source of ellagitannins and/or urolithins) may have in several models of PD, in relation with its probable induction of autophagy and/or /mitophagy. The modulation of autophagy by natural compounds from food would help to treat or slow neuronal death occurs in neurodegenerative process.

This research was partially supported by the “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. S.C-C and E.U-C were supported by a FPU fellowship [FPU19/04435 and FPU16/00684, respectively] from the Ministerio de Ciencia, Innovación y Universidades, Spain. G.D-G is supported by Program [RYC-2016–20883]. E.A-C is supported by a pre-doctoral fellowship of Valhondo Calaff Foundation. M.P-B and A.G-B received fellowships from “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (Universidad de Extremadura). M.N-S was supported by the “Ramon y Cajal” Program [RYC-2016–20883], and P.G-S, was funded by “Juan de la Cierva Incorporación” Program [IJC2019-039229-I], Spain. S.M.S.Y-D was supported by CIBERNED [CB06/05/0041]. RAGP received research support from “Junta de Extremadura”, financed by the European Union [IB20031]. JMF received research support from “Instituto de Salud Carlos III”, CIBERNED [CB06/05/0041]. Authors thank FUNDESALUD for helpful assistance.

Keywords: Autophagy, neurodegeneration, mitophagy.

Corresponding author: Saray Canales-Cortés.

P3-22

Differential role of Orai1α and Orai1β in agonist-induced NF-κB transcriptional activity

^*Joel Nieto Felipe, Sandra Alvarado, Isaac Jardín, Gines M Salido, Jose J López, Juan A. Rosado

Department of Physiology (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, Universidad de Extremadura, Caceres, Spain.

NF-κB is a protein complex, formed by homo/heterodimers consisting of a combination of five subunits (p50, p52, RelB, c-Rel and RelA/p65) and present in all mammalian cells, capable of acting as a transcription factor responsible for regulating many cellular functions, especially the immune response. In resting, this complex locates at the cytosol attached to an inhibitory heterodimer named IκB, which is phosphorylated by different kinase proteins and marked for its degradation by the proteosome. One of the mechanisms that regulate this phosphorylation is mediated by certain DAG and Ca²⁺-dependent PKC isoforms. Orai1 is the principal constituent of the prototypical CRAC (Ca²⁺ release-activated Ca²⁺) channel. Two variants of Orai1 are present in mammalian cells, Orai1α and Orai1β, generated by alternative translation initiation. While Orai1α is the full-length variant, Orai1β lacks the N-terminal 63 amino acids. Using WT HEK-293 cells and Orai1 knockout HEK-293 cells (O1KO), we have investigated the role of the Orai1 variants in agonist-induced NF-κB activation. Our results indicate that stimulation of HEK-293 cells with carbachol enhances NF-κB transcriptional activity; meanwhile, in O1KO cells this effect was partially but significantly attenuated. Expression of Orai1α in O1KO cells rescued the ability of carbachol to induce NF-κB transcriptional activity; by contrast, this was not achieved when we expressed Orai1β, despite the expression of Orai1β rescued store-operated Ca²⁺ influx in these cells. Experiments performed in control and BAPTA-loaded cells in a Ca²⁺-free medium revealed that NF-κB activation is entirely dependent on Ca²⁺ entry. Silencing TRPC1, TRPC6 and Orai3 using shRNA in O1KO cells, as well as analysis in Orai-triple KO HEK-293 cells, indicate that both, TRPC1 and Orai3, also participate in agonist-evoked NF-kB activation. Finally, the PKCβ selective inhibitor, ruboxistaurin, impaired agonist-induced NF-κB activation in O1KO cells expressing Orai1α. These findings indicate that Orai1α, but not Orai1β, is required for agonist-induced NF-κB transcriptional activity by a mechanism dependent on PKCβ activation.

Supported by Grants PID2019-104084GB-C21 funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe, and Junta de Extremadura-cofinanciado por la Unión Europea (Grants IB20007 and GR21008).

Keywords: Orai1α, Orai1β, Ca²⁺ influx, NF-κB.

Corresponding author: Juan A. Rosado, https://orcid.org/0000-0002-9749-2325.