Constitutive phosphorylation of serine 29 as a critical regulator of TRPM8 channel function

Resumen del trabajo presentado al VIII Congreso Red Española de Canales Iónicos, celebrado en Alicante del 24 al 27 de mayo de 2022.The main molecular entity involved in innocuous cold detection in mammals is TRPM8. This polymodal TRP channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. Basal kinase activity phosphorylates TRPM8 and modulates its function under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. We identified four serine residues within the N-terminal domain constitutively phosphorylated in the mouse ortholog by mass spectrometry. TRPM8 function was assessed by Ca2+-imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, increased cold- and mentholevoked responses of the channel. S29A mutation is sufficient to enhance TRPM8 activity, suggesting that phosphorylation of this residue is a critical molecular determinant of this negative regulation. Biophysical and TIRF-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: an increase in the number of active channels at the plasma membrane and a shift in the voltage activation curve towards more negative potentials. Notably, basal kinase activity downregulates TRPM8 function at cold thermoreceptor neurons, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.Supported by Grants Millennium Nucleus for the Study of Pain (MiNuSPain) (RM, MP), Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD) (RM, MP), DICYT VRIDeI-USACH 022143PP (MP, RM) and by VRIDeI-USACH 021843MM (RM).Peer reviewe

Negative Modulation of TRPM8 Channel Function by Protein Kinase C in Trigeminal Cold Thermoreceptor Neurons

TRPM8 is the main molecular entity responsible for cold sensing. This polymodal ion channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. In corneal cold thermoreceptor neurons (CTNs), TRPM8 expression determines not only their sensitivity to cold, but also their role as neural detectors of ocular surface wetness. Several reports suggest that Protein Kinase C (PKC) activation impacts on TRPM8 function; however, the molecular bases of this functional modulation are still poorly understood. We explored PKC-dependent regulation of TRPM8 using Phorbol 12-Myristate 13-Acetate to activate this kinase. Consistently, recombinant TRPM8 channels, cultured trigeminal neurons, and free nerve endings of corneal CTNs revealed a robust reduction of TRPM8-dependent responses under PKC activation. In corneal CTNs, PKC activation decreased ongoing activity, a key parameter in the role of TRPM8-expressing neurons as humidity detectors, and also the maximal cold-evoked response, which were validated by mathematical modeling. Biophysical analysis indicated that PKC-dependent downregulation of TRPM8 is mainly due to a decreased maximal conductance value, and complementary noise analysis revealed a reduced number of functional channels at the cell surface, providing important clues to understanding the molecular mechanisms of how PKC activity modulates TRPM8 channels in CTNs

Agreements in a decentralized matching party

Magíster en Economía Aplicada. Ingeniero Civil IndustrialEl presente trabajo estudia la infinita interacción descentralizada de un mercado two-sided, el cual se comporta de manera análoga a una fiesta, donde hombres y mujeres se emparejan a partir de propuestas de baile. En cada instante, cualquiera de los dos podría abandonar al otro para obtener una mejor asignación, teniendo todos un factor de descuento común $\delta$. Utilizando como refinamiento al equilibrio Markoviano perfecto (restringido en cuanto a la formación de nuevas parejas), se analiza la relación entre el conjunto de matchings estables \Matchingset^* y el conjunto de asignaciones \Agreeset_{\delta} ante el cual ya no se generen más ``cambios'' dentro de la fiesta, entendiendo a estos últimos como ``acuerdos''. Los resultados son que: (1) todo acuerdo debe ser estable, es decir, \Agreeset_{\delta} \subseteq \Matchingset^*; (2) en caso de existir más de un \textit{matching} estable, existe un $\hat{\delta}$ fijo tal que \Agreeset_{\delta \geq \hat{\delta}} \subset \Matchingset^*; (3) los incentivos opuestos del mercado son los generan que matchings estables puedan no ser acuerdos en la fiesta. La contribución de esta investigación viene dada por mostrar que los incentivos opuestos entre los dos lados del mercado, no permiten a los agentes alcanzar o mantener asignaciones estables de manera perpetua a lo largo del tiempo

Educación física y salud versus recreo : intensidad de actividad física en niños de 5to. básico

Tesis (Profesor Educación Física para Educación General Básica)El objetivo del presente estudio descriptivo trasversal, es medir y comparar la intensidad de la actividad física en la clase de Educación Física y Salud y Recreo en niños de 5° básico con el fin de relacionar su nivel de intensidad y comprobar si se alcanzan los parámetros óptimos (moderado/vigoroso) dictados por las organizaciones competentes. Dichos parámetros reportan numerosos beneficios para la salud y evitan considerablemente Enfermedades Crónicas no Transmisibles (O.M.S. 2010). El sedentarismo y la Inactividad física son una de las principales causas para contraer Diabetes tipo 2, Hipertensión, Obesidad, etc. Patologías que actualmente desencadenan un problema de salud pública a nivel mundial. La medición se realizó en dos establecimientos de la comuna de Quilpué, V región, Chile. Se seleccionaron 45 estudiantes de 5° año básico en total. El instrumento de evaluación fue a través de acelerometria (modelo Actigraph GT3X) y se midió dos veces a la semana, en dos sesiones cada día (clase de Educación Física y Salud/ Recreo) durante tres semanas. Se calcularon los niveles de intensidad en la actividad física tanto en la clase de Educación Física y Salud, como en el Recreo. Los resultados mostraron un predominio en el porcentaje de intensidad moderada en los niños (68,4%) respecto a las niñas (52,8%); Las niñas (21,8%) duplicaron el porcentaje de actividad sedentaria de los niños (11%); Respecto a la comparación entre el porcentaje total de actividad vigorosa, el recreo (5,4%) casi alcanza el cuádruple que la clase de Educación Física y Salud (1,3%) respectivamente.The purpose of this descriptive cross-sectional study is to measure and compare the intensity of physical activity in the Physical Education and Health and Recreation class in children of 5th basic in order to relate their level of intensity and check if the parameters are reached Optimal (moderate / vigorous) performance by the competent organizations. These parameters provide numerous health benefits and considerably avoid chronic noncommunicable diseases (O.M.S. 2010). Physical inactivity and physical inactivity are one of the main causes for contracting type 2 diabetes, hypertension, obesity, etc. Pathologies that currently trigger a public health problem worldwide. The measurement was made in two establishments of the commune of Quilpué, V region, Chile. We selected 45 students from the 5th basic year in total. The instrument of assessment was through accelerometry (model Actigraph GT3X) and measured twice a week, in two sessions each day (Physical Education and Health / Recreation class) for three weeks. The levels of intensity in physical activity were calculated in the Physical Education and Health class, as well as in the Recreo. The results showed a predominance in the percentage of moderate intensity in children (68.4%) compared to girls (52.8%); Girls (21.8%) doubled the percentage of children's sedentary activity (11%); Regarding the comparison between the total percentage of vigorous activity, recess (5.4%) almost reached quadruple that of Physical Education and Health (1.3%), respectively

Calidad de vida y bienestar en estudiantes de medicina de una universidad del sur de Chile

The quality of life is a relevant issue in the context of higher education in Chile. This work sought to characterize the quality of life of students of different levels of the Medicine program in Andrés Bello University, Concepción campus, Chile. A descriptive cross-sectional study was conducted in which 159 students, whose ages ranged from 18 to 25 years, answered the WHOQOL-BREF questionnaire. Within the findings of this study, a general perception of positive quality of life was found (45,57% considered it quite good). When analyzing the domains of WHOQOL-BREF, it was found that women reported lower values than men in physical health, social environment and psychological health, the latter being the worst qualified in both sexes, suggesting that psychological wellbeing is relatively deficitary. It was concluded that the students had a good perception of quality of life, however, sex was a variable that influenced their perception.La calidad de vida es un tema relevante en el contexto de la educación superior de Chile. Este trabajo buscó caracterizar la calidad de vida de los estudiantes de los distintos niveles de la carrera de Medicina de la Universidad Andrés Bello, sede Concepción, Chile. Se realizó un estudio descriptivo de corte transversal en el que 159 estudiantes, cuyas edades fluctuaron entre 18 y 25 años, respondieron el cuestionario WHOQOL-BREF. Dentro de los hallazgos de este estudio, se encontró una percepción general de calidad de vida positiva (45,57% la consideró bastante buena). Al analizar los dominios del WHOQOL-BREF, se encontró que las mujeres reportaron valores más bajos que los hombres en salud física, entorno social y salud psicológica; esta última fue el aspecto peor calificad en ambos sexos, sugiriendo que el bienestar psicológico es relativamente deficitario. Se concluyó que los estudiantes tenían una buena percepción de calidad de vida, no obstante, el sexo fue una variable que influyó en la percepción de esta

Physical Exercise Methods and Their Effects on Glycemic Control and Body Composition in Adults with Type 2 Diabetes Mellitus (T2DM): A Systematic Review

The prevalence of T2DM represents a challenge for health agencies due to its high risk of morbidity and mortality. Physical Activity (PA) is one of the fundamental pillars for the treatment of T2DM, so Physical Exercise (PE) programs have been applied to research their effectiveness. The objective of the study was to analyze the effects of PE methods on glycemic control and body composition of adults with T2DM. A systematic review without meta-analysis was performed, using the PubMed database. Quasi-experimental and pure experimental clinical trials were included, which were available free of charge and were published during 2010–2020. In the results, 589 articles were found and 25 passed the inclusion criteria. These were classified and analyzed according to the methods identified (AE, IE, RE, COM, and others), duration and variable(s) studied. It is concluded that PE is effective for glycemic control and body composition in adults with T2DM using different methods (AE, IE, RE, COM, and others), both in the short and long term. Adequate organization of PE components such as frequency, duration, volume, and intensity, is essential

Constitutive phosphorylation as a key regulator of TRPM8 channel function

In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca2+ imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.This work was supported by Grants DICYT VRIDeI-USACH 022143PP to M.P. and R.M.; FONDECYT 1161733 to R.M. and M.P.; FONDECYT 1181076 to P.O.; Centro Basal AC3E FB0008 to P.O.; VRIDeI-USACH AP-539MM to R.M.; National Research Foundation of Korea NRF-2019R1A2C2003642 to K.-S.P.; MINECO PID2019-108194RB and the Severo Ochoa Program for Centers of Excellence in R&D SEV-2017-0723 to F.V.; the Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD) to R.M. and M.P.; and the Millennium Nucleus for the Study of Pain (MiNuSPain) to R.M. and M.P. R.M. also thanks VRIDeI-USACH for postdoctoral support to B.R. (02183MM). B.R. (21161660) and B.L. (21181611) thank Comisión Nacional de Investigación Científica y Tecnológica de Chile PhD fellowships. MiNICAD is a Millennium Nucleus supported by the Millennium Science Initiative of the Ministry of Science, Technology, Knowledge and Innovation (Chile). MiNuSPain is a Millennium Nucleus supported by the Millennium Science Initiative of the Ministry of Science, Technology, Knowledge and Innovation (Chile). The CINV is a Millennium Science Institute funded by Agencia Nacional de Investigación y Desarrollo de Chile Grant ICN09_022 of the Ministry of Science, Technology, Knowledge and Innovation (Chile).Peer reviewe

Boldine inhibits the alveolar bone resorption during ligature-induced periodontitis by modulating the Th17/Treg imbalance

Background During periodontitis, tooth-supporting alveolar bone is resorbed when there is an increased expression of the pro-osteolytic factor termed receptor activator of nuclear factor kappa B ligand (RANKL), which is responsible for osteoclast differentiation and activation. In periodontitis-affected tissues, the imbalance between T-helper type-17 (Th17) and T-regulatory (Treg) lymphocyte activity favors this RANKL overexpression. In this context, immunotherapeutic strategies aimed at modulating this Th17/Treg imbalance could eventually arrest the RANKL-mediated alveolar bone loss. Boldine has been reported to protect from pathological bone loss during rheumatoid arthritis and osteoporosis, whose pathogenesis is associated with a Th17/Treg imbalance. However, the effect of boldine on alveolar bone resorption during periodontitis has not been elucidated yet. This study aimed to determine whether boldine inhibits alveolar bone resorption by modulating the Th17/Treg imbalance during periodontitis. Methods Mice with ligature-induced periodontitis were orally treated with boldine (10/20/40 mg/kg) for 15 consecutive days. Non-treated periodontitis-affected mice and non-ligated mice were used as controls. Alveolar bone loss was analyzed by micro-computed tomography and scanning electron microscopy. Osteoclasts were quantified by histological identification of tartrate-resistant acid phosphatase-positive cells. Production of RANKL and its competitive antagonist osteoprotegerin (OPG) were analyzed by ELISA, quantitative polymerase chain reaction (qPCR), and immunohistochemistry. The Th17 and Treg responses were analyzed by quantifying the T-cell frequency and number by flow cytometry. Also, the expression of their signature transcription factors and cytokines were quantified by qPCR. Results Boldine inhibited the alveolar bone resorption. Consistently, boldine caused a decrease in the osteoclast number and RANKL/OPG ratio in periodontal lesions. Besides, boldine reduced the Th17-lymphocyte detection and response and increased the Treg-lymphocyte detection and response in periodontitis-affected tissues. Conclusion Boldine, administered orally, inhibited the alveolar bone resorption and modulated the Th17/Treg imbalance during experimental periodontitis.Plataforma Experimental Bio-CT (FONDEQUIP, Faculty of Dentistry, Universidad de Chile) EQM150010 FONDECYT from the Chilean Governmental Agencia Nacional de Investigacion y Desarrollo (ANID) 1181780 Graduate School of the Faculty of Dentistry, Universidad de Chile ANID 2119221