TDC-2: Multimodal Foundation for Therapeutic Science.

Therapeutics Data Commons (tdcommons.ai) is an open science initiative with unified datasets, AI models, and benchmarks to support research across therapeutic modalities and drug discovery and development stages. The Commons 2.0 (TDC-2) is a comprehensive overhaul of Therapeutic Data Commons to catalyze research in multimodal models for drug discovery by unifying single-cell biology of diseases, biochemistry of molecules, and effects of drugs through multimodal datasets, AI-powered API endpoints, new multimodal tasks and model frameworks, and comprehensive benchmarks. TDC-2 introduces over 1,000 multimodal datasets spanning approximately 85 million cells, pre-calculated embeddings from 5 state-of-the-art single-cell models, and a biomedical knowledge graph. TDC-2 drastically expands the coverage of ML tasks across therapeutic pipelines and 10+ new modalities, spanning but not limited to single-cell gene expression data, clinical trial data, peptide sequence data, peptidomimetics protein-peptide interaction data regarding newly discovered ligands derived from AS-MS spectroscopy, novel 3D structural data for proteins, and cell-type-specific protein-protein interaction networks at single-cell resolution. TDC-2 introduces multimodal data access under an API-first design using the model-view-controller paradigm. TDC-2 introduces 7 novel ML tasks with fine-grained biological contexts: contextualized drug-target identification, single-cell chemical/genetic perturbation response prediction, protein-peptide binding affinity prediction task, and clinical trial outcome prediction task, which introduce antigen-processing-pathway-specific, cell-type-specific, peptide-specific, and patient-specific biological contexts. TDC-2 also releases benchmarks evaluating 15+ state-of-the-art models across 5+ new learning tasks evaluating models on diverse biological contexts and sampling approaches. Among these, TDC-2 provides the first benchmark for context-specific learning. TDC-2, to our knowledge, is also the first to introduce a protein-peptide binding interaction benchmark.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and TnI altering calcium sensitivity. We measured degradation of other myofilament proteins including cMyBP-C and actininshowing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

The impact of incorporating a simulation program into the undergraduate nursing curricula: A cross-sectional descriptive study.

AIM: To determine the degree of satisfaction for each academic year and according to the type of simulation performed (simulated patient actor/advanced simulator) among nursing students after the use of clinical simulation. INTRODUCTION: Clinical simulation is currently being incorporated in a cross-cutting manner throughout undergraduate nursing education. Its implementation requires a novel curricular design and educational changes throughout the academic subjects. DESIGN: A cross-sectional descriptive study was performed. METHODS: During the academic years 2018-2019 and 2019-2020, 425 students completed the High-Fidelity Simulation Satisfaction Reduced Scale for Students based on 25 questions and six factors, with a total score between 0 and 125. In total, 91 simulation sessions were performed among students who had different degrees of clinical and previous experience with simulation as well as standardized patient versus advanced simulator. A bivariate analysis was performed, comparing the total scores and the different subscales by sex, previous experience, academic year, and simulation methodology. Linear regression was used for both bivariate and multivariate analysis. RESULTS: The mean scale score was 116.8 (SD=7.44). The factor with the highest score was "F2: feedback or subsequent reflection", with a mean score of 14.71 (SD=0.73) out of 15. Fourth year students scored the highest (mean=119.17; SD=5.28). Students who underwent simulation training with a simulated patient actor presented a higher level of overall satisfaction (p<0.05) (Mean=120.31; SD=4.91), compared to students who used an advanced simulator (Mean=118.11; SD=5.75). CONCLUSIONS: Satisfaction with the simulation program was higher in fourth-year students compared to first-year students and was also higher when a simulated patient actor was used compared to an advanced simulator. The most highly valued aspect was the subsequent debriefing or reflective process.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Aims: Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs that involves decreased blood flow velocity and reduced atrial contractility. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. The aim of this project is to define the contractile remodeling that occurs in AFib. Methods and Results: To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction in skinned single cardiomyocyte calcium-force studies. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed decreased troponin I phosphorylation, consistent with the increased calcium sensitivity that was found within these cardiomyocytes. Conclusions: With these results it can be concluded that AFib causes alterations in contraction that can be explained by both molecular changes occurring in myofilament proteins and overall myofilament protein degradation. These results provide an understanding of the contractile remodeling that occurs in AFib and provides insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

Validation of a short version of the high-fidelity simulation satisfaction scale in nursing students.

BACKGROUND: Clinical simulation provides a practical and effective learning method during the undergraduate education of health professions. Currently there is only one validated scale in Spanish to assess nursing students' satisfaction with the use of high-fidelity simulation, therefore, our objective is to validate a brief version of this scale in undergraduate nursing students with or without clinical experience. METHOD: A cross-sectional descriptive study was performed. Between 2018 and 2020, the students from all academic courses of the Fundación Jiménez Díaz nursing school completed the satisfaction scale at the end of their simulation experiences. To validate this scale, composed of 33 items and eight dimensions, exploratory factor analysis (EFA) of the principal components was performed, the internal consistency was studied using Cronbach's alpha, and the corrected item-test correlation of each of the items of the total scale was reviewed. RESULTS: 425 students completed the scale, after the exploratory factor analysis, a scale consisting of 25 items distributed into six subscales, each containing between two and six items, explained a variance of 66.5%. The KMO test (Kaiser-Meyer-Olkin) obtained a value of 0.938, Bartlett's sphericity test was < 0.01 and Goodness of Fit Index (GFI) was 0.991. CONCLUSION: The modified ESSAF scale, reduced from 33 to 25 items and divided into six subscales, is as valid and reliable as the original scale for use in nursing students of different levels, with, or without clinical experience.

Mechanisms of Sarcomere Protein Mutation-Induced Cardiomyopathies.

PURPOSE OF REVIEW: The pace of identifying cardiomyopathy-associated mutations and advances in our understanding of sarcomere function that underlies many cardiomyopathies has been remarkable. Here, we aim to synthesize how these advances have led to the promising new treatments that are being developed to treat cardiomyopathies. RECENT FINDINGS: The genomics era has identified and validated many genetic causes of hypertrophic and dilated cardiomyopathies. Recent advances in our mechanistic understanding of sarcomere pathophysiology include high-resolution molecular models of sarcomere components and the identification of the myosin super-relaxed state. The advances in our understanding of sarcomere function have yielded several therapeutic agents that are now in development and clinical use to correct contractile dysfunction-mediated cardiomyopathy. New genes linked to cardiomyopathy include targets with limited clinical evidence and require additional investigation. Large portions of cardiomyopathy with family history remain genetically undiagnosed and may be due to polygenic disease.

Partial and complete loss of myosin binding protein H-like cause cardiac conduction defects.

A premature truncation of MYBPHL in humans and a loss of Mybphl in mice is associated with dilated cardiomyopathy, atrial and ventricular arrhythmias, and atrial enlargement. MYBPHL encodes myosin binding protein H-like (MyBP-HL). Prior work in mice indirectly identified Mybphl expression in the atria and in small puncta throughout the ventricle. Because of its genetic association with human and mouse cardiac conduction system disease, we evaluated the anatomical localization of MyBP-HL and the consequences of loss of MyBP-HL on conduction system function. Immunofluorescence microscopy of normal adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl heterozygous ventricles had a marked reduction of MyBP-HL-positive cells compared to controls. Lightsheet microscopy of normal perinatal day 5 mouse hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the contactin-2-positive ventricular conduction system, but this association was not apparent in Mybphl heterozygous hearts. Surface telemetry of Mybphl-null mice revealed atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a shorter atrial relative refractory period and atrial tachycardia. Calcium transient analysis of isolated Mybphl-null atrial cardiomyocytes demonstrated an increased heterogeneity of calcium release and faster rates of calcium release compared to wild type controls. Super-resolution microscopy of Mybphl heterozygous and homozygous null atrial cardiomyocytes showed ryanodine receptor disorganization compared to wild type controls. Abnormal calcium release, shorter atrial refractory period, and atrial dilation seen in Mybphl null, but not wild type control hearts, agree with the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene contributes to ventricular arrhythmias and ventricular dysfunction.

Inulin Improves Diet-Induced Hepatic Steatosis and Increases Intestinal Akkermansia Genus Level.

Hepatic steatosis is characterized by triglyceride accumulation within hepatocytes in response to a high calorie intake, and it may be related to intestinal microbiota disturbances. The prebiotic inulin is a naturally occurring polysaccharide with a high dietary fiber content. Here, we evaluate the effect of inulin on the intestinal microbiota in a non-alcoholic fatty liver disease model. Mice exposed to a standard rodent diet or a fat-enriched diet, were supplemented or not, with inulin. Liver histology was evaluated with oil red O and H&E staining and the intestinal microbiota was determined in mice fecal samples by 16S rRNA sequencing. Inulin treatment effectively prevents liver steatosis in the fat-enriched diet group. We also observed that inulin re-shaped the intestinal microbiota at the phylum level, were Verrucomicrobia genus significantly increased in the fat-diet group; specifically, we observed that Akkermansia muciniphila increased by 5-fold with inulin supplementation. The family Prevotellaceae was also significantly increased in the fat-diet group. Overall, we propose that inulin supplementation in liver steatosis-affected animals, promotes a remodeling in the intestinal microbiota composition, which might regulate lipid metabolism, thus contributing to tackling liver steatosis.

Formación de profesionales mediante simulación clínica durante la primera ola de covid-19 / Training professionals through clinical simulation during the first covid-19 wave

En este artículo se comparte la experiencia de la formación de profesionales sanitarios del Hospital General Universitario de Ciudad Real (HGUCR) mediante la formación teórico-práctica con simulación clínica y la valuación de sus resultados. Se plantearon sesiones con simulación de baja y alta compleji-dad para 40 personas y en grupos reducidos. La evolución de las necesidades hizo aumentar el número de sesiones y la supresión de la sala de simulación avanzada.Se realizó un programa de 64 sesiones entre marzo y abril de 2020 en turnos de mañana y tarde, en grupos de 15 personas.Participó el 41,5% (n= 852) del personal sanitario de la Gerencia de Atención Integrada de Ciudad Real. Respondieron el cuestionario de evaluación 91 personas que refirieron una alta satisfacción con la actividad (4,32 puntos sobre 5 a nivel global), valoraron especialmente la utilidad para el trabajo, la claridad de la información y la parte práctica de la sesión (simulación). Así mismo, el 80,6% manifestó haber aumentado su confianza y el 65,9% una disminución de la ansiedad en el manejo de pacientes con COVID. Si bien habría que analizar otras causas, el porcentaje de contagios entre los profesionales (10%) fue similar al de la población general. Disponer de espacios de formación con simulación y profesionales formados en el diseño de este tipo de actividades supone un recurso útil y flexible a las demandas sanitarias que puedan surgir.(AU)

In this article, we share the experience in training healthcare professionals from the Hospital General Universitario of Ciudad Real (HGUCR) through theoretical-practical training with clinical simulation, and the evaluation of its results.Sessions were set with high and low complexity simulation for 40 persons and in reduced groups. The evolution of needs led to an increase in the number of sessions, and the suppression of the Advanced Simulation Room.A 64-session program was held between March and April 2020, in morning and afternoon shifts and for groups of 15 persons, with participation by 41.5% (n= 852) of the healthcare staff from the Integrated Care Management of Ciudad Real. The evaluation questionnaire was answered by 91 persons, who reported high satisfaction with the activity (4.32 scores over 5 at overall level); they valued particularly its utility for work, the clarity of the information, and the practical aspect of the session (simulation). Likewise, 80.6% reported an increase in confidence, and 65.9% reported a reduction in anxiety regarding management of patients with COVID. Even though other causes should be analysed, the proportion of contagion among professionals (10%) was similar to that among the general population.The availability of training spaces with simulation and professionals trained in the design of this type of activities represents a useful and flexible resource for any healthcare demands that might arise.(AU)

Experiences and Perceptions of Nursing Students during the COVID-19 Crisis in Spain.

In the early stages of the 2019 coronavirus pandemic in Spain, the Spanish health system was overwhelmed, mainly due to a lack of personnel, and many hospital centers collapsed by following avalanche of patients with COVID-19; this meant that the National System of Health called for fourth-year nursing students to come to the hospital as health care personnel. Our aim was to describe the perceptions and experiences of a sample of nursing students during the early stages of the outbreak. We conducted a qualitative study with an empirical-phenomenological approach. Twenty nursing students in their final year of study in Spain were recruited using purposive and snowball sampling. They participated in in-depth interviews between 20 April and 10 May 2020. The interviews were transcribed and then analyzed using Haase's adaptation of Colaizzi's phenomenological method. Four main themes emerged from data analysis: "social responsibility and pride as a health worker", "pressure caused by working with COVID-19 patients", "feeling defenseless and let down", and "personal growth as a health worker". These main themes were further divided into 11 theme categories. Due to an intense work day for several days in a row, the students were tired and mentally exhausted. Even so, they managed to overcome any difficulties, demonstrating their professional dedication and resilience. Greater preparatory support should be provided to safeguard the well-being of these future healthcare providers. More intensive preparatory training is necessary for health sciences students to facilitate crisis preparedness and effective crisis management. It is necessary to implement support from healthcare systems, including sufficient personal protective equipment, as well as contracts that accurately reflect the work they do. It is necessary for nursing supervisors to have effective communication in the performance of their functions with nursing students; this dialogue helps to clearly explain which are the functions that students must perform when they are carrying out their internships. There is also a need for preparatory training in managing infectious diseases such as COVID-19.

Effectiveness of an Educational Intervention With High-Fidelity Clinical Simulation to Improve Attitudes Toward Teamwork Among Health Professionals.

BACKGROUND: Failures in teamwork are a common reason for adverse medical events. The goals of this study are to evaluate attitudes toward teamwork among an interprofessional group of health professionals and to analyze the effectiveness of an educational intervention with high-fidelity clinical simulation to improve these attitudes. METHOD: An educational intervention was developed that used a 6-hour session that included three simulated clinical cases. The Attitudes Toward Health Care Teams Scale was used for assessment. Mean difference before and after the intervention was calculated with the Wilcoxon test. RESULTS: Positive attitude toward teamwork after completion of the training activities was measured. The health care professionals who showed the most positive attitude toward teamwork were physicians (93.92, SD = 6.58) and resident physicians (95.01, SD = 6.33). The greatest increase was observed among orderlies (p < .001) and nursing assistants (p < .001). CONCLUSION: The use of clinical simulation for interprofessional training of health care professionals showed a positive effect on attitudes toward teamwork. [J Contin Educ Nurs. 2021;52(10):457-467.].

New prophylaxis regimen for SARS-CoV-2 infection in health professionals with low doses of hydroxychloroquine and bromhexine: a randomised, double-blind placebo clinical trial (ELEVATE Trial).

INTRODUCTION: SARS-CoV-2 infection in Mexico has caused ~2.7 million confirmed cases; around 20%-25% of health workers will be infected by the virus at their workplace, with approximately 4.4% of mortality. High infectivity of SARS-CoV-2 is related with cell entry mechanism, through the ACE receptor. SARS-CoV-2 requires transmembrane protease serine 2 to cleave its spike glycoprotein and ensure fusion of host cell and virus membrane. We propose studying prophylactic treatment with hydroxychloroquine (HCQ) and bromhexine (BHH), which have been shown to be effective in preventing SARS-CoV-2 infection progression when administered in early stages. The aim of this study is to assess the efficacy of HCQ and BHH as prophylactic treatments for SARS-CoV-2 infection in healthy health workers exposed to the virus. METHODS AND ANALYSIS: Double-blind randomised clinical trial, with parallel allocation at a 1:1 ratio with placebo, of low doses of HCQ plus BHH, for 60 days. Study groups will be defined as follows: (1) HCQ 200 mg/day+BHH 8 mg/8 hours versus (2) HCQ placebo plus BHH placebo. Primary endpoint will be efficacy of both interventions for the prevention of SARS-CoV-2 infection, determined by the risk ratio of infected personnel and the absolute risk. At least a 16% reduction in absolute risk is expected between the intervention and placebo groups; a minimum of 20% infection is expected in the placebo group. The sample size calculation estimated a total of 214 patients assigned: two groups of 107 participants each. ETHICS AND DISSEMINATION: This protocol has been approved by the local Medical Ethics Committee (National Institute of Rehabilitation 'Luis Guillermo Ibarra Ibarra', approval number INRLGII/25/20) and by the Federal Commission for Protection against Sanitary Risks (COFEPRIS, approval number 203 300 410A0058/2020). The results of the study will be submitted for publication in peer-reviewed journals and disseminated through conferences. TRIAL REGISTRATION NUMBER: NCT04340349.

Niveles de ansiedad de los equipos interprofesionales durante el entrenamiento con simulación clínica / Anxiety levels in interprofessional teams during training with clinical simulation

Objetivo: analizar el efecto de la simulación clínica en el nivel de ansiedad pre/post-actividad formativa en varios grupos profesionales y en estudiantes de Enfermería, y evaluar la autoconfianza percibida antes/después de la simulación en cada grupo.Métodos: estudio cuasi-experimental en profesionales sanitarios (enfermeras, técnicos en cuidados auxiliares de Enfermería o TCAE y médicos) y estudiantes de Enfermería (4º curso) sometidos por primera vez a una intervención de simulación clínica en el Hospital General Universitario de Ciudad Real. Se evaluó el nivel de ansiedad estado antes/después de la simulación mediante el cuestionario STAI de Spielberg, y se valoró la autoconfianza percibida antes/después de la simulación. Se realizó análisis descriptivo. Se usó la t de Student para la comparación de medias. Se estableció significación estadística si p< 0,05.Resultados: participaron 53 sujetos, 43 (81,2%) profesionales y 10 (18,8%) estudiantes de Enfermería. Edad media (DE) fue 39 (11,8) años; 84,9% (n= 45) mujeres. El nivel de ansiedad medio (DE) al inicio de la simulación fue mayor en los estudiantes de Enfermería 24,6 (5,4) y menor en los médicos 14,4 (5,8). Tras la intervención se redujo la media (DE) del nivel de ansiedad-estado en todos los grupos, siendo significativo (p< 0,001) en TCAE -13,1 (6,9) y enfermeras -12,5 (8,4). Hubo un aumento del nivel de autoconfianza de todos los participantes al finalizar la actividad.Conclusiones: los profesionales que participaron por primera vez en una actividad de simulación clínica presentaron alto grado de ansiedad, aunque en médicos fue inferior. Hubo un descenso de la ansiedad y aumento de la confianza al final de la simulación clínica.(AU)

Objective: to analyze the effect of clinical simulation in the level of anxiety pre- and post- training activity in different professional groups and Nursing students, and to assess the self-confidence perceived before/after the simulation in each group.Methods: a quasi-experimental study in healthcare professionals (nurses, assistant nursing technicians or TCAE, and doctors), and Nursing students (4th year), who underwent for the first time a clinical simulation intervention at the Hospital General Universitario de Ciudad Real. The level of anxiety was evaluated before/after the simulation, through Spielberger’s STAI questionnaire, and the level of self-confidence perceived was also evaluated before / after the simulation. Descriptive analysis was conducted, and Student’s t test was used for mean comparison. Statistical significance was established at p< 0.05.Results: the study included 53 subjects: 43 (81.2%) were professionals and 10 (18.8%) were Nursing students; their mean age (SD) was 39 (11.8) years; and 84.9% (n= 45) were female. The mean anxiety level (SD) at the start of the simulation was higher in Nursing students, with 24.6 (5.4), and lower in doctors, with 14.4 (5.8). After the intervention, the mean (SD) level of anxiety-status was reduced in all groups; it was significant (p< 0.001) in the TCAE group, with -13.1 (6.9) and nurses, with -12.5 (8.4). There was an increase in the level of self-confidence in all participants by the end of the activity.Conclusions: the professionals who participated for the first time in a clinical simulation activity presented a high level of anxiety, though this was lower among doctors. There was a reduction in anxiety and increase in confidence by the end of the clinical simulation.(AU)

Nursing students' experience and training in healthcare aid during the COVID-19 pandemic in Spain.

BACKGROUND: During the COVID-19 pandemic health crisis, in some countries such as Spain, nursing students have offered to provide health assistance, but the role they have played, their degree of preparedness to face the situation, and what must be improved in their training to be ready for these situations is unknown. OBJECTIVE: Describe the experience and perceptions of students of the Nursing university degree during their participation as health support in the COVID-19 health crisis in Spain. DESIGN: We conducted a cross-sectional survey study. METHOD: 503 students enrolled in the 4th course of the Nursing degree in Spain during the months of March and April 2020. An online questionnaire was developed, based on a pilot study and distributed through the Nursing Association, students' unions and students' associations. Variables were used to describe their participation, degree of preparedness and training needs to determine how to improve training through descriptive statistics, as well as nonparametric tests to analyse the relationship between training and degree of preparedness nursing students. Results are reported according to the STROBE Statement. RESULTS: 73.2% (368) of students offered to participate in healthcare aid, of which 225 were actively involved. 27.8% carried out nursing tasks without supervision, and 47.7% assisted COVID-19 patients as any other nurse. Only 3.4% felt very prepared to work in the field of intensive care, finding that those students who perceived a higher degree of preparedness had received previous training in personal protective equipment and mechanical ventilation (p < 0.005). The highest scores for training activities that may improve their preparedness were simulations to improve levels of anxiety and stress when managing critical patients, simulation in ventilatory support and mandatory practices in services where ventilators are used. CONCLUSIONS: Although three out of four students were willing to provide health assistance, they recognise that they were not specially prepared in the field of intensive care and demand training with simulation to improve anxiety and stress levels in the management of critical patients and simulation in ventilatory support. RELEVANCE TO CLINICAL PRACTICE: Students have been vital resources for our health system and society when they have been needed. It is now up to us, both teachers and health authorities, to share their efforts by implementing the necessary improvements in training and safety measures not only because these affects the health and safety of the patient, but because they will be essential parts in future pandemics.

The PKC-ζ pseudosubstrate peptide induces glutamate release from retinal pigment epithelium cells through kinase- independent activation of Best1.

AIMS: The retinal pigment epithelium (RPE) is a highly specialized cell monolayer, that plays a key role in the maintenance of photoreceptor function and the blood-retina barrier (BRB). In this study, we found that a myristoylated pseudosubstrate of PKC-ζ (PKCζ PS), considered as a PKC-ζ inhibitor, plays a distinct role in RPE. MAIN METHODS: We demonstrated that PKCζ PS stimulates the release of Glutamate (Glu) using in vitro3H-Glutamate release experiments. By western blot, kinase assays, and Fluoresence Ca+2 Concentration Measurements, we determined the cellular mechanisms involved in such release. KEY FINDINGS: Surprisingly, PKCζ PS has no effect on either phosphorylation of T560, essential for catalytic activity, nor it has an effect on kinase activity. It induces the dose-dependent release of Glu by increasing intracellular Ca+2 levels. Interestingly, this release was not observed upon stimulation by other non-competitive PKC-ζ inhibitors. We here demonstrated that the PKCζ PS stimulates the release of Glutamate from RPE by activating the Ca2+-dependent Cl channel Bestrophin 1 (Best1). SIGNIFICANCE: These results question PKCζ PS specificity as an inhibitor of this enzyme. Furthermore, the present results underline the relevance of clarifying the molecular mechanisms involved in glutamate release from the retina under conditions derived from excitotoxic stimuli.

Common opossum population density in an agroforestry system in Bolivia

The common opossum, Didelphis marsupialis thrives near human settlements. Understanding its ecology could help planning management decisions about this species, especially in anthropogenic landscapes. Yet, there are no density estimations for this species throughout its distribution range in Bolivia. We estimated the density of D. marsupialis in a rural agricultural community, where agroforestry plantations and fallows cover most of the land. We counted individuals in line transects and used DISTANCE software to calculate density. We covered a total of 70.21 km in 143 night counts, obtaining 38 records of D. marsupialis. We estimated a density of 0.30 individuals ha-1 (SE = 0.062; range: 0.20 - 0.45 individuals ha-1), with a mean encounter rate of 0.54 individuals km-1. Encounter rate varied between habitats, with mean values of 0.20 in secondary forests and 0.64 in agroforestry plantations. Our density estimate is near the lower range of previously reported values for the common opossum in other countries. We argue that our results may reflect the response to the availability of food resources and predation pressure in agroforestry plantations. (AU)

Thrombin-activated PAR1 membrane expression is regulated by Rab11a-RCP complex dissociation.

PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.

Thrombin induces Ca2+-dependent glutamate release from RPE cells mediated by PLC/PKC and reverse Na+/Ca2+ exchange.

Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.

Thrombin-Induced Calpain Activation Promotes Protease-Activated Receptor 1 Internalization.

The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1-4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca+2 concentration ([Ca+2]i) mediated by Gq11α coupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation of µ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of α-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel µ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin.

Home range and habitat use by pacas in a montane tropical forest in Bolivia / Ámbito de hogar y uso de hábitat del jochi pintado en un bosque montano tropical de Bolivia

ABSTRACT We studied the home range and habitat use of paca (Cuniculus paca) in a rural agricultural community in the Alto Beni region, in La Paz department, Bolivia, where game meat is the main source of protein for a large portion of local inhabitants. We captured and radio collared five individual paca (4 females and 1 male), which were followed for six months in 2012 to estimate home range of the individuals and assess their habitat use and selection. The total mean home range (95% Minimum Convex Polygon) was 2.26 ha (SD = 0.80), and ranged from 1.5 to 2.96 ha. The mean female home range (2.04 ha) was smaller than the male´s (2.95 ha). Pacas used two (crops and fallows) of the three available habitats (secondary forest patches), and appeared to positively select crops over the other two. The importance of crops may be linked to the type of agriculture conducted in the area (i.e. mainly cacao in agroforestry systems), which may offer more food and shelter for pacas. Given that, the importance of habitats may change seasonally; this topic should be examined in future studies. Our results suggest that agroforestry systems may be important for the species since they offer a variety of food resources throughout the year.

RESUMEN Hemos estudiado el ámbito de hogar y uso de hábitat del jochi pintado (Cuniculus paca) en una comunidad de Alto Beni en el Norte de La Paz-Bolivia, donde la carne obtenida por cacería es, como en muchas regiones de la Amazonia, una fuente principal de proteína para muchas poblaciones indígenas y campesinas. Hemos capturado y seguido con radio-collar a cinco individuos (4 hembras y 1 macho) por seis meses en 2012. El ámbito de hogar promedio de los individuos fue de 2,26 ha (ds=0,80), usando el estimador Mínimo Polígono Convexo (MPC al 95%) y los valores se encuentran entre 1,5 a 2,96 ha. El ámbito de hogar promedio de las hembras fue más pequeño (2,04 ha) que del macho (2,95 ha). Los jochis estudiados usaron 2 (cultivos y barbechos) de los 3 hábitats disponibles (parches de bosque secundario), aunque los cultivos parecen ser seleccionados positivamente respecto a los otros dos. La importancia de los cultivos podría estar ligada al tipo de cultivo que se realiza en la zona (sistemas agroforestales), que podrían estar ofreciendo mejores condiciones para ser usados por los jochis. Adicionalmente, la importancia de los hábitats podría cambiar de acuerdo a la época del año, lo cual podría ser abordado en siguientes estudios. Este trabajo es el primero en dar detalles sobre la ecología de C. paca en vida silvestre en Bolivia; sin embargo sería necesario contar con más individuos para tener más detalle sobre la ecología espacial de esta especie.