RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad. In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that b-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVb subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined. Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study

Impacts of Time Restricted Feeding on Peak Volume of Oxygen Uptake and Substrate Utilization

Time Restricted Feeding (TRF) is a type of Intermittent Fasting, which refers to the finite time to intake calories during the day. TRF has become a dietary approach that is used for weight loss and overall health. Individuals that partake in TRF may experience a decrease in peak volume of oxygen uptake (VO2peak) due to minimization of glycolytic stores. To date, few studies have compared the impact of TRF on VO2peak. PURPOSE: The current study aimed to further investigate the metabolic impact of TRF. METHODS: Twenty one participants, ages 18-60, completed an eleven week longitudinal study to examine differences in VO2peak, substrate utilization crossover, and resting substrate utilization. Participants self-reported diet, exercise, sleep, and medications over two separate four week periods. The first four weeks were without TRF and the following four were with TRF. A maximal exercise test and a resting metabolic test were performed three times, four weeks apart from each other. A repeated measures ANOVA was performed to determine within subject differences. A post-hoc analysis was performed to determine the time effect. RESULTS: VO2peak was significantly lower after implementing TRF (phttps://openriver.winona.edu/urc2019/1012/thumbnail.jp

Rad GTPase Deletion Atenuates Post-Ischemic Cardiac Dysfunction and Remodeling

The protein Rad interacts with the L-type calcium channel complex to modulate trigger Ca2+ and hence to govern contractility. Reducing Rad levels increases cardiac output. Ablation of Rad also attenuated the inflammatory response following acute myocardial infarction. Future studies to target deletion of Rad in the heart could be conducted to establish a novel treatment paradigm whereby pathologically stressed hearts would be given safe, stable positive inotropic support without arrhythmias and without pathological structural remodeling. Future investigations will also focus on establishing inhibitors of Rad and testing the efficacy of Rad deletion in cardioprotection relative to the time of onset of acute myocardial infarction

A site assessment tool for inpatient controlled human infection models for enteric disease pathogens

The use of the controlled human infection model to facilitate product development and to advance understanding of host-pathogen interactions is of increasing interest. While administering a virulent (or infective) organism to a susceptible host necessitates an ongoing evaluation of safety and ethical considerations, a central theme in conducting these studies in a safe and ethical manner that yields actionable data is their conduct in facilities well-suited to address their unique attributes. To that end, we have developed a framework for evaluating potential sites in which to conduct inpatient enteric controlled human infection model to ensure consistency and increase the likelihood of success.publishedVersio

Relationship Between the King-Devick Test and Commonly Used Concussion Tests at Baseline

Context: Comprehensive assessments are recommended to evaluate sport-related concussion (SRC). The degree to which the King-Devick (KD) test adds novel information to an SRC evaluation is unknown. Objective: To describe relationships at baseline among the KD and other SRC assessments and explore whether the KD provides unique information to a multimodal baseline concussion assessment. Design: Cross-sectional study. Setting: Five National Collegiate Athletic Association institutions participating in the Concussion Assessment, Research and Education (CARE) Consortium. Patients or other participants: National Collegiate Athletic Association student-athletes (N = 2258, age = 20 ± 1.5 years, 53.0% male, 68.9% white) in 11 men's and 13 women's sports. Main outcome measure(s): Participants completed baseline assessments on the KD and (1) the Symptom Inventory of the Sport Concussion Assessment Tool-3rd edition, (2) the Brief Symptom Inventory-18, (3) the Balance Error Scoring System, (4) the Standardized Assessment of Concussion (SAC), (5) the Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT) test battery, and (6) the Vestibular/Ocular Motor Screening tool during their first year in CARE. Correlation coefficients between the KD and the 6 other concussion assessments in isolation were determined. Assessments with ρ magnitude >0.1 were included in a multivariate linear regression analysis to evaluate their relative association with the KD. Results: Scores for SAC concentration, ImPACT visual motor speed, and ImPACT reaction time were correlated with the KD (ρ = -0.216, -0.276, and 0.164, respectively) and were thus included in the regression model, which explained 16.8% of the variance in baseline KD time (P < .001, Cohen f2 = 0.20). Better SAC concentration score (β = -.174, P < .001), ImPACT visual motor speed (β = -.205, P < .001), and ImPACT reaction time (β = .056, P = .020) were associated with faster baseline KD performance, but the effect sizes were small. Conclusions: Better performance on cognitive measures involving concentration, visual motor speed, and reaction time was weakly associated with better baseline KD performance. Symptoms, psychological distress, balance, and vestibular-oculomotor provocation were unrelated to KD performance at baseline. The findings indicate limited overlap at baseline among the CARE SRC assessments and the KD

The epigenetic landscape of T cell exhaustion.

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells

Development of Scientific Competences in Chemistry Courses

Enseñar química en los primeros años de educación universitaria es clave en la formación de futuros profesionales, puesto que, además de proveer los conocimientos que aportan las ciencias básicas, contribuye al desarrollo de competencias científicas para que el estudiante resuelva problemas reales, a partir de la búsqueda adecuada de información en fuentes confiables y su lectura, con el propósito de desarrollar habilidades analíticas, críticas y creativas. Es por esto que, como estrategia de innovación pedagógica, el Departamento de Ciencias Básicas de la Universidad de La Salle ha venido implementando desde el año 2016 la formulación de un proyecto de investigación en los cursos de química. En el marco de esta estrategia, desde el II-2018 las autoras se abocaron a la reflexión e implementación de la estrategia didáctica que propende por el desarrollo de competencias científicas en los cursos de Química General, Química Orgánica y Bioquímica desde tareas, investigación y aprendizaje de problemáticas vigentes en Colombia y en el mundo.Abstract: Teaching chemistry in the first years of university education is key in the training of future professionals since, in addition to providing the knowledge of the basic sciences, it contributes to the development of scientific skills that lead to the student to solve real problems, starting from the appropriate search of information in reliable sources and its reading with the purpose of developing analytical, critical and creative skills. Therefore, as a pedagogical innovation strategy, the formulation of a research project in chemistry courses has been implemented since 2016 by the Department of Basic Sciences of the Universidad de La Salle. Within the framework of this strategy, from II-2018 the authors focused on the reflection and implementation of the didactic strategy that depends on the development of scientific competences in the courses of General Chemistry, Organic Chemistry and Biochemistry from tasks, research and learning of current problems in Colombia or in the world

Measuring the predictability of life outcomes with a scientific mass collaboration.

How predictable are life trajectories? We investigated this question with a scientific mass collaboration using the common task method; 160 teams built predictive models for six life outcomes using data from the Fragile Families and Child Wellbeing Study, a high-quality birth cohort study. Despite using a rich dataset and applying machine-learning methods optimized for prediction, the best predictions were not very accurate and were only slightly better than those from a simple benchmark model. Within each outcome, prediction error was strongly associated with the family being predicted and weakly associated with the technique used to generate the prediction. Overall, these results suggest practical limits to the predictability of life outcomes in some settings and illustrate the value of mass collaborations in the social sciences