Guanylyl cyclase-A phosphorylation decreases cardiac hypertrophy and improves systolic function in male, but not female, mice.

Atrial natriuretic peptide (NP) and BNP increase cGMP, which reduces blood pressure and cardiac hypertrophy by activating guanylyl cyclase (GC)-A, also known as NPR-A or Npr1. Although GC-A is highly phosphorylated, and dephosphorylation inactivates the enzyme, the significance of GC-A phosphorylation to heart structure and function remains unknown. To identify in vivo processes that are regulated by GC-A phosphorylation, we substituted glutamates for known phosphorylation sites to make GC-A8E/8E mice that express an enzyme that cannot be inactivated by dephosphorylation. GC-A activity, but not protein, was increased in heart and kidney membranes from GC-A8E/8E mice. Activities were threefold higher in female compared to male cardiac ventricles. Plasma cGMP and testosterone were elevated in male and female GC-A8E/8E mice, but aldosterone was only increased in mutant male mice. Plasma and urinary creatinine concentrations were decreased and increased, respectively, but blood pressure and heart rate were unchanged in male GC-A8E/8E mice. Heart weight to body weight ratios for GC-A8E/8E male, but not female, mice were 12% lower with a 14% reduction in cardiomyocyte cross-sectional area. Subcutaneous injection of fsANP, a long-lived ANP analog, increased plasma cGMP and decreased aldosterone in male GC-AWT/WT and GC-A8E/8E mice at 15 min, but only GC-A8E/8E mice had elevated levels of plasma cGMP and aldosterone at 60 min. fsANP reduced ventricular ERK1/2 phosphorylation to a greater extent and for a longer time in the male mutant compared to WT mice. Finally, ejection fractions were increased in male but not female hearts from GC-A8E/8E mice. We conclude that increased phosphorylation-dependent GC-A activity decreases cardiac ERK activity, which results in smaller male hearts with improved systolic function.

Increasing reliability of axially compressed cylinders through stiffness tailoring and optimization.

The capabilities of the rapid tow shearing (RTS) process are explored to reduce the well-known imperfection sensitivity of axially compressed cylindrical shells. RTS deposits curvilinear carbon fibre tapes with a fibre-angle-thickness coupling that enables the in situ manufacturing of embedded rings and stringers. By blending the material's elastic modulus and wall thickness smoothly across the cylindrical surface, the load paths can be redistributed favourably with a minimal-design approach that contains part count and weight while ameliorating imperfection sensitivity. A genetic algorithm that incorporates realistic manufacturing imperfections and axial stiffness penalty is used to maximize the 99.9% reliability load of straight fibre (SF) and RTS cylinders. The axial stiffness penalty ensures that reliability does not come at the expense of stiffness. The first-order second-moment method is used to calculate statistical moments that enable an estimate of the 99.9% reliability load. Due to the fibre-angle-thickness coupling of RTS, buckling data are normalized by mass and thickness. Compared to a quasi-isotropic laminate, which corresponds to the optimal eight-layer design for a perfect cylinder, the optimized SF and RTS laminates have a 6% and 8% greater 99.9% normalized reliability load. By relaxing the axial stiffness penalty, the performance benefit can be increased such that SF and RTS cylinders exceed the 99.9% normalized reliability load of an eight-layer quasi-isotropic laminate by 23% and 37%, respectively. Both improvements (with and without penalty functions) stem largely from a reduction in the variance of the buckling-load distribution, thereby demonstrating the potential of fibre-steered cylinders in reducing the imperfection sensitivity of cylindrical shells. This article is part of the theme issue 'Probing and dynamics of shock sensitive shells'.

Citizen Science as an Ecosystem of Engagement: Implications for Learning and Broadening Participation.

The bulk of research on citizen science participants is project centric, based on an assumption that volunteers experience a single project. Contrary to this assumption, survey responses (n = 3894) and digital trace data (n = 3649) from volunteers, who collectively engaged in 1126 unique projects, revealed that multiproject participation was the norm. Only 23% of volunteers were singletons (who participated in only one project). The remaining multiproject participants were split evenly between discipline specialists (39%) and discipline spanners (38% joined projects with different disciplinary topics) and unevenly between mode specialists (52%) and mode spanners (25% participated in online and offline projects). Public engagement was narrow: The multiproject participants were eight times more likely to be White and five times more likely to hold advanced degrees than the general population. We propose a volunteer-centric framework that explores how the dynamic accumulation of experiences in a project ecosystem can support broad learning objectives and inclusive citizen science.

Guanylyl Cyclase-B Dependent Bone Formation in Mice is Associated with Youth, Increased Osteoblasts, and Decreased Osteoclasts.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B7E/7E mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28 days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts.

Greenspace and park use associated with less emotional distress among college students in the United States during the COVID-19 pandemic.

The COVID-19 pandemic has negatively affected many people's psychological health. Impacts may be particularly severe among socially vulnerable populations such as college students, a group predisposed to mental health problems. Outdoor recreation and visits to greenspaces such as parks offer promising pathways for addressing the mental health challenges associated with COVID-19. During the early stages of the pandemic (March-May 2020), we surveyed 1280 college students at four large public universities across the United States (U.S.) to assess how, and why, outdoor recreation and park use changed since the emergence of COVID-19. We also measured students' self-reported levels of emotional distress (a proxy for psychological health) and assessed potential demographic and contextual correlates of distress, including county-level per capita park area and greenness, using generalized linear models. We found that 67% of students reported limiting outdoor activities and 54% reported reducing park use during the pandemic. Students who reduced their use of outdoor spaces cited structural reasons (e.g., lockdowns), concerns about viral transmission, and negative emotions that obstructed active lifestyles. Students who maintained pre-pandemic park use levels expressed a desire to be outdoors in nature, often with the explicit goal of improving mental and physical health. Emotional distress among students was widespread. Models showed higher levels of emotional distress were associated with reducing park use during the pandemic and residing in counties with a smaller area of parks per capita. This study of U.S. college students supports the value of park-based recreation as a health promotion strategy for diverse populations of young adults during a time of crisis.

Citizen science as a tool for enhancing recreation research in protected areas: Applications and opportunities.

As the popularity of nature-based recreation and tourism grows, protected area (PA) managers around the world are faced with escalating monitoring and management challenges across spatial and temporal scales. Citizen science, an emerging research approach which involves active public participation and collaboration with scientists in the scientific process, is an innovative tool that could help managers address these challenges. This study applied the Preferred Reporting Items for Systematic Review Recommendations (PRISMA) protocol to review published studies that utilized citizen science methods in recreation research, examining the extent and nature of such applications and identifying future opportunities. We identified 20 peer-reviewed journal articles from the Web of Science, most of which were published since 2015. These studies utilized different citizen science approaches to examine recreation patterns, behaviors, and impacts in terrestrial and marine PAs. We found that citizen science was used most often in marine PAs, with specialized recreationists (e.g., SCUBA divers) as the most frequent contributors. The types of volunteers recruited differed by their sources (i.e., general public, recreation specialists, and organizational affiliates) and roles (i.e., volunteers as agents of data collection and volunteers as research subjects), with innovative technology (e.g., participatory GIS) creating new engagement opportunities. Despite these benefits, the accuracy and reliability of citizen science data remain important considerations for managers. Our review demonstrates how citizen science can inform management and enhance public participation in PA stewardship activities, and it reveals the need for more research to explore applications of citizen science in different recreation contexts.

Nature-based Pathways to Health Promotion: The Value of Parks and Greenspace.

The built environment is a key social determinant of health. Exposure to parks and greenspace can improve physical and mental health and provide other benefits that enhance well-being. Programs and initiatives that capitalize on nature-based opportunities offer health care providers with a cost-effective alternative for upstream health promotion.

"Messy transitions": Students' perspectives on the impacts of the COVID-19 pandemic on higher education.

The COVID-19 pandemic affected every area of students' lives, especially their education. Limited research has explored students' experiences during the pandemic. This study documents how students across seven United States universities viewed the impact of the COVID-19 pandemic on their educational experiences and how these students reacted to these impacts. We present qualitative data from an online survey conducted between March and May 2020 that resulted in 1267 respondents with relevant data. Conventional content analysis with an inductive approach was used to analyze open-ended responses to the question, "We are interested in the ways that the coronavirus (COVID-19) pandemic has changed how you feel and behave. What are the first three ways that come to mind?" Six categories emerged from the data: changes in instruction delivery mode, changes in schedule and everyday life, increased technology use, decreased academic opportunities and resources, negative reaction to the changes in higher education, and positive reactions to changes in higher education. Among our recommendations for practice are personalized approaches to material delivery and evaluation, synchronous classes and opportunities to connect with professors and students, and convenient support services.

Chronic respiratory disease disparity between American Indian/Alaska Native and white populations, 2011-2018.

BACKGROUND: American Indian/Alaska Native (AI/AN) populations have been disproportionately affected by chronic respiratory diseases for reasons incompletely understood. Past research into disease disparity using population-based surveys mostly focused on state-specific factors. The present study investigates the independent contributions of AI/AN racial status and other socioeconomic/demographic variables to chronic respiratory disease disparity in an 11-state region with historically high AI/AN representation. Using data from the Behavioral Risk Factor Surveillance System (BRFSS) spanning years 2011-2018, this work provides an updated assessment of disease disparity and potential determinants of respiratory health in AI/AN populations. METHODS: This cross-sectional study used data from the BRFSS survey, 2011-2018. The study population included AI/AN and non-Hispanic white individuals resident in 11 states with increased proportion of AI/AN individuals. The yearly number of respondents averaged 75,029 (62878-87,350) which included approximately 5% AI/AN respondents (4.5-6.3%). We compared the yearly adjusted prevalence for chronic respiratory disease, where disease status was defined by self-reported history of having asthma and/or chronic obstructive pulmonary disease (COPD). Multivariable logistic regression was performed to determine if being AI/AN was independently associated with chronic respiratory disease. Covariates included demographic (age, sex), socioeconomic (marital status, education level, annual household income), and behavioral (smoking, weight morbidity) variables. RESULTS: The AI/AN population consistently displayed higher adjusted prevalence of chronic respiratory disease compared to the non-Hispanic white population. However, the AI/AN race/ethnicity characteristic was not independently associated with chronic respiratory disease (OR, 0.93; 95% CI, 0.79-1.10 in 2017). In contrast, indicators of low socioeconomic status such as annual household income of <$10,000 (OR, 2.02; 95% CI, 1.64-2.49 in 2017) and having less than high school education (OR, 1.37; 95% CI, 1.16-1.63 in 2017) were positively associated with disease. These trends persisted for all years analyzed. CONCLUSIONS: This study highlighted that AI/AN socioeconomic burdens are key determinants of chronic respiratory disease, in addition to well-established risk factors such as smoking and weight morbidity. Disease disparity experienced by the AI/AN population is therefore likely a symptom of disproportionate socioeconomic challenges they face. Further promotion of public health and social service efforts may be able to improve AI/AN health and decrease this disease disparity.

Assessing Impact of Show-Me ECHO on the Health of Missourians: Two Examples.

Show Me ECHO is a model for interprofessional collaboration that utilizes telehealth technologies to share evidence-based medical knowledge to improve patient outcomes and minimize variation in care for underserved populations. To measure ECHO outcomes, Show Me ECHO develops both an evaluation of clinical outcomes for patients as well as assessing learner outcomes on the Kirkpatrick Typology of Evaluation. This paper describes evaluation models for Dermatology and Childhood Asthma ECHOs.

Telehealth and Telemedicine in Missouri.

Missouri is a national leader in telemedicine, and the Missouri Telehealth Network has led operational, legal and regulatory, and research and evaluation efforts since 1994. Telehealth and telemedicine have the potential to increase access to and efficiency of healthcare delivery, improve quality, and improve patient outcomes. Coverage and reimbursement rules vary by regulator, and Missouri enjoys a broad statutory definition of telehealth coverage and reimbursement parity (no distinction between in-person and telehealth services).

Accelerating Care Through ECHO: Case Examples from the Field.

In this article, we describe three life-changing patient cases demonstrating high-quality and timely care they received in their communities, thanks to the Show-Me ECHO project. Early autism diagnosis, a potentially deadly tumor manifesting as a benign-looking rash, a recalcitrant case of hepatitis C: rural and underserved Missourians now have access to state-of-the-art care through their local providers receiving interdisciplinary telementoring on evidence based practices.

Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr27A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr27A/7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.SIGNIFICANCE STATEMENT The branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.

Skeletal overgrowth-causing mutations mimic an allosterically activated conformation of guanylyl cyclase-B that is inhibited by 2,4,6,-trinitrophenyl ATP.

Activating mutations in the receptor for C-type natriuretic peptide (CNP), guanylyl cyclase B (GC-B, also known as Npr2 or NPR-B), increase cellular cGMP and cause skeletal overgrowth, but how these mutations affect GTP catalysis is poorly understood. The A488P and R655C mutations were compared with the known mutation V883M. Neither mutation affected GC-B concentrations. The A488P mutation decreased the EC50 5-fold, increased Vmax 2.6-fold, and decreased the Km 13-fold, whereas the R655C mutation decreased the EC50 5-fold, increased the Vmax 2.1-fold, and decreased the Km 4.7-fold. Neither mutation affected maximum activity at saturating CNP concentrations. Activation by R655C did not require disulfide bond formation. Surprisingly, the A488P mutant only activated the receptor when it was phosphorylated. In contrast, the R655C mutation converted GC-B-7A from CNP-unresponsive to CNP-responsive. Interestingly, neither mutant was activated by ATP, and the Km and Hill coefficient of each mutant assayed in the absence of ATP were similar to those of wild-type GC-B assayed in the presence of ATP. Finally, 1 mm 2,4,6,-trinitrophenyl ATP inhibited all three mutants by as much as 80% but failed to inhibit WT-GC-B. We conclude that 1) the A488P and R655C missense mutations result in a GC-B conformation that mimics the allosterically activated conformation, 2) GC-B phosphorylation is required for CNP-dependent activation by the A488P mutation, 3) the R655C mutation abrogates the need for phosphorylation in receptor activation, and 4) an ATP analog selectively inhibits the GC-B mutants, indicating that a pharmacologic approach could reduce GC-B dependent human skeletal overgrowth.

A Glutamate-Substituted Mutant Mimics the Phosphorylated and Active Form of Guanylyl Cyclase-A.

Multisite phosphorylation is required for activation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, by cardiac natriuretic peptides (NPs). Seven chemically identified sites (Ser-487, Ser-497, Thr-500, Ser-502, Ser-506, Ser-510, and Thr-513) and one functionally identified putative site (Ser-473) were reported. Single alanine substitutions for Ser-497, Thr-500, Ser-502, Ser-506, and Ser-510 reduced maximal velocity (Vmax), whereas glutamate substitutions had no effect or increased Vmax Ala but not Glu substitution for Ser-497 increased the Michaelis constant (Km) approximately 400%. A GC-A mutant containing Glu substitutions for all seven chemically identified sites (GC-A-7E) had a Km approximately 10-fold higher than phosphorylated wild-type (WT) GC-A, but one additional substitution for Ser-473 to make GC-A-8E resulted in the same Vmax, Km, and EC50 as the phosphorylated WT enzyme. Adding more glutamates to make GC-A-9E or GC-A-10E had little effect on activity, and sequential deletion of individual glutamates in GC-A-8E progressively increased the Km Double Ala substitutions for Ser-497 and either Thr-500, Ser-510 or Thr-513 in WT-GC-A increased the Km 23- to 70-fold but the same mutations in GC-A-8E only increased the Km 8-fold, consistent with one site affecting the phosphorylation of other sites. Phosphate measurements confirmed that single-site Ala substitutions reduced receptor phosphate levels more than expected for the loss of a single site. We conclude that a concentrated region of negative charge, not steric properties, resulting from multiple interdependent phosphorylation sites is required for a GC-A conformation capable of transmitting the hormone binding signal to the catalytic domain.

Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone.

The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2-7E). Npr2-7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events.

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes.

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

meso-Acetoxymethyl BODIPY dyes for photodynamic therapy: improved photostability of singlet oxygen photosensitizers.

We report two BODIPY based photosensitizers (Br2BOAc and I2BOAc) featuring an acetoxymethyl substituent at the meso-position. These photosensitizers show improved photostability against singlet oxygen, when compared to a BODIPY photosensitizer lacking the acetoxymethyl group. Both compounds were evaluated for photodynamic therapy against HeLa cells and photodynamic inactivation against E. coli bacteria. We show that the compounds readily embed in the lipid membranes of HeLa cervical cancer cells and efficiently induced light-dependent apoptosis at nanomolar concentration. Also, both compounds showed a substantial degree of photoinactivation of E. coli bacteria when used at low micromolar concentrations.