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Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. Gα o K46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, Gα o K46E forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gα o , without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells., (© 2024. The Author(s).)

Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial reactive oxygen species (mitoROS) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitoROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca 2+ ) via the mitochondrial Ca 2+ uniporter (MCU-1) that results in an upregulation of mitoROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion., Competing Interests: RD, KK, ED, FH No competing interests declared, (© 2024, Doser et al.)

Protein kinase C (PKC) functions are essential for synaptic plasticity, learning, and memory. However, the roles of specific members of the PKC family in synaptic function, learning, and memory are poorly understood. Here, we investigated the role of individual PKC homologs for synaptic plasticity in Caenorhabditis elegans and found a differential role for pkc-2 and tpa-1 , but not pkc-1 and pkc-3 in associative olfactory learning and memory. More specifically we show that PKC-2 is essential for associative learning and TPA-1 for short-term associative memory (STAM). Using endogenous labeling and cell-specific rescues, we show that TPA-1 and PKC-2 are required in AVA for their functions. Previous studies demonstrated that olfactory learning and memory in C. elegans are tied to proper synaptic content and trafficking of AMPA-type ionotropic glutamate receptor homolog GLR-1 in the AVA command interneurons. Therefore, we quantified synaptic content, transport, and delivery of GLR-1 in AVA and showed that loss of pkc-2 and tpa-1 leads to decreased transport and delivery but only a subtle decrease in GLR-1 levels at synapses. AVA-specific expression of both PKC-2 and TPA-1 rescued these defects. Finally, genetic epistasis showed that PKC-2 and TPA-1 likely act in the same pathway to control GLR-1 transport and delivery, while regulating different aspects of olfactory learning and STAM. Thus, our data tie together cell-specific functions of 2 PKCs to neuronal and behavioral outcomes in C. elegans , enabling comparative approaches to understand the evolutionarily conserved role of PKC in synaptic plasticity, learning, and memory., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Heterotrimeric G proteins transduce extracellular chemical messages to generate appropriate intracellular responses. Point mutations in GNAO1, encoding the G protein α o subunit, have been implicated in a pathogenic condition characterized by seizures, movement disorders, intellectual disability, and developmental delay (GNAO1 disorder). However, the effects of these mutations on G protein structure and function are unclear. Here, we report the effects of 55 mutations on Gα o conformation, thermostability, nucleotide binding, and hydrolysis, as well as interaction with Gβγ subunits, receptors, and effectors. Our effort reveals four functionally distinct groups of mutants, including one group that sequesters receptors and another that sequesters Gβγ, both acting in a genetically dominant manner. These findings provide a more comprehensive understanding of disease-relevant mutations and reveal that GNAO1 disorder is likely composed of multiple mechanistically distinct disorders that will likely require multiple therapeutic strategies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polypropylene meshes used in pelvic organ prolapse (POP) repair are hampered by complications. Most POP meshes are highly unstable after tensioning ex vivo, as evidenced by marked deformations (pore collapse and wrinkling) that result in altered structural properties and material burden. By intentionally introducing collapsed pores and wrinkles into a mesh that normally has open pores and remains relatively flat after implantation, we reproduce mesh complications in vivo. To do this, meshes were implanted onto the vagina of rhesus macaques in nondeformed (flat) vs deformed (pore collapse +/- wrinkles) configurations and placed on tension. Twelve weeks later, animals with deformed meshes had two complications, (1) mesh exposure through the vaginal epithelium, and (2) myofibroblast proliferation with fibrosis - a mechanism of pain. The overarching response to deformed mesh was vaginal thinning associated with accelerated apoptosis, reduced collagen content, increased proteolysis, deterioration of mechanical integrity, and loss of contractile function consistent with stress shielding - a precursor to mesh exposure. Regional differences were observed, however, with some areas demonstrating myofibroblast proliferation and matrix deposition. Variable mechanical cues imposed by deformed meshes likely induce these two disparate responses. Utilizing meshes associated with uniform stresses on the vagina by remaining flat with open pores after tensioning is critical to improving outcomes. STATEMENT OF SIGNIFICANCE: Pain and exposure are the two most reported complications associated with the use of polypropylene mesh in urogynecologic procedures. Most meshes have unstable geometries as evidenced by pore collapse and wrinkling after tensioning ex vivo, recapitulating what is observed in meshes excised from women with complications in vivo. We demonstrate that collapsed pores and wrinkling result in two distinct responses (1) mesh exposure associated with tissue degradation and atrophy and (2) myofibroblast proliferation and matrix deposition consistent with fibrosis, a tissue response associated with pain. In conclusion, mesh deformation leads to areas of tissue degradation and myofibroblast proliferation, the likely mechanisms of mesh exposure and pain, respectively. These data corroborate that mesh implantation in a flat configuration with open pores is a critical factor for reducing complications in mesh-augmented surgeries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We would like to draw the attention of the Editor to the following facts which may be considered as potential declaration of interests: Pamela A. Moalli reports equipment, drugs, or supplies was provided by Coloplast Corp. Katrina M. Knight reports financial support was provided by Magee-Womens Research Institute & Foundation. Pamela A. Moalli reports a relationship with Hologic Inc that includes: board membership. However, there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Although falls are common and can cause serious injury to older adults, many health care facilities do not have falls prevention resources available. Falls prevention resources can reduce injury and mortality rates. Using the Centers for Disease Control and Prevention's (CDC) Stopping Elderly Accidents, Deaths & Injuries (STEADI) model, a falls risk clinic was implemented in a rural Indian Health Service (IHS) facility., Methods: A Fall Risk Questionnaire was created and implemented into the Provider's Electronic Health Records system interface to streamline provider screening and referral of patients who may be at risk for falls to a group falls risk reduction class., Results: Participants exhibited average improvements in the Timed Up and Go (6.8 s) (P = 0.0001), Five-Time Sit-to-Stand (5.1 s) (P = 0.0002), and Functional Reach (3.6 inches) (P = 1.0) tests as compared to their own baseline. Results were analyzed via paired t test. 71% of participants advanced out of an "increased risk for falls" category in at least one outcome measure. Of the participants to complete the clinic, all were successfully contacted and three (18%) reported one or more falls at the 90-day mark, of which one (6%) required a visit to the Emergency Department but did not require hospital admission., Conclusions: In regards to reducing falls in the community, per the CDC STEADI model, an integrated approach is best. All clinicians can play a part in reducing elder falls., (© 2021. The Author(s).)

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gβγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gβγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation., Competing Interests: Declaration of interests R.H.J.O. is a co-inventor of the TRUPATH technology and could receive royalties. This relationship has been disclosed to and is under management by UNC-Chapel Hill., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.

Background: Due to large geographical distances, the telephone is central to enabling rural Australian communities to access care from their local health service. While there is a history of rural nurses providing care via the telephone, it has been a highly controversial practice that is not routinely documented and little is known about how the practice is governed. The lack of knowledge regarding governance extends to the role of Directors of Nursing as clinical leaders charged with the responsibility of ensuring practice safety, quality, regulation and risk management. The purpose of this study was to identify clinical governance processes related to managing telephone presentations, and to explore Directors of Nursing perceptions of processes and clinical practices related to the management of telephone presentations to health services in rural Victoria, Australia., Methods: Qualitative documentary analysis and semi structured interviews were used in the study to examine the content of health service policies and explore the perceptions of Directors of Nursing in eight rural health services regarding policy content and enactment when people telephone rural health services for care. Participants were purposively selected for their knowledge and leadership role in governance processes and clinical practice. Data from the interviews were analysed using framework analysis. The process of analysis resulted in the identification of five themes., Results: The majority of policies reviewed provided little guidance for managing telephone presentations. The Directors of Nursing perceived policy content and enactment to be largely inadequate. When organisational structures failed to provide appropriate governance for the context, the Directors of Nursing engaged in protective mechanisms to support rural nurses who manage telephone presentations., Conclusions: Rural Directors of Nursing employed intuitive behaviours to protect rural nurses practicing within a clinical governance context that is inadequate for the complexities of the environment. Protective mechanisms provided indicators of clinical leadership and governance effectiveness, which may assist rural nurse leaders to strengthen quality and safe care by unlocking the potential of intuitive behaviours. Kanter's theory of structural power provides a way of conceptualising these protective mechanisms, illustrating how rural nurse leaders enact power.

Plants respond to synthetic chemicals by eliciting a xenobiotic response (XR) that enhances the expression of detoxifying enzymes such as glutathione transferases (GSTs). In agrochemistry, the ability of safeners to induce an XR is used to increase herbicide detoxification in cereal crops. Based on the responsiveness of the model plant Arabidopsis thaliana to the rice safener fenclorim (4,6-dichloro-2-phenylpyrimidine), a series of related derivatives was prepared and tested for the ability to induce GSTs in cell suspension cultures. The XR in Arabidopsis could be divided into rapid and slow types depending on subtle variations in the reactivity (electrophilicity) and chemical structure of the derivatives. In a comparative microarray study, Arabidopsis cultures were treated with closely related compounds that elicited rapid (fenclorim) and slow (4-chloro-6-methyl-2-phenylpyrimidine) XRs. Both chemicals induced major changes in gene expression, including a coordinated suppression in cell wall biosynthesis and an up-regulation in detoxification pathways, whereas only fenclorim selectively induced sulfur and phenolic metabolism. These transcriptome studies suggested several linkages between the XR and oxidative and oxylipin signaling. Confirming links with abiotic stress signaling, suppression of glutathione content enhanced GST induction by fenclorim, whereas fatty acid desaturase mutants, which were unable to synthesize oxylipins, showed an attenuated XR. Examining the significance of these studies to agrochemistry, only those fenclorim derivatives that elicited a rapid XR proved effective in increasing herbicide tolerance (safening) in rice.

Objectives: We sought to determine whether unhealthy behaviors play a stress-buffering role in observed racial disparities in physical and mental health., Methods: We conducted logistic regressions by race on data from the first 2 waves of the Americans' Changing Lives Survey to determine whether unhealthy behaviors had buffering effects on the relationship between major stressors and chronic health conditions, and on the relationship between major stressors and meeting the criteria for major depression., Results: Among Whites, unhealthy behaviors strengthened the relationship between stressors and meeting major-depression criteria. Among Blacks, however, the relationship between stressors and meeting major-depression criteria was stronger among those who had not engaged in unhealthy behaviors than among those who had. Among both race groups there was a positive association between stressors and chronic health conditions. Among Blacks there was an additional positive association between number of unhealthy behaviors and number of chronic conditions., Conclusions: Those who live in chronically stressful environments often cope with stressors by engaging in unhealthy behaviors that may have protective mental-health effects. However, such unhealthy behaviors can combine with negative environmental conditions to eventually contribute to morbidity and mortality disparities among social groups.

Background: More people than ever are turning to the Internet for health-related information, and recent studies indicate that the information patients find online directly affects the decisions they make about their health care. Little is known about the information needs or actual search behavior of people who use the Internet for health information., Objective: This study analyzes what people search for when they use a health-education Web site offering information about arthritis, orthopaedics, and sports-medicine topics. Additionally, it determines who is performing these searches: is it patients, friends or relatives of patients, or neither? Finally, it examines the similarities and differences among questions submitted by Web site visitors from different countries., Methods: Content analysis was performed on 793 free-text search queries submitted to a patient-education Web site owned and operated by the Department of Orthopaedics and Sports Medicine at the University of Washington Medical Center. The 793-query data set was coded into 3 schemes: (1) the purpose of the query, (2) the topic of the query, and (3) the relationship between the asker of the query and the patient. We determined the country from which each query was submitted by analyzing the Internet Protocol addresses associated with the queries., Results: The 5 most frequent reasons visitors searched the Web site were to seek: (1) information about a condition, (2) information about treatment, (3) information about symptoms, (4) advice about symptoms, and (5) advice about treatment. We were able to determine the relationship between the person submitting the query and the patient in question for 178 queries. Of these, the asker was the patient in 140 cases, and the asker was a friend or relative of the patient in 38 cases. The queries were submitted from 34 nations, with most coming from the United States, Australia, the United Kingdom, and Canada. When comparing questions submitted from the United States versus those from all other countries, the 3 most frequent types of questions were the same for both groups (and were the top 3 question types listed above)., Conclusions: These results provide the University of Washington Department of Orthopaedics and Sports Medicine, as well as other organizations that provide health-information Web sites, with data about what people around the world are seeking when they turn to the Internet for health information. If Web site managers can adapt their health-information Web sites in response to these findings, patients may be able to find and use Internet-based health information more successfully, enabling them to participate more actively in their health care.

In a multicenter study, responses to a combined vaccine containing standard diphtheria-tetanus-pertussis (DTP) and polyribosylribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, were evaluated in 107 infants who received single doses at two, four, and six months of age and compared with those in 61 infants given single doses of DTP alone on the same schedule. Reaction rates were comparable in the two treatment groups. At seven months of age 61% of the subjects given the combined DTP-PRP vaccine and 8% and of those given DTP alone showed an antibody response to PRP, as defined as a twofold increase in titer over the lowest previous level. Among those given the DTP-PRP combined vaccine, 92% of the positive antibody responses occurred after the third dose. There was a variation in antibody response, possibly due to a difference in the vaccine lots administered.