Attitudes to Short-Term Staffing and Workforce Priorities of Community Users of Remote Aboriginal Community-Controlled Health Services: A Qualitative Study.

In recent years, there has been an increasing trend of short-term staffing in remote health services, including Aboriginal Community-Controlled Health Services (ACCHSs). This paper explores the perceptions of clinic users' experiences at their local clinic and how short-term staffing impacts the quality of service, acceptability, cultural safety, and continuity of care in ACCHSs in remote communities. Using purposeful and convenience sampling, community users (aged 18+) of the eleven partnering ACCHSs were invited to provide feedback about their experiences through an interview or focus group. Between February 2020 and October 2021, 331 participants from the Northern Territory and Western Australia were recruited to participate in the study. Audio recordings were transcribed verbatim, and written notes and transcriptions were analysed deductively. Overall, community users felt that their ACCHS provided comprehensive healthcare that was responsive to their health needs and was delivered by well-trained staff. In general, community users expressed concern over the high turnover of staff. Recognising the challenges of attracting and retaining staff in remote Australia, community users were accepting of rotation and job-sharing arrangements, whereby staff return periodically to the same community, as this facilitated trusting relationships. Increased support for local employment pathways, the use of interpreters to enhance communication with healthcare services, and services for men delivered by men were priorities for clinic users.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing.

Previous observations showed that chloride and osmotic stress regulate the autophosphorylation and activity of the kinase domains of WNK1 and WNK3. Further, prior crystallography on the asymmetric dimeric of the unphosphorylated WNK1 kinase domain (WNK1/S382A, WNK1/SA) revealed conserved waters in the active site. Here we show by crystallography that PEG400 applied to crystals of dimeric WNK1/SA grown in space group P1 induces de-dimerization with a change in space group to P2 1 . Both the conserved waters, referred to here as conserved water network 1 (CWN1) and the chloride binding site are disrupted by PEG400. CWN1 is surrounded and stabilized by a pan-WNK-conserved cluster of charged residues. Here we mutagenized these charges in WNK3 to probe the importance of the CWN1 to WNK regulation. Two mutations at E314 in the Activation Loop (WNK3/E314Q and WNK3/E314A) enhanced activity, consistent with the idea that the CWN1 is inhibitory. Mutations of other residues in the cluster had similar or less activity than wild-type. PEG400 activation of WNK3 was not significantly reduced in the point mutants tested. The crystallographic and assay data support a role for CWN1 and the charged cluster in stabilizing an inactive configuration of WNKs and suggest that water functions as an allosteric inhibitor of WNKs.

Hydrostatic Pressure Sensing by WNK kinases.

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

Comparison of urban and rural mortality rates across the lifespan in Aotearoa/New Zealand: a population-level study.

BACKGROUND: Previous studies undertaken in New Zealand using generic rurality classifications have concluded that life expectancy and age-standardised mortality rates are similar for urban and rural populations. METHODS: Administrative mortality (2014-2018) and census data (2013 and 2018) were used to estimate age-stratified sex-adjusted mortality rate ratios (aMRRs) for a range of mortality outcomes across the rural-urban spectrum (using major urban centres as the reference) for the total population and separately for Maori and non-Maori. Rural was defined according to the recently developed Geographic Classification for Health. RESULTS: Mortality rates were higher overall in rural areas. This was most pronounced in the youngest age group (<30 years) in the most remote communities (eg, all-cause, amenable and injury-related aMRRs (95% CIs) were 2.1 (1.7 to 2.6), 2.5 (1.9 to 3.2) and 3.0 (2.3 to 3.9) respectively. The rural:urban differences attenuated markedly with increasing age; for some outcomes in those aged 75 years or more, estimated aMRRs were <1.0. Similar patterns were observed for Maori and non-Maori. CONCLUSION: This is the first time that a consistent pattern of higher mortality rates for rural populations has been observed in New Zealand. A purpose-built urban-rural classification and age stratification were important factors in unmasking these disparities.

Habitat and climatic associations of climate-sensitive species along a southern range boundary.

Climate change and habitat loss are recognized as important drivers of shifts in wildlife species' geographic distributions. While often considered independently, there is considerable overlap between these drivers, and understanding how they contribute to range shifts can predict future species assemblages and inform effective management. Our objective was to evaluate the impacts of habitat, climatic, and anthropogenic effects on the distributions of climate-sensitive vertebrates along a southern range boundary in Northern Michigan, USA. We combined multiple sources of occurrence data, including harvest and citizen-science data, then used hierarchical Bayesian spatial models to determine habitat and climatic associations for four climate-sensitive vertebrate species (American marten [Martes americana], snowshoe hare [Lepus americanus], ruffed grouse [Bonasa umbellus] and moose [Alces alces]). We used total basal area of at-risk forest types to represent habitat, and temperature and winter habitat indices to represent climate. Marten associated with upland spruce-fir and lowland riparian forest types, hares with lowland conifer and aspen-birch, grouse with lowland riparian hardwoods, and moose with upland spruce-fir. Species differed in climatic drivers with hares positively associated with cooler annual temperatures, moose with cooler summer temperatures and grouse with colder winter temperatures. Contrary to expectations, temperature variables outperformed winter habitat indices. Model performance varied greatly among species, as did predicted distributions along the southern edge of the Northwoods region. As multiple species were associated with lowland riparian and upland spruce-fir habitats, these results provide potential for efficient prioritization of habitat management. Both direct and indirect effects from climate change are likely to impact the distribution of climate-sensitive species in the future and the use of multiple data types and sources in the modelling of species distributions can result in more accurate predictions resulting in improved management at policy-relevant scales.

Telehealth in remote Australia: a supplementary tool or an alternative model of care replacing face-to-face consultations?

BACKGROUND: The COVID-19 pandemic increased the use of telehealth consultations by telephone and video around the world. While telehealth can improve access to primary health care, there are significant gaps in our understanding about how, when and to what extent telehealth should be used. This paper explores the perspectives of health care staff on the key elements relating to the effective use of telehealth for patients living in remote Australia. METHODS: Between February 2020 and October 2021, interviews and discussion groups were conducted with 248 clinic staff from 20 different remote communities across northern Australia. Interview coding followed an inductive approach. Thematic analysis was used to group codes into common themes. RESULTS: Reduced need to travel for telehealth consultations was perceived to benefit both health providers and patients. Telehealth functioned best when there was a pre-established relationship between the patient and the health care provider and with patients who had good knowledge of their personal health, spoke English and had access to and familiarity with digital technology. On the other hand, telehealth was thought to be resource intensive, increasing remote clinic staff workload as most patients needed clinic staff to facilitate the telehealth session and complete background administrative work to support the consultation and an interpreter for translation services. Clinic staff universally emphasised that telehealth is a useful supplementary tool, and not a stand-alone service model replacing face-to-face interactions. CONCLUSION: Telehealth has the potential to improve access to healthcare in remote areas if complemented with adequate face-to-face services. Careful workforce planning is required while introducing telehealth into clinics that already face high staff shortages. Digital infrastructure with reliable internet connections with sufficient speed and latency need to be available at affordable prices in remote communities to make full use of telehealth consultations. Training and employment of local Aboriginal staff as digital navigators could ensure a culturally safe clinical environment for telehealth consultations and promote the effective use of telehealth services among community members.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

Evaluation of an open forecasting challenge to assess skill of West Nile virus neuroinvasive disease prediction.

BACKGROUND: West Nile virus (WNV) is the leading cause of mosquito-borne illness in the continental USA. WNV occurrence has high spatiotemporal variation, and current approaches to targeted control of the virus are limited, making forecasting a public health priority. However, little research has been done to compare strengths and weaknesses of WNV disease forecasting approaches on the national scale. We used forecasts submitted to the 2020 WNV Forecasting Challenge, an open challenge organized by the Centers for Disease Control and Prevention, to assess the status of WNV neuroinvasive disease (WNND) prediction and identify avenues for improvement. METHODS: We performed a multi-model comparative assessment of probabilistic forecasts submitted by 15 teams for annual WNND cases in US counties for 2020 and assessed forecast accuracy, calibration, and discriminatory power. In the evaluation, we included forecasts produced by comparison models of varying complexity as benchmarks of forecast performance. We also used regression analysis to identify modeling approaches and contextual factors that were associated with forecast skill. RESULTS: Simple models based on historical WNND cases generally scored better than more complex models and combined higher discriminatory power with better calibration of uncertainty. Forecast skill improved across updated forecast submissions submitted during the 2020 season. Among models using additional data, inclusion of climate or human demographic data was associated with higher skill, while inclusion of mosquito or land use data was associated with lower skill. We also identified population size, extreme minimum winter temperature, and interannual variation in WNND cases as county-level characteristics associated with variation in forecast skill. CONCLUSIONS: Historical WNND cases were strong predictors of future cases with minimal increase in skill achieved by models that included other factors. Although opportunities might exist to specifically improve predictions for areas with large populations and low or high winter temperatures, areas with high case-count variability are intrinsically more difficult to predict. Also, the prediction of outbreaks, which are outliers relative to typical case numbers, remains difficult. Further improvements to prediction could be obtained with improved calibration of forecast uncertainty and access to real-time data streams (e.g. current weather and preliminary human cases).

What progress can the Australian Journal of Rural Health celebrate on its thirtieth anniversary?

Thirty years ago the first edition of the Australian Journal of Rural Health (AJRH) was published. Following reviews published in 2002 and 2012, it is again time to review what progress has been made in bringing about improved health outcomes for residents of rural and remote Australia over the past decade. Compounded by the Covid-19 crisis that has affected the health and health care system throughout Australia, this review notes the significant lack of progress over the past decade in ameliorating ongoing problems of poor access to primary health care and associated avoidable hospitalisations, persistent poor health of Indigenous Australians, and the greater prevalence of a range of health risk factors. Following the findings of the recent New South Wales enquiry into rural health, this review highlights what is needed to implement the many recommendations that have emerged from the wealth of evidence-based research published in journals such as the AJRH to improve health outcomes and increase the parity and equity in health between metropolitan and non-metropolitan Australians.

Effects of turnover and stability of health staff on quality of care in remote communities of the Northern Territory, Australia: a retrospective cohort study.

OBJECTIVES: To evaluate the relationship between markers of staff employment stability and use of short-term healthcare workers with markers of quality of care. A secondary objective was to identify clinic-specific factors which may counter hypothesised reduced quality of care associated with lower stability, higher turnover or higher use of short-term staff. DESIGN: Retrospective cohort study (Northern Territory (NT) Department of Health Primary Care Information Systems). SETTING: All 48 government primary healthcare clinics in remote communities in NT, Australia (2011-2015). PARTICIPANTS: 25 413 patients drawn from participating clinics during the study period. OUTCOME MEASURES: Associations between independent variables (resident remote area nurse and Aboriginal Health Practitioner turnover rates, stability rates and the proportional use of agency nurses) and indicators of health service quality in child and maternal health, chronic disease management and preventive health activity were tested using linear regression, adjusting for community and clinic size. Latent class modelling was used to investigate between-clinic heterogeneity. RESULTS: The proportion of resident Aboriginal clients receiving high-quality care as measured by various quality indicators varied considerably across indicators and clinics. Higher quality care was more likely to be received for management of chronic diseases such as diabetes and least likely to be received for general/preventive adult health checks. Many indicators had target goals of 0.80 which were mostly not achieved. The evidence for associations between decreased stability measures or increased use of agency nurses and reduced achievement of quality indicators was not supported as hypothesised. For the majority of associations, the overall effect sizes were small (close to zero) and failed to reach statistical significance. Where statistically significant associations were found, they were generally in the hypothesised direction. CONCLUSIONS: Overall, minimal evidence of the hypothesised negative effects of increased turnover, decreased stability and increased reliance on temporary staff on quality of care was found. Substantial variations in clinic-specific estimates of association were evident, suggesting that clinic-specific factors may counter any potential negative effects of decreased staff employment stability. Investigation of clinic-specific factors using latent class analysis failed to yield clinic characteristics that adequately explain between-clinic variation in associations. Understanding the reasons for this variation would significantly aid the provision of clinical care in remote Australia.

An Efficient Symmetric Electrolyzer Based On Bifunctional Perovskite Catalyst for Ammonia Electrolysis.

Ammonia is a natural pollutant in wastewater and removal technique such as ammonia electro-oxidation is of paramount importance. The development of highly efficient and low-costing electrocatalysts for the ammonia oxidation reaction (AOR) and hydrogen evolution reaction (HER) associated with ammonia removal is subsequently crucial. In this study, for the first time, the authors demonstrate that a perovskite oxide LaNi0.5 Cu0.5 O3-δ after being annealed in Ar (LNCO55-Ar), is an excellent non-noble bifunctional catalyst towards both AOR and HER, making it suitable as a symmetric ammonia electrolyser (SAE) in alkaline medium. In contrast, the LNCO55 sample fired in air (LNCO55-Air) is inactive towards AOR and shows very poor HER activity. Through combined experimental results and theoretical calculations, it is found that the superior AOR and HER activities are attributed to the increased active sites, the introduction of oxygen vacancies, the synergistic effect of B-site cations and the different active sites in LNCO55-Ar. At 1.23 V, the assembled SAE demonstrates ≈100% removal efficiency in 2210 ppm ammonia solution and >70% in real landfill leachate. This work opens the door for developments towards bifunctional catalysts, and also takes a profound step towards the development of low-costing and simple device configuration for ammonia electrolysers.

N,N-Dimethylacetamide-Diluted Nitrate Electrolyte for Aqueous Zn//LiMn2O4 Hybrid Ion Batteries.

N,N-Dimethylacetamide (DMA) cooperated with LiNO3 salt has previously shown to be a promising electrolyte for a Li//O2 battery, showing good stability against both the O2 electrode reaction and Li stripping/plating. In this work, DMA is hybridized with a concentrated nitrate electrolyte [2.5 m Zn(NO3)2 + 13 m LiNO3 aqueous solution] for better electrochemical stability while using less dissolved salts. The widest electrochemical stability window for this DMA-diluted electrolyte is determined as 3.1 V, the negative critical stability potential of which is -1.6 V versus Ag/AgCl, indicating desirable stability against hydrogen evolution and Zn deposition. The findings can be attributed to the weakened Li+/Zn2+ solvation sheath caused by low permittivity of DMA, as revealed through Raman spectra characterization and molecular dynamics simulation. A Zn//Zn symmetrical cell and Zn//LiMn2O4 hybrid ion batteries are assembled in air directly, attributed to the stability of DMA toward O2. Zn stripping/plating with a dendrite-free morphology is delivered for 110 h and 200 charge/discharge cycles under 1 C rate, achieving 99.0% Coulombic efficiency. The maximum capacity of the battery is 121.0 mA h·g-1 under 0.2 C rate (based on the mass of LiMn2O4), delivering an energy density of 165.8 W h·kg-1 together with 2.0 V working voltage. This work demonstrates the feasibility and validity of utilizing a relatively dilute electrolyte dissolved in oxygen for a highly stable aqueous rechargeable battery.

Integrating Spatiotemporal Epidemiology, Eco-Phylogenetics, and Distributional Ecology to Assess West Nile Disease Risk in Horses.

Mosquito-borne West Nile virus (WNV) is the causative agent of West Nile disease in humans, horses, and some bird species. Since the initial introduction of WNV to the United States (US), approximately 30,000 horses have been impacted by West Nile neurologic disease and hundreds of additional horses are infected each year. Research describing the drivers of West Nile disease in horses is greatly needed to better anticipate the spatial and temporal extent of disease risk, improve disease surveillance, and alleviate future economic impacts to the equine industry and private horse owners. To help meet this need, we integrated techniques from spatiotemporal epidemiology, eco-phylogenetics, and distributional ecology to assess West Nile disease risk in horses throughout the contiguous US. Our integrated approach considered horse abundance and virus exposure, vector and host distributions, and a variety of extrinsic climatic, socio-economic, and environmental risk factors. Birds are WNV reservoir hosts, and therefore we quantified avian host community dynamics across the continental US to show intra-annual variability in host phylogenetic structure and demonstrate host phylodiversity as a mechanism for virus amplification in time and virus dilution in space. We identified drought as a potential amplifier of virus transmission and demonstrated the importance of accounting for spatial non-stationarity when quantifying interaction between disease risk and meteorological influences such as temperature and precipitation. Our results delineated the timing and location of several areas at high risk of West Nile disease and can be used to prioritize vaccination programs and optimize virus surveillance and monitoring.

Review of Vesicular Stomatitis in the United States with Focus on 2019 and 2020 Outbreaks.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by vesicular stomatitis New Jersey virus (VSNJV) or vesicular stomatitis Indiana virus (VSIV). The disease circulates endemically in northern South America, Central America, and Mexico and only occasionally causes outbreaks in the United States. Over the past 20 years, VSNJV outbreaks in the southwestern and Rocky Mountain regions occurred with incursion years followed by virus overwintering and subsequent expansion outbreak years. Regulatory response by animal health officials is deployed to prevent spread from lesioned animals. The 2019 VS incursion was the largest in 40 years, lasting from June to December 2019 with 1144 VS-affected premises in 111 counties in eight states (Colorado, Kansas, Nebraska, New Mexico, Oklahoma, Texas, Utah, and Wyoming) and was VSIV serotype, last isolated in 1998. A subsequent expansion occurred from April to October 2020 with 326 VS-affected premises in 70 counties in eight states (Arizona, Arkansas, Kansas, Missouri, Nebraska, New Mexico, Oklahoma, and Texas). The primary serotype in 2020 was VSIV, but a separate incursion of VSNJV occurred in south Texas. Summary characteristics of the outbreaks are presented along with VSV-vector sampling results and phylogenetic analysis of VSIV isolates providing evidence of virus overwintering.

Interventions for health workforce retention in rural and remote areas: a systematic review.

BACKGROUND: Attracting and retaining sufficient health workers to provide adequate services for residents of rural and remote areas has global significance. High income countries (HICs) face challenges in staffing rural areas, which are often perceived by health workers as less attractive workplaces. The objective of this review was to examine the quantifiable associations between interventions to retain health workers in rural and remote areas of HICs, and workforce retention. METHODS: The review considers studies of rural or remote health workers in HICs where participants have experienced interventions, support measures or incentive programs intended to increase retention. Experimental, quasi-experimental and observational study designs including cohort, case-control, cross-sectional and case series studies published since 2010 were eligible for inclusion. The Joanna Briggs Institute methodology for reviews of risk and aetiology was used. Databases searched included MEDLINE (OVID), CINAHL (EBSCO), Embase, Web of Science and Informit. RESULTS: Of 2649 identified articles, 34 were included, with a total of 58,188 participants. All study designs were observational, limiting certainty of findings. Evidence relating to the retention of non-medical health professionals was scant. There is growing evidence that preferential selection of students who grew up in a rural area is associated with increased rural retention. Undertaking substantial lengths of rural training during basic university training or during post-graduate training were each associated with higher rural retention, as was supporting existing rural health professionals to extend their skills or upgrade their qualifications. Regulatory interventions requiring return-of-service (ROS) in a rural area in exchange for visa waivers, access to professional licenses or provider numbers were associated with comparatively low rural retention, especially once the ROS period was complete. Rural retention was higher if ROS was in exchange for loan repayments. CONCLUSION: Educational interventions such as preferential selection of rural students and distributed training in rural areas are associated with increased rural retention of health professionals. Strongly coercive interventions are associated with comparatively lower rural retention than interventions that involve less coercion. Policy makers seeking rural retention in the medium and longer term would be prudent to strengthen rural training pathways and limit the use of strongly coercive interventions.

Understanding and responding to the cost and health impact of short-term health staffing in remote and rural Aboriginal and Torres Strait Islander community-controlled health services: a mixed methods study protocol.

INTRODUCTION: Access to high-quality primary healthcare is limited for remote residents in Australia. Increasingly, remote health services are reliant on short-term or 'fly-in, fly-out/drive-in, drive-out' health workforce to deliver primary healthcare. A key strategy to achieving health service access equity, particularly evident in remote Australia, has been the development of Aboriginal Community Controlled Health Services (ACCHSs). This study aims to generate new knowledge about (1) the impact of short-term staffing in remote and rural ACCHSs on Aboriginal and Torres Strait Islander communities; (2) the potential mitigating effect of community control; and (3) effective, context-specific evidence-based retention strategies. METHODS AND ANALYSIS: This paper describes a 3-year, mixed methods study involving 12 ACCHSs across three states. The methods are situated within an evidence-based programme logic framework for rural and remote primary healthcare services. Quantitative data will be used to describe staffing stability and turnover, with multiple regression analyses to determine associations between independent variables (population size, geographical remoteness, resident staff turnover and socioeconomic status) and dependent variables related to patient care, service cost, quality and effectiveness. Qualitative assessment will include interviews and focus groups with clinical staff, clinic users, regionally-based retrieval staff and representatives of jurisdictional peak bodies for the ACCHS sector, to understand the impact of short-term staff on quality and continuity of patient care, as well as satisfaction and acceptability of services. ETHICS AND DISSEMINATION: The study has ethics approval from the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (project number DR03171), Central Australian Human Research Ethics Committee (CA-19-3493), Western Australian Aboriginal Health Ethics Committee (WAAHEC-938) and Far North Queensland Human Research Ethics Committee (HREC/2019/QCH/56393). Results will be disseminated through peer-reviewed journals, the project steering committee and community/stakeholder engagement activities to be determined by each ACCHS.

Vector Surveillance, Host Species Richness, and Demographic Factors as West Nile Disease Risk Indicators.

West Nile virus (WNV) is the most common arthropod-borne virus (arbovirus) in the United States (US) and is the leading cause of viral encephalitis in the country. The virus has affected tens of thousands of US persons total since its 1999 North America introduction, with thousands of new infections reported annually. Approximately 1% of humans infected with WNV acquire neuroinvasive West Nile Disease (WND) with severe encephalitis and risk of death. Research describing WNV ecology is needed to improve public health surveillance, monitoring, and risk assessment. We applied Bayesian joint-spatiotemporal modeling to assess the association of vector surveillance data, host species richness, and a variety of other environmental and socioeconomic disease risk factors with neuroinvasive WND throughout the conterminous US. Our research revealed that an aging human population was the strongest disease indicator, but climatic and vector-host biotic interactions were also significant in determining risk of neuroinvasive WND. Our analysis also identified a geographic region of disproportionately high neuroinvasive WND disease risk that parallels the Continental Divide, and extends southward from the US-Canada border in the states of Montana, North Dakota, and Wisconsin to the US-Mexico border in western Texas. Our results aid in unraveling complex WNV ecology and can be applied to prioritize disease surveillance locations and risk assessment.

Osmosensing by WNK Kinases.

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

WNKs are potassium-sensitive kinases.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

A Phosphorylated Intermediate in the Activation of WNK Kinases.

WNK kinases autoactivate by autophosphorylation. Crystallography of the kinase domain of WNK1 phosphorylated on the primary activating site (pWNK1) in the presence of AMP-PNP reveals a well-ordered but inactive configuration. This new pWNK1 structure features specific and unique interactions of the phosphoserine, less hydration, and smaller cavities compared with those of unphosphorylated WNK1 (uWNK1). Because WNKs are activated by osmotic stress in cells, we addressed whether the structure was influenced directly by osmotic pressure. pWNK1 crystals formed in PEG3350 were soaked in the osmolyte sucrose. Suc-WNK1 crystals maintained X-ray diffraction, but the lattice constants and pWNK1 structure changed. Differences were found in the activation loop and helix C, common switch loci in kinase activation. On the basis of these structural changes, we tested for effects on in vitro activity of two WNKs, pWNK1 and pWNK3. The osmolyte PEG400 enhanced ATPase activity. Our data suggest multistage activation of WNKs.