CFTR function, pathology and pharmacology at single-molecule resolution.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4-6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.

Pediatric emergency asthma presentations in Southwest Victoria: a retrospective cross-sectional study 2017 to 2020.

OBJECTIVES: Australia has one of the highest prevalence of asthma globally, and accessible emergency asthma presentation data remains vital, however, is currently underreported in regional and rural areas. Utilizing the Rural Acute Hospital Data Register (RAHDaR) which includes previously non-reported data, the aim of the study is to provide a more accurate understanding of asthma emergency presentation events, while investigating the factors associated with these presentations. METHODS: A retrospective cross-sectional study collected de-identified emergency asthma presentation data from nine health services in regional Victoria for children aged 0 to 14 years between 2017 and 2020. Demographic and presentation data were collated along with government datasets. Asthma emergency presentations incidence rates and predictor variables were analyzed using hierarchical multiple regression after adjusting for smoking and sex. Significance was determined at p < 0.05. RESULTS: Of the 1090 emergency asthma presentations, n = 369 occurred at health services who did not previously report data. This represents a 33.86% increase in our understanding of emergency asthma presentations and demonstrating a rate of 16.06 presentations per 1000 children per year. Key factors such as age, population density, and private health insurance were associated with asthma emergency presentation events among both sexes, while socioeconomic status and rurality were not predictive. CONCLUSIONS: Although some findings are consistent with current research, the study highlights previously unrecognized specific factors that are predictive of asthma among 0-14-year-old children. These findings provide more accurate insights for healthcare workers and policymakers as they seek to support people with asthma and accurately address health inequities.

"Thank You for Listening": An Exploratory Study Regarding the Lived Experience and Perception of Medical Errors Among Those Who Receive Care.

The issue of medical errors, or adverse events caused within a health care context or by a health care provider, is largely under-researched. While the experience and perspective of health care professionals regarding medical errors have been explored, little attention is paid to the health care consumers regarding their perceptions and experiences. Therefore, there is a need to better understand the public's views on medical errors to enhance patient safety and quality care. The current study sought to examine Australian public perceptions and experiences, especially concerning what errors had occurred, the perceived sources of the errors, and if the errors had been reported. This paper reports the qualitative findings of an online survey for Australian residents who have accessed or received medical services at any time in Australia. Responses from 304 surveys were analyzed and discussed, including demographic information and key themes about medical errors, which were categorized into engagement and patients' voices being heard, the quality of care being provided, and the system's accountability. Based on the findings, the study highlights the importance of effective health professional-patient communication, enhanced capacity to deliver high quality care, and improved mechanism for error reporting and resolution where patients feel safe and confident about positive changes being made.

Factors contributing to COPD hospitalisations from 2010 to 2015: Variation among rural and metropolitan Australians.

INTRODUCTION: Rural and remote populations experience the greatest burden of chronic obstructive pulmonary disease (COPD), the fifth leading cause of death in Australia. Currently there is a need to prioritise health services to improve health outcomes among those at higher risk of COPD. OBJECTIVES: To investigate the differences in COPD hospitalisation between rural and urban populations and determine predictive factors contributing to COPD hospitalisation. METHODS: Statewide hospitalisation data from 2010 to 2015 were obtained through the Victorian Admitted Episodes Dataset and other key data sets. The rates of hospitalisation were analysed using hierarchical multiple regression to examine the association between COPD hospitalisations and a number of predictor variables. RESULTS: The highest COPD incidence occurred in metropolitan males aged 85 years of age and older (35.092 hospitalisations per 1000 population). Among metropolitan residents, smoking, population density and household income had a significant association with COPD hospitalisations for both sexes. Among rural males, smoking rates, household income and rural land use (farming) were significant predictors of COPD hospitalisations. There was an overall stability in statewide COPD hospitalisation over the 5 years to 2015, P = 0.420. CONCLUSION: This investigation highlights many rural and regional areas have much lower COPD hospitalisation rates than metropolitan areas. Between males and females, there are heterogenetic factors that contribute to the significant variation associated with COPD hospitalisation in metropolitan and rural areas, such as rural land use among rural males. This indicates that risk factor assessments, beyond smoking alone, need to be individualised and prioritised in practice to optimise care.

Dynamics of P-type ATPase transport revealed by single-molecule FRET.

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.

Asthma hospitalisation trends from 2010 to 2015: variation among rural and metropolitan Australians.

BACKGROUND: Asthma remains a leading cause of illness, where primary care can assist to reduce hospitalisations through prevention, controlling acute episodes, and overall management of asthma. In Victoria, Asthma hospitalisations were as high as 3.1 hospitalisations per 1000 population in 1993-94. The primary aims of this study are to: determine if changes in asthma hospitalisations have occurred between 2010 and 2015; determine the key factors that impact asthma hospitalisation over time; and verify whether rural and urban asthma hospitalisations are disparate. A secondary aim of the study is to compare 2010-2015 results with asthma data prior to 2010. METHODS: Hospital separation data from 1 July 2010 to 30 June 2015 were obtained through the Victorian Admitted Episodes Dataset and other agencies. Data included sex, age, Local Government Area, private or public patient, length of stay, and type of discharge. Asthma and predictor variables were analysed according to hospital separation rates after adjusting for smoking and sex. Hierarchical multiple regression examined the association between asthma and predictor variables. RESULTS: During the study period, 49,529 asthma hospital separations occurred, of which 77.5% were in metropolitan hospitals, 55.4% hospital separations were aged 0-14 years, and 21.7% were privately funded. State-wide hospital separations were 1.85 per 1000 population and were consistently higher in metropolitan compared to rural areas (1.93 vs 1.64 per 1000 population). When data among metropolitan adults aged 15 and over were analysed, an increase in the proportion of smokers in the population was reflected by an increase in the number of hospital separations (Adj OR 1.035). Further, among rural and metropolitan children aged 0-14 the only predictor of asthma hospital separations was sex, where metropolitan male children had higher odds of separation than metropolitan females of the same age (Adj OR 4.297). There was no statistically meaningful difference for separation rates between males and females in rural areas. CONCLUSIONS: We demonstrated a higher overall hospital separation rate in metropolitan Victoria. For children in metropolitan areas, males were hospitalised at higher rates than females, while the inverse was demonstrated for children residing in rural areas. Therefore, optimising asthma management requires consideration of the patient's age, gender and residential context. Primary health care may play a leading role in increasing health literacy for patients in order to improve self-management and health-seeking behaviour.

Engineering a Prototypic P-type ATPase Listeria monocytogenes Ca(2+)-ATPase 1 for Single-Molecule FRET Studies.

Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrolysis in the cytoplasmic domains to ion transport across the membrane. The Ca(2+)-ATPase from Listeria monocytogenes, LMCA1, was found to be a suitable model of P-type ATPases and was engineered to facilitate single-molecule FRET studies of transport-related structural changes. Mutational analyses of the endogenous cysteine residues in LMCA1 were performed to reduce background labeling without compromising activity. Pairs of cysteines were introduced into the optimized low-reactivity background, and labeled with maleimide derivatives of Cy3 and Cy5 resulting in site-specifically double-labeled protein with moderate activity. Ensemble and confocal single-molecule FRET studies revealed changes in FRET distribution related to structural changes during the transport cycle, consistent with those observed by X-ray crystallography for the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Notably, the cytosolic headpiece of LMCA1 was found to be distinctly more compact in the E1 state than in the E2 state. Thus, the established experimental system should allow future real-time FRET studies of the structural dynamics of LMCA1 as a representative P-type ATPase.

Workplace health and safety issues among community nurses: a study regarding the impact on providing care to rural consumers.

OBJECTIVES: The objective of the study was to investigate the types of workplace health and safety issues rural community nurses encounter and the impact these issues have on providing care to rural consumers. METHODS: The study undertook a narrative inquiry underpinned by a phenomenological approach. Community nursing staff who worked exclusively in rural areas and employed in a permanent capacity were contacted among 13 of the 16 consenting healthcare services. All community nurses who expressed a desire to participate were interviewed. Data were collected using semistructured interviews with 15 community nurses in rural and remote communities. Thematic analysis was used to analyse interview data. RESULTS: The role, function and structures of community nursing services varied greatly from site to site and were developed and centred on meeting the needs of individual communities. In addition, a number of workplace health and safety challenges were identified and were centred on the geographical, physical and organisational environment that community nurses work across. The workplace health and safety challenges within these environments included driving large distances between client's homes and their office which lead to working in isolation for long periods and without adequate communication. In addition, other issues included encountering, managing and developing strategies to deal with poor client and carer behaviour; working within and negotiating working environments such as the poor condition of patient homes and clients smoking; navigating animals in the workplace; vertical and horizontal violence; and issues around workload, burnout and work-related stress. CONCLUSIONS: Many nurses achieved good outcomes to meet the needs of rural community health consumers. Managers were vital to ensure that service objectives were met. Despite the positive outcomes, many processes were considered unsafe by community nurses. It was identified that greater training and capacity building are required to meet the needs among all staff.

Single-molecule dynamics of gating in a neurotransmitter transporter homologue.

Neurotransmitter:Na(+) symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na(+) gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.

Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging.

Organic fluorophores common to fluorescence-based investigations suffer from unwanted photophysical properties, including blinking and photobleaching, which limit their overall experimental performance. Methods to control such processes are particularly important for single-molecule fluorescence and fluorescence resonance energy transfer imaging where uninterrupted, stable fluorescence is paramount. Fluorescence and FRET-based assays have been carried out on dye-labeled DNA and RNA-based systems to quantify the effect of including small-molecule solution additives on the fluorescence and FRET behaviors of both cyanine and Alexa fluorophores. A detailed dwell time analysis of the fluorescence and FRET trajectories of more than 200,000 individual molecules showed that two compounds identified previously as triplet state quenchers, cyclooctatetraene, and Trolox, as well as 4-nitrobenzyl alcohol, act to favorably attenuate blinking, photobleaching, and influence the rate of photoresurrection in a concentration-dependent and context-dependent manner. In both biochemical systems examined, a unique cocktail of compounds was shown to be optimal for imaging performance. By simultaneously providing the most rapid and direct access to multiple photophysical kinetic parameters, smFRET imaging provides a powerful avenue for future investigations aimed at discovering new compounds, and effective combinations thereof. These efforts may ultimately facilitate tuning organic dye molecule performance according to each specific experimental demand.