GPCR-mediated ß-arrestin activation deconvoluted with single-molecule precision.

ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.

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.

Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation.

Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.

Author Correction: Quantifying secondary transport at single-molecule resolution.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Quantifying secondary transport at single-molecule resolution.

Secondary active transporters, which are vital for a multitude of physiological processes, use the energy of electrochemical ion gradients to power substrate transport across cell membranes1,2. Efforts to investigate their mechanisms of action have been hampered by their slow transport rates and the inherent limitations of ensemble methods. Here we quantify the activity of individual MhsT transporters, which are representative of the neurotransmitter:sodium symporter family of secondary transporters3, by imaging the transport of individual substrate molecules across lipid bilayers at both single- and multi-turnover resolution. We show that MhsT is active only when physiologically oriented and that the rate-limiting step of the transport cycle varies with the nature of the transported substrate. These findings are consistent with an extracellular allosteric substrate-binding site that modulates the rate-limiting aspects of the transport mechanism4,5, including the rate at which the transporter returns to an outward-facing state after the transported substrate is released.

Resonantly hybridized excitons in moiré superlattices in van der Waals heterostructures.

Atomically thin layers of two-dimensional materials can be assembled in vertical stacks that are held together by relatively weak van der Waals forces, enabling coupling between monolayer crystals with incommensurate lattices and arbitrary mutual rotation1,2. Consequently, an overarching periodicity emerges in the local atomic registry of the constituent crystal structures, which is known as a moiré superlattice3. In graphene/hexagonal boron nitride structures4, the presence of a moiré superlattice can lead to the observation of electronic minibands5-7, whereas in twisted graphene bilayers its effects are enhanced by interlayer resonant conditions, resulting in a superconductor-insulator transition at magic twist angles8. Here, using semiconducting heterostructures assembled from incommensurate molybdenum diselenide (MoSe2) and tungsten disulfide (WS2) monolayers, we demonstrate that excitonic bands can hybridize, resulting in a resonant enhancement of moiré superlattice effects. MoSe2 and WS2 were chosen for the near-degeneracy of their conduction-band edges, in order to promote the hybridization of intra- and interlayer excitons. Hybridization manifests through a pronounced exciton energy shift as a periodic function of the interlayer rotation angle, which occurs as hybridized excitons are formed by holes that reside in MoSe2 binding to a twist-dependent superposition of electron states in the adjacent monolayers. For heterostructures in which the monolayer pairs are nearly aligned, resonant mixing of the electron states leads to pronounced effects of the geometrical moiré pattern of the heterostructure on the dispersion and optical spectra of the hybridized excitons. Our findings underpin strategies for band-structure engineering in semiconductor devices based on van der Waals heterostructures9.

Author Correction: Resonantly hybridized excitons in moiré superlattices in van der Waals heterostructures.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

Nurse-led clinics in primary health care: A scoping review of contemporary definitions, implementation enablers and barriers and their health impact.

AIMS: To define nurse-led clinics in primary health care, identify barriers and enablers that influence their successful implementation, and understand what impact they have on patient and population health outcomes. BACKGROUND: Nurse-led clinics definitions remain inconsistent. There is limited understanding regarding what enablers and barriers impact successful nurse-led clinic implementation and their impact on patient health care. DESIGN: Scoping review using narrative synthesis. METHODS: PubMed, MEDLINE, Web of Science, Scopus, CINAHL and PsycINFO were searched to identify nurse-led clinic definitions and models of care between 2000 and 2023. Screening and selection of studies were based on eligibility criteria and methodological quality assessment. Narrative synthesis enabled to communicate the phenomena of interest and follows the PRISMA for Scoping Reviews (PRISMA-ScR) checklist. RESULTS: Among the 36 identified studies, key principles of what constitutes nurse-led clinics were articulated providing a robust definition. Nurse-led clinics are, in most cases, commensurate with standard care, however, they provide more time with patients leading to greater satisfaction. Enablers highlight nurse-led clinic success is achieved through champions, partners, systems, and clear processes, while barriers encompass key risk points and sustainability considerations. CONCLUSION: The review highlights several fundamental elements are central to nurse-led clinic success and are highly recommended when developing interventional nurse-led strategies. Nurse-led clinics within primary health care seek to address health care through community driven, health professional and policy supported strategies. Overall, a robust and contemporary definition of nurse-led care and the clinics in which they operate is provided. RELEVANCE TO CLINICAL PRACTICE: The comprehensive definition, clear mediators of success and the health impact of nurse-led clinics provide a clear framework to effectively build greater capacity among nursing services within primary health care. This, in addition, highlights the need for good health care policy to ensure sustainability. PATIENT OR PUBLIC CONTRIBUTION: No Patient or Public Contribution.

Self-efficacy, grit, and rural career aspirations among early career nurses: a repeated cross-sectional study.

BACKGROUND: Global nursing workforce shortage represents an impediment to the delivery of safe, evidence-based healthcare. Despite collective efforts, a consistent stream of nurses leaving the profession remains, particularly within the first five years of practice, which is exacerbated in rural communities. The aim of the study was to compare self-efficacy, grit, and rural career aspirations among nursing graduates between their second and fourth year of their nursing profession. METHODS: As part of a longitudinal investigation, a repeated cross-sectional design was utilised. Participants included, 117 (response rate 52.2%) who completed an online questionnaire 18-24 months after graduating, and 32 participants (response rate of 21.0%) who agree to repeat the questionnaire 36-48 months after graduating. The questionnaire included demographic, employment, and measures examining general and occupational self-efficacy, grit, and rural career aspirations. RESULTS: No differences between general and occupational self-efficacy or grit were identified between second- and fourth-year nurses. In addition, the importance placed on undertaking rural career also remains unchanged. However, a higher proportion of fourth year nurses were more likely to be in management or were considering leaving the profession. CONCLUSIONS: This examination of early career nurses, now in their second and fourth-year post-graduation highlights self-efficacy, grit, and rural career aspirations remains stable between two- and four-years following graduation, while nursing in their fourth year were more likely to consider leaving the profession. Nursing retention is a 'Wicked Problem' that is unavoidably a complex amalgam of macro, meso and micro factors that we are yet to fully appreciate.

Understanding rural pharmacists' perspectives: lived experiences and insights associated with rural recruitment and retention.

INTRODUCTION: Pharmacists serve an important role in rural communities, and in some cases they may be the only health professional available. Their recruitment and retention is a major concern for rural communities and health services; however, a deeper understanding regarding the advantages and challenges of sustaining a rural pharmacy workforce is somewhat limited. The aim of this study was to develop a deeper understanding of pharmacists' perspectives about factors influencing pharmacist recruitment and retention to rural and remote communities. METHODS: The exploratory study, carried out in rural Tasmania and rural Western Victoria, used a qualitative descriptive design. Structured interviews, lasting between 30-60 minutes, were conducted by a single researcher using the Pharmacist Community Apgar Questionnaire via face-to-face, telephone or videoconferencing technology. Data were analysed thematically using verbatim transcription, extraction of significant statements and identification of similarities in formulated meanings, grouping the similar meanings and significant statements that pertained to the phenomena of interest. Specifically, qualitative data were used to provide a deeper understanding of factors identified as key assets, capabilities, or those most challenging for pharmacist recruitment and retention. RESULTS: The advantages and disadvantages rural communities face in recruiting and retaining pharmacists are presented. These insights are linked to the advantages of financial income, incentives and moving allowance. Further advantages include the degree of practice autonomy, breadth of tasks, the perception of the community, loyalty to the pharmacy and its pharmacists, along with community recognition. Challenges associated with the recruitment and retention of pharmacists centred on the need for spousal or partner employment opportunities, having greater proximity to schools, access to social or cultural opportunities, along with good transport connections. Further challenges included housing, the cost of schooling for children, having adequate locum or peer coverage and opportunities to host interns. DISCUSSION: The study provides a deeper exploration of the meaning and experiences of factors that previous research has shown are considered advantageous or challenging to the recruitment and retention of pharmacists in rural areas. Through the voices of pharmacists living and working in a rural area, the findings further enlighten our understanding regarding how the multifaceted and complex nature of health workforce planning may be addressed. As such, greater pharmacist recruitment and retention is enabled through adequate financial compensation and incentives, along with additional tax incentives for business and health services. Further, innovation is required to enhance economic sustainability. Locum coverage and intern opportunities also require innovative approaches to address concerns among potential candidates. Lastly, efforts to enable and support social connections such as schooling and spousal employment, while building community connection and a sense of rural community belonging, remain essential to recruit and retain pharmacists. CONCLUSION: Rural pharmacist recruitment and retention is complex, requiring a multi-pronged approach to implement practical solutions. Given this complexity and the unique features of each rural community, solutions require whole-of-community ownership to create innovative solutions. Recognition of specific advantages and challenges can address key driving factors for pharmacist recruitment and retention in rural communities.

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+.

Single-molecule analysis of ligand efficacy in ß2AR-G-protein activation.

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the ß2 adrenergic receptor (ß2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound ß2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound ß2AR-Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis.

The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation.

Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A.

The increase in multi-drug-resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores.

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

Sustaining rural pharmacy workforce understanding key attributes for enhanced retention and recruitment.

OBJECTIVE: To pilot the Pharmacist Community Apgar Questionnaire (PharmCAQ) and evaluate its usability and capacity to develop a greater understanding of the unique factors that impact the rural recruitment and retention of pharmacists. DESIGN: Cross-sectional design involving face-to-face, telephone or video conferencing interviews. SETTING: Twelve rural communities across Tasmania and Western Victoria, Australia. PARTICIPANTS: Participants (n = 24) included pharmacists, a Director of Clinical Services, pharmacy practice managers and senior pharmacy assistants. MAIN OUTCOME MEASURES: Interviews enabled the completion of the PharmCAQ, which assigns quantitative values to 50 key factors to ascertain a community's strengths and challenges associated with recruitment and retention and their relative importance to the pharmacist workforce. RESULTS: The cumulative PharmCAQ scores indicated the tool was sensitive enough to differentiate high- and low-performing communities. Overall, the highest-rated factors considered most vital to pharmacist recruitment and retention were the reputation of the pharmacy, the ability of the pharmacist to be independent and autonomous, the loyalty of the community to the pharmacy, the level and stability of monetary compensation and the breadth of tasks available to a pharmacist. CONCLUSIONS: This study identified the strengths and challenges of participating communities and provided an insight into the shared factors to consider in recruiting and retaining pharmacists. Further, each community has unique strengths that can further be promoted in recruitment, flagging where limited resources are best used to address site specific challenges. This is more likely to ensure the matching of the right candidate with the right community.

Changes in grit and psychological capital at the time of major crisis: nursing students' perseverance, resources, and resilience.

OBJECTIVES: To examine changes in grit and psychological capital among nursing students prior to, during the height of the pandemic, and more than 12 months after the initial pandemic announcement. METHODS: A cross-sectional study design addressed the aim of the study. Nursing students undertaking a three-year baccalaureate degree between 2019 and 2021 were included. RESULTS: Mean grit levels among the n=818 unique student participants were significantly lower in 2020 than in 2019 and 2021; however, no significant difference was detected for psychological capital over the same period. CONCLUSIONS: Although normative day-to-day challenges may aid grit development, a major event has a negative impact yet has a buffering effect of negative life events at the time of a crisis. The study further placates that psychological capital remains malleable and open to change at the time of a crisis and may be an essential mechanism to mediate grit and has the capacity to influence student performance over time. It remains essential to develop grit through the mediating elements of psychological capital to enable nursing student to undertake academic studies, particularly in the event of major challenges, such approaches may further enable students' endurance to withstand major crises as they enter the workforce.