AlphaFold predictions are valuable hypotheses and accelerate but do not replace experimental structure determination.

Artificial intelligence-based protein structure prediction methods such as AlphaFold have revolutionized structural biology. The accuracies of these predictions vary, however, and they do not take into account ligands, covalent modifications or other environmental factors. Here, we evaluate how well AlphaFold predictions can be expected to describe the structure of a protein by comparing predictions directly with experimental crystallographic maps. In many cases, AlphaFold predictions matched experimental maps remarkably closely. In other cases, even very high-confidence predictions differed from experimental maps on a global scale through distortion and domain orientation, and on a local scale in backbone and side-chain conformation. We suggest considering AlphaFold predictions as exceptionally useful hypotheses. We further suggest that it is important to consider the confidence in prediction when interpreting AlphaFold predictions and to carry out experimental structure determination to verify structural details, particularly those that involve interactions not included in the prediction.

Outcomes of the EMDataResource cryo-EM Ligand Modeling Challenge.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein-nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.

The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

Small molecules disaggregate alpha-synuclein and prevent seeding from patient brain-derived fibrils.

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.

An expandable, modular de novo protein platform for precision redox engineering.

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

A synthesis and subgroup analysis of the eosinophilic esophagitis tissue transcriptome.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic immune mediated inflammatory disorder of the esophagus. It is still unknown why children and adults present differently, and there is little evidence about why it is more common in men than women. OBJECTIVE: Our aim was to synthesize published and unpublished esophageal bulk RNA-sequencing (RNA-seq) data to gain novel insights into the pathobiology of EoE and examine the differences in EoE transcriptome by sex and age group. METHODS: Esophageal bulk RNA-seq data from 5 published and 2 unpublished studies resulting in 137 subjects (EoE: N = 76; controls: N = 61) were analyzed. For overall analysis, combined RNA-seq data of patients with EoE were compared with those of controls and subgroup analysis was conducted in patients with EoE by age of the patient (children [<18 years] vs adults [≥18 years]) and sex (female vs male). Gene-set enrichment analysis, ingenuity pathway analysis (IPA), cell-type analysis, immunohistochemistry, and T-cell or B-cell receptor analysis were performed. RESULTS: Overall analysis identified dysregulation of new genes in EoE compared with controls. IPA revealed that EoE is characterized by a mixed inflammatory response compared with controls. Cell-type analysis showed that cell composition varied with age: children had more mast cells, whereas adults had more macrophages. Finally, gene-set enrichment analysis and IPA revealed pathways that were differentially regulated in adults versus children and male versus female patients with EoE. CONCLUSIONS: Using a unique approach to analyze bulk RNA-seq data, we found that EoE is characterized by a mixed inflammatory response, and the EoE transcriptome may be influenced by age and sex. These findings enhance insights into the molecular mechanisms of EoE.

Hyperextended telomeres promote formation of C-circle DNA in telomerase positive human cells.

Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect circular DNA with extrachromosomal telomere repeats. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (∼23 kb telomeres) and HeLaE1 (∼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.

MCC is a centrosomal protein that relocalizes to non-centrosomal apical sites during intestinal cell differentiation.

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

A neural network potential based on pairwise resolved atomic forces and energies.

Molecular simulations have become a key tool in molecular and materials design. Machine learning (ML)-based potential energy functions offer the prospect of simulating complex molecular systems efficiently at quantum chemical accuracy. In previous work, we have introduced the ML-based PairF-Net approach to neural network potentials, that adopts a pairwise interatomic scheme to predicting forces within a molecular system. Here, we further develop the PairF-Net model to intrinsically incorporate energy conservation and couple the model to a molecular mechanical (MM) environment within the OpenMM package. The updated PairF-Net model yields energy and force predictions and dynamical distributions in good agreement with the rMD17 dataset of ten small organic molecules in the gas-phase. We further show that these in vacuo ML models of small molecules can be applied to force predictions in aqueous solution via hybrid ML/MM simulations. We present a new benchmark dataset for these ten molecules in solution, obtained from QM/MM simulations, which we denote as rMD17-aq (https://zenodo.org/records/10048644); and assess the ability of PairF-Net to reproduce the molecular energy, atomic forces and dynamical distributions of these solution conformations via ML/MM simulations.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Epigenetic Regulation of F2RL3 Associates With Myocardial Infarction and Platelet Function.

BACKGROUND: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. METHODS: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. RESULTS: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. CONCLUSIONS: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

SNAP: Supportive non-invasive ventilation for acute chest syndrome prevention for hospitalized children with sickle cell disease: Perspectives of patients, parents, and the healthcare team.

RATIONALE: Acute chest syndrome (ACS) often develops during hospitalizations for sickle cell disease (SCD) vaso-occlusive episodes and may be triggered by a combination of chest wall splinting, opioid use, hypoventilation, and atelectasis. In 2017, Boston Medical Center's general pediatric inpatient unit instituted the novel use of bi-level positive airway pressure (BiPAP) as "supportive non-invasive ventilation for ACS prevention" (SNAP) to prevent ACS and respiratory decompensation. OBJECTIVE: The goals of this qualitative study were to identify perceived benefits, harms, facilitators, and barriers to use of SNAP. METHODS: We conducted semi-structured key informant interviews at three sites with different levels of SNAP implementation (Site 1: extensive implementation; Site 2: limited implementation; Site 3: not yet implemented) regarding experiences with and/or perceptions of SNAP. Interviews and coding were guided by the Promoting Action on Research Implementation in Health Services (PARiHS) framework. RESULTS: Thirty-four participants (physicians, nurses, respiratory therapists, child life specialists, psychologists, youth with SCD, and parents) completed interviews. Major themes included: (i) participants perceive BiPAP as effective at preventing ACS, and for those with medically stable ACS, for preventing respiratory decompensation. (ii) BiPAP use is appropriate on the general pediatric inpatient unit for medically stable patients with SCD. (iii) Improving the patient experience is the most important factor to optimize acceptance of BiPAP by patients and families. CONCLUSION/FUTURE DIRECTIONS: SNAP is perceived as effective and appropriate for hospitalized pediatric patients with SCD. Improving the patient experience is the biggest challenge. These data will inform a future protocol for a multicenter hybrid effectiveness/implementation trial of SNAP.

Strong π-stacking causes unusually large anisotropic thermal expansion and thermochromism.

π-stacking in ground-state dimers/trimers/tetramers of N-butoxyphenyl(naphthalene)diimide (BNDI) exceeds 50 kcal ⋅ mol-1 in strength, drastically surpassing that for the *3[pyrene]2 excimer (∼30 kcal ⋅ mol-1; formal bond order = 1) and similar to other weak-to-moderate classical covalent bonds. Cooperative π-stacking in triclinic (BNDI-T) and monoclinic (BNDI-M) polymorphs effects unusually large linear thermal expansion coefficients (α a , α b , α c , ß) of (452, -16.8, -154, 273) × 10-6 ⋅ K-1 and (70.1, -44.7, 163, 177) × 10-6 ⋅ K-1, respectively. BNDI-T exhibits highly reversible thermochromism over a 300-K range, manifest by color changes from orange (ambient temperature) toward red (cryogenic temperatures) or yellow (375 K), with repeated thermal cycling sustained for over at least 2 y.

Receive coil quality assurance procedure and automated analysis for ViewRay MRIdian MR-Linac.

PURPOSE: Regular receiving coil quality assurance (QA) is required to ensure image quality of an MRIdian Linac system. The manufacturer provides a spherical phantom and positioning tube for single-slice signal-to-noise ratio (SNR) and uniformity assessments. We aimed to improve imaging setup and coverage and eliminate inter-scan variability by employing multi-slice imaging of a stable phantom. Additionally, we strived to expedite analysis by developing objective, automated analysis software. METHODS: A 5300 mL cylindrical plastic bottle placed in plastic bins was scanned at isocenter using a spin-echo sequence with NEMA-recommended parameters and 18 axial slices, avoiding phantom repositioning. Acquisition was repeated with and without prescan normalization filtering and by saving uncombined element images. Obtained data were analyzed using custom open-source MATLAB code. Signal and noise images were automatically assigned, and ROIs for SNR and uniformity calculations were defined using image thresholding. SNR and uniformity pass/fail decisions were made using baseline comparisons. RESULTS: The proposed method was successfully implemented as monthly coil QA for 3.5 years. Setup and scanning took 41 min on average for a coil set. Automated image analysis was completed in a few minutes. Signal intensity peaked around +90 or -90 mm for Torso or Head/Neck coil unfiltered images. Noise peaked and minimized SNR inside ±30 mm from isocenter, while maximizing it around ±130 mm. Prescan normalization smoothed signal response, reduced SNR and increased uniformity. Individual coil element image analysis identified their position, signal or noise response and SNR. SNR and uniformity pass/fail thresholds were set for already tested and new coils. Conspicuous and subtle Torso coil malfunctions were detected considering baseline deviations of combined and individual element results. CONCLUSIONS: Our QA method eliminated observer bias and provided insights into coil function, image filtering performance and coil element location. It provided SNR and uniformity thresholds and identified faulty coil elements.

An integrative review of the impact of allied health student placements on current staff's knowledge and procedural skills in acute and primary care settings.

BACKGROUND: Staff shortages limit access to health services. The bidirectional benefits of allied health clinical placements are understood in the domains of student learning, health service delivery, and future workforce development. Still, the benefits to current workforce outcomes remain unknown. This review provides insights into the effects of allied health student placements in acute and primary care settings, particularly on healthcare staff's knowledge and procedural skills. METHODS: This search was based on the integrative review process established by Whittemore and Knafl in 2005. In October 2023, the first author (MH) searched five major electronic databases: Medline-EBSCO, PubMed, CINAHL, Embase, and Scopus. The CLUSTER model was used to track additional references. The first three authors (MH, SM, and SC) were involved in screening, quality appraisal, and synthesis of the studies. Data were thematically synthesised and analysed. RESULTS: MeSH headings and keywords were used in key search areas: health education, health professional training, clinical placements, and allied health professions. The systematic search yielded 12 papers on allied health student placements across various healthcare settings in rural and metropolitan areas, with no high-quality methodologies measuring student placements' impact on staff knowledge and skills. Four main themes were identified from the analysis: meaningful student integration in service delivery, targeted educational support to healthcare staff, development of staff procedural skills and confidence, and the mechanisms of why student placements work in this aspect. CONCLUSIONS: This review suggests that offering allied health student placement could be a promising approach to supporting rural healthcare staff in performing patient assessments and treatments proficiently and collaboratively. However, this requires further investigation to confirm.

Adipose Tissue in Breast Cancer Microphysiological Models to Capture Human Diversity in Preclinical Models.

Female breast cancer accounts for 15.2% of all new cancer cases in the United States, with a continuing increase in incidence despite efforts to discover new targeted therapies. With an approximate failure rate of 85% for therapies in the early phases of clinical trials, there is a need for more translatable, new preclinical in vitro models that include cellular heterogeneity, extracellular matrix, and human-derived biomaterials. Specifically, adipose tissue and its resident cell populations have been identified as necessary attributes for current preclinical models. Adipose-derived stromal/stem cells (ASCs) and mature adipocytes are a normal part of the breast tissue composition and not only contribute to normal breast physiology but also play a significant role in breast cancer pathophysiology. Given the recognized pro-tumorigenic role of adipocytes in tumor progression, there remains a need to enhance the complexity of current models and account for the contribution of the components that exist within the adipose stromal environment to breast tumorigenesis. This review article captures the current landscape of preclinical breast cancer models with a focus on breast cancer microphysiological system (MPS) models and their counterpart patient-derived xenograft (PDX) models to capture patient diversity as they relate to adipose tissue.

Association Between the United States Department of Health and Human Services' COVID-19 Public Education Campaign and Initial Adult COVID-19 Vaccination Uptake by Race and Ethnicity in the United States, 2020-2022.

Non-Hispanic Black (Black) and Hispanic/Latino (Latino) populations face an increased risk of COVID-19 infection, hospitalization, and death from COVID-19 relative to non-Hispanic White (White) populations. When COVID-19 vaccines became available in December 2020, Black and Latino adults were less likely than White adults to get vaccinated due to factors such as racial discrimination and structural barriers to uptake. In April 2021, the U.S. HHS COVID-19 public education campaign (the Campaign) was launched to promote vaccination through general and audience-tailored messaging. As of March 2022, Black and Latino adults had reached parity with White adults in COVID-19 vaccine uptake. This study evaluated the relationship between Campaign exposure and subsequent vaccine uptake among Black, Latino, and White adults in the United States and assessed whether participant race/ethnicity moderated the relationship between Campaign exposure and vaccine uptake. Campaign media delivery data was merged with survey data collected from a sample of U.S. adults (n = 2,923) over four waves from January 2021 to March 2022. Logistic regression analysis showed that cumulative Campaign digital impressions had a positive, statistically significant association with COVID-19 vaccine uptake, and that participant race/ethnicity moderated this association. Compared with White adults, the magnitude of the relationship between cumulative impressions and vaccination was greater among Black and Latino adults. Results from a simulation model suggested that the Campaign may have been responsible for closing 5.0% of the gap in COVID-19 vaccination by race/ethnicity from April to mid-September 2021. We discuss implications for future public education campaigns that aim to reduce health disparities.

Implementation of a day-stay joint replacement pathway in an Australian regional public hospital: A descriptive study.

OBJECTIVE: To describe the implementation, feasibility and safety of a day-stay joint replacement pathway in a regional public hospital in Australia. METHOD: Over a 12-month pilot period, a prospective descriptive analysis of consecutive patients undergoing total knee and hip arthroplasty was conducted. The number of eligible day-stay patients, proportion of successful same-day discharges and reasons for same-day failure to discharge were recorded. Outcome measures captured for all joint replacements across this period included length of stay (LoS), patient reported outcomes, complications and patient satisfaction. The implementation pathway as well as patient and staff identified success factors derived from interviews were outlined. RESULTS: Forty-one/246 (17%) patients booked for joint replacement surgery were eligible for day-stay and 21/41 (51%) achieved a successful same-day discharge. Unsuccessful same-day discharges were due to time of surgery too late in the day (7/20), no longer meeting same-day discharge criteria (11/20) and declined discharge same-day (2/20). Over the implementation period 65% (162/246) of all patients were discharged with a LoS of 2 days or less. Patient satisfaction for the day-stay pathway was high. Complication rates and patient-reported outcomes were equivalent across LoS groups. CONCLUSION: The day-stay joint replacement surgery pathway was feasible to implement, safe and acceptable to patients. Day-stay pathways have potential patient and system-level efficiency benefits.