Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.
IntroductionSubstantial variation in the delivery of hip fracture care, and patient outcomes persists between hospitals, despite established UK national standards and guidelines. Patients’ outcomes are partly explained by patient-level risk factors, but it is hypothesised that organisational-level factors account for the persistence of unwarranted variation in outcomes. The mixed-methods REducing unwarranted variation in the Delivery of high qUality hip fraCture services in England and Wales (REDUCE) study, aims to determine key organisational factors to target to improve patient care.Methods and analysisQuantitative analysis will assess the outcomes of patients treated at 172 hospitals in England and Wales (2016–2019) using National Hip Fracture Database data combined with English Hospital Episodes Statistics; Patient Episode Database for Wales; Civil Registration (deaths) and multiple organisational-level audits to characterise each service provider. Statistical analyses will identify which organisational factors explain variation in patient outcomes, and typify care pathways with high-quality consistent patient outcomes. Documentary analysis of 20 anonymised British Orthopaedic Association hospital-initiated peer-review reports, and qualitative interviews with staff from four diverse UK hospitals providing hip fracture care, will identify barriers and facilitators to care delivery. The COVID-19 pandemic has posed a major challenge to the resilience of services and interviews will explore strategies used to adapt and innovate. This system-wide understanding will inform the development, in partnership with key national stakeholders, of an ‘Implementation Toolkit’ to inform and improve commissioning and delivery of hip fracture services.Ethics and disseminationThis study was approved: quantitative study by London, City and East Research Ethics Committee (20/LO/0101); and qualitative study by Faculty of Health Sciences University of Bristol Research Ethics Committee (Ref: 108284), National Health Service (NHS) Health Research Authority (20/HRA/71) and each NHS Trust provided Research and Development approval. Findings will be disseminated through scientific conferences, peer-reviewed journals and online workshops.
Objectives Despite established standards and guidelines, substantial variation remains in the delivery of hip fracture care across the United Kingdom. We aimed to determine which hospital-level organisational factors predict adverse patient outcomes in the months following hip fracture. Methods We examined a national record-linkage cohort of 178,757 patients aged ≥60 years who sustained a hip fracture in England and Wales in 2016–19. Patient-level hospital admissions datasets, National Hip Fracture Database and mortality data were linked to metrics from 18 hospital-level organisational-level audits and reports. Multilevel models identified organisational factors, independent of patient case-mix, associated with three patient outcomes: length of hospital stay (LOS), 30-day all-cause mortality and emergency 30-day readmission. Results Across hospitals mean LOS ranged from 12 to 41.9 days, mean 30-day mortality from 3.7 to 10.4% and mean readmission rates from 3.7 to 30.3%, overall means were 21.4 days, 7.3% and 15.3%, respectively. In all, 22 organisational factors were independently associated with LOS; e.g. a hospital’s ability to mobilise >90% of patients promptly after surgery predicted a 2-day shorter LOS (95% confidence interval [CI]: 1.2–2.6). Ten organisational factors were independently associated with 30-day mortality; e.g. discussion of patient experience feedback at clinical governance meetings and provision of prompt surgery to >80% of patients were each associated with 10% lower mortality (95%CI: 5–15%). Nine organisational factors were independently associated with readmissions; e.g. readmissions were 17% lower if hospitals reported how soon community therapy would start after discharge (95%CI: 9–24%). Conclusions Receipt of hip fracture care should be reliable and equitable across the country. We have identified multiple, potentially modifiable, organisational factors associated with important patient outcomes following hip fracture.
The technique of electron microscopy (EM) has been fundamental to muscle research since the days of Huxley and Hanson. Direct observation of how proteins in the sarcomere are arranged and visualising the changes that occur upon activation have greatly increased our understanding of function. In the 1980s specimen preparation techniques for biological EM moved away from traditional fixing and staining. The technique known as cryo-electron microscopy (Cryo-EM) was developed, which involves rapidly freezing proteins in liquid ethane which maintains them in a near native state. Within the last 5 years there has been a step change in the achievable resolution using Cryo-EM. This ‘resolution revolution’ can be attributed to advances in detector technology, microscope automation and maximum likelihood image processing. In this article we look at how Cryo-EM has contributed to the field of muscle research in this post revolution era, focussing on recently published high resolution structures of sarcomeric proteins.
Ischaemic cardiac injury results in a complex coordinated cellular response that enables effective damage control and recovery. Intercellular communication is integral to the coordination of these repair responses in complex tissue contexts. Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive in vitro studies have described the biogenesis, cargos and functional roles of EV populations but the characterisation of endogenously produced EVs in vivo is still in its infancy. Here we use larval and adult transgenic zebrafish to characterize endogenous cardiovascular EVs. Using a membranetethered fluorophore reporter system, cell-type specific EVs can be tracked in vivo by high spatiotemporal resolution light sheet imaging and modified flow cytometry methods allows these EVs to be extracted and assessed ex vivo. Importantly, we show that ischaemic cardiac injury models dynamically alter EV production during repair.
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