Single-cell analysis of chromatin and expression reveals age- and sex-associated alterations in the human heart.

Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex and find epigenetic signatures of variation in ATAC-Seq data in this discovery cohort. We then scale up our single-cell RNA-Seq analysis by combining our data with five recently published single nucleus RNA-Seq datasets of healthy adult hearts. We find variation such as metabolic alterations by sex and immune changes by age in differential expression tests, as well as alterations in abundance of cardiomyocytes by sex and neurons with age. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs. Our analysis represents the largest single-cell analysis of cardiac variation by age and sex to date and provides a resource for further study of healthy cardiac variation and transcriptional regulation at single-cell resolution.

Implementation of low-intensity thrombolysis monitoring care in routine practice: process evaluation of the Optimal Post rtPA-IV Monitoring in Acute Ischemic Stroke (OPTIMISTmain) study in the United States.

INTRODUCTION: The ongoing OPTIMISTmain study, an international, multicenter, stepped-wedge cluster randomized trial, aims to determine effectiveness and safety of low-intensity versus standard monitoring in thrombolysis-treated patients with mild-to-moderate acute ischemic stroke (AIS). An embedded process evaluation explored integration and impact of the intervention on care processes at participating US sites. METHODS: A mixed-methods approach with quantitative and qualitative data were collected between September 2021 and November 2022. Implementer surveys were undertaken at pre- and post-intervention phases to understand the perceptions of low-intensity monitoring strategy. A sample of stroke care nurses were invited to participate in semi-structured interviews at an early stage of post-intervention. Qualitative data were analyzed deductively using the normalization process theory; quantitative data were tabulated. RESULTS: Interviews with 21 nurses at 8 hospitals have shown low-intensity monitoring was well accepted, as there were less time constraints and reduced workload for each patient. There were initial safety concerns over missing deteriorating patients and difficulties in changing established routines. Proper training, education, and communication, and changing the habits and culture of care, were key elements to successfully adopting the new monitoring care into routine practice. Similar results were found in the post-intervention survey (42 nurses from 13 hospitals). Nurses reported time being freed up to provide patient education (56%), daily living care (50%), early mobilization (26%), mood/cognition assessment (44%), and other aspects (i.e. communication, family support). CONCLUSIONS: Low-intensity monitoring for patients with mild-to-moderate acute ischemic stroke, facilitated by appropriate education and organizational support, appears feasible and acceptable at US hospitals.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

A single-cell multi-omic atlas spanning the adult rhesus macaque brain.

Cataloging the diverse cellular architecture of the primate brain is crucial for understanding cognition, behavior, and disease in humans. Here, we generated a brain-wide single-cell multimodal molecular atlas of the rhesus macaque brain. Together, we profiled 2.58 M transcriptomes and 1.59 M epigenomes from single nuclei sampled from 30 regions across the adult brain. Cell composition differed extensively across the brain, revealing cellular signatures of region-specific functions. We also identified 1.19 M candidate regulatory elements, many previously unidentified, allowing us to explore the landscape of cis-regulatory grammar and neurological disease risk in a cell type-specific manner. Altogether, this multi-omic atlas provides an open resource for investigating the evolution of the human brain and identifying novel targets for disease interventions.

Cross-species imputation and comparison of single-cell transcriptomic profiles.

Cross-species comparison and prediction of gene expression profiles are important to understand regulatory changes during evolution and to transfer knowledge learned from model organisms to humans. Single-cell RNA-seq (scRNA-seq) profiles enable us to capture gene expression profiles with respect to variations among individual cells; however, cross-species comparison of scRNA-seq profiles is challenging because of data sparsity, batch effects, and the lack of one-to-one cell matching across species. Moreover, single-cell profiles are challenging to obtain in certain biological contexts, limiting the scope of hypothesis generation. Here we developed Icebear, a neural network framework that decomposes single-cell measurements into factors representing cell identity, species, and batch factors. Icebear enables accurate prediction of single-cell gene expression profiles across species, thereby providing high-resolution cell type and disease profiles in under-characterized contexts. Icebear also facilitates direct cross-species comparison of single-cell expression profiles for conserved genes that are located on the X chromosome in eutherian mammals but on autosomes in chicken. This comparison, for the first time, revealed evolutionary and diverse adaptations of X-chromosome upregulation in mammals.

The main Optimal Post rTpa-Iv Monitoring in Ischemic Stroke Trial (OPTIMISTmain): protocol for a pragmatic, stepped wedge, cluster randomized controlled trial.

Introduction Careful monitoring of patients who receive intravenous thrombolysis (IVT) for acute ischemic stroke (AIS) is resource-intensive, and potentially less relevant in those with mild degrees of neurological impairment who are at low-risk of symptomatic intracerebral hemorrhage (sICH) and other complications. \ Methods OPTIMISTmain is an international, multicenter, prospective, stepped wedge, cluster randomized, blinded outcome assessed trial aims to determine whether a less-intensity monitoring protocol is at least as effective, safe and efficient as standard post-IVT monitoring in patients with mild deficits post-AIS. Clinically-stable adult patients with mild AIS (defined by a NIHSS <10) who do not require intensive care within 2 hours post-IVT are recruited at hospitals in Australia, Chile, China, Malaysia, Mexico, UK, US and Vietnam. An average of 15 patients recruited per period (overall 60 patient participants) at 120 sites for a total of 7200 IVT-treated AIS patients will provide 90% power (one-sided α 0.025). The initiation of eligible hospitals is based on a rolling process whenever ready, stratified by country. Hospitals are randomly allocated using permuted blocks into 3 sequences of implementation, stratified by country and the projected number of patients to be recruited over 12 months. These sequences have four periods that dictate the order in which they are to switch from control (usual care) to intervention (implementation of low intensity monitoring protocol) to different clusters of patients in a stepped manner. Compared to standard monitoring, the low-intensity monitoring protocol includes assessments of neurological and vital signs every 15 minutes for 2 hours, 2 hourly (versus every 30 minutes) for 8 hours, and 4 hourly (versus every 1 hour) until 24 hours, post-IVT. The primary outcome measure is functional recovery, defined by the modified Rankin scale (mRS) at 90 days, a seven-point ordinal scale (0 [no residual symptom] to 6 [death]). Secondary outcomes include death or dependency, length of hospital stay, and health-related quality of life, sICH and serious adverse events. Conclusion OPTIMISTmain will provide Level I evidence for the safety and effectiveness of a low-intensity post-IVT monitoring protocol in patients with mild severity of AIS.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

A single-cell transcriptional timelapse of mouse embryonic development, from gastrula to pup.

The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.

Hyperacute stroke thrombolysis via telemedicine: a multicentre study of performance, safety and clinical efficacy.

OBJECTIVES: Timely thrombolysis of ischaemic stroke improves functional recovery, yet its delivery nationally is challenging due to shortages in the stroke specialist workforce and large geographical areas. One solution is remote stroke specialist input to regional centres via telemedicine. This study evaluates the usage and key metrics of performance of the East of England Stroke Telemedicine Partnership-the largest telestroke service in the UK-in providing hyperacute stroke care. DESIGN: Prospective observational study. SETTING: The East of England Stroke Telemedicine Partnership provides a horizontal 'hubless' model of out-of-hours hyperacute stroke care to a population of 6.2 million across a 7500 square mile semirural region. PARTICIPANTS: All (2709) telestroke consultations between 1 January 2014 and 31 December 2019. MAIN OUTCOME MEASURES: Thrombolysis decision, pre-thrombolysis and post-thrombolysis stroke severity (National Institutes of Health Stroke Scale, NIHSS), haemorrhagic complications, and hyperacute pathway timings. RESULTS: Over the period, 1149 (42.4%) individuals were thrombolysed. Thrombolysis rates increased from 147/379 (38.8%) in 2014 to 225/490 (45.9%) in 2019. Median (IQR) pre-thrombolysis NIHSS was 10 (6-17), reducing to 6 (2-14) 24-hour post-thrombolysis (p<0.001). Post-thrombolysis haemorrhage occurred in 27 cases (2.3%). Over the period, median (IQR) door-to-needle time reduced from 85 (65-108) min to 68 (55-97.5) min (p<0.01), driven by improved imaging-to-needle times from 52.5 (38-72.25) min to 42 (30.5-62.5) min (p<0.01). However, the same period saw an increase in median onset-to-hospital arrival time from 77.5 (60-109.25) min to 95 (70-135) min (p<0.001). CONCLUSIONS: The results from this large hyperacute telestroke cohort indicate two important points for clinical practice. First, telemedicine via a hubless horizontal model provides a clinically effective and safe method for delivering hyperacute stroke thrombolysis. Second, improved door-to-needle times were offset by a concerning rise in prehospital timings. These findings indicate that although telemedicine may benefit in-hospital hyperacute stroke care, improvements across the whole stroke pathway are essential.

Spatial and cell type transcriptional landscape of human cerebellar development.

The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.

Modified early warning score and risk of mortality after acute stroke.

OBJECTIVE: An accurate prediction tool may facilitate optimal management of patients with acute stroke from an early stage. We evaluated the association between admission modified early warning score (MEWS) and mortality in patients with acute stroke. METHOD: Data from the Anglia Stroke Clinical Network Evaluation Study (ASCNES) were analysed. We evaluated the association between admission MEWS and four outcomes; in-patient, 7-day, 30-day and 1-year mortality. Logistic regression models were used to calculate the odds of all mortality timeframes, whereas Cox proportional hazards models were used to calculate mortality at 1 year. Five univariate and multivariate models were constructed, adjusting for confounders. Patients with a moderate (2-3) or high (≥4) scores were compared to patients with a low score (0-1). RESULTS: The study population consisted of 2006 patients. A total of 1196 patients had low MEWS, 666 had moderate MEWS and 144 had a high MEWS. A high MEWS was associated with increased mortality as an in-patient (OR 4.93, 95 % CI: 2.88-8.42), at 7 days (OR 7.53, 95 % CI: 4.24-13.38), at 30 days (OR 5.74, 95 % CI: 3.38-9.76) and 1-year (HR 2.52, 95 % CI 1.88-3.39). At 1 year, model 5 had a 1.02 OR (95 % CI 0.83-1.24) with moderate MEWS and 2.52 (95 % CI 1.88-3.39) with high MEWS. CONCLUSION: Elevated MEWS on admission is a potential marker for acute-stroke mortality and may therefore be a useful risk prediction tool, able to guide clinicians attempting to prognosticate outcomes for patients with acute-stroke.

A human cell atlas of fetal chromatin accessibility.

The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells. We leveraged cell types defined by gene expression to annotate these data and cataloged hundreds of thousands of candidate regulatory elements that exhibit cell type-specific chromatin accessibility. We investigated the properties of lineage-specific transcription factors (such as POU2F1 in neurons), organ-specific specializations of broadly distributed cell types (such as blood and endothelial), and cell type-specific enrichments of complex trait heritability. These data represent a rich resource for the exploration of in vivo human gene regulation in diverse tissues and cell types.

A human cell atlas of fetal gene expression.

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

Variations in Rates of Discharges to Nursing Homes after Acute Hospitalization for Stroke and the Influence of Service Heterogeneity: An Anglia Stroke Clinical Network Evaluation Study.

Nursing home placement after stroke indicates a poor outcome but numbers placed vary between hospitals. The aim of this study is to determine whether between-hospital variations in new nursing home placements post-stroke are reliant solely on case-mix differences or whether service heterogeneity plays a role. A prospective, multi-center cohort study of acute stroke patients admitted to eight National Health Service acute hospitals within the Anglia Stroke and Heart Clinical Network between 2009 and 2011 was conducted. We modeled the association between hospitals (as a fixed-effect) and rates of new discharges to nursing homes using multiple logistic regression, adjusting for important patient risk factors. Descriptive and graphical data analyses were undertaken to explore the role of hospital characteristics. Of 1335 stroke admissions, 135 (10%) were discharged to a nursing home but rates varied considerably from 6% to 19% between hospitals. The hospital with the highest adjusted odds ratio of nursing home discharges (OR 4.26; 95% CI 1.69 to 10.73), was the only hospital that did not provide rehabilitation beds in the stroke unit. Increasing hospital size appeared to be related to an increased odds of nursing home placement, although attenuated by the number of hospital stroke admissions. Our results highlight the potential influence of hospital characteristics on this important outcome, independently of patient-level factors.

Whole-Genome and RNA Sequencing Reveal Variation and Transcriptomic Coordination in the Developing Human Prefrontal Cortex.

Gene expression levels vary across developmental stage, cell type, and region in the brain. Genomic variants also contribute to the variation in expression, and some neuropsychiatric disorder loci may exert their effects through this mechanism. To investigate these relationships, we present BrainVar, a unique resource of paired whole-genome and bulk tissue RNA sequencing from the dorsolateral prefrontal cortex of 176 individuals across prenatal and postnatal development. Here we identify common variants that alter gene expression (expression quantitative trait loci [eQTLs]) constantly across development or predominantly during prenatal or postnatal stages. Both "constant" and "temporal-predominant" eQTLs are enriched for loci associated with neuropsychiatric traits and disorders and colocalize with specific variants. Expression levels of more than 12,000 genes rise or fall in a concerted late-fetal transition, with the transitional genes enriched for cell-type-specific genes and neuropsychiatric risk loci, underscoring the importance of cataloging developmental trajectories in understanding cortical physiology and pathology.

Marijuana use differentially affects cannabinoid receptor expression in early gestational human endometrium and placenta.

Marijuana is one of the most abused drugs among pregnant women leading to maternal and fetal abnormalities. Cannabinoids are the active ingredients of marijuana, which interact with cannabinoid receptors such as CNR1 and CNR2 to activate cellular signaling pathways. Human endometrium and placenta are known to express CNR1 and CNR2 and can respond to cannabinoid signaling. In this study, we show that marijuana use significantly increases mRNA or protein expression of CNR1 and CNR2 in human endometrium from the first and early second trimester pregnancies, with minor effects on placental expression of CNRs.

Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation.

Despite frequent herpes simplex virus (HSV) reactivation, peripheral nerve destruction and sensory anesthesia are rare. We discovered that skin biopsies obtained during asymptomatic human HSV-2 reactivation exhibit a higher density of nerve fibers relative to biopsies during virological and clinical quiescence. We evaluated the effects of HSV infection on keratinocytes, the initial target of HSV replication, to better understand this observation. Keratinocytes produced IL-17c during HSV-2 reactivation, and IL-17RE, an IL-17c-specific receptor, was expressed on nerve fibers in human skin and sensory neurons in dorsal root ganglia. In ex vivo experiments, exogenous human IL-17c provided directional guidance and promoted neurite growth and branching in microfluidic devices. Exogenous murine IL-17c pretreatment reduced apoptosis in HSV-2-infected primary neurons. These results suggest that IL-17c is a neurotrophic cytokine that protects peripheral nerve systems during HSV reactivation. This mechanism could explain the lack of nerve damage from recurrent HSV infection and may provide insight to understanding and treating sensory peripheral neuropathies.

Pre-Stroke Modified Rankin Scale: Evaluation of Validity, Prognostic Accuracy, and Association with Treatment.

BACKGROUND AND PURPOSE: The modified Rankin Scale (mRS) was designed to measure poststroke recovery but is often used to describe pre-stroke disability. We sought to evaluate three aspects of pre-stroke mRS: validity as a measure of pre-stroke disability; prognostic accuracy and association of pre-stroke mRS scores, and process of care. METHODS: We used data from a large, UK clinical registry. For analysis of validity, we compared pre-stroke mRS against other markers of pre-stroke function (age, comorbidity index, care needs). For analysis of prognostic accuracy, we described univariable and multivariable models comparing pre-stroke mRS and other prognostic variables against a variety of outcomes (early and late mortality, length of stay, institutionalization, incident complications). Finally, we described association of pre-stroke mRS and components of evidence-based stroke care (early neuroimaging, admission to stroke unit, assessment of swallow). RESULTS: We analyzed data of 2,491 stroke patients. Concurrent validity analyses suggested statistically significant, but modest correlations between pre-stroke mRS and chosen variables (rho >0.40; p < 0.0001 for all). Every point increase of pre-stroke mRS was associated with poorer outcomes for our prognostic variables (unadjusted p < 0.001). This association held when corrected for other covariates. For example, pre-stroke mRS 4-5 odds ratio (OR): 6.84 (95% CI: 4.24-11.03) for 1 year mortality compared to mRS 0 in adjusted model. There was a difference between pre-stroke mRS and treatment, with higher pre-stroke mRS more likely to receive evidence-based care. CONCLUSION: Results suggest that pre-stroke mRS has some concurrent validity and is a robust predictor of prognosis. This association is not explained by the influence of pre-stroke mRS on care pathways.

Abnormal glycosylation in Joubert syndrome type 10.

BACKGROUND: The discovery of disease pathogenesis requires systematic agnostic screening of multiple homeostatic processes that may become deregulated. We illustrate this principle in the evaluation and diagnosis of a 5-year-old boy with Joubert syndrome type 10 (JBTS10). He carried the OFD1 mutation p.Gln886Lysfs*2 (NM_003611.2: c.2656del) and manifested features of Joubert syndrome. METHODS: We integrated exome sequencing, MALDI-TOF mass spectrometry analyses of plasma and cultured dermal fibroblasts glycomes, and full clinical evaluation of the proband. Analyses of cilia formation and lectin staining were performed by immunofluorescence. Measurement of cellular nucleotide sugar levels was performed with high-performance anion-exchange chromatography with pulsed amperometric detection. Statistical analyses utilized the Student's and Fisher's exact t tests. RESULTS: Glycome analyses of plasma and cultured dermal fibroblasts identified abnormal N- and O-linked glycosylation profiles. These findings replicated in two unrelated males with OFD1 mutations. Cultured fibroblasts from affected individuals had a defect in ciliogenesis. The proband's fibroblasts also had an abnormally elevated nuclear sialylation signature and increased total cellular levels of CMP-sialic acid. Ciliogenesis and each glycosylation anomaly were rescued by expression of wild-type OFD1. CONCLUSIONS: The rescue of ciliogenesis and glycosylation upon reintroduction of WT OFD1 suggests that both contribute to the pathogenesis of JBTS10.

Important factors in predicting mortality outcome from stroke: findings from the Anglia Stroke Clinical Network Evaluation Study.

Background: Although variation in stroke service provision and outcomes have been previously investigated, it is less well known what service characteristics are associated with reduced short- and medium-term mortality. Methods: Data from a prospective multicentre study (2009­12) in eight acute regional NHS trusts with a catchment population of about 2.6 million were used to examine the prognostic value of patient-related factors and service characteristics on stroke mortality outcome at 7, 30 and 365 days post stroke, and time to death within 1 year. Results: A total of 2,388 acute stroke patients (mean (standard deviation) 76.9 (12.7) years; 47.3% men, 87% ischaemic stroke) were included in the study. Among patients characteristics examined increasing age, haemorrhagic stroke, total anterior circulation stroke type, higher prestroke frailty, history of hypertension and ischaemic heart disease and admission hyperglycaemia predicted 1-year mortality. Additional inclusion of stroke service characteristics controlling for patient and service level characteristics showed varying prognostic impact of service characteristics on stroke mortality over the disease course during first year after stroke at different time points. The most consistent finding was the benefit of higher nursing levels; an increase in one trained nurses per 10 beds was associated with reductions in 30-day mortality of 11­28% (P < 0.0001) and in 1-year mortality of 8­12% (P < 0.001). Conclusions: There appears to be consistent and robust evidence of direct clinical benefit on mortality up to 1 year after acute stroke of higher numbers of trained nursing staff over and above that of other recognised mortality risk factors.