Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism.

Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA-editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here, we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA-binding-defective mutants demonstrate clear separation of function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H while, at the same time, positively regulating its antiviral activity.

Five-Year Outcomes among U.S. Bronchiectasis and NTM Research Registry Patients.

Rationale: Nontuberculous mycobacteria (NTM) are prevalent among patients with bronchiectasis. However, the long-term natural history of patients with NTM and bronchiectasis is not well described. Objectives: To assess the impact of NTM on 5-year clinical outcomes and mortality in patients with bronchiectasis. Methods: Patients in the Bronchiectasis and NTM Research Registry with ⩾5 years of follow-up were eligible. Data were collected for all-cause mortality, lung function, exacerbations, hospitalizations, and disease severity. Outcomes were compared between patients with and without NTM at baseline. Mortality was assessed using Cox proportional hazards models and the log-rank test. Measurements and Main Results: In total, 2,634 patients were included: 1,549 (58.8%) with and 1,085 (41.2%) without NTM at baseline. All-cause mortality (95% confidence interval) at Year 5 was 12.1% (10.5%, 13.7%) overall, 12.6% (10.5%, 14.8%) in patients with NTM, and 11.5% (9.0%, 13.9%) in patients without NTM. Independent predictors of 5-year mortality were baseline FEV1 percent predicted, age, hospitalization within 2 years before baseline, body mass index, and sex (all P < 0.01). The probabilities of acquiring NTM or Pseudomonas aeruginosa were approximately 4% and 3% per year, respectively. Spirometry, exacerbations, and hospitalizations were similar, regardless of NTM status, except that annual exacerbations were lower in patients with NTM (P < 0.05). Conclusions: Outcomes, including exacerbations, hospitalizations, rate of loss of lung function, and mortality rate, were similar across 5 years in patients with bronchiectasis with or without NTM.

2024 AHA/ASA Performance and Quality Measures for Spontaneous Intracerebral Hemorrhage: A Report From the American Heart Association/American Stroke Association.

The American Heart Association/American Stroke Association released a revised spontaneous intracerebral hemorrhage guideline in 2022. A working group of stroke experts reviewed this guideline and identified a subset of recommendations that were deemed suitable for creating performance measures. These 15 performance measures encompass a wide spectrum of intracerebral hemorrhage patient care, from prehospital to posthospital settings, highlighting the importance of timely interventions. The measures also include 5 quality measures and address potential challenges in data collection, with the aim of future improvements.

Measuring the Implementation Preferences of Emergency Medical Services Clinicians Using Discrete Choice Experiments.

INTRODUCTION: Prehospital research and evidence-based guidelines (EBGs) have grown in recent decades, yet there is still a paucity of prehospital implementation research. While recent studies have revealed EMS agency leadership perspectives on implementation, the important perspectives and opinions of frontline EMS clinicians regarding implementation have yet to be explored in a systematic approach. The objective of this study was to measure the preferences of EMS clinicians for the process of EBG implementation and whether current agency practices align with those preferences. METHODS: This study was a cross-sectional survey of National Registry of Emergency Medical Technicians registrants. Eligible participants were certified paramedics who were actively practicing EMS clinicians. The survey contained discrete choice experiments (DCEs) for three EBG implementation scenarios and questions about rank order preferences for various aspects of the implementation process. For the DCEs, we used multinomial logistic regression to analyze the implementation preference choices of EMS clinicians, and latent class analysis to classify respondents into groups by their preferences. RESULTS: A total of 183 respondents completed the survey. Respondents had a median age of 39 years, were 74.9% male, 89.6% White, and 93.4% of non-Hispanic ethnicity. For all three DCE scenarios, respondents were significantly more likely to choose options with hospital feedback and individual-level feedback from EMS agencies. Respondents were significantly less likely to choose options with email/online only education, no feedback from hospitals, and no EMS agency feedback to clinicians. In general, respondents' preferences favored classroom-based training over in-person simulation. For all DCE questions, most respondents (66.2%-77.1%) preferred their survey DCE choice to their agency's current implementation practices. In the rank order preferences, most participants selected "knowledge of the underlying evidence behind the change" as the most important component of the process of implementation. CONCLUSIONS: In this study of EMS clinicians' implementation preferences using DCEs, respondents preferred in-person education, feedback on hospital outcomes, and feedback on their individual performance. However, current practice at EMS agencies rarely matched those expressed EMS clinician preferences. Collectively, these results present opportunities for improving EMS implementation from the EMS clinician perspective.

Numerical model of pressure generated by elastic compression garments on a compressible human limb analogue.

OBJECTIVE: This study aimed to formulate a numerical approach (finite element modelling (FEM)) to calculate pressure values generated by compression garments on a compressible limb analogue, and to validate the numerical approach using experimental measurements. Existing models were also compared. METHOD: Experimentally measured pressure values and deformation caused by compression bands on a compressible human limb analogue were compared with values predicted using the Young-Laplace equation, a previously formulated analytical model and the FEM. RESULTS: The FEM provided greater accuracy in predicting the pressure generated by compression bands compared to existing models. The FEM also predicted deformation of the limb analogue with good agreement relative to experimental values. CONCLUSION: It was concluded that modelling the non-uniform manner in which the way a limb analogue is compressed should be incorporated into future modelling of the pressures generated by compression garments on a compressible limb analogue. DECLARATION OF INTEREST: The authors have no conflicts of interest to declare.

Prehospital Stroke Care Part 1: Emergency Medical Services and the Stroke Systems of Care.

Acute stroke care begins before hospital arrival, and several prehospital factors are critical in influencing overall patient care and poststroke outcomes. This topical review provides an overview of the state of the science on prehospital components of stroke systems of care and how emergency medical services systems may interact in the system to support acute stroke care. Topics include layperson recognition of stroke, prehospital transport strategies, networked stroke care, systems for data integration and real-time feedback, and inequities that exist within and among systems.

Prehospital Stroke Care Part 2: On-Scene Evaluation and Management by Emergency Medical Services Practitioners.

The prehospital phase is a critical component of delivering high-quality acute stroke care. This topical review discusses the current state of prehospital acute stroke screening and transport, as well as new and emerging advances in prehospital diagnosis and treatment of acute stroke. Topics include prehospital stroke screening, stroke severity screening, emerging technologies to aid in the identification and diagnosis of acute stroke in the prehospital setting, prenotification of receiving emergency departments, decision support for destination determination, and the capabilities and opportunities for prehospital stroke treatment in mobile stroke units. Further evidence-based guideline development and implementation of new technologies are critical for ongoing improvements in prehospital stroke care.

No virologic resistance to bulevirtide monotherapy detected in patients through 24 weeks treatment in phase II and III clinical trials for chronic hepatitis delta.

BACKGROUND & AIMS: Bulevirtide (BLV) is a HDV/HBV entry inhibitor that is associated with virologic response (responders, HDV-RNA undetectable or ≥2 log10 IU/ml decrease from baseline) in >50% of patients after a 24-week treatment. However, some patients only achieve a <1 log10 IU/ml decline in HDV-RNA after the 24-week treatment (non-responders). Here, we report a viral resistance analysis in participants receiving BLV monotherapy who were non-responders or experienced virologic breakthrough (VB, i.e., two consecutive increases in HDV-RNA of ≥1 log10 IU/ml from nadir or two consecutive HDV-RNA detectable results if previously undetectable) from the phase II MYR202 and phase III MYR301 study. METHODS: Deep-sequencing of the BLV-corresponding region in HBV PreS1 and of the HDV HDAg gene, as well as in vitro phenotypic testing, were performed for the participant with VB (n = 1) and non-responders (n = 20) at baseline (BL) and Week 24 (WK24). RESULTS: No amino acid exchanges associated with reduced susceptibility to BLV within the BLV-corresponding region or within HDAg were identified in isolates from any of the 21 participants at BL or at WK24. Although variants (HBV n = 1; HDV n = 13) were detected at BL in some non-responders or in the participant with VB, none were associated with reduced sensitivity to BLV in vitro. Furthermore, the same variant was detected in virologic responders. A comprehensive phenotypic analysis demonstrated that the BLV EC50 values from 116 BL samples were similar across non-responders, partial responders (HDV RNA decline ≥1 but <2 log10 IU/ml), and responders regardless of the presence of HBV and/or HDV polymorphisms. CONCLUSIONS: No amino acid substitutions associated with reduced sensitivity to BLV monotherapy were detected at BL or WK24 in non-responders or the participant with VB after 24-week BLV treatment. IMPACT AND IMPLICATIONS: This is the first study investigating the development of resistance in patients treated with BLV. Excluding resistance to BLV as an explanation for an insufficient decrease in HDV-RNA levels during BLV therapy is an important finding for patients, clinicians, and researchers. It demonstrates that BLV has a high barrier to resistance, indicating it is safe and suitable for long-term treatment, although long-term surveillance for resistance should be performed. Our results hint at other still unknown mechanisms as an explanation for the persistence of serum HDV-RNA during inhibition of viral entry. CLINICAL TRIAL NUMBERS: NCT03546621 and NCT03852719.

Locomotor, ecological and phylogenetic drivers of skeletal proportions in frogs.

Frogs exhibit complex anatomical features of the pelvis, limbs and spine, long assumed to represent specialisations for jumping. Yet frogs employ a wide range of locomotor modes, with several taxa featuring primary locomotor modes other than jumping. Using a combination of techniques (CT imaging and 3D visualization, morphometrics, phylogenetic mapping), this study aims to determine the link between skeletal anatomy and locomotor style, habitat type and phylogenetic history, shedding new light on how functional demands impact morphology. Body and limb measurements for 164 taxa from all the recognised anuran families are extracted from digitally segmented CT scans of whole frog skeletons and analysed using various statistical techniques. We find that the expansion of the sacral diapophyses is the most important variable for predicting locomotor mode, which was more closely correlated with frog morphology than either habitat type or phylogenetic relationships. Predictive analyses suggest that skeletal morphology is a useful indicator of jumping but less so for other locomotor modes, suggesting that there is a wide range of anatomical solutions to performing locomotor styles such as swimming, burrowing or walking.

Nontuberculous mycobacterial (NTM) infections in bronchiectasis patients: A retrospective US registry cohort study.

RATIONALE: Longitudinal epidemiological and clinical data are needed to improve the management of patients with bronchiectasis developing nontuberculous mycobacterial (NTM) pulmonary disease. OBJECTIVES: To describe the epidemiology, patient management, and treatment outcomes of NTM infections in patients with bronchiectasis enrolled in the United States Bronchiectasis and NTM Research Registry (US BRR). METHODS: This was a retrospective cohort study of patients with bronchiectasis and NTM infections enrolled with follow-up in the US BRR in 2008-2019. The study included patients with ≥1 positive NTM respiratory culture in the 24-month baseline period (baseline NTM cohort) and/or during the annual follow-up visits (incident NTM cohort). Incidence, prevalence, baseline patient characteristics, treatment exposure, treatment outcomes, and respiratory clinical outcomes were described in the baseline NTM cohort, incident NTM cohort, and both cohorts combined (prevalent NTM cohort). RESULTS: Between 2008 and 2019, 37.9% (1457/3840) of patients with bronchiectasis in the US BRR met the inclusion criteria for this study and were reported to have Mycobacterium avium complex (MAC) and/or Mycobacterium abscessus complex (MABSC) infections. MAC prevalence increased steadily in the US BRR during 2009-2019; incidence was relatively stable, except for a peak in 2011 followed by a slow decrease. MABSC and mixed MAC/MABSC infections were rare. Most patients with bronchiectasis and NTM infections in the registry were female, White, and aged >65 years. The antibiotics administered most commonly reflected current guidelines. In the prevalent cohort, 44.9% of MAC infections and 37.1% of MABSC infections remained untreated during follow-up, and MAC treatment was initiated with delay (>90 days after positive NTM respiratory culture) twice as frequently as promptly (≤90 days after positive NTM respiratory culture) (68.6% vs 31.4%, respectively). The median time from diagnosis to treatment was shorter for MABSC versus MAC infections (194.0 days [interquartile range (IQR) 8.0, 380.0] vs 296.0 days [IQR 35.0, 705.0], respectively). Among patients with MAC infections who completed treatment, 27.6% were classified as cured and 29.6% as treatment failure during the annual follow-up visit window. For MABSC, these proportions were 25.0% and 28.0%, respectively. CONCLUSIONS: A considerable proportion of MAC and MABSC infections were untreated or treated after initial delay/observation. MABSC infections were more likely to be treated and start treatment sooner than MAC infections. Further longitudinal studies are warranted to evaluate the monitor-with-delay approach and inform clinical guidelines.

Double asymmetric synthesis: faster reactions are more selective and a model to estimate relative rate.

The catalysed reaction of an enantiopure substrate with formation of a new chirality element may result in higher diastereoselectivity with one enantiomer of a catalyst (matched pair) than with the other (mismatched pair). The hypothesis that the matched reaction is faster was investigated using literature examples of kinetic resolution procedures that result in the formation of a new stereogenic centre. With one exception from fifteen examples, the selectivity factor (s = kfast/kslow) = kmatched/kmismatched. A model to estimate the relative rate of a fast-matched reaction vs. the corresponding slow-mismatched reaction is proposed. This model also provides insight into the basis of the selectivity displayed in the kinetic resolution procedures studied.

An expedient copper-catalysed asymmetric synthesis of γ-lactones and γ-lactams. Application to the synthesis of lucidulactone A.

The parent Josiphos ligand gave excellent ee values (95-99%) and good yields (60-97%) in the copper-catalysed asymmetric conjugate reduction of ß-aryl α,ß-unsaturated lactones and lactams with PMHS. The substrates were obtained from stereospecific copper-catalysed addition of arylboronic acids to alkynoates followed by deprotection and cyclisation. The acyclic lactam precursors also underwent reduction with good ee values (83-85%) and yields (79-95%). Application of this asymmetric reduction methodology included the synthesis of natural product lucidulactone A.

2022 Systematic Review of Evidence-Based Guidelines for Prehospital Care.

INTRODUCTION: Multiple national organizations and federal agencies have promoted the development, implementation, and evaluation of evidence-based guidelines (EBGs) for prehospital care. Previous efforts have identified opportunities to improve the quality of prehospital guidelines and highlighted the value of high-quality EBGs to inform initial certification and continued competency activities for EMS personnel. OBJECTIVES: We aimed to perform a systematic review of prehospital guidelines published from January 2018 to April 2021, evaluate guideline quality, and identify top-scoring guidelines to facilitate dissemination and educational activities for EMS personnel. METHODS: We searched the literature in Ovid Medline and EMBASE from January 2018 to April 2021, excluding guidelines identified in a prior systematic review. Publications were retained if they were relevant to prehospital care, based on organized reviews of the literature, and focused on providing recommendations for clinical care or operations. Included guidelines were appraised to identify if they met the National Academy of Medicine (NAM) criteria for high-quality guidelines and scored across the six domains of the Appraisal of Guidelines for Research and Evaluation (AGREE) II tool. RESULTS: We identified 75 guidelines addressing a variety of clinical and operational aspects of EMS medicine. About half (n = 39, 52%) addressed time/life-critical conditions and 33 (44%) contained recommendations relevant to non-clinical/operational topics. Fewer than half (n = 35, 47%) were based on systematic reviews of the literature. Nearly one-third (n = 24, 32%) met all NAM criteria for clinical practice guidelines. Only 27 (38%) guidelines scored an average of >75% across AGREE II domains, with content relevant to guideline implementation most commonly missing. CONCLUSIONS: This interval systematic review of prehospital EBGs identified many new guidelines relevant to prehospital care; more than all guidelines reported in a prior systematic review. Our review reveals important gaps in the quality of guideline development and the content in their publications, evidenced by the low proportion of guidelines meeting NAM criteria and the scores across AGREE II domains. Efforts to increase guideline dissemination, implementation, and related education may be best focused around the highest quality guidelines identified in this review.

Recommendations for Improving the Quality of Prehospital Evidence-Based Guidelines.

Evidence-based guidelines that provide recommendations for clinical care or operations are increasingly being published to inform the EMS community. The quality of evidence evaluation and methodological rigor undertaken to develop and publish these recommendations vary. This can negatively affect dissemination, education, and implementation efforts. Guideline developers and end users could be better informed by efforts across medical specialties to improve the quality of guidelines, including the use of specific criteria that have been identified within the highest quality guidelines. In this special contribution, we aim to describe the current state of published guidelines available to the EMS community informed by two recent systematic reviews of existing prehospital evidenced based guidelines (EBGs). We further aim to provide a description of key elements of EBGs, methods that can be used to assess their quality, and concrete recommendations for guideline developers to improve the quality of evidence evaluation, guideline development, and reporting. Finally, we outline six key recommendations for improving prehospital EBGs, informed by systematic reviews of prehospital guidelines performed by the Prehospital Guidelines Consortium.

Emergency Medical Services Leadership Perspectives on Implementation of Evidence-Based Guidelines: A Qualitative Study.

Introduction: Prehospital evidence-based guidelines (EBGs) are developed to optimize clinical outcomes for emergency medical services (EMS) patients. However, widespread implementation of EBGs is often inconsistent. Therefore, this study aimed to assess the baseline knowledge and practices of EMS leaders related to EBG implementation.Methods: This was a qualitative study using focus groups to assess prehospital implementation awareness and knowledge. Participants were EMS EBG authors, EMS medical directors, and EMS professional organization leaders. Focus groups were held via video conference, audio recorded, and transcribed. Thematic coding used domains and constructs of the Consolidated Framework for Implementation Research (CFIR).Results: Six focus groups were conducted with a total of 18 participants. A total of 1,044 codes were analyzed. "Process" was the CFIR domain with the most codes (n = 350, 33.5%), followed by the "inner setting" (the EMS agency; n = 250, 23.9%), "characteristics of the intervention" (n = 203, 19.4%), "outer setting" (the health care system and community the EMS agency serves, and the broader national EMS professional context; n = 141, 13.5%), and "characteristics of individuals" (n = 100, 9.6%). The ten most frequent constructs across all domains were: reflecting and evaluating, executing, cosmopolitanism, planning, external policy and incentives, design quality and packaging, learning climate, culture, complexity, and other personal attributes.Conclusion: EMS leadership and stakeholder views on EBG implementation identified dominant themes related to the process of implementation and the culture and learning/implementation climate of EMS agencies. Opinions were mixed on the utility of the CFIR as a potential guide for EMS implementation. Further work is required to gain the frontline EMS clinician perspective on implementation and tie key themes to quantitative prehospital implementation outcomes.

Outcomes of Patients Receiving Thrombolysis in a Mobile Stroke Unit: A 4-Year Retrospective, Observational, Single-Center Study.

BACKGROUND AND PURPOSE: Patients with acute ischemic stroke (AIS) eligible for thrombolysis benefit when thrombolysis is administered quickly, and mobile stroke units (MSU) can facilitate timely thrombolysis. We sought to compare time metrics and clinical outcomes of AIS patients receiving thrombolysis in an MSU compared with patients arriving via local emergency medical services (EMS). METHODS: We performed a retrospective, non-randomized, cohort study comparing MSU-arriving to EMS-arriving AIS patients from January 20, 2017 through November 30, 2020. The primary outcome was rate of return to baseline functional status as measured by the modified Rankin Score (mRS) 90 days after thrombolysis. Secondary outcomes included evaluation and treatment intervals from last known well, treatment rate in the first hour of symptoms, hospital length of stay, and mortality. Chi square and Student's t-test were used to compare groups. RESULTS: Of 1752 total patients with prehospital suspected stroke, 975 (55.7%) were transported via MSU, of whom 431 (44.2%) were diagnosed with stroke, including 368 (85.4%) with AIS, and 69 AIS patients (18.8%) received thrombolysis. Of 777 (44.3%) EMS-arriving patients, 373 (48%) were diagnosed with stroke, including 305 (81.8%) with AIS, and 74 (24.3%) received thrombolysis. Though not statistically significant, point estimates of the proportion of AIS patients treated with thrombolysis returning to baseline functional status were more commonly observed for MSU than for EMS transports when the baseline mRS was 0-2 (45.8% vs 33.3%), 0-3 (41.9% vs 33.3%), and 4-5 (71.4% vs 20.0%). MSU patients were more likely to receive thrombolysis in the first 60 minutes of symptom onset (31.9% vs 12.2%, p = 0.006). Overall mortality rates regardless of baseline mRS were similar between groups. CONCLUSIONS: AIS patients received thrombolysis faster in the MSU compared with EMS and more frequently within 60 minutes of stroke onset. Point estimates for 90-day clinical outcomes of AIS patients treated with thrombolysis favored MSU without a statistically significant difference.

Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria.

OBJECTIVES: This study aimed to elucidate mechanistic explanation(s) for compositional changes to enteric microbiota by determining the impacts of continuous nicotine/cotinine exposure on representative gastrointestinal bacteria and how these alterations impact innate immune cell plasticity. METHODS: In vitro cultures of the gastrointestinal bacteria (Bacteroides fragilis 25285, Prevotella bryantii B14, and Acetoanaerobium sticklandii SR) were continuously exposed to nicotine or cotinine. Supernatant samples were collected for fermentation acid analysis. Vesicles were collected and analyzed for physiological changes in number, size, and total protein cargo. Cultured macrophages were stimulated to a tolerogenic phenotype, exposed to control or altered (nicotine or cotinine - exposed) vesicles, and inflammatory plasticity assessed via inflammatory cytokine production. RESULTS: Nicotine/cotinine exposure differentially affected metabolism of all bacteria tested in a Gram (nicotine) and concentration-dependent (cotinine) manner. Physiological studies demonstrated changes in vesiculation number and protein cargo following nicotine/cotinine exposures. Continuous exposure to 1 µM nicotine and 10 µM cotinine concentrations reduced total protein cargo of Gram (-) - 25285 and B14 vesicles, while cotinine generally increased total protein in Gram (+) - SR vesicles. We found that theses physiological changes to the vesicles of 25285 and SR formed under nicotine and cotinine, respectively, challenged the plasticity of tolerogenic macrophages. Tolerogenic macrophages exposed to vesicles from 1 µM nicotine, and 5 or 10 µΜ cotinine cultures produced significantly less IL-12p70, TNFα, or KC/GRO, regardless of macrophage exposure to nicotine/cotinine. CONCLUSIONS: Nicotine/cotinine exposure differentially alters bacterial metabolism and vesicle physiology, ultimately impacting the inflammatory response of tolerogenic macrophages.

Emergency Medical Service Time Intervals for Patients With Suspected Stroke in the United States.

BACKGROUND: Optimal care for patients with stroke relies on timely recognition and rapid transport to appropriate treatment, often by emergency medical services (EMS). Our primary objective was to describe EMS time intervals for patients with suspected stroke in the United States. We also sought to quantify the variation in EMS time intervals by geographic location and urbanicity. METHODS: We conducted a cross-sectional evaluation of EMS 9-1-1 activations (ie, calls for service) included in the 2018 and 2019 National EMS Information System datasets. We included ground or air EMS activations for a 9-1-1 scene response where a patient aged ≥18 years with suspected stroke was treated and transported by EMS. Time intervals for dispatch, response, scene, transport, and total prehospital time (ie, from dispatch to hospital arrival) were calculated, stratified by ground and air transport type. RESULTS: A total of 410 187 activations for suspected stroke were included, of which 98% were a ground transport. The median total prehospital time for ground transports was 35 minutes (interquartile range, 27-45, 90th percentile 58). Median total prehospital time for air transports was substantially longer at 56 minutes (interquartile range, 43-70, 90th percentile 86). Times varied by Census division and urbanicity with the shortest ground total prehospital times in the East North Central division and urban areas and longest times in the East South Central and rural and frontier areas. CONCLUSIONS: Timely EMS response and transport is critical for optimizing care of patients with suspected stroke. Using a large, national dataset of EMS activations, we found variations by geographic location and urbanicity in total prehospital time for ground and air EMS activations for patients with stroke.

ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes.

Ca2+ is a major second messenger involved in cellular and subcellular signaling in a wide range of cells, including astrocytes, which use calcium ions to communicate with other cells in the brain. Even though a variety of genetically encoded Ca2+ indicators have been developed to study astrocyte calcium signaling, understanding the dynamics of endoplasmic reticulum calcium signaling is greatly limited by the currently available tools. To address this, we developed an endoplasmic reticulum-targeted calcium indicator, ER-GCaMP6f, which is anchored to the cytosolic side of the organelle and measures signaling that occurs in close proximity to the endoplasmic reticulum of astrocytes. Using a combination of confocal and super-resolution microscopy techniques, we demonstrate the localization of the indicator in the endoplasmic reticulum in both cell soma and processes of astrocytes. Combining ER-GCaMP6f with total internal reflection fluorescence microscopy, we show that Ca2+ fluctuations in small astrocytic processes can be detected, which are otherwise not observable with existing indicators and standard wide-field and confocal techniques. We also compared the ER-GCaMP6f indicator against currently used plasma membrane-tethered and cytosolic GCaMP6f indicators. ER-GCaMP6f identifies dynamics in calcium signaling of endoplasmic reticulum resident receptors that are missed by plasma membrane-anchored indicators. We also generated an adeno-associated virus (AAV5) and demonstrate that ER-GCaMP6f can be expressed in vivo and by measured calcium activity in brain slices. ER-GCaMP6f provides a powerful tool to study calcium signaling in close proximity to the endoplasmic reticulum in astrocyte cell soma and processes both in culture and in brain slices.