A multi-biomarker micronucleus assay using imaging flow cytometry.

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against É£H2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer's software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, É£H2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay's ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.

A protocol for the ERICA-ARREST feasibility study of Emergency Resuscitative Endovascular Balloon occlusion of the Aorta in Out-of-Hospital Cardiac Arrest.

Background: Fewer than one in ten out-of-hospital cardiac arrest (OHCA) patients survive to hospital discharge in the UK. For prehospital teams to improve outcomes in patients who remain in refractory OHCA despite advanced life support (ALS); novel strategies that increase the likelihood of return of spontaneous circulation, whilst preserving cerebral circulation, should be investigated. Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) has been shown to improve coronary and cerebral perfusion during cardiopulmonary resuscitation. Early, prehospital initiation of REBOA may improve outcomes in patients who do not respond to standard ALS. However, there are significant clinical, technical, and logistical challenges with rapidly delivering prehospital REBOA in OHCA; and the feasibility of delivering this intervention in the UK urban-rural setting has not been evaluated. Methods: The Emergency Resuscitative Endovascular Balloon Occlusion of the Aorta in Out-of-Hospital Cardiac Arrest (ERICA-ARREST) study is a prospective, single-arm, interventional feasibility study. The trial will enrol 20 adult patients with non-traumatic OHCA. The primary objective is to assess the feasibility of performing Zone I (supra-coeliac) aortic occlusion in patients who remain in OHCA despite standard ALS in the UK prehospital setting. The trial's secondary objectives are to describe the hemodynamic and physiological responses to aortic occlusion; to report key time intervals; and to document adverse events when performing REBOA in this context. Discussion: Using compressed geography, and targeted dispatch, alongside a well-established femoral arterial access programme, the ERICA-ARREST study will assess the feasibility of deploying REBOA in OHCA in a mixed UK urban and rural setting.Trial registration.ClinicalTrials.gov (NCT06071910), registration date October 10, 2023, https://classic.clinicaltrials.gov/ct2/show/NCT06071910.

Increased survival for resuscitated Utstein-comparator group patients conveyed directly to cardiac arrest centres in a large rural and suburban population in England.

AIM: The cohort of patients in which cardiac arrest centres (CAC) in rural and suburban populations confer the greatest survival benefit remains unclear. The aim of this study was to assess whether the transfer of resuscitated Utstein-comparator out-of-hospital cardiac arrest (OHCA) patients direct to a CAC was associated with improved survival to hospital discharge compared to patients conveyed to non-specialist centres. METHODS: A consecutive sample of adult (≥18 years old) Utstein-comparator patients (witnessed collapse and initial shockable rhythm) were included from the East of England Ambulance Service NHS Trust Utstein resuscitation registry; 2018-2022. Logistic regression was used to compare survival to discharge in patients transported to CACs compared with patients transported to non-specialist centres. RESULTS: During the study period, resuscitation was attempted in 18,276 OHCA patients. N = 2448 (13.4%) met the Utstein-comparator definition and 1151 patients were included in the final analysis; per protocol. Survival was greater for patients conveyed directly to a CAC (n = 768, 60.7%) compared to non-specialist centres (n = 383, 47.3%); adjusted OR 1.44 (95%CI 1.07-1.94),p = 0.017. Amongst the centres analysed in this study, there was significant inter-hospital variability in survival between CACs (p = 0.017). There was no association between patient volume and survival (p = 0.850). CONCLUSION: Direct transport to a cardiac arrest centre was associated with a 44% increase in the odds of survival compared to conveyance to a non-specialist centre for resuscitated adult patients presenting with witnessed collapse and initial shockable OHCA rhythm.

The association between prehospital post-return of spontaneous circulation core temperature and survival after out-of-hospital cardiac arrest.

BACKGROUND AND IMPORTANCE: Following the return of spontaneous circulation (ROSC) after out-of-hospital cardiac arrest (OHCA), a low body temperature on arrival at the hospital and on admission to the ICU is reportedly associated with increased mortality. Whether this association exists in the prehospital setting, however, is unknown. OBJECTIVE: The objective of this study was to investigate whether the initial, prehospital core temperature measured post-ROSC is independently associated with survival to hospital discharge in adult patients following OHCA. DESIGN, SETTING AND PARTICIPANTS: This retrospective observational study was conducted at East Anglian Air Ambulance, a physician-paramedic staffed Helicopter Emergency Medical Service in the East of England, UK. Adult OHCA patients attended by East Anglian Air Ambulance from 1 February 2015 to 30 June 2023, who had post-ROSC oesophageal temperature measurements were included. OUTCOME MEASURE AND ANALYSIS: The primary outcome measure was survival to hospital discharge. Core temperature was defined as the first oesophageal temperature recorded following ROSC. Multivariable logistic regression evaluated the adjusted association between core temperature and survival to hospital discharge. MAIN RESULTS: Resuscitation was attempted in 3990 OHCA patients during the study period, of which 552 patients were included in the final analysis. The mean age was 61 years, and 402 (72.8%) patients were male. Among them, 194 (35.1%) survived to hospital discharge. The mean core temperature was lower in nonsurvivors compared with those who survived hospital discharge; 34.6 and 35.2 °C, respectively (mean difference, -0.66; 95% CI, -0.87 to -0.44; P < 0.001). The adjusted odds ratio for survival was 1.41 (95% CI, 1.09-1.83; P = 0.01) for every 1.0 °C increase in core temperature between 32.5 and 36.9 °C. CONCLUSION: In adult patients with ROSC following OHCA, early prehospital core temperature is independently associated with survival to hospital discharge.

In the Murine and Bovine Maternal Mammary Gland Signal Transducer and Activator of Transcription 3 is Activated in Clusters of Epithelial Cells around the Day of Birth.

Signal transducers and activators of transcription (STAT) proteins regulate mammary development. Here we investigate the expression of phosphorylated STAT3 (pSTAT3) in the mouse and cow around the day of birth. We present localised colocation analysis, applicable to other mammary studies requiring identification of spatially congregated events. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. Arginase-1 expressing cells, consistent with macrophages, exhibit distinct clustering within the periparturient mammary gland. These findings represent a new facet of mammary STAT3 biology, and point to the presence of mammary sub-microenvironments.

Implementation of advanced vascular access, physiological monitoring and goal-directed resuscitation during OHCA in a helicopter emergency medical service.

Outcomes after out-of-hospital cardiac arrest (OHCA) remain poor in the UK. In order to increase the chances of successful resuscitation, international society guidelines on cardiopulmonary resuscitation quality have recommended titration of chest compression parameters and vasopressor administration to arterial diastolic blood pressure if invasive catheters are in situ at the time of cardiac arrest. However, prehospital initiation of arterial and central venous catheterisation is seldom undertaken due to the risks and significant technical challenges in the context of ongoing resuscitation in this environment. In 2019, a dedicated programme was started at East Anglian Air Ambulance (EAAA) to enable the safe introduction of contemporary emergency vascular access devices, in order to improve physiological monitoring intra-arrest and deliver nuanced, goal-directed resuscitation in OHCA patients. This programme was entitled Specialist Percutaneous Emergency Aortic Resuscitation (SPEAR). This article details the EAAA SPEAR technique; and the development, implementation and governance of this novel endovascular strategy in our UK physician-paramedic staffed helicopter emergency medical service.

Prehospital invasive arterial blood pressure monitoring in critically ill patients attended by a UK helicopter emergency medical service- a retrospective observational review of practice.

BACKGROUND: Accurate haemodynamic monitoring in the prehospital setting is essential. Non-invasive blood pressure measurement is susceptible to vibration and motion artefact, especially at extremes of hypotension and hypertension: invasive arterial blood pressure (IABP) monitoring is a potential solution. This study describes the largest series to date of cases of IABP monitoring being initiated prehospital. METHODS: This retrospective observational study was conducted at East Anglian Air Ambulance (EAAA), a UK helicopter emergency medical service (HEMS). It included all patients attended by EAAA who underwent arterial catheterisation and initiation of IABP monitoring between 1st February 2015 and 20th April 2023. The following data were retrieved for all patients: sex; age; aetiology (medical cardiac arrest, other medical emergency, trauma); site of arterial cannulation; operator role (doctor/paramedic); time of insertion and, where applicable, times of pre-hospital emergency anaesthesia, and return of spontaneous circulation following cardiac arrest. Descriptive analyses were performed to characterise the sample. RESULTS: 13,556 patients were attended: IABP monitoring was initiated in 1083 (8.0%) cases, with a median age 59 years, of which 70.8% were male. 546 cases were of medical cardiac arrest: in 22.4% of these IABP monitoring was initiated during cardiopulmonary resuscitation. 322 were trauma cases, and the remaining 215 were medical emergencies. The patients were critically unwell: 981 required intubation, of which 789 underwent prehospital emergency anaesthesia; 609 received vasoactive medication. In 424 cases IABP monitoring was instituted en route to hospital. CONCLUSION: This study describes over 1000 cases of prehospital arterial catheterisation and IABP monitoring in a UK HEMS system and has demonstrated feasibility at scale. The high-fidelity of invasive arterial blood pressure monitoring with the additional benefit of arterial blood gas analysis presents an attractive translation of in-hospital critical care to the prehospital setting.

Bronchogenic cyst: a rare cause of palpitations and atrial fibrillation.

Bronchogenic cysts are rare congenital lesions found primarily in the mediastinum. Most patients are asymptomatic and can be treated with minimally invasive resection. We present a case of a middle-aged patient who presented to a district general hospital with palpitations and shortness of breath. She underwent a computerised tomographic pulmonary angiogram that showed a likely bronchogenic cyst and was subsequently transferred to our hospital. She developed atrial fibrillation during admission requiring therapy with beta-blockers and digoxin. Cardiac MRI revealed a large cyst posterior to the left atrium, a moderate circumferential pericardial effusion and bilateral pleural effusions. There was significant left atrial compression. The patient underwent surgical removal of the cyst and was discharged. She returned to the hospital within a week with palpitations and was treated with intravenous antibiotics for sepsis. She was discharged a week later and remained clinically stable.

OPTIMAL: An OPTimized Imaging Mass cytometry AnaLysis framework for benchmarking segmentation and data exploration.

Analysis of imaging mass cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single-cell segmentation and suboptimal approaches for data visualization and exploration. This can lead to inaccurate identification of cell phenotypes, states, or spatial relationships compared to reference data from single-cell suspension technologies. To this end we have developed the "OPTimized Imaging Mass cytometry AnaLysis (OPTIMAL)" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualization/clustering, and spatial neighborhood analysis. Using a panel of 27 metal-tagged antibodies recognizing well-characterized phenotypic and functional markers to stain the same Formalin-Fixed Paraffin Embedded (FFPE) human tonsil sample tissue microarray over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, 5 different dimensionality reduction algorithms, and 2 clustering methods. Finally, we assessed the optimal approach for performing neighborhood analysis. We found that single-cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bivariate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximized the statistical separation between negative and positive signal distributions and a simple Z-score normalization step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing phenograph in terms of cell type identification. We also found that neighborhood analysis was influenced by the method used for finding neighboring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly, OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output and allows for single-cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19.

BACKGROUND: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. METHODS: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. FINDINGS: Forty patients (32M:8F, age: 22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. INTERPRETATION: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. FUNDING: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.

Selective aortic arch perfusion: a first-in-human observational cadaveric study.

BACKGROUND: Selective aortic arch perfusion (SAAP) is a novel endovascular technique that combines thoracic aortic occlusion with extracorporeal perfusion of the brain and heart. SAAP may have a role in both haemorrhagic shock and in cardiac arrest due to coronary ischaemia. Despite promising animal studies, no data is available that describes SAAP in humans. The primary aim of this study was to assess the feasibility of selective aortic arch perfusion in humans. The secondary aim of the study was to assess the feasibility of achieving direct coronary artery access via the SAAP catheter as a potential conduit for salvage percutaneous coronary intervention. METHODS: Using perfused human cadavers, a prototype SAAP catheter was inserted into the descending aorta under fluoroscopic guidance via a standard femoral percutaneous access device. The catheter balloon was inflated and the aortic arch perfused with radio-opaque contrast. The coronary arteries were cannulated through the SAAP catheter. RESULTS: The procedure was conducted four times. During the first two trials the SAAP catheter was passed rapidly and without incident to the intended descending aortic landing zone and aortic arch perfusion was successfully delivered via the device. The SAAP catheter balloon failed on the third trial. On the fourth trial the left coronary system was cannulated using a 5Fr coronary guiding catheter through the central SAAP catheter lumen. CONCLUSIONS: For the first time using a perfused cadaveric model we have demonstrated that a SAAP catheter can be easily and safely inserted and SAAP can be achieved using conventional endovascular techniques. The SAAP catheter allowed successful access to the proximal aorta and permitted retrograde perfusion of the coronary and cerebral circulation.

Deductive automated pollen classification in environmental samples via exploratory deep learning and imaging flow cytometry.

Pollen and tracheophyte spores are ubiquitous environmental indicators at local and global scales. Palynology is typically performed manually by microscopic analysis; a specialised and time-consuming task limited in taxonomical precision and sampling frequency, therefore restricting data quality used to inform climate change and pollen forecasting models. We build on the growing work using AI (artificial intelligence) for automated pollen classification to design a flexible network that can deal with the uncertainty of broad-scale environmental applications. We combined imaging flow cytometry with Guided Deep Learning to identify and accurately categorise pollen in environmental samples; here, pollen grains captured within c. 5500 Cal yr BP old lake sediments. Our network discriminates not only pollen included in training libraries to the species level but, depending on the sample, can classify previously unseen pollen to the likely phylogenetic order, family and even genus. Our approach offers valuable insights into the development of a widely transferable, rapid and accurate exploratory tool for pollen classification in 'real-world' environmental samples with improved accuracy over pure deep learning techniques. This work has the potential to revolutionise many aspects of palynology, allowing a more detailed spatial and temporal understanding of pollen in the environment with improved taxonomical resolution.

Survivorship With Incrementally Faster Times to Primary Percutaneous Coronary Intervention (SWIFT-PPCI): A Systematic Review and Meta-Analysis.

In the United States, there are approximately 750,000 ST-elevation myocardial infarction cases each year. Streamlined care and rapid delivery for primary percutaneous coronary intervention (PPCI) is associated with improved survival. This systematic review and meta-analysis aimed to generate a practical estimate of mortality savings for every notional 30-minute decrease in the time to achieving PPCI. Included studies were those that provided a specific absolute risk reduction for a specific reduction in pre-PPCI time. The eligible studies evaluated the survival benefit from pre-PPCI time savings measured in any interval ending with PPCI and commencing with objectively recorded timing, such as initial emergency call, first medical contact, or hospital arrival. Study planning called for the reporting of data as individual study results, with a pooled effect estimate of relative risk calculated with random-effects meta-analysis. A total of 1,088 records were eligible for review; 52 were reviewed in full text, with 4 studies (total patient n = 235,814, overall mortality 4.7% to 7.8%) included in the final analysis. All 4 studies reported significant time-related survival benefit over the study focus window of 60 to 180 minutes pre-PPCI. The number of lives saved per 100 cases for each 30-minute pre-PPCI time savings ranged from 0.8 to 1.9. The overall effect estimate generated was 0.753 (95% confidence interval 0.712 to 0.796), with acceptable heterogeneity (I2 = 36%). In conclusion, a pooled effect calculation estimated a 24.7% relative risk reduction for each 30 minutes of time savings. For cases that underwent PPCI within 60 to 180 minutes of initial presentation with known baseline mortality risk, the time savings in 30-minute epochs can be leveraged to estimate a specific number of lives saved; this may be useful for those involved in the organization of medical care who make systemwide plans and individual patient triage decisions.

Pericardial Fluid Analysis in Diagnosis and Prognosis of Patients Who Underwent Pericardiocentesis.

In this study, we aimed to examine the diagnostic yield of pericardial fluid biochemistry and cytology and their prognostic significance in patients with percutaneously drained pericardial effusions, with and without malignancy. This is a single-center, retrospective study of patients who underwent pericardiocentesis between 2010 and 2020. Data were extracted from electronic patient records, including procedural information, underlying diagnosis, and laboratory results. Patients were grouped into those with and without underlying malignancy. A Cox proportional hazards model was used to analyze the association of variables with mortality. The study included 179 patients; 50% had an underlying malignancy. There were no significant differences in pericardial fluid protein and lactate dehydrogenase between the 2 groups. Diagnostic yield from pericardial fluid analysis was greater in the malignant group (32% vs 11%, p = 0.002); 72% of newly diagnosed malignancies had positive fluid cytology. The 1-year survival was 86% and 33% in nonmalignant and malignant groups, respectively (p <0.001). Of 17 patients who died within the nonmalignant group, idiopathic effusions were the largest group (n = 6). In malignancy, lower pericardial fluid protein and higher serum C-reactive protein were associated with increased risk of mortality. In conclusion, pericardial fluid biochemistry has limited value in determining the etiology of pericardial effusions; fluid cytology is the most important diagnostic test. Mortality in malignant pericardial effusions may be associated with lower pericardial fluid protein levels and a higher serum C-reactive protein. Nonmalignant pericardial effusions do not have a benign prognosis and close follow-up is required.

Label-free cell segmentation of diverse lymphoid tissues in 2D and 3D.

Unlocking and quantifying fundamental biological processes through tissue microscopy requires accurate, in situ segmentation of all cells imaged. Currently, achieving this is complex and requires exogenous fluorescent labels that occupy significant spectral bandwidth, increasing the duration and complexity of imaging experiments while limiting the number of channels remaining to address the study's objectives. We demonstrate that the excitation light reflected during routine confocal microscopy contains sufficient information to achieve accurate, label-free cell segmentation in 2D and 3D. This is achieved using a simple convolutional neural network trained to predict the probability that reflected light pixels belong to either nucleus, cytoskeleton, or background classifications. We demonstrate the approach across diverse lymphoid tissues and provide video tutorials demonstrating deployment in Python and MATLAB or via standalone software for Windows.

Spatial statistics is a comprehensive tool for quantifying cell neighbor relationships and biological processes via tissue image analysis.

Automated microscopy and computational image analysis has transformed cell biology, providing quantitative, spatially resolved information on cells and their constituent molecules from the sub-micron to the whole-organ scale. Here we explore the application of spatial statistics to the cellular relationships within tissue microscopy data and discuss how spatial statistics offers cytometry a powerful yet underused mathematical tool set for which the required data are readily captured using standard protocols and microscopy equipment. We also highlight the often-overlooked need to carefully consider the structural heterogeneity of tissues in terms of the applicability of different statistical measures and their accuracy and demonstrate how spatial analyses offer a great deal more than just basic quantification of biological variance. Ultimately, we highlight how statistical modeling can help reveal the hierarchical spatial processes that connect the properties of individual cells to the establishment of biological function.

Dinaciclib as an effective pan-cyclin dependent kinase inhibitor in platinum resistant ovarian cancer.

Background: Ovarian cancer (OC) is amongst the most lethal of common cancers in women. Lacking in specific symptoms in the early stages, OC is predominantly diagnosed late when the disease has undergone metastatic spread and chemotherapy is relied on to prolong life. Platinum-based therapies are preferred and although many tumors respond initially, the emergence of platinum-resistance occurs in the majority of cases after which prognosis is very poor. Upregulation of DNA damage pathways is a common feature of platinum resistance in OC with cyclin dependent kinases (CDKs) serving as key regulators of this process and suggesting that CDK inhibitors (CDKis) could be effective tools in the treatment of platinum resistant and refractory OC. Aim: The aim of this study was to evaluate the efficacy of CDKis in platinum resistant OC models and serve as a predictor of potential clinical utility. Methods: The efficacy of CDKi, dinaciclib, was determined in wildtype and platinum resistant cell line pairs representing different OC subtypes. In addition, dinaciclib was evaluated in primary cells isolated from platinum-sensitive and platinum-refractory tumors to increase the clinical relevance of the study. Results and conclusions: Dinaciclib proved highly efficacious in OC cell lines and primary cells, which were over a thousand-fold more sensitive to the CDKi than to cisplatin. Furthermore, cisplatin resistance in these cells did not influence sensitivity to dinaciclib and the two drugs combined additively in both platinum-sensitive and platinum-resistant OC cells suggesting a potential role for pan-CDKis (CDKis targeting multiple CDKs), such as dinaciclib, in the treatment of advanced and platinum-resistant OC.

Out-of-hospital cardiac arrest complicated by hyperthermia.

Background: The aims of this study were to establish epidemiology, clinical management and outcomes in cases of adult out-of-hospital cardiac arrest complicated by hyperthermia attended by the London Ambulance Service NHS Trust between January 2018 and December 2019. Where evidence is available in relation to this sub-set of cardiac arrest patients it is generally limited to small case series and we therefore we sought to improve knowledge and target therapeutic interventions. Methods and results: Retrospective analysis of 253 cases was undertaken following abstraction from an established cardiac arrest database. Age ranged from 18-99 years with a median of 72 years (IQR 28) and 53.4% (n = 135) of patients were female. Overall thirty-day mortality was 94.5% (n = 239), with 48.2% (n = 122) of patients recognised life extinct in the out-of-hospital phase following termination of resuscitation. No significant differences in clinical characteristics stratified according to temperature group were identified. The presumed aetiology was infective in 62.8% (n = 159) of patients, and due to drug ingestion or heat illness in 7.5% (n = 19) and 2% (n = 5) respectively. In the remaining cases (27.7%, n = 70) it was not possible to determine the likely cause of the arrest. Conclusions: Previous research relating to cardiac arrest complicated by hyperthermia is limited to case reports and small case series, suggesting that the current study represents the most comprehensive analysis of this sub-group of out-of-hospital cardiac arrest patients currently available. Most cases were associated with evidence of infection compared with drug related aetiologies and heat illness. Where indicated, cooling was applied infrequently using inconsistent methods.

A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle.

We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.