Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells.

ABSTRACT: Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

Assessing mid-career female physician burnout in the military health system: finding joy in practice after the COVID-19 pandemic.

BACKGROUND: Rates of physician burnout increased during the COVID-19 pandemic and are expected to continue to rise. Mid-career physicians, female physicians, and military physicians have all been identified as potentially vulnerable populations to experience burnout. We examine factors associated with physician burnout among this intersectional group through a qualitative key informant interview study. METHODS: We developed a semi-structured interview guide using the Institute for Healthcare Improvement's Improving Joy in Work Framework and recruited military, mid-career female physicians who worked in the Military Health System(MHS) during the COVID-19 pandemic, (March 2020 -December 2021). Notes were collated and deductive thematic analysis was conducted. RESULTS: We interviewed a total of 22 mid-career female physician participants. Participants were between 30 and 44 years of age and 7 were mothers during the pandemic. Most were White and served in the Army. All participants discussed the importance of building rapport and having a good relationship with coworkers. All participants also described their discontentment with the new MHS GENESIS electronic health record system. An emerging theme was military pride as most participants were proud to serve in and support the military population. Additionally, participants discussed the negative impact from not feeling supported and not feeling heard by leadership. CONCLUSIONS: Much like providers in other health systems during the pandemic, MHS physicians experienced burnout. This study allowed us to gather key insights to improve policies for active duty service mid-career female military physicians. Provider inclusion, autonomy, and work culture play critical roles in future systems improvement and workforce retention.

Healthcare professional views about a prehospital redirection pathway for stroke thrombectomy: a multiphase deductive qualitative study.

BACKGROUND: Mechanical thrombectomy for stroke is highly effective but time-critical. Delays are common because many patients require transfer between local hospitals and regional centres. A two-stage prehospital redirection pathway consisting of a simple ambulance screen followed by regional centre assessment to select patients for direct admission could optimise access. However, implementation might be challenged by the limited number of thrombectomy providers, a lack of prehospital diagnostic tests for selecting patients and whether finite resources can accommodate longer ambulance journeys plus greater central admissions. We undertook a three-phase, multiregional, qualitative study to obtain health professional views on the acceptability and feasibility of a new pathway. METHODS: Online focus groups/semistructured interviews were undertaken designed to capture important contextual influences. We purposively sampled NHS staff in four regions of England. Anonymised interview transcripts underwent deductive thematic analysis guided by the NASSS (Non-adoption, Abandonment and Challenges to Scale-up, Spread and Sustainability, Implementation) Implementation Science framework. RESULTS: Twenty-eight staff participated in 4 focus groups, 2 group interviews and 18 individual interviews across 4 Ambulance Trusts, 5 Hospital Trusts and 3 Integrated Stroke Delivery Networks (ISDNs). Five deductive themes were identified: (1) (suspected) stroke as a condition, (2) the pathway change, (3) the value participants placed on the proposed pathway, (4) the possible impact on NHS organisations/adopter systems and (5) the wider healthcare context. Participants perceived suspected stroke as a complex scenario. Most viewed the proposed new thrombectomy pathway as beneficial but potentially challenging to implement. Organisational concerns included staff shortages, increased workflow and bed capacity. Participants also reported wider socioeconomic issues impacting on their services contributing to concerns around the future implementation. CONCLUSIONS: Positive views from health professionals were expressed about the concept of a proposed pathway while raising key content and implementation challenges and useful 'real-world' issues for consideration.

A qualitative exploration of ambulance clinician behaviour and decision making to identify factors influencing on-scene times for suspected stroke patients in North East England.

Aims/objectives: Ambulance clinician assessment of suspected stroke patients aims to provide rapid access to specialist care, however regional and national data show increasing pre-hospital times. This study explored paramedic views about factors contributing to on-scene time (OST) for suspected stroke patients, with a view to identifying opportunities for future interventions, to reduce OST. Methods: Views of paramedics from one regional service on factors influencing OST were explored using a qualitative approach. Semi-structured interviews with volunteers were recorded, transcribed and analysed using thematic analysis. Results: Interviews were conducted with 13 paramedics between August and November 2021. Five interlinked themes were identified and described a range of factors influencing OST: 'Initial assessment and sources of information' describes how clinicians make assessments based on initial presentation, influenced by pre-arrival information from ambulance control and family members / bystanders at the scene, and how this influences OST.'Suitability for treatment and interventions' describes how paramedics consider actions such as the face, arms, speech test, cannulation, electrocardiograms and neurological assessments while recognising that pre-hospital interventions for suspected stroke are limited.'The environment' describes the influence of incident setting on OST, including the overall process needed to transport the patient to appropriate care.'Hospital interactions' describes how interactions with hospital staff influenced paramedic actions and OST.'Changing practice' describes the influence of experience and interaction with hospital staff leading to changes in paramedic practice over time. Conclusion: This study provides insight into how UK paramedics spend time on scene with stroke patients. Multiple factors influencing OST were identified which signpost opportunities for interventions designed to reduce OST. Standardising on-scene assessments for stroke patients, refining communication processes between ambulance services and hospital stroke services and increasing availability of stroke continuing professional development for paramedics were all identified as potential targets for improving OST.

Stroke survivor views on ambulance redirection as a strategy to increase access to thrombectomy in England.

Introduction: Intravenous thrombolysis and mechanical thrombectomy are effective time-sensitive treatments for selected cases of acute ischaemic stroke. While thrombolysis is widely available, thrombectomy can only be provided at facilities with the necessary equipment and interventionists. Suitable patients admitted to other hospitals require secondary transfer, causing delays to treatment. Pre-hospital ambulance redirection to thrombectomy facilities may improve access but treatment eligibility cannot be confirmed pre-hospital. Some redirected patients would travel further and be displaced without receiving thrombectomy. This study aimed to elicit stroke survivor and carer/relative views about the possible consequences of introducing a conceptual, idealised ambulance redirection pathway. Methods: Focus groups were undertaken using a topic guide describing four hypothetical ambulance redirection scenarios and their possible consequences: earlier treatment with thrombectomy; delayed diagnosis of non-stroke 'mimic' conditions; delayed thrombolysis treatment; and delayed diagnosis of haemorrhagic stroke. Meetings were audio recorded, transcribed verbatim and data analysed thematically using emergent coding. Results: Fifteen stroke survivors and carers/relatives participated in three focus groups. There was wide acceptance of possible low-risk consequences of ambulance redirection, including extended travel time, being further from home and experiencing longer hospital stays. Participants were more uncertain about higher-risk consequences, including delays in diagnosis/treatment for patients unsuitable for thrombectomy, but remained positive about ambulance redirection overall. Participants rationalised acceptance of higher-risk consequences by recognising that redirected patients would still access appropriate treatment, even if delayed. In addition, acceptance of ambulance redirection would be increased if there were robust clinical evidence showing net benefit over secondary transfer pathways. Conclusions: Participant views were generally supportive of ambulance redirection to facilitate access to thrombectomy. Further research is needed to demonstrate overall benefit in an NHS context.

Rapid Assay Diagnostic for Acute Stroke Recognition (RADAR): study protocol for a diagnostic accuracy study.

INTRODUCTION: Large-vessel occlusion (LVO) stroke is effectively treated by time-critical thrombectomy, a highly specialised procedure only available in a limited number of centres. Many patients with suspected stroke are admitted to their nearest hospital and require transfer to access treatment, with resulting delays. This study is evaluating the accuracy of a new rapid portable test for LVO stroke which could be used in the future to select patients for direct admission to a thrombectomy centre. METHODS AND ANALYSIS: Rapid Assay Diagnostic for Acute Stroke Recognition (RADAR) is a prospective observational cohort study taking place in stroke units in England. Participants are adults with a new suspected stroke with at least one face, arm or speech (FAST) symptom(s) and known onset within 6 hours or last known to be well 6-24 hours ago. The index test ('LVOne test' (Upfront Diagnostics)), consists of two portable lateral flow assays which use fingerprick capillary blood to detect d-dimer and glial fibrillary acidic protein concentrations. Reference standards comprise independently adjudicated standard CT/MRI brain±CT/MR angiography with senior clinician opinion to establish: ischaemic stroke±LVO; intracerebral haemorrhage; transient ischaemic attack; stroke mimic. Analyses will report sensitivity, specificity and negative and positive predictive values for identification of LVO stroke. Powered using a primary analysis population (≥2 FAST symptoms and known onset within 6 hours), 276 participants will detect a test specificity of 92%. The broader total study population which allows evaluation of the test for milder symptoms and unknown onset times is estimated to be 552 participants. ETHICS AND DISSEMINATION: Ethical (North East-Newcastle & North Tyneside 2 Research Ethics Committee (reference: 23/NE/0043), Health Research Authority and participating National Health Service Trust approvals are granted. Consent is required for enrolment. Dissemination of results will include presentations at conferences, publication in journals and plain English summaries. TRIAL REGISTRATION NUMBER: ISRCTN12414986.

Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells.

Actin cytoskeleton remodeling sustains the ability of cytotoxic T cells to search for target cells and eliminate them. We here investigated the relationship between energetic status, actin remodeling, and functional fitness in human CD8+ effector T cells. Cell spreading during migration or immunological synapse assembly mirrored cytotoxic activity. Morphological and functional fitness were boosted by interleukin-2 (IL-2), which also stimulated the transcription of glycolytic enzymes, actin isoforms, and actin-related protein (ARP)2/3 complex subunits. This molecular program scaled with F-actin content and cell spreading. Inhibiting glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility, and adhesion, while mitochondrial oxidative phosphorylation blockade impacted cell elongation during confined migration. The severe morphological and functional defects of ARPC1B-deficient T cells were only partially corrected by IL-2, emphasizing ARP2/3-mediated actin polymerization as a crucial energy state integrator. The study therefore underscores the tight coordination between metabolic and actin remodeling programs to sustain the cytotoxic activity of CD8+ T cells.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.