Research priorities for the management of major trauma: an international priority setting partnership with the James Lind Alliance.

OBJECTIVE: The objective of this study is to determine research priorities for the management of major trauma, representing the shared priorities of patients, their families, carers and healthcare professionals. DESIGN/SETTING: An international research priority-setting partnership. PARTICIPANTS: People who have experienced major trauma, their carers and relatives, and healthcare professionals involved in treating patients after major trauma. The scope included chest, abdominal and pelvic injuries as well as major bleeding, multiple injuries and those that threaten life or limb. METHODS: A multiphase priority-setting exercise was conducted in partnership with the James Lind Alliance over 24 months (November 2021-October 2023). An international survey asked respondents to submit their research uncertainties which were then combined into several indicative questions. The existing evidence was searched to ensure that the questions had not already been sufficiently answered. A second international survey asked respondents to prioritise the research questions. A final shortlist of 19 questions was taken to a stakeholder workshop, where consensus was reached on the top 10 priorities. RESULTS: A total of 1572 uncertainties, submitted by 417 respondents (including 132 patients and carers), were received during the initial survey. These were refined into 53 unique indicative questions, of which all 53 were judged to be true uncertainties after reviewing the existing evidence. 373 people (including 115 patients and carers) responded to the interim prioritisation survey and 19 questions were taken to a final consensus workshop between patients, carers and healthcare professionals. At the final workshop, a consensus was reached for the ranking of the top 10 questions. CONCLUSIONS: The top 10 research priorities for major trauma include patient-centred questions regarding pain relief and prehospital management, multidisciplinary working, novel technologies, rehabilitation and holistic support. These shared priorities will now be used to guide funders and teams wishing to research major trauma around the globe.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Adrenaline-induced hypotension in the context of significant quetiapine overdose.

In patients presenting with hypotension due to significant quetiapine or mixed overdose, adrenaline should be avoided and consider medications such as noradrenaline or vasopressin as an alternative. If using noradrenaline peripherally, use a lower concentration (4 mg in 50 mL 5% glucose) and use a large bore cannula in a large vein.

Comparison of in silico strategies to prioritize rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders.

The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 249 variants of uncertain significance (VUSs) that underwent splicing functional analyses. The capability of algorithms to differentiate VUSs away from the immediate splice site as being 'pathogenic' or 'benign' is likely to have substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as 'pathogenic' or 'likely pathogenic'; one in five of these cases could lead to new or refined diagnoses.

Dysfunctional Bronchial Cilia Are a Feature of Chronic Obstructive Pulmonary Disease (COPD).

Impaired mucociliary clearance may increase COPD exacerbation risk. We aimed to compare bronchial ciliary function and epithelial ultrastructure of COPD patients to healthy controls and explore its relationship to exacerbator phenotypes (frequent [FE] and infrequent [IFE] exacerbator). In this cross-sectional study, 16 COPD patients and 12 controls underwent bronchial brushings. Ciliary beat frequency (CBF) and dyskinesia index (DI; % of dyskinetic cilia) were assessed using digital high-speed video microscopy, and epithelial ultrastructure using transmission electron microscopy (TEM). Bronchial epithelium in COPD showed lower CBF and higher DI, compared to controls (median [IQR] CBF: 6.8 (6.1-7.2) Hz vs 8.5 (7.7-8.9) Hz, p<0.001 and DI: 73.8 (60.7-89.8) % vs 14.5 (11.2-16.9) %, p<0.001, respectively). This was true for FE and IFE phenotypes of COPD, which were similar in terms of bronchial CBF or DI. Subgroup analyses demonstrated lower CBF and higher DI in FE and IFE COPD phenotypes compared to controls, irrespective of smoking status. TEM showed more loss of cilia, extrusion of cells, cytoplasmic blebs and dead cells in COPD patients versus controls. Profound dysfunction of bronchial cilia is a feature of COPD irrespective of exacerbation phenotype and smoking status, which is likely to contribute to poor mucus clearance in COPD.Supplemental data for this article is available online at https://doi.org/10.1080/15412555.2021.1963695 .

BET 1: The Ottawa Subarachnoid Hemorrhage Clinical Decision Rule shows high sensitivity but limited evidence of patient benefit.

A short-cut review of the literature was carried out to examine the diagnostic test characteristics and potential patient benefits through the use of the Ottawa Subarachnoid Haemorrhage Clinical Decision Rule. Nine papers were identified as suitable for inclusion using the reported search strategy. The author, date and country of publication, patient group studied, study type, relevant outcomes, results and study weaknesses of the best papers are tabulated. It is concluded that the Ottawa Clinical Decision Rule has a high sensitivity for the diagnosis of subarachnoid haemorrhage; however, there is limited robust evidence of international generalisability and no evidence of improved patient outcomes following implementation. Further prospective research is required in populations with variable prevalence to evaluate the safety and effectiveness of this intervention, compared with routine evaluation strategies.

Psychological distress and trauma in doctors providing frontline care during the COVID-19 pandemic in the United Kingdom and Ireland: a prospective longitudinal survey cohort study.

OBJECTIVES: The psychological impact of the COVID-19 pandemic on doctors is a significant concern. Due to the emergence of multiple pandemic waves, longitudinal data on the impact of COVID-19 are vital to ensure an adequate psychological care response. The primary aim was to assess the prevalence and degree of psychological distress and trauma in frontline doctors during the acceleration, peak and deceleration of the COVID-19 first wave. Personal and professional factors associated with psychological distress are also reported. DESIGN: A prospective online three-part longitudinal survey. SETTING: Acute hospitals in the UK and Ireland. PARTICIPANTS: Frontline doctors working in emergency medicine, anaesthetics and intensive care medicine during the first wave of the COVID-19 pandemic in March 2020. PRIMARY OUTCOME MEASURES: Psychological distress and trauma measured using the General Health Questionnaire-12 and the Impact of Events-Revised. RESULTS: The initial acceleration survey distributed across networks generated a sample of 5440 doctors. Peak and deceleration response rates from the original sample were 71.6% (n=3896) and 56.6% (n=3079), respectively. Prevalence of psychological distress was 44.7% (n=1334) during the acceleration, 36.9% (n=1098) at peak and 31.5% (n=918) at the deceleration phase. The prevalence of trauma was 23.7% (n=647) at peak and 17.7% (n=484) at deceleration. The prevalence of probable post-traumatic stress disorder was 12.6% (n=343) at peak and 10.1% (n=276) at deceleration. Worry of family infection due to clinical work was the factor most strongly associated with both distress (R2=0.06) and trauma (R2=0.10). CONCLUSION: Findings reflect a pattern of elevated distress at acceleration and peak, with some natural recovery. It is essential that policymakers seek to prevent future adverse effects through (a) provision of vital equipment to mitigate physical and psychological harm, (b) increased awareness and recognition of signs of psychological distress and (c) the development of clear pathways to effective psychological care. TRIAL REGISTRATION NUMBER: ISRCTN10666798.

Ciliopathy genes are required for apical secretion of Cochlin, an otolith crystallization factor.

Here, we report that important regulators of cilia formation and ciliary compartment-directed protein transport function in secretion polarity. Mutations in cilia genes cep290 and bbs2, involved in human ciliopathies, affect apical secretion of Cochlin, a major otolith component and a determinant of calcium carbonate crystallization form. We show that Cochlin, defective in human auditory and vestibular disorder, DFNA9, is secreted from small specialized regions of vestibular system epithelia. Cells of these regions secrete Cochlin both apically into the ear lumen and basally into the basal lamina. Basally secreted Cochlin diffuses along the basal surface of vestibular epithelia, while apically secreted Cochlin is incorporated into the otolith. Mutations in a subset of ciliopathy genes lead to defects in Cochlin apical secretion, causing abnormal otolith crystallization and behavioral defects. This study reveals a class of ciliary proteins that are important for the polarity of secretion and delineate a secretory pathway that regulates biomineralization.

Higher throughput drug screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia.

BACKGROUND: Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. METHODS: We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. RESULTS: Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.

Psychological distress during the acceleration phase of the COVID-19 pandemic: a survey of doctors practising in emergency medicine, anaesthesia and intensive care medicine in the UK and Ireland.

OBJECTIVE: To quantify psychological distress experienced by emergency, anaesthetic and intensive care doctors during the acceleration phase of COVID-19 in the UK and Ireland. METHODS: Initial cross-sectional electronic survey distributed during acceleration phase of the first pandemic wave of COVID-19 in the UK and Ireland (UK: 18 March 2020-26 March 2020 and Ireland: 25 March 2020-2 April 2020). Surveys were distributed via established specialty research networks, within a three-part longitudinal study. Participants were doctors working in emergency, anaesthetic and intensive medicine during the first pandemic wave of COVID-19 in acute hospitals across the UK and Ireland. Primary outcome measures were the General Health Questionnaire-12 (GHQ-12). Additional questions examined personal and professional characteristics, experiences of COVID-19 to date, risk to self and others and self-reported perceptions of health and well-being. RESULTS: 5440 responses were obtained, 54.3% (n=2955) from emergency medicine and 36.9% (n=2005) from anaesthetics. All levels of doctor seniority were represented. For the primary outcome of GHQ-12 score, 44.2% (n=2405) of respondents scored >3, meeting the criteria for psychological distress. 57.3% (n=3045) had never previously provided clinical care during an infectious disease outbreak but over half of respondents felt somewhat prepared (48.6%, n=2653) or very prepared (7.6%, n=416) to provide clinical care to patients with COVID-19. However, 81.1% (n=4414) either agreed (31.1%, n=2709) or strongly agreed (31.1%, n=1705) that their personal health was at risk due to their clinical role. CONCLUSIONS: Findings indicate that during the acceleration phase of the COVID-19 pandemic, almost half of frontline doctors working in acute care reported psychological distress as measured by the GHQ-12. Findings from this study should inform strategies to optimise preparedness and explore modifiable factors associated with increased psychological distress in the short and long term. TRIAL REGISTRATION NUMBER: ISRCTN10666798.