Unquantifiably low aldosterone concentrations are prevalent in hospitalised COVID-19 patients but may not be revealed by chemiluminescent immunoassay.

Objective: Previous studies have reported conflicting findings regarding aldosterone levels in patients hospitalised with COVID-19. We therefore used the gold-standard technique of liquid chromatography-tandem mass spectrometry (LCMSMS) to address this uncertainty. Design: All patients admitted to Cambridge University Hospitals with COVID-19 between 10 March 2020 and 13 May 2021, and in whom a stored blood sample was available for analysis, were eligible for inclusion. Methods: Aldosterone was measured by LCMSMS and by immunoassay; cortisol and renin were determined by immunoassay. Results: Using LCMSMS, aldosterone was below the limit of detection (<70 pmol/L) in 74 (58.7%) patients. Importantly, this finding was discordant with results obtained using a commonly employed clinical immunoassay (Diasorin LIAISON®), which over-estimated aldosterone compared to the LCMSMS assay (intercept 14.1 (95% CI -34.4 to 54.1) + slope 3.16 (95% CI 2.09-4.15) pmol/L). The magnitude of this discrepancy did not clearly correlate with markers of kidney or liver function. Solvent extraction prior to immunoassay improved the agreement between methods (intercept -14.9 (95% CI -31.9 to -4.3) and slope 1.0 (95% CI 0.89-1.02) pmol/L) suggesting the presence of a water-soluble metabolite causing interference in the direct immunoassay. We also replicated a previous finding that blood cortisol concentrations were often increased, with increased mortality in the group with serum cortisol levels > 744 nmol/L (P = 0.005). Conclusion: When measured by LCMSMS, aldosterone was found to be profoundly low in a significant proportion of patients with COVID-19 at the time of hospital admission. This has likely not been detected previously due to high levels of interference with immunoassays in patients with COVID-19, and this merits further prospective investigation.

Development and validation of a dynamic 48-hour in-hospital mortality risk stratification for COVID-19 in a UK teaching hospital: a retrospective cohort study.

OBJECTIVES: To develop a disease stratification model for COVID-19 that updates according to changes in a patient's condition while in hospital to facilitate patient management and resource allocation. DESIGN: In this retrospective cohort study, we adopted a landmarking approach to dynamic prediction of all-cause in-hospital mortality over the next 48 hours. We accounted for informative predictor missingness and selected predictors using penalised regression. SETTING: All data used in this study were obtained from a single UK teaching hospital. PARTICIPANTS: We developed the model using 473 consecutive patients with COVID-19 presenting to a UK hospital between 1 March 2020 and 12 September 2020; and temporally validated using data on 1119 patients presenting between 13 September 2020 and 17 March 2021. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome is all-cause in-hospital mortality within 48 hours of the prediction time. We accounted for the competing risks of discharge from hospital alive and transfer to a tertiary intensive care unit for extracorporeal membrane oxygenation. RESULTS: Our final model includes age, Clinical Frailty Scale score, heart rate, respiratory rate, oxygen saturation/fractional inspired oxygen ratio, white cell count, presence of acidosis (pH <7.35) and interleukin-6. Internal validation achieved an area under the receiver operating characteristic (AUROC) of 0.90 (95% CI 0.87 to 0.93) and temporal validation gave an AUROC of 0.86 (95% CI 0.83 to 0.88). CONCLUSIONS: Our model incorporates both static risk factors (eg, age) and evolving clinical and laboratory data, to provide a dynamic risk prediction model that adapts to both sudden and gradual changes in an individual patient's clinical condition. On successful external validation, the model has the potential to be a powerful clinical risk assessment tool. TRIAL REGISTRATION: The study is registered as 'researchregistry5464' on the Research Registry (www.researchregistry.com).

Recognition and management of sepsis by junior doctors.

There is growing evidence that outcomes in sepsis are improved by early recognition and treatment. In this study, we assessed junior doctors' ability to recognise and manage sepsis. We also explored junior doctors' perceptions regarding barriers to delivering timely sepsis care. From 46 respondents, only 4% were able to list the systemic inflammatory response syndrome (SIRS) criteria, 50% could define sepsis and 46% could list the Sepsis Six. Following further teaching on sepsis, 35% could list the SIRS criteria, 87% correctly defined sepsis, and 91% could state the Sepsis Six. Junior doctors perceived time pressure when on call to be the greatest barrier in treating sepsis, and their own knowledge to be the least important barrier. Our data suggest that knowledge of sepsis among junior doctors is poor and that there is a lack of insight into this competency gap.

The burden of sepsis in the Emergency Department: an observational snapshot.

The primary aim of our study was to establish what proportion of patients in the Emergency Department (ED) fulfill the criteria for sepsis. All adult patients presenting to ED in two 1-week periods, 6 months apart, were included. Notes were reviewed retrospectively to identify which patients fulfilled the criteria for sepsis and severe sepsis. The proportion of patients with sepsis was 4.3% (95% confidence interval 3.3-5.2%) and the proportion with severe sepsis was 2.2% (95% confidence interval 1.5-2.8%). In conclusion our results suggest that sepsis is more common than previously reported and this represents a significant burden on ED.

Identification and initial functional characterization of a human vascular cell-enriched long noncoding RNA.

OBJECTIVE: Long noncoding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. APPROACH AND RESULTS: RNA sequencing (RNA-seq) of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell-enriched lncRNA (Smooth muscle and Endothelial cell-enriched migration/differentiation-associated long NonCoding RNA [SENCR]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5' end of the FLI1 gene and exists as 2 splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-seq experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. CONCLUSIONS: SENCR is a new vascular cell-enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.

Mitogen-activated protein kinase 14 is a novel negative regulatory switch for the vascular smooth muscle cell contractile gene program.

OBJECTIVE: Several studies have shown through chemical inhibitors that p38 mitogen-activated protein kinase (MAPK) promotes vascular smooth muscle cell (VSMC) differentiation. Here, we evaluate the effects of knocking down a dominant p38MAPK isoform on VSMC differentiation. METHODS AND RESULTS: Knockdown of p38MAPKα (MAPK14) in human coronary artery SMCs unexpectedly increases VSMC differentiation genes, such as miR145, ACTA2, CNN1, LMOD1, and TAGLN, with little change in the expression of serum response factor (SRF) and 2 SRF cofactors, myocardin (MYOCD) and myocardin-related transcription factor A (MKL1). A variety of chemical and biological inhibitors demonstrate a critical role for a RhoA-MKL1-SRF-dependent pathway in mediating these effects. MAPK14 knockdown promotes MKL1 nuclear localization and VSMC marker expression, an effect partially reversed with Y27632; in contrast, MAP2K6 (MKK6) blocks MKL1 nuclear import and VSMC marker expression. Immunostaining and Western blotting of injured mouse carotid arteries reveal elevated MAPK14 (both total and phosphorylated) and reduced VSMC marker expression. CONCLUSIONS: Reduced MAPK14 expression evokes unanticipated increases in VSMC contractile genes, suggesting an unrecognized negative regulatory role for MAPK14 signaling in VSMC differentiation.

Expression and promoter analysis of a highly restricted integrin alpha gene in vascular smooth muscle.

Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity, indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArG-SRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region.

Smooth muscle calponin: an unconventional CArG-dependent gene that antagonizes neointimal formation.

OBJECTIVE: Smooth muscle calponin (CNN1) contains multiple conserved intronic CArG elements that bind serum response factor and display enhancer activity in vitro. The objectives here were to evaluate these CArG elements for activity in transgenic mice and determine the effect of human CNN1 on injury-induced vascular remodeling. METHODS AND RESULTS: Mice carrying a lacZ reporter under control of intronic CArG elements in the human CNN1 gene failed to show smooth muscle cell (SMC)-restricted activity. However, deletion of the orthologous sequences in mice abolished endogenous Cnn1 promoter activity, suggesting their necessity for in vivo Cnn1 expression. Mice carrying a 38-kb bacterial artificial chromosome (BAC) harboring the human CNN1 gene displayed SMC- restricted expression of the corresponding CNN1 protein, as measured by immunohistochemistry and Western blotting. Extensive BAC recombineering studies revealed the absolute necessity of a single intronic CArG element for correct SMC-restricted expression of human CNN1. Overexpressing human CNN1 suppressed neointimal formation following arterial injury. Mice with an identical BAC carrying mutations in CArG elements that inhibit human CNN1 expression showed outward remodeling and neointimal formation. CONCLUSIONS: A single intronic CArG element is necessary but insufficient for proper CNN1 expression in vivo. CNN1 overexpression antagonizes arterial injury-induced neointimal formation.