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Background: In patients with coronavirus disease 2019 (COVID-19) requiring supplemental oxygen, dexamethasone reduces acute severity and improves survival, but longer-term effects are unknown. We hypothesised that systemic corticosteroid administration during acute COVID-19 would be associated with improved health-related quality of life (HRQoL) 1 year after discharge., Methods: Adults admitted to hospital between February 2020 and March 2021 for COVID-19 and meeting current guideline recommendations for dexamethasone treatment were included using two prospective UK cohort studies (Post-hospitalisation COVID-19 and the International Severe Acute Respiratory and emerging Infection Consortium). HRQoL, assessed by the EuroQol-Five Dimensions-Five Levels utility index (EQ-5D-5L UI), pre-hospital and 1 year after discharge were compared between those receiving corticosteroids or not after propensity weighting for treatment. Secondary outcomes included patient-reported recovery, physical and mental health status, and measures of organ impairment. Sensitivity analyses were undertaken to account for survival and selection bias., Findings: Of the 1888 participants included in the primary analysis, 1149 received corticosteroids. There was no between-group difference in EQ-5D-5L UI at 1 year (mean difference 0.004, 95% CI -0.026-0.034). A similar reduction in EQ-5D-5L UI was seen at 1 year between corticosteroid exposed and nonexposed groups (mean±sd change -0.12±0.22 versus -0.11±0.22). Overall, there were no differences in secondary outcome measures. After sensitivity analyses modelled using a cohort of 109 318 patients admitted to hospital with COVID-19, EQ-5D-5L UI at 1 year remained similar between the two groups., Interpretation: Systemic corticosteroids for acute COVID-19 have no impact on the large reduction in HRQoL 1 year after hospital discharge. Treatments to address the persistent reduction in HRQoL are urgently needed., Competing Interests: Conflict of interest: O.C. Leavy declares that their institute received joint funding from UKRI and NIHR (MR/V027859/1 and COV0319) to complete this work. A.B. Docherty declares that they were awarded funding from a Wellcome Clinical Research Career Development Fellowship (216606/Z/19/Z) to complete this work. A Sheikh declares that their institute was awarded grant funding from NIHR and UKRI to complete this work; participation on a Data Safety Monitoring Board or Advisory Board for AstraZeneca's Thrombotic Thrombocytopenic Taskforce; and leadership or fiduciary roles for UK and Scottish Government COVID-19 advisory groups. C.E. Bolton declares that their institute received grant funding from NIHR/UKRI and NIHR to complete this work; and their institute received grant funding from Nottingham Hospitals Charity and University of Nottingham. G. Choudhury declares funding from GlaxoSmithKline and AstraZeneca; received honoraria for delivering talks from GSK, AZ, Chiesi and BI; participation on a Data Safety Monitoring Board or Advisory Board as Chair on the Act on COPD Programme for AZ in Scotland; and a leadership or fiduciary role as Chair for the Lothian Respiratory Managed Clinical Network. N.D. Bakerly declares they have received nonrestrictive educational grants from Chiesi, AZ and Teva for attending conferences; honoraria from Teva, AZ and GSK; support for attending meetings and/or travel from Chiesi and AZ; participation on a Data Safety Monitoring Board or Advisory Board for Teva; and receipt of equipment from Global Access Diagnostics (previously Mologic Inc). A. Shikotra declares that their institute was awarded joint funding from UKRI and the NIHR (MR/V027859/1 and COV0319) to complete this work. R. Aul declares lecture fees and support for attending a meeting from Boehringer Ingelheim. C. Echevarria declares a grant from GSK. J.R. Hurst declares funding from AstraZeneca; consulting fees from AstraZeneca and GSK; payment for lectures and presentations from AstraZeneca, Boehringer Ingelheim, Chiesi, Sanofi and Takeda; support for attending meetings and/or travel from AstraZeneca; participation on a Data Safety Monitoring Board or Advisory Board for AstraZeneca; and receipt of equipment from Nonin. M. Jones declares funding from the MRC to complete this work; funding from the MRC, British Lung Foundation and Boehringer Ingelheim; consulting fees from Skyhawk Therapeutics; and a leadership or fiduciary role in the AAIR Charity Scientific Committee. P. Pfeffer declares funding from NIHR. S. Rowland-Jones declares that their institute received funding from UKRI to complete this work; and their institute received funding from NIHR Sheffield Biomedical Research centre, Bill and Melinda Gates Foundation, UKRI (MRC) and EDCTP. D. Parekh declares funding from NIHR and MRC; and a leadership or fiduciary role for the Faculty of Intensive Care Medicine Board. A.A.R. Thompson declares that their institute was awarded a fellowship from the British Heart Foundation, and grant funding from Heart Research UK and the National Institute for Health and Care Research; payment for lectures and presentations from Janssen-Cilag Ltd; and support for attending meetings from Janssen-Cilag Ltd. M.J. Davies declares grant funding from Novo Nordisk, Sanofi-Aventis, Lilly, Boehringer Ingelheim, AstraZeneca and Janssen; consulting fees from Eli Lilly, Boehringer Ingelheim, Novo Nordisk and Sanofi; payment for speaking for Boehringer Ingelheim, Lilly, Novo Nordisk, Sanofi, AstraZeneca, Amgen, Napp Pharmaceuticals and Novartis; and is an advisory board member for Boehringer Ingelheim, Lilly, Novo Nordisk, Sanofi, Lexicon, Pfizer, Medtronic and ShouTi Pharma Inc., and Zealand Pharma. A. De Soyza declares that their institute was awarded grant funding from AstraZeneca, Bayer, GSK, Chiesi, Novartis and Pfizer, outside the submitted manuscript; consulting fees from AstraZeneca, Bayer, GSK, Chiesi, Novartis, Pfizer, Insmed and Gilead; payment for lectures and presentations from AstraZeneca, Bayer, GSK, Chiesi, Novartis, Pfizer, Insmed, Gilead and 30T; participation on a Data Safety Monitoring Board or Advisory Board for Bayer; and receipt of drugs from GSK outside the submitted manuscript. S. Heller declares consulting fees from NovoNordisk; and participation on a Data Safety Monitoring Board or Advisory Board for Eli Lilly with payments made to their institution. J. Jacob declares funding from Gilead, Microsoft Research and GlaxoSmithKline; consulting fees from Boehringer Ingelheim, Roche, GlaxoSmithKline and NHSX; payment for lectures and presentations received from Boehringer Ingelheim, Roche, GlaxoSmithKline and Takeda; support for attending meetings and/or travel from Boehringer Ingelheim; patents planned, issued or pending (UK patent application number 2113765.8 and UK patent application number GB2211487.0); and participation on a Data Safety Monitoring Board or Advisory Board for Boehringer Ingelheim and Roche. R.G. Jenkins declares that their institute received funding from AstraZeneca, Biogen, Galecto, GlaxoSmithKline, Nordic Biosciences, RedX and Pliant; consulting fees from AstraZeneca, Brainomix, Bristol Myers Squibb, Chiesi, Cohbar, Daewoong, GlaxoSmithKline, Veracyte, Resolution Therapeutics and Pliant; payment for lectures and presentations received from Boehringer Ingelheim, Chiesi, Roche, PatientMPower and AstraZeneca; payment for expert testimony from Pinsent Masons LLP; participation on a Data Safety Monitoring Board or Advisory Board for Boehringer Ingelheim, Galapagos and Vicore; and a leadership or fiduciary role for NuMedii and is president for Action for Pulmonary Fibrosis. W.D-C. Man declares that their institute received funding from National Institute for Health Research and NHS Accelerated Access Collaborative; and is Honorary President of the Association for Respiratory Technology and Physiology, and an associate editor of ERJ Open Research. G.P. McCann declares funding from NIHR (RP-2017-ST2-007) to complete this work; funding from the British Heart Foundation, Wellcome Trust and NIHR; and research support from Resonance Health, Circle CVi and Perspectum. S. Neubauer declares grant funding from Oxford NIHR Biomedical Research centre. P.J.M. Openshaw declares funding from UKRI-MRC/DHSC NIHR and UKRI-BEIS. M.J. Rowland declares support for attending meetings and/or travel from Novartis Pharmaceuticals; stock or stock options from Novartis Pharmaceuticals and Roche Pharmaceuticals; and is employed full time as a Senior Clinical Development Medical Director at Novartis Pharmaceuticals. J. Porter declares funding from Breathing Matters and UCL/H BRC (NIHR), and consulting fees from The Limbic. L.G. Heaney declares that their institute received funding from GSK, AstraZeneca and Roche/Genentech; payment for lectures received from AstraZeneca, Novartis, Roche/Genentech, Sanofi, Circassia, GlaxoSmithKline, Chiesi and Teva; support to travel to meetings from AstraZeneca and GSK; participation on a Data Safety Monitoring Board or Advisory Board for Novartis, Roche/Genentech, GSK, Teva and Celltrion; and funding from the NIHR (RfPB grant PB-PG-0317-20032). J.T. Scott declares funding from UKRI. M.G. Semple declares grant funding from National Institute of Health Research UK, Medical Research Council UK and Health Protection Research Unit in Emerging and Zoonotic Infections, and University of Liverpool to complete this work; participation on a Data Safety Monitoring Board or Advisory Board for Pfizer; leadership or fiduciary roles as Chair of Infectious Disease Scientific Advisory Board Integrum Scientific LLC and Director of MedEx Solutions Ltd; stock or stock options as minority owner of Integrum Scientific LLC and majority owner of MedEx Solutions Ltd; receipt of equipment, materials, drugs, medical writing, gifts or other services from Chiesi Farmaceutici SpA; and is a nonremunerated independent member of HMG UK Scientific Advisory Group for Emergencies (SAGE), COVID-19 Response (March 2020 to March 2022) and a nonremunerated independent member of HMG UK New Emerging Respiratory Virus Threats Advisory Group (NERVTAG) (2014 to July 2023). S.J. Singh declares grants or contracts from NIHR (programme grant (NIHR 202020), Wellcome Doctoral Training Programme, HTA Project Grant (NIHR 131015), NIHR DHSC/UKRI COVID-19 Rapid Response Initiative, NIHR Global Research Group (NIHR 17/63/20)), Actegy Limited and NIHR Senior Investigator; payment for presentations for GSK, Ministry of Justice, CIPLA, Sherbourne Gibbs; participation on NICE Expert Adviser Panel (long COVID), Wales Long COVID Advisory Board and NHS-E Long Covid Your Covid Recovery working group; and leadership or fiduciary roles as ATS Pulmonary Rehabilitation Assembly Chair, Clinical Lead RCP Pulmonary Rehabilitation Accreditation Scheme and Clinical Lead NACAP Audit for Pulmonary Rehabilitation. M. Toshner declares grant funding from the NIHR Cambridge BRC and NIHR HTA to complete this work; consulting fees from Janssen; support for attending meetings and/or travel from GSK and Janssen; and participation on a Data Safety Monitoring Board or Advisory Board for ComCov and FluCov. A. Horsley declares that their institute was awarded funding from UK Research and Innovation (MR/V027859/1), the National Institute of Health Research (NIHR) (COV0319) and NIHR Manchester BRC; and is Chair for the NIHR Translational Research Collaboration. M. Marks declares that their institute received joint funding from UKRI and NIHR to complete this work. K. Poinasamy declares funding from UKRI and NIHR to complete this work. A. Briggs declares consulting fees from Roche, Merck, Sanofi and GSK. J.K. Quint declares that their institute received funding from the Industrial Strategy Challenge Fund, the Medical Research Council, Health Data Research, GSK, BI, Asthma+Lung UK and AZ; and consulting fees from GlaxoSmithKline, Evidera, Chiesi, AstraZeneca and Insmed. J.D. Chalmers declares funding from AstraZeneca, Boehringer Ingelheim, Grifols, Gilead Sciences, Insmed, Genentech and GlaxoSmithKline; consulting fees from AstraZeneca, Boehringer Ingelheim, Grifols, Gilead Sciences, Insmed, Genentech, Glaxosmithkline, Antabio, Zambon and Trudell; and leadership or fiduciary roles as Chief Editor of the European Respiratory Journal and associate editor of ERJ Open Research, Chair of the British Thoracic Society Science and Research Committee, and Trustee of the British Thoracic Society. L.V. Wain declares funding from UK Research and Innovation (MR/V027859/1), GSK/Asthma+Lung UK (Professorship (C17-1)) and National Institute of Health Research (COV0319) to complete this work; funding from Orion Pharma, GSK, Genentech, AstraZeneca, Nordic Bioscience and Sysmex (OGT); consulting fees Galapagos, Boehringer Ingelheim and GSK; support for attending meetings and/or travel Genentech; participation on Advisory Board for Galapagos; leadership or fiduciary roles as an associate editor for the European Respiratory Journal, and Medical Research Council Board member and Deputy Chair. C.E. Brightling declares that their institute received grant funding from MRC/NIHR and NIHR to complete this work; their institute received grant funding from GSK, AZ, Sanofi, Regeneron, Roche, Genentech, BI, Novartis, Chiesi, 4Dpharma and Mologic; and consulting fees from GSK, AZ, Sanofi, Regeneron, Roche, Genentech, BI, Novartis, Chiesi, 4Dpharma, Mologic and Areteia. R.A. Evans declares funding from UKRI/MRC/NIHR to complete this work; funding from Wolfson Foundation and Genentech/Roche; consulting fees from AstraZeneca/Evidera; speaking fees from Boehringer and Moderna; support for attending meetings from Chiesi; and leadership or fiduciary roles as ERS Group 01.02 Pulmonary Rehabilitation and Chronic Care Secretary, and ATS Pulmonary Rehabilitation Assembly Chair. All other authors declare no conflicts of interest., (Copyright ©The authors 2024.)

Asthma is a disease of heterogeneous pathology, typically characterised by excessive inflammatory and bronchoconstrictor responses to the environment. The clinical expression of the disease is a consequence of the interaction between environmental factors and host factors over time, including genetic susceptibility, immune dysregulation and airway remodelling. As a critical interface between the host and the environment, the airway epithelium plays an important role in maintaining homeostasis in the face of environmental challenges. Disruption of epithelial integrity is a key factor contributing to multiple processes underlying asthma pathology. In this review, we first discuss the unmet need in asthma management and provide an overview of the structure and function of the airway epithelium. We then focus on key pathophysiological changes that occur in the airway epithelium, including epithelial barrier disruption, immune hyperreactivity, remodelling, mucus hypersecretion and mucus plugging, highlighting how these processes manifest clinically and how they might be targeted by current and novel therapeutics., Competing Interests: Conflict of interest: R.J. Russell has received support for conference registration fees and expenses from Chiesi. L-P. Boulet has received grants and/or consultancy fees from Amgen, AstraZeneca, BioHaven, Cipla, Covis, GSK, Merck, Novartis, Sanofi-Regeneron and Teva Pharmaceuticals. C.E. Brightling has received grants and consultancy fees from 4D Pharma, AstraZeneca, Chiesi, Genentech, GSK, Mologic, Novartis, Regeneron Pharmaceuticals, Roche and Sanofi. I.D. Pavord has received speaker fees from Aerocrine AB, Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi, GSK, Novartis, Regeneron Pharmaceuticals, Sanofi and Teva Pharmaceuticals, payments for organisation of educational events from AstraZeneca, GSK, Regeneron Pharmaceuticals, Sanofi and Teva Pharmaceuticals, consultancy fees from Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi, Circassia, Dey Pharma, Genentech, GSK, Knopp Biosciences, Merck, MSD, Napp Pharmaceuticals, Novartis, Regeneron Pharmaceuticals, RespiVert, Sanofi, Schering-Plough and Teva Pharmaceuticals, international scientific meeting sponsorship from AstraZeneca, Boehringer Ingelheim, Chiesi, GSK, Napp Pharmaceuticals, Regeneron Pharmaceuticals, Sanofi and Teva Pharmaceuticals, and a research grant from Chiesi. C. Porsbjerg has received grants and consultancy fees from ALK-Abelló, AstraZeneca, Chiesi, GSK, Novartis, Sanofi and Teva Pharmaceuticals. D. Dorscheid has received grants and clinical trial support from AstraZeneca, British Columbia Lung Association, Canadian Institutes of Health Research, Michael Smith Foundation for Health Research, Regeneron Pharmaceuticals, Sanofi and Teva Pharmaceuticals, and speaking and consultancy fees, travel grants, unrestricted project grants and writing fees from AstraZeneca, GSK, Novartis Canada, Regeneron Pharmaceuticals, Sanofi and Valeo Pharma. A. Sverrild has received grants and consultancy fees from Amgen, AstraZeneca, Chiesi, GSK and Sanofi., (Copyright ©The authors 2024.)

Background: Understanding how asthma biomarkers relate to gene expression signatures could help identify drivers of pathogenesis., Objective: This post hoc exploratory analysis of the phase II tralokinumab trial MESOS (ClinicalTrials.gov identifier NCT02449473) aimed to profile baseline airway inflammation in patients with moderate-to-severe asthma., Methods: The T2 and T17 gene expression signatures, 3-gene mean and 5-gene mean, were calculated through transcriptomic analysis of baseline bronchial brushing samples. Clustering analysis using these signatures identified 3 distinct inflammatory subgroups: T2 LOW /T17 HIGH (n = 33), T2 HIGH /T17 LOW (n = 10), and T2 LOW /T17 LOW (n = 27)., Results: Fractional exhaled nitric oxide (Feno) levels were highest for T2 HIGH /T17 LOW and lowest for T2 LOW /T17 HIGH (median = 52.0 [range 42.5-116.3] and median = 18.8 [range 6.6-128.6] ppb, respectively; P = .003). High Feno levels were strongly correlated with high T2 gene expression (Spearman ρ = 0.5537; P < .001). Individual genes differentially expressed in patients with elevated levels of Feno, blood and bronchial submucosal eosinophil counts, and IgE level were explored, with cystatin SN (CST1) being the most upregulated gene in all subgroups (4.49- to 34.42-fold upregulation across clinically defined subgroups with high biomarker expression)., Conclusion: Feno level may be useful to differentiate patients with T2 or T17 gene expression. Elevated Feno levels are associated with high CST1 expression., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous inflammatory airway disease. The epithelial-derived IL-33 and its receptor ST2 have been implicated in airway inflammation and infection. We aimed to determine whether astegolimab, a selective ST2 IgG2 monoclonal antibody, reduces exacerbations in COPD., Methods: COPD-ST2OP was a single-centre, randomised, double-blinded, placebo-controlled phase 2a trial in moderate-to-very severe COPD. Participants were randomly assigned (1:1) with a web-based system to received 490 mg subcutaneous astegolimab or subcutaneous placebo, every 4 weeks for 44 weeks. The primary endpoint was exacerbation rate assessed for 48 weeks assessed with a negative binomial count model in the intention-to-treat population, with prespecified subgroup analysis by baseline blood eosinophil count. The model was the number of exacerbations over the 48-week treatment period, with treatment group as a covariate. Safety was assessed in the whole study population until week 60. Secondary endpoints included Saint George's Respiratory Questionnaire for COPD (SGRQ-C), FEV 1 , and blood and sputum cell counts. The trial was registered with ClinicalTrials.gov, NCT03615040., Findings: The exacerbation rate at 48 weeks in the intention-to-treat analysis was not significantly different between the astegolimab group (2·18 [95% CI 1·59 to 2·78]) and the placebo group (2·81 [2·05 to 3·58]; rate ratio 0·78 [95% CI 0·53 to 1·14]; p=0·19]). In the prespecified analysis stratifying patients by blood eosinophil count, patients with 170 or fewer cells per μL had 0·69 exacerbations (0·39 to 1·21), whereas those with more than 170 cells per μL had 0·83 exacerbations (0·49 to 1·40). For the secondary outcomes, the mean difference between the SGRQ-C in the astegolimab group versus placebo group was -3·3 (95% CI -6·4 to -0·2; p=0·039), and mean difference in FEV 1 between the two groups was 40 mL (-10 to 90; p=0·094). The difference in geometric mean ratios between the two groups for blood eosinophil counts was 0·59 (95% CI 0·51 to 0·69; p<0·001) and 0·25 (0·19 to 0·33; p<0·001) for sputum eosinophil counts. Incidence of treatment-emergent adverse events was similar between groups., Interpretation: In patients with moderate-to-very severe COPD, astegolimab did not significantly reduce exacerbation rate, but did improve health status compared with placebo., Funding: Funded by Genentech and National Institute for Health Research Biomedical Research Centres., Competing Interests: Declaration of interests CEB reports funding from Genentech to support this study; grants from AstraZeneca, GlaxoSmithKline, Roche/Genentech, Boehringer Ingelheim, Novartis, Chiesi, Merck Sharp & Dohme, Sanofi, Regeneron, 4DPharma, and Mologic; and consulting fee paid to institution from AstraZeneca, GlaxoSmithKline, Roche/Genentech, Boehringer Ingelheim, Novartis, Chiesi, Merck Sharp & Dohme, Sanofi, Regeneron, and 4DPharma. NJG reports an investigator led grant from GlaxoSmithKline; National Institute for Health Research fellowship; and lecture honoraria from GlaxoSmithKline and Chiesi. AW reports a research grant from Sanofi Genzyme. CJ reports a Medical Research Council clinical research training fellowship. MAG, DFC, and DC are employed by Genentech and receive stock options. All other authors declare no competing interests., (Crown Copyright © 2022 Published by Elsevier Ltd. All rights reserved.)

Background: Asthma and non-asthmatic eosinophilic bronchitis (NAEB) are among the commonest causes of chronic cough in adults. We sought to determine the role of non-invasive measurements of airway inflammation, including induced sputum and fractional exhaled nitric oxide, in the evaluation of cough associated with asthma, and what the best treatment is for cough due to asthma or NAEB., Methods: We undertook three systematic reviews of randomized controlled trials and observational trials of adults and adolescents > 12 years of age with a chronic cough due to asthma or NAEB. Eligible studies were identified in MEDLINE, CENTRAL, and SCOPUS and assessed for relevance and quality. Guidelines were developed and voted upon using CHEST guideline methodology., Results: Of the citations reviewed, 3/1,175, 53/656, and 6/134 were identified as being eligible for inclusion in the three systematic reviews, respectively. In contrast to established guidelines for asthma therapies in general and the inclusion in some guidelines for a role of biomarkers of airway inflammation to guide treatment in severe disease, the evidence of specific benefit related to the use of non-invasive biomarkers in patients with chronic cough due to asthma was weak. The best therapeutic option for cough in asthma or NAEB is inhaled corticosteroids followed by leukotriene receptor antagonism., Conclusions: This guideline offers recommendations on the role of non-invasive measurements of airway inflammation and treatment for cough due to asthma or NAEB based on the available literature, and identifies gaps in knowledge and areas for future research., (Copyright © 2020 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Despite the use of effective medications to control asthma, severe exacerbations in asthma are still a major health risk and require urgent action on the part of the patient and physician to prevent serious outcomes such as hospitalisation or death. Moreover, severe exacerbations are associated with substantial healthcare costs and psychological burden, including anxiety and fear for patients and their families. The European Academy of Allergy and Clinical Immunology (EAACI) and the European Respiratory Society (ERS) set up a task force to search for a clear definition of severe exacerbations, and to also define research questions and priorities. The statement includes comments from patients who were members of the task force., Competing Interests: Conflict of interest: A. Bourdin reports personal and institutional fees for advisory board work from AstraZeneca, Novartis, GSK, Boehringher Ingelheim, Chiesi, Actelion, Pfizer and Teva, outside the submitted work. Conflict of interest: L. Bjermer has nothing to disclose. Conflict of interest: C. Brightling reports grants and personal fees for consultancy from GlaxoSmithKline, AstraZeneca/Medimmune, Novartis, Chiesi, Roche/Genentech and Boehringer Inglheim, personal fees for consultancy from Vectura, Theravance, PreP, Gilead, Sanofi/Regeneron, Teva, Gossamer and 4DPharma, grants from Pfizer and Mologic, outside the submitted work. Conflict of interest: G.G. Brusselle reports personal fees for advisory board work and lectures from AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Novartis and Teva, personal fees for advisory board work from Sanofi, outside the submitted work. Conflict of interest: P. Chanez reports research grants and personal fees for consultancy, advisory board work and lectures from ALK, Almirall, Boehringer Ingelheim, GSK, AstraZeneca, Novartis, TEVA and Chiesi, grants from AMU, outside the submitted work. Conflict of interest: K.F. Chung has received honoraria for participating in advisory board meetings of GSK, AZ, Novartis, Merck, BI and TEVA regarding treatments for asthma and COPD, and has also been renumerated for speaking engagements. Conflict of interest: A. Custovic reports personal fees for consultancy from Novartis, Regeneron/Sanofi, Boehringer Ingelheim and Philips, personal fees for lectures from Thermo Fisher Scientific and Novartis, outside the submitted work. Conflict of interest: Z. Diamant reports personal fees from AstraZeneca and Sanofi-Genzyme, during the conduct of the study; personal fees from Aquilon, ALK, Boehringer Ingelheim, Gilead, Hal Allergy and MSD, outside the submitted work; and in addiction to academic affiliations, also works at a phase I/II unit performing clinical studies for different biotech and pharma companies. Conflict of interest: S. Diver has nothing to disclose. Conflict of interest: R. Djukanovic reports receiving fees for lectures at symposia organised by Novartis, AstraZeneca and TEVA, consultation for TEVA and Novartis as member of advisory boards, and participation in a scientific discussion about asthma organised by GlaxoSmithKline; in addition, is a co-founder and current consultant, and has shares in, Synairgen, a University of Southampton spin out company. Conflict of interest: D. Hamerlijnck has nothing to disclose. Conflict of interest: I. Horvath reports personal fees from AstraZeneca, Berlin-Chemie, Boehringer Ingelheim, Chiesi, GSK, Novartis, CSL-Behring and Roche, outside the submitted work. Conflict of interest: S.L. Johnston reports personal fees for advisory board work from Therapeutic Frontiers and Virtus Respiratory Research, personal fees for consultancy from Myelo Therapeutics GmbH, Concert Pharmaceuticals, Bayer, Gerson Lehrman Group, resTORbio, Bioforce, Materia Medical Holdings, PrepBio Pharma, Pulmotect, Virion Health and Lallemand Pharma, personal and insititutional fees for consultancy from Synairgen, Novartis, Boehringer Ingelheim and Chiesi; and has received personal fees for the following patents planned, issued or pending: transgenic animal models of HRV with human ICAM-1 sequences (UK patent application number 02 167 29.4, and international patent application number PCT/EP2003/007939); anti-virus therapy for respiratory diseases (UK patent application number GB 0405634.7); interferon-beta for anti-virus therapy for respiratory diseases (international patent application number PCT/GB05/50031); interferon lambda therapy for the treatment of respiratory disease (UK patent application number 6779645.9, granted); induction of cross-reactive cellular response against rhinovirus antigens (European patent number 13305152), outside the submitted work. Conflict of interest: F. Kanniess reports personal fees for lectures and advisory board work from AstraZeneca, Novartis, Mundipharma and TEVA, outside the submitted work. Conflict of interest: N. Papadopoulos reports personal fees for advisory board work and lectures from Novartis, Nutricia, HAL, personal fees from Menarini/Faes Farma and Mylan/Meda, personal fees for lectures from Sanofi, Biomay, MSD, ASIT Biotech and Boehringer Ingelheim, personal fees for advisory board work from AstraZeneca and GSK, grants from Gerolymatos International SA and Capricare, outside the submitted work. Conflict of interest: A. Papi reports grants, personal fees for lectures, advisory board work and consultancy, and travel expenses reimbursement from AstraZeneca, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline and Teva, personal fees for advisory board work and consultancy from Sanofi/Regeneron, personal fees for lectures and travel expenses reimbursement from Zambon and Novartis, personal fees for lectures, advisory board work and consultancy, and travel expenses reimbursement from Mundipharma, personal fees for lectures and advisory board work, and travel expenses reimbursement Almirall, grants, personal fees for lectures and travel expenses reimbursement from Menarini, grants from Fondazione Maugeri, grants from Fondazione Chiesi Farmaceutici, outside the submitted work. Conflict of interest: R.J. Russell has nothing to disclose. Conflict of interest: D. Ryan reports personal fees for advisory board work from GSK and Trudell Medical, personal fees for advisory board work and lectures from AZ, personal fees for lectures from Mylan and Chiesi, personal fees for consultancy from Optimum Patient Care, outside the submitted work. Conflict of interest: K. Samitas has nothing to disclose. Conflict of interest: T. Tonia acts as ERS methodologist. Conflict of interest: E. Zervas reports personal fees consultancy and lectures from Astra, Bristol-Myers Squibb, Chiesi, GSK, Elpen, Merck, MSD, Novartis, Menarini and Pfizer, non-financial support for travel, accommodation and meeting expenses from Astra, Bristol-Myers Squibb, Galenica, Chiesi, Elpen, Novartis, Menarini and Roche, outside the submitted work. Conflict of interest: M. Gaga reports grants and personal fees from AZ, grants from BI, Elpen, Novartis and Menarini, personal fees from BMS, MSD, Chiesi and Pharmaten, outside the submitted work., (Copyright ©ERS 2019.)

Eosinophilic airway inflammation is observed in 30% to 50% of chronic cough sufferers. It is a common feature of asthma and upper airway cough syndrome, and it is required in the diagnosis of nonasthmatic eosinophilic bronchitis. Our understanding of the mechanisms underlying allergic and nonallergic eosinophilic inflammation have evolved tremendously in the last 2 decades, but the cause of this inflammation in any individual is often uncertain. Inhaled corticosteroids are the mainstay therapy for cough due to asthma or nonasthmatic eosinophilic bronchitis, and response is related to the presence of biomarkers of eosinophilic airway inflammation. In upper airway cough syndrome, nasal topical corticosteroids are beneficial in allergic rhinitis and chronic rhinosinusitis with polyposis. This review will describe the diagnosis, current and possible future treatments, and prognosis of chronic cough in adults with eosinophilic inflammation., (Copyright © 2019. Published by Elsevier Inc.)

Mutations in the gene encoding collagen VII cause the devastating blistering disease recessive dystrophic epidermolysis bullosa (RDEB). RDEB is characterized by severe skin fragility and nonhealing wounds aggravated by scarring and fibrosis. We previously showed that TSP1 is increased in RDEB fibroblasts. Because transforming growth factor-β (TGF-β) signaling is also increased in RDEB, and TSP1 is known to activate TGF-β, we investigated the role of TSP1 in TGF-β signaling in RDEB patient cells. Knockdown of TSP1 reduced phosphorylation of smad3 (a downstream target of TGF-β signaling) in RDEB primary fibroblasts, whereas overexpression of collagen VII reduced phosphorylation of smad3. Furthermore, inhibition of TSP1 binding to the LAP/TGF-β complex decreased fibrosis in engineered extracellular matrix formed by RDEB fibroblasts, as evaluated by picrosirius red staining and analyses of birefringent collagen fibrillar deposits. We show that collagen VII binds TSP1, which could potentially limit TSP1-LAP association and subsequent TGF-β activation. Our study suggests a previously unreported mechanism for increased TGF-β signaling in the absence of collagen VII in RDEB patient skin. Moreover, these data identify TSP1 as a possible target for reducing fibrosis in the tumor-promoting dermal microenvironment of RDEB patients., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The role of interleukin 13 in airway inflammation and remodelling in asthma is unclear. Tralokinumab is a human monoclonal antibody that neutralises interleukin 13. We aimed to evaluate whether tralokinumab would have an effect on airway eosinophilic infiltration, blood and sputum eosinophil concentrations, eosinophil activation, and airway remodelling., Methods: We did a multicentre, double-blind, randomised, placebo-controlled phase 2 trial at 15 centres across the UK, Denmark, and Canada. We enrolled participants of either sex aged 18-75 years with inadequately controlled moderate-to-severe asthma for 12 months or more, requiring treatment with inhaled corticosteroids at a stable dose. We randomly assigned participants (1:1) to receive tralokinumab (300 mg) or placebo by an interactive web-based system or voice response system. Participants and study personnel were masked to treatment allocation. Both tralokinumab and placebo were administered subcutaneously every 2 weeks. The primary outcome measure was change from baseline to week 12 in bronchial biopsy eosinophil count. Secondary outcome measures included change in blood and sputum eosinophil counts. Exploratory outcomes included fractional exhaled nitric oxide (FENO) and blood IgE concentrations. Safety analyses were carried out in all participants who received study drug. This trial is registered with ClinicalTrials.gov, number NCT02449473, and with the European Clinical Trials Database, EudraCT 2015-000857-19., Findings: Between Sept 25, 2015, and June 21, 2017, 224 participants were enrolled and screened. Of these participants, 79 were randomly assigned to receive tralokinumab (n=39) or placebo (n=40). Tralokinumab did not significantly affect bronchial eosinophil count compared with placebo at week 12 (treatment effect ratio 1·43, 95% CI 0·63-3·27; p=0·39). Compared with placebo, tralokinumab did not significantly affect blood eosinophil count (treatment effect ratio 1·21, 95% CI 1·00-1·48; p=0·055) or sputum eosinophil count (0·57, 0·06-6·00; p=0·63), but FENO concentration (0·78, 0·63-0·96; p=0·023) and total blood IgE concentration (0·86, 0·77-0·97; p=0·014) were significantly reduced. 33 (85%) of 39 patients receiving tralokinumab and 32 (80%) of 40 receiving placebo reported at least one adverse event during the treatment period. No deaths in either treatment group were observed. Treatment-related adverse events occurred more frequently in the tralokinumab group than in the placebo group (11 [28%] of 39 vs seven [18%] of 40)., Interpretation: Tralokinumab did not significantly affect eosinophilic inflammation in bronchial submucosa, blood, or sputum compared with placebo, but did reduce FENO and IgE concentrations. These results suggest interleukin 13 is not crucial for eosinophilic airway inflammation control in moderate-to-severe asthma., Funding: AstraZeneca., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bronchial thermoplasty is a treatment for asthma. It is currently unclear whether its histopathological impact is sufficiently explained by the proportion of airway wall that is exposed to temperatures necessary to affect cell survival.Airway smooth muscle and bronchial epithelial cells were exposed to media (37-70°C) for 10 s to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution post-thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 patients with severe asthma. In vitro airway smooth muscle and epithelial cell number decreased significantly following the addition of media heated to ≥65°C. In silico simulations showed a heterogeneous heat distribution that was amplified in larger airways, with <10% of the airway wall heated to >60°C in airways with an inner radius of ∼4 mm. In vivo at 6 weeks post-thermoplasty, there was an improvement in asthma control (measured via Asthma Control Questionnaire-6; mean difference 0.7, 95% CI 0.1-1.3; p=0.03), airway smooth muscle mass decreased (absolute median reduction 5%, interquartile range (IQR) 0-10; p=0.03) and epithelial integrity increased (14%, IQR 6-29; p=0.007). Neither of the latter two outcomes was related to improved asthma control.Integrated in vitro and in silico modelling suggest that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating, and nor did this reduction confer a greater improvement in asthma control., Competing Interests: Conflict of interest: R.M. Saunders reports grants from 7th EU Framework, Wellcome Trust and National Institute for Health Research, during the conduct of the study. Conflict of interest: A.H. Mansur has received an educational grant for service support from AstraZeneca Pharmaceuticals, and received fees for talks and advisory group contribution and conference attendance from Novartis, GlaxoSmithKline, AstraZeneca, Napp Pharmaceuticals, Boehringer Ingelheim and others, outside the submitted work. Conflict of interest: P.H. Howarth reports grants from the European Union (AirPROM collaborative grant), during the conduct of the study. Conflict of interest: R. Chaudhuri reports being an advisory board member for GlaxoSmithKline, AstraZeneca, Teva Pharmaceutical Industries and Novartis and receiving educational grants for her institute from Novartis; receiving fees for speaking at meetings organised by GlaxoSmithKline, AstraZeneca, Chiesi and for attending international conferences sponsored by Novartis, Teva Pharmaceutical Industries, AstraZeneca and Boehringer Ingelheim. Conflict of interest: S. Siddiqui reports personal fees for advisory board participation from AstraZeneca and Boehringer Ingelheim, personal fees for advisory/consulting from Owlstone Nanotech and Mundipharma, speaker fees from Novartis, grants for imaging research in asthma from Napp Pharmaceuticals, and speaker fees from the European Respiratory Society, outside the submitted work. Conflict of interest: C.E. Brightling has received, paid to his institution, grants and consultancy fees from GlaxoSmithKline, Novartis, Chiesi, MedImmune/AstraZeneca, Boehringer Ingelheim, MSD Pharmaceuticals, PrEP Biopharm, Vectura, Teva Pharmaceutical Industries, Sanofi, Regeneron and Roche/Genentech. Conflict of interest: I.L. Chernyavsky reports research fellowship support from the European Commission (FP7 AirPROM) and a grant from Medical Research Council UK (New Investigator Research Grant), during the conduct of the study., (Copyright ©ERS 2018.)

Asthma is a common chronic inflammatory condition of the airways affecting over 300 million people world-wide. In 5%-10% of cases, it is severe, with disproportionate healthcare resource utilization including costs associated with frequent exacerbations and the long-term health effects of systemic steroids. Characterization of inflammatory pathways in severe asthma has led to the development of targeted biological and small molecule therapies which aim to achieve disease control while minimizing corticosteroid-associated morbidity. Herein, we review currently licensed agents and those in development, and speculate how drug therapy for severe asthma might evolve and impact on clinical outcomes in the near future., (© 2018 John Wiley & Sons Ltd.)

The pathogenesis of asthma is complex and multi-faceted. Asthma patients have a diverse range of underlying dominant disease processes and pathways despite apparent similarities in clinical expression. Here, we present the current understanding of asthma pathogenesis. We discuss airway inflammation (both T2 HIGH and T2 LOW ), airway hyperresponsiveness (AHR) and airways remodelling as four key factors in asthma pathogenesis, and also outline other contributory factors such as genetics and co-morbidities. Response to current asthma therapies also varies greatly, which is probably related to the inter-patient differences in pathogenesis. Here, we also summarize how our developing understanding of detailed pathological processes potentially translates into the targeted treatment options we require for optimal asthma management in the future., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F 420 . Studies have shown that actinobacterial F 420 H 2 -dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis . These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F 420 H 2 . Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F 420 H - to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F 420 H - ) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F 420 H 2 -dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.

A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F 420 This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F 420 enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium Mycobacterium smegmatis Mutant strains unable to synthesize or reduce F 420 were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F 420 mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F 420 H 2 -dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F 420 H 2 lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F 420 loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F 420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F 420 H 2 -dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify. IMPORTANCE This study reveals that a unique microbial cofactor, F 420 , is critical for antimicrobial resistance in the environmental actinobacterium Mycobacterium smegmatis We show that a superfamily of redox enzymes, the F 420 H 2 -dependent reductases, can reduce diverse antimicrobials in vitro and in vivo M. smegmatis strains unable to make or reduce F 420 become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F 420 to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F 420 H 2 -dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis., (© Crown copyright 2016.)

Objectives: Combat casualty care is a complex system involving multiple clinicians, medical interventions and casualty transfers. Improving the performance of this system requires examination of potential weaknesses. This study reviewed the cause and timing of death of casualties deemed to have died from their injuries after arriving at a medical treatment facility during the recent conflicts in Iraq and Afghanistan, in order to identify potential areas for improving outcomes., Methods: This was a retrospective review of all casualties who reached medical treatment facilities alive, but subsequently died from injuries sustained during combat operations in Afghanistan and Iraq. It included all deaths from start to completion of combat operations. The UK military joint theatre trauma registry was used to identify cases, and further data were collected from clinical notes, postmortem records and coroner's reports., Results: There were 71 combat-related fatalities who survived to a medical treatment facility; 17 (24%) in Iraq and 54 (76%) in Afghanistan. Thirty eight (54%) died within the first 24 h. Thirty-three (47%) casualties died from isolated head injuries, a further 13 (18%) had unsurvivable head injuries but not in isolation. Haemorrhage following severe lower limb trauma, often in conjunction with abdominal and pelvic injuries, was the cause of a further 15 (21%) deaths., Conclusions: Severe head injury was the most common cause of death. Irrespective of available medical treatment, none of this group had salvageable injuries. Future emphasis should be placed in preventative strategies to protect the head against battlefield trauma., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: Washbasin U-bends are reservoirs of microbial contamination in healthcare environments. U-Bends are constantly full of water and harbour microbial biofilm., Aim: To develop an effective automated cleaning and disinfection system for U-bends using two solutions generated by electrochemical activation of brine including the disinfectant anolyte (predominantly hypochlorous acid) and catholyte (predominantly sodium hydroxide) with detergent properties., Methods: Initially three washbasin U-bends were manually filled with catholyte followed by anolyte for 5min each once weekly for five weeks. A programmable system was then developed with one washbasin that automated this process. This U-bend had three cycles of 5min catholyte followed by 5min anolyte treatment per week for three months. Quantitative bacterial counts from treated and control U-bends were determined on blood agar (CBA), R2A, PAS, and PA agars following automated treatment and on CBA and R2A following manual treatment., Findings: The average bacterial density from untreated U-bends throughout the study was >1×10(5) cfu/swab on all media with Pseudomonas aeruginosa accounting for ∼50% of counts. Manual U-bend electrochemically activated (ECA) solution treatment reduced counts significantly (<100cfu/swab) (P<0.01 for CBA; P<0.005 for R2A). Similarly, counts from the automated ECA-treatment U-bend were significantly reduced with average counts for 35 cycles on CBA, R2A, PAS, and PA of 2.1±4.5 (P<0.0001), 13.1±30.1 (P<0.05), 0.7±2.8 (P<0.001), and 0 (P<0.05) cfu/swab, respectively. P. aeruginosa was eliminated from all treated U-bends., Conclusion: Automated ECA treatment of washbasin U-bends consistently minimizes microbial contamination., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Large blast injuries during dismounted operations in southwest Afghanistan causing major limb amputations and perineal injuries associated with large blood volume resuscitation were associated with invasive fungal, primarily mold, infections. This article outlines the interventions undertaken to mitigate excess morbidity and mortality associated with invasive fungal infection. These interventions include defining the problem and associated risk with systemically collected and analyzed information, developing improved protective body armor for the thigh and perineal region, standardizing management through clinical practice guidelines that outlined risk, diagnostic and treatment recommendations with enhanced discussions on the weekly Theater Combat Casualty Care Conference that includes personnel from the combat zone, Germany, and the United States. The article concludes by explaining the key way forward with regarding an inner-war approach to sustained knowledge and skills.

Objectives: Decontaminating dental chair unit (DCU) suction systems in a convenient, safe and effective manner is problematic. This study aimed to identify and quantify the extent of the problems using 25 DCUs, methodically eliminate these problems and develop an efficient approach for reliable, effective, automated disinfection., Methods: DCU suction system residual contamination by environmental and human-derived bacteria was evaluated by microbiological culture following standard aspiration disinfection with a quaternary ammonium disinfectant or alternatively, a novel flooding approach to disinfection. Disinfection of multicomponent suction handpieces, assembled and disassembled, was also studied. A prototype manual and a novel automated Suction Tube Cleaning System (STCS) were developed and tested, as were novel single component suction handpieces., Results: Standard aspiration disinfection consistently failed to decontaminate DCU suction systems effectively. Semi-confluent bacterial growth (101-500 colony forming units (CFU) per culture plate) was recovered from up to 60% of suction filter housings and from up to 19% of high and 37% of low volume suction hoses. Manual and automated flood disinfection of DCU suction systems reduced this dramatically (ranges for filter cage and high and low volume hoses of 0-22, 0-16 and 0-14CFU/plate, respectively) (P<0.0001). Multicomponent suction handpieces could not be adequately disinfected without prior removal and disassembly. Novel single component handpieces, allowed their effective disinfection in situ using the STCS, which virtually eliminated contamination from the entire suction system., Conclusion: Flood disinfection of DCU suction systems and single component handpieces radically improves disinfection efficacy and considerably reduces potential cross-infection and cross-contamination risks., Clinical Significance: DCU suction systems become heavily contaminated during use. Conventional disinfection does not adequately control this. Furthermore, multicomponent suction handpieces cannot be adequately disinfected without disassembly, which is costly in time, staff and resources. The automated STCS DCU suction disinfection system used with single component handpieces provides an effective solution., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The current Tactical Combat Casualty Care (TCCC) Guidelines recommend parenteral promethazine as the single agent for the treatment of opioid-induced nausea and/or vomiting and give a secondary indication of "synergistic analgesic effect." Promethazine, however, has a well-documented history of undesired side effects relating to impairment and dysregulation of the central and autonomic nervous systems, such as sedation, extrapyramidal symptoms, dystonia, impairment of psychomotor function, neuroleptic malignant syndrome, and hypotension. These may be particularly worrisome in the combat casualty. Additionally, since 16 September 2009, there has been a US Food and Drug Administration (FDA) black box warning for the injectable form of promethazine, due to "the risk of serious tissue injury when this drug is administered incorrectly." Conversely, ondansetron, which is now available in generic form, has a well-established favorable safety profile and demonstrated efficacy in undifferentiated nausea and vomiting in the emergency department and prehospital settings. It has none of the central and autonomic nervous system side effects noted with promethazine and carries no FDA black box warning. Ondansetron is available in parenteral form and an orally disintegrating tablet, providing multiple safe and effective routes of administration. Despite the fact that it is an off-label use, ondansetron is being increasingly given for acute, undifferentiated nausea and vomiting and is presently being used in the field on combat casualties by some US and Allied Forces. Considering the risks involved with promethazine use, and the efficacy and safety of ondansetron and ondansetron?s availability in a generic form, we recommend removing promethazine from the TCCC Guidelines and replacing it with ondansetron., (2015.)

The low aqueous solubility and chiral complexity of synthetic pyrethroids, together with large differences between isomers in their insecticidal potency, have hindered the development of meaningful assays of their metabolism and metabolic resistance to them. To overcome these problems, Shan and Hammock (2001) [7] therefore developed fluorogenic and more water-soluble analogues of all the individual isomers of the commonly used Type 2 pyrethroids, cypermethrin and fenvalerate. The analogues have now been used in several studies of esterase-based metabolism and metabolic resistance. Here we test the validity of these analogues by quantitatively comparing their hydrolysis by a battery of 22 heterologously expressed insect esterases with the hydrolysis of the corresponding pyrethroid isomers by these esterases in an HPLC assay recently developed by Teese et al. (2013) [14]. We find a strong, albeit not complete, correlation (r = 0.7) between rates for the two sets of substrates. The three most potent isomers tested were all relatively slowly degraded in both sets of data but three esterases previously associated with pyrethroid resistance in Helicoverpa armigera did not show higher activities for these isomers than did allelic enzymes derived from susceptible H. armigera. Given their amenability to continuous assays at low substrate concentrations in microplate format, and ready detection of product, we endorse the ongoing utility of the analogues in many metabolic studies of pyrethroids., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H2) found in the Earth’s atmosphere.This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of Actinobacteria, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H2 to the reduction of ambient O2. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H2. In addition, it provides insight into the wider significance of hydrogen scavenging in global H2 cycling and soil microbial ecology.

Objectives: To use the UK Joint Theatre Trauma Registry (UK-JTTR) to identify service personnel sustaining traumatic brain injury (TBI) in recent conflicts and to examine injury characteristics, outcomes, and severity measures predictive of survival., Setting: Operations HERRICK (Afghanistan) and TELIC (Iraq)., Design: The UK-JTTR records data for every UK service person either killed on operations or treated by Defence Medical Services after a trauma call, including those evacuated for inpatient care following traumatic injury. UK-JTTR data were retrospectively analyzed to identify those who sustained TBI., Main Measures: The Mayo system was used to define TBI. Glasgow Coma Scale score, injury severity score, new injury severity score, trauma injury severity score, abbreviated injury scale, and a severity characterization of trauma were used to predict survival., Results: In total, 464 UK service personnel sustained TBI, representing 19% of the 2440 casualties in Afghanistan and Iraq, recorded in the UK-JTTR. Most TBI casualties had moderate-severe TBI (402, 87%). There were 181 (39%) survivors, 56% of these received neurorehabilitation. Improvised explosive devices accounted for 55% of TBIs sustained in Afghanistan and 31% of TBIs in Iraq. Logistic regression analyses were performed using the 412 cases (149 survivors: 263 fatalities) with scores on all severity measures. The best-fitting model was based on trauma injury severity score. A trauma injury severity score more than 11.13 indicates a more than 95% probability of survival., Conclusion: This is the first study of UK combat TBIs between 2003 and 2011. Almost 1 in 5 UK service personnel recorded in the UK-JTTR had TBI; most were moderate-severe. However, mild TBI is likely to be underrepresented in the UK-JTTR. These findings may be used to plan future rehabilitation needs, as almost half the survivors did not receive neurorehabilitation.

The administration of blood products to battlefield casualties in the prehospital arena has contributed significantly to the survival of critically injured patients in Afghanistan over the past 5 years. Given as part of an established military "chain of survival," blood product administration has represented a step-change improvement in capability for both UK and US tactical aeromedical evacuation (TACEVAC) platforms. The authors explore current concepts, analyzing and exploring themes associated with early use of blood products (fresh frozen plasma [FFP] and red blood cells [RBCs]), and they compare and evaluate a US/UK study analyzing the differences and recommending future strategy. The subject matter expert (SME) consensus guidelines developed for use by the US Army Air Ambulance units commonly known as call sign "DUSTOFF." These TACEVAC assets in Afghanistan were validated in this retrospective study. Using statistical analysis, the authors were able to ascertain that the current DUSTOFF SME-derived guidelines offer a sensitivity of 63.04% and a specificity of 89.07%. By adjusting the indicators to include a single above-ankle amputation with a systolic blood pressure (SBP) less than 90 mmHg and pulse greater than 120/min, the sensitivity could be increased to 67.39% while maintaining the specificity at 89.07%. In our data set, a single amputation above the ankle, in combination with an SBP of less than 100 mmHg and a pulse of greater than 120/min, increased the sensitivity to 76% but with a slight drop in specificity to 86%. Further study of military prehospital casualty data is under way to identify additional physiological parameters that will allow simple scoring tools in the remote setting to guide the administration of prehospital blood products., (2014.)

The metabolism of volatile signal molecules by odorant degrading enzymes (ODEs) is crucial to the ongoing sensitivity and specificity of chemoreception in various insects, and a few specific esterases, cytochrome P450s, glutathione S-transferases (GSTs) and UDP-glycosyltransferases (UGTs) have previously been implicated in this process. Significant progress has been made in characterizing ODEs in Lepidoptera but very little is known about them in Diptera, including in Drosophila melanogaster, a major insect model. We have therefore carried out a transcriptomic analysis of the antennae of D. melanogaster in order to identify candidate ODEs. Virgin male and female and mated female antennal transcriptomes were determined by RNAseq. As with the Lepidoptera, we found that many esterases, cytochrome P450 enzymes, GSTs and UGTs are expressed in D. melanogaster antennae. As olfactory genes generally show selective expression in the antennae, a comparison to previously published transcriptomes for other tissues has been performed, showing preferential expression in the antennae for one esterase, JHEdup, one cytochrome P450, CYP308a1, and one GST, GSTE4. These largely uncharacterized enzymes are now prime candidates for ODE functions. JHEdup was expressed heterologously and found to have high catalytic activity against a chemically diverse group of known ester odorants for this species. This is a finding consistent with an ODE although it might suggest a general role in clearing several odorants rather than a specific role in clearing a particular odorant. Our findings do not preclude the possibility of odorant degrading functions for other antennally expressed esterases, P450s, GSTs and UGTs but, if so, they suggest that these enzymes also have additional functions in other tissues., (Copyright © 2014 CSIRO. Published by Elsevier Ltd.. All rights reserved.)

Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Aim: The aim of the study was to explore how useful clinicians deployed to the Field Hospital in Afghanistan found using the four quadrant approach (4QA) as a tool to aid ethical decision-making. In addition, the study aimed to determine whether the 4QA needed to be amended to make it more effective in assisting the ethical decision-making process for military health professionals on deployment., Method: A qualitative pilot study in two phases was undertaken between September 2012 and January 2013. In Phase I, senior deployed clinicians completed a pro forma of the 4QA on cases that potentially raised ethical issues. Thirteen pro formas were submitted on four cases; the Deployed Medical Director submitted a log of 14 cases that had involved using the 4QA. Phase II consisted of interviews with five senior clinicians who had recently returned from deployment in Afghanistan to discuss their experiences and perceptions of using the 4QA., Results: Phase I identified a variation in the level of detail recorded and where that information was placed on the quadrant. Four themes were generated from Phase II. These included the characteristics of ethical decisions; the processes used to make ethical decisions; use, usefulness and limitations of the 4QA; and views about training in ethics. The findings suggested that amendments to the pro forma may improve its utility., Conclusions: The 4QA is a useful tool within an operational setting but amending its diagrammatic presentation could improve its effectiveness. Pre-deployment training should include practising using the quadrant as described in Clinical Guidelines for Operations. This is particularly important as the participants relied heavily on experience to help them make ethical decisions, and this experience may not be available in future operations outside Afghanistan., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.

The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this review, HDX-MS based workflows designed for protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS in protein therapeutics characterization are also described., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.

The influenza virus neuraminidase (NA) is essential for the virus life cycle. The rise of resistance mutations against current antiviral therapies has increased the need for the development of novel inhibitors. Recent efforts have targeted a cavity adjacent to the catalytic site (the 150-cavity) in addition to the primary catalytic subsite in order to increase specificity and reduce the likelihood of resistance. This study details structural and in vitro analyses of a class of inhibitors that bind uniquely in both subsites. Crystal structures of three inhibitors show occupation of the 150-cavity in two distinct and novel binding modes. We believe these are the first nanomolar inhibitors of NA to be characterized in this way. Furthermore, we show that one inhibitor, binding within the catalytic site, offers reduced susceptibility to known resistance mutations via increased flexibility of a pendant pentyloxy group and the ability to pivot about a strong hydrogen-bonding network.

Cleaning-in-place (CIP) for column chromatography plays an important role in therapeutic protein production. A robust and efficient CIP procedure ensures product quality, improves column life time and reduces the cost of the purification processes, particularly for those using expensive affinity resins, such as MabSelect protein A resin. Cleaning efficiency, resin compatibility, and facility compatibility are the three major aspects to consider in CIP process design. Cleaning MabSelect resin with 50mM sodium hydroxide (NaOH) along with 1M sodium chloride is one of the most popular cleaning procedures used in biopharmaceutical industries. However, high concentration sodium chloride is a leading cause of corrosion in the stainless steel containers used in large scale manufacture. Corroded containers may potentially introduce metal contaminants into purified drug products. Therefore, it is challenging to apply this cleaning procedure into commercial manufacturing due to facility compatibility and drug safety concerns. This paper reports a safe, effective and environmental and facility-friendly cleaning procedure that is suitable for large scale affinity chromatography. An alternative salt (sodium sulfate) is used to prevent the stainless steel corrosion caused by sodium chloride. Sodium hydroxide and salt concentrations were optimized using a high throughput screening approach to achieve the best combination of facility compatibility, cleaning efficiency and resin stability. Additionally, benzyl alcohol is applied to achieve more effective microbial control. Based on the findings, the recommended optimum cleaning strategy is cleaning MabSelect resin with 25 mM NaOH, 0.25 M Na2SO4 and 1% benzyl alcohol solution every cycle, followed by a more stringent cleaning using 50 mM NaOH with 0.25 M Na2SO4 and 1% benzyl alcohol at the end of each manufacturing campaign. A resin life cycle study using the MabSelect affinity resin demonstrates that the new cleaning strategy prolongs resin life time and consistently delivers high purity drug products., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The widely accepted paradigm for the development of insecticide resistance in field populations of insects is of selection for one or a very few genes of major effect. Limited genetic mapping data for organophosphate and pyrethroid resistance in heliothine and spodopteran pests generally agrees with this paradigm. However, other biochemical and transcriptomic data suggest a more complex set of changes in multiple P450 and esterase gene/enzyme systems in resistant strains of these species. We discuss possible explanations for this paradox, including the likely embedding of these genes in regulatory cascades and emerging evidence for their arrangement in large clusters of closely related genes. We conclude that there could indeed be an unusually large number of genetic options for evolving resistance in these species., (© 2013 Society of Chemical Industry.)

Ralstonia sp. strain GA3-3 is a hexachlorocyclohexane (HCH)-degrading bacterial strain isolated from suburban soil in Canberra, Australia. The genome of strain GA3-3 was sequenced to investigate its ability to degrade α-HCH. Here, we report the annotated genome sequence of this strain.

Pandoraea sp. strain SD6-2 is a δ-hexachlorocyclohexane-degrading bacterial strain isolated from lindane-contaminated soil in Queensland, Australia. The genome of SD6-2 was sequenced to investigate its ability to degrade δ-hexachlorocyclohexane. Here we report the annotated genome sequence of this strain.

Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10(6) M(-1) s(-1)) indicative of a natural substrate.

Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.

Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L⁻¹ of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC₅₀ values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: The objective of this study is to characterize modern point-of-injury (POI) en-route care platforms and to compare mortality among casualties evacuated with conventional military retrieval (CMR) methods to those evacuated with an advanced medical retrieval (AMR) capability., Background: Following a decade of war in Afghanistan, the impact of en-route care capabilities from the POI on mortality is unknown., Methods: Casualties evacuated from POI to one level III facility in Afghanistan (July 2008-March 2012) were identified from UK and US trauma registries. Groups comprised those evacuated by a medically qualified provider-led, AMR and those by a medic-led CMR capability. Outcomes were compared per incremental Injury Severity Score (ISS) bins., Results: Most casualties (n = 1054; 61.2%) were in the low-ISS (1-15) bracket in which there was no difference in en-route care time or mortality between AMR and CMR. Casualties in the mid-ISS bracket (16-50) (n = 583; 33.4%) experienced the same median en-route care time (minutes) on AMR and CMR platforms [78 (58) vs 75 (93); P = 0.542] although those on AMR had shorter time to operation [110 (95) vs 117 (126); P < 0.001]. In this mid-ISS bracket, mortality was lower in the AMR than in the CMR group (12.2% vs 18.2%; P = 0.035). In the high-ISS category (51-75) (n = 75; 4.6%), time to operation was lower in the AMR than the CMR group (66 ± 77 vs 113 ± 122; P = 0.013) but there was no difference in mortality., Conclusions: This study characterizes en-route care capabilities from POI in modern combat. Conventional platforms are effective in most casualties with low injury severity. However, a definable injury severity exists for which evacuation with an AMR capability is associated with improved survival.

Conserved tryptophan-187 facilitates homodimerization of the influenza A virus NS1 protein effector domain. We generated a mutant influenza virus strain expressing NS1-W187R to destabilize this self-interaction. NS1-W187R protein exhibited lower double-stranded RNA (dsRNA)-binding activity, showed a temporal redistribution during infection, and was minimally compromised for interferon antagonism. The mutant virus replicated similarly to the wild type in vitro, but it was slightly attenuated for replication in mice, causing notably reduced morbidity and mortality. These data suggest biological relevance for the W187-mediated homotypic interaction of NS1.

Human norovirus (NoV) is a major cause of acute gastroenteritis in closed settings such as hospitals, hotels and cruise ships. The virus survives on inanimate surfaces for extended periods of time, and environmental contamination has been implicated in its transmission. The disinfection of contaminated areas is important in controlling the spread of NoV infections. Neutral solutions of electrochemically activated (ECA)-anolyte have been shown to be powerful disinfectants against a broad range of bacterial pathogens. The active chemical ingredient is hypochlorous acid (HOCl), which is registered as an approved food contact surface sanitizer in the United States by the Environmental Protection Agency, pursuant to 40 CFR 180.940. We evaluated the antiviral activity of Ecasol (an ECA-anolyte) against feline calicivirus (FCV), a surrogate of NoV. FCV dried on plastic surfaces was exposed to Ecasol for 1, 2, or 5min. After exposure to Ecasol, the virus titers were compared with untreated controls to determine the virus inactivation efficacy after different contact times. Ecasol was found to decrease the FCV titer by >5log(10) within 1min of contact, indicating its suitability for inactivation of NoV on surfaces., (Copyright © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.)

Novel 3-C-alkylated-Neu5Ac2en derivatives have been designed to target the expanded active site cavity of influenza virus sialidases with an open 150-loop, currently seen in X-ray crystal structures of influenza A virus group-1 (N1, N4, N5, N8), but not group-2 (N2, N9), sialidases. The compounds show selectivity for inhibition of H5N1 and pdm09 H1N1 sialidases over an N2 sialidase, providing evidence of the relative 150-loop flexibility of these sialidases. In a complex with N8 sialidase, the C3 substituent of 3-phenylally-Neu5Ac2en occupies the 150-cavity while the central ring and the remaining substituents bind the active site as seen for the unsubstituted template. This new class of inhibitors, which can 'trap' the open 150-loop form of the sialidase, should prove useful as probes of 150-loop flexibility.

Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants' levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A series of C3 O-functionalized 2-acetamido-2-deoxy-Δ⁴-β-D-glucuronides were synthesized to explore noncharge interactions in subsite 2 of the influenza virus sialidase active site. In complex with A/N8 sialidase, the parent compound (C3 OH) inverts its solution conformation to bind with all substituents well positioned in the active site. The parent compound inhibits influenza virus sialidase at a sub-μM level; the introduction of small alkyl substituents or an acetyl group at C3 is also tolerated.

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation., (Copyright © 2012 The Protein Society.)

Esterases have been implicated in metabolic resistance to synthetic pyrethroids in several insect species but little is yet known of the molecular basis for these effects. In this work modern directed evolution technology was used to test to what extent it is possible to genetically enhance the pyrethroid hydrolytic activity of the E3 carboxylesterase from the blowfly Lucilia cuprina. High throughput screening of a random mutant library with individual stereoisomers of fluorogenic analogues of two type II pyrethroids identified 17 promising variants that were then also tested with the commercial pyrethroid deltamethrin. Between them, these variants displayed significantly improved activities for all the substrates tested. Amino acid substitutions at ten different residues were clearly implicated in the improvements, although most only enhanced activity for a subset of the stereoisomers. Several new combinations of the most promising amino acid substitutions were then made, and negative epistatic effects were found in most of the combinations, but significant improvements were also found in a minority of them. The best mutant recovered contained three amino acid changes and hydrolysed deltamethrin at more than 100 times the rate of wild-type E3. Structural analysis shows that nine of the ten mutated residues improving pyrethroid or analogue activities cluster in putative substrate binding pockets in the active site, with the three mutations of largest effect all increasing the volume of the acyl pocket., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Background: Contaminated washbasin taps and output water are an important source of bacteria that may cause nosocomial infection. A five-week pretreatment study of hot and cold water from 15 washbasin taps at Dublin Dental Hospital showed consistently heavy contamination by aerobic heterotrophic bacteria: mean bacterial counts of 482.5 [standard deviation (SD) 293] colony-forming units (cfu)/mL and 5022 (SD 4322) cfu/mL, respectively., Aim: To minimize microbial contamination of washbasin taps and output water in the long term using the electrochemically generated, pH-neutral disinfectant, Ecasol., Methods: Initially, the 15,000-L water tank providing cold and hot water to washbasins, calorifiers and the distribution network were drained and sediment was removed. The system was shock-dosed with Ecasol 100 ppm to eradicate gross contamination and biofilms. Thereafter, tank water was automatically maintained at Ecasol 2.5 ppm prior to distribution. The microbiological quality of water from five sentinel washbasin taps was monitored weekly for 54 weeks using R2A agar., Findings: The mean counts for hot, cold, mains and tank water during the 54-week study period were 1 (SD 4) cfu/mL, 2 (SD 4) cfu/mL, 205 (SD 160) cfu/mL and 0 cfu/mL, respectively. Swab samples of 33/40 taps, each tested on three separate occasions, yielded no growth on R2A agar, while five samples yielded <20 cfu/swab and two samples yielded >200 cfu/swab. No detrimental effects due to Ecasol were observed in the water network., Conclusion: Ecasol consistently minimized bacterial contamination of washbasin taps and output water in a dental hospital setting., (Copyright © 2012 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Objectives: Recent military campaigns in Iraq and Afghanistan have resulted in the treatment of children in British Medical facilities. In order to determine how care for children may develop in the future, it is necessary to understand the current situation. The aim of this article is to examine the pattern of paediatric trauma on recent operations in Iraq and Afghanistan., Methods: Data was requested from the Joint Theatre Trauma Registry, held at the Royal Centre for Defence Medicine in Birmingham, on all trauma calls for patients aged under 16 between the dates 21/3/03 and 31/8/09. Data included age, gender, theatre of operation, injury mechanism and type, trauma scores and destination of the child., Results: 176 children were identified with 16.5% from Iraq and 83.5% from Afghanistan. The overall survival rate was 88.6% with survival rates in Iraq of 89.7% and in Afghanistan of 88.4%. Males accounted for 66.5% of admissions and the commonest age group was age 6-8 years. In 59.1% of total admissions the mechanism of injury was related to explosives. This differed between theatres with explosive injury causing 27.6% of admissions in Iraq and 63.5% in Afghanistan. Injury Severity Scores (ISS) showed equal numbers of minor and severe injuries with fewer moderately injured patients. The median ISS of all data was nine. The median ISS from Iraq was 16 and the median ISS from Afghanistan was nine., Conclusions: The treatment of children in British medical facilities whilst deployed on operations is likely to continue. An assessment of the injury patterns of paediatric patients on current deployments allows development of training and an understanding of logistic requirements. Data collection will also need to be adapted to meet the needs of paediatric patients. These remain issues that are being addressed by the Defence Medical Services.