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Purpose: Asthma is a clinical syndrome with various underlying pathomechanisms and clinical phenotypes. Genetic, ethnic, and geographic factors may influence the differences in clinical presentation, severity, and prognosis. We compared the characteristics of asthma based on the geographical background by analyzing representative cohorts from the United States, Europe, South America, and Asia using the Severe Asthma Research Program (SARP), Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED), Program for Control of Asthma in Bahia (ProAR), and Cohort for Reality and Evolution of Adult Asthma in Korea (COREA), respectively., Methods: The clinical characteristics and medications for the SARP (n = 669), U-BIOPRED (n = 509), ProAR (n = 996), and COREA (n = 3,748) were analyzed. Subgroup analysis was performed for severe asthma., Results: The mean age was highest and lowest in the COREA and SARP, respectively. The asthma onset age was lowest in the ProAR. The mean body mass index was highest and lowest in the SARP and COREA, respectively. Baseline pulmonary function was lowest and highest in the U-BIOPRED and COREA, respectively. The number of patients with acute exacerbation in the previous year was highest in U-BIOPRED. The mean blood eosinophil count was highest in COREA. The total immunoglobulin E was highest in the ProAR. The frequency of atopy was highest in the SARP. The principal component analysis plot revealed differences among all cohorts., Conclusions: The cohorts from 4 different continents exhibited different clinical and physiological characteristics, probably resulting from the interplay between genetic susceptibility and geographical factors., Competing Interests: There are no financial or other issues that might lead to conflict of interest., (Copyright © 2024 The Korean Academy of Asthma, Allergy and Clinical Immunology • The Korean Academy of Pediatric Allergy and Respiratory Disease.)

Fibroblast growth factor receptor (FGFR) kinase inhibitors have been shown to be effective in the treatment of intrahepatic cholangiocarcinoma and other advanced solid tumors harboring FGFR2 alterations, but the toxicity of these drugs frequently leads to dose reduction or interruption of treatment such that maximum efficacy cannot be achieved. The most common adverse effects are hyperphosphatemia caused by FGFR1 inhibition and diarrhea due to FGFR4 inhibition, as current therapies are not selective among the FGFRs. Designing selective inhibitors has proved difficult with conventional approaches because the orthosteric sites of FGFR family members are observed to be highly similar in X-ray structures. In this study, aided by analysis of protein dynamics, we designed a selective, covalent FGFR2 inhibitor. In a key initial step, analysis of long-timescale molecular dynamics simulations of the FGFR1 and FGFR2 kinase domains allowed us to identify differential motion in their P-loops, which are located adjacent to the orthosteric site. Using this insight, we were able to design orthosteric binders that selectively and covalently engage the P-loop of FGFR2. Our drug discovery efforts culminated in the development of lirafugratinib (RLY-4008), a covalent inhibitor of FGFR2 that shows substantial selectivity over FGFR1 (~250-fold) and FGFR4 (~5,000-fold) in vitro, causes tumor regression in multiple FGFR2 -altered human xenograft models, and was recently demonstrated to be efficacious in the clinic at doses that do not induce clinically significant hyperphosphatemia or diarrhea., Competing Interests: Competing interests statement:Some authors are currently or have in the past been Relay employees. Some authors may own Relay stock. Relay (and some of the authors) have pending patents containing claims for lirafugratinib: World Intellectual Property Organization, WO2022109577 A1 2022-05-27; World Intellectual Property Organization, WO2020231990 A1 2020-11-19; US 11780845 B2 2023-10-10.

Allele-specific gene expression evolves rapidly on heteromorphic sex chromosomes. Over time, the accumulation of mutations on the Y chromosome leads to widespread loss of gametolog expression, relative to the X chromosome. It remains unclear if expression evolution on degrading Y chromosomes is primarily driven by mutations that accumulate through processes of selective interference, or if positive selection can also favor the down-regulation of coding regions on the Y chromosome that contain deleterious mutations. Identifying the relative rates of cis-regulatory sequence evolution across Y chromosomes has been challenging due to the limited number of reference assemblies. The threespine stickleback (Gasterosteus aculeatus) Y chromosome is an excellent model to identify how regulatory mutations accumulate on Y chromosomes due to its intermediate state of divergence from the X chromosome. A large number of Y-linked gametologs still exist across 3 differently aged evolutionary strata to test these hypotheses. We found that putative enhancer regions on the Y chromosome exhibited elevated substitution rates and decreased polymorphism when compared to nonfunctional sites, like intergenic regions and synonymous sites. This suggests that many cis-regulatory regions are under positive selection on the Y chromosome. This divergence was correlated with X-biased gametolog expression, indicating the loss of expression from the Y chromosome may be favored by selection. Our findings provide evidence that Y-linked cis-regulatory regions exhibit signs of positive selection quickly after the suppression of recombination and allow comparisons with recent theoretical models that suggest the rapid divergence of regulatory regions may be favored to mask deleterious mutations on the Y chromosome., Competing Interests: Conflict of Interest The authors declare no conflicts of interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Rationale: Patients with severe asthma are dependent upon treatment with high doses of inhaled corticosteroids (ICS) and often also oral corticosteroids (OCS). The extent of endogenous androgenic anabolic steroid (EAAS) suppression in asthma has not previously been described in detail. The objective of the present study was to measure urinary concentrations of EAAS in relation to exogenous corticosteroid exposure., Methods: Urine collected at baseline in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease outcomes) study of severe adult asthmatics (SA, n=408) was analysed by quantitative mass spectrometry. Data were compared to that of mild-to-moderate asthmatics (MMA, n=70) and healthy subjects (HC, n=98) from the same study., Measurements and Main Results: The concentrations of urinary endogenous steroid metabolites were substantially lower in SA than in MMA or HC. These differences were more pronounced in SA patients with detectable urinary OCS metabolites. Their dehydroepiandrosterone sulfate (DHEA-S) concentrations were <5% of those in HC, and cortisol concentrations were below the detection limit in 75% of females and 82% of males. The concentrations of EAAS in OCS-positive patients, as well as patients on high-dose ICS only, were more suppressed in females than males (p<0.05). Low levels of DHEA were associated with features of more severe disease and were more prevalent in females (p<0.05). The association between low EAAS and corticosteroid treatment was replicated in 289 of the SA patients at follow-up after 12-18 months., Conclusion: The pronounced suppression of endogenous anabolic androgens in females might contribute to sex differences regarding the prevalence of severe asthma., Competing Interests: Conflict of interest: V. Yasinska reports participation in advisory boards for AZ and GSK, and lecture honoraria from Sanofi and GSK. Conflict of interest: C. Gómez has nothing to disclose. Conflict of interest: J. Kolmert has nothing to disclose. Conflict of interest: M. Ericsson has nothing to disclose. Conflict of interest: A. Pohanka has nothing to disclose. Conflict of interest: A. James reports personal grant from Swedish Heart-Lung Foundation. Conflict of interest: L.I. Andersson has nothing to disclose. Conflict of interest: M. Sparreman-Mikus has nothing to disclose. Conflict of interest: A.R. Sousa reports employment and stocks or stock options from GSK. Conflict of interest: J.H. Riley has nothing to disclose. Conflict of interest: S. Bates has nothing to disclose. Conflict of interest: P.S. Bakke reports lecture honoraria from AstraZeneca and Boehringer Ingelheim. Conflict of interest: N. Zounemat Kermani has nothing to disclose. Conflict of interest: M. Caruso has nothing to disclose. Conflict of interest: P. Chanez reports participation in advisory boards, honoraria for consultancy, lectures fees and support for attending and/or travel from ALK, Almirall, AZ, Chiesi, GSK, Menarini, Novartis and Sanofi. Conflict of interest: S.J. Fowler has nothing to disclose. Conflict of interest: T. Geiser has nothing to disclose. Conflict of interest: P.H. Howarth reports employment and stocks or stock options from GSK. Conflict of interest: I. Horváth reports participation on an advisory board for AZ and Chiesi, honoraria for lectures from Chiesi and Roche, and support for attending and/or travel from Roche. Conflict of interest: N. Krug has nothing to disclose. Conflict of interest: P. Montuschi has nothing to disclose. Conflict of interest: M. Sanak has nothing to disclose. Conflict of interest: A. Behndig has nothing to disclose. Conflict of interest: D.E. Shaw has nothing to disclose. Conflict of interest: R.G. Knowles has nothing to disclose. Conflict of interest: B. Dahlén reports grant from GSK and Novartis. Conflict of interest: A-H. Maitland-van der Zee reports grants from BI, Vertex Innovation Award, Dutch Lung Foundation, Stichting Astma Bestrijding, IMI/3TR, EU grant ONELAB and EUROSTARS grant with Respiq, consulting fees from AZ and BI, and lecture honoraria from GSK. Conflict of interest: P.J. Sterk reports a grant from the Innovative Medicines Initiative. Conflict of interest: R. Djukanovic reports consulting fees from Synairgen, lecture honoraria from Regeneron, GSK and Kymab, and an advisory board for Synairgen. Conflict of interest: I.M. Adcock reports grant from EU-IMI, grants from GSK, MRK, EPSRC and Sanofi, consulting fees from GSK, Sanofi, Chiesi and Kinaset, lecture honoraria from AZ, Sanofi and Eurodrug, and payment for an educational event from Sunovion. Conflict of interest: K.F. Chung reports lectures honoraria from Novartis, AZ and Merck, advisory boards for GSK, AZ, Novartis, Roche, Merck, Rickett-Beckinson, Nocion and Shionogi, the Scientific Advisory Board of the Clean Breathing Institute supported by Haleon, grants from GSK, MRC and EPSRC, and support for travel from AZ. Conflict of interest: C.E. Wheelock has nothing to disclose. Conflict of interest: S-E. Dahlén reports research grants, consulting fees or lecture honoraria from AZ, Cayman Chemicals, GSK, Regeneron, Sanofi and Teva. Conflict of interest: E. Wikström Jonsson reports a research grant and expert assignment by Region Stockholm, and an expert appointment by the Swedish Medical Product Agency., (Copyright ©The authors 2023.)

To perform their physiological functions, amino methyl propionic acid receptors (AMPARs) cycle through active, resting, and desensitized states, and dysfunction in AMPAR activity is associated with various neurological disorders. Transitions among AMPAR functional states, however, are largely uncharacterized at atomic resolution and are difficult to examine experimentally. Here, we report long-timescale molecular dynamics simulations of dimerized AMPAR ligand-binding domains (LBDs), whose conformational changes are tightly coupled to changes in AMPAR functional states, in which we observed LBD dimer activation and deactivation upon ligand binding and unbinding at atomic resolution. Importantly, we observed the ligand-bound LBD dimer transition from the active conformation to several other conformations, which may correspond with distinct desensitized conformations. We also identified a linker region whose structural rearrangements heavily affected the transitions to and among these putative desensitized conformations, and confirmed, using electrophysiology experiments, the importance of the linker region in these functional transitions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable., (© 2023. The Author(s).)

Inward-rectifier potassium channels (Kirs) are lipid-gated ion channels that differ from other K+ channels in that they allow K+ ions to flow more easily into, rather than out of, the cell. Inward rectification is known to result from endogenous magnesium ions or polyamines (e.g., spermine) binding to Kirs, resulting in a block of outward potassium currents, but questions remain regarding the structural and dynamic basis of the rectification process and lipid-dependent channel activation. Here, we present the results of long-timescale molecular dynamics simulations starting from a crystal structure of phosphatidylinositol 4,5-bisphosphate (PIP2)-bound chicken Kir2.2 with a non-conducting pore. After introducing a mutation (G178R) that is known to increase the open probability of a homologous channel, we were able to observe transitions to a stably open, ion-conducting pore, during which key conformational changes occurred in the main activation gate and the cytoplasmic domain. PIP2 binding appeared to increase stability of the pore in its open and conducting state, as PIP2 removal resulted in pore closure, with a median closure time about half of that with PIP2 present. To investigate structural details of inward rectification, we simulated spermine binding to and unbinding from the open pore conformation at positive and negative voltages, respectively, and identified a spermine-binding site located near a previously hypothesized site between the pore cavity and the selectivity filter. We also studied the effects of long-range electrostatics on conduction and spermine binding by mutating charged residues in the cytoplasmic domain and found that a finely tuned charge density, arising from basic and acidic residues within the cytoplasmic domain, modulated conduction and rectification., (© 2022 Jogini et al.)

Electronic monitoring devices (EMDs) have been trialled in interventions to improve inhaled corticosteroid adherence and clinical outcomes. This study sought to understand the perceptions and experiences of EMD end-users. Participants recruited into a six-month EMD study were invited to a semi-structured interview. Interviews were audio-recorded, transcribed verbatim and analysed using the framework approach. Twenty-eight participants (68% female, median age 47) were interviewed. Individuals described feeling responsible for their asthma control. Recent attacks motivated a desire to maintain control. Study participation led to increased awareness of asthma status and medication use. Several individuals were open to integrating digital monitoring data with other mHealth inputs, perceiving the potential to enhance communication with clinicians and empower self-management. Openness to data sharing was tied to expectations of transparent data use. Data supported integrating beliefs and habit formation to achieve behaviour change. There was a willingness for an integrated, platform-based approach to digital self-management., (© 2022. The Author(s).)

Proteins that are expressed on membrane surfaces or secreted are involved in all aspects of cellular and organismal life, and as such require extremely high fidelity during their synthesis and maturation. These proteins are synthesized at the endoplasmic reticulum (ER) where a dedicated quality control system (ERQC) ensures only properly matured proteins reach their destinations. An essential component of this process is the identification of proteins that fail to pass ERQC and their retrotranslocation to the cytosol for proteasomal degradation. This study by Wu et al . reports a cryo-electron microscopy (cryo-EM) structure of the five-protein channel through which aberrant proteins are extracted from the ER, providing insights into how recognition of misfolded proteins is coupled to their transport through a hydrophobic channel that acts to thin the ER membrane, further facilitating their dislocation to the cytosol 1 ., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2022 Faculty Opinions Ltd.)

Background: Airway smooth muscle (ASM) cells are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyperresponsiveness and airway remodelling. The extracellular matrix (ECM) can influence tissue remodelling pathways; however, to date no study has investigated the effect of ASM ECM stiffness and cross-linking on the development of asthmatic airway remodelling. We hypothesised that transforming growth factor-β (TGF-β) activation by ASM cells is influenced by ECM in asthma and sought to investigate the mechanisms involved., Methods: This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF-β activation and expression of ECM cross-linking enzymes. Human bronchial biopsies from asthmatic and nonasthmatic donors were used to confirm lysyl oxidase like 2 (LOXL2) expression in ASM. A chronic ovalbumin (OVA) model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling., Results: We found that asthmatic ASM cells activated more TGF-β basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-β activated. Our data demonstrate that the ECM cross-linking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGF-β activation in vitro , and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an OVA mouse model of asthma., Conclusion: These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma., Competing Interests: Conflict of interest: J. Ramis has a patent “Industrial Synthesis of Modified Crosslinkable Biopolymer” pending. Conflict of interest: R. Middlewick has nothing to disclose. Conflict of interest: F. Pappalardo has nothing to disclose. Conflict of interest: J.T. Cairns has nothing to disclose. Conflict of interest: I.D. Stewart has nothing to disclose. Conflict of interest: A.E. John has nothing to disclose. Conflict of interest: S-U-N. Naveed has nothing to disclose. Conflict of interest: R. Krishnan has nothing to disclose. Conflict of interest: S. Miller has nothing to disclose. Conflict of interest: D.E. Shaw has nothing to disclose. Conflict of interest: C.E. Brightling has nothing to disclose. Conflict of interest: L. Buttery has nothing to disclose. Conflict of interest: F. Rose has nothing to disclose. Conflict of interest: G. Jenkins reports personal fees and other (sponsored research agreement paid to institution) from Biogen, GlaxoSmithKline and MedImmune, personal fees from Galapagos, Heptares, Boehringer Ingelheim, Pliant, Roche/InterMune, PharmAkea, Bristol Myers Squibb, Chiesi and Roche/Promedior, other (sponsored research agreement paid to institution) from Galecto, other (collaborative awards) from RedX and Nordic Biosciences, other (advisory board membership) from NuMedii, outside the submitted work; is supported by a National Institute of Health Research Professorship (RP-2017-08-ST2-014); and is a trustee for Action for Pulmonary Fibrosis. Conflict of interest: S.R. Johnson reports grants from the Medical Research Council (MRC), during the conduct of the study; grants from the National Institute of Health Research, MRC and Pfizer, personal fees from AstraZeneca, outside the submitted work. Conflict of interest: A.L. Tatler reports personal fees for consultancy from Pliant Therapeutics, outside the submitted work., (Copyright ©The authors 2022.)

Introduction: Asthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication., Methods: Baseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods., Results: A total of 90 metabolites were identified, with 40 significantly altered (p<0.05, false discovery rate <0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and patients with mild-to-moderate asthma differed significantly from those in patients with severe asthma (p=2.6×10 -20 ), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10 -4 ), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings., Conclusions: This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma., Competing Interests: Conflict of interest: S.N. Reinke reports grants from Canadian Institutes of Health Research, during the conduct of the study. Conflict of interest: S. Naz has nothing to disclose. Conflict of interest: R. Chaleckis has nothing to disclose. Conflict of interest: H. Gallart-Ayala has nothing to disclose. Conflict of interest: J. Kolmert reports personal fees for consultancy from Gesynta Pharma AB, outside the submitted work. Conflict of interest: N.Z. Kermani has nothing to disclose. Conflict of interest: A. Tiotiu has nothing to disclose. Conflict of interest: D.I. Broadhurst has nothing to disclose. Conflict of interest: A. Lundqvist has nothing to disclose. Conflict of interest: H. Olsson is an employee and shareholder of AstraZeneca. Conflict of interest: M. Ström has nothing to disclose. Conflict of interest: Å.M. Wheelock has nothing to disclose. Conflict of interest: C. Gómez has nothing to disclose. Conflict of interest: M. Ericsson has nothing to disclose. Conflict of interest: A.R. Sousa has nothing to disclose. Conflict of interest: J.H. Riley works for and own shares in GlaxoSmithKline. Conflict of interest: S. Bates is an employee of Johnson & Johnson and has previously worked for and holds stock in GlaxoSmithKline. Conflict of interest: J. Scholfield reports grants from Innovative Medicines Initiative, during the conduct of the study; and is director and employee of TopMD Precision Medicine Ltd. Conflict of interest: M. Loza is an employee of and owns stock in Johnson & Johnson. Conflict of interest: F. Baribaud is a shareholder of Johnson & Johnson and a current employee of Bristol Myers Squibb. Conflict of interest: P.S. Bakke reports personal fees for advisory board work and lectures from AstraZeneca, and personal fees for lectures from Novartis and Boehringer Ingelheim, outside the submitted work. Conflict of interest: M. Caruso has nothing to disclose. Conflict of interest: P. Chanez reports grants and personal fees from AstraZeneca, ALK, Boehringer Ingelheim, Chiesi, Sanofi-Aventis, Novartis and GlaxoSmithKline, outside the submitted work. Conflict of interest: S.J. Fowler reports personal fees from AstraZeneca, Novartis, TEVA and Chiesi, outside the submitted work. Conflict of interest: T. Geiser has nothing to disclose. Conflict of interest: P. Howarth has nothing to disclose. Conflict of interest: I. Horvath has nothing to disclose. Conflict of interest: N. Krug has nothing to disclose. Conflict of interest: P. Montuschi has nothing to disclose. Conflict of interest: A. Behndig has nothing to disclose. Conflict of interest: F. Singer reports personal fees from Vertex Pharmaceuticals (CH) and Novartis, outside the submitted work. Conflict of interest: J. Musial has nothing to disclose. Conflict of interest: D.E. Shaw has nothing to disclose. Conflict of interest: B. Dahlén reports personal fees for advisory board work and lectures from AstraZeneca, TEVA and Sanofi, and grants from Novartis and GlaxoSmithKline, outside the submitted work. Conflict of interest: S. Hu has nothing to disclose. Conflict of interest: J. Lasky-Su has nothing to disclose. Conflict of interest: P.J. Sterk reports a public private grant from the Innovative Medicines Initiative (IMI) covered by the EU and EFPIA, during the conduct of the study. Conflict of interest: K.F. Chung has received honoraria for participating in advisory board meetings of GlaxoSmithKline, AstraZeneca, Roche, Novartis, Merck, Nocion and Shionogi regarding treatments for asthma, COPD and chronic cough and has also been remunerated for speaking engagements. 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; and is a co-founder and current consultant, and has shares in Synairgen, a University of Southampton spin out company. Conflict of interest: S-E. Dahlén reports personal fees for consultancy from AstraZeneca, Cayman Chemical, GlaxoSmithKline, Novartis, Merck, Regeneron, Sanofi and TEVA, outside the submitted work. Conflict of interest: I.M. Adcock has nothing to disclose. Conflict of interest: C.E. Wheelock has nothing to disclose., (Copyright ©The authors 2022.)

Although molecular dynamics (MD) simulations have been used extensively to study the structural dynamics of proteins, the role of MD simulation in studies of nucleic acid based systems has been more limited. One contributing factor to this disparity is the historically lower level of accuracy of the physical models used in such simulations to describe interactions involving nucleic acids. By modifying nonbonded and torsion parameters of a force field from the Amber family of models, we recently developed force field parameters for RNA that achieve a level of accuracy comparable to that of state-of-the-art protein force fields. Here we report force field parameters for DNA, which we developed by transferring nonbonded parameters from our recently reported RNA force field and making subsequent adjustments to torsion parameters. We have also modified the backbone charges in both the RNA and DNA parameter sets to make the treatment of electrostatics compatible with our recently developed variant of the Amber protein and ion force field. We name the force field resulting from the union of these three parameter sets (the new DNA parameters, the revised RNA parameters, and the existing protein and ion parameters) DES-Amber . Extensive testing of DES-Amber indicates that it can describe the thermal stability and conformational flexibility of single- and double-stranded DNA systems with a level of accuracy comparable to or, especially for disordered systems, exceeding that of state-of-the-art nucleic acid force fields. Finally, we show that, in certain favorable cases, DES-Amber can be used for long-timescale simulations of protein-nucleic acid complexes.

Background: Exacerbation-prone asthma is a feature of severe disease. However, the basis for its persistency remains unclear., Objectives: To determine the clinical and transcriptomic features of frequent exacerbators (FEs) and persistent FEs (PFEs) in the U-BIOPRED cohort., Methods: We compared features of FE (≥2 exacerbations in past year) to infrequent exacerbators (IE, <2 exacerbations) and of PFE with repeat ≥2 exacerbations during the following year to persistent IE (PIE). Transcriptomic data in blood, bronchial and nasal epithelial brushings, bronchial biopsies and sputum cells were analysed by gene set variation analysis for 103 gene signatures., Results: Of 317 patients, 62.4% had FE, of whom 63.6% had PFE, while 37.6% had IE, of whom 61.3% had PIE. Using multivariate analysis, FE was associated with short-acting beta-agonist use, sinusitis and daily oral corticosteroid use, while PFE was associated with eczema, short-acting beta-agonist use and asthma control index. CEA cell adhesion molecule 5 (CEACAM5) was the only differentially expressed transcript in bronchial biopsies between PE and IE. There were no differentially expressed genes in the other four compartments. There were higher expression scores for type 2, T-helper type-17 and type 1 pathway signatures together with those associated with viral infections in bronchial biopsies from FE compared to IE, while there were higher expression scores of type 2, type 1 and steroid insensitivity pathway signatures in bronchial biopsies of PFE compared to PIE., Conclusion: The FE group and its PFE subgroup are associated with poor asthma control while expressing higher type 1 and type 2 activation pathways compared to IE and PIE, respectively., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are "cryptic": When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)-which performs its function by forming a PPI with its receptor-without incorporating any prior structural information about the ligands' binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein-small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2-small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: YS, VPM, AEL, ETK, and SS are former paid employees of D. E. Shaw Research; DES is the sole beneficial owner and Chief Scientist of D. E. Shaw Research.

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex or RTC) associated with two copies of nsp13 (nsp13 2 -RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp13 2 -RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp13 2 -RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Rationale: Asthma phenotyping requires novel biomarker discovery., Objectives: To identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, COPD subjects and healthy controls (HCs)., Methods: An antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HCs in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a 2-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED., Results: In U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HCs. 10 proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, TNF receptor superfamily member 11a, transforming growth factor-β and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, high-sensitivity C-reactive protein and body mass index, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation., Conclusions: The plasma proteomic panel revealed previously unexplored yet potentially useful Type-2-independent biomarkers and validated several proteins with established involvement in the pathophysiology of SA., Competing Interests: Conflict of interest: M. Sparreman Mikus has nothing to disclose. Conflict of interest: J. Kolmert reports personal fees from Gesynta Pharma AB. Conflict of interest: L.I. Andersson has nothing to disclose. Conflict of interest: J. Östling has nothing to disclose. Conflict of interest: R.G. Knowles has nothing to disclose. Conflict of interest: C. Gómez has nothing to disclose. Conflict of interest: M. Ericsson has nothing to disclose. Conflict of interest: J-O. Thörngren has nothing to disclose. Conflict of interest: P. Emami Khoonsari has nothing to disclose. Conflict of interest: B. Dahlén reports personal fees from AstraZeneca, Teva, Sanofi and grants from Novartis and GlaxoSmithKline outside the submitted work. Conflict of interest: M. Kupczyk has nothing to disclose. Conflict of interest: B. De Meulder report grants from the Innovative Medicines Initiative during the conduct of the study. Conflict of interest: C. Auffray report grants from the Innovative Medicines Initiative during the conduct of the study. Conflict of interest: P.S. Bakke reports personal fees from GlaxoSmithKline, AstraZeneca, Boehringer Ingelheim and Chiesi outside the submitted work. Conflict of interest: B. Beghe reports personal fees from AstraZeneca, Boehringer Ingelheim, Menarini and GlaxoSmithKline outside the submitted work. Conflict of interest: E.H. Bel reports grants and personal fees from GlaxoSmithKline, AstraZeneca, Novartis, TEVA, Sanofi/Regeneron, Chiesi, and Sterna outside the submitted work. Conflict of interest: M. Caruso has nothing to disclose. Conflict of interest: P. Chanez has nothing to disclose. Conflict of interest: B. Chawes has nothing to disclose. Conflict of interest: S.J. Fowler has nothing to disclose. Conflict of interest: M. Gaga reports grants and personal fees from Novartis, Menarini, Merck Sharp & Dohme, BMS, Galapagos, and AstraZeneca outside the submitted work. Conflict of interest: T. Geiser has nothing to disclose. Conflict of interest: M. Gjomarkaj has nothing to disclose. Conflict of interest: I. Horváth reports grants from EFPIA during the conduct of the study, and personal fees from AstraZeneca, GlaxoSmithKline, Novartis, Boehringer-Ingelheim, Sandoz, Teva and Chiesi outside the submitted work. Conflict of interest: P.H. Howarth has nothing to disclose. Conflict of interest: S.L. Johnston reports personal fees from Virtus Respiratory Research, Myelo Therapeutics GmbH, Concert Pharmaceuticals, Bayer, Synairgen, Novartis, Boehringer Ingelheim, Chiesi, Gerson Lehrman Group, resTORbio, Bioforce, Materia Medical Holdings, PrepBio Pharma, Pulmotect, Virion Health, Lallemand Pharma and AstraZeneca outside the submitted work. In addition, Sebastian L. Johnston also has three patents (Anti-virus therapy for respiratory diseases, UK patent application No. GB 0405634.7, Interferon-Beta for Anti-Virus Therapy for Respiratory Diseases, International Patent Application No. PCT/ GB05/50031 and Interferon Lambda therapy for the treatment of respiratory disease, UK patent application No.6779645.9). Conflict of interest: G. Joos reports grants and personal fees from AstraZeneca, Bayer, Chiesi, Eureca vzw, GlaxoSmithKline and Teva outside the submitted work. Conflict of interest: N. Krug has nothing to disclose. Conflict of interest: P. Montuschi has nothing to disclose. Conflict of interest: J. Musial has nothing to disclose. Conflict of interest: E. Niżankowska-Mogilnicka has nothing to disclose. Conflict of interest: H.K. Olsson reports other support from AstraZeneca outside the submitted work. Conflict of interest: A. Papi reports grants, personal fees, non-financial support and others from GlaxoSmithKline, AstraZeneca, Boehringer Ingelheim, Chiesi, Teva, Mundipharma, Zambon, Novartis, Menarini, Sanofi/Regeneron, Roche, Fondazione Salvatore Maugeri, Chiesi and Edmond pharma outside the submitted work. Conflict of interest: K.F. Rabe reports grants and personal fees from AstraZeneca, Boehringer Ingelheim, Sanofi Aventis, MERCK SHARP & DOHME, Novartis, Orion Cooperation, Berlin Chemie, Roche, Chiesi and grants for research from the Ministry of Education and Science, Germany. Conflict of interest: T. Sandström has nothing to disclose. Conflict of interest: D.E. Shaw reports personal fees and non-financial support from GlaxoSmithKline, Novartis and AstraZeneca outside the submitted work. Conflict of interest: N.M. Siafakas has nothing to disclose. Conflict of interest: M. Uhlen has nothing to disclose. Conflict of interest: J.H. Riley is an employee and shareholder in GlaxoSmithKline. Conflict of interest: S. Bates is an employee and shareholder in GlaxoSmithKline. Conflict of interest: R.J.M. Middelveld reports grants from the Swedish Strategic Research Foundation, AstraZeneca, the Swedish Heart Lung Foundation and the Swedish Asthma and Allergy Association outside the submitted work. Conflict of interest: C.E. Wheelock has nothing to disclose. Conflict of interest: K.F. Chung reports grants and personal fees from GlaxoSmithKline, AstraZeneca, Novartis, Merck, Boehringer Ingelheim, Roche and Shionogi outside the submitted work. Conflict of interest: I.M. Adcock has nothing to disclose. Conflict of interest: P.J. Sterk reports grants to Amsterdam UMC from the public private Innovative Medicines Initiative (IMI) covered by the European Union (EU) and the European Federation of Pharmaceutical Industries and Associations (EFPIA) during the conduct of the study. Conflict of interest: R. Djukanovic reports receiving fees for lectures at symposia organised by Novartis, GlaxoSmithKline, AstraZeneca and Teva, consultation fees from Teva and Novartis; he is a co-founder and current consultant and has shares in Synairgen. Conflict of interest: P. Nilsson has nothing to disclose. Conflict of interest: S-E. Dahlén reports personal fees from AstraZeneca, Cayman Chemicals, GlaxoSmithKline, Novartis, Regeneron, Sanofi and Teva outside the submitted work. Conflict of interest: A. James has nothing to disclose., (Copyright ©The authors 2022.)

Effective inactivation of the HER2-HER3 tumor driver has remained elusive because of the challenging attributes of the pseudokinase HER3. We report a structure-function study of constitutive HER2-HER3 signaling to identify opportunities for targeting. The allosteric activation of the HER2 kinase domain (KD) by the HER3 KD is required for tumorigenic signaling and can potentially be targeted by allosteric inhibitors. ATP binding within the catalytically inactive HER3 KD provides structural rigidity that is important for signaling, but this is mimicked, not opposed, by small molecule ATP analogs, reported here in a bosutinib-bound crystal structure. Mutational disruption of ATP binding and molecular dynamics simulation of the apo KD of HER3 identify a conformational coupling of the ATP pocket with a hydrophobic AP-2 pocket, analogous to EGFR, that is critical for tumorigenic signaling and feasible for targeting. The value of these potential target sites is confirmed in tumor growth assays using gene replacement techniques., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Inhaled corticosteroids (ICS), prednisolone and antibiotics all play a crucial role in the management of respiratory diseases. The aim of this study was to analyse whether the declaration of the COVID-19 pandemic affected prescribing rates, as public health measures were implemented to reduce transmission of SARS-CoV-2. Monthly practise-level prescribing data published by NHS Digital were analysed. At the point, the COVID-19 outbreak was declared a pandemic, ICS prescriptions rose significantly. This was followed by a decrease in ICS and prednisolone prescribing in the following months. There was no difference in the antibiotic prescribing trend., (© The Author(s) 2021. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs., (© 2021. The Author(s).)

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Understanding the potential deleterious effects of corticosteroids on bone health in people with asthma is important when making treatment decisions. There is a need for clearer evidence to better quantify the risk and effect size., Methods: Databases were systematically searched to identify studies reporting on bone mineral density (BMD) measurement and risk of osteoporosis or fracture, comparing people with asthma exposed to inhaled (ICS) or oral (OCS) corticosteroids, with nonexposed people with asthma and healthy controls. Data were narratively synthesized, and a series of meta-analyses were performed using the random-effects inverse variance method., Results: This review consists of 28 studies (six randomized control trials and 22 observational). There was no effect of ICS on bone loss both at spine and femoral neck in asthma. People with asthma receiving OCS were at greater risk of osteoporosis than nonexposed people with asthma (pooled HR = 1.76; 95%CI: 1.48 to 2.09; I 2 =68%). Similarly, higher ICS exposure was associated with higher risk of osteoporosis (OR = 1.63; 95%CI: 1.33 to 1.99) and fracture (pooled OR = 1.19; 95%CI: 1.05 to 1.35; I 2 =0%) when comparing people with asthma receiving ICS and not., Conclusion: Patients with asthma exposed to OCS or high ICS doses become more susceptible to bone comorbidities. Striking the right balance between efficacy and safety of steroids in asthma is important to improve patients' quality of life., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A minority of patients presenting to hospital with COVID-19 have bacterial co-infection. Procalcitonin testing may help identify patients for whom antibiotics should be prescribed or withheld. This study describes the use of procalcitonin in English and Welsh hospitals during the first wave of the COVID-19 pandemic. A web-based survey of antimicrobial leads gathered data about the use of procalcitonin testing. Responses were received from 148/151 (98%) eligible hospitals. During the first wave of the COVID-19 pandemic, there was widespread introduction and expansion of PCT use in NHS hospitals. The number of hospitals using PCT in emergency/acute admissions rose from 17 (11%) to 74/146 (50.7%) and use in Intensive Care Units (ICU) increased from 70 (47.6%) to 124/147 (84.4%). This increase happened predominantly in March and April 2020, preceding NICE guidance. Approximately half of hospitals used PCT as a single test to guide decisions to discontinue antibiotics and half used repeated measurements. There was marked variation in the thresholds used for empiric antibiotic cessation and guidance about interpretation of values. Procalcitonin testing has been widely adopted in the NHS during the COVID-19 pandemic in an unevidenced, heterogeneous way and in conflict with relevant NICE guidance. Further research is needed urgently that assesses the impact of this change on antibiotic prescribing and patient safety.

Advances in computational chemistry create an ongoing need for larger and higher-quality datasets that characterize noncovalent molecular interactions. We present three benchmark collections of quantum mechanical data, covering approximately 3,700 distinct types of interacting molecule pairs. The first collection, which we refer to as DES370K, contains interaction energies for more than 370,000 dimer geometries. These were computed using the coupled-cluster method with single, double, and perturbative triple excitations [CCSD(T)], which is widely regarded as the gold-standard method in electronic structure theory. Our second benchmark collection, a core representative subset of DES370K called DES15K, is intended for more computationally demanding applications of the data. Finally, DES5M, our third collection, comprises interaction energies for nearly 5,000,000 dimer geometries; these were calculated using SNS-MP2, a machine learning approach that provides results with accuracy comparable to that of our coupled-cluster training data. These datasets may prove useful in the development of density functionals, empirically corrected wavefunction-based approaches, semi-empirical methods, force fields, and models trained using machine learning methods.

While the cost and time for assembling a genome has drastically decreased, it still remains a challenge to assemble a highly contiguous genome. These challenges are rapidly being overcome by the integration of long-read sequencing technologies. Here, we use long-read sequencing to improve the contiguity of the threespine stickleback fish (Gasterosteus aculeatus) genome, a prominent genetic model species. Using Pacific Biosciences sequencing, we assembled a highly contiguous genome of a freshwater fish from Paxton Lake. Using contigs from this genome, we were able to fill over 76.7% of the gaps in the existing reference genome assembly, improving contiguity over fivefold. Our gap filling approach was highly accurate, validated by 10X Genomics long-distance linked-reads. In addition to closing a majority of gaps, we were able to assemble segments of telomeres and centromeres throughout the genome. This highlights the power of using long sequencing reads to assemble highly repetitive and difficult to assemble regions of genomes. This latest genome build has been released through a newly designed community genome browser that aims to consolidate the growing number of genomics datasets available for the threespine stickleback fish., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Introduction: Osteoporosis and fragility fractures are associated with corticosteroids which are the mainstay treatment for asthma; however, these bone comorbidities within asthma need to be better described., Methods: A matched cohort study was conducted using the UK Clinical Practice Research Database (CPRD). Adults with an incident asthma code were identified and matched, with up to four randomly selected people without asthma, by age, sex and practice. Osteoporosis and fragility fracture incidence rates were calculated, and Cox regression was performed comparing hazard rates to the general population. We report the impact of age, sex, glucocorticoids and the risk of specific fractures., Results: Patients with asthma had a higher risk of osteoporosis (adjusted hazard ratio (aHR) 1.18, 95% CI 1.13-1.23) and were 12% (aHR 1.12, 95% CI 1.07-1.16) more likely to sustain fragility fractures than the general population. Age modified the effect of asthma on osteoporosis and fragility fractures, such that the effect was stronger in younger people (p interaction <0.0001). The vertebra (aHR 1.40, 95% CI 1.33-1.48) and forearm/wrist (aHR 1.27, 95% CI 1.22-1.32) were the sites linked with a larger incidence. A dose-response relationship between oral corticosteroids (OCS) and osteoporosis was observed, whereas the risk of fragility fractures increased in those with six or more OCS courses per year. Regular use of inhaled corticosteroids (ICS) increased the risk of both bone conditions., Conclusions: Patients with asthma are more likely to develop osteoporosis or sustain fragility fractures than the general population, with a particular concern in younger people and those more frequently using OCS and ICS., Competing Interests: Conflict of interest: C.V. Chalitsios has nothing to disclose. Conflict of interest: T.M. McKeever has nothing to disclose. Conflict of interest: D.E. Shaw has nothing to disclose., (Copyright ©ERS 2021.)

Exposure to oral corticosteroids (OCS) is associated with an increased risk of osteoporosis and fragility fractures. Guidelines suggest bisphosphonate (BP) therapy as the first-line treatment of glucocorticoid-induced osteoporosis (GIOP). This population study used publicly available data, including prescription annual cost analysis and monthly practice-level data. Our aim was to examine the prescribing of OCS and BP at practice level and investigate reasons for variation using a mixed-effect negative binomial regression analysis. There was a rise in OCS and BP prescriptions of 55% and 1200% from 1998 to 2018, respectively. Of the 6586 included practices, the median (IQR) of OCS and BP prescriptions were 120.8 (84.8-160.4) and 107.7 (73.8-147.4) per 1000 patients, respectively. Asthma and chronic obstructive pulmonary disease (COPD) were significantly associated with OCS use (p < 0.0001), but only COPD was associated with BP use (p < 0.0001). Higher OCS prescribing rates were associated with higher BP prescribing rates (5th to 1st quintile-IRR = 1.99; 95% CI: 1.88-2.10). Practice list size, deprivation and advanced age were all associated with both drugs (p < 0.0001). In conclusion, although OCS use is positively associated with BP prescription, variation among practices and CCGs exists. The variation in prescribing suggests there is still a need to improve GIOP prevention.

Bruton's tyrosine kinase (Btk) is critical for B cell proliferation and activation, and the development of Btk inhibitors is a vigorously pursued strategy for the treatment of various B cell malignancies. A detailed mechanistic understanding of Btk activation has, however, been lacking. Here, inspired by a previous suggestion that Btk activation might depend on dimerization of its lipid-binding PH-TH module on the cell membrane, we performed long-timescale molecular dynamics simulations of membrane-bound PH-TH modules and observed that they dimerized into a single predominant conformation. We found that the phospholipid PIP 3 stabilized the dimer allosterically by binding at multiple sites, and that the effects of PH-TH mutations on dimer stability were consistent with their known effects on Btk activity. Taken together, our simulation results strongly suggest that PIP 3 -mediated dimerization of Btk at the cell membrane is a critical step in Btk activation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Gastro-oesophageal reflux disease (GORD) has long been associated with poor asthma control without an established cause-effect relationship. 610 asthmatics (421 severe/88 mild-moderate) and 101 healthy controls were assessed clinically and a subset of 154 severe asthmatics underwent proteomic analysis of induced sputum using untargeted mass spectrometry, LC-IMS-MS E . Univariate and multiple logistic regression analyses (MLR) were conducted to identify proteins associated with GORD in this cohort. When compared to mild/moderate asthmatics and healthy individuals, respectively, GORD was three- and ten-fold more prevalent in severe asthmatics and was associated with increased asthma symptoms and oral corticosteroid use, poorer quality of life, depression/anxiety, obesity and symptoms of sino-nasal disease. Comparison of sputum proteomes in severe asthmatics with and without active GORD showed five differentially abundant proteins with described roles in anti-microbial defences, systemic inflammation and epithelial integrity. Three of these were associated with active GORD by multiple linear regression analysis: Ig lambda variable 1-47 (p = 0·017) and plasma protease C1 inhibitor (p = 0·043), both in lower concentrations, and lipocalin-1 (p = 0·034) in higher concentrations in active GORD. This study provides evidence which suggests that reflux can cause subtle perturbation of proteins detectable in the airways lining fluid and that severe asthmatics with GORD may represent a distinct phenotype of asthma., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Despite the biological importance of protein-protein complexes, determining their structures and association mechanisms remains an outstanding challenge. Here, we report the results of atomic-level simulations in which we observed five protein-protein pairs repeatedly associate to, and dissociate from, their experimentally determined native complexes using a molecular dynamics (MD)-based sampling approach that does not make use of any prior structural information about the complexes. To study association mechanisms, we performed additional, conventional MD simulations, in which we observed numerous spontaneous association events. A shared feature of native association for these five structurally and functionally diverse protein systems was that if the proteins made contact far from the native interface, the native state was reached by dissociation and eventual reassociation near the native interface, rather than by extensive interfacial exploration while the proteins remained in contact. At the transition state (the conformational ensemble from which association to the native complex and dissociation are equally likely), the protein-protein interfaces were still highly hydrated, and no more than 20% of native contacts had formed., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Type-2 (T2) immune responses in airway epithelial cells (AECs) classifies mild-moderate asthma into a T2-high phenotype. We examined whether currently available clinical biomarkers can predict AEC-defined T2-high phenotype within the U-BIOPRED cohort.The transcriptomic profile of AECs obtained from brushings of 103 patients with asthma and 44 healthy controls was obtained and gene set variation analysis used to determine the relative expression score of T2 asthma using a signature from interleukin (IL)-13-exposed AECs.37% of asthmatics (45% nonsmoking severe asthma, n=49; 33% of smoking or ex-smoking severe asthma, n=18; and 28% mild-moderate asthma, n=36) were T2-high using AEC gene expression. They were more symptomatic with higher exhaled nitric oxide fraction ( F eNO ) and blood and sputum eosinophils, but not serum IgE or periostin. Sputum eosinophilia correlated best with the T2-high signature. F eNO (≥30 ppb) and blood eosinophils (≥300 cells·µL -1 ) gave a moderate prediction of T2-high asthma. Sputum IL-4, IL-5 and IL-13 protein levels did not correlate with gene expression.T2-high severe asthma can be predicted to some extent from raised levels of F eNO , blood and sputum eosinophil counts, but serum IgE or serum periostin were poor predictors. Better bedside biomarkers are needed to detect T2-high., Competing Interests: Conflict of interest: S. Pavlidis has nothing to disclose. Conflict of interest: K. Takahashi reports personal fees from Asahi General Hospital, during the conduct of the study. Conflict of interest: F. Ng Kee Kwong has nothing to disclose. Conflict of interest: J. Xie has nothing to disclose. Conflict of interest: U. Hoda has nothing to disclose. Conflict of interest: K. Sun has nothing to disclose. Conflict of interest: V. Elyasigomari has nothing to disclose. Conflict of interest: P. Agapow has nothing to disclose. Conflict of interest: M. Loza is employed by Janssen R&D and owns stock in parent company Johnson & Johnson, outside the submitted work. Conflict of interest: F. Baribaud reports being an employee of Janssen R&D and being a share holder of Johnson & Johnson. Conflict of interest: P. Chanez has provided consultancy services for Boehringer Ingelheim, Johnson & Johnson, GlaxoSmithKline, Merck Sharp & Dohme, AstraZeneca, Novartis, Teva, Chiesi, Sanofi and SNCF; served on advisory boards for Almirall, Boehringer Ingelheim, Johnson & Johnson, GlaxoSmithKline, AstraZeneca, Novartis, Teva, Chiesi and Sanofi; received lecture fees from Boehringer Ingelheim, Centocor, GlaxoSmithKline, AstraZeneca, Novartis, Teva, Chiesi, Boston Scientific and ALK; and received industry-sponsored grants from Roche, Boston Scientific, Boehringer Ingelheim, Centocor, GlaxoSmithKline, AstraZeneca, ALK, Novartis, Teva, ABscience and Chiesi. Conflict of interest: S.J. Fowler reports grants from IMI, during the conduct of the study; personal fees from GlaxoSmithKline, AstraZeneca, Boehringer Ingelheim and Novartis, outside the submitted work. Conflict of interest: D.E. Shaw reports personal fees for advisory board work from GSK, AZ, Teva and Boehringer Ingelheim, outside the submitted work. Conflict of interest: L.J. Fleming reports personal fees for advisory board work from Novartis, Vectura and Boehringer Ingelheim, grants from Asthma UK and BLF, and personal fees for speaking engagements from Novartis, outside the submitted work. Conflict of interest: P.H. Howarth reports grants from IMI, during the conduct of the study; part-time employment by GSK as Global Medical Expert, outside the submitted work. Conflict of interest: A.R. Sousa has nothing to disclose. Conflict of interest: J. Corfield has nothing to disclose. Conflict of interest: C. Auffray reports grants (U-BIOPRED Consortium IMI number 115010) from Innovative Medicine Initiative, during the conduct of the study. Conflict of interest: B. De Meulder reports grants (U-BIOPRED Consortium IMI number 115010) from Innovative Medicine Initiative, during the conduct of the study. Conflict of interest: R. Knowles is an employee of Knowles Consulting Ltd, outside the submitted work; and is a former employee (to 2011) and current shareholder in GlaxoSmithKline Ltd, and drug discovery consultant for Knowles Consulting Ltd, Peptinnovate Ltd and Imperial College London. Conflict of interest: P.J. Sterk reports grants from Innovative Medicines Initiative (IMI), during the conduct of the study. Conflict of interest: Y. Guo has nothing to disclose. Conflict of interest: I.M. Adcock reports personal fees for advisory board work from GSK, A-Z, Novartis, Boehringer Ingelheim and Vectura, grants from Pfizer, GSK, MRC, EU, BI and IMI, and personal fees for speaking from AZ and BI, outside the submitted work. Conflict of interest: R. Djukanovic reports personal fees for lectures at company sponsored symposia and consulting on advisory boards from TEVA, grants and personal fees for lectures at company sponsored symposia and consulting on advisory boards from Novartis, and personal fees for consultancy from and hold shares in Synairgen, outside the submitted work. Conflict of interest: K.F. Chung reports grants and personal fees for advisory board work from GlaxoSmithKline, personal fees for advisory board work and speaking from AstraZeneca and Novartis, grants and personal fees for advisory board work and speaking from Merck, and personal fees for advisory board work from Boehringer Ingelheim, TEVA and Menlo Therapeutics, outside the submitted work., (Copyright ©ERS 2019.)

Chaperonins (ubiquitous facilitators of protein folding) sequester misfolded proteins within an internal cavity, thus preventing protein aggregation during the process of refolding. GroEL, a tetradecameric bacterial chaperonin, is one of the most studied chaperonins, but the role of the internal cavity in the refolding process is still unclear. It has been suggested that rather than simply isolating proteins while they refold, the GroEL cavity actively promotes protein folding. A detailed characterization of the folding dynamics and thermodynamics of protein substrates encapsulated within the cavity, however, has been difficult to obtain by experimental means, due to the system's complexity and the many steps in the folding cycle. Here, we examine the influence of the GroEL cavity on protein folding based on the results of unbiased, atomistic molecular dynamics simulations. We first verified that the computational setup, which uses a recently developed state-of-the-art force field that more accurately reproduces the aggregation propensity of unfolded states, could recapitulate the essential structural dynamics of GroEL. In these simulations, the GroEL tetradecamer was highly dynamic, transitioning among states corresponding to most of the structures that have been observed experimentally. We then simulated a small, unfolded protein both in the GroEL cavity and in bulk solution and compared the protein's folding process within these two environments. Inside the GroEL cavity, the unfolded protein interacted strongly with the disordered residues in GroEL's C-terminal tails. These interactions stabilized the protein's unfolded states relative to its compact states and increased the roughness of its folding free-energy surface, resulting in slower folding compared to the rate in solution. For larger proteins, which are more typical GroEL substrates, we speculate that these interactions may allow substrates to more quickly escape kinetic traps associated with compact, misfolded states, thereby actively promoting folding.

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.

Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF2α and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF2α; IS-transcriptomics; BB-transcriptomics; BBr-transcriptomics). Urinary 8-iso-PGF2α was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF2α was increased in SAs/ex, median (IQR) = 31.7 (24.5-44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6-36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4-47.7), vs. ESA, median (IQR) = 29.4 (22.3-40.5), and NSA, median (IQR) = 26.5 (19.6-16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922&#95;PM&#95;s&#95;at fold-change = 1.05 p = 0.006; 203923&#95;PM&#95;s&#95;at fold-change = 1.06, p = 0.003; 233538&#95;PM&#95;s&#95;at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF2α in the smokers/ex-smokers group. Trial registration ClinicalTrials.gov-Identifier: NCT01976767., Competing Interests: M. Caruso, R. Emma, A.T. Bansal, C.E. Wheelock, M.J. Loza, L. Fabbella, T. Sandstrom, F. Singer, A.R. Sousa, D.E. Shaw, R. Lutter, S.J. Fowler, I. Horvath, I. Pandis, P. Montuschi declare non conflict of interest. J. Kolmert, B. De Meulder, D. Lefaudeux, C. Auffray, P.J. Sterk, N. Krug, M. Sanak, J. Corfield declare grants from IMI (Innovative Medicine Initiatives) during the conduct of the study. S.E. Dahlen declares financial supporting by Swedish Research Academic Foundations, RSPS Incentive, AZ, Teva and GSK consultancies. B. Dahlen declares consultancies for TEVA. P. Chanez reports grants and personal fees from Almirall, BI, Centocor, GSK, MSD, AstraZeneca, Novartis, Teva, Chiesi, Schering Plough, outside the submitted work. P.S. Bakke reports personal fees from GlaxoSmithKline, Boehringer-Ingelheim, AstraZeneca, Orionpharma, Mundipharma, outside the submitted work. R. Djukanovic reports receiving fees for lectures at symposia organised by Novartis and TEVA and consultation for these two companies as member of advisory boards. He is a co-founder and current consultant, and has shares in Synairgen, a University of Southampton spin out company. P.H. Howarth reports fees from GSK, outside the submitted work. L. Fleming reports personal fees and other from Vectura, Novartis, Boehringer ingelheim, outside the submitted work. I. Adcock reports grants from EU-IMI, during the conduct of the study; grants from MRC, BHF, Dunhill Medical Trust, personal fees from Chiesi, GSK, Boehringer Ingelheim, outside the submitted work. K.F. Chung reports personal fees from Advisory Board membership, grants for research, and personal fees from payments for lectures, outside the submitted work. ATB is employed by Acclarogen Ltd., MJL by Janssen Research & Development, LLC., ARS by GSK and JC by Astra Zeneca R&D and Areteva R&D. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Objective: Early Warning Scores (EWSs) are used to monitor patients for signs of imminent deterioration. Although used in respiratory disease, EWSs have not been well studied in this population, despite the underlying cardiopulmonary pathophysiology often present. We examined the performance of two scoring systems in patients with respiratory disease., Design: Retrospective cohort analysis of vital signs observations of all patients admitted to a respiratory unit over a 2-year period. Scores were linked to outcome data to establish the performance of the National EWS (NEWS) compared results to a locally adapted EWS., Setting: Nottingham University Hospitals National Health Service Trust respiratory wards. Data were collected from an integrated electronic observation and task allocation system employing a local EWS, also generating mandatory referrals to clinical staff at set scoring thresholds., Outcome Measures: Projected workload, and sensitivity and specificity of the scores in predicting mortality based on outcome within 24 hours of a score being recorded., Results: 8812 individual patient episodes occurred during the study period. Overall, mortality was 5.9%. Applying NEWS retrospectively (vs local EWS) generated an eightfold increase in mandatory escalations, but had higher sensitivity in predicting mortality at the protocol cut points., Conclusions: This study highlights issues surrounding use of scoring systems in patients with respiratory disease. NEWS demonstrated higher sensitivity for predicting death within 24 hours, offset by reduced specificity. The consequent workload generated may compromise the ability of the clinical team to respond to patients needing immediate input. The locally adapted EWS has higher specificity but lower sensitivity. Statistical evaluation suggests this may lead to missed opportunities for intervention, however, this does not account for clinical concern independent of the scores, nor ability to respond to alerts based on workload. Further research into the role of warning scores and the impact of chronic pathophysiology is urgently needed., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Cancer survival in the UK has doubled in the last 40 years; however, 1-year and 5-year survival rates are still lower than other countries. One cause may be a delay between referral into secondary care and subsequent investigation. We set out to evaluate the impact of a straight to test pathway (STTP) on time to diagnosis for upper gastrointestinal (UGI) cancer., Methods: Six hospital Trusts across the East Midlands Clinical Network introduced a STTP enabling general practitioners to refer patients with suspected UGI cancer (oesophageal/gastric) for immediate investigation, without the need to see a hospital specialist first. Data were collected for all patients referred between 2013 and 2015 with suspected UGI cancer and stratified by STTP or traditional referral pathway. Overall time from referral to diagnosis was compared. Data from two Trusts who did not implement STTP acted as control., Results: 340 patients followed the STTP pathway and 495 followed the traditional route. STTP saved a mean of 7 days from referral to treatment (with a 95% CI of 3 to 11 days, p<0.008) and a mean of 16 days from referral to diagnosis, when compared with a traditional referral pathway. The number of diagnostic tests performed using STTP or traditional referral pathways were similar., Conclusion: A STTP is associated with an overall reduction of 1 week from referral to treatment for UGI cancer. The approach is feasible and did not require more resource. Larger studies are required to assess whether this time saving translates into improved cancer outcomes., Competing Interests: Competing interests: None declared.

Molecular dynamics (MD) simulation is a valuable tool for characterizing the structural dynamics of folded proteins and should be similarly applicable to disordered proteins and proteins with both folded and disordered regions. It has been unclear, however, whether any physical model (force field) used in MD simulations accurately describes both folded and disordered proteins. Here, we select a benchmark set of 21 systems, including folded and disordered proteins, simulate these systems with six state-of-the-art force fields, and compare the results to over 9,000 available experimental data points. We find that none of the tested force fields simultaneously provided accurate descriptions of folded proteins, of the dimensions of disordered proteins, and of the secondary structure propensities of disordered proteins. Guided by simulation results on a subset of our benchmark, however, we modified parameters of one force field, achieving excellent agreement with experiment for disordered proteins, while maintaining state-of-the-art accuracy for folded proteins. The resulting force field, a99SB- disp , should thus greatly expand the range of biological systems amenable to MD simulation. A similar approach could be taken to improve other force fields., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Severe asthma patients with a significant smoking history have airflow obstruction with reported neutrophilia. We hypothesise that multi-omic analysis will enable the definition of smoking and ex-smoking severe asthma molecular phenotypes.The U-BIOPRED cohort of severe asthma patients, containing current-smokers (CSA), ex-smokers (ESA), nonsmokers and healthy nonsmokers was examined. Blood and sputum cell counts, fractional exhaled nitric oxide and spirometry were obtained. Exploratory proteomic analysis of sputum supernatants and transcriptomic analysis of bronchial brushings, biopsies and sputum cells was performed.Colony-stimulating factor (CSF)2 protein levels were increased in CSA sputum supernatants, with azurocidin 1, neutrophil elastase and CXCL8 upregulated in ESA. Phagocytosis and innate immune pathways were associated with neutrophilic inflammation in ESA. Gene set variation analysis of bronchial epithelial cell transcriptome from CSA showed enrichment of xenobiotic metabolism, oxidative stress and endoplasmic reticulum stress compared to other groups. CXCL5 and matrix metallopeptidase 12 genes were upregulated in ESA and the epithelial protective genes, mucin 2 and cystatin SN, were downregulated.Despite little difference in clinical characteristics, CSA were distinguishable from ESA subjects at the sputum proteomic level, with CSA patients having increased CSF2 expression and ESA patients showing sustained loss of epithelial barrier processes., Competing Interests: Conflict of interest: K. Takahashi received personal fees from Asahi General Hospital, during the conduct of the study. Conflict of interest: M. Loza is employed by and owns stock in Johnson & Johnson, the parent company of Janssen R&D. Conflict of interest: F. Baribaud is an employee and shareholder of Janssen R&D. Conflict of interest: P. Chanez has provided consultancy services for Boehringer Ingelheim, Johnson & Johnson, GlaxoSmithKline, Merck Sharp & Dohme, AstraZeneca, Novartis, Teva, Chiesi, Sanofi and SNCF; has served on advisory boards for Almirall, Boehringer Ingelheim, Johnson & Johnson, GlaxoSmithKline, AstraZeneca, Novartis, Teva, Chiesi and Sanofi; has received lecture fees from Boehringer Ingelheim, Centocor, GlaxoSmithKline, AstraZeneca, Novartis, Teva, Chiesi, Boston Scientific and ALK; and has received industry-sponsored grants from Roche, Boston Scientific, Boehringer Ingelheim, Centocor, GlaxoSmithKline, AstraZeneca, ALK, Novartis, Teva and Chiesi. Conflict of interest: I. Horvath has received personal fees from Astra Zeneca, Boehringer Ingelheim, Novartis, CSL, Chiesi, Roche, GSK, Berlin-Chemie and Sandoz, outside the submitted work. Conflict of interest: P. Montuschi reports personal fees from advisory board meetings with AstraZeneca, outside the submitted work. Conflict of interest: F. Singer has received personal fees and honoraria for speaking engagements from Vertex and Novartis, outside the submitted work. Conflict of interest: S-E. Dahlén has received research grants from several Swedish funding bodies such as MRC, Heart-Lung-Foundation and the Strategic Research Foundation, during the conduct of the study; and has received personal fees for advisory board meetings from GSK, AZ, Novartis, Teva, Regeneron/Sanofi, Merck and RSPR AB, grants on asthma phenotyping from AZ, and honoraria for speaking engagements from GSK and Teva, outside the submitted work. Conflict of interest: B. Dahlén has received personal fees from Teva (for advisory board membership) and AstraZeneca (payments for lectures), outside the submitted work. Conflict of interest: N. Krug reports grants from IMI (U-BIOPRED consortium IMI number 115010), during the conduct of the study. Conflict of interest: T. Sandström has received personal fees from advisory board meetings with pharmaceutical companies GSK, AZ, Novartis, Teva and Boehringer Ingelheim, and honoraria for speaking engagements for AZ, Novartis, Boehringer Ingelheim, outside the submitted work. Conflict of interest: D.E. Shaw has received personal fees for advisory board work from GSK, AZ, Teva and Boehringer Ingelheim, outside the submitted work. Conflict of interest: R. Lutter has received personal fees for advisory board meetings from GSK and Boehringer Ingelheim, and grants on asthma and COPD from GSK, Lung Foundation and MedImmune, outside the submitted work. Conflict of interest: P. Bakke has received personal fees for advisory board meetings from GSK, AZ, Novartis and Teva, and honoraria for speaking engagements from AZ and Boehringer Ingelheim, outside the submitted work. Conflict of interest: L.J. Fleming has received personal fees for advisory board meetings from Novartis, Vectura and Boehringer Ingelheim, grants from Asthma UK and BLF, and honoraria for speaking engagements for Novartis, outside the submitted work. Conflict of interest: P.H. Howarth was employed part time by GSK as Global Medical Expert. Conflict of interest: M. Caruso received grants (paid to institute) from the Innovative Medicines Initiative (IMI), during the conduct of the study. Conflict of interest: C. Auffray received grants from the Innovative Medicine Initiative (U-BIOPRED Consortium IMI number 115010 and eTRIKS Consortium IMI number 115446), during the conduct of the study. Conflict of interest: B. De Meulder received grants from the Innovative Medicine Initiative (U-BIOPRED Consortium IMI number 115010 and eTRIKS Consortium IMI number 115446), during the conduct of the study. Conflict of interest: D. Lefaudeux received grants from the Innovative Medicine Initiative (U-BIOPRED Consortium IMI number 115010 and eTRIKS Consortium IMI number 115446), during the conduct of the study. Conflict of interest: R. Djukanovic has received personal fees for lectures at company sponsored symposia and consulting in advisory boards from TEVA, grants (for an investigator led study of mechanisms of action of omalizumab) and personal fees (for lectures at company sponsored symposia and consulting in advisory boards) from Novartis, and is a consultant to, co-founder of, and holds shares in Synairgen, outside the submitted work. Conflict of interest: P.J. Sterk has received a public-private grant (paid to institute) from Innovative Medicines Initiative (IMI) covered by the European Union (EU) and the European Federation of Pharmaceutical Industries and Associations (EFPIA), during the conduct of the study. Conflict of interest: I.M. Adcock has received personal fees for advisory board meetings from GSK, AZ, Novartis, Boehringer Ingelheim and Vectura, grants on asthma and COPD from Pfizer, GSK, MRC, EU, BI and IMI, and honoraria for speaking engagements for AZ and BI, outside the submitted work. Conflict of interest: K.F. Chung has received personal fees for advisory board meetings from GSK, AZ, Novartis, Teva, Boehringer Ingelheim and J&J, grants on asthma and COPD from Pfizer, GSK, MRC, EU IMI and NIH, and honoraria for speaking engagements for AZ and Merck, outside the submitted work., (Copyright ©ERS 2018.)