Mesoscale structural gradients in human tooth enamel.

The outstanding mechanical and chemical properties of dental enamel emerge from its complex hierarchical architecture. An accurate, detailed multiscale model of the structure and composition of enamel is important for understanding lesion formation in tooth decay (dental caries), enamel development (amelogenesis) and associated pathologies (e.g., amelogenesis imperfecta or molar hypomineralization), and minimally invasive dentistry. Although features at length scales smaller than 100 nm (individual crystallites) and greater than 50 µm (multiple rods) are well understood, competing field of view and sampling considerations have hindered exploration of mesoscale features, i.e., at the level of single enamel rods and the interrod enamel (1 to 10 µm). Here, we combine synchrotron X-ray diffraction at submicrometer resolution, analysis of crystallite orientation distribution, and unsupervised machine learning to show that crystallographic parameters differ between rod head and rod tail/interrod enamel. This variation strongly suggests that crystallites in different microarchitectural domains also differ in their composition. Thus, we use a dilute linear model to predict the concentrations of minority ions in hydroxylapatite (Mg2+ and CO32-/Na+) that plausibly explain the observed lattice parameter variations. While differences within samples are highly significant and of similar magnitude, absolute values and the sign of the effect for some crystallographic parameters show interindividual variation that warrants further investigation. By revealing additional complexity at the rod/interrod level of human enamel and leaving open the possibility of modulation across larger length scales, these results inform future investigations into mechanisms governing amelogenesis and introduce another feature to consider when modeling the mechanical and chemical performance of enamel.

Persistent polyamorphism in the chiton tooth: From a new biomineral to inks for additive manufacturing.

Engineering structures that bridge between elements with disparate mechanical properties are a significant challenge. Organisms reap synergy by creating complex shapes that are intricately graded. For instance, the wear-resistant cusp of the chiton radula tooth works in concert with progressively softer microarchitectural units as the mollusk grazes on and erodes rock. Herein, we focus on the stylus that connects the ultrahard and stiff tooth head to the flexible radula membrane. Using techniques that are especially suited to probe the rich chemistry of iron at high spatial resolution, in particular synchrotron Mössbauer and X-ray absorption spectroscopy, we find that the upper stylus of Cryptochiton stelleri is in fact a mineralized tissue. Remarkably, the inorganic phase is nano disperse santabarbaraite, an amorphous ferric hydroxyphosphate that has not been observed as a biomineral. The presence of two persistent polyamorphic phases, amorphous ferric phosphate and santabarbaraite, in close proximity, is a unique aspect that demonstrates the level of control over phase transformations in C. stelleri dentition. The stylus is a highly graded material in that its mineral content and mechanical properties vary by a factor of 3 to 8 over distances of a few hundred micrometers, seamlessly bridging between the soft radula and the hard tooth head. The use of amorphous phases that are low in iron and high in water content may be key to increasing the specific strength of the stylus. Finally, we show that we can distill these insights into design criteria for inks for additive manufacturing of highly tunable chitosan-based composites.

Genetic and clinical characteristics of treatment-resistant depression using primary care records in two UK cohorts.

Treatment-resistant depression (TRD) is a major contributor to the disability caused by major depressive disorder (MDD). Primary care electronic health records provide an easily accessible approach to investigate TRD clinical and genetic characteristics. MDD defined from primary care records in UK Biobank (UKB) and EXCEED studies was compared with other measures of depression and tested for association with MDD polygenic risk score (PRS). Using prescribing records, TRD was defined from at least two switches between antidepressant drugs, each prescribed for at least 6 weeks. Clinical-demographic characteristics, SNP-based heritability (h2SNP) and genetic overlap with psychiatric and non-psychiatric traits were compared in TRD and non-TRD MDD cases. In 230,096 and 8926 UKB and EXCEED participants with primary care data, respectively, the prevalence of MDD was 8.7% and 14.2%, of which 13.2% and 13.5% was TRD, respectively. In both cohorts, MDD defined from primary care records was strongly associated with MDD PRS, and in UKB it showed overlap of 71-88% with other MDD definitions. In UKB, TRD vs healthy controls and non-TRD vs healthy controls h2SNP was comparable (0.25 [SE = 0.04] and 0.19 [SE = 0.02], respectively). TRD vs non-TRD was positively associated with the PRS of attention deficit hyperactivity disorder, with lower socio-economic status, obesity, higher neuroticism and other unfavourable clinical characteristics. This study demonstrated that MDD and TRD can be reliably defined using primary care records and provides the first large scale population assessment of the genetic, clinical and demographic characteristics of TRD.

LabPipe: an extensible bioinformatics toolkit to manage experimental data and metadata.

BACKGROUND: Data handling in clinical bioinformatics is often inadequate. No freely available tools provide straightforward approaches for consistent, flexible metadata collection and linkage of related experimental data generated locally by vendor software. RESULTS: To address this problem, we created LabPipe, a flexible toolkit which is driven through a local client that runs alongside vendor software and connects to a light-weight server. The toolkit allows re-usable configurations to be defined for experiment metadata and local data collection, and handles metadata entry and linkage of data. LabPipe was piloted in a multi-site clinical breathomics study. CONCLUSIONS: LabPipe provided a consistent, controlled approach for handling metadata and experimental data collection, collation and linkage in the exemplar study and was flexible enough to deal effectively with different data handling challenges.

Resistome analyses of sputum from COPD and healthy subjects reveals bacterial load-related prevalence of target genes.

BACKGROUND: Antibiotic resistance is a major global threat. We hypothesised that the chronic obstructive pulmonary disease (COPD) airway is a reservoir of antimicrobial resistance genes (ARGs) that associate with microbiome-specific COPD subgroups. OBJECTIVE: To determine the resistance gene profiles in respiratory samples from COPD patients and healthy volunteers. METHODS: Quantitative PCR targeting 279 specific ARGs was used to profile the resistomes in sputum from subjects with COPD at stable, exacerbation and recovery visits (n=55; COPD-BEAT study), healthy controls with (n=7) or without (n=22) exposure to antibiotics in the preceding 12 months (EXCEED study) and in bronchial brush samples from COPD (n=8) and healthy controls (n=7) (EvA study). RESULTS: ARG mean (SEM) prevalence was greater in stable COPD samples (35.2 (1.6)) than in healthy controls (27.6 (1.7); p=0.004) and correlated with total bacterial abundance (r2=0.23; p<0.001). Prevalence of ARG positive signals in individuals was not related to COPD symptoms, lung function or their changes at exacerbation. In the COPD subgroups designated High γProteobacteria and High Firmicutes, ARG prevalence was not different at stable state but significantly declined from stable through exacerbation to recovery in the former (p=0.011) without changes in total bacterial abundance. The ARG patterns were similar in COPD versus health, COPD microbiome-subgroups and between sputum and bronchoscopic samples independent of antibiotic exposure in the last 12 months. CONCLUSIONS: ARGs are highly prevalent in sputum, broadly in proportion to bacterial abundance in both healthy and COPD subjects. Thus, COPD appears to be an ARG reservoir due to high levels of bacterial colonisation.

Automating and Extending Comprehensive Two-Dimensional Gas Chromatography Data Processing by Interfacing Open-Source and Commercial Software.

Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful analytical tool for both nontargeted and targeted analyses. However, there is a need for more integrated workflows for processing and managing the resultant high-complexity datasets. End-to-end workflows for processing GC×GC data are challenging and often require multiple tools or software to process a single dataset. We describe a new approach, which uses an existing underutilized interface within commercial software to integrate free and open-source/external scripts and tools, tailoring the workflow to the needs of the individual researcher within a single software environment. To demonstrate the concept, the interface was successfully used to complete a first-pass alignment on a large-scale GC×GC metabolomics dataset. The analysis was performed by interfacing bespoke and published external algorithms within a commercial software environment to automatically correct the variation in retention times captured by a routine reference standard. Variation in 1tR and 2tR was reduced on average from 8 and 16% CV prealignment to less than 1 and 2% post alignment, respectively. The interface enables automation and creation of new functions and increases the interconnectivity between chemometric tools, providing a window for integrating data-processing software with larger informatics-based data management platforms.

The sputum microbiome is distinct between COPD and health, independent of smoking history.

BACKGROUND: Airway bacterial dysbiosis is a feature of chronic obstructive pulmonary disease (COPD). However, there is limited comparative data of the lung microbiome between healthy smokers, non-smokers and COPD. METHODS: We compared the 16S rRNA gene-based sputum microbiome generated from pair-ended Illumina sequencing of 124 healthy subjects (28 smokers and 96 non-smokers with normal lung function), with single stable samples from 218 COPD subjects collected from three UK clinical centres as part of the COPDMAP consortium. RESULTS: In healthy subjects Firmicutes, Bacteroidetes and Actinobacteria were the major phyla constituting 88% of the total reads, and Streptococcus, Veillonella, Prevotella, Actinomyces and Rothia were the dominant genera. Haemophilus formed only 3% of the healthy microbiome. In contrast, Proteobacteria was the most dominant phylum accounting for 50% of the microbiome in COPD subjects, with Haemophilus and Moraxella at genus level contributing 25 and 3% respectively. There were no differences in the microbiome profile within healthy and COPD subgroups when stratified based on smoking history. Principal coordinate analysis on operational taxonomic units showed two distinct clusters, representative of healthy and COPD subjects (PERMANOVA, p = 0·001). CONCLUSION: The healthy and COPD sputum microbiomes are distinct and independent of smoking history. Our results underline the important role for Gammaproteobacteria in COPD.

A nested case-control study of predictors for tuberculosis recurrence in a large UK Centre.

BACKGROUND: Tuberculosis (TB) recurrence represents a challenge to control programs. In low incidence countries, the prevailing risk factors leading to recurrence are poorly characterised. METHODS: We conducted a nested case-control study using the Leicester TB service TBIT database. Cases were identified from database notifications between 1994 and 2014. Controls had one episode and were matched to cases on a ratio of two to one by the date of notification. Multiple imputation was used to account for missing data. Multivariate conditional logistic regression analysis was employed to identify clinical, sociodemographic and TB specific risk factors for recurrence. RESULTS: From a cohort of 4628 patients, 82 TB recurrences occurred (1.8%). Nineteen of 82 patients had paired isolates with MIRU-VNTR strain type profiles available, of which 84% were relapses and 16% reinfections. On multivariate analysis, smoking (OR 3.8; p = 0.04), grade 3/4 adverse drug reactions (OR 5.6; p = 0.02), ethnicity 'Indian subcontinent' (OR 8.5; p = <0.01), ethnicity 'other' (OR 31.2; p = 0.01) and receipt of immunosuppressants (OR 6.8; p = <0.01) were independent predictors of TB recurrence. CONCLUSIONS: Within this UK setting, the rate of TB recurrence was low, predominantly due to relapse. The identification of an elevated recurrence risk amongst the ethnic group contributing most cases to the national TB burden presents an opportunity to improve individual and population health.

The relationship between the Leicester cough questionnaire, eosinophilic airway inflammation and asthma patient related outcomes in severe adult asthma.

BACKGROUND: Severe asthma is characterised by a variety of symptoms, which include chronic cough, however the mechanisms responsible for cough reflex hypersensitivity in asthma remain poorly elucidated. Current asthma patient-related outcome instruments such as the six-point Juniper Asthma Control Score (ACQ-6) and Asthma Quality of Life Questionnaire (AQLQ) were not primarily designed to capture cough and its related morbidity in asthma. The Leicester Cough Questionnaire (LCQ) is a patient-related outcome instrument designed to capture the health-related quality of life associated with cough. To date the LCQ has not been evaluated in a severe asthma population. METHODS: We evaluated 262 extensively characterised adult patients with severe asthma attending the Leicester Severe Asthma Service. All patients had a clinician diagnosis of asthma and objective physiological evidence and met the ATS/ERS criterion for servere asthma. In all patients we evaluated a) the LCQ distribution and b) the relationships between the LCQ and ACQ-6, AQLQ, airway inflammation in sputum. RESULTS: The LCQ demonstrated the following properties; mean: 15.0, standard deviation: 4.54, median: 15.48, and range: 11.6-19.2. We found a moderate correlation between LCQ and ACQ-6 (r = - 0.605, p < 0.0001) and a LCQ and AQLQ (r = 0.710, p < 0.0001). There was no relationship between LCQ and log10 sputum percentage eosinophils (%). CONCLUSION: A proportion of patients with severe asthma have a significant degree of cough-related morbidity that appears independent of eosinophilic airway inflammation and is not captured fully by existing asthma patient-reported outcome instruments. Our preliminary findings suggest that further research is now required to validate the LCQ and its responsiveness in severe asthma populations to capture cough-related morbidity and response to specific interventions.

The BioMart community portal: an innovative alternative to large, centralized data repositories.

The BioMart Community Portal (www.biomart.org) is a community-driven effort to provide a unified interface to biomedical databases that are distributed worldwide. The portal provides access to numerous database projects supported by 30 scientific organizations. It includes over 800 different biological datasets spanning genomics, proteomics, model organisms, cancer data, ontology information and more. All resources available through the portal are independently administered and funded by their host organizations. The BioMart data federation technology provides a unified interface to all the available data. The latest version of the portal comes with many new databases that have been created by our ever-growing community. It also comes with better support and extensibility for data analysis and visualization tools. A new addition to our toolbox, the enrichment analysis tool is now accessible through graphical and web service interface. The BioMart community portal averages over one million requests per day. Building on this level of service and the wealth of information that has become available, the BioMart Community Portal has introduced a new, more scalable and cheaper alternative to the large data stores maintained by specialized organizations.

Automated design of paralogue ratio test assays for the accurate and rapid typing of copy number variation.

MOTIVATION: Genomic copy number variation (CNV) can influence susceptibility to common diseases. High-throughput measurement of gene copy number on large numbers of samples is a challenging, yet critical, stage in confirming observations from sequencing or array Comparative Genome Hybridization (CGH). The paralogue ratio test (PRT) is a simple, cost-effective method of accurately determining copy number by quantifying the amplification ratio between a target and reference amplicon. PRT has been successfully applied to several studies analyzing common CNV. However, its use has not been widespread because of difficulties in assay design. RESULTS: We present PRTPrimer (www.prtprimer.org) software for automated PRT assay design. In addition to stand-alone software, the web site includes a database of pre-designed assays for the human genome at an average spacing of 6 kb and a web interface for custom assay design. Other reference genomes can also be analyzed through local installation of the software. The usefulness of PRTPrimer was tested within known CNV, and showed reproducible quantification. This software and database provide assays that can rapidly genotype CNV, cost-effectively, on a large number of samples and will enable the widespread adoption of PRT. AVAILABILITY: PRTPrimer is available in two forms: a Perl script (version 5.14 and higher) that can be run from the command line on Linux systems and as a service on the PRTPrimer web site (www.prtprimer.org).

Prognostication of co-morbidity clusters on hospitalisation and mortality in advanced COPD.

RATIONALE: As the prevalence of multimorbidity increases, understanding the impact of isolated comorbidities in people COPD becomes increasingly challenging. A simplified model of common comorbidity patterns may improve outcome prediction and allow targeted therapy. OBJECTIVES: To assess whether comorbidity phenotypes derived from routinely collected clinical data in people with COPD show differences in risk of hospitalisation and mortality. METHODS: Twelve clinical measures related to common comorbidities were collected during annual reviews for people with advanced COPD and k-means cluster analysis performed. Cox proportional hazards with adjustment for covariates was used to determine hospitalisation and mortality risk between clusters. MEASUREMENTS AND MAIN RESULTS: In 203 participants (age 66 ± 9 years, 60 % male, FEV1%predicted 31 ± 10 %) no comorbidity in isolation was predictive of worse admission or mortality risk. Four clusters were described: cluster A (cardiometabolic and anaemia), cluster B (malnourished and low mood), cluster C (obese, metabolic and mood disturbance) and cluster D (less comorbid). FEV1%predicted did not significantly differ between clusters. Mortality risk was higher in cluster A (HR 3.73 [95%CI 1.09-12.82] p = 0.036) and B (HR 3.91 [95%CI 1.17-13.14] p = 0.027) compared to cluster D. Time to admission was highest in cluster A (HR 2.01 [95%CI 1.11-3.63] p = 0.020). Cluster C was not associated with increased risk of mortality or hospitalisation. CONCLUSIONS: Despite presence of advanced COPD, we report striking differences in prognosis for both mortality and hospital admissions for different co-morbidity phenotypes. Objectively assessing the multi-system nature of COPD could lead to improved prognostication and targeted therapy for patients.

Single-step QuantiFERON screening of adult contacts: a prospective cohort study of tuberculosis risk.

BACKGROUND: The efffectiveness of tuberculosis (TB) contact screening programmes using interferon γ release assays remains uncertain as prospective contact TB risk is not well characterised. OBJECTIVES: To quantify 2-year TB risk and evaluate screening performance with single-step QuantiFERON TB Gold-In Tube (QFT) in adult contacts. To compare TB risk between QFT tested subgroups stratified by exposure type (smear positive pulmonary (SP) versus non-smear positive (NSP) TB) and age (younger (16-35 years) versus older (≥36 years)). METHODS: Screening involved QFT testing in older contacts of SP and all younger contacts, 8-12 weeks after index notification. Chemoprevention (3RH) was offered to QFT positive (+) younger adults. TB risk was determined in a prospective cohort study. RESULTS: 43 TB events occurred in 1769 adult contacts observed for median 717 days (2-year rate (95% CI)=2·5% (1.7 to 3.2)). Index-contact strain matching was demonstrable for 18 of 22 (82%) paired samples. No contacts (0/98) receiving 3RH developed TB. 215 of 817 appropriately tested adults (26.3%) were QFT+. 14 of 112 untreated QFT+ adults developed TB (2-year rate (95% CI)=13·4% (7.7 to 21.1)). The model required 35 contacts screened with QFT to identify one contact developing TB at 2 years. TB rates were comparable in QFT+ contacts of SP and NSP (rate ratio (RR)=0.98, p=0·962). For QFT+ older contacts, the disease rate was lower (8.9% (3.3 to 19.1)) and similar to the overall group rate (RR=1.4, p=0.503). CONCLUSIONS: QFT based single-step contact screening is effective in young adults.

A data-driven framework for clinical decision support applied to pneumonia management.

Despite their long history, it can still be difficult to embed clinical decision support into existing health information systems, particularly if they utilise machine learning and artificial intelligence models. Moreover, when such tools are made available to healthcare workers, it is important that the users can understand and visualise the reasons for the decision support predictions. Plausibility can be hard to achieve for complex pathways and models and perceived "black-box" functionality often leads to a lack of trust. Here, we describe and evaluate a data-driven framework which moderates some of these issues and demonstrate its applicability to the in-hospital management of community acquired pneumonia, an acute respiratory disease which is a leading cause of in-hospital mortality world-wide. We use the framework to develop and test a clinical decision support tool based on local guideline aligned management of the disease and show how it could be used to effectively prioritise patients using retrospective analysis. Furthermore, we show how this tool can be embedded into a prototype clinical system for disease management by integrating metrics and visualisations. This will assist decision makers to examine complex patient journeys, risk scores and predictions from embedded machine learning and artificial intelligence models. Our results show the potential of this approach for developing, testing and evaluating workflow based clinical decision support tools which include complex models and embedding them into clinical systems.

Genome-wide association study of thyroid-stimulating hormone highlights new genes, pathways and associations with thyroid disease.

Thyroid hormones play a critical role in regulation of multiple physiological functions and thyroid dysfunction is associated with substantial morbidity. Here, we use electronic health records to undertake a genome-wide association study of thyroid-stimulating hormone (TSH) levels, with a total sample size of 247,107. We identify 158 novel genetic associations, more than doubling the number of known associations with TSH, and implicate 112 putative causal genes, of which 76 are not previously implicated. A polygenic score for TSH is associated with TSH levels in African, South Asian, East Asian, Middle Eastern and admixed American ancestries, and associated with hypothyroidism and other thyroid disease in South Asians. In Europeans, the TSH polygenic score is associated with thyroid disease, including thyroid cancer and age-of-onset of hypothyroidism and hyperthyroidism. We develop pathway-specific genetic risk scores for TSH levels and use these in phenome-wide association studies to identify potential consequences of pathway perturbation. Together, these findings demonstrate the potential utility of genetic associations to inform future therapeutics and risk prediction for thyroid diseases.

Characterization of mass and swelling of hydrogel microstructures using MEMS resonant mass sensor arrays.

The use of hydrogels for biomedical engineering, and for the development of biologically inspired cellular systems at the microscale, is advancing at a rapid pace. Microelectromechanical system (MEMS) resonant mass sensors enable the mass measurement of a range of materials. The integration of hydrogels onto MEMS resonant mass sensors is demonstrated, and these sensors are used to characterize the hydrogel mass and swelling characteristics. The mass values obtained from resonant frequency measurements of poly(ethylene glycol)diacrylate (PEGDA) microstructures match well with the values independently verified through volume measurements. The sensors are also used to measure the influence of fluids of similar and greater density on the mass measurements of microstructures. The data show a size-dependent increase in gel mass when fluid density is increased. Lastly, volume comparisons of bulk hydrogels with a range polymer concentration (5% to 100% (v/v)) show a non-linear swelling trend.

The stoic tooth root: how the mineral and extracellular matrix counterbalance to keep aged dentin stable.

Aging is a physiological process with profound impact on the biology and function of biosystems, including the human dentition. While resilient, human teeth undergo wear and disease, affecting overall physical, psychological, and social human health. However, the underlying mechanisms of tooth aging remain largely unknown. Root dentin is integral to tooth function in that it anchors and dissipates mechanical load stresses of the tooth-bone system. Here, we assess the viscoelastic behavior, composition, and ultrastructure of young and old root dentin using nano-dynamic mechanical analysis, micro-Raman spectroscopy, small angle X-ray scattering, atomic force and transmission electron microscopies. We find that the root dentin overall stiffness increases with age. Unlike other mineralized tissues and even coronal dentin, however, the ability of root dentin to dissipate energy during deformation does not decay with age. Using a deconstruction method to dissect the contribution of mineral and organic matrix, we find that the damping factor of the organic matrix does deteriorate. Compositional and ultrastructural analyses revealed higher mineral-to-matrix ratio, altered enzymatic and non-enzymatic collagen cross-linking, increased collagen d-spacing and fibril diameter, and decreased abundance of proteoglycans and sulfation pattern of glycosaminoglycans . Therefore, even in the absence of remodeling, the extracellular matrix of root dentin shares traits of aging with other tissues. To explain this discrepancy, we propose that altered matrix-mineral interactions, possibly mediated by carbonate ions sequestered at the mineral interface and/or altered glycosaminoglycans counteract the deleterious effects of aging on the structural components of the extracellular matrix. STATEMENT OF SIGNIFICANCE: Globally, a quarter of the population will be over 65 years old by 2050. Because many will retain their dentition, it will become increasingly important to understand and manage how aging affects teeth. Dentin is integral to the protective, biomechanical, and regenerative features of teeth. Here, we demonstrate that older root dentin not only has altered mechanical properties, but shows characteristic shifts in mineralization, composition, and post-translational modifications of the matrix. This strongly suggests that there is a mechanistic link between mineral and matrix components to the biomechanical performance of aging dentin with implications for efforts to slow or even reverse the aging process.

Culturally adapted pulmonary rehabilitation for adults living with post-tuberculosis lung disease in Kyrgyzstan: protocol for a randomised controlled trial with blinded outcome measures.

INTRODUCTION: Pulmonary rehabilitation (PR) is a programme of individually prescribed physical exercise, education and self-management activities. PR is recommended in international guidelines for managing chronic obstructive pulmonary disease (COPD) and other chronic respiratory diseases. PR is still under-recognised in tuberculosis (TB) guidelines and PR is not available in many low and middle-income countries and for people with post-TB lung disease (PTBLD). The main aims of the study are to adapt and define a culturally appropriate PR programme in Kyrgyzstan for people living with PTBLD and to test, in a fully powered randomised controlled trial (RCT), the effectiveness of PR in improving exercise capacity for people living with PTBLD. METHODS AND ANALYSIS: The study will be divided into three stages: stage 1: focus group discussions with patients living with PTBLD and interviews with PR referrers will be conducted to explore initial perceptions and inform the cultural adaptation, structure and content of PR. Stage 2a: a single-blind RCT evaluating the effectiveness of a culturally adapted 6-week PR programme on maximal exercise capacity, assessed by the incremental shuttle walking test, before and after PR. Participants will be additionally followed-up 12 weeks postbaseline. Additional outcomes will include health-related quality of life, respiratory symptoms, psychological well-being and physical function. Stage 2b: participants' experience of PR will be collected through interviews and using a log book and a patient evaluation form. Staff delivering PR will be interviewed to explore their experience of delivering the intervention and refining the delivery for future implementation. ETHICS AND DISSEMINATION: The study was approved 22/07/2019 by Ethics Committee National Center for Cardiology and Internal Medicine (reference number 17) and by University of Leicester ethics committee (reference number 22293). Study results will be disseminated through appropriate peer-reviewed journals, national and international respiratory/physiotherapy conferences, social media, and through patient and public involvement events in Kyrgyzstan and in the UK. TRIAL REGISTRATION NUMBER: ISRCTN11122503.

Protocol for a single-centre mixed-method pre-post single-arm feasibility trial of a culturally appropriate 6-week pulmonary rehabilitation programme among adults with functionally limiting chronic respiratory diseases in Malawi.

INTRODUCTION: Malawi has a substantial burden of chronic respiratory diseases (CRDs) which cause significant morbidity and loss of economic productivity, affecting patients, families and health systems. Pulmonary rehabilitation (PR) is a highly recommended non-pharmacological intervention in the clinical management of people with CRDs. However, Malawi lacks published evidence on the implementation of PR for people with CRDs. This trial will test the feasibility and acceptability of implementing a culturally appropriate hospital-based PR programme among adults with functionally limiting CRDs at Queen Elizabeth Central Hospital in Blantyre, Malawi. METHODS AND ANALYSIS: This is a single-centre mixed-methods pre-post single-arm feasibility trial. Ten patients aged ≥18 years, with a spirometry confirmed diagnosis of a CRD and breathlessness of ≥2 on the modified Medical Research Council dyspnoea scale, will be consecutively recruited. Their baseline lung function, exercise tolerance and health status will be assessed; including spirometry, Incremental Shuttle Walk Test and Chronic Obstructive Pulmonary Disease Assessment Test, respectively. Pretrial semistructured in-depth interviews will explore their experiences of living with CRD and potential enablers and barriers to their PR uptake. Along with international PR guidelines, these data will inform culturally appropriate delivery of PR. We initially propose a 6-week, twice-weekly, supervised centre-based PR programme, with an additional weekly home-based non-supervised session. Using combination of researcher observation, interaction with the participants, field notes and informal interviews with the participants, we will assess the feasibility of running the programme in the following areas: participants' recruitment, retention, engagement and protocol adherence. Following programme completion (after 6 weeks), repeat assessments of lung function, exercise tolerance and health status will be conducted. Quantitative changes in clinical outcomes will be described in relation to published minimal clinically important differences. Post-trial semistructured interviews will capture participants' perceived impact of the PR programme on their quality of life, enablers, and barriers to fully engaging with the programme, and allow iteration of its design. ETHICS AND DISSEMINATION: Ethical approval for this trial was obtained from University of Malawi College of Medicine Research and Ethics Committee (COMREC), Blantyre, Malawi (protocol number: P.07/19/2752) and University of Leicester Research Ethics Committee, Leicester, UK (ethics reference: 31574). The results of the trial will be disseminated through oral presentations at local and international scientific conferences or seminars and publication in a peer-reviewed journal. We will also engage the participants who complete the PR trial and the Science Communication Department at Malawi-Liverpool-Wellcome Trust Clinical Research Programme to organise community outreach activities within Blantyre to educate communities about CRDs and PR. We will also broadcast our trial results through national radio station programmes such as the weekly "Thanzi la Onse" (Health of All) programme by Times Radio Malawi. We will formally present our trial results to Blantyre District Health Office and Malawi Ministry of Health. TRIAL REGISTRATION NUMBER: ISRCTN13836793.

Visualization of exhaled breath metabolites reveals distinct diagnostic signatures for acute cardiorespiratory breathlessness.

Acute cardiorespiratory breathlessness accounts for one in eight of all emergency hospitalizations. Early, noninvasive diagnostic testing is a clinical priority that allows rapid triage and treatment. Here, we sought to find and replicate diagnostic breath volatile organic compound (VOC) biomarkers of acute cardiorespiratory disease and understand breath metabolite network enrichment in acute disease, with a view to gaining mechanistic insight of breath biochemical derangements. We collected and analyzed exhaled breath samples from 277 participants presenting acute cardiorespiratory exacerbations and aged-matched healthy volunteers. Topological data analysis phenotypes differentiated acute disease from health and acute cardiorespiratory exacerbation subtypes (acute heart failure, acute asthma, acute chronic obstructive pulmonary disease, and community-acquired pneumonia). A multibiomarker score (101 breath biomarkers) demonstrated good diagnostic sensitivity and specificity (≥80%) in both discovery and replication sets and was associated with all-cause mortality at 2 years. In addition, VOC biomarker scores differentiated metabolic subgroups of cardiorespiratory exacerbation. Louvain clustering of VOCs coupled with metabolite enrichment and similarity assessment revealed highly specific enrichment patterns in all acute disease subgroups, for example, selective enrichment of correlated C5-7 hydrocarbons and C3-5 carbonyls in heart failure and selective depletion of correlated aldehydes in acute asthma. This study identified breath VOCs that differentiate acute cardiorespiratory exacerbations and associated subtypes and metabolic clusters of disease-associated VOCs.