Agrobacterium-Mediated Transient Gene Expression Optimized for the Bioenergy Crop Camelina sativa.

Camelina sativa, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N-terminal ER membrane signal anchor region (1-32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system. Key features • This method is based on the approach of Zhang et al. [1] and has been optimized for bioenergy crop Camelina species. • A constitutive and inducible transient gene expression in the hexaploid species Camelina sativa cultivar DH55. • Requires only 16-18 days to complete with high efficacy. Graphical overview.

In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development.

Protein translational control is critical for ensuring that the fetus develops correctly and that necessary organs and tissues are formed and functional. We developed an in utero method to quantify tissue-specific protein dynamics by monitoring amino acid incorporation into the proteome after pulse injection. Fetuses of pregnant mice were injected with isotopically labeled lysine and arginine via the vitelline vein at various embyonic days, and organs and tissues were harvested. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates were calculated between 3.81E-5 and 0.424 h-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver vs. brain); however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates, which correlated with known physiological changes during mouse development.

Racial Disparity among Youth Decedents of Legal Intervention Trauma.

BACKGROUND: Legal intervention trauma (LIT) is defined as injury due to any encounter with law enforcement. This study investigates associations between demographics, violent status, and law enforcement tactics among youth decedents of LIT. STUDY DESIGN: Decedents of LIT age 26 years or younger were identified using the CDC's National Violent Death Reporting System from 2003 to 2018. Decedents were classified as "violent" if they possessed a weapon, were committing a violent crime, or if law enforcement reported justified use of force. All others were classified as "nonviolent." Law enforcement tactics were stratified into "lethal" (firearm with standard ammunition) or "less lethal" (any other) force. Differences in the racial distribution across these classifications were assessed using chi-square tests of proportions. RESULTS: We identified 1,281 youth decedents of LIT; of which, 92.5% met violent criteria. Black youths were less likely than White youths to possess a weapon (71.6% vs 77.4%, p = 0.02) and were not more likely to be committing a violent crime (63.6% vs 60.4%, p = 0.27). They were, however, more likely than White youths to experience force reported as justified by law enforcement (89.9% vs 82.4%, p = 0.002) and to experience exclusively lethal force not preceded by less-lethal tactics (94.0% vs 88.7%, p = 0.001). Among the subset of 85 cases where law enforcement reported justified use of force despite the decedent not possessing a weapon or committing a violent crime, the precipitating event was more often a traffic stop for Black youths than for White youths (28.5% vs 6.66%, p = 0.02). CONCLUSIONS: These findings indicate a racial disparity among youth decedents of LIT.

Low levels of endogenous anabolic androgenic steroids in females with severe asthma taking corticosteroids.

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.

Development of the Preferred Components for Co-Design in Research Guideline and Checklist: Protocol for a Scoping Review and a Modified Delphi Process.

BACKGROUND: There is increasing evidence that co-design can lead to more engaging, acceptable, relevant, feasible, and even effective interventions. However, no guidance is provided on the specific designs and associated methods or methodologies involved in the process. We propose the development of the Preferred Components for Co-design in Research (PRECISE) guideline to enhance the consistency, transparency, and quality of reporting co-design studies used to develop complex health interventions. OBJECTIVE: The aim is to develop the first iteration of the PRECISE guideline. The purpose of the PRECISE guideline is to improve the consistency, transparency, and quality of reporting on studies that use co-design to develop complex health interventions. METHODS: The aim will be achieved by addressing the following objectives: to review and synthesize the literature on the models, theories, and frameworks used in the co-design of complex health interventions to identify their common elements (components, values or principles, associated methods and methodologies, and outcomes); and by using the results of the scoping review, prioritize the co-design components, values or principles, associated methods and methodologies, and outcomes to be included in the PRECISE guideline. RESULTS: The project has been funded by the Canadian Institutes of Health Research. CONCLUSIONS: The collective results of this project will lead to a ready-to-implement PRECISE guideline that outlines a minimum set of items to include when reporting the co-design of complex health interventions. The PRECISE guideline will improve the consistency, transparency, and quality of reports of studies. Additionally, it will include guidance on how to enact or enable the values or principles of co-design for meaningful and collaborative solutions (interventions). PRECISE might also be used by peer reviewers and editors to improve the review of manuscripts involving co-design. Ultimately, the PRECISE guideline will facilitate more efficient use of new results about complex health intervention development and bring better returns on research investments. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/50463.

Extracting microscopic insight from transient dielectric measurements during large amplitude oscillatory shear.

Probing the transient microstructure of soft matter far from equilibrium is an ongoing challenge to understanding material processing. In this work, we investigate inverse worm-like micelles undergoing large amplitude oscillatory shear using time-resolved dielectric spectroscopy. By controlling the Weissenburg number, we compare the non-linear microstructure response of branched and unbranched worm-like micelles and isolate distinct elastic effects that manifest near flow reversal. We validate our dielectric measurements with small angle neutron scattering and employ sequence of physical processes to disentangle the elastic and viscous contributions of the stress.

A Novel VACTERL Assessment Tool to Facilitate Counseling for Expectant Families.

INTRODUCTION: VACTERL is defined as 3 or more of the following congenital defects: vertebral, anorectal, cardiac, tracheoesophageal (TE), renal, and limb. The purpose of this study was to create an easy-to-use assessment tool to help providers counsel expecting families regarding the likelihood of additional anomalies and postnatal outcomes. METHODS: Employing the Kids' Inpatient Database from 2003-2016, neonates (<29 days old) with VACTERL were identified using ICD-9-CM and ICD-10-CM codes. For each unique combination of VACTERL, multivariable logistic regression was used to estimate inpatient mortality, and Poisson regression was used to estimate length-of-stay during the initial hospitalization. RESULTS: The assessment tool used in this study is available at https://choc-trauma.shinyapps.io/VACTERL. 1,886 of 11,813,782 (0.016%) neonates presented with VACTERL. 32% weighed <1,750 g, and 239 (12.7%) died prior to discharge. Associated with mortality were limb anomaly (1.8 [1.01-3.22], p < 0.05), prematurity (1.99 [1.14-3.47], p < 0.02), and weight <1,750 g (2.19 [1.25-3.82], p < 0.01). Median length-of-stay was 14 days (IQR: 7-32). Associated with increased length-of-stay were cardiac defect (1.47 [1.37-1.56], p < 0.001), vertebral anomaly (1.1 [1.05-1.14], p < 0.001), TE fistula (1.73 [1.66-1.81], p < 0.001), anorectal malformation (1.12 [1.07-1.16], p < 0.001), and weight <1,750 g (1.65 [1.57-1.73], p < 0.001). CONCLUSION: This novel assessment tool may help providers counsel families confronting a VACTERL diagnosis.

Haemophilus influenzae and Moraxella catarrhalis in sputum of severe asthma with inflammasome and neutrophil activation.

BACKGROUND: Because of altered airway microbiome in asthma, we analysed the bacterial species in sputum of patients with severe asthma. METHODS: Whole genome sequencing was performed on induced sputum from non-smoking (SAn) and current or ex-smoker (SAs/ex) severe asthma patients, mild/moderate asthma (MMA) and healthy controls (HC). Data were analysed by asthma severity, inflammatory status and transcriptome-associated clusters (TACs). RESULTS: α-diversity at the species level was lower in SAn and SAs/ex, with an increase in Haemophilus influenzae and Moraxella catarrhalis, and Haemophilus influenzae and Tropheryma whipplei, respectively, compared to HC. In neutrophilic asthma, there was greater abundance of Haemophilus influenzae and Moraxella catarrhalis and in eosinophilic asthma, Tropheryma whipplei was increased. There was a reduction in α-diversity in TAC1 and TAC2 that expressed high levels of Haemophilus influenzae and Tropheryma whipplei, and Haemophilus influenzae and Moraxella catarrhalis, respectively, compared to HC. Sputum neutrophils correlated positively with Moraxella catarrhalis and negatively with Prevotella, Neisseria and Veillonella species and Haemophilus parainfluenzae. Sputum eosinophils correlated positively with Tropheryma whipplei which correlated with pack-years of smoking. α- and ß-diversities were stable at one year. CONCLUSIONS: Haemophilus influenzae and Moraxella catarrhalis were more abundant in severe neutrophilic asthma and TAC2 linked to inflammasome and neutrophil activation, while Haemophilus influenzae and Tropheryma whipplei were highest in SAs/ex and in TAC1 associated with highest expression of IL-13 type 2 and ILC2 signatures with the abundance of Tropheryma whipplei correlating positively with sputum eosinophils. Whether these bacterial species drive the inflammatory response in asthma needs evaluation.

In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development.

Protein translational control is highly regulated step in the gene expression program during mammalian development that is critical for ensuring that the fetus develops correctly and that all of the necessary organs and tissues are formed and functional. Defects in protein expression during fetal development can lead to severe developmental abnormalities or premature death. Currently, quantitative techniques to monitor protein synthesis rates in a developing fetus (in utero) are limited. Here, we developed a novel in utero stable isotope labeling approach to quantify tissue-specific protein dynamics of the nascent proteome during mouse fetal development. Fetuses of pregnant C57BL/6J mice were injected with isotopically labeled lysine (Lys8) and arginine (Arg10) via the vitelline vein at various gestational days. After treatment, fetal organs/tissues including brain, liver, lung, and heart were harvested for sample preparation and proteomic analysis. We show that the mean incorporation rate for injected amino acids into all organs was 17.50 ± 0.6%. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates (kobs) were calculated between 3.81E-5 and 0.424 hour-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver versus brain), however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates which correlated with known physiological changes during mouse development.

Structural change in feedlot cattle death loss rates.

Introduction: Industry reports and anecdotal evidence indicate that the death loss rate in cattle feedlots has increased over time. Such increases in death loss rates impact feedlot cost and thus profitability. Objectives: The primary objective of this study is to examine whether feedlot death loss rates in cattle have changed over time, to analyze the nature of any identified structural change, and to identify possible catalysts for that change. Methods: Data from the Kansas Feedlot Performance and Feed Cost Summary from 1992 through 2017 is used to model feedlot death loss rate as a function of feeder cattle placement weight, days on feed, time, and seasonality in the form of monthly dummy variables. Commonly used tests of structural change, including the CUSUM, CUSUMSQ, and Bai and Perron methods, are implemented to examine the existence and nature of any structural changes in the proposed model. All tests indicate the presence of structural breaks in the model, including both systematic change and abrupt change. Following a synthesis of structural test results, the final model is modified to include a structural shift parameter for the period from December 2000 to September 2010. Results: Models indicate that days on feed has a significant positive influence on death loss rate. Trend variables indicate that death loss rates have increased systematically over the period studied. However, the structural shift parameter in the modified model is positive and significant for December 2000 to September 2010, indicating that death loss is higher on average during this period. Variance of death loss percentage is also higher during this period. Parallels between evidence of structural change and possible industry and environmental catalysts are also discussed. Conclusions: Statistical evidence does indicate changes in the structure of death loss rates. Ongoing factors such as changes in feeding rations prompted by market forces and feeding technologies may have contributed to systematic change. Other events, such as weather events and beta agonist use could result in abrupt changes. No clear evidence directly connects these factors to death loss rates and disaggregated data would be required to facilitate such a study.

Training and capacity development in patient-oriented research: Ontario SPOR SUPPORT Unit (OSSU) initiatives.

BACKGROUND: In Canada, the Canadian Institutes of Health Research launched the Strategy for Patient-Oriented Research (SPOR) in 2011. The strategy defines 'patient-oriented research' as a continuum of research that engages patients as partners, focuses on patient priorities, and leads to improved patient outcomes. The overarching term 'patient' is inclusive of individuals with personal experience of a health issue as well as informal caregivers including family and friends. The vision for the strategy is improved patient experiences and outcomes through the integration of patient-oriented research findings into practice, policy, and health system improvement. Building capacity in patient-oriented research among all relevant stakeholders, namely patients, practitioners, organizational leaders, policymakers, researchers, and research funders is a core element of the strategy. MAIN BODY: The objective of this paper is to describe capacity building initiatives in patient-oriented research led by the Ontario SPOR SUPPORT Unit in Ontario, Canada over the period 2014-2020. CONCLUSION: The Ontario SPOR SUPPORT Unit Working Group in Training and Capacity Development has led numerous capacity building initiatives: developed a Capacity Building Compendium (accessed greater than 45,000 times); hosted Masterclasses that have trained hundreds of stakeholders (patients, practitioners, organizational leaders, policymakers, researchers, and trainees) in the conduct and use of patient-oriented research; funded the development of online curricula on patient-oriented research that have reached thousands of stakeholders; developed a patient engagement resource center that has been accessed by tens of thousands of stakeholders; identified core competencies for research teams and research environments to ensure authentic and meaningful patient partnerships in health research; and shared these resources and learnings with stakeholders across Canada, North America, and internationally.

In 2011, Canada developed a Strategy for Patient-Oriented Research. The aim of the strategy was to ensure that patients were included as equal partners in research, with the goal to improve the patient experience and enhance health outcomes using research findings to influence clinical care, policy, and health system improvement. Building capacity in patient-oriented research is a core element of the strategy. Since 2014, the Ontario SPOR SUPPORT Unit has led numerous initiatives to build capacity in patient-oriented research. Successes include a Capacity Building Compendium (a catalogue of resources that has been accessed greater than 45,000 times); courses on how to do and how to use patient-oriented research that have trained hundreds of patients, practitioners, organizational leaders, policymakers, and researchers; created online patient-oriented research materials; developed a patient engagement resource center; identified what is required to ensure authentic and meaningful patient partnerships in research; and shared these resources and learnings widely.

Fetal allotransplant recipients are resistant to graft-versus-host disease.

In utero hematopoietic cell transplantation (IUHCT) is an experimental treatment for congenital hemoglobinopathies, including Sickle cell disease and thalassemias. One of the principal advantages of IUHCT is the predisposition of the developing fetus toward immunologic tolerance. This allows for engraftment across immune barriers without immunosuppression and, potentially, decreased susceptibility to graft-versus-host disease (GVHD). We demonstrate fetal resistance to GVHD following T cell-replete allogeneic hematopoietic cell transplantation compared with the neonate. We show that this resistance is associated with elevated fetal serum interleukin-10 conducive to the induction of regulatory T cells (Tregs). Finally, we demonstrate that the adoptive transfer of Tregs from IUHCT recipients to neonates uniformly prevents GVHD, recapitulating the predisposition to tolerance observed after fetal allotransplantation. These findings demonstrate fetal resistance to GVHD following hematopoietic cell transplantation and elucidate Tregs as important contributors.

IL1RAP expression and the enrichment of IL-33 activation signatures in severe neutrophilic asthma.

BACKGROUND: Interleukin (IL)-33 is an upstream regulator of type 2 (T2) eosinophilic inflammation and has been proposed as a key driver of some asthma phenotypes. OBJECTIVE: To derive gene signatures from in vitro studies of IL-33-stimulated cells and use these to determine IL-33-associated enrichment patterns in asthma. METHODS: Signatures downstream of IL-33 stimulation were derived from our in vitro study of human mast cells and from public datasets of in vitro stimulated human basophils, type 2 innate lymphoid cells (ILC2), regulatory T cells (Treg) and endothelial cells. Gene Set Variation Analysis (GSVA) was used to probe U-BIOPRED and ADEPT sputum transcriptomics to determine enrichment scores (ES) for each signature according to asthma severity, sputum granulocyte status and previously defined molecular phenotypes. RESULTS: IL-33-activated gene signatures were cell-specific with little gene overlap. Individual signatures, however, were associated with similar signalling pathways (TNF, NF-κB, IL-17 and JAK/STAT signalling) and immune cell differentiation pathways (Th17, Th1 and Th2 differentiation). ES for IL-33-activated gene signatures were significantly enriched in asthmatic sputum, particularly in patients with neutrophilic and mixed granulocytic phenotypes. IL-33 mRNA expression was not elevated in asthma whereas the expression of mRNA for IL1RL1, the IL-33 receptor, was up-regulated in the sputum of severe eosinophilic asthma. The mRNA expression for IL1RAP, the IL1RL1 co-receptor, was greatest in severe neutrophilic and mixed granulocytic asthma. CONCLUSIONS: IL-33-activated gene signatures are elevated in neutrophilic and mixed granulocytic asthma corresponding with IL1RAP co-receptor expression. This suggests incorporating T2-low asthma in anti-IL-33 trials.

Mapping atopic dermatitis and anti-IL-22 response signatures to type 2-low severe neutrophilic asthma.

BACKGROUND: Transcriptomic changes in patients who respond clinically to biological therapies may identify responses in other tissues or diseases. OBJECTIVE: We sought to determine whether a disease signature identified in atopic dermatitis (AD) is seen in adults with severe asthma and whether a transcriptomic signature for patients with AD who respond clinically to anti-IL-22 (fezakinumab [FZ]) is enriched in severe asthma. METHODS: An AD disease signature was obtained from analysis of differentially expressed genes between AD lesional and nonlesional skin biopsies. Differentially expressed genes from lesional skin from therapeutic superresponders before and after 12 weeks of FZ treatment defined the FZ-response signature. Gene set variation analysis was used to produce enrichment scores of AD and FZ-response signatures in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes asthma cohort. RESULTS: The AD disease signature (112 upregulated genes) encompassing inflammatory, T-cell, TH2, and TH17/TH22 pathways was enriched in the blood and sputum of patients with asthma with increasing severity. Patients with asthma with sputum neutrophilia and mixed granulocyte phenotypes were the most enriched (P < .05). The FZ-response signature (296 downregulated genes) was enriched in asthmatic blood (P < .05) and particularly in neutrophilic and mixed granulocytic sputum (P < .05). These data were confirmed in sputum of the Airway Disease Endotyping for Personalized Therapeutics cohort. IL-22 mRNA across tissues did not correlate with FZ-response enrichment scores, but this response signature correlated with TH22/IL-22 pathways. CONCLUSIONS: The FZ-response signature in AD identifies severe neutrophilic asthmatic patients as potential responders to FZ therapy. This approach will help identify patients for future asthma clinical trials of drugs used successfully in other chronic diseases.

Plasma proteins elevated in severe asthma despite oral steroid use and unrelated to Type-2 inflammation.

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-ß 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.

Urinary metabotype of severe asthma evidences decreased carnitine metabolism independent of oral corticosteroid treatment in the U-BIOPRED study.

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.