Journal of Cystic Fibrosis

BackgroundNonsense variants in CFTR account for ~10% of cystic fibrosis (CF) variants and cannot be treated with approved CFTR modulators. Translational readthrough agents such as ELX-02 offer a potential therapeutic strategy, but clinical trials evaluated mainly in G542X CFTR nonsense variant and underlined limited efficacy. This study aimed to evaluate ELX-02-mediated CFTR rescue across a broad range of nonsense variants using patient-derived intestinal organoids (PDIOs) to define variant-specific determinants of readthrough efficacy and assess its potential across a genetically diverse CF population. MethodThe ex vivo response to ELX-02 was assessed in 206 PDIOs carrying heterogeneous nonsense variants. CFTR function was quantified using forskolin-induced swelling (FIS) assay after 48-hour exposure to ELX-02. Responses were analysed by genotype and stop codon identity, with secondary validation performed in a selected subset of PDIOs (n = 60). ResultsELX-02-mediated CFTR rescue varied markedly, ranging from responses approaching those observed with approved CFTR modulators (LUM/IVA) to responses at or below detection limit. Overall, maximal responses were modest and at the lower end of the functional range for CFTR modulators. Rescue was dose-dependent and higher in PDIOs carrying two nonsense variants when compared with PDIOs carrying a single nonsense variant combined with a residual or minimal function variant. Nonsense variants in nucleotide-binding domain 1, including G542X, S466X, G550X and R553X, showed relatively higher responsiveness. ConclusionELX-02 induces limited and highly heterogeneous CFTR rescue across nonsense variants. PDIO-based functional screening provides a framework to guide patient selection and stratification for future readthrough therapy trials.

Background: Cystic fibrosis (CF) is associated with chronic inflammation and progressive multisystem disease, which may contribute to accelerated biological aging. Elexacaftor/tezacaftor/ivacaftor (ETI) substantially improves clinical outcomes in people with CF (pwCF), but its effects on epigenetic aging and their relationship with inflammation remain poorly understood. Methods: We performed a pilot longitudinal study of 8 modulator-naive pwCF. Blood DNA methylation was profiled before ETI initiation and after 1 year of treatment. DNAmGrimAge2 age acceleration was calculated adjusting for chronological age and blood cell composition. Associations with inflammatory biomarkers and clinical measures were assessed using repeated measures correlation. Results: DNAmGrimAge2 was strongly correlated with chronological age at baseline and follow-up. DNAmGrimAge2 age acceleration decreased after ETI treatment, from a median of 1.92 years before treatment to -1.70 years after 1 year (p=0.008). Greater age acceleration was associated with higher IL-6, IL-1{beta}, and calprotectin concentrations. Higher FEV1pp was associated with lower age acceleration, whereas higher sweat chloride was associated with greater age acceleration. Conclusions: DNAmGrimAge2 age acceleration decreased following ETI initiation and was associated with markers of innate and neutrophilic inflammation and CF disease activity. These findings suggest that epigenetic aging in CF may be dynamic and responsive to improvements in CFTR function and inflammatory disease burden. Larger longitudinal studies are needed to validate these findings and determine their relationship with long-term clinical outcomes.

Diabetes is a highly prevalent complication of cystic fibrosis (CF), affecting 50% of adults with CF and over 80% of those with exocrine pancreatic insufficiency (PI) by age 50 years. Development of cystic fibrosis-related diabetes (CFRD) is associated with increased morbidity and mortality mostly due to advancement of chronic obstructive lung disease. Highly effective modulator therapy (HEMT), using precision medications targeting the cystic fibrosis transmembrane conductance regulator (CFTR), improves CFTR function and CF lung disease, but its impact on diabetes pathogenesis remains uncertain. We sought to determine whether two types of HEMT, ivacaftor and elexacaftor/tezacaftor/ivacaftor (ETI), alter diabetes prevalence in two large cohorts of individuals with CF and exocrine PI. For comparison, a non-highly-effective modulator, lumacaftor/ivacaftor (LUM/IVA), was also assessed. Data were provided by the CFTR2 project, a multinational CF registry (for ivacaftor and LUM-IVA), and by the CF Genome Project (CFGP), a predominantly US-based CF cohort (for ETI). Among 32,753 individuals with CF (2,803 treated), ivacaftor was associated with reduced diabetes prevalence (age-adjusted OR=0.55). In contrast, lumacaftor/ivacaftor (not highly effective) was not associated with diabetes prevalence (n=32,749). Among 2,854 individuals with CF (2,458 treated), ETI was associated with reduced diabetes prevalence (age-adjusted OR=0.47). Overall, HEMT (ivacaftor and ETI) was associated with a 25-39% reduction in diabetes prevalence in CF, while a non-highly-effective modulator (lumacaftor/ivacaftor) showed no difference. Precision targeted amelioration of CFTR dysfunction can delay onset of diabetes in a high-risk CF population.

Background Elexacaftor-tezacaftor-ivacaftor (ETI) has been demonstrated to improve lung function in clinical trials; however, evidence describing effects on trajectories and whether long-term improvements are sustained (>1-year) is lacking. We estimated within-person lung clearance index (LCI) trajectories before and after ETI initiation, assessing changes in level and rate of change, alongside acute LCI change, up to three years after ETI initiation. Methods Prospective observational study of children at a tertiary hospital. Children aged 3-17 years with [&ge;]2 LCI testing occasions (i) before and (ii) after starting ETI were used to describe lung function trajectories. Children with [&ge;]1 pre-ETI and [&ge;]1 post-ETI LCI occasion(s) were used to describe acute LCI change after ETI initiation. Age-adjusted LCI trajectories for time periods (i) before and (ii) after ETI initiation were estimated using linear mixed-effects models, and pre- and post-ETI LCIs were compared using paired Wilcoxon tests. Results Mean pre-ETI and post-ETI longitudinal changes in LCI were -0.007 (95% CI: -0.28, 0.27; n=35) and 0.12 (95% CI: -0.17, 0.41; n=20) turnovers per year, respectively. Before ETI initiation, 57% (30/53) of patients had an LCI[&ge;]7.1 turnovers (indicating impaired lung function), compared to 26% (14/53) post-ETI, with a median LCI difference of -0.70 (95% CI -0.84, -0.46; p<0.001) turnovers. Within-individual variability in LCI decreased post-ETI. Conclusions Our real-world data within a unique longitudinal study provide a comprehensive picture of ETI benefit by outlining not only acute improvement in LCI but maintained stability in LCI trajectories and improved LCI stability sustained up to three years post-initiation.

Research on Pseudomonas aeruginosa adaptation in cystic fibrosis (CF) has historically relied on comparing chronic isolates to laboratory reference strains, or evolving reference strains in environments simulating chronic CF. This work has established a small set of genes, including lasR, mucA, and mexZ, as canonical markers of CF patho-adaptation. However, without broad non-CF comparators, it remains unclear how specific these signatures are to CF. We used a structured literature review to define 20 historically emphasized "canonical CF genes", then evaluated their mutational patterns across 4,475 genetically distinct P. aeruginosa genomes from seven defined clinical and environmental contexts. We tested four competing hypotheses: (1) enrichment in adult CF alone, (2) in adult and pediatric CF combined, (3) in chronic lung infections broadly (including non-CF bronchiectasis), or (4) no strong environment-specific enrichment. We found little evidence that canonical gene mutations were specifically enriched in adult CF or CF more broadly. Instead, loss-of-function and individual mutations in genes including mucA, mexB, and mexZ were enriched across chronic lung infections, while most canonical genes (including lasR) showed no strong environment-specific enrichment. These results demonstrate that a canon of genes believed to drive patho-adaptation in CF instead largely reflects the narrow comparative framework of past studies rather than CF-exclusive selection. Our findings emphasize shared evolutionary pressures between CF and non-CF bronchiectasis, highlighting opportunities to exchange research and therapeutic insights across chronic infection clinical contexts.

Pseudomonas aeruginosa is an important agent of acute or chronic airway infections. A complex set of genetic and environmental factors determine the outcome of a P. aeruginosa airway infection. P. aeruginosa can undergo genetic adaptation to fine tune the expression of a type III secretion system (T3SS) and other virulence factors in response to selective pressures encountered during infection. Genetic and environmental factors predispose many patient groups for P. aeruginosa infections, including people with cystic fibrosis (pwCF). CF is a genetic disorder resulting in areas of hypoxia and thick mucus that fosters P. aeruginosa airway infection. Resident alveolar macrophages (AMs) help coordinate immune responses to P. aeruginosa in airways by producing proinflammatory cytokines such as IL-1{beta}, which can be important for infection resistance. AMs infected with laboratory strains of P. aeruginosa detect the T3SS and activate the NLRC4 inflammasome, resulting in IL-1{beta} release. Studies of inflammasome responses in AMs to a clinical CF isolate of P. aeruginosa have not been reported. Here, we characterized the CF clinical isolate DH1137 and found that it has a downregulated but functional T3SS, is adapted to grow in hypoxia, and induces significant production of the inflammasome cytokines IL-1{beta} and IL-1 during lung infection in a CF mouse model. Inflammasome gene expression was primed in AMs by LPS stimulation, and upon DH1137 infection these cells released IL-1[beta]. Intriguingly, we found that infection of AMs with DH1137 in hypoxia resulted in significantly dampened inflammasome activation compared to normoxia. These data describe a new mechanism that P. aeruginosa may exploit to evade immune detection by tissue-resident lung macrophages in the context of CF.

Changes in the gut microbiome in cystic fibrosis (CF) are well characterized, yet their causes and downstream effects remain largely unknown. Well-documented alterations include reduced overall complexity (i.e. alpha diversity) of the gut microbiota and increased relative abundance of E. coli, which are associated with greater inflammation and shorter stature in infants. Previous results from our laboratory using a germ-free cystic fibrosis transmembrane conductance regulator (Cftr) mutant mouse model (CF mouse) demonstrated that the observed fecal microbiome dysbiosis is driven by mutated Cftr independent of factors such as diet or antibiotic treatment. We expand on these results in this report by using the defined 8-member community Altered Schaedler Flora (ASF) with and without E. coli, to show that E. coli is pathogenic in the context of the CF gut microbiome, resulting in increased intestinal permeability. We also show that Cftr deletion in intestinal epithelial cells alone, using a Villin-Cre targeted model, is sufficient to raise E. coli abundance in the fecal microbiome, increase intestinal permeability, and amplify the number of TH17 cells in the mesenteric lymph nodes. Together, our results demonstrate that the intestinal epithelium plays a dominant role in fecal microbiome alterations in CF and that the resultant dysbiosis contributes to CF pathogenesis.

Background: GINA-based clinical assessment of asthma control provides limited insight into the molecular mechanisms driving disease progression and treatment response. Circulating microRNAs (miRNAs) are implicated in immune regulation and airway remodeling, but their relationship to longitudinal, treatment-specific asthma control is not well characterized. We aimed to identify treatment-specific miRNAs associated with longitudinal asthma control and evaluate their ability to discriminate well-controlled from uncontrolled asthma. Methods: Baseline serum miRNA sequencing data from 491 children in the Childhood Asthma Management Program (CAMP), a randomized trial of budesonide versus placebo, were analyzed, with GINA-based composite symptom scores assessed at baseline and 2, 4, 8, and 12 months. Cumulative link mixed models were fitted across 266 miRNAs to identify associations with longitudinal ordinal asthma control, adjusting for time, baseline status, and treatment. Random Forest classifiers were trained within each treatment group using Group K-Fold cross-validation. Pathway enrichment of validated miRNA targets was performed with DAVID. Results: In the budesonide group, hsa-miR-1224-5p was associated with lower symptom severity and hsa-miR-199a-3p|hsa-miR-199b-3p with higher severity; both associations persisted at 12 months. The placebo group showed a broader pattern, with ten miRNAs associated with symptoms. Random Forest classifiers achieved mean AUC of 0.776 (budesonide) and 0.714 (placebo) for 12-month control status. Budesonide-associated targets were enriched for glucocorticoid-responsive and MAPK/Ras signaling, while placebo-associated targets showed broad enrichment for general regulatory processes. Conclusion: Treatment-specific circulating miRNAs distinguish asthma control over time and implicate distinct signaling pathways, supporting their potential as complementary molecular markers for asthma monitoring in children.

ABSTRACT Background: The esophageal String Test (EST) is emerging as a minimally invasive alternative for assessing disease activity in eosinophilic esophagitis (EoE), yet the molecular composition of its protein content remains unknown. Objective: To comprehensively characterize the esophageal EST proteome, define an EoE-associated luminal protein signature, and evaluate its biological and clinical relevance. Methods: EST samples were obtained from patients with active EoE, healthy controls (C) and patients with gastroesophageal reflux disease (GERD). Label-free proteomic profiling was performed by liquid chromatography-tandem mass spectrometry. Differential protein accumulation, principal component analysis (PCA), pathway annotation, comparison with published esophageal tissue and salivary proteomes, receiver operating characteristic (ROC) analyses and correlations with clinical disease activity were performed. Results: More than 3,000 proteins were identified in esophageal EST samples. Global proteomic profiling clearly separated patients with EoE from both C and GERD, whereas only limited differences were observed between the latter two groups. This enabled the identification of an EoE-associated luminal protein signature comprising inflammatory, epithelial, and tissue remodeling proteins. Comparison with published proteomic datasets indicated that the EST samples a complex esophageal luminal protein microenvironment containing proteins represented in esophageal mucosa and saliva, together with inflammatory and extracellular components. Several individual proteins showed high discriminatory performance in this exploratory cohort for distinguishing EoE from non-EoE subjects, and multiple proteins correlated with eosinophil counts and endoscopic measures of disease activity. Conclusions: This study demonstrates that the EST provides access to a complex esophageal luminal protein microenvironment and establishes a molecular framework for its minimally invasive characterization in EoE. KEYWORDS: eosinophilic esophagitis, esophageal string test, esophageal luminal protein signature

Rationale: The role of DNA methylation (DNAm) markers in the upper-airway microbiome related to asthma exacerbations despite inhaled corticosteroid (ICS) treatment is unexplored. Objectives: To identify whole-blood DNAm markers associated with the upper-airway microbiome related to asthma exacerbations despite ICS treatment. Methods: We analyzed salivary, nasal, and pharyngeal microbiome in 242 asthma patients (GEMAS) for discovery. We tested associations between whole-blood DNAm at 703,505 CpGs and 21 exacerbation-related microbiome traits. Replication for the nasal microbiome was evaluated in 272 (Project Viva) and 66 (SysPharmPediA) children with and without asthma. We analyzed differentially methylated regions (DMRs), gene enrichment, associations with blood gene expression (RNA-seq, n=119), and DNAm predictors of plasma proteins. Measurements and Main Results: Whole-blood DNAm at 73 CpGs and 550 DMRs was associated with 17 exacerbation-related microbiome traits (false discovery rate [FDR]<0.05), including genes implicated in immune and inflammatory responses and the 17q12-q21 asthma locus ORMDL3. Most CpGs were associated with Campylobacter (pharyngeal) and Leptotrichia (nasal) abundance. Ten DNAm markers were replicated (p<0.05), including the corticosteroid insensitivity-related gene PTPRR associated with nasal bacterial richness. Enrichment analyses identified genes related to diabetes, obesity, smoking, TGF-{beta}, and Wnt signaling and regulated by trichostatin A, NF-{kappa}B, and the glucocorticoid receptor (FDR<0.05). The CpG cg18994033, associated with pharyngeal Tannerella, was associated with ITM2C whole-blood expression. Nine DNAm-predicted plasma proteins involved in inflammation, tissue remodeling, and TGF-{beta} signaling were associated with seven exacerbation-related microbiome traits, including Streptococcus and Selenomonas. Conclusions: Whole-blood DNAm markers implicated in inflammation, immunity, and asthma comorbidities are associated with the upper-airway microbiome linked to asthma exacerbations.

Abstract The upper respiratory tract (URT) microbiome plays a vital role in respiratory health, with older children (5-14 years old) serving as key reservoirs for the transmission of bacteria to younger individuals (< 5 years old). However, the URT microbiome in this age group remains poorly characterised, particularly in relation to colonisation by the pathobiont Streptococcus pneumoniae. To address this gap, we analysed URT samples collected over eight visits from 134 healthy children aged 5-14 years in The Gambia, using culture-dependent methods to detect S. pneumoniae and culture-independent 16S rRNA sequencing to characterise the microbiome in parallel. 16S rRNA sequencing identified Streptococcus and Prevotella as the dominant microbiome constituents. When integrating longitudinal sampling data, we observed temporal instability with frequent switches in microbiome profiles over time. URT samples colonised with S. pneumoniae were associated with increased abundance of Moraxella, Streptococcus and Haemophilus, whereas non-colonised URT samples were enriched with Granulicatella and Filifactor. S. pneumoniae colonisation was also associated with lower Shannon diversity compared with non-colonised samples. Age and season were the strongest determinants of microbiome composition; increasing age was associated with decreasing prevalence and abundance of taxa including Haemophilus and Moraxella, while gram-positive taxa were predominantly enriched in the dry season. These findings provide new insight into the URT microbiome of older children in The Gambia and highlight Granulicatella and Filifactor as potential contributors to colonisation resistance against S. pneumoniae.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling, which may extend to the central airways and alter tracheal morphology. Whether tracheal shape relates to disease severity, quality of life, functional capacity, or survival is not established. Objective: To determine whether CT-derived tracheal lumen eccentricity is associated with cross-sectional and longitudinal clinical outcomes in COPD. Methods: We analyzed 8,383 participants from the multicenter COPDGene study. At each axial cross-section of the trachea, lumen eccentricity ({epsilon}) was computed from the major and minor axis lengths, and the participant-level value was taken as the arithmetic mean across all tracheal slices; {epsilon} = 0 denotes a circular lumen and values approaching 1 denote progressively elongated ellipses. Baseline outcomes were spirometry, 6-minute walk distance (6MWD), St. George's Respiratory Questionnaire (SGRQ) total score, and modified Medical Research Council (mMRC) dyspnea score. Longitudinal outcomes were annualized change in FEV1 and all-cause mortality. Multivariable linear and Cox proportional hazards models were adjusted for [age, sex, race, BMI, smoking status, pack-years, scanner model]. Results: Tracheal eccentricity rose monotonically with severity of airflow obstruction (0.60{+/-}0.07, 0.61{+/-}0.07, 0.62{+/-}0.07, 0.64{+/-}0.08, and 0.64{+/-}0.08 in GOLD stages 0 through 4, respectively). Higher eccentricity was independently associated with lower FEV1 ({beta} = -0.30 L, 95% CI -0.47 to -0.14; p < 0.001), lower FEV1/FVC ({beta} = -0.06, 95% CI -0.09 to -0.04; p < 0.001), shorter 6MWD ({beta} = -15.7 m, 95% CI -25.2 to -6.2; p = 0.001), higher SGRQ score ({beta} = 16.9, 95% CI 11.2 to 22.5; p < 0.001), higher mMRC dyspnea score ({beta} = 0.4, 95% CI 0.1 to 0.6; p = 0.007), and accelerated lung function decline ({beta} = -23.8 mL/year, 95% CI -45.13 to -2.48; p = 0.029). Eccentricity was also associated with all-cause mortality (adjusted HR 1.81, 95% CI 1.01 to 3.26; p = 0.047). Conclusions: Tracheal lumen eccentricity is associated with worse quality of life, reduced functional capacity, accelerated lung function decline, and mortality in COPD, supporting central airway morphology as a candidate imaging biomarker.

Background and Aims: Fibrostenotic stricture is a severe complication of Crohn's disease (CD) that affect up to 50% of patients over their lifetime, causing irreversible narrowing of the intestinal lumen and bowel obstruction, but the molecular programs that distinguish stenotic from non-stenotic inflammation and the relative contributions of fibrosis and smooth-muscle remodeling remain incompletely characterized. We aimed to dissect bowel-wall compartment-specific and histology-defined remodeling programs in fibrostenotic CD. Methods: Matched full-thickness terminal ileal tissues from 4 patients with fibrostenotic CD were sampled from stenotic stricture (SS), non-stenotic inflamed bowel (NS), and non-inflamed bowel (NI). Histomorphology-guided NanoString GeoMx whole-transcriptome profiling was performed across mucosa, muscularis mucosae, submucosa, and muscularis propria. GeoMx data were processed by standard software provided by GeoMx platform. DESeq2 models included patient identity as a blocking factor, and the analysis was performed in 74 histologically valid regions of interest (ROIs) representing full spectrum of CD histopathologies. DEGs were defined as adjusted P < .05 and |log2FC| >= 1.0. Results: Out of 18,677 genes analyzed in each of the ROIs, direct SS-versus-NS comparison identified 0 significant DEGs in mucosa, 18 in muscularis mucosae, 43 in submucosa, and 0 in muscularis propria. NS-versus-NI submucosa showed 132 DEGs (131 increased, 1 decreased), dominated by humoral/plasma-cell genes with a modest stromal-remodeling component. SS-versus-NI submucosa showed 144 DEGs (138 increased, 6 decreased) with coordinated contractile, extracellular-matrix/remodeling and cell-matrix adhesion programs. Muscularized versus fibrotic SS submucosa showed 219 DEGs, with marked enrichment of contractile and remodeling genes in muscularized regions. Excluding 5 follicle-containing submucosal ROIs markedly altered the NS comparison but not the core SS fibromuscular program. Exploratory follicular-niche analyses showed that lymphoid follicle-rich (LFR) submucosa was lymphoid/B-cell enriched relative to lymphoid follicle-null (LFN) areas; SS-LFR nevertheless acquired a stronger fibroblast/ECM program relative to NS-LFR, while CCL19 was the only significant LFR-versus-LFN DEG within SS. Conclusions: Fibrostenotic CD is characterized by spatially concentrated submucosal fibromuscular remodeled inflammatory program. Non-stenotic submucosa is heterogeneous and may already show regional stromal remodeling, whereas established stenosis displays a broader and more robust ECM-contractile-adhesion phenotype with molecularly distinct fibrotic and muscularized niches.

Background: Although the lung is of low biomass, it harbors a diverse and dynamic microbiome that may influence disease and healing. Existing studies have used diverse sampling methods with high propensities for contamination and sampling error, leading to diverse and unclear results. Here, we characterized the lung microbiome via airway and parenchymal samples to determine variation across patients and sampling methods. Methods: We recruited adult patients undergoing lung resection for suspected or confirmed malignancy. After resection and under sterile conditions, a 1 cm cubic piece of non-cancerous lung parenchyma and a swab from the specimen's bronchus were collected and sent for microbiome analysis via 16S rRNA gene amplicon (V4) sequencing on an Illumina platform. An established bioinformatics pipeline was used to determine taxonomic identification. Baseline clinical and demographic data were compared to microbiome composition. Results: A total of 86 patients were included in the study. Beta diversity (microbial composition) varied significantly by sampling method (biopsy of lung parenchyma versus airway swabs), so all further results were analyzed within sample types. Further analyses revealed significant differences in beta diversity by lobe of the lung, indicating a different microbial composition by anatomic location. Analyses of patient demographics revealed significant differences by age and comorbidities, including chronic obstructive pulmonary disease and atrial fibrillation. Conclusions: The lung harbors a diverse microbiome that differs by anatomic location and patient characteristics. This study provides a framework for more accurate future lung microbiome sampling and characterization.

Background. Sarcopenia is common in chronic obstructive pulmonary disease (COPD) and contributes to adverse outcomes. Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause of COPD, but its impact on skeletal muscle remains to be determined. Methods. We performed a retrospective analysis of 487,207 participants in the UK Biobank with available SERPINA1 genotyping. Associations between AAT genotypes and measures of skeletal muscle mass were determined using multivariable linear and logistic regression models. Models adjusted for age, sex, smoking status, genetic principal components, lung function (FEV% predicted), and liver fibrosis risk (FIB-4). We determined that the Z-allele was associated with sarcopenia and mortality using regression models and mediation analysis. Results. Compared with PI*MM individuals, carriers of the Z allele (PI*MZ, PI*SZ, PI*ZZ) demonstrated significantly lower fat free mass index (FFMI) and appendicular skeletal muscle index (ASMI), as well as higher odds of sarcopenia by binary definition. Associations persisted after adjustment for lung and liver function. A dose-dependent relationship was observed, with the greatest reductions in muscle mass and highest sarcopenia associated with PI*ZZ individuals (adjusted OR for FFMI-defined sarcopenia 2.02, 95% CI 1.16-3.32). Each additional Z allele was associated with increased mortality risk (adjusted OR 1.09, 95% CI 1.02-1.16), with sarcopenia mediating a modest proportion (~2.6%) of this association. Conclusions. The SERPINA1 Z allele is associated with reduced muscle mass and increased risk for sarcopenia independent of lung and liver disease. Our findings expand the phenotypic spectrum of AATD-related diseases to include skeletal muscle as a modifiable target for intervention.

Delafloxacin (DLX) is a novel fluoroquinolone with enhanced antibacterial activity in acidic environments, a property that may be advantageous for treating Pseudomonas aeruginosa infections in cystic fibrosis (CF), where airway surface liquid pH is typically reduced (pH 5.5-6.7). However, the propensity for resistance development and the underlying mechanisms in P. aeruginosa remain incompletely defined. We conducted serial passage experiments on six clinical P. aeruginosa isolates (three CF-derived and three non-CF-derived) exposed to sub-inhibitory concentrations of DLX or ciprofloxacin (CIP) at pH 6.0 and 7.3 over nine days. Susceptibility was assessed by broth microdilution (BMD), and resistance mechanisms were characterized by whole-genome sequencing (WGS), efflux pump expression analysis (qRT-PCR), and functional validation using CRISPR/Cas9-mediated genome editing and complementation assays. DLX minimal inhibitory concentrations (MICs) rose only 10.1- to 28.5-fold over 9 days, compared with 77.6- to 97.8-fold for CIP, indicating a substantially higher genetic barrier to resistance. This barrier was most pronounced under acidic conditions: only 38.9% of DLX-passaged samples crossed the resistance breakpoint, compared with 94.4% at neutral pH, whereas CIP resistance reached 100% regardless of pH. Cross-resistance was asymmetric: exposure to DLX consistently selected for CIP cross-resistance (97.2% of samples), whereas exposure to CIP induced DLX cross-resistance efficiently at neutral pH but only partially under acidic conditions. A previously undescribed gyrA mutation (p.Ala51Val) conferred a 4-fold increase in DLX MIC when introduced by CRISPR/Cas9, and upregulation of the MexEF-OprN efflux pump, reversible by mexS complementation, emerged as a prominent resistance mechanism. Overall, DLX exhibited a markedly higher genetic barrier to resistance than CIP in P. aeruginosa, particularly under the acidic conditions characteristic of the CF airway. However, its use may co-select for CIP cross-resistance through efflux upregulation, underscoring the need for careful stewardship in CF.

Objectives Genetic testing is increasingly used in the diagnosis and management of respiratory conditions, including pulmonary fibrosis (PF). The perspectives of people with PF and healthcare professionals (HCP) on the use of genetic testing remain largely unexplored. Methods A qualitative study was undertaken. People living with PF, their caregivers, and HCP were invited to undertake a semi-structured interview. Interviews were conducted via videoconference or telephone, audio-recorded, and transcribed verbatim. Data were analysed by two researchers using inductive thematic analysis. Results Thirty-eight participants; 15 people living with PF, 1 caregiver, and 22 HCPs were interviewed. Analysis revealed three key themes. Genetic testing in PF was valued by all groups; people with PF wanted testing now, whilst respiratory physicians were cautious, citing their uncertainty regarding clinical value. All groups desired more information and support; people with PF desired a better understanding of terminology, whilst genetic counsellors wanted to better understand PF. No single model for returning genetic results in PF was identified, however resources, multidisciplinary care, and timely return of results was considered important. Conclusion Genetic testing is valued by people with PF and their HCP, but uncertainties remain regarding whether it should be offered and how results should be best communicated.

Abstract Background. Enteric hyperoxaluria (EH) causes calcium oxalate kidney stones, oxalate nephropathy, and chronic kidney disease (CKD), and can progress to end-stage kidney disease. It occurs in Crohn's disease (CD), cystic fibrosis (CF), chronic pancreatitis, celiac disease, short bowel syndrome, and after bariatric surgery. Its prevalence in these populations has not been synthesized. Methods. PROSPERO CRD420261364424. PubMed and Embase were searched to 24 August 2026 for studies reporting 24-hour urinary oxalate. Prevalence was pooled using the Freeman-Tukey double arcsine transformation with a DerSimonian-Laird random-effects model; unweighted crude prevalence is also reported. Because heterogeneity was high, the narrative synthesis stratified by testing condition is the primary result. Prespecified subgroups addressed oxalate load before collection, diagnostic threshold, ileal resection, age, and urine collection format. Results. Forty-seven studies were included. In CD (17 studies, 471 patients; 204 with EH), crude prevalence was 43.3% and pooled prevalence 49.3% (95% CI 38.9-59.8; I^2 = 95.3%). With an oxalate load, prevalence was 64.7% (pooled 74.0%, 55.9-88.6) versus 38.6% without (pooled 39.1%, 28.7-50.0). Among CD patients, EH was more frequent with ileal resection (48.8% vs 31.7%; OR 1.62, 0.91-2.89) and with steatorrhea (62.5% vs 29.3%; OR 9.88, 3.81-25.61). In CF (7 studies, 348 patients; 179 with EH), pooled prevalence was 49.8% (40.5-59.1; I^2 = 91.9%). After bariatric surgery (15 studies, 849 patients; 474 with EH), pooled prevalence was 54.7% (45.4-63.9; I^2 = 96.7%). Conclusions. Roughly half of these patients have EH, but the proportion depends heavily on whether an oxalate load precedes urine collection (39% unprovoked vs 65%-74% provoked); because most studies do not state this condition, published prevalence estimates are not directly comparable. Ileal resection and steatorrhea identify those who should be screened.

Background Idiopathic Pulmonary Fibrosis (IPF) is a fatal chronic lung disease with limited therapeutic options. While alveolar epithelial injury and fibroblast activation are well-studied, endothelial-mesenchymal transition (EndoMT) is emerging as a critical pathogenic mechanism. The regulatory role of exosomal miRNAs in pulmonary fibrosis remains unclear. This study investigates serum exosomal miRNAs, particularly let-7a-5p, in modulating EndoMT during the onset of pulmonary fibrosis. Methods Clinical cohorts of IPF patients and healthy controls were enrolled. Serum exosomal miRNAs were profiled, followed by differential expression and functional enrichment analyses. In vitro experiments involved human pulmonary artery endothelial cells (HPAECs) transfected with let-7a-5p mimic or inhibitor. Dual-luciferase reporter assays confirmed the binding between let-7a-5p and TGFBR1. HPAECs were co-cultured with lung epithelial cells to examine paracrine signaling. In vivo studies used a bleomycin-induced mouse model with let-7a-5p agomir administration. Assessments included histopathological staining, hydroxyproline content, Western blot, qPCR, micro-CT, and pulmonary function tests. Results Let-7a-5p was significantly downregulated in serum exosomes from IPF patients, correlating with clinical indicators. Mechanistically, let-7a-5p directly bound the TGFBR1 3'UTR to inhibit its expression. Inhibition of let-7a-5p upregulated -SMA, FN1, smad2/3 phosphorylation, and collagen I, while downregulating CD31 and VE-cadherin. Therapeutically, let-7a-5p mimic reversed bleomycin-induced EndoMT and suppressed epithelial-mesenchymal transition (EMT) via paracrine signaling. Mice administered agomir showed reduced fibrosis, improved lung function, and suppressed TGF-{beta}/Smad signaling. Conclusion Serum exosomal let-7a-5p suppresses pulmonary fibrosis by targeting TGFBR1 to inhibit EndoMT. Its downregulation in IPF patients correlates with disease progression, highlighting its biomarker potential.

Primary cilia coordinate signaling pathways that regulate tissue homeostasis and development, and defects in cilia contribute to numerous ciliopathies. However, the transcriptional consequences of disrupting ciliary protein localization remain poorly defined. ARL13B is a cilia-enriched regulatory GTPase required for ciliary trafficking and signaling. The ARL13BV358A variant is undetectable in cilia yet retains known biochemical functions, providing a unique model to investigate the functions of ciliary ARL13B independently of ciliogenesis. To define transcriptional programs associated with loss of ciliary ARL13B, we generated two independent Arl13bV358A/V358A kidney epithelial cell lines and matched rescue lines. The ARL13BV358A mutation did not affect ciliation frequency or cilia length but altered ciliary protein composition, including loss of ARL3 and INPP5E localization and increased accumulation of GPR161 and TULP3. RNA sequencing revealed expression changes in genes associated with ciliary biology, mechanotransduction, epithelial organization, and kidney-related phenotypes. Despite similar ciliary phenotypes, the independently-derived, mutant clones displayed substantial transcriptomic heterogeneity, highlighting a potential source of variation in CRISPR-based transcriptional studies. By integrating data from the independent mutant and rescue clones, we identified a high-confidence set of 131 genes whose expression reproducibly tracked with loss and restoration of ciliary ARL13B. Together, these findings demonstrate that ciliary ARL13B is required to maintain normal ciliary composition and gene expression programs and underscores the value of multi-clone, rescue-based experimental designs for robust transcriptomic analyses. Summary for ReviewersThis study examined how excluding the protein ARL13B from primary cilia affects kidney epithelial cells. The researchers created two independent cell lines carrying a modified form of ARL13B,along with matched rescue cell lines. The findings show that ciliary ARL13B helps maintain normal ciliary composition. By comparing the cell lines, the researchers identified a high-confidence set of genes associated with loss of ciliary ARL13B. By highlighting the importance of using independent gene-edited clones and rescue-based controls, these results advance understanding of how cilia regulate kidney cell function and provide guidance for designing robust transcriptomic analyses.