Journal of Cystic Fibrosis

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.

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.

Using an analysis pipeline that combines machine learning-based feature selection with classical statistical modelling, we aimed to predict incident inflammatory bowel disease (IBD) and to identify candidate proteins associated with disease development. We used UK Biobank data in an observational cohort study of 52,331 participants with proteomics data and without a history of IBD. The cohort was split into an 80/20 train/test dataset, and three separate extreme gradient boosting models were trained on the training dataset. Using Shapley values, we ranked the predictive importance of 2,912 proteins for incident IBD, Crohn's disease (CD), and ulcerative colitis (UC). The top 20 proteins per outcome served as features in multivariate Cox models predicting 350 incident cases over 14 years. All three proteomics Cox models showed moderate discrimination in the test dataset, with the UC model performing the best (C-index: 0.68), followed by IBD (C-index: 0.65), and CD (C-index: 0.60). All proteomics models outperformed clinical models. Smoking remained the only significant clinical predictor in the combined proteomics + clinical model. 48 unique proteins were identified across the three models, with 16 reaching statistical significance (p< 0.05). Machine learning-based feature selection with statistical survival modelling identifies plasma proteins that predict incident IBD and highlight biologically plausible, potentially targetable pathways in the preclinical phase of IBD.

Fibrotic strictures are among the most disabling complications of Crohn's disease, permanently narrowing the bowel and impairing motility, yet no approved therapy reverses them. Chronic inflammation alters pacemaker-network coupling, smooth-muscle excitability, and calcium-dependent contractility, while fibrosis thickens the bowel wall, narrows the lumen, and changes tissue mechanics. The relative contributions of these coupled electrical, contractile, and structural alterations to motility loss remain unclear. To address this gap, we develop an integrated electromechanical finite-element framework for fibrostenosing Crohn's disease that couples a fibrosis-driven growth model with a FitzHugh-Nagumo electromechanical model. A full-factorial 25 design of experiments is used to quantify the relative effects of electrical diffusivity, excitation threshold, peak active stress, wall stiffness, and hypertrophic remodeling on cyclic lumen-volume deformation. Motility is quantified by the standard deviation of lumen volume over one contraction cycle. Within the parameter ranges examined, increased wall stiffness emerged as the dominant contributor to motility loss, followed by impaired smooth-muscle contractility. Changes in excitation threshold, hypertrophic remodeling, and electrical diffusivity produced substantially smaller effects. Pairwise interactions were small relative to the dominant main effects, indicating that the mechanisms contributed largely through their individual effects. Our findings suggest that limiting wall stiffening while preserving smooth-muscle contractile function may provide a therapeutic strategy for maintaining intestinal motility in fibrostenosing Crohn's disease.

Abstract Background. Tuberous Sclerosis Complex (TSC) is caused by inactivating mutations in either the TSC1 or TSC2 genes, leading to activation of the mammalian target of rapamycin complex 1 (mTORC1) and unhindered cell growth and proliferation. The epithelium of TSC renal cysts in both mice and humans is composed of proliferating A-intercalated (A-IC) cells. The exact molecular mechanism of kidney cystogenesis in TSC remains speculative. Hypothesis.Superfluous cell proliferation driven by mTORC1 activation increases metabolic demand, causing oxidative stress and excess reactive oxygen species. If unchecked, this overwhelms antioxidant defenses and causes cell death. In TSC kidney cystogenesis, Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2), also known as NRF2, serves as the "master regulator" of antioxidant and anti-inflammatory responses, enabling cells to survive and proliferate by clearing the toxic environment and supplying nutrients and fuel. Results. RNA-seq and proteomics, along with western blot analysis, showed robust downregulation of Fumarate Hydrase 1 (FH1) and upregulation of NRF2, STAT3, and HIF1 in kidneys from TSC mice with moderate or heavy cyst burden. Confocal microscopy and immunohistochemical staining on kidney sections, and/or western blot studies on nuclear and cytoplasmic fractions, showed nuclear localization of NRF2, STAT3, and HIF1. In cyst-lining cells in TSC mouse models, FH1 downregulation was associated with inactivating succination of KEAP1 in immunoprecipitation experiments, promoting NRF2 nuclear localization in A-IC cells lining the cysts. NRF2 nuclear localization was associated with ectopic induction of the glutamine transporter SLC38A3 (SNAT3) on the basolateral membrane and activation of the NH3/NH4+ transporters RHCG and RHBG in A-IC cells lining the cysts. Twenty-four h urine NH3/NH4+ excretion rates increased significantly in Tsc1 KO vs. WT mice. The activation of NRF2, STAT3, and HIF1 can drive metabolic reprogramming and activate survival genes in proliferating cells. Together with SLC38A3 induction and upregulation of other glutamine and NH3/NH4+ transporters, these factors activate glutaminolysis and aerobic glycolysis, supplying nutrients to proliferating cystic epithelial cells and supporting cyst expansion in TSC. Consistent with this central role for glutaminolysis in kidney cystic epithelium and TSC cystogenesis, we find a significant reduction in kidney cyst burden in Tsc1 KO mice on a glutamine-free diet. Conclusions. NRF2 plays a critical role in antioxidant defense. Along with STAT3 and HIF1, NRF2 is a key player in metabolic reprogramming through glutaminolysis, which supplies nutrients to proliferating cystic epithelial cells and supports cyst expansion in TSC. These findings suggest that inhibiting or inactivating NRF2, alone or in combination with HIF1 or STAT3, may represent a potential treatment strategy for kidney lesions in TSC.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease associated with failed alveolar epithelial repair with an expansion of aberrant airway-like epithelium in the alveolar space leading to lung function decline usually resulting in death within 3-5 years of diagnosis. While single-cell and spatial transcriptomic approaches have been used to characterize disease-emergent cell populations, less is known about the regulation of transcriptional programs that drive failed alveolar epithelial cell repair in IPF. DNA methylation is a fundamental layer of gene regulation that stabilizes differentiated cell identity; however, changes in methylation in the epithelial compartment in IPF have not been studied. To identify novel epigenetic mediators of epithelial cell dysfunction, we performed high-resolution DNA methylation profiling of purified distal lung epithelial cells from 10 age-matched control and 14 IPF lungs using Oxford Nanopore Technologies (ONT) whole-genome, long-read sequencing. We identified widespread methylome remodeling in the IPF lung epithelium, with 84% of differentially methylated regions (DMRs) hypomethylated. DMRs were largely found outside of promoters, with 88% outside of 3 kb from the transcription start site (TSS), consistent with altered distal regulatory element activity. DMRs were enriched for transcription factor (TF) binding site motifs and gene associations consistent with dysregulation of polycomb repressive complex 2 (PRC2) and increases in p63 activity. DNA methylation at DMRs associated with p63 target genes, including KRT5, and genes implicated in failed epithelial repair, including MUC5B, MMP7, and S100A2 inversely correlated with gene expression. Consistent with the observed dysregulation of the IPF epithelial methylome, an experimental co-culture model of alveolar type II epithelial cell (AT2s) to basal-like epithelial cell transdifferentiation revealed widespread hypomethylation. Further, sites associated with NKX2.1 and FOXA1/2 binding, TFs involved in alveolar fate maintenance, were hypermethylated, suggesting loss of epigenetic regulation of alveolar identity. Together, these data implicate DNA methylation in the failed alveolar epithelial repair processes in IPF, potentially providing future therapeutic strategies by identifying putative regulatory elements associated with aberrant transcriptional programs.

Acute respiratory distress syndrome (ARDS) is a severe lung disorder influenced by the interplay between the intestinal microbiome, host metabolism, and immune function. We previously found that Bacteroides acidifaciens (BA) has opposing effects on staphylococcal enterotoxin B (SEB)-induced ARDS, worsening the condition in female mice while alleviating it in males. This study aimed to examine whether these outcomes are linked to sex-dependent changes in the intestinal microbiome and gut-lung metabolome. C3H/HeJ mice of both sexes were orally colonized with Bacteroides acidifaciens before SEB exposure. Stool and bronchoalveolar lavage fluid (BALF) were collected for analysis using 16S rRNA sequencing and untargeted liquid chromatography mass spectrometry metabolomics. Our findings revealed distinct sex-dependent metabolic and microbial responses. In females, disease exacerbation correlated with elevated lipid mediators (leukotriene B4, 13-oxoODE, and 13S-HODE) alongside shifts in bile acid and redox metabolism. The intestinal microbiome showed changes in Bacteroides, Akkermansia, and Lachnospiraceae. Conversely, males showed a protective phenotype characterized by increased butyrate and alterations in sphingolipid, amino acid, redox, and nucleotide pathways. Integrated analyses identified sex-associated microbial communities and metabolomic profiles, highlighting distinct patterns of lipid and redox associations in females versus sphingolipid and short-chain fatty acid patterns in males. These results suggest that host sex influences how a commensal bacterium affects inflammatory lung injury and point to pathways for future research.

Background. Polygenic risk scores (PRS) capture inherited susceptibility to asthma, whereas methylation risk scores (MRS) may reflect disease-associated epigenetic variation. We assessed whether these scores differ in patients requiring biologic therapy and whether MRS change after mepolizumab initiation. Methods. Cross-sectional analyses in the GEMAS study included 699 participants with genotype data (428 with asthma, 271 controls) and 668 with blood DNA methylation data (408 with asthma, 260 controls). We evaluated 35 existing asthma PRS and derived six MRS from published epigenomic asthma studies. Longitudinal MRS changes after mepolizumab initiation were assessed in 16 GEMAS patients and were replicated in 20 patients from the MEGA study. Results. Twenty-eight PRS differentiated biologic-treated patients from controls (areas under the curve [AUC], 0.68-0.75), but none distinguished biologic-treated and non-treated patients after multiple-testing correction. MRS was higher in asthma without biologic than in controls (odds ratio [OR], 1.64; 95% CI, 1.34-1.99) and lower in biologic-treated than in asthma without biologic patients (OR, 0.54; 95% CI, 0.35-0.81). MRS did not differ significantly between biologic-treated patients and controls. Following mepolizumab initiation, MRS decreased at 6 and 12 months in GEMAS (p=4.81x10-5 and p=2.40x10-5, respectively). The 6-month decrease was also observed in MEGA (p=3.25x10-9). MRS epigenetic markers were predominantly hypermethylated, opposite to their described pattern in asthma risk. Conclusions. Asthma PRS did not differ significantly between biologic-treated and non-biologic-treated patients, but asthma MRS were lower in biologic-treated patients. The MRS decreased after mepolizumab initiation in two independent cohorts, consistent with a reversal of asthma-associated epigenetic patterns.

Objectives: To investigate whether the PneumoRator wearable sensor could accurately assess Respiratory Rate (RR) and signs of air hunger (SAH) compared with clinician-observed assessment in adults with and without breathing pattern disorder (BrPD). Design: A pilot cross-sectional observational study. Setting: A research laboratory on a university campus. Participants: Healthy adults aged 18-40, who were not pregnant, no smokers or history of cardiovascular, respiratory or neurological illness. Thirty consented to the study, Twenty-nine had full data for analysis. Interventions: Participants wore the PneumoRator chest-worn sensor continuously for 10 minutes while seated at rest. Main outcome measures: Respiratory Rate was compared between PneumoRator-derived measurements and randomised 1-minute observer manual counts. Clinician-observed SAH and BrPD were assessed using the Breathing Pattern Assessment Tool (BPAT). Participants also completed the Nijmegen Questionnaire. PneumoRator signals were analysed to identify large-volume breaths as potential SAH events. Results: Thirty participants were recruited. Twelve participants met BPAT criteria for BrPD and four met Nijmegen Questionnaire criteria. There was no significant difference between manual and PneumoRator RR measurements (p=0.89). No significant difference was observed between methods for SAH detection (p=0.17). Participants with clinician-observed SAH demonstrated significantly higher PneumoRator signal amplitudes than those without SAH (p=0.035). Conclusion: The PneumoRator produced RR measurements comparable to clinician assessment and demonstrated potential for identifying signs of air hunger. Findings from this pilot study indicate that the PneumoRator device shows promise as a tool for continuous RR monitoring and as an adjunct to BrPD assessment. Larger clinical validation studies are now warranted.

Study Question. COPD exacerbations with respiratory failure have an outsized impact on morbidity, mortality, and healthcare costs. Physicians treat these severe events with high doses of corticosteroids out of concern for intrinsic corticosteroid resistance. This study was designed to determine whether people who suffer from exacerbations with respiratory failure have intrinsic steroid resistance that could affect that decision. Patients and Methods. A matched, case-control study was performed at University of Colorado. Subjects were enrolled into the Exacerbation group if they were hospitalized for an exacerbation requiring ventilation, or into the Stable COPD group if they had no inpatient exacerbation within 12 months. Stable COPD subjects were frequency-matched to Exacerbation subjects for age, smoking status and FEV1%. Corticosteroid sensitivity was assessed ex vivo and in vivo >45 days following the last exacerbation during a period of relative quiescence. The primary outcome was the group difference in methylprednisolone IC50 for LPS-stimulated IL-8 release by mononuclear cells. Measurements and Main Results. The Exacerbation group (N=8) had more exacerbations in the prior year (6.0 vs. 0.5; p<0.01) and had higher baseline blood lymphocytes (p<0.01) versus Stable controls (N=8). Ex vivo, methylprednisolone equally suppressed IL-8, IL-6, TNFalpha and IL-1bets release by mononuclear cells and whole blood across groups. In vivo, methylprednisolone decreased plasma mediators and mononuclear cells in both groups but had a small differential effect on mononuclear cell gene expression. Answer to the Question. Exacerbations with respiratory failure have persistent lymphocytosis, an altered gene response to corticosteroids, but no corticosteroid resistance to suppress inflammation.

Background: Moderate-to-severe asthma affects 15-30% of asthma patients, but accounts for over 50% of healthcare costs and disproportionate morbidity. However, the genetic mechanisms underlying moderate-to-severe versus mild asthma remain poorly understood. Methods: We performed separate pooled multi-ancestry GWAS of mild (N = 14,372) and moderate-to-severe asthma (N = 7,096) versus non-asthma controls (N = 28,816) among adult participants in the NIH All of Us Research Program (version 8). European, African, and Latino/admixed ancestry-specific GWAS were combined using fixed-effect meta-analysis. Lung expression quantitative trait loci (eQTL) and pathway analyses were performed to identify candidate genes and biological pathways across asthma phenotypes. Results: Mild and moderate-to-severe asthma shared susceptibility loci at IL1RL1, IKZF3, and the GTF3AP1-IL33, GTF3AP1-RANBP6, LINC02757-EMSY and HLA-DRB1-HLA-DQA1 regions. Shared lung eQTLs implicated IL18R1, IL18RAP, HLA genes, IL33, LRRC32, GRB7, MIEN1 and GSDMB. In contrast, the phenotypes exhibited distinct genetic architectures. Mild asthma was characterized by broader HLA class II signals and associations at SMAD3 and GSDMB, with enrichment of antigen presentation, T-helper cell differentiation, and TGF-{beta} regulation, consistent with adaptive immune mechanisms. Moderate-to-severe asthma was characterized by WDR36 and PTCH1, with lung eQTLs implicating TSLP, CAMK4, and FANCC. Pathway analysis identified enrichment of IL-13, IL-6, and IL-10 production, myeloid leukocyte differentiation, and oxidative stress responses, consistent with innate inflammation and pathways implicated in steroid resistance. The IL33 signals showed a severity-gradient effect, with stronger associations in moderate-to-severe asthma. Conclusion: Mild and moderate-to-severe asthma exhibit shared and distinct genetic architectures that support existing biologic targets and suggest novel therapeutic candidates.

Background: Sex differences in asthma are well recognized, but sex-associated airway DNA methylation (DNAm) patterns and their relationships with gene expression remain incompletely characterized. We therefore conducted cis- and trans-expression quantitative trait methylation (eQTM) analyses using sex-stratified and CpG-by-sex interaction models. Methods: We analyzed nasal airway epithelial DNAm and transcriptomic data from 298 participants in the CAAPA discovery cohort. Asthma-associated differentially methylated CpGs (DMCs) were identified using sex-combined and sex-stratified models. Cis- and trans-eQTM analyses were then performed in participants with asthma using sex-combined, sex-stratified, and CpG-by-sex interaction models. To prioritize asthma-associated regulatory relationships, eQTMs that were also significant in non-asthmatic controls were excluded from downstream analyses. Replication in an independent bronchial epithelial cohort was defined by concordant effect direction and P < 0.05. Replicated targets underwent pathway and network analyses. Findings: Asthma-associated DMCs were predominantly hypomethylated in females and hypermethylated in males. Among trans-eQTMs, 379 associations were shared across all four models, whereas the majority were model-specific, indicating extensive sex-dependent regulatory architecture. We replicated 3, 1, 9, and 2 cis-eQTMs and 96, 84, 216, and 41 trans-eQTMs in the sex-combined, female-stratified, male-stratified, and interaction models, respectively. Sex-combined targets implicated innate immune and inflammatory signaling (TNF, IRF8); female-stratified targets implicated cytoskeletal remodeling, focal adhesion, and PI3K-Akt signaling (ITGA10); male-stratified targets implicated transcriptional regulation and post-translational modification (MOV10, HDAC7, CSF1, KAT2A); and interaction targets implicated lipid-mediator metabolism and inflammatory signaling and resolution (CYP2C9, LTB4R, CCL19). Our sex-dependent analyses showed that the majority of eQTMs resided within the trans-regulatory architecture, suggesting that sex-dependent gene expression differences are associated predominantly with trans rather than cis regulatory mechanisms. Pathway analyses revealed enrichment of innate immunity in the sex-combined model, structural remodeling in females, transcriptional control in males, and inflammatory resolution in the sex-interaction model. Interpretation: Sex-specific and sex-dependent airway methylation-expression associations were largely undetected by sex-combined analysis. Sex-aware analytical models may help resolve epigenetic mechanisms underlying sex differences in asthma biology.

Background and Aims: As a microbiome/diet-derived vitamin, queuine (q) is a micronutrient that eukaryotes acquire from microbiota or diet. Vitamin q, the free base form of queuosine (Q), is used for Q-modification of transfer RNA (tRNA). Although tRNA-Q-modification in regulating eukaryotic cell physiology is recognized, the relationship between Q-tRNA-modification and the human microbiome in health and inflammatory bowel diseases (IBD) remains unknown. Which bacteria produce q and how the q-producing bacteria contributing to human health and IBD are not explored. Here, we hypothesize that disrupted microbial Q-biosynthesis contributes to the dysbiosis of human IBD. We performed comprehensive multi-omics integrative analyses using publicly available datasets. Specifically, we re-analyzed publicly available shotgun metagenomic, single-cell RNA sequencing (scRNA-seq), metabolic datasets, and completed meta-analysis. Methods: To systematically investigate microbial Q-biosynthesis in healthy individuals and IBD patients, we performed a systematic secondary analysis by integrating 9 publicly available metagenomic datasets, 15 scRNA-seq datasets and one metabolic dataset published prior to 2026. We present a novel secondary bioinformatic reanalysis of sequence outputs mapped to the latest microbial taxonomy, functional pathways and microbial Q-related genes. Longitudinal study of shotgun metagenomics across the human lifespan (3146 samples) enabled taxonomic and functional profiling. Results: We first identified key q-producing bacteria (e.g., Bacteroides and Alistipes) in healthy populations. We then found that patients with IBD had significantly reducation of q-producing bacteria and a coordinated downregulation of the Q-related pathways, including Q, preQ0, de novo guanosine ribonucleotide biosynthesis and de novo guanosine deoxyribonucleotide biosynthesis pathways. This functional impairment was primarily driven by reduced contributions from Bacteroides, Alistipes and Akkermansia. The longitudinal stability of the q-producing bacteria community across the human lifespan (1638 samples). We observed a significant negative correlation between host age and the abundance of q-producing bacteria in patients with UC. Moreover, Queuine tRNA-ribosyltransferase 1 (QTRT1) expression in human intestinal epithelial cells was significantly reduced in patients with IBD compared to healthy controls. Conclusions: Our study used multi-omics datasets to define a novel functional dysbiosis characterized by a reduction of microbial Q-biosynthesis in patients with IBD. Vitamin q and its tRNA modification is lesser-recognized in human IBD. Understanding the cellular and organismal mechanisms of this microbiome-dependent micronutrient will advance the prevention of IBD and improve the quality of life of patients with IBD.

Background: Cardiovascular-kidney-metabolic (CKM) syndrome integrates cardiac, renal, and metabolic abnormalities that drive multi-organ injury. In pulmonary arterial hypertension (PAH), CKM manifests as systemic metabolic derangements, right ventricular dysfunction, and renal compromise. Renal dysfunction strongly predicts mortality in PAH, yet current therapies provide little renal benefit. Moreover, the mechanisms driving PAH nephropathy remain poorly understood, limiting our ability to effectively treat PAH-CKM. Methods: Single-nucleus RNA sequencing identified cell-type-specific transcriptional alterations in autopsy-derived kidneys from control (n=5) and PAH (n=4) patients. Mitochondrial, cytoplasmic, and phosphoproteomic analyses profiled proteomic alterations. AlphaFold 3/ChimeraX modeling defined the predicted structural consequences of altered protein phosphorylation. Histological analysis assessed renal fibrosis, glomerular structure, immune cell infiltration, and nephron segment density. Results: 3 of 4 PAH patients exhibited renal impairment, with a cohort mean estimated glomerular filtration rate of 61 plus or minus 36 mL/min/1.73m squared. snRNA-seq identified a distinct cellular architecture in PAH kidneys, marked by an increase in thick ascending limb, proximal tubule, and immune cell nuclei and depletion of collecting duct nuclei. Transcriptional profiling demonstrated proximal tubule and thick ascending limb cells both upregulated fatty acid oxidation, ferroptosis, and cuproptosis pathways. PAH lymphocytes displayed heightened T87 cell receptor signaling, natural killer cell-mediated cytotoxicity, and Th17 differentiation. Histological analyses demonstrated increased perivascular fibrosis, glomerular T-cell infiltration, and a reduction in glomerular basement membrane density in PAH kidneys. Mitochondrial and cytoplasmic proteomics revealed broad metabolic dysfunction characterized by impaired {beta}-oxidation, TCA cycle activity, amino acid metabolism, transsulfuration, and urea cycle pathways. Phosphoproteomics predicted increased GSK3{beta}, STK, and casein kinase activity in PAH kidneys. Finally, using the totality of our data, we nominated multiple druggable targets that could be evaluated to counteract PAH nephropathy. Conclusions: Integrated multi-omic profiling demonstrates PAH nephropathy is defined by glomerular structural remodeling, proximal nephron ferroptotic and cuproptotic signaling, amino acid metabolic dysregulation, and innate and adaptive leukocyte activation. Future studies intervening on these pathways could lead to the development of novel therapeutics to augment renal function in PAH.

Introduction: Fighting the ventilator (FV) is characterized by a patients active counteraction of respiratory gas flow across multiple breaths during mechanical ventilation. An effective ventilation is prevented during such episodes. The objective of this exploratory study was to evaluate clinicians knowledge, perception and agreement regarding this condition. Methods: In a cross-sectional online survey, medical professionals with experience in intensive care medicine (mostly neonatal and pediatric patients) responded to 11 questions covering various clinical aspects of FV. Results: All respondents were familiar with the phenomenon and assessed it to be clinically relevant. Neonates were perceived to be predominantly affected. Promoting factors of FV included inadequate sedation and ventilation-associated factors. In clinical practice, the participants identified FV by clinical evaluation, analysis of ventilator waveforms and capnography and used both pharmacological and non-pharmacological intervention strategies. There was no consensus concerning the English terminology of the condition. Only few respondents were familiar with local guidelines to manage FV. Conclusion: Although there was consensus on the relevance of FV, diagnostics and management strategies were highly heterogeneous. Many participants agreed that poor patient-ventilator synchrony was an important cause of FV. Future work should focus on characterizing and generating evidence on FV.

Pancreatic ductal adenocarcinoma (PDAC) arises within an extensively remodeled microenvironment, yet tissue markers that distinguish invasive carcinoma from precursor lesions remain limited. Integrating four single cell RNA sequencing atlases comprising 324,662 cells from 26 patients, we identified cancer associated fibroblast expansion, accumulation of C1QA+ immunoregulatory macrophages, and depletion of a dendritic cell compartment expressing plasmacytoid markers. Fibroblasts were enriched for extracellular matrix and PI3K AKT programs and macrophages for an antigen-presentation program. We then examined the protein level correlates of these populations in a tissue proteomic reference framework of 356 samples spanning non diseased pancreas, normal-adjacent tissue, low and high grade intraductal papillary mucinous neoplasms (IPMNs), IPMN associated invasive carcinoma, and PDAC, prioritizing proteins that changed monotonically across the six histopathologic reference groups or marked tumor enriched cell types. Despite depletion of plasmacytoid dendritic cells, the principal source of type I interferon, the interferon stimulated protein ISG15, expressed by both fibroblasts and macrophages, increased monotonically toward PDAC. In exploratory cross-validated benchmarking of PDAC versus low and high grade IPMN, excluding IPMN-associated invasive carcinoma (145 versus 56 samples), CATB+EPIPL achieved an AUC of 0.98, with a sensitivity of 92% at an achieved specificity of 92%, exceeding protein abundance models of diagnostic IHC-associated markers (S100P+IMP3+maspin+MSLN, 0.79) and refitted published IPMN progression combinations (<0.91). Against non diseased pancreas, candidate and comparator panels performed similarly (AUC 0.99 1.00). These biologically informed tissue protein panels warrant independent validation, including immunohistochemical evaluation of EUS guided fine needle biopsy specimens and measurement in blood.

Background and Aims: Fibroblast-neutrophil interactions are implicated in ulcerative colitis (UC) treatment resistance, but whether fibroblasts modify neutrophil inflammatory activity remains unclear. We investigated responses to fibroblast exposure, stromal depletion, and 5{beta}1-directed treatment. Methods: We integrated human and mouse colonic single-cell RNA sequencing (scRNA-seq), spatial protein profiling (CODEX), fibroblast-neutrophil coculture, and dextran sulfate sodium (DSS) mouse colitis with fibroblast activation protein (FAP) ablation or 5{beta}1-directed treatment. Readouts included cellular representation and protein fluorescence via flow cytometry, neutrophil extracellular trap (NET)-associated elastase activity, RNA programs and epithelial responses using scRNA-seq. Results: Inflamed UC tissue contained higher fractions of inflammatory fibroblasts and oncostatin M (OSM)- and CXCR4-associated neutrophils. Inflammatory fibroblasts had higher FAP-5{beta}1 module scores, and proximity to FAP-high fibroblasts was associated with higher neutrophil OSM and CXCR4 fluorescence. UC fibroblasts increased neutrophil OSM, CXCR4 and myeloperoxidase (MPO) fluorescence and NET-associated elastase activity relative to control fibroblasts, while 5{beta}1-directed treatment attenuated these responses. Both mouse FAP ablation and 5{beta}1 blockade in DSS colitis reduced histologic inflammation and overall neutrophil frequency. Ablation broadly reduced recovered neutrophil representation, whereas blockade increased OSM-positive and PADI4-positive percentages within neutrophils despite lower MPO fluorescence within these subsets. Both interventions were associated with lower epithelial chemokine scores but distinct absorptive and mucus/secretory responses. Conclusions: Fibroblast exposure modifies neutrophil inflammatory phenotype and effector-associated activity, identifying candidate therapeutic pathways in UC. Evaluating these responses alongside neutrophil representation and tissue inflammation could inform pharmacodynamic assessment of stromal-directed therapies.