American Journal of Respiratory Cell and Molecular Biology

Purpose: Airway remodeling is a convergent feature across respiratory diseases, yet current CT tools provide limited characterization of the airway tree. We present Radiomics of the Airway (RadAr), an automated framework for multi-scale airway phenotyping from routine chest CT. Methods: RadAr extracts multi-scale, interpretable airway measurements capturing luminal dimensions, tapering, architectural distortion, and global morphology and provides an interactive web portal for analysis and visualization. It was evaluated across four settings: 63-week mortality prediction in fibrotic interstitial lung disease (fILD; N=147), COVID-19 severity prediction (N=1164), structure-function association in progressive pulmonary fibrosis (PPF; N=9) and structure-inflammation markers in pediatric cystic fibrosis (CF; N=11). Unsupervised clustering identified airway phenotypes across the fILD and COVID-19 cohorts. Results: In fILD, lower-lobe architectural distortion was associated with mortality (balanced accuracy 0.654). In COVID-19, severe disease was independently associated with luminal dilation (AUC 0.719, odds ratio 2.32, p=0.017). In PPF, airway phenotypes correlated with forced vital capacity ({rho}=0.83), mid-expiratory flow ({rho}=0.87), and 129Xe MRI alveolar gas exchange impairment ({rho}=0.70). In pediatric CF, reduced tapering and increased cylindricity were associated with prior exacerbations and bronchoalveolar lavage neutrophilia ({rho}=-0.64 to -0.78). Five phenotypes were identified from extensive, tapered airway trees to sparse, dilated, thick-walled, tortuous trees, with increasing COVID-19 severity and fILD mortality across this spectrum. Conclusions: RadAr identified interpretable, disease-specific airway signatures associated with function and outcomes across restrictive, obstructive, and mixed lung diseases in adult and pediatric settings. It provides a scalable framework that may support diagnosis, risk stratification, and longitudinal monitoring across pulmonary diseases.

Background: Randomized controlled trials have shown that computed tomographic (CT) screening reduces lung cancer mortality. Improved identification of at-risk groups, by leveraging non-smoking risk factors, could help refine screening selection. Aim: To evaluate polygenic risk scores (PRSs) and ambient air pollution (AAP) exposure for risk stratification in the NELSON lung cancer screening cohort. Methods: Two PRSs (PRS-McKay/PRS-Byun) and several AAPs (including nitrogen dioxide, ozone, and particulate matter [PM]) were assessed in the NELSON lung cancer screening trial (N=7,364). PRSs were validated in the Rotterdam Study (N=11,493). Associations with lung cancer, mortality, screening results, and discriminative ability to distinguish lung cancer were evaluated. Results: PRS-McKay and PRS-Byun were associated with lung cancer (odds ratio [OR] per SD [95%CI]: 1.22 [1.08-1.37] and 1.28 [1.13-1.44], respectively) and lung cancer-specific mortality (OR [95%CI]: 1.24 [1.05-1.47], for both), but not with non-lung cancer mortality (OR [95%CI]: 1.01 [0.94-1.10] and 1.03 [0.95-1.12], respectively). Exposure to PM2.5 was associated with lung cancer (OR [95%CI]: 1.11 [1.01-1.22]). PM constituents were associated with adenocarcinoma, particularly PM10 (OR [95%CI]: 1.16 [1.01-1.32]) and ultra-fine particles (OR [95%CI]: 1.16 [1.04-1.30]). PRS and AAP added modestly to the discriminative ability for lung cancer on top of pack-years, age, and sex (area under the curve [95%CI]: 0.659 [0.624-0.695] vs. 0.643 [0.608-0.679]). Conclusions: PRSs and exposure to PM were associated with lung cancer in a high-risk screening population. The primary potential of PRSs may reside in refining lung cancer screening selection toward individuals at higher risk of dying from lung cancer specifically.

Residual congestion is the principal driver of heart failure readmission, and reliable serial assessment of volume status remains an unmet clinical need. This study asked whether a wrist-worn, machine-learning-based device for non-invasive venous waveform analysis in heart failure (the NIVAHF device), which produces an integer-scaled estimate of pulmonary capillary wedge pressure termed the NIVA Score, responds to acute changes in volume status. Agreement between the NIVA Score and invasively measured pulmonary capillary wedge pressure at single time points has been established in a separate prospective, multi-site study; however, such static agreement does not establish whether the measure tracks dynamic decongestion. We therefore evaluated the directional responsiveness of the locked NIVA Score in two prespecified cohorts: hospitalized adults with acute decompensated heart failure undergoing routine intravenous diuresis, and a controlled porcine model of volume overload followed by diuresis. In eleven patients contributing thirteen paired measurements (mean net fluid balance -2.1 {+/-} 1.0 L), NIVA Scores decreased significantly after diuresis (paired t-test, P = 0.04). In five pigs contributing twenty-four paired measurements, NIVA Scores decreased significantly after intravenous furosemide following crystalloid loading (P < 0.01), and the direction of change was concordant with measured urine output in every animal. Statistical significance was reached in both cohorts despite modest sample sizes, indicating a measurable NIVA Score reduction with volume removal. In an exploratory analysis, the discharge NIVA Score yielded an area under the receiver-operating-characteristic curve of 0.85 (95% confidence interval 0.575-1.00; P = 0.04) for thirty-day readmission. Together, the significant, directionally concordant NIVA Score reductions across independent clinical and preclinical cohorts demonstrate that the device tracks acute decongestion and support its use for serial, non-invasive congestion monitoring; an adequately powered prospective study is the planned next step.

Stress-related psychiatric disorders and inflammatory bowel diseases share high co-morbidity and contribute to the symptom severity of one another. In mice, ten days of Chronic Social Defeat Stress (CSDS) is sufficient to reduce gut microbiome diversity and the relative abundance of Firmicutes, which are hallmarks of inflammatory bowel diseases. However, mechanisms by which stress causes gut microbiome dysbiosis are largely unknown. Here, we demonstrate that pharmacologically inhibiting {beta}-adrenergic receptors (ARs), which are activated by (nor)adrenaline during stress, mitigates gut dysbiosis otherwise caused by CSDS. Compared to vehicle-treated mice following CSDS, propranolol-treated mice displayed a modest increase in sociability, increased alpha diversity, and increased abundance of anaerobic commensal Clostridia. Abundance of short-chain fatty acid-producing anaerobic Firmicutes abundance correlated with sociability following CSDS across all treatments. Pharmacologically blocking -ARs during stress increased subsequent sociability, but had little effect on gut microbiome composition. Together, our findings support the hypothesis that {beta}-AR activation contributes to stress-induced changes of the gut microbiome. One Sentence SummaryPharmacologically inhibiting beta-adrenergic receptors during chronic stress mitigates reductions in anaerobic, short-chain fatty acid-producing bacteria in the gut.

Background: Neighborhood-level social determinants of health influence cardiovascular outcomes; however, their association with post-discharge healthcare utilization in heart failure with preserved ejection fraction (HFpEF) remains incompletely defined. Methods: We conducted a retrospective cohort study of 6,702 adults hospitalized for HFpEF (2014 to 2022). Patients were assigned to one of four neighborhood environments (NEnv-1 to NEnv-4) using a validated clustering framework based on ZIP code-level socioeconomic variables. The primary outcome was time to first HF readmission, evaluated within prespecified post-discharge intervals (0-30 days, >30-90 days, and >90-365 days). Secondary outcomes included HF-related healthcare re-encounters and HF hospitalization burden (0, 1, or [&ge;]2 admissions). Cox proportional hazards and multinomial logistic regression models were used. Results: Neighborhood environment was independently associated with post-discharge outcomes with distinct temporal patterns. Early (0-30 days) HF readmission risk was higher in NEnv-3 (aHR, 1.63) and NEnv-4 (aHR, 1.76), with similar increases in HF-related re-encounters (aHR, 1.72 and 1.84) persisting through the >30-90-day interval. In contrast, NEnv-2 demonstrated a delayed-risk pattern, with the highest risk occurring in the >90-365-day interval (readmission aHR, 3.42; re-encounter aHR, 3.45). All non-reference environments were associated with a higher likelihood of at least one post-index HF admission (aOR range, 1.84-2.24). NEnv-4 uniquely demonstrated higher odds of recurrent hospitalization ([&ge;]2 vs. 1 admission; aOR, 1.64). Conclusions: Neighborhood environment is associated with distinct, time-dependent patterns of HF utilization in HFpEF, including early, delayed, and recurrent risks. Incorporating neighborhood context may help identify when patients with HFpEF are most vulnerable after discharge and guide the timing of post-discharge interventions.

Efficient systemic delivery to the lung remains a major barrier for adeno-associated virus (AAV)-mediated pulmonary gene therapy, particularly when pre-existing immunity limits the use of conventional capsids. Here, we evaluated Bovine AAV, a phylogenetically divergent capsid, as candidate vector for lung-directed gene transfer. In adult C57BL/6J mice, intravenous delivery of Bovine AAV resulted in robust and preferential lung transduction comparable to AAV4, with predominant targeting of alveolar type I pneumocytes and pulmonary endothelial cells. In primary human lung-resident cells, Bovine AAV was particularly effective in microvascular endothelial cells, a target poorly transduced by AAV4 in vitro. Bovine AAV demonstrated scalable production with yield, purification performance, capsid quality, and genome integrity comparable to AAV9. In sera from healthy adults from the United States and Switzerland, Bovine AAV showed intermediate neutralization frequencies, lower than AAV2 and AAV4 but higher than AAV5 and AAV9. Of relevance, Bovine AAV maintained in vivo transduction efficiency in mice previously immunized with a pool of human and non-human primate-derived AAV capsids, including AAV4. Together, these results position Bovine AAV as a promising lung-tropic and immune-distinct vector for pulmonary gene therapy, with particular relevance for applications requiring systemic delivery in the presence of pre-existing immunity to conventional serotypes.

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

Background: Short-chain fatty acids (SCFAs) are microbial metabolites with immunoregulatory properties. Human milk contains SCFAs which have been proposed as potential modulators of infant immune development. We aimed to examine associations between human milk SCFA concentrations and infant allergic disease outcomes in a high-risk cohort of infants of atopic mothers. Methods: SCFAs were measured by targeted liquid chromatography-mass spectrometry in human milk samples collected at 3 and 6 months postpartum from atopic mothers enrolled in the Infant Fish Oil Supplementation (IFOS) Study (n=147). Associations between milk SCFA concentrations and early childhood allergic disease outcomes (atopic dermatitis, food allergy, allergic rhinitis, and allergen sensitisation at 1 and 2-3 years) were examined using logistic regression. Results: Human milk SCFA concentrations were broadly stable between 3 and 6 months postpartum, except for acetate which was significantly elevated at 6 months. No significant associations were observed between human milk SCFA concentrations and any allergic disease outcome after correction for multiple comparisons (all p>0.05). Conclusions: Human milk SCFA concentrations are not associated with allergic disease outcomes up to 4 years of age. These findings suggest that oral SCFA exposure via human milk is insufficient to reduce infant allergy risk, and that gut SCFA production may be a more relevant target for future allergy prevention research.

Background Early-life respiratory syncytial virus (RSV) infection is associated with childhood recurrent wheeze or asthma (RW/A), but causality and shared genetic liability remain unclear. Methods We combined Finnish nationwide registries and Nordic genetic cohorts. First, in 965 312 Finnish children born between 1998 and 2014, we defined severe RSV as RSV hospitalisation before age 1 year, and recurrent wheezing or asthma (RW/A) as inhaled medication reimbursement between ages 1 and 7 years, and compared medication and eosinophil trajectories by RSV history. Second, we assessed familial confounding in 527 776 full siblings and 15 667 RW/A-discordant pairs. Third, we performed a genome-wide association study (GWAS) of RSV susceptibility with meta-analysis across six Nordic cohorts (3 107 cases, 92 031 controls) and two-sample Mendelian randomisation (2SMR) using 155 asthma-associated variants. Findings RSV-associated RW/A showed higher inhaled medication use at ages 1-2 years but lower use after age 4, and lower mean blood eosinophils (0.34 vs 0.39*10e9/L; p=0.003) than RW/A without RSV hospitalisation. In RW/A-discordant sibling pairs, RSV hospitalisation was associated with RW/A (OR 2.8; 95% CI 2.4-3.2), while unaffected siblings also had elevated RW/A prevalence. GWAS identified an RSV association at APBB1IP (rs787036; beta=0.209; p=8.80*10e-9). 2SMR provided no evidence that asthma genetic liability influenced RSV susceptibility. Interpretation The RSV-asthma association is unlikely to be explained by shared genetic or environmental factors, and RSV-associated RW/A shows a distinct trajectory. These findings help prioritise long-term outcomes for RSV prevention trials and monitoring. Funding: Paivikki and Sakari Sohlberg Foundation, Foundation for Pediatric Research, Sigrid Juselius Foundation, Orion Research Foundation, the Research Council of Norway.

TEXT ABSTRACTCatalytic-RNA (cat-RNA) expressed from mobile DNA can record cellular events, such as the uptake of plasmids via horizontal gene transfer, by splicing a barcode onto 16S ribosomal RNA (rRNA) - a system termed RNA addressable modification (RAM). However, scaling RAM to record multiple simultaneous biological events requires large numbers of orthogonal cat-RNA whose signals reflect the biological features under investigation rather than variability arising from the barcode sequence. Here, we explore how to design orthogonal cat-RNA to record information about multiple plasmid-encoded traits in parallel. We show that cat-RNA having tRNA-derived barcodes with sequence variation in the anticodon stem-loop present greater signal consistency within Escherichia coli than mRNA-derived barcodes. When orthogonal cat-RNA designs harboring tRNA-derived barcodes were evaluated in Vibrio natriegens and Pseudomonas putida, increased variance was observed compared with Escherichia coli. Nevertheless, the signal consistency was sufficient to use these orthogonal cat-RNAs to report on the relative activities of four promoters and two origins of replication by sequencing barcoded-rRNA derived from the three organisms. These results show how RAM can be multiplexed to report on mobile DNA features in microbial communities and illustrate the importance of accounting for variability in RNA outputs when designing and interpreting multiplexed RNA barcoding data. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/738544v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@406ebaorg.highwire.dtl.DTLVardef@259751org.highwire.dtl.DTLVardef@1f1512corg.highwire.dtl.DTLVardef@8384b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Introduction Hospitalised community-acquired pneumonia (CAP) is heterogeneous in aetiology, severity, and outcome. Phenotyping and endotyping approaches offer potential to stratify patients biologically and guide targeted therapy, but require well-characterised cohorts with linked biosamples. We describe the PARIS (Pneumonia: Acute Respiratory Infection +/- Sepsis) study: a prospective observational cohort of hospitalised patients with pneumonia, designed to characterise functional outcomes and to provide a biobank for translational immunological research. Methods Adults admitted with CAP to a single NHS district general hospital were enrolled within 24 hours of admission between December 2020 and March 2022. Clinical, functional, and physiological data were collected at enrolment, hospital discharge, and 6-8 week follow-up. Serial blood samples were collected for flow cytometry, transcriptomics, pathogen DNA detection, and plasma biobanking. Results Forty-seven patients were enrolled (15 without and 32 with sepsis [SOFA >=2] at enrolment); 87% met sepsis criteria by 24 hours post enrolment. Most patients (30/47, 64%) were managed as COVID-19, microbiologically confirmed in 27. Mean age was 57 years (SD 16), 70% were male, and baseline comorbidity burden was low. Severity was moderate (median NEWS2 4 at enrolment, rising to 6 by 24 hours post enrolment; p<0.001). Mortality was 4/47 (8.5%), with 44/47 (94%) alive at hospital discharge. Median length of stay was 8 days (IQR 5.5-11). Translational samples were collected from the majority: fresh flow cytometry (44/47, 94%), transcriptomics from the sepsis subgroup (31/32, 97%), pathogen DNA sampling (35 samples received across study timepoints; see Table 5), and stored plasma (29/47, 62%). The primary outcome of functional decline (Barthel score decrease >=1.85) occurred in only 1/29 patients with paired assessments (3.4%). Persistent CRP elevation (>3 mg/L) at 6-8 week follow-up was present in 16/31 (52%) survivors with available data. Conclusions The PARIS cohort provides a well-characterised clinical platform and linked biobank to support translational studies of pneumonia and sepsis. The low rate of functional decline reflects the younger, lower-comorbidity, COVID-predominant population recruited. Primary protocol endpoints were not achieved owing to pandemic-related disruption. Data and samples underpin a programme of linked translational studies.

Background: Urinary tract infection (UTI) is the most common secondary condition among people with spinal cord injury/disease (SCI/D). Intravesical Lacticaseibacillus rhamnosus GG (LGG) is an antibiotic-sparing approach to managing urinary symptoms. Objective: Determine the optimal number of doses of intravesical LGG for urinary symptom reduction. Design: Prospective, randomized, two-arm dosing trial. Setting: National recruitment with a local subsample providing urine samples in Washington, DC, USA. Participants: Adults with SCI/D and neurogenic lower urinary tract dysfunction (NLUTD) who use intermittent catheterization (IC); 177 enrolled and randomized (intention-to-treat), with 76 compliant instillers (39 low-dose, 37 high-dose) in the per-protocol analytic sample. Interventions: Two (2 doses/24 hours) or four (4 doses/36 hours) intravesical LGG regimens, self-initiated in response to cloudier or malodorous urine per the Self-Management Protocol using Probiotics (SMP-Pro). Main Outcome Measures: Primary: proportion achieving [&ge;]20% reduction on the Urinary Symptom Questionnaire for Neurogenic Bladder-Intermittent Catheter version (USQNB-IC). Secondary: urinary biomarkers (leukocyte esterase, nitrite, white blood cells, urinary neutrophil gelatinase-associated lipocalin [uNGAL]) and standard urine culture (SUC) in a local subsample. Results: By Day 2, 57.9% (63.8% low-dose; 51.2% high-dose) achieved [&ge;]20% total symptom reduction; high-dose success rose to 70.0% by Day 4. Thirty percent of high-dose participants did not respond at either time point and could not be distinguished from responders by demographics or urine biomarkers. Urinary biomarkers and SUC were unchanged pre- to post-instillation. No serious adverse events were adjudicated as attributable to intravesical LGG by an independent Data Safety Monitoring Board (DSMB). Conclusions: A two-dose course of intravesical LGG yields clinically meaningful symptom improvement in the majority of people with SCI/D and NLUTD who use IC; four doses benefits a meaningful subgroup of two-day non-responders, while a small cohort remains nonresponsive. These results provide preliminary dosing guidance and support progression to a definitive trial.

Importance: Heart failure readmissions remain common following hospitalization, but accurately identifying which patients will be readmitted after discharge remains challenging. Improved prediction could support targeted transitional care interventions and more efficient allocation of clinical resources. Objective: In this study we attempted to improve readmission prediction after heart failure hospitalization by using variables chosen through a modified Delphi process, and using inpatient Electronic Medical Record (EMR) data, focusing on clinical notes. Design: This prognostic study developed competing risk survival models to predict readmission after heart failure hospitalization. Variables were chosen using a modified Delphi process, and extracted from EMR notes using various natural language processing techniques or from other EMR elements where appropriate. Patients were admitted between 2011 through 2019, and at least one year of follow-up was available for all patients. Models were evaluated using C-statistics, as well as sensitivity, specificity, positive and negative predictive values. Setting: During the study period, all acute-care facilities in Calgary, Alberta used the same EMR system, from which patients were selected. Participants: Patients were 18 years or older, resided in Alberta, and were admitted to a Calgary hospital. All corresponding admissions with a most responsible diagnosis of heart failure were included (n=15,160). Main Outcomes and Measures: The main outcome of interest was readmission within 30 days, though 90- and 365-day time frames were also analyzed. Death was treated as a competing risk and analysed at those time frames as well.

Background: Unplanned emergency readmissions remain common following hospitalisation for heart failure (HF). Residual congestion, atrial fibrillation, frailty, and other comorbidities contribute to adverse outcomes after discharge. Identifying patients at high risk of readmission or death may help target post-discharge management. Methods: We conducted a retrospective cohort study of patients hospitalised with HF in selected New South Wales hospitals who were discharged alive and not documented as receiving end-of-life care. Clinical, laboratory, medication, and text-derived variables extracted from electronic health records were used to develop predictive models and corresponding risk scores for emergency readmission and all-cause mortality within 180 days of discharge. Feature importance methods were used to identify key predictors and explain individual risk estimates. To illustrate model predictions while preserving patient privacy, we generated representative synthetic patient profiles by summarising the characteristics of groups of patients with similar predicted risk patterns and visualised the major contributors to their predicted risks using Shapley values. Results: The study included 5,202 hospitalisations among 3,933 patients. Within 180 days of discharge, 45.2% of patients experienced at least one emergency readmission and 12.4% died. The most common causes of emergency readmission were recurrent HF, followed by atrial fibrillation, chest pain, and pneumonia. Predictive performance was moderate for emergency readmission (AUC 0.70; calibration slope 1.30) and good for mortality (AUC 0.84; calibration slope 1.01). Emergency readmission risk was primarily associated with greater prior healthcare utilisation, a higher number of active medical problems, high risk of falls, older age, and impaired kidney function. Mortality risk was most strongly associated with abnormal red blood cell distribution width, elevated blood urea, older age, and lower systolic blood pressure. A lower number of discharge medications, particularly cardiovascular therapies, was associated with a higher risk of emergency readmission and a lower risk of mortality. Representative synthetic patient profiles demonstrated heterogeneity in the factors contributing to predicted risks, illustrating the value of patient-level risk visualisation. Conclusions: Predictive models identified clinically meaningful predictors of emergency readmission and mortality following HF hospitalisation. Patient-level visualisation of individual risk drivers may support more personalised post-discharge management.

Background: Prior analyses have demonstrated an inverse association between transcatheter aortic valve replacement (TAVR) procedural volume and short-term outcomes. However, less is known regarding the relationship between procedural volume and 1-year outcomes in the contemporary TAVR era. Objectives: To evaluate the association between annual hospital and operator TAVR procedural volumes and 1-year clinical outcomes in a contemporary national cohort. Methods: Clinical records from the Society of Thoracic Surgeons (STS)/American College of Cardiology (ACC) Transcatheter Valve Therapies (TVT) Registry for patients undergoing commercial TAVR between January 2020 and December 2022 were linked to Centers for Medicare & Medicaid Services administrative claims. Annualized hospital and operator TAVR volumes were modeled continuously and categorized into tertiles. Primary outcomes included 1-year all-cause mortality, stroke, the composite of mortality or stroke, and all-cause readmissions. Hierarchical risk-adjusted models accounting for patient clustering within sites were used to evaluate associations between procedural volume and outcomes. Results: Among 215,335 patients undergoing TAVR at 788 hospitals by 3,444 operators between 2020 and 2022, median annual hospital and operator volumes were 74 (IQR: 43-115) and 16 (IQR: 10-32), respectively. Volume was then categorized into tertiles (low, medium and high). Compared with high-volume hospitals ([&ge;]102/year), low-volume hospitals ([&le;]52/year) had higher adjusted rates of 1-year all-cause mortality (Odds Ratio (OR): 1.10 [95% CI: 1.05-1.16]), stroke (OR: 1.10 [95% CI: 1.01-1.19]), mortality or stroke (OR: 1.10 [95% CI: 1.05-1.15]), and all-cause readmissions (OR: 1.05 [95% CI: 1.00-1.09]). Compared with high-volume operators ([&ge;]25/year), low-volume operators ([&le;]11/year) had higher adjusted rates of stroke (OR: 1.16 [95% CI: 1.05-1.28]) and mortality or stroke (OR: 1.09 [95% CI: 1.03-1.15]) but not other endpoints. Conclusions: In a large, contemporary national TAVR registry, lower annual hospital ([&le;] 52/year) and operator ([&le;] 11/year) procedural volumes were independently associated with worse 1-year clinical outcomes. These findings suggest that procedural experience continues to influence outcomes despite maturation of contemporary TAVR practice.

Macrophages are innate immune cells of embryonic or adult origin with tissue specific roles in homeostasis, disease surveillance, and wound repair that can be co-opted to promote tumor growth and spread1-11. An understanding of the specific roles of macrophage subsets in lung tumor initiation and progression could promote new therapeutic approaches for this deadly disease. Here, we show that KRASG12D mutations in lung epithelium drive proliferation of resident, embryonically-derived alveolar macrophages, which then promote tumor cell proliferation and protection from ferroptosis, leading to tumor progression. Using genetically engineered mouse models of mutant KRASG12D non-small cell lung cancer12,13, we found that alveolar macrophages accumulate by proliferation in response to tumor cell-secreted IL-34, recapitulating events observed in late embryonic lung development. Tumor alveolar macrophages in turn drive IGF-1-dependent tumor cell proliferation. Neutralization or deletion of IL-34 suppresses IGF-1 expression, reduces macrophage and tumor cell proliferation and inhibits tumor progression. High IL34 and IGF1 correlate with poor survival in KRASG12D/V lung adenocarcinomas and in other solid tumors, indicating that bi-directional proliferative signaling between resident macrophages and tumor cells can drive human lung tumor progression. These studies identify resident macrophage-tumor cell interactions as key interception points for lung cancer therapy.

Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction. HighlightsO_LIAccumulation of metabolic intermediates was alleviated by adaptive laboratory evolution C_LIO_LISequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation C_LIO_LIIncreased CatA1 expression reduced bottlenecks and improved muconate production C_LI

Background: Apical sparing of left ventricular longitudinal strain (LS) is an echocardiographic clue to cardiac amyloidosis but may also occur in hypertensive heart disease (HHD). Objectives: To determine whether apical sparing in HHD is associated with regional left ventricular wall stress estimated according to Laplace's law. Methods: We retrospectively studied 1,559 patients with HHD, 47 with light-chain cardiac amyloidosis (ALCA), and 409 normotensive controls. Artificial intelligence-assisted echocardiography quantified segmental LS, wall thickness, and cavity radius at the basal, midventricular, and apical levels. Wall stress was estimated as mean blood pressure (MBP) x radius/(2 x wall thickness). Apical sparing was defined as a relative regional strain ratio (RRSR)[&ge;]1.0. Results: Apical sparing was present in 14 patients with HHD (0.9%), 13 with ALCA (27.7%), and no controls. Among HHD patients with apical sparing, RRSR decreased from 1.11{+/-}0.13 to 0.72{+/-}0.10 after antihypertensive treatment (P<0.001), accompanied by reduced wall stress and improved basal and midventricular LS, with resolution of apical sparing in all 14 patients. In the overall HHD cohort, changes in MBP and left ventricular mass index were independently associated with changes in RRSR. In an exploratory analysis of HHD patients with apical sparing, a reduction in basal wall stress was associated with a reduction in RRSR ({beta}=0.267 for {bigtriangleup}RRSRx100, 95% CI 0.023-0.511; P=0.036). In ALCA, favorable hematologic response was the only determinant of RRSR reduction. Conclusions: Apical sparing in HHD was uncommon but reversible and may represent a load-sensitive deformation pattern associated with regional wall stress, consistent with Laplace's law.

Engineering heterologous utilization of substrates requires selection of catabolic pathways that balance strain performance and product biosynthesis. Here, we compare the oxidative and isomerase arabinose utilization pathways in Pseudomonas putida strains engineered for cis,cis-muconic acid production from glucose and xylose. Based on the point of entry into central carbon metabolism, we hypothesized that the oxidative arabinose pathway would enable higher productivity while the arabinose isomerase pathway would enable higher muconate yield. In both strains, additional modifications were engineered to improve muconic acid production including sugar transporter tuning, catechol 1,2-dioxygenase overexpression, a feedback-resistant DAHP synthase, and a flux-stabilizing gltA variant. Consistent with our hypothesis, the oxidative arabinose pathway supported faster growth and higher productivity (0.58 g/L/h), whereas the arabinose isomerase pathway improved carbon efficiency, achieving muconate yields of up to 50 C-mol% in fed-batch bioreactors. Process modeling indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid to $2.74/kg and greenhouse gas emissions to 1.31 kg CO2e/kg, approaching cost parity and reducing emissions by 86% relative to fossil carbon-derived adipic acid. Overall, this study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield.

PurposeExtracellular vesicles (EVs) are increasingly explored as natural vehicles for drug delivery and gene therapy approaches. However, reproducible yield and scalability of EV production still pose major challenges in the clinical translation of EV-based therapies. In this study, we sought to quantify and characterize EVs released by suspension-cultured HEK293 cells (Expi293F cells) grown in shaker flasks or small-scale bioreactors, to investigate how the culturing environment affects EV production yield. MethodsExpi293F cells were cultivated (N=3) in either shaker flasks or a bioreactor system, and total cell density, viability, and size were monitored. Supernatants were drawn daily post-cell seeding and were analyzed for EV quantity, size, morphology, and CD63 expression. ResultsNo significant differences were observed in terms of total cell density, viability, and cell size between both cultivation settings. However, cultivation of Expi293F cells in the bioreactor environment significantly increased EV yield by 3-fold compared to shaker flask cultivation (p < 0.01). Other parameters such as average nanoparticle size, EV morphology, and CD63 expression remained comparable between both cultivation methods. ConclusionThese results demonstrate that Expi293F-derived EV yield can be increased by culturing cells in a scalable bioreactor system. These findings pave the way towards the production of therapeutic-based EVs in a scalable and reproducible manner suitable for future (pre-)clinical applications.