Identifying drugs that reverse disease-associated transcriptomic features has been widely explored for drug repurposing, but its potential for de novo drug discovery remains underexplored. Here, we present gene expression profile predictor on chemical structures (GPS), a deep-learning-based drug discovery platform, guided by transcriptomic features, that screens large compound libraries and optimizes lead molecules. We first develop a model that captures transcriptomic perturbation signatures solely from chemical structures and deploy it to library compounds. We refine scoring methods and employ a tree-search method for optimization. By incorporating structure-gene-activity relationships, we uncover drug mechanisms from transcriptomic data. We evaluate GPS across multiple diseases and conduct extensive validation in two cases. In hepatocellular carcinoma, we discover two unique compound series with favorable cellular selectivity and in vivo efficacy. In idiopathic pulmonary fibrosis, we identify one repurposing candidate and one novel anti-fibrotic compound by reversing gene expression of multiple distinct cell types derived from single-cell transcriptomics.

Pulmonary fibrosis (PF) is a chronic and progressive lung disease characterized by repetitive alveolar injury leading to parenchymal thickening, scarring, and respiratory impairment. Current rodent models, particularly those utilizing bleomycin (BLEO), have limited translational relevance to human idiopathic pulmonary fibrosis (IPF) due to anatomical differences such as the absence of respiratory bronchioles. To address this limitation, we developed a large-animal model of pulmonary fibrosis in ferrets, which possess respiratory bronchioles similar to those in humans. Three-month-old wild-type ferrets received intratracheal instillation of BLEO. Lungs were harvested 8 weeks post-treatment for micro-computed tomography (micro-CT) and histological evaluation. Owing to the limited spatial resolution of multidetector CT (MDCT) in visualizing the ferret small airways, excised lungs were imaged ex vivo using a micro-CT scanner at controlled airway pressures (0 cmH2O and 25 cmH2O) to simulate expiratory and inspiratory conditions. Micro-CT imaging revealed distinct structural differences between normal and BLEO-treated ferret lungs. Normal lungs displayed low attenuation and preserved architecture, whereas BLEO-treated lungs showed markedly increased attenuation consistent with fibrosis. Features characteristic of human fibrotic lung disease, including honeycombing with cystic spaces, thickened interlobular septa, and ground-glass opacities indicative of early fibrotic or inflammatory changes, were evident. Using quantitative micro-CT analysis, it was observed that airways in BLEO-treated ferret lungs exhibited significantly reduced radial expansion, longitudinal stretching, and volume change at both applied air pressures. Furthermore, generation-matched analysis revealed significant thickening of airway walls in regions with visually apparent fibrosis. Histological assessment, including Masson's trichrome staining, confirmed extensive collagen deposition and fibrosis. This study demonstrates that BLEO-treated ferrets develop radiologic, mechanical, and histopathologic features resembling human pulmonary fibrosis. Furthermore, micro-CT enables high-resolution assessment of fibrotic distribution, airway dynamics, and lung volume changes. These findings highlight the potential of the ferret as a translational model for studying PF pathogenesis and evaluating novel therapeutic strategies.

Public transcriptomic repositories contain millions of samples, yet their large-scale reuse is hindered by heterogeneous and inconsistently reported metadata. In the Gene Expression Omnibus (GEO), key biological information is often distributed across study- and sample-level records, requiring context-dependent interpretation. Here we present GEOMeta, a large language model (LLM)-based multi-stage workflow with task-specialized agents for automated GEO metadata curation. The pipeline separates metadata retrieval, task-specific information extraction, field standardization, ontology mapping and quality control. Using GEOMeta, we generated standardized annotations for approximately 600,000 human bulk RNA-seq samples. To demonstrate its utility, we benchmarked transcriptome representation models for predicting sex, age, tissue and disease from transcriptome embeddings. We further prospectively annotated newly submitted GEO studies and evaluated 22 frontier LLMs. Recent open-source Flash models achieved annotation quality comparable to leading reasoning models while reducing costs by an order of magnitude. GEOMeta provides a scalable resource and reproducible framework for metadata curation.

People with cystic fibrosis (pwCF) have reduced mucociliary clearance in their airways, leading to the build-up of thick, sticky mucus susceptible to opportunistic infection. A new treatment, comprised of three small molecule drugs called Elexacaftor/Tezacaftor/Ivacaftor (ETI), has improved mucociliary clearance and lung function in pwCF, but how this therapy alters lung infections is poorly understood. This study experimentally modeled the biochemical changes in airway mucus caused by ETI to determine its effect on the CF lung microbiome structure and function. We prepared Artificial Sputum Medium (ASM) with reduced primary carbon sources (amino acids, deoxyribonucleic acid DNA, and mucin) to mimic the effects of ETI on mucus biochemistry due to improved mucociliary clearance and reduced pulmonary inflammation. The control and modified ASM were inoculated with pure CF pathogens or mixed-species communities and then grown in oxic and anoxic conditions, followed by multi-omics data analysis. Although oxygen strongly altered the community structure, the nutrient depletions in ASM had little effect. Instead, the reduced carbon sources altered the physiology of the collective community and its individual pathogens. This included modified growth kinetics in addition to altered nitrogen and nucleotide metabolism. Under reduced amino acid concentrations, a known effect of ETI on the sputum metabolome, the production of both Pseudomonas aeruginosa’s quinolones and rhamnolipids was significantly reduced. This indirect effect of ETI translates to reduced killing of competing pathogens and reduced toxicity to epithelial cells isolated from the airways of explanted human lung tissues. These findings indicate that ETI may provide further benefit to pwCF by reducing the competition and virulence of its principal pathogen and highlight how microenvironmental effects can have powerful impacts on polymicrobial infections.

Monogenic syndromes are associated with neurodevelopmental changes that result in cognitive impairments, neurobehavioral phenotypes including autism and seizures. Limited studies and resources are available to make meaningful headway into the underlying molecular mechanisms that result in these symptoms. One such example is DeSanto-Shinawi Syndrome (DESSH), a rare disorder caused by pathogenic variants in the WAC gene. Individuals with DESSH syndrome exhibit a recognizable craniofacial gestalt, developmental delay/intellectual disability, neurobehavioral symptoms that include autism, ADHD, behavioral difficulties and seizures. However, no thorough studies from a vertebrate model exist to understand how these changes occur. To overcome this, we developed both murine and zebrafish Wac/wac deletion mutants and studied whether their phenotypes recapitulate those described in individuals with DESSH syndrome. We first show that the two Wac models exhibit craniofacial and behavioral changes, reminiscent of abnormalities found in DESSH syndrome. In addition, each model revealed impacts to GABAergic neurons and further studies showed that the mouse mutants are susceptible to seizures, changes in brain volumes that are different between sexes and relevant behaviors. Finally, we uncovered transcriptional impacts of Wac loss of function in mice that will pave the way for future molecular studies into DESSH. These studies present two new animals that begin to uncover some biological underpinnings of DESSH syndrome and elucidate the biology of Wac.

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal lung disease with mucus accumulation in distal small airways. Our recent studies have identified an overexpression of ATP12A, the alpha subunit of the non-gastric proton pump, in the small airways of IPF patients. In murine models, overexpression of ATP12A exacerbates bleomycin-induced lung fibrosis, which is blocked by vonoprazan (VON), a potassium-competitive proton pump inhibitor. These findings suggest that ATP12A plays a profibrotic role in lung fibrosis. ATP12A is expressed in multiple epithelial cell types within the distal lungs of IPF patients, including basal cells and goblet cells. Airway basal cells (ABCs) normally function as stem cells for airway regeneration. However, the precise role of ABCs in the progression of IPF remains poorly understood. We hypothesize that ATP12A expression in ABCs contributes to their profibrotic functions, driving disease progression. METHODS and RESULTS: Airway basal cells (ABCs) were isolated from the small airways of explanted lungs from patients with IPF (IPF-SABCs). Single-cell RNA sequencing confirmed their basal cell identity through the expression of Krt5 and TP63. When cultured on transwells with an air-liquid interface, IPF-SABCs differentiated into various airway epithelial cell types, including goblet cells expressing MUC5AC and MUC5B and ciliated cells. ATP12A was overexpressed in IPF small airway cultures compared to normal controls, as confirmed by immunofluorescence and immunoblotting. With VON treatment blocking ATP12A activity, normal IPF-SABCs differentiation patterns were restored. This included increased ciliated cells and reduced mucus production, with a significant upregulation of FOXJ1 expression following VON treatment. In an in vivo model, intratracheal administration of human IPF-SABCs into NSG mice with minimal bleomycin-induced lung injury resulted in cellular engraftment and proliferation within the lungs. This led to the formation of cellular clusters, increased extracellular matrix deposition, and mucus accumulation. In contrast, normal lung (NL)-SABCs showed minimal engraftment and proliferation and had limited effects on bleomycin-induced fibrosis. To further investigate the role of ATP12A, an adenoviral vector expressing Sh-RNA against ATP12A was used to silence ATP12A expression in IPF-SABCs. This silencing reduced cellular engraftment, collagen deposition, and mucus accumulation in lung tissue, suggesting that ATP12A plays a significant role in driving the profibrotic behavior of IPF-SABCs. CONCLUSIONS: Overexpression of ATP12A in IPF-SABCs may contribute to the development of IPF by promoting mucociliary dysfunction in the small airways. Targeting ATP12A in IPF-SABCs presents a promising therapeutic strategy for treating IPF.

Rationale: Elevated levels of both plasma donor-derived cell-free DNA (dd-cfDNA) and various cytokines in bronchoalveolar lavage fluid (BALF) have been associated with acute lung allograft dysfunction (ALAD) in lung transplant recipients and predict worse long-term outcomes. Data regarding the association between dd-cfDNA and BALF cytokines is lacking. We sought to compare select BALF cytokines in lung transplant recipients with marked elevations in dd-cfDNA levels to those with stable allograft and normal dd-cfDNA. Methods: BALF from 11 patients (14 samples) with high (≥3.0%) dd-cfDNA at time of collection were matched with 14 control recipients with normal (<1%) dd-cfDNA based on age and time post-transplant. Plasma dd-cfDNA was measured with the Prospera (Natera) assay. Values for single lung transplant recipients were adjusted by doubling the measured percentage. A multiplex assay was performed for the quantitative analysis of eight cytokines (MIP-1a, IL-1b, IP-10, IL-6, IL-8, IL-17a, RANTES, TNFa) in BALF using the Luminex MAGPIX system and a custom Thermo Fisher Scientific 8-plex panel on a 96-well plate. Sterile saline, used the perform the lavages, was used as the background media for the assay. Analysis was performed with paired T-test. Results: The high dd-cfDNA group included 5 males, median age 68 (range: 41-74), nine bilateral transplants with median post-transplant interval of 11 months (range: 2-36). Median dd-cfDNA was 5.3% (range: 3.1-14.2). The clinical diagnoses at the time of BAL were infection in 11 cases (4 aspergillus, 3 COVID, 2 other viral, 2 bacterial), two acute cellular rejection and one fibrothorax. The control group included nine males, median age 67 (range: 35-74) with median post-operative time of 12 months (range: 1-84). All were bilateral transplants with normal bronchoscopy findings and stable pulmonary function. Median dd-cfDNA was 0.25% (range: 0.08-0.81). BALF cytokine profiling in the elevated dd-cfDNA compared with the control group demonstrated elevated concentrations of IL-6 (33.6 pg/mL vs. 11.2 pg/mL; P=0.04) and MIP-1α (31.7 pg/mL vs. 12.3 pg/mL; P=0.04). Strong trends were noted for higher IP-10 (P=0.06) and IL-8 (P=0.1). RANTES, IL-1β, IL-17α and TNFα were numerically greater in the high dd-cfDNA group but not significant. Conclusions: IL-6, MIP-1α, IP-10 and IL-8 are elevated in BALF of lung transplant recipients with high dd-cfDNA compared with subjects with low dd-cfDNA. Additional research is required to further characterize the BALF cytokine profile associated with ALAD and injury indicated by dd-cfDNA. There is potential to use cytokine profiles in conjunction with dd-cfDNA testing to improve diagnostic performance.

Pulmonary fibrosis (PF) is a progressive and chronic lung disease characterized by repeated alveolar epithelial injury that leads to excessive extracellular matrix deposition, resulting in tissue thickening, scarring, and impaired gas exchange, leading to respiratory dysfunction. In the United States, around 50,000 new cases are reported annually, with patients facing serious complications such as pneumothorax, pulmonary hypertension, respiratory failure, and an increased risk of lung cancer. Current therapeutic options are limited in efficacy and primarily aim to slow disease progression. Cell therapy has emerged as a promising intervention, offering the potential to regenerate damaged lung tissue, modulate inflammation, and improve pulmonary function. However, the effectiveness of these therapies depends significantly on the ability to monitor the distribution, survival, and integration of transplanted cells within the host lungs. Magnetic Particle Imaging (MPI) is a novel, non-invasive, preclinical imaging modality that utilizes superparamagnetic iron oxide nanoparticles (SPIONs) as tracers. MPI offers high sensitivity, specificity, and no background signal, allowing for real-time and quantitative tracking of labeled cells in vivo. In this study, we investigated the use of MPI for monitoring human distal lung epithelial progenitor cells transplanted into the lungs of immunocompromised mice. Cells were labeled with varying SPION concentrations to optimize the signal, confirmed by immunostaining and iron quantification. After intratracheal instillation, 2D MPI scans were acquired to track the spatial distribution of transplanted cells. Longitudinal imaging over 2 weeks enabled visualization of cell integration and retention within lung tissue. Successful instillation exhibited MPI signals in both left and right lungs, which decreased (~65%) over time. Mice were subsequently sacrificed for histological validation. This study demonstrates the utility of MPI for noninvasive, longitudinal tracking of cell therapy in pulmonary fibrosis, and pivots around the intricate techniques utilized during the procedures.

Gene therapy holds promise as a life-changing option for individuals with genetic variants that give rise to disease. FDA-approved gene therapies for Spinal Muscular Atrophy (SMA), cerebral adrenoleukodystrophy, β-Thalassemia, hemophilia A/B, retinal dystrophy, and Duchenne Muscular Dystrophy have generated buzz around the ability to change the course of genetic syndromes. However, this excitement risks over-expansion into areas of genetic disease that may not fit the current state of gene therapy. While in situ (targeted to an area) and ex vivo (removal of cells, delivery, and administration of cells) approaches show promise, they have a limited target ability. Broader in vivo gene therapy trials have shown various continued challenges, including immune response, use of immune suppressants correlating to secondary infections, unknown outcomes of overexpression, and challenges in driving tissue-specific corrections. Viral delivery systems can be associated with adverse outcomes such as hepatotoxicity and lethality if uncontrolled. In some cases, these risks are far outweighed by the potentially lethal syndromes for which these systems are being developed. Therefore, it is critical to evaluate the field of genetic diseases to perform cost-benefit analyses for gene therapy. In this work, we present the current state while setting forth tools and resources to guide informed directions to avoid foreseeable issues in gene therapy that could prevent the field from continued success.

In contrast to pig large airways, the pH of airway surface liquid (ASL) in pig small airways is regulated by CFTR-mediated HCO-3 secretion and the vacuolar-type H+ ATPase (V-ATPase) proton secretion. We hypothesized that, in cystic fibrosis (CF), the ASL pH of small airways is acidic, and the V-ATPase is internalized. We quantified proton secretion during the addition of an alkaline test solution by measuring changes in a pH-dependent fluorescent dye generated by porcine small airway epithelia in the absence and presence of bafilomycin A1. The pH-dependent translocation of V-ATPase in ex vivo and in vivo preparations was measured using immunolocalization of V-ATPase. We found that bafilomycin-sensitive proton secretion stopped when the ASL pH was less than 7.10. In non-CF pigs and mice, we found that V-ATPase was localized in the apical membrane, and internalized when the lungs were instilled with a pH 6.8 solution. Studies in which we immediately fixed lungs from pigs revealed apical V-ATPase detection in non-CF piglets and less apical detection in CF piglets. Our data suggest that V-ATPase in small airways is internalized when the ASL pH is acidic. The decrease in apical localization of V-ATPase in CF pigs is consistent with an acidic ASL pH.NEW & NOTEWORTHY In this study, we describe that vacuolar-type H+ ATPase (V-ATPase) internalizes when the airway surface liquid (ASL) pH in pig small airways is less than 7.10. Furthermore, we found that V-ATPase is not localized to the apical membrane in the small airways of newborn cystic fibrosis pigs.

Idiopathic pulmonary fibrosis (IPF) is a pathological condition wherein lung injury precipitates the deposition of scar tissue, ultimately leading to a decline in pulmonary function. Existing research indicates a notable exacerbation in the clinical prognosis of IPF patients following infection with COVID-19. This investigation employed bulk RNA-sequencing methodologies to describe the transcriptomic profiles of small airway cell cultures derived from IPF and post-COVID fibrosis patients. Differential gene expression analysis unveiled heightened activation of pathways associated with microtubule assembly and interferon signaling in IPF cell cultures. Conversely, post-COVID fibrosis cell cultures exhibited distinctive characteristics, including the upregulation of pathways linked to extracellular matrix remodeling, immune system response, and TGF-β1 signaling. Notably, BMP signaling levels were elevated in cell cultures derived from IPF patients compared to non-IPF control and post-COVID fibrosis samples. These findings underscore the molecular distinctions between IPF and post-COVID fibrosis, particularly in the context of signaling pathways associated with each condition. A better understanding of the underlying molecular mechanisms holds the promise of identifying potential therapeutic targets for future interventions in these diseases.

Idiopathic pulmonary fibrosis (IPF) is a pathological condition of unknown etiology that results from injury to the lung and an ensuing fibrotic response that leads to the thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear, and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to cystic fibrosis lung disease. The ATP12A gene encodes the α-subunit of the nongastric H+, K+-ATPase, which functions to acidify the airway surface fluid and impairs mucociliary transport function in patients with cystic fibrosis. It is hypothesized that the ATP12A protein may play a role in the pathogenesis of IPF. The authors' studies demonstrate that ATP12A protein is overexpressed in distal small airways from the lungs of patients with IPF compared with normal human lungs. In addition, overexpression of the ATP12A protein in mouse lungs worsened bleomycin induced experimental pulmonary fibrosis. This was prevented by a potassium competitive proton pump blocker, vonoprazan. These data support the concept that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has potential as a novel therapeutic strategy in IPF treatment.

IPF is a condition in which an injury to the lung leads to the accumulation of scar tissue. This fibrotic tissue reduces lung compliance and impairs gas exchange. Studies have shown that infection with COVID-19 significantly worsens the clinical outcomes of IPF patients. The exact etiology of IPF is unknown, but recent evidence suggests that the distal small airways, (those having a diameter less than 2 mm in adults), play a role in the early pathogenesis of IPF. TGF-β1 is a main driver of fibrosis in a variety of tissues; the binding of TGF-β1 to its receptor triggers a signaling cascade that results in inflammatory signaling, accumulation of collagen and other components of the extracellular matrix, and immune system activation. This study aimed to investigate possible mechanisms that contribute to worsening lung fibrosis in IPF patients after being diagnosed with COVID-19, with a particular focus on the role of TGF-β1. Small airway cell cultures derived from IPF and post-COVID-19 IPF patient transplant tissues were submitted for RNA-sequencing and differential gene expression analysis. The genetic signatures for each disease state were determined by comparing the differentially expressed genes present in the cells cultured under control conditions to cells cultured with TGF-β1. The genes shared between the culture conditions laid the framework for determining the genetic signatures of each disease. Our data found that genes associated with pulmonary fibrosis appeared to be more highly expressed in the post-COVID fibrosis samples, under both control and TGF-β1-treated conditions. A similar trend was noted for genes involved in the TGF-β1 signaling pathway; the post-COVID fibrosis cell cultures seemed to be more responsive to treatment with TGF-β1. Gene expression analysis, RT-PCR, and immunohistochemistry confirmed increased levels of BMP signaling in the IPF small airway cell cultures. These findings suggest that TGF-β1 signaling in IPF small airway cells could be inhibited by BMP signaling, leading to the differences in genetic signatures between IPF and post-COVID fibrosis.

Background: Idiopathic Pulmonary Fibrosis (IPF) is a pathological condition of unknown etiology which results from injury to the lung and an ensuing fibrotic response that leads to the thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to Cystic Fibrosis (CF) lung disease. The ATP12A gene encodes the alpha-subunit of the non-gastric H+, K+-ATPase, which functions to acidify the airway surface liquid (ASL) and impairs mucociliary transport function in cystic fibrosis patients. Hypothesis: We hypothesize that the ATP12A protein may play a role in the pathogenesis of IPF. Methods: ATP12A expression level was evaluated by immunohistochemical staining and RNAscope in situ hybridization in normal and IPF human distal lungs. Primary human small airway culture model was used to elucidate the potential roles of ATP12A in the activation of latent TGF-beta activation. Viral vector mediated ATP12A overexpression was used in the bleomycin-induced lung fibrosis mouse model. A potassium-competitive proton pump blocker, vonoprazan was used to block ATP12A functions both in vitro and in vivo. Results: Our studies demonstrate that ATP12A protein is overexpressed in distal small airways from IPF patient lungs compared to normal human lungs. Potassium competitive proton pump blocker vonoprazan decreased airway surface liquid (ASL) pH and TGF-β1 activation in IPF small airway epithelial cells. In addition, overexpression of the ATP12A protein in mouse lungs worsened the Bleomycin (BLEO)-induced experimental pulmonary fibrosis. This was prevented by a potassium-competitive proton pump blocker, vonoprazan. Conclusion: Those data support that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has the potential as a novel therapeutic strategy in IPF. This work was funded by the National Institutes of Health (HL153165-01A1 to X.L.), the Cystic Fibrosis Foundation (LI19XX0), Cystic Fibrosis Research Institute, and Spectrum Health-MSU Alliance Corporation funds. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

The small GTPase family is well-studied in cancer and cellular physiology. With 162 annotated human genes, the family has a broad expression throughout cells of the body. Members of the family have multiple exons that require splicing. Yet, the role of splicing within the family has been underexplored. We have studied the splicing dynamics of small GTPases throughout 41,671 samples by integrating Nanopore and Illumina sequencing techniques. Within this work, we have made several discoveries. 1). Using the GTEx long read data of 92 samples, each small GTPase gene averages two transcripts, with 83 genes (51%) expressing two or more isoforms. 2). Cross-tissue analysis of GTEx from 17,382 samples shows 41 genes (25%) expressing two or more protein-coding isoforms. These include protein-changing transcripts in genes such as RHOA, RAB37, RAB40C, RAB4B, RAB5C, RHOC, RAB1A, RAN, RHEB, RAC1, and KRAS. 3). The isolation and library technique of the RNAseq influences the abundance of non-sense-mediated decay and retained intron transcripts of small GTPases, which are observed more often in genes than appreciated. 4). Analysis of 16,243 samples of "Blood PAXgene " identified seven genes (3.7%; RHOA, RAB40C, RAB4B, RAB37, RAB5B, RAB5C, RHOC) with two or more transcripts expressed as the major isoform (75% of the total gene), suggesting a role of genetics in altering splicing. 5). Rare (ARL6, RAB23, ARL13B, HRAS, NRAS) and common variants (GEM, RHOC, MRAS, RAB5B, RERG, ARL16) can influence splicing and have an impact on phenotypes and diseases. 6). Multiple genes (RAB9A, RAP2C, ARL4A, RAB3A, RAB26, RAB3C, RASL10A, RAB40B, and HRAS) have sex differences in transcript expression. 7). Several exons are included or excluded for small GTPase genes (RASEF, KRAS, RAC1, RHEB, ARL4A, RHOA, RAB30, RHOBTB1, ARL16, RAP1A) in one or more forms of cancer. 8). Ten transcripts are altered in hypoxia (SAR1B, IFT27, ARL14, RAB11A, RAB10, RAB38, RAN, RIT1, RAB9A) with RHOA identified to have a transient 3'UTR RNA base editing at a conserved site found in all of its transcripts. Overall, we show a remarkable and dynamic role of splicing within the small GTPase family that requires future explorations.

The small airways of humans are affected early in several lung diseases. However, because they are relatively inaccessible, little is known about the epithelial cells that line these airways. We performed a single cell RNA-seq census of small and large airways of wild-type pigs and pigs with disrupted cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. The sequencing data showed that small airway epithelia had similar major cell types as large airways but no ionocytes; moreover, lack of CFTR expression had minimal effect on the transcriptome. Small airway epithelial cells expressed a different transcriptome than large airway cells. Quantitative immunohistochemistry showed that small airway basal cells participate in epithelial barrier function. Finally, sequencing data and in vitro electrophysiologic studies suggest that small airway epithelia have a water and ion transport advantage. Our data highlight the archetypal nature of basal, secretory, and ciliated airway cells with location-dependent gene expression and function. ### Competing Interest Statement The authors have declared no competing interest.

We recently identified epigallocatechin gallate (EGCG), a trihydroxyphenolic compound, as a dual inhibitor of lysyl oxidase-like2 and transforming growth factor-β1 (TGFβ1) receptor kinase that when given orally to patients with idiopathic pulmonary fibrosis (IPF) reversed profibrotic biomarkers in their diagnostic biopsies. Here, we extend these findings to advanced pulmonary fibrosis using cultured precision-cut lung slices from explants of patients with IPF undergoing transplantation. During these experiments, we were surprised to discover that not only did EGCG attenuate TGFβ1 signalling and new collagen accumulation but also activated matrix metalloproteinase-dependent collagen I turnover, raising the possibility of slow fibrosis resolution with continued treatment.