Biology subjects

Lin, Y.-C.

Publications and source records attributed to Lin, Y.-C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Combination of mitomycin C and low-dose metronidazole synergistically against Clostridioides difficile infection and recurrence prevention

Clostridioides difficile is an anaerobic, spore-forming, Gram-positive pathogen responsible for various conditions from mild diarrhea to severe toxic megacolon and potentially death. Current treatments for C. difficile infection (CDI) rely on antibiotics like vancomycin and metronidazole (MTZ); however, the high doses required often disrupt gut microbiota, leading to a high recurrence rate. Mitomycin C (MMC), a chemotherapy drug approved by the FDA, is known for inducing phage production in lysogenic bacterial strains, effectively targeting the host bacteria. Given that 70% of C. difficile strains harbor prophages, this study investigates MMCs potential to enhance antibiotic efficacy against CDI. Our in vitro experiments indicate that MMC acts synergistically with MTZ to inhibit the growth of C. difficile strain R20291. Furthermore, this combination decreases biofilm-resident vegetative cell resistance and reduces the MTZ concentration needed to kill C. difficile in stool samples ex vivo. In a CDI mouse relapse model, in vivo results show that MMC combined with a low dose of MTZ significantly improves survival rates and reduces fecal spore counts after antibiotic treatment. Overall, these findings suggest that a low-dose combination of MMC and MTZ offers enhanced therapeutic efficacy for CDI, both in vitro and in vivo, and may provide a promising new approach for treatment.

microbiology↗

bioRxiv2024

Dynamic positioning of a TFIIE-related winged-helix domain in RNA polymerase elongation complex III underpins its multi-functionality

The tandem-winged-helix (tWH) domain of RNA polymerase III (Pol III) Rpc34 subunit is a multi-function hub in Pol III apparatus while the structure basis remains elusive. To probe tWH in Pol III elongation complex (EC), we engineered an azide-bearing unnatural amino acid into tWH and implemented a thiol-capping scheme to enable strain- promoted click reaction for selective dye labeling to suit single-molecule Forster resonance energy transfer (smFRET). Our discrete smFRET results and nano-positioning analysis reveal multiple docking sites of Rpc34 WH2 on Pol III EC with characteristic dwell-times, reflecting promiscuous and weak interactions underpinning tWHs multi-functionality. The docking sites include one overlapping with that during initiation and others on transcription bubble and downstream DNA, previously unreported. This work provides mechanistic insights into Pol III transcription re-initiation and elongation, with useful bio-orthogonal strategies for studying structural dynamics of large native protein complexes.

biophysics↗

bioRxiv2024

StomaVision: stomatal trait analysis through deep learning

StomaVision is an automated tool designed for high-throughput detection and measurement of stomatal traits, such as stomatal number, pore size, and closure rate. It provides insights into plant responses to environmental cues, streamlining the analysis of micrographs from field-grown plants across various species, including monocots and dicots. Enhanced by a novel collection method that utilizes video recording, StomaVision increases the number of captured images for robust statistical analysis. Accessible via an intuitive web interface at and available for local use in a containerized environment at , this tool ensures long-term usability by minimizing the impact of software updates and maintaining functionality with minimal setup requirements. The application of StomaVision has provided significant physiological insights, such as variations in stomatal density, opening rates, and total pore area under heat stress. These traits correlate with critical physiological processes, including gas exchange, carbon assimilation, and water use efficiency, demonstrating the tools utility in advancing our understanding of plant physiology. The ability of StomaVision to identify differences in responses to varying durations of heat treatment highlights its value in plant science research. Plain language summaryStomaVision is a tool that automatically counts and measures tiny openings on plant leaves, helping us learn how plants deal with their surroundings. It is easy to use and works well with various plant species. This tool helps scientists see how plants change under stress, making plant research easier and more accurate.

plant biology↗

bioRxiv2024

The L-lactate dehydrogenases of Pseudomonas aeruginosa are conditionally regulated but both contribute to survival during macrophage infection

Pseudomonas aeruginosa is an opportunistic pathogen that thrives in environments associated with human activity, including soil and water altered by agriculture or pollution. Because L-lactate is a significant product of plant and animal metabolism, it is available to serve as a carbon source for P. aeruginosa in the diverse settings it inhabits. Here, we evaluate P. aeruginosas production and use of its redundant L-lactate dehydrogenases, termed LldD and LldA. We confirm that the protein LldR represses lldD and identify a new transcription factor, called LldS, that activates lldA; these distinct regulators and the genomic contexts of lldD and lldA contribute to their differential expression. We demonstrate that the lldD and lldA genes are conditionally controlled in response to lactate isomers as well as to glycolate and {square}-hydroxybutyrate, which, like lactate, are {square}-hydroxycarboxylates. We also show that lldA is induced when iron availability is low. Our examination of lldD and lldA expression across depth in biofilms indicates a complex pattern that is consistent with the effects of glycolate production, iron availability, and cross-regulation on enzyme preference. Finally, macrophage infection assays revealed that both lldD and lldA contribute to persistence within host cells, underscoring the potential role of L-lactate as a carbon source during P. aeruginosa-eukaryote interactions. Together, these findings help us understand the metabolism of a key resource that may promote P. aeruginosas success as a resident of contaminated environments and animal hosts. ImportancePseudomonas aeruginosa is a major cause of lung infections in people with cystic fibrosis, hospital-acquired infections, and wound infections. It consumes L-lactate, which is found at substantial levels in human blood and tissues. In this study, we investigated the spatial regulation of two redundant enzymes, called LldD and LldA, which enable L-lactate metabolism in P. aeruginosa biofilms. We uncovered mechanisms and identified compounds that control P. aeruginosas LldD/LldA preference. We also showed that both enzymes contribute to its ability to survive within macrophages, a behavior that is thought to augment the chronicity and recalcitrance of infections. Our findings shed light on a key metabolic strategy used by P. aeruginosa and have the potential to inform the development of therapies targeting bacterial metabolism during infection.

microbiology↗

bioRxiv2024

Lipophilic compounds restore wt function of neurodevelopmental-associated KCNQ3 mutations.

A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their slow activation and deactivation kinetics produces a current that regulates membrane potential and impedes repetitive firing. Mutations in KCNQ2 and KCNQ3 lead to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these mutations on KCNQ channel function remains poorly understood and existing treatments have unpleasant side effects. Here we use voltage clamp fluorometry and molecular dynamic simulations to investigate how R227Q and R236C, two novel NDD-causing mutations in the voltage sensor of KCNQ3, impair channel function. We show that the two mutations perturb channel gating by two distinct mechanisms: R227Q altering voltage sensor movement and R236C altering voltage sensor-to-gate coupling. Our study further shows that polyunsaturated fatty acids (PUFAs), a novel class of ion channel modulators, primarily target the voltage sensor domain in its activated conformation and yield partial and complete restoration of wt function in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.

biophysics↗

bioRxiv2024

Cross-institutional HER2 assessment via a computer-aided system using federated learning and stain composition augmentation

The rapid advancement of precision medicine and personalized healthcare has heightened the demand for accurate diagnostic tests. These tests are crucial for administering novel treatments like targeted therapy. To ensure the widespread availability of accurate diagnostics with consistent standards, the integration of computer-aided systems has become essential. Specifically, computer-aided systems that assess biomarker expression have thrusted through the widespread application of deep learning for medical imaging. However, the generalizability of deep learning models has usually diminished significantly when being confronted with data collected from different sources, especially for histological imaging in digital pathology. It has therefore been challenging to effectively develop and employ a computer-aided system across multiple medical institutions. In this study, a biomarker computer-aided framework was proposed to overcome such challenges. This framework incorporated a new approach to augment the composition of histological staining, which enhanced the performance of federated learning models. A HER2 assessment system was developed following the proposed framework, and it was evaluated on a clinical dataset from National Taiwan University Hospital and a public dataset coordinated by the University of Warwick. This assessment system showed an accuracy exceeding 90% for both institutions, whose generalizability outperformed a baseline system developed solely through the clinical dataset by 30%. Compared to previous works where data across different institutions were mixed during model training, the HER2 assessment system achieved a similar performance while it was developed with guaranteed patient privacy via federated learning.

pathology↗

bioRxiv2023

Microscopy-guided subcellular proteomic discovery by high-speed ultra-content photo-biotinylation

Microscopy-guided proteomics at an organelle-dimension resolution is desired for revealing unknown protein constituents at specific disease- or functional-associated regions at the molecular-molecular interactions level. Here, we achieve protein spatial purification by introducing a firmware-integrated microscopy platform that triggers in situ subcellular photo-biotinylation of proteins at user-defined regions of interest (ROIs) one field of view (FOV) at a time for thousands of FOVs fully automatically. An illumination pattern at the analogous ROIs of each FOV is calculated on the fly by either machine learning or traditional image processing. Photoactivatable amino acid crosslinkers are activated by a two-photon focal light one spot at a time at a sub-millisecond illumination duration per spot. Imaging, pattern generation, targeted illumination, and FOV movement are coordinated and cycled with high-speed mechatronic control to complete illumination on millions of ROI spots within hours. Once enough proteins are biotinylated in a cell or tissue sample, the sample is scraped and lysed, and avidin pulldown is used to enrich proteins to achieve spatial protein scooping at a 240-nm precision. Subsequent LC-MS/MS is implemented to reveal the subcellular proteome in high sensitivity, specificity, and resolution. Using this technology termed optoproteomics, we have revealed novel stress granule-localized and amyloid {beta}-localized proteins validated by immunostaining. Together, spatial purification by ultra-content, high-speed microscopy-targeted photo-biotinylation enables unprecedented subcellular spatial proteomics discovery in any microscopically recognizable regions.

cell biology↗

bioRxiv2022

Binding Free Energies of Piezo1 Channel Agonists at Protein-Membrane Interface

Mechanosensitive Piezo channels convert mechanical stimuli into biological signals in vertebrates. Piezo1 chemical modulators are anticipated to yield many clinical benefits. To date, Yoda1 is the most potent and widely used Piezo1-selective agonist, yet how Yoda1 interacts with Piezo1 at the protein-membrane interface and stabilizes Piezo1s open state remains elusive. Here, using a previously identified putative Yoda1 binding site and three molecular dynamics (MD)-based methods, we computed the binding free energies of Yoda1 and its analogs in a Piezo1 cryo-EM closed state and an in silico open state. Our computed absolute binding free energy of Yoda1 in the closed state agrees well with the experimental Kd in which Piezo1 is expected to be in a closed state. More importantly, Yoda1 binds the open state better than the closed state, in agreement with its agonist effects. All three methods predicted that Dooku1, a Yoda1 analog, binds the closed state stronger than Yoda1, but binds the open state weaker than Yoda1. These results are consistent with the fact that Dooku1 antagonizes the effects of Yoda1 but lacks the ability to activate Piezo1. The relative binding free energies of seven Yoda1 analogs recapitulate key experimental structure-activity-relationships (SAR). Based on the state-dependent binding free energies, we were able to predict whether a molecule is an agonist or inhibitor and whether a chemical modification will lead to a change in affinity or efficacy. These mechanistic insights and computational workflow designed for transmembrane binders open an avenue to structural-based screening and design of novel Piezo1 agonists and inhibitors.

biophysics↗

bioRxiv2022

Subtyping of major SARS-CoV-2 variants reveals different transmission dynamics

SARS-CoV-2 continues to evolve, causing waves of the pandemic. Up to March 2022, eight million genome sequences have accumulated, which are classified into five major variants of concern. With the growing number of sequenced genomes, analysis of the big dataset has become increasingly challenging. Here we developed systematic approaches for comprehensive subtyping and pattern recognition for transmission dynamics. By analyzing the first two million viral genomes as of July 2021, we found that different subtypes of the same variant exhibited distinct temporal trajectories. For example, some Delta subtypes did not spread rapidly, while others did. We identified sets of characteristic single nucleotide variations (SNVs) that appeared to enhance transmission or decrease efficacy of antibodies for some subtypes of the Delta and Alpha variants. We also identified a set of SNVs that appeared to suppress transmission or increase viral sensitivity to antibodies. These findings are later confirmed in an analysis of six million genomes as of December 2021. For the Omicron variant, the dominant type in the world, we identified the subtypes with enhanced and suppressed transmission in an analysis of seven million genomes as of January 2022 and further confirmed the findings in a later analysis of eight million genomes as of March 2022. While the "enhancer" SNVs exhibited an enriched presence on the spike protein, the "suppressor" SNVs are mainly elsewhere. Disruption of the SNV correlation largely destroyed the enhancer-suppressor phenomena. These results suggest the importance of fine subtyping of variants, and point to potential complex interactions among SNVs.

bioinformatics↗

bioRxiv2022

Anti-spike antibody response to natural infection with SARS-CoV-2 and its activity against emerging variants

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has substantially impacted human health globally. Spike-specific antibody response plays a major role in protection against SARS-CoV-2. Here, we demonstrated that acute SARS-CoV-2 infection elicits rapid and robust spike-binding and ACE2-blocking antibody responses, which wane approximately 11 months after infection. Serological responses were found to be correlated with the frequency of spike-specific memory B cell responses to natural infections. Further, significantly higher spike-binding, ACE2-blocking, and memory B cell responses were detected in patients with fever and pneumonia. Spike-specific antibody responses were found to be greatly affected by spike mutations in emerging variants, especially the Beta and Omicron variants. These results warrant continued surveillance of spike-specific antibody responses to natural infections and highlight the importance of maintaining functional anti-spike antibodies through immunization. ImportanceAs spike protein-specific antibody responses play a major role in protection against SARS-CoV-2, we examined the spike-binding and ACE2-blocking antibody responses in SARS-CoV-2 infection at different time points. We found robust responses following acute infection, which waned approximately 11 months after infection. Further, the serological responses were correlated with the frequency of spike-specific memory B cell responses to natural infections. Patients with fever and pneumonia showed significantly stronger spike-binding, ACE2-blocking antibody, and memory B cell responses. Moreover, the spike-specific antibody responses were substantially affected by the emerging variants, especially the Beta and Omicron variants. These results warrant continued surveillance of spike-specific antibody responses to natural infections and highlight the importance of maintaining functional anti-spike antibodies through immunization.

immunology↗

bioRxiv2022

Development of a Novel Peptide Aptamer that Interacts with the eIF4E Capped-mRNA Binding Site using Peptide Epitope Linker Evolution (PELE).

Identifying new binding sites and poses that modify biological function are an important step towards drug discovery. We have identified a novel disulphide constrained peptide that interacts with the cap-binding site of eIF4E, an attractive therapeutic target that is commonly overexpressed in many cancers and plays a significant role in initiating a cancer specific protein synthesis program though binding the 5cap (7methyl-guanoisine) moiety found on mammalian mRNAs. The use of disulphide constrained peptides to explore intracellular biological targets is limited by their lack of cell permeability and the instability of the disulphide bond in the reducing environment of the cell, loss of which results in abrogation of binding. To overcome these challenges, the cap-binding site interaction motif was placed in a hypervariable loop on an VH domain, and then selections performed to select a molecule that could recapitulate the interaction of the peptide with the target of interest in a process termed Peptide Epitope Linker Evolution (PELE). A novel VH domain was identified that interacted with the eIF4E cap binding site with a nanomolar affinity and that could be intracellularly expressed in mammalian cells. Additionally, it was demonstrated to specifically modulate eIF4E function by decreasing cap-dependent translation and cyclin D1 expression, common effects of eIF4F complex disruption.

biochemistry↗

bioRxiv2022

The erythrocyte membrane properties of beta thalassaemia heterozygotes and their consequences for Plasmodium falciparum invasion

Malaria parasites such as Plasmodium falciparum have exerted formidable selective pressures on the human genome. Of the human genetic variants associated with malaria protection, beta thalassaemia (a haemoglobinopathy) was the earliest to be associated with malaria prevalence. However, the malaria protective properties of beta thalassaemic erythrocytes remain unclear. Here we studied the mechanics and surface protein expression of beta thalassaemia heterozygous erythrocytes, measured their susceptibility to P. falciparum invasion, and calculated the energy required for merozoites to invade them. We found invasion-relevant differences in beta thalassaemic cells versus matched controls, specifically: elevated membrane tension, reduced bending modulus, and higher levels of expression of the major invasion receptor basigin. However, these differences acted in opposition to each other with respect to their likely impact on invasion, and overall we did not observe beta thalassaemic cells to have lower P. falciparum invasion efficiency for any of the strains tested.

microbiology↗

bioRxiv2022

Optogenetic Manipulation of Cell Migration with High Spatiotemporal Resolution Using Lattice Lightsheet Microscopy

Lattice lightsheet microscopy (LLSM) is modified with the aim of manipulating cellular behavior with subcellular resolution through three-dimensional (3D) optogenetic activation. In this study, we report a straightforward implementation of the activation source in LLSM in which the stimulating light can be generated by changing the spatial light modulator (SLM) patterns and the annual masks. As a result, a Bessel beam as a stimulation source is integrated into the LLSM without changing the optical configuration, achieving better spatiotemporal control of photoactivation. We show that the energy power required for optogenetic reactions is lower than 1 nW (or 24 mW/cm2), and membrane ruffling can be activated at different locations within a cell with subcellular resolution. We also demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable damage to cells.

cell biology↗

bioRxiv2021

Single-Channel Permeability from Markovian Milestoning, Umbrella Sampling, and Voltage Simulations

Various all-atom molecular dynamics (MD) simulation methods have been developed to compute free energies and crossing rates of ions and small molecules through ion channels. However, a systemic comparison across different methods is scarce. Using a carbon nanotube as a model of small conductance ion channel, we computed the single-channel permeability for potassium ion using umbrella sampling, Markovian milestoning, and steady-state flux under applied voltage. We show that a slightly modified inhomogeneous solubility-diffusion equation yields a single-channel permeability consistent with the mean first passage time (MFPT)-based method. For milestoning, applying cylindrical and spherical bulk boundary conditions yield consistent MFPT if factoring in the effective bulk concentration. The sensitivity of the MFPT to the output frequency of collective variables is highlighted using the convergence and symmetricity of the inward and outward MFPT profiles. The consistent transport kinetic results from all three methods demonstrated the robustness of MD-based methods in computing ion channel permeation. The advantages and disadvantages of each technique are discussed, focusing on the future applications of milestoning in more complex systems.

biophysics↗

bioRxiv2021

DNA-free CRISPR-Cas9 gene editing of tetraploid tomatoes using protoplast regeneration

Wild tomatoes are important genomic resources for tomato research and breeding. Development of a foreign DNA-free CRISPR-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free protoplast regeneration and CRISPR-Cas9 genome editing system for Solanum peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs (siRNA) biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6) and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProsys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-calcium-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type stock and pollination with wild-type pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the wild type. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding. One-sentence summaryDNA-free CRISPR-Cas9 genome editing in wild tomatoes creates stable and inheritable diploid and tetraploid regenerants.

plant biology↗

bioRxiv2021

Phosphatidylglycerol synthesis facilitates plastid gene expression and light induction of nuclear photosynthetic genes

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development with decreased chlorophyll accumulation, impaired thylakoid formation, and downregulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects the gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated the growth and transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species-responsive genes were upregulated in pgp1-2. Decreased growth light did not alleviated the impaired leaf development and the downregulation of photosynthesis-associated genes in pgp1-2, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered downregulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor regulating chloroplast development, in pgp1-2 upregulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in the plastid gene expression and plant development.

plant biology↗

bioRxiv2021

Tumor Suppressor p53 Restrains Cancer Cell Dissemination by Modulating Mitochondrial Dynamics

Tumor suppressor p53 plays a central role in preventing tumorigenesis. Here, we unravel how p53 modulates mitochondrial dynamics to restrain the metastatic properties of cancer cells. p53 inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling to attenuate the protein level of mitochondrial fission process 1 (MTFP1), which fosters the pro-fission dynamin-related protein 1 (Drp1) phosphorylation. This regulatory mechanism allows p53 to restrict cell migration and invasion governed by Drp1-mediated mitochondrial fission. Downregulating p53 expression or elevating the molecular signature of mitochondrial fission correlates with aggressive tumor phenotypes and poor prognosis in cancer patients. Upon p53 loss, exaggerated mitochondrial fragmentation stimulates the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling resulting in epithelial-to-mesenchymal transition (EMT)-like changes in cell morphology, accompanied by accelerated matrix metalloproteinase-9 (MMP9) expression and invasive cell migration. Notably, blocking the activation of mTORC1/MTFP1/Drp1/ERK1/2 axis completely abolishes the p53 deficiency-driven cellular morphological switch, MMP9 expression, and cancer cell dissemination. Our findings unveil a hitherto unrecognized mitochondria-dependent molecular mechanism underlying the metastatic phenotypes of p53-compromised cancers.

molecular biology↗

bioRxiv2021

Isolation, omics characterization and organotypic culture of alveolar type II pulmonary epithelial cells

The alveolar type II (AT2) epithelial cell fraction includes the stem cells of the pulmonary alveoli, functioning in lung homeostasis and post-injury repair. AT2 cells have been characterized primarily in situ, in transgenic mouse models. We report a new methodology for their isolation, their "omics" characterization and stroma-cell-free organotypic culture. Our multi-omics analysis identified high expression of genes involved in oxidative phosphorylation and of AP-1 components, as well as new phosphorylation sites in AT2 biomarkers. Furthermore, we show that supplementation with KGF, FGF10 & HGF suffices for the in vitro proliferation of AT2 cells and formation of alveolar organoids, suggesting that AT2-based organotypic development depends on ligands of the c-Met and FGFR2 receptors. The reported methodology and in-depth molecular characterization provide new tools for the in vitro and in vivo functional analysis of pulmonary cells and of mouse models of lung disease.

cell biology↗