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PubMed1978

Phospholipid biosynthesis and secretion by a cell line (A549) which resembles type II aleveolar epithelial cells.

The A549 cell line is a continuous cell line derived from a human adenocarcinoma of the lung. At low cell population density the cells contain relatively few lamellar bodies, but in mature cells in very confluent cultures lamellar bodies are abundant. The lamellar bodies from these cells are enriched for phosphatidylcholine and disaturated phosphatidylcholine. In mature cells, 45% of newly synthesized phosphatidylcholine is disaturated. Stimulation with the calcium ionophore A23187 produces exocytosis of phosphatidylcholine (46% disaturated). The A549 cell synthesizes, stores in lamellar bodies, and secretes phosphatidylcholine, and thus has many important biological properties of the alveolar epithelial type II cell.

Adenocarcinoma↗
PubMedDate not supplied

In vitro biological effects of glass fibers.

Two glass fiber samples, which had previously been tested for their ability to induce mesotheliomata in rats, were subjected to size fractionation and the respirable fibers from each were collected. The sizes of the fibers in the total and respirable fractions were measured and all four materials were tested in vitro for their cytotoxic activities against V79-4 cells, A549 cells, and mouse peritoneal macrophages. The respirable fraction from the coarser material had considerably enhanced activity (on a mass basis) compared with its parent (total) material. The respirable fraction of the other fiber was only slightly more active than the corresponding total sample. The fiber size distributions are discussed in relation to the observed biological activity of all four samples, and it is concluded that the cytotoxic activity of fibrous glass is determined by the number of fibers within certain size ranges. It is likely that in the systems used in this present paper, fibers less than 10 micrometer long are inactive. The relationship between these observations and those obtained with fibrous glass in vivo by Stanton et al. (1977) are discussed.

Animals↗
PubMed1979

Cell line A549 as a model of the type II pneumocyte. Phospholipid biosynthesis from native and organometallic precursors.

1. A549 is a continuous cell line derived from a human pulmonary adenocarcinoma. To evaluate the suitability of this cell line as a model of the type II pneumocyte, the morphology and the composition and biosynthesis of phosphatidylcholine was examined under control culture conditions and during fatty acid supplementation with palmitate. A number of the ultrastructural characteristics of A549 cells were similar to the in situ type II pneumocyte and were unchanged by fatty acid supplementation. The phospholipid composition of the cell line was similar to that of primary isolates of type II cells in total phosphatidylcholine, disaturated phosphatidylcholine, and palmitate and saturated fatty acid. Phospholipid biosynthetic results were also consistent with those reported for isolated type II cell models. These included: (i) the pattern of incorporation of choline, palmitate and acetate into phosphatidylcholines; (ii) the effect of palmitate supplementation, which resulted in stimulation of the rate of phosphatidylcholine biosynthesis and in increased percentage of labeled precursor in disaturated phosphatidylcholine; and (iii) the preferential synthesis from labeled choline and palmitate of a highly disaturated phosphatidylcholine in short-term incubations. 2. The incorporation of an organometallic palmitate analog, 12,12-dimethyl-12-stannahexadecanoate, into A549 cell lipids was examined and compared to that of palmitate. These date demonstrate for the first time the incorporation of an organometallic substrate into the phospholipids of a mammalian cell line. This analog substitutes selectively for the native fatty acid at a rate similar to that of the native fatty acid with no cytotoxic effects. The organotin probe, coupled with spectroscopic detection and electron microscopy, may be useful for examining ultrastructural aspects of phospholipid synthesis, translocation and assembly.

Acetates↗
PubMed1976

A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells.

The A549 tumor-cell line, initiated from a human alveolar cell carcinoma, has been continuously propagated in vitro for more than 3 years (more than 1,000 cell generations). These cells have a human karyotype and appear to have been derived from a single parent cell. All A549 cells examined by electron microscopy at both early and late passage levels contain multilamellar cytoplasmic inclusion bodies typical of those found in type II alveolar epithelial cells of the lung. At early and late passage levels, the cells synthesize lecithin with a high percentage of disaturated fatty acids utilizing the cytidine diphosphocholine pathway; such a pattern of phospholipid synthesis is expected for cells believed to be responsible for pulmonary surfactant synthesis. The A549 cell line should permit in vitro analysis of human surfactant synthesis and secretion and possibly provide a source of human surfactant for therapeutic intervention in pulmonary disease states characterized by surfactant deficiency.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed2026

Genome mining and metabolomics unveil new napyradiomycin antibiotics from Streptomyces sp. 0H2M.

Napyradiomycins are a family of meroterpenoid natural products known for their promising antibiotic activities. In this study, four new napyradiomycins derivatives were identified, SF2415B4 (1), SF2415B5 (2), SF2415B6 (3), and SF2415B7 (4) from Streptomyces sp. 0H2M, alongside a known molecule, A80915A (5) through the synergy between genome mining and metabolomics analysis. Their structures were elucidated through a combination of spectroscopic and spectrometric analyses, including HRMS-ESI, NMR, and DP4+. Genome sequencing identified a putative biosynthetic gene cluster, and subsequent analyses revealed a distinct biosynthetic pathway with an unprecedented tailoring mechanism mediated by novel hydroxylases and halogenases. Biological assays demonstrated significant activity against Bacillus subtilis, Bacillus cereus and methicillin-resistant Staphylococcus aureus due to perturbation of cell membrane integrity, and minimum inhibitory concentration (MIC) values ranged from 0.24 to 30.7 μM. Additionally, in vitro cytotoxicity experiments indicated that compounds 2-5 very mildly inhibited the viability of human non-small cell lung cancer (NSCLC) cell line A549 in a concentration-dependent manner, with IC50 values of 16.7, 39.1, 65.0, and 32.8 μM, respectively. Moreover, they were shown to induce apoptosis and autophagy in A549 cells, evidenced by increased levels of cleaved PARP, decreased expression of anti-apoptotic proteins (Bcl-2, Bcl-xL, and Survivin), and accumulation of LC3-II. These findings offer new insights into the natural product chemistry in Streptomyces and the pharmacology of napyradiomycin class antibiotics.

Streptomyces↗
PubMed2024

Adaptation for Staphylococcus aureus to hosts via insertion mutation in the accessory gene regulator agrC gene: decreased virulence and enhanced persistence capacity.

UNLABELLED: Staphylococcus aureus is an important human pathogen due to its vast array of virulence factors regulated by multiple regulatory mechanisms, including the accessory gene regulator. In this study, two S. aureus strains were simultaneously isolated from the blood of a febrile patient, belonging to the same clone, designated as 23H with a complete hemolytic phenotype, and 23B, exhibiting an incomplete hemolytic phenotype. The genomic comparison between strains 23B and 23H revealed that 23B had a single adenine base insertion at position 923 in the agrC gene, leading to a functional loss of the encoded AgrC. Experimental findings showed that strain 23B had decreased hemolytic activity, lower cytotoxicity against human alveolar epithelial A549 cells and in the Galleria mellonella model, and a reduced ability to survive intracellularly after infecting macrophages, in comparison to 23H. Conversely, 23B exhibited enhanced biofilm formation, greater adherence to A549 cells, and increased persistence in the face of vancomycin and daptomycin treatment. Transcriptomic analysis revealed that 23B upregulated surface protein-encoding genes while simultaneously reducing the expression levels of virulence factors, highlighting the intricate regulatory adjustments facilitating its persistence and reducing pathogenic potential. ATP assay results indicated that 23B maintained elevated ATP levels during the exponential phase yet exhibited reduced levels in the stationary phase when compared with 23H. Our findings suggested that the mutation in the agrC gene of S. aureus results in diminished virulence but markedly enhances persistence. This mutated strain warrants clinical attention because it may lead to treatment failures and persist in patients. IMPORTANCE: In clinical antimicrobial therapy, bacterial strains often develop resistance to antimicrobial agents. Additionally, mutations in their gene regulatory networks can increase their persistence, especially in immunocompromised patients. This study identified an insertion mutation in the accessory gene regulator, agrC gene, carried by a Staphylococcus aureus strain isolated from the blood of a febrile patient, leading to the functional loss of AgrC. Further research revealed that despite the reduced virulence of the mutated strain, it significantly bolstered the capacity to adapt and endure within the host during prolonged infections. This was evidenced by increased adhesion and biofilm formation capabilities, development of antimicrobial tolerance, and decreased ATP levels linked to persistence. Therefore, monitoring these mutations in S. aureus is crucial clinically, as they can complicate treatment strategies.

Staphylococcus aureus↗
PubMed2026

Epigenetic activation of NK-cell effector programs and caspase-8-dependent apoptosis mediates the antitumor activity of LGP in NSCLC.

BACKGROUND: Effective activation of natural killer (NK) cell cytotoxicity and caspase-8-dependent extrinsic apoptosis remains a major challenge in non-small cell lung cancer (NSCLC). Epigenetic mechanisms regulating NK cell function within the tumor microenvironment are poorly understood and rarely targeted therapeutically. METHODS: The antitumor activity of Li-Ginseng Powder (LGP), a specifically processed Panax ginseng formulation enriched in rare ginsenosides (Rh4, Rg3, Rg5, Rk1, and Rk3), was evaluated in human lung cancer A549 cells and A549 xenograft mouse models. NK cell infiltration and activation were assessed by flow cytometry, immunoblotting, and immunohistochemistry. Whole-genome bisulfite sequencing (WGBS) was performed to analyze DNA methylation changes. The effects of LGP ginsenosides (LGG) on tumor cell apoptosis and death receptor signaling were examined in vitro. RESULTS: LGP significantly suppressed tumor growth and enhanced systemic and intratumoral NK cell activation. Promoter demethylation of NK cell effector genes, including Ncr1, Gzmb, Nktr, and Itgal, was associated with increased NK cell infiltration and activation, elevated granule-mediated cytotoxicity, and enhanced IFN-γ signaling. In parallel, LGP treatment induced caspase-8-dependent apoptosis associated with increased expression of membrane death receptors, their ligands, FADD, and procaspase-8 in tumor tissues. In vitro, LGG upregulated these apoptosis-initiating proteins and triggered caspase-8 activation in A549 cells independent of promoter methylation changes. Collectively, these immune-associated and tumor-intrinsic responses contributed to robust tumor suppression with a favorable systemic safety profile. CONCLUSIONS: LGP exerts dual antitumor effects characterized by enhanced NK-cell activation and increased sensitivity of tumor cells to caspase-8-dependent extrinsic apoptosis. These coordinated immune-associated and apoptosis-sensitizing effects underscore the therapeutic potential of LGP for the treatment of NSCLC.

DNA methylation↗
PubMed2025

Identification of ECE2 signaling in promoting non-small lung cancer progression through ET1/YAP1/MAGEA3 axis.

Non-small cell lung cancer (NSCLC) is a major cause of cancer-related mortality worldwide with high heterogeneity. However, the molecular basis for NSCLC development remains poorly understood. In this study, we analyzed endothelin converting enzyme 2 (ECE2) expression in NSCLC using transcriptome data from 59 normal and 515 NSCLC tissues obtained from The cancer genome atlas (TCGA) database. Additionally, we investigated the role of ECE2 in metastasis using 30 clinical NSCLC specimens. In vitro cell proliferation and migration assays were conducted using CCK8 and Transwell assays in NSCLC cells overexpressing ECE2. We employed Western blotting and immunostaining to assess activation of the endothelin-1 (ET1)/YAP1/MAGEA3 pathway. Furthermore, in vivo studies using subcutaneous xenograft mouse models with vector and ECE2-overexpressing A549 cells evaluated the anticancer effects. Our findings revealed elevated ECE2 expression in NSCLC tissues associated with poor prognosis. Moreover, overexpression of ECE2 enhanced both the proliferative and metastatic potential of NSCLC cells. Mechanistically, ECE2 promoted the production of ET1 in NSCLC cells. Subsequently, increased ET1 levels activated the YAP1/MAGEA3 pathway, thereby facilitating tumor progression. Our study uncovered the oncogenic role of ECE2 in promoting NSCLC growth through the ET1/YAP1/MAGEA3 pathway. Inhibiting ET1 signaling markedly enhanced the anticancer effectiveness of paclitaxel (PTX), providing a promising approach for managing NSCLC.

Humans↗
PubMed2026

Desmoplakin loss in alveolar epithelium drives Wnt/β-Catenin-mediated extracellular matrix remodeling and fibrotic signaling in vitro.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease characterized by aberrant extracellular matrix (ECM) remodeling, epithelial dysfunction, and limited therapeutic options. Genetic studies implicate Desmoplakin (DSP), a desmosomal adhesion protein, in IPF susceptibility; however, its mechanistic role remains unclear. This study aimed to investigate the role of DSP in regulating fibrotic and ECM remodeling pathways in alveolar epithelial cells. METHODS AND RESULTS: DSP was silenced using siRNA in adenocarcinoma-derived human alveolar epithelial A549 cells. DSP loss induced epithelial-to-mesenchymal transition, enhanced cell migration, and increased epithelial permeability, along with upregulation of fibrotic and ECM-associated genes. Pathway enrichment analysis of DSP interactors (STRING database) identified the Wnt/β-catenin signaling as a potential key pathway. Mechanistic validation using cycloheximide chase assays, qPCR, western blotting, immunofluorescence, and luciferase-reporter assays suggested that DSP loss destabilizes desmosomal complexes, promoting plakoglobin (γ-catenin) degradation while reducing β-catenin turnover. This was associated with increased nuclear accumulation of β-catenin and enhanced TCF/LEF-dependent transcription, leading to elevated expression of ECM-related genes, including COL1A1 and MMP9. DSP overexpression suppressed Wnt/β-catenin signaling and fibrotic gene expression, while pharmacological inhibition of this pathway attenuated DSP-dependent increases in ECM-associated gene expression. CONCLUSION: These findings suggest that DSP may function as a regulator of alveolar epithelial homeostasis and extracellular matrix remodeling in an in vitro epithelial model. Loss of DSP is associated with activation of Wnt/β-catenin-mediated fibrotic signaling, correlating with reduced plakoglobin stability. This study provides mechanistic insight into epithelial-matrix crosstalk in vitro and identifies a candidate pathway that may contribute to ECM dysregulation in IPF, the disease relevance of which will require validation in primary human alveolar epithelial cells and in vivo models.

Humans↗
PubMed2026

IFNL1 gene promoter single nucleotide polymorphism rs7247086 enhances transcription through a STAT-binding site.

A single nucleotide polymorphism (SNP) within the human interferon lambda 1 (IFN-L1, IFN-λ1) gene promoter, rs7247086 (C/T) has been reported to be associated with severe dengue and possibly with psoriasis and COVID-19. However, its functional nature is unknown. The present study was undertaken to examine the effect of rs7247086 on transcription, by utilizing promoter and enhancer-reporter assays. We see that the T allele completes a consensus signal transducer and activator of transcription (STAT)-binding site. While we did not find strong evidence to show that the STAT-binding site drove transcription from the IFNL1 gene promoter, we saw that it acts like an enhancer in reporter assays. The T allele of rs7247086 within the STAT-binding site significantly increased transcription of the reporter gene compared to the C allele when incorporated into enhancer-reporter constructs in both HEK293 and A549 cell lines. Mechanistically, we obtained evidence from electrophoretic mobility shift assays to show that the T allele binds to STAT proteins more strongly than the C allele. In a cohort of healthy individuals, we saw that the T allele carriers, specifically males but not females, had significantly increased secretion of IFN-λ1 from their peripheral blood mononuclear cells after stimulation. Lastly, rs7247086 significantly associated with psoriasis, only in males but not in females.

Humans↗
PubMed2026

DORSSAA: Drug-Target interactOmics Resource Based on Stability/Solubility Alteration Assay.

Advancements in high-throughput techniques such as Thermal Proteome Profiling and the high-throughput Proteome Integral Solubility Alteration assay have revolutionized our understanding of drug-protein interactions. Despite these innovations, the absence of an integrative platform for cross-study analysis of stability and solubility alteration data represents a significant bottleneck. To address this gap, we introduce Drug-target interactOmics Resource based on Stability/Solubility Alteration Assay (DORSSAA), an interactive and expandable web-based platform for the systematic analysis and visualization of proteome stability and solubility alteration assay datasets. Currently, DORSSAA features 1,135,985 records spanning 38 cell lines and organisms, 135 compounds, and 40,742 protein targets. Through its user-friendly interface, the resource supports comparative drug-protein interaction analysis and facilitates the discovery of actionable therapeutic targets. Through two case studies, methotrexate target profiling in A549 cells and combinatorial-therapy drug-target interactions in leukemia cell lines, we demonstrate DORSSAA's utility for identifying protein-drug interactions across diverse experimental contexts. This resource empowers researchers to accelerate drug discovery and enhance our understanding of protein behavior. Compared with data repositories and interaction databases, DORSSAA provides direct protein-level evidence of mechanisms of action with strict statistical control for each study. This enables more reliable identification of drug targets, off-target effects, and potential drug combinations.

Humans↗
PubMed1979

Expression of feline xenotropic RNA tumor virus in hybrids between permissive human and non-permissive mouse cells.

Somatic cell hybrids were generated by fusing human (A549) cells, cloned after infection with the feline xenotropic CCC virus, to mouse (3T3) cells which are non-permissive for this virus. Hybrid clones were found to be capable of expressing infectious virus. CCC virus expression, however, was regulated in the hybrid cells in such a way that 20-200 times less virus was released into the culture fluid than by the human parental line. Thus human permissiveness for this virus is co-expressed with murine restriction. Markers for twenty human chromosomes were assayed in the hybrid clones. No single human chromosome was found to be essential and sufficient for CCC virus production, since none of them was consistently present or lost in virus-positive and virus-negative clones, respectively.

Animals↗
PubMed2025

Genome-scale evolution and phylodynamics of swine influenza A viruses in China: a genomic epidemiology study.

BACKGROUND: Pigs are recognised as crucial intermediate hosts for the emergence of influenza viruses of pandemic potential. As the largest pork-producing nation, China hosts a complex ecosystem of swine influenza viruses (SIVs). We aimed to investigate the evolutionary processes, spatiotemporal dynamics, and biological characteristics of SIVs in China. METHODS: From Jan 15, 2016, to Dec 22, 2020, we collected nasal swabs from pigs at eight abattoirs and 16 swine farms in the Guangdong, Henan, and Shandong provinces of China, as part of SIV surveillance. SIVs were detected with RT-PCR. Positive samples underwent viral isolation and genome sequencing. We analysed evolution and spatiotemporal dynamics using the whole genomes of isolated SIVs, as well as genome sequences of SIV isolates from human infections worldwide retrieved from the Global Initiative on Sharing All Influenza Data and GenBank Flu databases up to April 28, 2024. Viral sequences without a sample collection area or date were excluded from the analysis. Viral receptor-binding properties and in-vitro replication of strains isolated in this study were evaluated with a solid-phase binding assay and various cell lines, including Madin-Darby canine kidney cells, porcine alveolar macrophages, primary porcine trachea epithelial cells, human bronchial epithelioid, and human lung adenocarcinoma epithelial (A549) cells. Viral replication and transmission studies were conducted in 33 guinea pigs and 13 pigs. Additionally, we collected serum samples from pig farm workers and members of the general public recruited by the Third Affiliated Hospital of Sun Yat-sen University between Feb 28 and May 11, 2023, to detect specific antibodies against Eurasian avian-like A(H1) and human-like A(H3N2) SIVs using the haemagglutination inhibition assay. FINDINGS: 23 (1·3%) of 1818 nasal swabs collected in abattoirs had SIVs; 22 (0·9%) of 2375 swabs from swine farms had SIVs. Further viral isolation yielded 39 strains of SIV. We identified 534 A(H1N1), 69 A(H1N2), and 92 A(H3N2) SIVs, representing 20 genotypes within the Eurasian avian-like lineage, 14 within the classical swine A(H1) lineage, and 16 within the human-like A(H3N2) lineage. The introduction of the A(H1N1)pdm/09 virus significantly influenced the internal gene pool of SIVs, enhancing genotypic diversity in China. Notably, the Eurasian avian-like A(H1), classical swine A(H1), and human-like A(H3N2) lineages showed human-mediated spread over long distances between provinces, with the Eurasian avian-like A(H1) lineage showing the most prevalent spread pathways. Eurasian avian-like A(H1) SIVs showed a preference for binding to sialic acid α-2,6 glycan receptors, predominantly found in humans, resulting in an increased production of progeny viruses in human airway epithelial cells, as well as effective transmission and infectivity among guinea pigs and pigs. Among 54 eligible serum samples collected from pig farm workers (24 from slaughterhouses and 30 from swine farms), 23 (43%) were seropositive for Eurasian avian-like A(H1) SIVs and 46 (85%) for human-like A(H3N2) SIVs. Among 100 eligible samples from members of the general public, 14 (14%) were seropositive for Eurasian avian-like A(H1) SIVs and 85 (85%) for human-like A(H3N2) SIVs. INTERPRETATION: This study elucidates the evolutionary processes and spatiotemporal patterns of SIVs, highlighting potential risks to public health. These findings are crucial for informing public health interventions that aim to prevent future SIV epidemics in China and other countries worldwide. FUNDING: Scientific Innovation Strategy-Construction of High-Level Academy of Agriculture Science-Distinguished Scholar (R2020PY-JC001).

Animals↗
PubMed2026

Machine learning-integrated multi-omics risk prediction for pulmonary fungal infection in COPD and lung cancer: a transcriptomic and immune profiling study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) and lung cancer are major risk factors for invasive pulmonary fungal infection (IPFI), carrying an attributable mortality of 30%-80%. Their coexistence further amplifies immunosuppression, while current diagnostic criteria remain inadequate for early risk identification. METHODS: Transcriptomic data from the GEO dataset GSE296912 (scRNA-seq; 12,078 cells from normal and COPD lung tissue) and The Cancer Genome Atlas (TCGA)-lung adenocarcinoma (LUAD) bulk RNA-seq cohort (539 tumor and 59 normal samples) underwent differential expression and cross-omics integration analysis. Five machine learning models were constructed: logistic regression, SVM, random forest, XGBoost, and LASSO. Candidate genes were validated by qRT-PCR in A549 cells and THP-1-derived macrophages stimulated with heat-inactivated Aspergillus fumigatus conidia, a protocol selected to ensure BSL-2 biosafety compliance and isolate PAMP-mediated innate immune signaling. Model performance was evaluated using 5-fold stratified cross-validation with AUC, calibration curves, and decision curve analysis. RESULTS: Single-cell transcriptomic analysis of 12,078 cells identified 14 distinct cell populations, with marked myeloid expansion and immune dysregulation in COPD lung tissue. Cross-omics integration with TCGA-LUAD data identified 1,145 shared genes (79 immune-related), converging on NF-κB, TLR4, and cytokine receptor signaling. The random forest model achieved excellent discriminative performance (5-fold CV AUC = 0.988), with Treg infiltration, TLR4, and MMP9 as the top predictors. qRT-PCR confirmed significant upregulation of all five candidate genes (DEFB4A, S100A8, IL-8, MMP9, and TLR4) in both A549 and THP-1 cells following fungal stimulation. CONCLUSION: This multi-omics machine learning model integrating scRNA-seq and TCGA transcriptomic data demonstrates excellent discriminative performance (AUC = 0.988), with mechanistic convergence of NF-κB, TLR4, and oncogenic signaling pathways identified across shared immune gene signatures. In vitro qRT-PCR validation confirms the biological relevance of five key antifungal immune genes, providing a transcriptomic foundation for future prospective IPFI risk stratification in patients with COPD and lung cancer.

TLR4↗
PubMed2026

The cellular protein TIAR mediates rapid initiation of West Nile virus genome RNA synthesis.

During the intracellular replication cycle of West Nile virus (WNV), genome RNA synthesis is initially inefficient but increases exponentially as viral replication complexes are sequestered in invaginations in the endoplasmic reticulum. In this study, we investigated the functional role of the cellular protein TIAR (T-cell intracellular antigen-related protein) in the transcription of WNV genome RNA. Close colocalization of cytoplasmic TIAR with viral double-stranded RNA was detected by a proximity ligation assay in WNV-infected cells. TIAR binds specifically to the WNV 3'(-) SL but not to the complementary WNV 5'(+) SL in in vitro RNA binding assays. Only the 3' end of the WNV minus-strand RNA was enriched by immunoprecipitation of infected cell lysates with anti-TIAR antibody. Stable overexpression of TIAR in clonal A549 cells increased the ratio of intracellular viral plus-strand to minus-strand RNA in a dose-dependent manner. TIAR contains three RNA recognition motifs (RRMs). Biophysical data indicated that only RRM2 directly contacts RNA and that up to three TIAR molecules can bind cooperatively to the WNV 3'(-) SL RNA. These data provide additional evidence that TIAR functions as a proviral host factor facilitating exponential amplification of WNV genome production in infected cells.IMPORTANCEWest Nile virus (WNV) is a mosquito-borne orthoflavivirus associated with increasing global human disease incidence. The molecular mechanisms underlying viral replication are not fully understood. In early stages of infection, viral genome transcription is inefficient; however, in late stages, viral genome transcription increases exponentially. T-cell intracellular antigen-related (TIAR) protein is a cellular protein that has been shown to interact with the 3' end of the WNV negative-sense antigenomic RNA. We obtained data showing colocalization of cellular TIAR with viral replication complexes in infected cells and an increased ratio of intracellular genomic to antigenomic viral RNA in TIAR-overexpressing cells, and confirmed preferential binding of TIAR to the 3' end of the WNV antigenome both in vitro and in infected cell extracts. We also demonstrated that multiple TIAR proteins can bind cooperatively to the WNV 3'(-) stem-loop RNA. These data provide supporting evidence for a model of TIAR-mediated rapid initiation of nascent genome RNA synthesis in infected cells.

TIAR↗
PubMed2026

Epigenetic Repression of TP53 Transcription Underlies Cancer Cell Persistence for Carboplatin Resistance in Non-Small Cell Lung Cancer.

While chemoresistance in non-small cell lung cancer (NSCLC) cells has historically been attributed to permanent genetic mutations, emerging evidence highlights the role of nongenetic transcriptional plasticity and 'drug-tolerant persister' cells. To systematically map these epigenetic vulnerabilities, we utilized a genome-wide CRISPR interference library to screen wild-type TP53 NSCLC (A549) cells under carboplatin selection. Using the DrugZ algorithm and subsequent pathway enrichment analyses, this screen revealed that transcriptional suppression of interstrand crosslink DNA repair networks, including the Fanconi anemia pathway, markedly sensitized cells to carboplatin. Unexpectedly, transcriptional silencing of TP53 and its downstream target CDKN1A emerged as the strongest drivers of resistance, enabling cells to bypass therapy-induced senescence and maintain their proliferative potential later. To validate these findings in a clinically relevant context, we established a chronic carboplatin-resistant cell model (A549CarboR cells). A549CarboR exhibited a reduction in TP53 transcripts, along with decreased H3K27 acetylation and increased DNA hypermethylation on its promoter. Epigenetic remodeling using the DNA methyltransferase inhibitor (DNMTi) was associated with unblocking TP53 transcription, restored p53 signaling, and resensitization of resistant cells to carboplatin. Conversely, histone deacetylase inhibitors induced CDKN1A transcription to bypass TP53, indicating distinct epigenetic circuits. Collectively, the results demonstrate for the first time that TP53 expression is dynamically regulated at the transcriptional level through promoter methylation related to the drug tolerance. These insights emphasize that epigenetic silencing, rather than exclusive genetic loss-of-function, contribute to platinum resistance and underscore the therapeutic potential of pairing platinum regimens with DNMTi to target the transcriptomic plasticity of persistent cancer cell populations.

CRISPR interference screening↗
PubMed2024

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Animals↗
PubMed2026

Identification and validation of an alternatively spliced novel isoform of maspin that modulate genes involved in inflammatory and apoptotic pathways.

Maspin regulates cellular adhesion, migration, apoptosis, angiogenesis, and tumor suppression in a tissue and context-dependent manner. Its functional diversity is governed largely thorough extracellular matrix interactions, subcellular localization, and the reactive center loop (RCL), although the structural details are not well understood. To examine whether alternative splicing contributes to this heterogeneity, we analysed the SERPINB5 gene using a computational genomics approach and identified a novel 80 bp coding exon upstream of the first coding exon (E1). The alternatively spliced transcript was validated in human skin and esophagus by semi-nested touchdown PCR, quantitative real-time PCR, and Sanger sequencing. Recombinant B5N displayed a red-shifted fluorescence emission spectrum, indicating a more solvent-exposed conformation, which was supported by molecular dynamics simulations showing greater exposure of the nuclear localization signal (NLS) and the reactive center loop. Enzyme kinetic assays demonstrated concentration-dependent enhancement of tissue plasminogen activator (tPA) activity by both isoforms. In HaCaT cells, wildtype maspin produced stronger antiproliferative and anti-migratory effects, whereas B5N was only mildly antiproliferative. Annexin V/7-AAD staining revealed that wildtype maspin induced higher early apoptosis and cell death, while B5N produced lower overall cell death but a greater proportion of late apoptotic cells. RNA-seq of transfected HaCaT cells identified differentially expressed genes enriched in inflammatory, antiviral, and apoptotic pathways, which was validated by qPCR, and several of these were markedly upregulated in SARS-CoV-2 infected A549 cells. Thus, a novel N-terminally extended maspin isoform with differentially regulated gene profile is identified and validated in this study.

Apoptosis↗