Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

High-resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD.

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

Characteristics and anatomic location of PD-1+TCF1+ stem-like CD8 T cells in chronic viral infection and cancer.

CD8 T cells play an essential role in antitumor immunity and chronic viral infections. Recent findings have delineated the differentiation pathway of CD8 T cells in accordance with the progenitor-progeny relationship of TCF1+ stem-like and Tim-3+TCF1- more differentiated T cells. Here, we investigated the characteristics of stem-like and differentiated CD8 T cells isolated from several murine tumor models and human lung cancer samples in terms of phenotypic and transcriptional features as well as their location compared to virus-specific CD8 T cells in the chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. We found that CD8 tumor-infiltrating lymphocytes (TILs) in both murine and human tumors exhibited overall similar phenotypic and transcriptional characteristics compared to corresponding subsets in the spleen of chronically infected mice. Moreover, stem-like CD8 TILs exclusively responded and produced effector-like progeny CD8 T cells in vivo after antigenic restimulation, confirming their lineage relationship and the proliferative potential of stem-like CD8 TILs. Most importantly, similar to the preferential localization of PD-1+ stem-like CD8 T cells in T cell zones of the spleen during chronic LCMV infection, we found that the PD-1+ stem-like CD8 TILs in lung cancer samples are preferentially located not in the tumor parenchyma but in tertiary lymphoid structures (TLSs). The stem-like CD8 T cells are present in TLSs located within and at the periphery of the tumor, as well as in TLSs closely adjacent to the tumor parenchyma. These findings suggest that TLSs provide a protective niche to support the quiescence and maintenance of stem-like CD8 T cells in the tumor.

Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts.

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-ß-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5ß1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

Rationale: Pseudomonas aeruginosa is the major bacterial pathogen colonizing the airways of adult patients with cystic fibrosis (CF) and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evade the immune system and antibiotic therapy. Although the ability of P. aeruginosa to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. Objectives: To detect and characterize intracellular P. aeruginosa in CF airway epithelium from human lung explant tissues. Methods: We sampled lung explant tissues from patients with CF undergoing lung transplantation and non-CF lung donor control tissue. We analyzed lung tissue sections for the presence of intracellular P. aeruginosa using quantitative culture and microscopy, in parallel to histopathology and airway morphometry. Measurements and Main Results: P. aeruginosa was isolated from the lungs of seven patients with CF undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P. aeruginosa within airway epithelial cells in three of the seven patients analyzed at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. Conclusions: This is the first study describing the presence of intracellular P. aeruginosa in CF lung tissues. Although intracellular P. aeruginosa in airway epithelial cells is likely relatively rare, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

Transcriptomics Analysis Identifies the Decline in the Alveolar Type II Stem Cell Niche in Aged Human Lungs.

Aging poses a global public health challenge, which is linked to the rise of age-related lung diseases. The precise understanding of the molecular and genetic changes in the aging lung that elevate the risk of acute and chronic lung diseases remains incomplete. Alveolar type II (AT2) cells are stem cells that maintain epithelial homeostasis and repair the lung after injury. AT2 progenitor function decreases with aging. The maintenance of AT2 function requires niche support from other cell types, but little has been done to characterize alveolar alterations with aging in the AT2 niche. To systematically profile the genetic changes associated with age, we present a single-cell transcriptional atlas comprising nearly half a million cells from the healthy lungs of human subjects spanning various ages, sexes, and smoking statuses. Most annotated cell lineages in aged lungs exhibit dysregulated genetic programs. Specifically, the aged AT2 cells demonstrate loss of epithelial identities, heightened inflammaging characterized by increased expression of AP-1 (Activator Protein-1) transcription factor and chemokine genes, and significantly increased cellular senescence. Furthermore, the aged mesenchymal cells display a remarkable decrease in collagen and elastin transcription and a loss of support to epithelial cell stemness. The decline of the AT2 niche is further exacerbated by a dysregulated genetic program in macrophages and dysregulated communications between AT2 and macrophages in aged human lungs. These findings highlight the dysregulations observed in both AT2 stem cells and their supportive niche cells, potentially contributing to the increased susceptibility of aged populations to lung diseases.

Functional and Proteomic Dissection of the Contributions of CodY, SigB and the Hibernation Promoting Factor HPF to Interactions of Staphylococcus aureus USA300 with Human Lung Epithelial Cells.

Staphylococcus aureus is a leading cause of severe pneumonia. Our recent proteomic investigations into S. aureus invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant S. aureus USA300. Interestingly, disruption of codY resulted in a "small colony variant" phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

Piezo channels modulate human lung fibroblast function.

Bronchial airways and lung parenchyma undergo both static and dynamic stretch in response to normal breathing as well as in the context of insults such as mechanical ventilation (MV) or in diseases such as asthma and chronic obstructive pulmonary disease (COPD) which lead to airway remodeling involving increased extracellular matrix (ECM) production. Here, the role of fibroblasts is critical, but the relationship between stretch- and fibroblast-induced ECM remodeling under these conditions is not well-explored. Piezo (PZ) channels play a role in mechanotransduction in many cell and organ systems, but their role in mechanical stretch-induced airway remodeling is not known. To explore this, we exposed human lung fibroblasts to 10% static stretch on a background of 5% oscillations for 48 h, with no static stretch considered controls. Collagen I, fibronectin, alpha-smooth muscle actin (α-SMA), and Piezo 1 (PZ1) expression was determined in the presence or absence of Yoda1 (PZ1 agonist) or GsMTx4 (PZ1 inhibitor). Collagen I, fibronectin, and α-SMA expression was increased by stretch and Yoda1, whereas pretreatment with GsMTx4 or knockdown of PZ1 by siRNA blunted this effect. Acute stretch in the presence and absence of Yoda1 demonstrated activation of the ERK pathway but not Smad. Measurement of [Ca2+]i responses to histamine showed significantly greater responses following stretch, effects that were blunted by knockdown of PZ1. Our findings identify an essential role for PZ1 in mechanical stretch-induced production of ECM mediated by ERK phosphorylation and Ca2+ influx in lung fibroblasts. Targeting PZ channels in fibroblasts may constitute a novel approach to ameliorate airway remodeling by decreasing ECM deposition.NEW & NOTEWORTHY The lung is an inherently mechanosensitive organ that can respond to mechanical forces in adaptive or maladaptive ways, including via remodeling resulting in increased fibrosis. We explored the mechanisms that link mechanical forces to remodeling using human lung fibroblasts. We found that mechanosensitive Piezo channels increase with stretch and mediate extracellular matrix formation and the fibroblast-to-myofibroblast transition that occurs with stretch. Our data highlight the importance of Piezo channels in lung mechanotransduction toward remodeling.

Type-II IFN inhibits SARS-CoV-2 replication in human lung epithelial cells and ex vivo human lung tissues through indoleamine 2,3-dioxygenase-mediated pathways.

Interferons (IFNs) are critical for immune defense against pathogens. While type-I and -III IFNs have been reported to inhibit SARS-CoV-2 replication, the antiviral effect and mechanism of type-II IFN against SARS-CoV-2 remain largely unknown. Here, we evaluate the antiviral activity of type-II IFN (IFNγ) using human lung epithelial cells (Calu3) and ex vivo human lung tissues. In this study, we found that IFNγ suppresses SARS-CoV-2 replication in both Calu3 cells and ex vivo human lung tissues. Moreover, IFNγ treatment does not significantly modulate the expression of SARS-CoV-2 entry-related factors and induces a similar level of pro-inflammatory response in human lung tissues when compared with IFNß treatment. Mechanistically, we show that overexpression of indoleamine 2,3-dioxygenase 1 (IDO1), which is most profoundly induced by IFNγ, substantially restricts the replication of ancestral SARS-CoV-2 and the Alpha and Delta variants. Meanwhile, loss-of-function study reveals that IDO1 knockdown restores SARS-CoV-2 replication restricted by IFNγ in Calu3 cells. We further found that the treatment of l-tryptophan, a substrate of IDO1, partially rescues the IFNγ-mediated inhibitory effect on SARS-CoV-2 replication in both Calu3 cells and ex vivo human lung tissues. Collectively, these results suggest that type-II IFN potently inhibits SARS-CoV-2 replication through IDO1-mediated antiviral response.

It takes two peroxisome proliferator-activated receptors (PPAR-ß/δ and PPAR-γ) to tango idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung epithelial phenotypes, fibroblast activation, and increased extracellular matrix deposition. Transforming growth factor-beta (TGF-ß)1-induced Smad signaling and downregulation of peroxisomal genes are involved in the pathogenesis and can be inhibited by peroxisome proliferator-activated receptor (PPAR)-α activation. However, the three PPARs, that is PPAR-α, PPAR-ß/δ, and PPAR-γ, are known to interact in a complex crosstalk. METHODS: To mimic the pathogenesis of lung fibrosis, primary lung fibroblasts from control and IPF patients with comparable levels of all three PPARs were treated with TGF-ß1 for 24 h, followed by the addition of PPAR ligands either alone or in combination for another 24 h. Fibrosis markers (intra- and extracellular collagen levels, expression and activity of matrix metalloproteinases) and peroxisomal biogenesis and metabolism (gene expression of peroxisomal biogenesis and matrix proteins, protein levels of PEX13 and catalase, targeted and untargeted lipidomic profiles) were analyzed after TGF-ß1 treatment and the effects of the PPAR ligands were investigated. RESULTS: TGF-ß1 induced the expected phenotype; e.g. it increased the intra- and extracellular collagen levels and decreased peroxisomal biogenesis and metabolism. Agonists of different PPARs reversed TGF-ß1-induced fibrosis even when given 24 h after TGF-ß1. The effects included the reversals of (1) the increase in collagen production by repressing COL1A2 promoter activity (through PPAR-ß/δ activation); (2) the reduced activity of matrix metalloproteinases (through PPAR-ß/δ activation); (3) the decrease in peroxisomal biogenesis and lipid metabolism (through PPAR-γ activation); and (4) the decrease in catalase protein levels in control (through PPAR-γ activation) and IPF (through a combined activation of PPAR-ß/δ and PPAR-γ) fibroblasts. Further experiments to explore the role of catalase showed that an overexpression of catalase protein reduced collagen production. Additionally, the beneficial effect of PPAR-γ but not of PPAR-ß/δ activation on collagen synthesis depended on catalase activity and was thus redox-sensitive. CONCLUSION: Our data provide evidence that IPF patients may benefit from a combined activation of PPAR-ß/δ and PPAR-γ.

Thorium Alters Lung Surfactant Protein Expression in Alveolar Epithelial Cells: In Vitro and In Vivo Investigation.

Thorium-232 (Th), the most abundant naturally occurring nuclear fuel, has been identified as a sustainable source of energy. In view of its large-scale utilization and human evidence of lung disorders and carcinogenicity, it is imperative to understand the effect of Th exposure on lung cells. The present study investigated the effect of Th-dioxide (1-100 µg/mL, 24-48 h) on expression of surfactant proteins (SPs) (SP-A, SP-B, SP-C, and SP-D, which are essential to maintain lung's surface tension and host-defense) in human lung cells (WI26 and A549), representative of alveolar cell type-I and type-II, respectively. Results demonstrated the inhibitory effect of Th on transcriptional expression of SP-A, SP-B, and SP-C. However, Th promoted the mRNA expression of SP-D in A549 and reduced its expression in WI26. To a significant extent, the effect of Th on SPs was found to be in accordance with their protein levels. Moreover, Th exposure altered the extracellular release of SP-D/A from A549, which remained unaltered in WI26. Our results suggested the differential role of oxidative stress and ATM and HSP90 signaling in Th-induced alterations of SPs. These effects of Th were found to be consistent in lung tissues of mice exposed to Th aerosols, suggesting a potential role of SPs in Th-associated lung disorders.

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Screening human lung cancer with predictive models of serum magnetic resonance spectroscopy metabolomics.

The current high mortality of human lung cancer stems largely from the lack of feasible, early disease detection tools. An effective test with serum metabolomics predictive models able to suggest patients harboring disease could expedite triage patient to specialized imaging assessment. Here, using a training-validation-testing-cohort design, we establish our high-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS)-based metabolomics predictive models to indicate lung cancer presence and patient survival using serum samples collected prior to their disease diagnoses. Studied serum samples were collected from 79 patients before (within 5.0 y) and at lung cancer diagnosis. Disease predictive models were established by comparing serum metabolomic patterns between our training cohorts: patients with lung cancer at time of diagnosis, and matched healthy controls. These predictive models were then applied to evaluate serum samples of our validation and testing cohorts, all collected from patients before their lung cancer diagnosis. Our study found that the predictive model yielded values for prior-to-detection serum samples to be intermediate between values for patients at time of diagnosis and for healthy controls; these intermediate values significantly differed from both groups, with an F1 score = 0.628 for cancer prediction. Furthermore, values from metabolomics predictive model measured from prior-to-diagnosis sera could significantly predict 5-y survival for patients with localized disease.

Effects of 630 nm laser on apoptosis, metastasis, invasion and epithelial-to-mesenchymal transition of human lung squamous cell carcinoma H520 cells mediated by hematoporphyrin derivatives.

Photodynamic therapy (PDT) has significant advantages in the treatment of malignant lung tumors. The research on the mechanism of PDT mediated by hematoporphyrin derivatives (HPD) and its cytotoxic effects on lung cancer cells has primarily focused on lung adenocarcinoma cells. However, the impact of HPD-PDT on lung squamous cell carcinoma has not been thoroughly studied. This study aimed to investigate the effects of 630 nm laser on apoptosis, metastasis, invasion, and epithelial-mesenchymal transition (EMT) in human lung squamous cell carcinoma H520 cells mediated by HPD. H520 cells were divided into four groups: control group, photosensitizer group, irradiation group, and HPD-PDT group. Cell proliferation was assessed using CCK8 assay; cell apoptosis was detected by Hoechst 33258 staining and flow cytometry; cell migration and invasion abilities were evaluated using wound-healing and invasion assays; and protein and mRNA expressions were analyzed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively. Results showed that HPD-PDT significantly inhibited cell proliferation, promoted apoptosis (P < 0.05), suppressed cell migration and invasion (P < 0.05), decreased Bcl-2 mRNA expression, and increased Bax and Caspase-9 mRNA expression(P < 0.05). Western blotting analysis indicated increased expression of Bax, Caspase-9, and E-cadherin, and decreased expression of Bcl-2, N-cadherin, and Vimentin (P < 0.05). In conclusion, 630 nm laser mediated by HPD promoted cell apoptosis via upregulation of Bax and caspase-9, and downregulation of Bcl-2, and inhibited cell migration and invasion by regulating EMT in H520 cells.

Synthesis of selenophene-containing flavonols and 2-styrylchromones: Evaluation of their activities compared with selenophene-containing chalcones as potential anticancer agents.

Previously, we documented the synthesis and assessed the biological effects of chalcones containing selenium against HT-29 human colorectal adenocarcinoma cells, demonstrating their significant potential. As research on selenium-containing flavonoids remains limited, this article outlines our design and synthesis of three selenium-based flavonols and three 2-styrylchromones. We conducted evaluations of these compounds to determine their impact on human lung cancer cells (A549, H1975, CL1-0, and CL1-5) and their influence on normal lung fibroblast MRC5 cells. Additionally, we included selenium-based chalcones in our testing for comparative purposes. Our findings highlight that the simplest compound, designated as compound 1, exhibited the most promising performance among the tested molecules.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Liposomal Formulation Reduces Transport and Cell Uptake of Colistin in Human Lung Epithelial Calu-3 Cell and 3D Human Lung Primary Tissue Models.

Respiratory tract infections caused by multi-drug-resistant (MDR) bacteria have been a severe risk to human health. Colistin is often used to treat the MDR Gram-negative bacterial infections as a last-line therapy. Inhaled colistin can achieve a high concentration in the lung but none of aerosolized colistin products has been approved in the USA. Liposome has been reported as an advantageous formulation strategy for antibiotics due to its controlled release profile and biocompatibility. We have developed colistin liposomal formulations in our previous study. In the present study, the cellular uptake and transport of colistin in colistin liposomes were examined in two human lung epithelium in vitro models, Calu-3 monolayer and EpiAirway 3D tissue models. In both models, cellular uptake (p < 0.05) and cellular transport (p < 0.01) of colistin were significantly reduced by the colistin liposome compared to the colistin solution. Our findings indicate that inhaled colistin liposomes could be a promising treatment for extracellular bacterial lung infections caused by MDR Pseudomonas aeruginosa (P. aeruginosa).

Global profiling of lysine lactylation in human lungs.

Lactate is closely related to various cellular processes, such as angiogenesis, responses to hypoxia, and macrophage polarization, while regulating natural immune signaling pathways and promoting neurogenesis and cognitive function. Lysine lactylation (Kla) is a novel posttranslational modification, the examination of which may lead to new understanding of the nonmetabolic functions of lactate and the various physiological and pathological processes in which lactate is involved, such as infection, tumorigenesis and tumor development. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), researchers have identified lactylation in human gastric cancer cells and some other species, but no research on lactylation in human lungs has been reported. In this study, we performed global profiling of lactylation in human lungs under normal physiological conditions, and 724 Kla sites in 451 proteins were identified. After comparing the identified proteins with those reported in human lactylation datasets, 141 proteins that undergo lactylation were identified for the first time in this study. Our work expands the database on human lactylation and helps advance the study on lactylation function and regulation under physiological and pathological conditions.

Replication of Novel Zoonotic-Like Influenza A(H3N8) Virus in Ex Vivo Human Bronchus and Lung.

Human infection with avian influenza A(H3N8) virus is uncommon but can lead to acute respiratory distress syndrome. In explant cultures of the human bronchus and lung, novel H3N8 virus showed limited replication efficiency in bronchial and lung tissue but had a higher replication than avian H3N8 virus in lung tissue.