Human Lung Epithelial A549 Cells Research Articles

In-Depth Characterization of Zika Virus Inhibitors Using Cell-Based Electrical Impedance.

ABSTRACTIn this study, we use electric cell-substrate impedance sensing (ECIS), an established cell-based electrical impedance (CEI) technology, to decipher the kinetic cytopathic effect (CPE) induced by Zika virus (ZIKV) in susceptible human A549 lung epithelial cells and to evaluate several classes of compounds with reported antiviral activity (two entry inhibitors and two replication inhibitors). To validate the assay, we compare the results with those obtained with more traditional in vitro methods based on cell viability and viral yield readouts. We demonstrate that CEI can detect viral infection in a sensitive manner and can be used to determine antiviral potency. Moreover, CEI has multiple benefits compared to conventional assays: the technique is less laborious and better at visualizing the dynamic antiviral activity profile of the compounds, while also it has the ability to determine interesting time points that can be selected as endpoints in assays without continuous readout. We describe several parameters to characterize the compounds’ cytotoxicity and their antiviral activity profile. In addition, the CEI patterns provide valuable additional information about the presumed mechanism of action of these compounds. Finally, as a proof of concept, we used CEI to evaluate the antiviral activity of a small series of compounds, for which we demonstrate that the sulfonated polymer PRO2000 inhibits ZIKV with a response profile representative for a viral entry inhibitor. Overall, we demonstrate for the first time that CEI is a powerful technology to evaluate and characterize compounds against ZIKV replication in a real-time, label-free, and noninvasive manner.IMPORTANCE Zika virus can cause serious disease in humans. Unfortunately, no antiviral drugs are available to treat infection. Here, we use an impedance-based method to continuously monitor virus infection in—and damage to—human cells. We can determine the Zika viral dose with this technique and also evaluate whether antiviral compounds protect the cells from damage caused by virus replication. We also show that this technique can be used to further unravel the characteristics of these compounds, such as their toxicity to the cells, and that it might even give further insight in their mechanism of antiviral action. Finally, we also find a novel Zika virus inhibitor, PRO2000. Overall, in this study, we use the impedance technology to—for the first time—evaluate compounds with anti-Zika virus properties, and therefore it can add valuable information in the further search for antiviral drugs.

Interlaboratory evaluation of a digital holographic microscopy–based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.Graphical abstract

Time-resolved proteome and transcriptome of paraquat-induced pulmonary fibrosis

BackgroundsPulmonary fibrosis (PF) is a pathological state presenting at the progressive stage of heterogeneous interstitial lung disease (ILD). The current understanding of the molecular mechanisms involved is incomplete. This clinical toxicology study focused on the pulmonary fibrosis induced by paraquat (PQ), a widely-used herbicide. Using proteo-transcriptome analysis, we identified differentially expressed proteins (DEPs) derived from the initial development of fibrosis to the dissolved stage and provided further functional analysis. MethodsWe established a mouse model of progressive lung fibrosis via intratracheal instillation of paraquat. To acquire a comprehensive and unbiased understanding of the onset of pulmonary fibrosis, we performed time-series proteomics profiling (iTRAQ) and RNA sequencing (RNA-Seq) on lung samples from paraquat-treated mice and saline control. The biological functions and pathways involved were evaluated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis. Correlation tests were conducted on comparable groups 7 days and 28 days post-exposure. Differentially expressed proteins and genes following the same trend on the protein and mRNA levels were selected for validation. The functions of the selected molecules were identified in vitro. The protein level was overexpressed by transfecting gene-containing plasmid or suppressed by transfecting specific siRNA in A549 cells. The levels of endothlial-mesenchymal transition (EMT) markers, including E-cadherin, vimentin, FN1, and α-SMA, were determined via western blot to evaluate the fibrotic process. ResultsWe quantified 1358 DEPs on day 7 and 426 DEPs on day 28 post exposure (Fold change >1.2; Q value < 0.05). The top 5 pathways – drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, complement and coagulation cascades, chemical carcinogenesis, protein digestion and absorption — were involved on both day 7 and day 28. Several pathways, including tight junction, focal adhesion, platelet activation, and ECM-receptor interaction, were more enriched on day 28 than on day 7. Integrative analysis of the proteome and transcriptome revealed a moderate correlation of quantitative protein abundance ratios with RNA abundance ratios (Spearman R = 0.3950 and 0.2477 on days 7 and 28, respectively), indicating that post-transcriptional regulation plays an important role in lung injury and repair. Western blot identified that the protein expressions of FN1, S100A4, and RBM3 were significantly upregulated while that of CYP1A1, FMO3, and PGDH were significantly downregulated on day 7. All proteins generally recovered to baseline on day 28. qPCR showed the mRNA levels of Fn1, S100a4, Rbm3, Cyp1a1, Fmo3, and Hpgd changed following the same trend as the levels of their respective proteins. Further, in vitro experiments showed that RBM3 was upregulated while PGDH was downregulated in an EMT model established in human lung epithelial A549 cells. RBM3 overexpression and PGDH knockout could both induce EMT in A549 cells. RBM3 knockout or PGDH overexpression had no reverse effect on EMT in A549 cells. ConclusionsOur proteo-transcriptomic study determined the proteins responsible for fibrogenesis and uncovers their dynamic regulation from lung injury to repair, providing new insights for the development of biomarkers for diagnosis and treatment of fibrotic diseases.

Abstract 1632: An orthotopic model for luciferase-labeled human lung epithelial carcinoma with response to standard of care treatment

Abstract The human lung epithelial carcinoma A549 cell line was first developed in 1972 after culturing pulmonary carcinoma tissue from a 58-year-old Caucasian male. Similar to human non-small cell lung carcinoma (NSCLC), this cell line carries a KRAS mutation making an attractive model for drug discovery/oncology research applications. NSCLC tumors grow slower and less aggressively than small cell lung cancers, yet the 5-year survival rate for NSCLC is 25%. Thus, more relevant preclinical models are still needed to evaluate the potential therapeutic activities of agents that can be used in the clinic. Orthotopic implantation of luciferase-labeled cancer cell lines facilitate the evaluation of disease progression using bioluminescence in vivo imaging. Here we report the development of a novel model of orthotopically implanted Luciferase-labeled A549-luc cells, its tumor growth kinetics and response to standard of care therapy. To establish the orthotopic model, we implanted cells by direct injection into the left lung lobe through a small incision on the lateral thorax between the 6th and 7th rib. Disease progression was evaluated by testing cell implants at multiple cell inoculums and following each group for tumor growth using an IVIS Spectrum CT. In line with the observed slow growth of NSCLC, A549 tumor growth kinetics revealed long latency with a median survival of 69.5-days post-implant. Mean flux values correlated with the number of cells inoculated and clinical observations of respiratory distress. Gross necropsies provided visual evidence of masses in the left lung lobe and thoracic cavity. Following the A549-luc orthotopic model development, we sought to evaluate the survival benefit in response to standard of care agents Paclitaxel and the tyrosine kinase inhibitors Erlotinib and Afatinib. Paclitaxel monotherapy was the most active agent in a 101-day study. A single treatment cycle showed robust decrease tumor burden as determined via imaging during the first few weeks following treatment, but disease progression was apparent despite the overall improved survival over the vehicle control group. To establish durable responses, we evaluated the efficacy of Paclitaxel administered for two dosing cycles. Palbociclib, a cyclin-dependent kinase 4/6 inhibitor that has been recently identified as potential target in KRAS-mutant (NSCLC) was also evaluated. Our preliminary results show significant activity for both Palbociclib and Paclitaxel therapies in the A549-luc lung orthotopic xenograft model. This lung orthotopic model provides a sensible alternative that recapitulates the difficulties of treating NCSL tumors in addition to establishing a more clinically relevant model to evaluate cancer therapies compared to standard subcutaneous implanted models. Histopathological analysis of Ki67, Cyclin D1, and CDK4 will be presented from lungs of treated and control groups. Citation Format: Elizabeth Rainbolt, Andrew Wong, Chassidy Hall, Patrick Wood, Paula L. Miliani De Marval. An orthotopic model for luciferase-labeled human lung epithelial carcinoma with response to standard of care treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1632.

MiR-181a-5p restrains non-small cell lung cancer cell invasion and migration by regulating the GTSE1/p53/NF-κB axis

microRNAs (miRNAs) are relevant to metastasis and invasion of non-small cell lung cancer (NSCLC). This study investigated the role of miR-181a-5p in lung cancer. Expression patterns of miR-181a-5p and GTSE1 in the human NSCLC cell line A549 and normal lung epithelial cell line BASE-2B were detected. miR-181a-5p mimic was delivered into A549 cells utilizing Lipofectamine 2000 to overexpress miR-181a-5p, followed by analysis of cell viability, proliferation, apoptosis, invasion, and migration. GTSE1 (G2 and S phase-expressed-1) was predicted as the downstream target gene of miR-181a-5p using bioinformatics analysis software. Targeting relationship between miR-181a-5p and GTSE1 was validated via dual-luciferase assay, RIP assay, and RNA pull-down. Activation of the p53/NF-κB pathway was determined. miR-181a-5p was weakly-expressed in NSCLC cells relative to normal lung epithelial cells. miR-181a-5p overexpression prevented NSCLC cell proliferation, migration, and invasion. Mechanically, miR-181a-5p targeted GTSE1. GTSE1 overexpression partly annulled repression of miR-181a-5p overexpression on NSCLC cell malignant behavior. miR-181a-5p activated the p53 pathway and inhibited the NF-κB pathway by targeting GTSE1. Overall, this study for the first time validated that miR-181a-5p impeded NSCLC cell invasion and migration through activation of the p53 pathway and inhibition of the NF-κB pathway by targeting GTSE1, which may provide a potential novel insight into NSCLC treatment.

A Myosin Vb Splice Variant Regulates Coronavirus M Protein Trafficking in Polarized Epithelial Cells

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two‐hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV‐CoV) M protein with Myosin Vb (MYO5B) (Figure 1A). MHV‐CoV M interacts specifically with the alternative splice variant of cellular MYO5B including Exon D (MYO5B+D), that also mediates interaction with cellular Rab10. When co‐expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co‐localized with MHV‐CoV M protein, as well as with M proteins from porcine epidemic diarrhea Virus (PEDV‐CoV), Middle East Respiratory Syndrome (MERS‐CoV) and Severe Acute Respiratory Syndrome 2 (SARS‐CoV‐2) (Figure 2). M‐GFP chimeric proteins co‐expressed with mCherry‐MYO5B+D also co‐localized with endogenous Rab10 and Rab11a (Figure 1B,C). We identified point mutations in MHV‐CoV M that blocked the interaction with MYO5B+D in yeast 2‐hybrid assays (Figure 1B). One of these point mutations (E121K) was previously shown to block MHV‐CoV virion assembly, and it blocked interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV‐CoV, MERS‐CoV and SARS‐CoV‐2 M proteins also blocked colocalization with MYO5B+D. Knockdown of Rab10 blocked the co‐localization of the M proteins with MYO5B+D (Figure 2). Re‐expression of Cerulein‐Rab10 in Rab10 KD cells re‐established colocalization between M proteins and MYO5B+D (Figure 2). Our results suggest that the interaction of CoV M proteins with MYO5B+D may play a role in regulating their trafficking through Rab10‐containing membrane systems in epithelial cells.

CHIKV strains Brazil (wt) and Ross (lab-adapted) differ with regard to cell host range and antiviral sensitivity and show CPE in human glioblastoma cell lines U138 and U251.

Chikungunya virus (CHIKV), a (re)emerging arbovirus, is the causative agent of chikungunya fever. To date, no approved vaccine or specific antiviral therapy are available. CHIKV has repeatedly been responsible for serious economic and public health impacts in countries where CHIKV epidemics occurred. Antiviral tests in vitro are generally performed in Vero-B4 cells, a well characterised cell line derived from the kidney of an African green monkey. In this work we characterised a CHIKV patient isolate from Brazil (CHIKVBrazil) with regard to cell affinity, infectivity, propagation and cell damage and compared it with a high-passage lab strain (CHIKVRoss). Infecting various cell lines (Vero-B4, A549, Huh-7, DBTRG, U251, and U138) with both virus strains, we found distinct differences between the two viruses. CHIKVBrazil does not cause cytopathic effects (CPE) in the human hepatocarcinoma cell line Huh-7. Neither CHIKVBrazil nor CHIKVRoss caused CPE on A549 human lung epithelial cells. The human astrocyte derived glioblastoma cell lines U138 and U251 were found to be effective models for lytic infection with both virus strains and we discuss their predictive potential for neurogenic CHIKV disease. We also detected significant differences in antiviral efficacies regarding the two CHIKV strains. Generally, the antivirals ribavirin, hydroxychloroquine (HCQ) and T-1105 seem to work better against CHIKVBrazil in glioblastoma cells than in Vero-B4. Finally, full genome analyses of the CHIKV isolates were done in order to determine their lineage and possibly explain differences in tissue range and antiviral compound efficacies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11262-022-01892-x.

Polymyxin Induces Significant Transcriptomic Perturbations of Cellular Signalling Networks in Human Lung Epithelial Cells.

Inhaled polymyxins are increasingly used to treat pulmonary infections caused by multidrug-resistant Gram-negative pathogens. We have previously shown that apoptotic pathways, autophagy and oxidative stress are involved in polymyxin-induced toxicity in human lung epithelial cells. In the present study, we employed human lung epithelial cells A549 treated with polymyxin B as a model to elucidate the complex interplay of multiple signalling networks underpinning cellular responses to polymyxin toxicity. Polymyxin B induced toxicity (1.0 mM, 24 h) in A549 cells was assessed by flow cytometry and transcriptomics was performed using microarray. Polymyxin B induced cell death was 19.0 ± 4.2% at 24 h. Differentially expressed genes (DEGs) between the control and polymyxin B treated cells were identified with Student’s t-test. Pathway analysis was conducted with KEGG and Reactome and key hub genes related to polymyxin B induced toxicity were examined using the STRING database. In total we identified 899 DEGs (FDR < 0.01), KEGG and Reactome pathway analyses revealed significantly up-regulated genes related to cell cycle, DNA repair and DNA replication. NF-κB and nucleotide-binding oligomerization domain-like receptor (NOD) signalling pathways were identified as markedly down-regulated genes. Network analysis revealed the top 5 hub genes (i.e., degree) affected by polymyxin B treatment were PLK1 (48), CDK20 (46), CCNA2 (42), BUB1 (40) and BUB1B (37). Overall, perturbations of cell cycle, DNA damage and pro-inflammatory NF-κB and NOD-like receptor signalling pathways play key roles in polymyxin-induced toxicity in human lung epithelial cells. Noting that NOD-like receptor signalling represents a group of key sensors for microorganisms and damage in the lung, understanding the mechanism of polymyxin-induced pulmonary toxicity will facilitate the optimisation of polymyxin inhalation therapy in patients.

MicroRNAs Associated with Chronic Mucus Hypersecretion in COPD Are Involved in Fibroblast-Epithelium Crosstalk.

We recently identified microRNAs (miRNAs) associated with chronic mucus hypersecretion (CMH) in chronic obstructive pulmonary disease (COPD), which were expressed in both airway epithelial cells and fibroblasts. We hypothesized that these miRNAs are involved in communication between fibroblasts and epithelium, contributing to airway remodeling and CMH in COPD. Primary bronchial epithelial cells (PBECs) differentiated at the air–liquid interface, and airway fibroblasts (PAFs) from severe COPD patients with CMH were cultured alone or together. RNA was isolated and miRNA expression assessed. miRNAs differentially expressed after co-culturing were studied functionally using overexpression with mimics in mucus-expressing human lung A549 epithelial cells or normal human lung fibroblasts. In PBECs, we observed higher miR-708-5pexpression upon co-culture with fibroblasts, and miR-708-5p expression decreased upon mucociliary differentiation. In PAFs, let-7a-5p, miR-31-5p and miR-146a-5p expression was significantly increased upon co-culture. miR-708-5p overexpression suppressed mucin 5AC (MUC5AC) secretion in A549, while let-7a-5poverexpression suppressed its target gene COL4A1 in lung fibroblasts. Our findings suggest that let-7a-5p, miR-31-5p and miR-146a-5p may be involved in CMH via fibroblasts–epithelium crosstalk, including extracellular matrix gene regulation, while airway epithelial expression of miR-708-5p may be involved directly, regulating mucin production. These findings shed light on miRNA-mediated mechanisms underlying CMH, an important symptom in COPD.

Estradiol reduces ACE2 and TMPRSS2 mRNA levels in A549 human lung epithelial cells.

Epidemiologic studies suggest slightly higher risk of severe Covid‐19 symptoms and fatalities following SARS‐CoV‐2 infection in men compared with women from similar age groups. This bias was suggested to reflect differences in the male and female immune system regulation, driven by different sex hormone levels in men and women, in particular, higher plasma estradiol in women. SARS‐CoV‐2 infects respiratory tract epithelial cells by binding to their cell membrane ACE2, followed by priming for cell entry by the host cell membrane serine protease TMPRSS2. The cell protease FURIN facilitates cell exit of mature SARS‐CoV‐2 virions. Our study examined the effects of in vitro treatment of A549 human lung epithelial cells with 17‐β‐estradiol on mRNA expression of genes coding for these proteins. Treatment of A549 human lung epithelial cells with 17‐β‐estradiol reduced the cellular mRNA levels of ACE2 and TMPRSS2 mRNA, while not affecting FURIN expression. Our findings suggest that 17‐β‐estradiol may reduce SARS‐CoV‐2 infection of lung epithelial cells, which may in part explain the reduced incidence of severe Covid‐19 and fatalities among women compared with men of similar age. Studies into the molecular pathways by which 17‐β‐estradiol reduces ACE2 and TMPRSS2 mRNA expression in lung epithelial cells are needed for assessing its potential protective value against severe Covid‐19.

Chlorine exposure induces Caspase-3 independent cell death in human lung epithelial cells

Chlorine (Cl2) is a common toxic industrial gas and human inhalation exposure causes tissue damage with symptoms ranging from wheezing to more severe symptoms such as lung injury or even death. Because the mechanism behind Cl2-induced cell death is not clearly understood, the present study aimed to study the cellular effects in vitro after Cl2 exposure of human A549 lung epithelial cells. In addition, the possible treatment effects of the anti-inflammatory antioxidant N-acetyl cysteine (NAC) were evaluated.Exposure of A549 cells to Cl2 (100–1000 ppm) in the cell medium induced cell damage and toxicity within 1 h in a dose-dependent manner. The results showed that 250 ppm Cl2 increased cell death and formation of apoptotic-like bodies, while 500 ppm Cl2 exposure resulted in predominantly necrotic death. Pre-treatment with NAC was efficient to prevent cell damage at lower Cl2 concentrations in part by averting the formation of apoptotic-like bodies and increasing the expression of the anti-apoptotic proteins clusterin and phosphorylated tumour protein p53(S46). Analysis showed that Cl2 induced cell death by a possibly caspase-independent mechanism, since no cleavage of caspase-3 could be detected after exposure to 250 ppm. Currently, these results justifies further research into new treatment strategies for Cl2-induced lung injury.

Arctigenin attenuates paraquat-induced human lung epithelial A549 cell injury by suppressing ROS/p38 mitogen-activated protein kinases-mediated apoptosis.

Paraquat (PQ)-induced acute lung injury (ALI) and pulmonary fibrosis are common diseases with high mortality but without effective antidotes in emergency medicine. Our previous study has proved that arctigenin suppressed pulmonary fibrosis induced by PQ. We wondered whether arctigenin could also have a protective effect on PQ-induced ALI. A PQ-induced A549 cell injury model was used, and the effect of arctigenin was determined by a cell counting kit-8 (CCK-8) cell viability assay. In addition, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining assays and mitochondrial membrane potential assays were performed to evaluate the level of cell apoptosis. The generation of reactive oxygen species (ROS) was reflected by dihydroethidium (DHE) staining and a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Moreover, immunoblotting studies were used to assess the expression of mitogen-activated protein kinases (MAPKs) and p38 MAPK. Arctigenin attenuated PQ-induced inhibition of A549 cell viability in a dose-dependent manner. Arctigenin also significantly reduced PQ-induced A549 cell apoptosis, as reflected by the TUNEL assay and mitochondrial membrane potential assay, which may result from suppressed ROS/p38 MAPK signaling because we found that arctigenin dramatically suppressed ROS generation and p38 MAPK phosphorylation. Arctigenin could attenuate PQ-induced lung epithelial A549 cell injury in vitro by suppressing ROS/p38 MAPK-mediated cell apoptosis, and arctigenin might be considered a potential candidate drug for PQ-induced ALI.

Djulis (Chenopodium formosanum) and Its Bioactive Compounds Protect Human Lung Epithelial A549 Cells from Oxidative Injury Induced by Particulate Matter via Nrf2 Signaling Pathway.

The protective effects of water extracts of djulis (Chenopodium formosanum) (WECF) and their bioactive compounds on particulate matter (PM)-induced oxidative injury in A549 cells via the nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling were investigated. WECF at 50–300 µg/mL protected A549 cells from PM-induced cytotoxicity. The cytoprotection of WECF was associated with decreases in reactive oxygen species (ROS) generation, thiobarbituric acid reactive substances (TBARS) formation, and increases in superoxide dismutase (SOD) activity and glutathione (GSH) contents. WECF increased Nrf2 and heme oxygenase-1 (HO-1) expression in A549 cells exposed to PM. SP600125 (a JNK inhibitor) and U0126 (an ERK inhibitor) attenuated the WECF-induced Nrf2 and HO-1 expression. According to the HPLC-MS/MS analysis, rutin (2219.7 µg/g) and quercetin derivatives (2648.2 µg/g) were the most abundant bioactive compounds present in WECF. Rutin and quercetin ameliorated PM-induced oxidative stress in the cells. Collectively, the bioactive compounds present in WECF can protect A549 cells from PM-induced oxidative injury by upregulating Nrf2 and HO-1 via activation of the ERK and JUN signaling pathways.

Application of three-dimensional Raman imaging to determination of the relationship between cellular localization of diesel exhaust particles and the toxicity

A diesel exhaust particle (DEP) is a type of particulate matter that is easily produced from combustion in a diesel power engine. It has been reported that DEPs can cause short- and long-term health problems. This is because DEPs are complex mixtures that are highly inhalable through the airways due to their small particle size. However, the relationship between intracellular localization of DEPs after their deposition in the lungs and the subsequent biological responses remains to be clarified. This is due to difficulties in distinguishing particles that are inside the cells from those that are outside. In this study, A549 human lung epithelial cells were exposed to DEPs at concentrations of 0, 25, 75, or 200 µg/mL for different periods, after that particles in the A549 cells were analyzed by three-dimensional (3D) images obtained from a Raman microscope. The cytotoxic effects of DEPs on the A549 cells were investigated by measuring cell viability, the levels of intracellular reactive oxygen species (ROS) and cell death. The Raman microscopy revealed that the particles invaded the A549 cells, and at a concentration of 200 µg/mL, they markedly decreased cell viability, increased intracellular ROS production, triggered late apoptosis/necrosis and induced nuclear damage. These results suggest that intracellular DEPs exposed at a high concentration may be highly toxic and can impair the viability of A549 cells. Furthermore, the 3D images from the Raman microscopy can be used to evaluate intracellular particle dynamics.

New β-Lactam Antibiotics and Ceragenins - A Study to Assess Their Potential in Treatment of Infections Caused by Multidrug-Resistant Strains of Pseudomonas aeruginosa.

BackgroundThe increasing number of infections caused by antibiotic resistant strains of Pseudomonas aeruginosa posed a very serious challenge for clinical practice. This standing is driving scientists to develop new antibiotics against these microorganisms.MethodsIn this study, we measured the MIC/MBC values and estimated the ability of tested molecules to prevent bacterial biofilm formation to explore the effectiveness of β-lactam antibiotics ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, and ceragenins CSA-13, CSA-44, and CSA-131 against 150 clinical isolates of Pseudomonas aeruginosa that were divided into five groups, based on their antibiotic resistance profiles to beta-lactams. Selected strains of microorganisms from each group were also subjected to prolonged incubations (20 passages) with ceragenins to probe the development of resistance towards those molecules. Cytotoxicity of tested ceragenins was evaluated using human red blood cell (RBCs) hemolysis and microscopy observations of human lung epithelial A549 cells after ceragenin treatment. Poloxamer 407 (pluronic F-127) at concentrations ranging from 0.5% to 5% was tested as a potential drug delivery substrate to reduce ceragenin toxicity.ResultsCollected data proved that ceragenins at low concentrations are highly active against clinical strains of Pseudomonas aeruginosa regardless of their resistance mechanisms to conventional antibiotics. Ceragenins also show low potential for resistance development, high antibiofilm activity, and controlled toxicity when used together with poloxamer 407.ConclusionThis data strongly supports the need for further study directed to develop this group of molecules as new antibiotics to fighting infections caused by antibiotic resistant strains of Pseudomonas aeruginosa.

D-Galactose treatment increases ACE2, TMPRSS2, and FURIN and reduces SERPINA1 mRNA expression in A549 human lung epithelial cells.

Several comorbidities including diabetes, immune deficiency, and chronic respiratory disorders increase the risk of severe Covid-19 and fatalities among SARS-CoV-2 infected individuals. Severe Covid-19 risk among diabetes patients may reflect reduced immune response to viral infections. SARS-CoV-2 initially infects respiratory tract epithelial cells by binding to the host cell membrane ACE2, followed by proteolytic priming for cell entry by the host cell membrane serine protease TMPRSS2. Additionally, the protease FURIN facilitates cell exit of mature SARS-CoV-2 virions. Alpha-1 antitrypsin (AAT), the major plasma serine protease inhibitor, encoded by SERPINA1, is known to promote immune response to viral infections. AAT inhibits neutrophil elastase, a key inflammatory serine protease implicated in alveolar cell damage during respiratory infections, and AAT deficiency is associated with susceptibility to lung infections. AAT is implicated in Covid-19 as it inhibits TMPRSS2, a protease essential for SARS-CoV-2 cell entry. Here we show that treatment of A549 human lung epithelial cells for 7 days with 25 mM d-galactose, an inducer of diabetic-like and oxidative stress cellular phenotypes, leads to increased mRNA levels of ACE2, TMPRSS2, and FURIN, along with reduced SERPINA1 mRNA. Together, the dysregulated transcription of these genes following d-galactose treatment suggests that chronic diabetic-like conditions may facilitate SARS-CoV-2 infection of lung epithelial cells. Our findings may in part explain the higher severe Covid-19 risk in diabetes, and highlight the need to develop special treatment protocols for diabetic patients.

FLASH Dose-Rate Helium Ion Beams: First In Vitro Investigations

<h3>Purpose/Objective(s)</h3> To establish and investigate the impact of LET and O₂ concentration on biological response to ultra-high dose-rate (uHDR, FLASH) helium ion beams compared to standard dose-rate (SDR) irradiation. <h3>Materials/Methods</h3> Beam delivery settings for raster-scanned helium ions at both uHDR and SDR were tuned to achieve > 100 Gy/s and 0.15 Gy/s, respectively. For both SDR and uHDR, 8 Gy and 12 Gy plan optimization and calibration for 10 × 10mm² fields was performed to assess <b>in vitro</b> response at lower LET (4.5 keV/μm) and higher LET (16 keV/μm) conditions. Clonogenic assay and DNA damage response (γH2AX) of human lung epithelial cancer cells A549 cells were used as biological indicators for the cellular response to uHDR and SDR irradiation for specified oxygen concentrations levels (1%, 21%). <h3>Results</h3> Average dose-rates for both lower and higher LET were 193 Gy/s and 0.15 Gy/s for uHDR and SDR, respectively. Cell survival was higher and γH2AX, a surrogate for DNA damage response, was lower for cells irradiated with uHDR as compared to SDR at 1% O₂ for both dose and LET levels. This difference in cellular response was not seen at 21% O₂ where cell survival and DNA damage response were unchanged at studied dose-rates. <h3>Conclusion</h3> The first uHDR delivery of raster-scanned particle beams was achieved using helium ions, reaching FLASH-level dose-rates of > 100 Gy/s. Baseline oxygen levels play a pivotal role, irrespective of investigated LET, for observation of a sparing effect for helium ions.

Microbiological and Toxicological Hazards in Sewage Treatment Plant Bioaerosol and Dust.

Despite the awareness that work in the sewage treatment plant is associated with biological hazards, they have not been fully recognised so far. The research aims to comprehensively evaluate microbiological and toxicological hazards in the air and settled dust in workstations in a sewage treatment plant. The number of microorganisms in the air and settled dust was determined using the culture method and the diversity was evaluated using high-throughput sequencing. Endotoxin concentration was assessed with GC-MS (gas chromatography-mass spectrometry) while secondary metabolites with LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry). Moreover, cytotoxicity of settled dust against a human lung epithelial lung cell line was determined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and UHPLC-Q-ToF-UHRMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight ultrahigh-resolution mass spectrometry) analysis was performed to determine the source of cytotoxicity. The total dust concentration in the sewage treatment plant was low and ranged from 0.030 mg m−3 to 0.044 mg m−3. The highest microbiological contamination was observed in sludge thickening building and screenings storage. Three secondary metabolites were detected in the air and sixteen in the settled dust. They were dominated by compounds typical of lichen and plants and Aspergillus, Penicillium and Fusarium genera mould. The settled dust from the sludge thickening building revealed high cytotoxicity to human lung epithelial cells A-549 (IC50 = 6.98 after 72 h). This effect can be attributed to a biocidal compound—didecyldimethylammonium chloride (DDAC-C10) and seven toxic compounds: 4-hydroxynonenal, carbofuran, cerulenin, diethylphosphate, fenpropimorph, naphthalene and onchidal. The presence of DDAC-C10 and other biocidal substances in the sewage treatment plant environment may bring negative results for biological sewage treatment and the natural environment in the future and contribute to microorganisms’ increasing antibiotics resistance. Therefore, the concentration of antibiotics, pesticides and disinfectants in sewage treatment plant workstations should be monitored.

Synthesis and investigation of in vitro cytotoxic activities and thermal stability of novel pyridine derivative platinum (II) complexes vis a vis DFT studies

Bis(2-amino-5-nitropyridine)dichloroplatinum(II), [PtCl2L12] and bis(3,4-dimethylpyridine)dichloroplatinum(II), [PtCl2L22] complexes were synthesized and characterized via FT-IR, UV–Vis, 1H NMR, 13C NMR spectrometers. Single crystal X-ray diffraction measurements were conducted to determine the crystal structure of the [PtCl2L22] complex. The [PtCl2L22] was crystallized in the monoclinic crystal system with P21/c space group. The crystal parameters were Z = 2, a = 9.5881(5) Å, b = 12.5863(7) Å and c = 13.5363(7) Å. Experimental data are supported by the theoretical calculations. LanL2DZ based DFT/B3LYP method was used determine the molecular structures of the complexes for their probable most stable status. Potential energy distribution analysis was made for to confer the vibrational transitions for the complexes. Molecular electrostatic potential maps, frontier molecular orbitals and Mulliken charge distribution were calculated and the active regions of the molecules were determined. In this study, for to investigate the interaction mechanisms of [PtCl2L12] and [PtCl2L22] complexes with colon cancer protein molecular docking study was performed. The thermal analyses of the complexes were carried out via DTA/TGA/TG combined system. The activation energies of the complexes were calculated to be 134.31–175.40 kJ/mol via Flynn-Wall-Ozawa (FWO) and 132.42–179.41 kJ/mol via Kissenger-Akahira-Sunose (KAS) methods. The cytotoxic effects of the complexes against the colon cancer cell line (DLD-1) were investigated. The complexes were found to be cytotoxic against DLD-1 colon cancer cell lines, A549 human lung cancer cells and Beas-2Bhealthy human lung epithelial cells. The results indicate that the [PtCl2L12] complex, in which amino and nitro groups are bound as ligand on the pyridine ring, have higher cytotoxic effect.

Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term “periodontal medicine” is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.