A549 Lung Epithelial Cells Research Articles

Influence of the polysaccharide capsule on virulence and fitness of Klebsiella pneumoniae

IntroductionThe capsular polysaccharide (CPS) of pathogenic bacteria is a critical virulence factor, often evading phagocytosis by host immune cells, while also interfering with the contact of the pathogen with host cells and contributing to biofilm formation. Klebsiella pneumoniae, a Gram-negative human pathogen associated with high antimicrobial resistances, produces 77 CPS serotypes. The CPS masks proteinaceous factors but also protects K. pneumoniae from uptake by host phagocytic cells and activation of the complement system. In addition to nosocomial, urinary tract and bloodstream infections or pneumonia hypervirulent strains have a highly mucoid phenotype and can cause soft tissue infections, liver abscesses, and meningitis as well. The CPS is therefore crucial for both escaping detection by the immune system and enhancing the virulence potential.MethodsIn this study, we generated a non-encapsulated mutant (Kpn2146∆wza) to observe how the CPS interferes with K. pneumoniae adhesion, survival in blood, and invasiveness in an experimental infection model.ResultsInfection of A549 lung epithelial cells showed similar adherence levels for the wild-type and non-capsulated strain, while our data showed a moderately higher internalization of Kpn2146Δwza when compared to the wild-type. In whole blood killing assays, we demonstrate that the K. pneumoniae capsule is essential for survival in human blood, protecting K. pneumoniae against recognition and clearance by the human immune system, as well as complement-mediated opsonization and killing. The non-encapsulated mutant, in contrast, was unable to survive in either whole blood or human plasma. Infections of Galleria mellonella larvae showed a significantly decreased virulence potential of the CPS-deficient mutant.DiscussionIn conclusion, our data indicate a crucial role of CPS in vivo.

P-1827. Novel Evaluation System for Aspergillus fumigatus Interactions with Human Cells via Combination of Imaging and Electrical Impedance

Abstract Background Aspergillus fumigatus is a major fungal pathogen causing pulmonary aspergillosis, an important disease associated with transplantation and immunosuppression. However, the interactions between A. fumigatus and human cells are not well understood. This study evaluated the interaction of A. fumigatus with THP-1 macrophages and A549 lung epithelial cells using fluorescence imaging and electrical impedance measurements to monitor fungal growth and cellular damage continuously.Figure 1.Schematic diagram of the relationship between electrical impedance and cell damage Methods The experimental setup used xCELLigence eSight (Agilent) and a specialized 96-well plate with electrodes at the bottom to measure impedance changes as cells adhered or detached (Figure 1). THP-1 and A549 cells were cultured, then infected with fluorescent A. fumigatus conidia. In addition, antifungal drugs (micafungin, caspofungin, voriconazole, isavuconazole, amphotericin B) were added to this experimental system at broth microdilution method and tested.Figure 2.Macrophage and A. fumigatus (MOI 4.0) in relation to each antifungal agentA:Voriconazole(VRCZ), B : Amphotericin B (AMPH-B) Results Results showed A. fumigatus filament growth leading to decreased impedance indicative of cell damage in both cell types, with faster damage at higher conidial loads. Fluorescence showed echinocandins inhibited hyphal growth but did not prevent impedance drops. Voriconazole and isavuconazole prevented impedance drops at clinically relevant concentrations (Figure 2A). Amphotericin B caused rapid impedance drops at high doses, suggesting direct cytotoxicity (Figure 2B). Conclusion A549 epithelial cells survived longer than macrophages, suggesting they play a defensive role against A. fumigatus. Impedance changes correlated with visual antifungal effects for azoles and amphotericin B but not echinocandins. This system enables evaluating fungal-host interactions using different cell types and fungi. Combining impedance with fluorescence imaging provides a comprehensive and objective assessment of antifungal activity and cellular responses. Disclosures Hiroshi Kakeya, MD, PhD, Asahikasei Phrma: Honoraria|GSK: Honoraria|MSD: Honoraria|Pfizer: Honoraria|Shionogi: Honoraria|Sumitomo Parma: Honoraria

Comparison of immune responses to respiratory syncytial virus in infancy, childhood, and adulthood using an in vitro model of human respiratory infection.

Respiratory syncytial virus (RSV) is a major contributor to morbidity and mortality in infants. We developed an in vitro model of human respiratory infection to study cellular immune responses to RSV in infants, children, and adults. The model includes human lung epithelial A549 cells or human fetal lung fibroblasts infected with a clinical strain of RSV at a multiplicity of infection of 0.3, cocultured with human cord blood mononuclear cells (CBMCs) or peripheral blood mononuclear cells (PBMCs). Mononuclear cells were collected at multiple ages ranging from birth to adulthood. After 20 h of incubation, flow cytometry was used to measure CBMC/PBMC responses to RSV. A549s were more permissive to RSV and when infected produced more CCL5, CCL11, and CXCL9; less CSF-3, CXCL10, interleukin (IL)-1α, IL-1RA, and IL-6; and similar CCL2, CCL3, CCL4, CCL7, CXCL1, CXCL11, IL-1β, IL-7, IL-8, and tumor necrosis factor α compared with fibroblasts; A594s were used for subsequent experiments. CBMCs/PBMCs upregulated multiple markers of activation, maturation, and degranulation upon exposure to RSV-infected A549s. Interferon γ expression in natural killer, CD4, and CD8 cells and CD107a expression in natural killer cells showed a gradual increase from infancy to adulthood. IL-12 expression in dendritic cells and monocytes was highest in adult PBMCs. Our in vitro model of human RSV infection recapitulated the expected bias away from T helper 1 and effector responses to RSV infection in infancy and revealed changes in innate and adaptive RSV-specific cellular immune responses over time.

TLR10 overexpression modulates immune response in A549 lung epithelial cells challenged with SARS-CoV-2 S and N proteins.

Toll-like receptors (TLRs) play an important role in the recognition of viral particles and activation of the innate immune system, but their role in SARS-CoV-2 infection is still poorly characterized. In the present study, we investigated the role of Toll-like receptor 10 (TLR10) in modulating the immune response during SARS-CoV-2 infection. The results showed that overexpression of TLR10 in A549 lung epithelial cells, immunostimulated with SARS-CoV-2 proteins S and N mainly downregulated proinflammatory cytokines and interferons and affected gene expression in the cocultured THP-1 monocytes. Our results suggest that TLR10 could mediate the extent of SARS-CoV-2 infection by downregulating the release of inflammatory cytokines and chemokines such as CXCL10, IL6, IL8, and IFNβ. Modulation of TLR10 expression could have implications for the treatment of patients with severe COVID-19, in whom excessive inflammation leading to the development of acute respiratory distress syndrome (ARDS) is a key feature. However, further research is needed to fully understand the impact of modulating TLR10 expression on the antiviral response and the overall balance of the immune response during SARS-CoV-2 infection.

PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

Idiopathic pulmonary fibrosis (IPF) is an irreversible lung condition that progresses over time, which ultimately results in respiratory failure and mortality. In this study, we found that PLAC8 was downregulated in the lungs of IPF patients based on GEO data, in bleomycin (BLM)-induced lungs of mice, and in primary murine alveolar epithelial type II (pmATII) cells and human lung epithelial cell A549 cells. Overexpression of PLAC8 facilitated autophagy and inhibited apoptosis of pmATII cells and A549 cells in vitro. Moreover, inhibition of autophagy or overexpression of p53 partially abolished the effects of PLAC8 on cell apoptosis. ATII cell-specific overexpression of PLAC8 alleviated BLM-induced pulmonary fibrosis in mice. Mechanistically, PLAC8 interacts with VCP-UFD1-NPLOC4 complex to promote p53 degradation and facilitate autophagy, resulting in inhibiting apoptosis of alveolar epithelial cells and attenuating pulmonary fibrosis. In summary, these findings indicate that PLAC8 may be a key target for therapeutic interventions in pulmonary fibrosis.

Design, synthesis, biological evaluation and multi spectroscopic studies of novel 2-styrylquinoline-carboxamide derivatives as potential DNA intercalating anticancer agents.

In this study, novel 2-styrylquinoline derivatives possessing a planar aromatic system and a flexible side chain with an amino substituent were designed and synthesized as DNA-intercalating antitumor agents. The cytotoxic activity of the synthesized compounds was evaluated against four cancer cell lines including MCF-7 (breast cancer cells), A549 (lung epithelial cancer cells), HCT116 (colon cancer cells) and normal cell line L929 (mouse fibroblast cell line). The results displayed that the anti-cancer activity of the target quinolines is sensitive to the lipophilic nature of the C-6 and C-7 quinoline substituents. The anticancer activity of most of the target quinolines against MCF-7 and A549 cells was more than those of HCT116. Compound 3h possessing two methyl groups at the C-6 and C-7 of quinoline ring displayed the most cytotoxicity with IC50 value of 5.7µM against A549 cancer cells. Interaction of compound 3h with calf thymus DNA (ctDNA) was investigated by means of UV absorption spectrophotometry, fluorescence spectroscopy, circular dichroism (CD), Resonance light scattering (RLS), viscos metric techniques and also by docking and molecular dynamic studies. RLS intensity, fluorescence quenching of ctDNA and fluorescence quenching EtBr-ctDNA and AO-ctDNA complexes augmented with increasing of compound 3h concentration, significant increasing in viscosity and melting point of ctDNA in the presence of compound 3h, absorbance increasing of ctDNA-compound 3h complex by increasing of NaCl and KI concentrations, higher Ksv value for dsctDNA (3.03×104 M-1) compared to ssctDNA (1.31×104 M-1), circular dichroism (CD) studies, docking and molecular dynamic studies revealed that compound 3h can interact with ctDNA through intercalation into DNA.

An improvised one-step OptiPrep cushion ultracentrifugation method for outer membrane vesicles isolation of Klebsiella pneumoniae

BackgroundExtracellular vesicles (EVs) play a crucial role in intraspecies and interspecies communication, significantly influencing physiological and pathological processes. Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria are rich in components from the parent cells and are important for bacterial communication, immune evasion, and pathogenic mechanisms. However, the extraction and purification of OMVs face numerous challenges due to their small size and heterogeneity.ResultsThis study proposes an innovative strategy that combines traditional differential centrifugation (DC) with one-step ultracentrifugation (ODG) to develop a dual differential gradient centrifugation (DDGC) method for extracting outer membrane vesicles from Klebsiella pneumoniae. By comparing the DC and DDGC extraction methods, we found that OMVs extracted by DDGC exhibited more typical morphology, clearer backgrounds, and more uniform particle size distribution. The lipid polysaccharide (LPS) content in OMVs extracted by DDGC was significantly higher than that obtained by DC, and the outer membrane protein content was also greater, demonstrating enhanced biological activity. Biological activity assays indicated that OMVs extracted by DDGC showed stronger cytotoxicity to A549 lung epithelial cells, a significant decrease in cell viability, and higher levels of inflammatory factor expression(IL-6, TNF-α, IL-1β, and IL-8).ConclusionOur study demonstrates the advantages of the DDGC method in extracting K. pneumoniae OMVs, showing improvements in morphology, particle size distribution, protein content, and biological activity. This provides a solid foundation for further exploration of the biological functions of OMVs and their potential applications in the biomedical field.

Synthesis and In Vitro Anticancer Activity of Pyrrolidone Derivatives Bearing 3,4,5-Trimethoxyphenyl Moiety as a Promising Anticancer Scaffold

A series of 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives–hydrazones, N-ethylhydrazones, pyrrole, pyrazole, oxadiazole, and triazole were synthesized and evaluated for their anticancer activity using human A549 pulmonary epithelial cells (ATCC CCl-185). The in vitro viability inhibitory effects of the compounds were assessed using the MTT assay. The characterization of the anticancer activity of the synthesized compounds showed that the incorporation of 1,3,4-oxadiazolethione and 4-aminotriazolethione rings into the molecular structures obviously enhances the anticancer activity against human A549 lung epithelial cells, reducing their viability to 28.0% and 29.6%, respectively. The anticancer activity of these azole derivatives was significantly higher than that of cytarabine. Further studies are needed to better optimize 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives and enhance their in vitro anticancer activity.

Toward Standardization of a Lung New Approach Model for Toxicity Testing of Nanomaterials.

This study represents an attempt toward the standardization of pulmonary NAMs and the development of a novel approach for toxicity testing of nanomaterials. Laboratory comparisons are challenging yet essential for identifying existing limitations and proposing potential solutions. Lung cells cultivated and exposed at the air-liquid interface (ALI) more accurately represent the physiology of human lungs and pulmonary exposure scenarios than submerged cell and exposure models. A triculture cell model system was used, consisting of human A549 lung epithelial cells and differentiated THP-1 macrophages on the apical side, with EA.hy926 endothelial cells on the basolateral side. The cells were exposed to silver nanoparticles NM-300K for 24 h. The model used here showed to be applicable for assessing the hazards of nanomaterials and chemicals, albeit with some limitations. Cellular viability was measured using the alamarBlue assay, DNA damage was assessed with the enzyme-modified comet assay, and the expression of 40 genes related to cell viability, inflammation, and DNA damage response was evaluated through RT2 gene expression profiling. Despite harmonized protocols used in the two independent laboratories, however, some methodological challenges could affect the results, including sensitivity and reproducibility of the model.

The polysaccharide from purple sweet potato (Ipomoea batatas (L.) Lam) alleviates lipopolysaccharide-induced acute lung injury in mice via the VIP/cAMP/PKA/AQPs signaling pathway

BackgroundThe polysaccharide (PSP) from purple sweet potato has great potential for regulating apoptosis, but its regulatory role in acute lung injury (ALI) is unknown. MethodsThe objective of this study was to investigate the protective effect of PSP on lipopolysaccharide (LPS)-induced ALI in mice and lung epithelial A549 cells and its mechanism. Moreover, subacute toxicity evaluation of PSP was carried out on ICR mice. ResultsThe results showed that compared with the ALI group, PSP significantly reduced the total protein content, wet-to-dry (W/D) ratio, the number of neutrophils, lymphocytes, and monocytes. Moreover, PSP was able to reduce cell apoptosis, the levels of macrophage inflammatory protein-2 (MIP-2), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA) and myeloperoxidase (MPO) and increase the level of superoxide (SOD). In addition, PSP could up-regulate the levels of VIP, cAMP, p-PKA/PKA and AQP1 in mice and A549 cells. And PSP exhibited no apparent adverse effects on the mice. ConclusionsPSP had a protective effect on LPS-induced ALI in mice and lung epithelial A549 cells, which may be related to the inflammatory response and via VIP/cAMP/PKA/AQPs signaling pathway. Thus, PSP may be a promising pharmacologic agent for ALI therapy.

Mutational landscape induced by chronic exposure to environmental PM10 and PM2.5 in A549 lung epithelial cell.

Exposure to particulate matter (PM) has been linked to an increased risk of multiple diseases, primarily lung cancer, through various molecular mechanisms. However, the mutagenic potential of PM remains unclear. This study aimed to provide a comprehensive description of genetic mutations and mutagenic signatures resulting from chronic exposure to PM10 or PM2.5. Using whole exome sequencing, we identified driver mutations and mutational signatures in A549cells, a lung epithelial cell model subjected to weekly exposure to either PM10 or PM2.5, for a period of 28 weeks. The number of single nucleotide variations, insertions, and deletions increased depending on the duration of exposure. PM10 generated the highest number of genomic alterations. Amplifications in SYK (oncogene) and mutations in NCOR1 (tumor suppressor gene) were prevalent in cells exposed to either PM10 or PM2.5; however, other mutations were exclusive, such as TP53 and ANK3 for PM10, and ERCC1 and ERCC2 for PM2.5. Different p53-related signaling pathways were most enriched by driver mutations upon exposure to both PM10 and PM2.5, particularly the glucose deprivation pathway. Exposure to either PM10 or PM2.5 resulted in high frequencies of C>A substitutions and one-base insertions/deletions in microhomology sites. The single-base substitution (SBS) signature SBS05, related to the nucleotide excision DNA repair pathway, contributed the most to both PM10-and PM2.5-exposed cells. The contribution of signature SBS18, related to oxidative stress, was observed in cells exposed to either PM10 or PM2.5, but a greater contribution was observed in PM2.5-exposed cells. In addition, SBS03 and SBS36, which are related to different DNA damage repair mechanisms, were observed more frequently in PM10-exposed cells. We assessed the mutagenic potential of PM10 and PM2.5, as a complete mixture, identifying mutated driver genes and mutational signatures generated by chronic PM exposure, which could contribute to the development of cancer, cardiovascular, and digestive diseases.

Modulating ferroptosis and mycobactericidal activity in lung epithelial cells via YY1/iNOS pathway

BackgroundMycobacterium tuberculosis infection triggers various forms of host cell death, including ferroptosis in lung epithelial cells; YY1, a critical transcription factor, plays a pivotal role in regulating ferroptosis, however, the underlying mechanisms are not fully understood. MethodsTo investigate Mycobacterium marinum (M.marinum) infection in lung epithelial cells A549 and H1299, we utilized flow cytometry to evaluate cell death and measure reactive oxygen species (ROS). Colony-forming unit (CFU) assays determined the intracellular bacterial load. Ferroptosis was analyzed using a specific detection kit to measure malondialdehyde (MDA) and glutathione (GSH) levels. The interaction between the transcription factor YY1 and the iNOS promoter was assessed through a dual-luciferase reporter assay. ResultsM.marinum induced ferroptosis in lung epithelial cells through invasion. This effect is most pronounced at 8 h of infection and decreases over time but increased with a higher multiplicity of infection (MOI). YY1 knockdown decreases the expression of SLC7A11 and GPX4, attenuates cellular ferroptosis, while YY1 overexpression has the opposite phenomenon, enhancing the expression of bactericidal molecules such as iNOS and MPEG1, thereby markedly reducing the intracellular bacterial load. We identified substantial binding of YY1 to the iNOS promoter region (−655 to −1018 bp), enhancing mycobactericidal activity in YY1-overexpressing cells. ConclusionsOur study demonstrates that YY1 inhibits ferroptosis induced by Mycobacterium marinum infection and reduces intracellular bacterial proliferation in lung epithelial cells. These findings provide a crucial basis for developing anti-tuberculosis therapies that target YY1 modulation, potentially offering new clinical avenues for the treatment of tuberculosis.

Survival and virulence of Acinetobacter baumannii in microbial mixtures

Acinetobacter species such as A. venetianus and A. guillouiae have been studied for various biotechnology applications, including bioremediation of recalcitrant and harmful environmental contaminants, as well as bioengineering of enzymes and diagnostic materials. Bacteria used in biotechnology are often combined with other microorganisms in mixtures to formulate efficacious commercial products. However, if the mixture contained a closely related Acinetobacter pathogen such as A. baumannii (Ab), it remains unclear whether the survival and virulence of Ab would be masked or augmented. This uncertainty poses a challenge in ensuring the safety of such biotechnology products, since Ab is one of the most significant pathogens for both hospital and community -acquired infections. This research aimed to investigate the growth and virulence of Ab within a mixture of 11 bacterial species formulated as a mock microbial mixture (MM). Growth challenges with environmental stressors (i.e., temperature, pH, sodium, iron, and antibiotics) revealed that Ab could thrive under diverse conditions except in the presence of ciprofloxacin. When cultured alone, Ab exhibited significantly more growth in the presence of almost all the environmental stressors than when it was co-incubated with the MM. During the exposure of A549 lung epithelial cells to the MM, Ab growth was stimulated compared to that in standard mammalian culture media. Cytotoxicity caused by Ab was suppressed in the presence of the MM. Lymphocytes were significantly reduced in mice exposed to Ab with or without MM via intravenous injection. The levels of the splenic cytokines IL-1α, IL-1β, MCP-1, and MIP-1α were significantly reduced 24 h after exposure to Ab + MM. This study demonstrated that the presence of the MM marginally but significantly reduced the growth and virulence of Ab, which has implications for the safety of mixtures of microorganisms for biotechnological applications. Furthermore, these findings expand our understanding of the virulence of Ab during host–pathogen interactions.

MDM2 Is Essential to Maintain the Homeostasis of Epithelial Cells by Targeting p53

Introduction: MDM2 is known as the primary negative regulator of p53, and MDM2 promotes lung cancer fibrosis and lung injury through p53-dependent and p53-independent pathways. However, the mechanism by which MDM2 influences the pathogenesis of asthma is unknown. In this study, we investigated the function of MDM2 in lung epithelial cells in type 2 lung inflammation. Methods: We used type II alveolar epithelial cell-specific heterozygous knockout of Mdm2 mice to validate its function. Then papain-induced asthma model was established, and changes in inflammation were observed by measuring immunohistochemistry and flow cytometry analysis. Results: In this study, we knockdown the mouse Mdm2 gene in type 2 alveolar epithelial cells. We demonstrated that heterozygous Mdm2 gene-deleted mice were highly susceptible to protease allergen papain-induced pulmonary inflammation characterized by increased ILC2 numbers, IL-5 and IL-13 cytokine levels, and lung pathology. A mechanistic study showed that following the decreased expression of Mdm2 in lung epithelial cells and A549 cell line, p53 was overactivated, and the expression of its downstream genes p21, Puma, and Noxa was elevated, which resulted in apoptosis. After Mdm2 knockdown, the mRNA expression of inflammation-related gene IL-25, HMGB1, and TNF-α were increased, which further amplified the downstream ILC2 response and lung inflammation. Conclusion: These results indicate that Mdm2 maintains the homeostasis of lung epithelial cells by targeting P53 and regulates the function of lung epithelial cells under type 2 lung inflammation.

IL-1β-activated PI3K/AKT and MEK/ERK pathways coordinately promote induction of partial epithelial–mesenchymal transition

Epithelial–mesenchymal transition (EMT) is a cellular process in embryonic development, wound healing, organ fibrosis, and cancer metastasis. Previously, we and others have reported that proinflammatory cytokine interleukin-1β (IL-1β) induces EMT. However, the exact mechanisms, especially the signal transduction pathways, underlying IL-1β-mediated EMT are not yet completely understood. Here, we found that IL-1β stimulation leads to the partial EMT-like phenotype in human lung epithelial A549 cells, including the gain of mesenchymal marker (vimentin) and high migratory potential, without the complete loss of epithelial marker (E-cadherin). IL-1β-mediated partial EMT induction was repressed by PI3K inhibitor LY294002, indicating that the PI3K/AKT pathway plays a significant role in the induction. In addition, ERK1/2 inhibitor FR180204 markedly inhibited the IL-1β-mediated partial EMT induction, demonstrating that the MEK/ERK pathway was also involved in the induction. Furthermore, we found that the activation of the PI3K/AKT and MEK/ERK pathways occurred downstream of the epidermal growth factor receptor (EGFR) pathway and the IL-1 receptor (IL-1R) pathway, respectively. Our findings suggest that the PI3K/AKT and MEK/ERK pathways coordinately promote the IL-1β-mediated partial EMT induction. The inhibition of not one but both pathways is expected yield clinical benefits by preventing partial EMT-related disorders such as organ fibrosis and cancer metastasis.

Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10.

Antiviral signaling, immune response and cell metabolism are dysregulated by SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical roles in mitochondrial function and morphology. On the other hand, all four ORFs altered mitochondrial dynamics and function, but only ORF3a and ORF9c induced a marked alteration in mitochondrial cristae structure. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features both shared across all accessory proteins and specific for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

Investigating the Effectiveness of Ceragenins against Acinetobacter baumannii to Develop New Antimicrobial and Anti-Adhesive Strategies.

A growing body of experimental data indicates that ceragenins (CSAs), which mimic the physicochemical properties of the host's cationic antimicrobial peptide, hold promise for the development of a new group of broad-spectrum antimicrobials. Here, using a set of in vivo experiments, we assessed the potential of ceragenins in the eradication of an important etiological agent of nosocomial infections, Acinetobacter baumannii. Assessment of the bactericidal effect of ceragenins CSA-13, CSA-44, and CSA-131 on clinical isolates of A. baumannii (n = 65) and their effectiveness against bacterial cells embedded in the biofilm matrix after biofilm growth on abiotic surfaces showed a strong bactericidal effect of the tested molecules regardless of bacterial growth pattern. AFM assessment of bacterial cell topography, bacterial cell stiffness, and adhesion showed significant membrane breakdown and rheological changes, indicating the ability of ceragenins to target surface structures of A. baumannii cells. In the cell culture of A549 lung epithelial cells, ceragenin CSA-13 had the ability to inhibit bacterial adhesion to host cells, suggesting that it interferes with the mechanism of bacterial cell invasion. These findings highlight the potential of ceragenins as therapeutic agents in the development of antimicrobial strategies against bacterial infections caused by A. baumannii.

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.

Betacyanins from red pitahaya (Hylocereus polyrhizus) exhibit antiviral response against influenza A virus

Seasonal influenza affects millions of lives worldwide, with the influenza A virus (IAV) responsible for pandemics and annual epidemics, causing the most severe illnesses resulting in patient hospitalizations or death. With IAV threatening the next global influenza pandemic, it is a race against time to search for antiviral drugs. Betacyanins are unique nitrogen-containing and water-soluble reddish-violet pigments that have been reported to possess antiviral properties against the dengue virus. This study aimed to examine the antiviral effect of betacyanins from red pitahaya (Hylocereus polyrhizus) on IAV-infected lung epithelial A549 cells. HPLC and LC-MS analysis of extracted betacyanin showed four betacyanins in the betacyanin fraction: phyllocactin, hylocerenin, betanin, and isobetanin. Cytotoxicity assay showed that betacyanin fractions were not cytotoxic to A549 cells at concentrations below 100 μg/mL. Betacyanin fraction concentrations of 12.5, 25.0, and 50.0 μg/mL prevented the formation of viral cytopathic effect and reduced virus titer in IAV-infected cells up to 72 h. A downregulation of protein and mRNA nucleoprotein expression levels was observed after treatment with 25.0 and 50.0 μg/mL of betacyanin fraction after 24 h, thereby providing evidence for the antiviral activity of betacyanin from red pitahaya against IAV in vitro.

Advancing Nanomaterial Toxicology Screening Through Efficient and Cost-Effective Quantitative Proteomics.

Proteomic investigations yield high-dimensional datasets, yet their application to large-scale toxicological assessments is hindered by reproducibility challenges due to fluctuating measurement conditions. To address these limitations, this study introduces an advanced tandem mass tag (TMT) labeling protocol. Although labeling approaches shorten data acquisition time by multiplexing samples compared to traditional label-free quantification (LFQ) methods in general, the associated costs may surge significantly with large sample sets, for example, in toxicological screenings. However, the introduced advanced protocol offers an efficient, cost-effective alternative, reducing TMT reagent usage (by a factor of ten) and requiring minimal biological material (1µg), while demonstrating increased reproducibility compared to LFQ. To demonstrate its effectiveness, the advanced protocol is employed to assess the toxicity of nine benchmark nanomaterials (NMs) on A549 lung epithelial cells. While LFQ measurements identify 3300 proteins, they proved inadequate to reveal NM toxicity. Conversely, despite detecting 2600 proteins, the TMT protocol demonstrates superior sensitivity by uncovering alterations induced by NM treatment. In contrast to previous studies, the introduced advanced protocol allows simultaneous and straightforward assessment of multiple test substances, enabling prioritization, ranking, and grouping for hazard evaluation. Additionally, it fosters the development of New Approach Methodologies (NAMs), contributing to innovative methodologies in toxicological research.