Search Results (42)

Background/Objectives: The BRG1 loss-of-function (LOF) mutation is found in ~10% of non-small cell lung cancer (NSCLC) cases, but its role in lung tumorigenesis is unclear and so it is investigated in this study. Methods: BRG1-knockout (KO) lines were generated from various non-malignant, pre-malignant, and malignant human lung epithelium-derived cell lines using CRISPR. The effects of BRG1-KO on cell growth, the transcriptome, the methylome, and epigenetic therapy were compared with those of wild-type (BRG1-WT) isogenic controls using standard in vitro and in vivo assays. Results: The BRG1 protein was expressed in all non-/pre-malignant lung epithelial cells but lost in 47% (14/30) of NSCLC cell lines. BRG1-KO and cigarette smoke (CS) exposure individually transformed human bronchial epithelial cell lines (HBECs), as evidenced by anchorage-independent growth. BRG1-KO and CS produced additive to synergistic effects on sensitivity to transformation that differed across HBECs. RNA-seq analysis revealed that BRG1-KO significantly changed the expression of over 8500 genes on average, impacting lung development, function, damage repair, and cancer pathways, including axonal guidance, pulmonary wound healing, and epithelial-to-mesenchymal transition (EMT). BRG1-KO also led to the hypermethylation of >47,000 promoter CpGs within ~9500 genes on average in different HBECs, including silencing of epithelial genes involved in EMT and tumor suppressor genes. BRG1-KO also moderately increased the in vitro and in vivo sensitivity of NSCLC cells to some epigenetic drugs. Conclusions: BRG1-LOF is frequent in NSCLC; can drive the transformation of lung epithelial cells such that they acquire properties of pre-malignant cells, indicating a potential role in lung cancer initiation; and sensitizes lung tumors to epigenetic therapy. Full article

Background: The proportion of people suffering from neurodegenerative conditions, such as Alzheimer’s disease (AD), is increasing in the population year on year. Despite the constant effort of researchers, these conditions remain incurable and can only be managed by alleviation or delaying of symptoms. The lack of suitable treatment is caused by constricted access to the brain, limited by the brain-blood barrier. The aim of this work was to investigate two pegylated gold nanoparticles as potential carriers of therapeutic siRNA and their impact on the cellular functions of Human Brain Endothelial Cells. Methods and Results: Nanoparticles AuNP14a and AuNP14b complexed with siRNA were internalized by HBEC-5i cells and located in the cytoplasm. The genotoxicity assay proved that the nucleus was not affected and complexed nanoparticles did not cause DNA damage. The reactive oxygen species formation and mitochondrial membrane potential changes were measured and showed an adaptive response of cells after compound administration. Results obtained in a cytotoxicity assay conducted on astrocytes and pericytes, which are components of the blood–brain barrier, confirmed the biosafety of tested nanoparticles. Conclusions: In summary, it was shown that AuNP14a and AuNP14b are promising candidates as nanocarriers for therapeutic nucleic acids through biological barriers. Full article

Background: Cell-penetrating peptides cross cell membrane barriers while carrying cargoes in a functional form. Our work identified two novel lung-targeting peptides, S7A and R11A. Here, we present studies on biodistribution, the cell types targeted, and an in vitro proof of application. Methods: Studies were performed in human bronchial epithelial cells (HBECs) with and without various endocytic inhibitors, and coincubation with fluorescently labeled transferrin or endocytic markers. Cyclic R11A (cR11A) was conjugated to siRNA duplexes and anti-viral activity against SARS-CoV-2 was tested. Biodistribution studies were performed by injecting wild-type mice with fluorescently labeled peptides, and various circulation times were allowed for, as well as cross-staining of lung sections or isolated single cells with various cellular markers, followed by fluorescence-activated cell sorting or confocal microscopy. Results: cR11A showed peak uptake in 15 min, with the highest uptake in airway epithelial type II (ATII) cells, followed by p63+ basal cells and ionocytes. Cyclization increased transduction efficiencies ~100-fold. Endocytosis studies showed a decrease in peptide uptake by pre-treatment with Pitstop2 but not Amiloride or Nystatin. Endocytic marker Lamp1 showed colocalization at the earliest time point, with the escape of the peptide from endocytic vesicles later. cR11A conjugated to ant-spike and anti-envelop proteins showed anti-viral effects with an EC₉₀ of 0.6 μM and 1.0 µM, respectively. Conclusions: We have identified a novel peptide, cR11A, that targets ATII, basal cells, and ionocytes, the cyclization of which increased transduction efficiency in vitro and in vivo. The uptake mechanism appears to be via clathrin-mediated endocytosis with escape from endocytic vesicles. cR11A can act as a vector to deliver anti-viral siRNA to epithelial cells. Full article

Neonatal Meningitis-causing Escherichia coli (NMEC) is the leading cause of neonatal meningitis and exhibits remarkable adaptability to diverse host environments. Understanding its transcriptional responses across different host niches is crucial for deciphering pathogenesis and identifying potential therapeutic targets. We performed a comparative transcriptomic analysis of NMEC RS218, the prototype strain of NMEC, under four distinct host-mimicking conditions: colonic fluid (CF), serum (S), human brain endothelial cells (HBECs), and cerebrospinal fluid (CSF). Differential gene expression analysis was conducted to assess metabolic shifts, virulence factor regulation, and niche-specific adaptation strategies, in which RS218 demonstrated niche-specific transcriptional reprogramming. In CF, genes associated with biofilm formation, motility, efflux pumps, and cell division regulation were upregulated, aiding gut colonization. The serum environment triggered the expression of siderophore-mediated iron acquisition, enterobactin biosynthesis, and heme utilization genes, facilitating immune evasion and bacterial persistence. In HBECs, NMEC upregulated genes linked to nucleoside metabolism, membrane remodeling, pilus organization, and blood–brain barrier (BBB) traversal. In CSF, genes related to oxidative stress resistance, chemotaxis, DNA repair, biofilm formation, and amino acid biosynthesis were enriched, reflecting NMEC’s adaptive mechanisms for survival under nutrient-depleted conditions. Energy-intensive pathways were consistently downregulated across all niches, highlighting the need for an energy conservation strategy. This study provides novel insights into NMEC’s adaptive strategies across different host environments, emphasizing its metabolic flexibility, virulence regulation, and immune evasion mechanisms, offering potential targets for therapeutic intervention. Full article

Background: Hypertension is a major risk factor for cerebrovascular diseases due to its damaging effects on the blood–brain barrier (BBB) and associated pathologies. Oxidative stress-induced endothelial damage plays a critical role in BBB disruption, potentially leading to cognitive impairment and neurodegeneration. In this study, we investigated the protective effects of two essential trace elements, zinc (Zn) and selenium (Se), against oxidative stress in human brain endothelial cells (HBCE5i) exposed to hypertensive shear stress. Using an innovative millifluidic system (LiveBox2), which allows for the precise simulation of continuous flow conditions, we replicated the hemodynamic forces associated with hypertension. Methods: Cells were treated with ZnCl₂ (5–50 µM) or Na₂SeO₃ (50–500 nM) at concentrations selected based on previous studies and confirmed by cytotoxicity assays. Results: Our results demonstrated that shear stress significantly altered the localization of the tight junction protein zonula occludens-1 (ZO-1) and induced the nuclear translocation of the transcription factor NRF2, a hallmark of oxidative stress. Importantly, treatment with 10 µM ZnCl₂ preserved ZO-1 membrane localization and prevented NRF2 translocation, as confirmed by quantitative image analysis. In contrast, Na₂SeO₃ did not provide comparable protection, although modest improvements in ZO-1 localization were observed in some replicates. Discussion: We discuss potential reasons for selenium’s limited efficacy, including differences in bioavailability and cellular uptake. Our findings underscore zinc’s promising role as a neurovascular protector and suggest that further investigation into more complex in vitro models and in vivo studies is warranted. Full article

Background and Objectives: The rising popularity of new-generation electronic cigarettes (e-cig) like JUUL necessitates a better understanding of their impact on respiratory and other body systems, as the effects of JUUL’s components remain unclear. This study aimed to investigate the effects of JUUL components on ion channels and airway surface liquid (ASL) height in human bronchial epithelial cells (HBECs). Furthermore, the cytotoxic effects of these components were investigated in human embryonic kidney 293T (HEK293T) cells. Materials and Methods: The components tested included nicotine salt (NicSalt), benzoic acid (BA), sodium hydrogen tartrate (NaTar), propylene glycol/vegetable glycerin (PG/VG), freebase nicotine (FBNic) and nicotine salt+benzoic acid (NicSalt+BA). Each component was prepared at 100 µM, and HBECs were exposed for 24 h to measure ASL height, short-circuit current (I_sc), and transepithelial electrical resistance (TEER). Results: Initial exposure (0 h) to these substances did not significantly alter ASL height. However, after 2 h, FBNic-treated HBECs exhibited a significant reduction in ASL height compared to NicSalt and other tested substances, with the most pronounced decrease observed at the 6th hour. This effect persisted over prolonged exposure, suggesting a cumulative impact on airway hydration and epithelial function. Additionally, adenosine administration did not induce a significant increase in ASL height. NicSalt, BA, and FBNic were found to disrupt ion balance in HBECs, affecting ion channels and ASL homeostasis while significantly decreasing TEER. In terms of cytotoxicity, NicSalt, and benzoic acid demonstrated minimal cytotoxicity at low concentrations, whereas FBNic showed significantly higher cytotoxicity at moderate levels. Conclusions: In conclusion, this study highlights that e-cigarette components can disrupt airway surface liquid homeostasis by affecting ion channel activity, compromise epithelial barrier integrity by reducing transepithelial electrical resistance, and emphasize the importance of their cytotoxic effects. Full article

Microfluidic systems offer controlled microenvironments for cell-to-cell and cell-to-stroma interactions, which have precise physiological, biochemical, and mechanical features. The optimization of their conditions to best resemble tumor microenvironments constitutes an experimental modeling challenge, particularly regarding carcinogenesis in the central nervous system (CNS), given the specific features of the blood–brain barrier (BBB). Gel-free 3D microfluidic cell culture systems (gel-free 3D-mFCCSs), including features such as self-production of extracellular matrices, provide significant benefits, including promoting cell–cell communication, interaction, and cell polarity. The proposed microfluidic system consisted of a gel-free culture device inoculated with human brain microvascular endothelial cells (HBEC5i), glioblastoma multiforme cells (U87MG), and astrocytes (ScienCell 1800). The gel-free 3D-mFCCS showed a diffusion coefficient of 4.06 × 10⁻⁹ m²·s⁻¹, and it reconstructed several features and functional properties that occur at the BBB, such as the vasculogenic ability of HBEC5i and the high duplication rate of U87MG. The optimized conditions of the gel-free 3D-mFCCS allowed for the determination of cellular proliferation, invasion, and migration, with evidence of both physical and biochemical cellular interactions, as well as the production of pro-inflammatory cytokines. In conclusion, the proposed gel-free 3D-mFCCSs represent a versatile and suitable alternative to microfluidic systems, replicating several features that occur within tumor microenvironments in the CNS. This research contributes to the characterization of microfluidic approaches and could lead to a better understanding of tumor biology and the eventual development of personalized therapies. Full article

This review delves into the molecular complexities underpinning the epithelial-to-mesenchymal transition (EMT) induced by cigarette smoke (CS) in human bronchial epithelial cells (HBECs). The complex interplay of pathways, including those related to WNT//β-catenin, TGF-β/SMAD, hypoxia, oxidative stress, PI3K/Akt, and NF-κB, plays a central role in mediating this transition. While these findings significantly broaden our understanding of CS-induced EMT, the research reviewed herein leans heavily on 2D cell cultures, highlighting a research gap. Furthermore, the review identifies a stark omission of genetic and epigenetic factors in recent studies. Despite these shortcomings, the findings furnish a consolidated foundation not only for the academic community but also for the broader scientific and industrial sectors, including large tobacco companies and manufacturers of related products, both highlighting areas of current understanding and identifying areas for deeper exploration. The synthesis herein aims to propel further research, hoping to unravel the complexities of the EMT in the context of CS exposure. This review not only expands our understanding of CS-induced EMT but also reveals critical limitations in current methodologies, primarily the reliance on 2D cell cultures, which may not adequately simulate more complex biological interactions. Additionally, it highlights a significant gap in the literature concerning the genetic and epigenetic factors involved in CS-induced EMT, suggesting an urgent need for comprehensive studies that incorporate these types of experiments. Full article

This study reports for the first time the isolation of four diterpenoid compounds: 15-Hydroxy-12-oxo-abietic acid (1), 12α-hydroxyabietic acid (2), (−)-Jolkinolide E (3), and 15-Hydroxydehydroabietic acid (4) from Clinopodium bolivianum (C. bolivianum). The findings demonstrate that both the dichloromethane/methanol (DCMECB) extract of C. bolivianum and the isolated compounds exhibit significant anti-inflammatory (inhibition of NF-κB activation), antibacterial (primarily against Gram-positive bacteria), and anti-biofilm (primarily against Gram-negative bacteria) activities. Among the isolated diterpenes, compounds 3 and 4 showed notable anti-inflammatory effects, with IC₅₀ values of 17.98 μM and 23.96 μM for compound 3, and 10.79 μM and 17.37 μM for compound 4, in the HBEC3-KT and MRC-5 cell lines. Regarding their antibacterial activity, compounds 3 and 4 were particularly effective, with MIC values of 0.53–1.09 μM and 2.06–4.06 μM, respectively, against the S. pneumoniae and S. aureus Gram-positive bacteria. Additionally, these compounds demonstrated significant anti-biofilm and anti-quorum sensing activities, especially against Gram-negative bacteria (H. influenzae and L. pneumophila). We also explain how compound 3 (BIC = 1.50–2.07 μM, Anti-QS = 0.31–0.64 μM) interferes with quorum sensing due to its structural homology with AHLs, while compound 4 (BIC = 4.65–7.15 μM, Anti-QS = 1.21–2.39 μM) destabilises bacterial membranes due to the presence and position of its hydroxyl groups. These results support the traditional use of C. bolivianum against respiratory infections caused by both Gram-positive and Gram-negative bacteria. Furthermore, given the increasing antibiotic resistance and biofilm formation by these bacteria, there is a pressing need for the development of new, more active compounds. In this context, compounds 3 and 4 isolated from C. bolivianum offer promising potential for the development of a library of new, more potent, and selective drugs. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to raise concerns worldwide. Numerous host factors involved in SARS-CoV-2 infection have been identified, but the regulatory mechanisms of these host factor remain unclear. Here, we report the role of G-quadruplexes (G4s) located in the host factor promoter region in SARS-CoV-2 infection. Using bioinformatics, biochemical, and biological assays, we provide evidence for the presence of G4 structures in the promoter regions of SARS-CoV-2 host factors NRP1. Specifically, we focus on two representative G4s in the NRP1 promoter and highlight its importance in SARS-CoV-2 pathogenesis. The presence of the G4 structure greatly increases NRP1 expression, facilitating SARS-CoV-2 entry into cells. Utilizing published single-cell RNA sequencing data obtained from simulated SARS-CoV-2 infection in human bronchial epithelial cells (HBECs), we found that ciliated cells with high levels of NRP1 are prominently targeted by the virus during infection. Furthermore, our study identifies E2F1 act as a transcription factor that binds to G4s. These findings uncover a previously unknown mechanism underlying SARS-CoV-2 infection and suggest that targeting G4 structures could be a potential strategy for COVID-19 prevention and treatment. Full article

Recent evidence indicates that the SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of the SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVECs), coronary artery endothelial (HCAECs), and bronchial epithelial cells (HBECs). Live Mito Orange staining, STED microscopy, and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry; an Agilent XFp analyser for mitochondrial/glycolytic activity; MitoSOX fluorescence for mitochondrial ROS; and qRT-PCR plus Luminex for cytokines. HLMVEC exposure to SARS-CoV-2 RBD resulted in the fragmentation of the mitochondrial network, mitochondrial swelling, increased cristae area, reduced cristae density, and suppressed mitochondrial oxygen consumption and glycolysis. No significant mitochondrial morphology or oxygen consumption changes were observed in HCAECs and HBECs. SARS-CoV-2 RBD induced mitoROS-mediated expression of cytokines GM-CSF and IL-1β in all three investigated cell types, along with IL-8 expression in both endothelial cell types. The findings suggest mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVECs, HCAECs, and HBECs, with the mitochondria of HLMVECs being more sensitive to SARS-CoV-2 RBD. Full article

St. Louis encephalitis virus (SLEV) is a neglected mosquito-borne Flavivirus that may cause severe neurological disease in humans and other animals. There are no specific treatments against SLEV infection or disease approved for human use, and drug repurposing may represent an opportunity to accelerate the development of treatments against SLEV. Here we present a scalable, medium-throughput phenotypic cell culture-based screening assay on Vero CCL81 cells to identify bioactive compounds that could be repurposed against SLEV infection. We screened eighty compounds from the Medicines for Malaria Venture (MMV) COVID Box library to identify nine (11%) compounds that protected cell cultures from SLEV-induced cytopathic effects, with low- to mid-micromolar potencies. We validated six hit compounds using viral plaque-forming assays to find that the compounds ABT-239, Amiodarone, Fluphenazine, Posaconazole, Triparanol, and Vidofludimus presented varied levels of antiviral activity and selectivity depending on the mammalian cell type used for testing. Importantly, we identified and validated the antiviral activity of the anti-flavivirus nucleoside analog 7DMA against SLEV. Triparanol and Fluphenazine reduced infectious viral loads in both Vero CCL81 and HBEC-5i cell cultures and, similar to the other validated compounds, are likely to exert antiviral activity through a molecular target in the host. Full article

Hemolytic disorders, like malaria and sickle cell disease (SCD), are responsible for significant mortality and morbidity rates globally, specifically in the Americas and Africa. In both malaria and SCD, red blood cell hemolysis leads to the release of a cytotoxic heme that triggers the expression of unique inflammatory profiles, which mediate the tissue damage and pathogenesis of both diseases. MicroRNAs (miRNAs), such as miR-451a and let-7i-5p, contribute to a reduction in the pro-inflammatory responses induced by circulating free hemes. MiR-451a targets both IL-6R (pro-inflammatory) and 14-3-3ζ (anti-inflammatory), and when this miRNA is present, IL-6R is reduced and 14-3-3ζ is increased. Let-7i-5p targets and reduces TLR4, which results in anti-inflammatory signaling. These gene targets regulate inflammation via NFκB regulation and increase anti-inflammatory signaling. Additionally, they indirectly regulate the expression of key heme scavengers, such as heme-oxygenase 1 (HO-1) (coded by the HMOX1 gene) and hemopexin, to decrease circulating cytotoxic heme concentration. MiRNAs can be transported within extracellular vesicles (EVs), such as exosomes, offering insights into the mechanisms of mitigating heme-induced inflammation. We tested the hypothesis that miR-451a- or let-7i-5p-loaded artificial EVs (liposomes) will reduce heme-induced inflammation in brain vascular endothelial cells (HBEC-5i, ATCC: CRL-3245) and macrophages (THP-1, ATCC: TIB-202) in vitro. We completed arginase and nitric oxide assays to determine anti- and pro-inflammatory macrophage presence, respectively. We also assessed the gene expression of IL-6R, TLR4, 14-3-3ζ, and NFκB by RT-qPCR for both cell lines. Our findings revealed that the exposure of HBEC-5i and THP-1 to liposomes loaded with miR-451a or let-7i-5p led to a reduced mRNA expression of IL-6R, TLR4, 14-3-3ζ, and NFκB when treated with a heme. It also resulted in the increased expression of HMOX1 and hemopexin. Finally, macrophages exhibited a tendency toward adopting an anti-inflammatory differentiation phenotype. These findings suggest that miRNA-loaded liposomes can modulate heme-induced inflammation and can be used to target specific cellular pathways, mediating inflammation common to hematological conditions, like malaria and SCD. Full article

The pathogenesis of cerebral small vessel disease (CSVD) is largely unknown. Endothelial disfunction has been suggested as the turning point in CSVD development. In this study, we tested the effect of plasma from CSVD patients on human cerebral microvascular endothelial cells with the aim of describing the pattern of endothelial activation. Plasma samples from three groups of young subjects have been tested: PTs (subjects affected by early stage CSVD); CTRLs (control subjects without abnormalities at MRI scanning); BDs (blood donors). Human Brain Endothelial Cells 5i (HBEC5i) were treated with plasma and total RNA was extracted. RNAs were pooled to reduce gene expression-based variability and NGS analysis was performed. Differentially expressed genes were highlighted comparing PTs, CTRLs and BDs with HBEC5i untreated cells. No significantly altered pathway was evaluated in BD-related treatment. Regulation of p38 MAPK cascade (GO:1900744) was the only pathway altered in CTRL-related treatment. Indeed, 36 different biological processes turned out to be deregulated after PT treatment of HBEC5i, i.e., the cytokine-mediated signaling pathway (GO:0019221). Endothelial cells activate inflammatory pathways in response to stimuli from CSVD patients’ plasma, suggesting the pathogenetic role of neuroinflammation from the early asymptomatic phases of cerebrovascular disease. Full article

Proteases such as neutrophil elastase cleave and activate the epithelial sodium channel (ENaC), causing airway dehydration. Our current study explores the impact of increased protease levels in vapers’ airways on ENaC activity and airway dehydration. Human bronchial epithelial cultures (HBECs) were exposed to bronchoalveolar lavage fluid (BALF) from non-smokers, smokers and vapers. Airway surface liquid (ASL) height was measured by confocal microscopy as a marker of hydration. ENaC cleavage was measured by Western blotting. Human peripheral blood neutrophils were treated with a menthol-flavored e-liquid (Juul), and the resulting secretions were added to HBECs. BALF from smokers and vapers significantly and equally increased ENaC activity and decreased ASL height. The ASL height decrease was attenuated by protease inhibitors. Non-smokers’ BALF had no effect on ENaC or ASL height. BALF from smokers and vapers, but not non-smokers, induced ENaC cleavage. E-liquid-treated neutrophil secretions cleaved ENaC and decreased ASL height. Our study demonstrated that elevated protease levels in vapers’ airways have functional significance since they can activate ENaC, resulting in airway dehydration. Lung dehydration contributes to diseases like cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. Thus, our data predict that vaping, like smoking, will cause airway surface dehydration that likely leads to lung disease. Full article