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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2022)

The relationship between electronic cigarette (EC) use and SARS-CoV-2 infection is poorly understood and contradictory with claims that vaping both increases and decreases the likelihood of contracting COVID-19. The objective of this dissertation was to determine how EC fluids and aerosols affect SARS-CoV-2 infection of human cells and tissues. Responses of human bronchial epithelial cells to EC fluids and aerosols and their individual constituents were evaluated using submerged cultures, air liquid interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex® system, which produces authentic heated EC aerosols. Both monolayers of BEAS-2B cells and 3D organotypic cultures of EpiAirway tissues were studied. Results were generally in agreement across the three exposure platforms and the two cell models. EC aerosols made from fluids with nicotine, BLU™ EC aerosols, and pure nicotine increased ACE2 (the viral receptor) and TMPRSS2 activity, a protease essential for viral entry. In contrast to BLU™ EC, aerosols from JUUL™ “Virginia Tobacco” decreased TMPRSS2 activity. Using SARS-CoV-2 viral pseudoparticles, we demonstrated that exposure to EC fluids or aerosols with nicotine or to pure nicotine increased infection dose dependently. PG/VG also increased infection but only when aerosols were made in the Cultex® exposure system, suggesting reaction products contributed to increased infection. Aerosols produced at both low (8 watt) and high (20 watt) powers increased nicotine-enhanced infection. EC fluid ingredients and aerosol nicotine dosimetry modulated the response of human bronchial epithelial cells to infection. Specifically, inclusion of benzoic acid reduced EC fluid pH, which reduced TMPRSS2 activity, providing protection against the enhanced infection produced by PG/VG and nicotine. This protection lasted at least 48 hours. Finally, JUUL™ “Virginia Tobacco” aerosols, which contain benzoic acid, protected against pseudoparticle infection, while BLU™ EC aerosols, which lack benzoic acid, did not. Our investigation demonstrates that the effect of ECs on SARS-CoV-2 infection are complex, and infection can either be increased or not affected depending on the ingredients and the pH of EC fluids, TMPRSS2 activity, and nicotine concentration.

    Cover page: A Vaper Paradox: Evaluation of the Relationship Between Electronic Cigarette Use and SARS-CoV-2 InfectionCreative Commons 'BY-ND' version 4.0 license
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2023)

    Introduction

    Both spill over and spill back of SARS-CoV-2 virus have been reported on mink farms in Europe and the United States. Zoonosis is a public health concern as dangerous mutated forms of the virus could be introduced into the human population through spillback.

    Methods

    The purpose of our study was to determine the SARS-CoV-2 entry mechanism using the mink lung epithelial cell line (Mv1Lu) and to block entry with drug inhibitors.

    Results

    Mv1Lu cells were susceptible to SARS-CoV-2 viral pseudoparticle infection, validating them as a suitable disease model for COVID-19. Inhibitors of TMPRSS2 and of endocytosis, two pathways of viral entry, were tested to identify those that blocked infection. TMPRSS2 inhibitors had minimal impact, which can be explained by the apparent lack of activity of this enzyme in the mink and its localization within the cell, not on the cell surface.

    Discussion

    Dyngo4a, a small molecule endocytosis inhibitor, significantly reduced infection, supporting the conclusion that the entry of the SARS-CoV-2 virus into Mv1Lu cells occurs primarily through endocytosis. The small molecule inhibitors that were effective in this study could potentially be used therapeutically to prevent SARS-CoV-2 infection in mink populations. This study will facilitate the development of therapeutics to prevent zoonotic transmission of SARS-CoV-2 variants to other animals, including humans.
      Cover page: Endocytosis inhibitors block SARS-CoV-2 pseudoparticle infection of mink lung epithelium
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2023)
      The relationship between the use of tobacco products and SARS-CoV-2 infection is poorly understood and controversial. Few studies have examined the effect of electronic cigarettes (ECs) on SARS-CoV-2 infection. We tested the hypothesis that EC fluids and aerosols with nicotine promote SARS-COV-2 infection by increasing viral entry into human respiratory epithelial cells. Responses of BEAS-2B cells to JUUL aerosols or their individual constituents were compared using three exposure platforms: submerged culture, air-liquid-interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex system, which produces authentic heated EC aerosols. In general, nicotine and nicotine + propylene glycol/vegetable glycerin aerosols increased ACE2 (angiotensin converting enzyme 2) levels, the SARS-CoV-2 receptor; and increased the activity of TMPRSS2 (transmembrane serine protease 2), an enzyme essential for viral entry. Lentivirus pseudoparticles with spike protein were used to test viral penetration. Exposure to nicotine, EC fluids, or aerosols altered the infection machinery and increased viral entry into cells. While most data were in good agreement across the three exposure platforms, cells were more responsive to treatments when exposed at the ALI in the Cultex system, even though the exposures were brief and intermittent. While both nicotine and JUUL aerosols increased SARS-CoV-2 infection, JUUL significantly decreased the effect of nicotine alone. These data support the idea that vaping can increase the likelihood of contracting COVID-19 and that e-liquid composition may modulate this effect.
        Cover page: New insights into how popular electronic cigarette aerosols and aerosol constituents affect SARS-CoV-2 infection of human bronchial epithelial cells
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2018)

        Objectives

        Epithelial-to-mesenchymal transition (EMT) is the initial step enabling the metastasis of cancer cells, which often leads to death. Although smoking is a major risk factor for lung cancer, there is still widespread use of conventional cigarettes. Recently, the tobacco industry has been transformed by the introduction of electronic cigarettes (ECs), which have lower levels of carcinogens and may provide a safer alternative. Here, we investigate the ability of EC liquids and aerosols to induce an EMT in A549 lung cancer cells.

        Materials and methods

        Human adenocarcinoma alveolar basal epithelial cells (A549) were exposed to EC liquids and aerosols from a popular product for 3-8 days. Live cell imaging, EMT biomarker analysis, and machine learning/image processing algorithms were used to characterize changes associated with EMT.

        Results

        Long-term exposure of A549 cells to menthol or tobacco-flavored EC liquids or aerosols induced an EMT that was characterized by acquisition of a fibroblast-like morphology, loss of cell-to-cell junctions, internalization of E-cadherin, increased motility, and upregulation of other EMT markers. The EMT was concurrent with plasma membrane to nuclear translocation of active β-catenin.

        Conclusion

        This is the first known study to show an EMT of lung cancer cells during exposure to EC products. Because an EMT is an initial step leading to metastasis, an intractable problem that often leads to patient death, this critical finding has significant implications for former or heavy cigarette smokers who are using EC and may be at risk for lung cancer or who may already have a lung tumor.
          Cover page: Epithelial-to-mesenchymal transition of A549 lung cancer cells exposed to electronic cigarettes
          • Peer Reviewed
          UC Riverside Previously Published Works (2015)
          Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities.
            Cover page: Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2018)
            There is a critical need for better analytical methods to study mitochondria in normal and diseased states. Mitochondrial image analysis is typically done on still images using slow manual methods or automated methods of limited types of features. MitoMo integrated software overcomes these bottlenecks by automating rapid unbiased quantitative analysis of mitochondrial morphology, texture, motion, and morphogenesis and advances machine-learning classification to predict cell health by combining features. Our pixel-based approach for motion analysis evaluates the magnitude and direction of motion of: (1) molecules within mitochondria, (2) individual mitochondria, and (3) distinct morphological classes of mitochondria. MitoMo allows analysis of mitochondrial morphogenesis in time-lapse videos to study early progression of cellular stress. Biological applications are presented including: (1) establishing normal phenotypes of mitochondria in different cell types; (2) quantifying stress-induced mitochondrial hyperfusion in cells treated with an environmental toxicant, (3) tracking morphogenesis in mitochondria undergoing swelling, and (4) evaluating early changes in cell health when morphological abnormalities are not apparent. MitoMo unlocks new information on mitochondrial phenotypes and dynamics by enabling deep analysis of mitochondrial features in any cell type and can be applied to a broad spectrum of research problems in cell biology, drug testing, toxicology, and medicine.
              Cover page: Deep Analysis of Mitochondria and Cell Health Using Machine Learning
              • Peer Reviewed
              UC Riverside Previously Published Works (2015)
              Commercial software is available for performing video bioinformatics analysis on cultured cells. Such software is convenient and can often be used to create suitable protocols for quantitative analysis of video data with relatively little background in image processing. This chapter demonstrates that CL-Quant software, a commercial program produced by DRVision, can be used to automatically analyze cell spreading in time-lapse videos of human embryonic stem cells (hESC). Two cell spreading protocols were developed and tested. One was professionally created by engineers at DRVision and adapted to this project. The other was created by an undergraduate student with 1 month of experience using CL-Quant. Both protocols successfully segmented small spreading colonies of hESC, and, in general, were in good agreement with the ground truth which was measured using ImageJ. Overall the professional protocol performed better segmentation, while the user-generated protocol demonstrated that someone who had relatively little background with CL-Quant can successfully create protocols. The protocols were applied to hESC that had been treated with ROCK inhibitors or blebbistatin, which tend to cause rapid attachment and spreading of hESC colonies. All treatments enabled hESC to attach rapidly. Cells treated with the ROCK inhibitors or blebbistatin spread more than controls and often looked stressed. The use of the spreading analysis protocol can provide a very rapid method to evaluate the cytotoxicity of chemical treatment and reveal effects on the cytoskeleton of the cell. While hESC are presented in this chapter, other cell types could also be used in conjunction with the spreading protocol.
                Cover page: A Video Bioinformatics Method to Quantify Cell Spreading and Its Application to Cells Treated with Rho-Associated Protein Kinase and Blebbistatin
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2016)
                Thirdhand cigarette smoke (THS) was recently recognized as an environmental health hazard; however, little is known about it effects on cells. Mitochondria are sensitive monitors of cell health and report on environmentally induced stress. We tested the effects of low levels of THS extracted from terry cloth on mitochondrial morphology and function using stem cells with well-defined mitochondria. Concentrations of THS that did not kill cells caused stress-induced mitochondrial hyperfusion (SIMH), which was characterized by changes in mitochondrial morphology indicative of fusion, increased mitochondrial membrane potential (MMP), increased ATP levels, increased superoxide production, and increased oxidation of mitochondrial proteins. SIMH was accompanied by a decrease in Fis1 expression, a gene responsible for mitochondrial fission, and a decrease in apoptosis-related genes, including Aifm2, Bbc3, and Bid There was also down regulation of Ucp2, Ucp4, and Ucp5, genes that decrease MMP thereby reducing oxidative phosphorylation, while promoting glycolysis. These effects, which collectively accompany SIMH, are a prosurvival mechanism to rescue damaged mitochondria and protect cells from apoptosis. Prolonged exposure to THS caused a reduction in MMP and decreased cell proliferation, which likely leads to apoptosis.
                  Cover page: From the Cover: Thirdhand Cigarette Smoke Causes Stress-Induced Mitochondrial Hyperfusion and Alters the Transcriptional Profile of Stem Cells
                  • Article
                  • Peer Reviewed
                  UC Riverside Previously Published Works (2019)
                  Stem cells provide a sensitive model to study exposure to toxicants, such as cigarette smoke. Electronic cigarettes (ECs) are popular nicotine delivery devices, often targeted to youth and pregnant mothers. However, little is known about how chemicals in ECs might affect neural stem cells, and in particular their mitochondria, organelles that maintain cell functionality and health. Here we show that the mechanism underlying EC-induced stem cell toxicity is stress-induced mitochondrial hyperfusion (SIMH), a transient survival response accompanied by increased mitochondrial oxidative stress. We identify SIMH as a survival response to nicotine, now widely available in EC refill fluids and in pure form for do-it-yourself EC products. These observed mitochondrial alterations combined with autophagy dysfunction to clear damaged mitochondria could lead to faulty stem cell populations, accelerate cellular aging, and lead to acquired mitochondriopathies. Any nicotine-containing product may likewise stress stem cells with long-term repercussions for users and passively exposed individuals. VIDEO ABSTRACT.
                    Cover page: Mitochondrial Stress Response in Neural Stem Cells Exposed to Electronic Cigarettes
                    • Article
                    • Peer Reviewed
                    UC Riverside Previously Published Works (2022)
                    Lung cancer is the leading cause of cancer-related deaths worldwide, with increased risk being associated with unresolved or chronic inflammation. Agricultural and livestock workers endure significant exposure to agricultural dusts on a routine basis; however, the chronic inflammatory and carcinogenic effects of these dust exposure is unclear. We have developed a chronic dust exposure model of lung carcinogenesis in which mice were intranasally challenged three times a week for 24 weeks, using an aqueous dust extract (HDE) made from dust collected in swine confinement facilities. We also treated mice with the omega-3-fatty acid lipid mediator, aspirin-triggered resolvin D1 (AT-RvD1) to provide a novel therapeutic strategy for mitigating the inflammatory and carcinogenic effects of HDE. Exposure to HDE resulted in significant immune cell influx into the lungs, enhanced lung tumorigenesis, severe tissue pathogenesis, and a pro-inflammatory and carcinogenic gene signature, relative to saline-exposed mice. AT-RvD1 treatment mitigated the dust-induced inflammatory response but did not protect against HDE + NNK-enhanced tumorigenesis. Our data suggest that chronic HDE exposure induces a significant inflammatory and pro-carcinogenic response, whereas treatment with AT-RvD1 dampens the inflammatory responses, providing a strong argument for the therapeutic use of AT-RvD1 to mitigate chronic inflammation.
                      Cover page: Aspirin-Triggered Resolvin D1 Reduces Chronic Dust-Induced Lung Pathology without Altering Susceptibility to Dust-Enhanced Carcinogenesis
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