1,721,122 research outputs found
A Video Bioinformatics Method to Quantify Cell Spreading and Its Application to Cells Treated with Rho-Associated Protein Kinase and Blebbistatin
Get PDFCommercial 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
Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools
Get PDFJust 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. CL-Quant 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
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Analytical and Toxicological Assessment of Chemicals and Elements in Multiple Generations of Electronic Cigarette Products
Get PDFElectronic cigarettes (e-cigarettes), which remain popular among consumers, have been associated with attractive flavored products, especially among adolescents and young adults. This dissertation aimed to evaluate the chemical composition of e-cigarettes and determine the toxic effects of e-cigarettes and chemicals used in high amounts. First, high-performance liquid chromatography and gas chromatography-mass spectrometry were used to identify and quantify nicotine, solvents, flavor chemicals, and synthetic coolant in over 400 products. Nicotine concentrations varied in one brand of refill fluids purchased worldwide. Concentrations of total flavor chemicals ranged from 0 - 343 mg/ml and were generally greater than nicotine concentrations. Most frequently occurring flavor chemicals (e.g., ethyl maltol, furaneol, benzyl alcohol, ethyl vanillin, corylone, triacetin, menthol, vanillin, and cinnamaldehyde, were categorized as fruity, floral, caramellic, vanilla and minty. These results showed the flavor chemicals in e-cigarettes, which often exceed levels used in edible or household products, are sufficiently high enough to make them attractive to youth and adolescents. Next, we evaluated the cytotoxicity of e-cigarette liquids, aerosols, and authentic chemicals standards using multiple endpoint assays (MTT, NRU, and live-cell imaging) and cell lines. E-liquid and aerosol cytotoxicities ranged from 0.01 - 10%, and 0.2 - 1.8%, respectively. Lower concentrations of pure flavor chemicals and WS-23 than in e-cigarettes significantly affected cells and correlated with toxicity. In some products, the flavor chemical concentrations were 30 (menthol), 100 (ethyl maltol), and 100,000 (cinnamaldehyde) times greater than their cytotoxic concentration. The WS-23 concentration that produced cytotoxic effects was 90 times lower than in an e-cigarette fluid and exceeded levels used in consumer products. Flavor chemicals have profound cytotoxic effects in acute in vitro assays, emphasizing the potential to impact human health negatively with chronic use. Finally, the Margin of Exposure (MOE) was used to calculate the cancer risk of pulegone and the health risk of synthetic coolants and flavor chemicals. Pulegone and synthetic coolant levels in e-cigarettes present a significantly calculated risk for cancer and health hazard, contributing to increased harm to consumers. The work in this dissertation emphasizes the need for continuous monitoring of e-cigarette constituents and the enactment of effective regulation to reduce unwanted toxicological effects
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Using Embryonic Stem Cells as a Novel Model to Compare the Toxicological Effects of Harm Reduction and Conventional Cigarette Smoke on Early Embryo Development
Get PDFEmbryonic stem cells are derived from the inner cell mass (ICM) of blastocysts. Before implantation, cells in the ICM undergo multiple divisions, and after implantation, the entire ICM develops into the embryo. Here, we are interested in the effects of environmental toxicants, such as cigarette smoke, on blastocysts during pre-implantation development. Our lab used mouse and human embryonic stem cells (mESC, hESC) as a pre-implantation model to examine the effects of mainstream (MS) or sidestream (SS) smoke from conventional (Marlboro Red) and harm reduction (Advance Lights, Quest, and Marlboro Lights) cigarettes on early development. Harm reduction tobacco products, contain genetically modified tobacco to produce lower nicotine concentration or are equipped with advanced filters designed to remove carcinogens. These products are often advertised as safer with fewer toxins. In our mESC study, stem cells were cultured in suspension with cigarette smoke solutions and allowed to attach over 24 hours. All brands tested inhibited mESC attachment, survival, and proliferation, and surprisingly, harm reduction cigarette smoke was more potent than conventional smoke (Lin et al., 2009). To further investigate the effects of cigarette products on human embryos, hESC colonies were treated with conventional and harm reduction cigarette smoke solutions for 48 hours. To overcome technical challenges with the hESC system, BioStation technology combined with video bioinformatics tools was used to develop assays based time-lapse video data of dynamic cellular processes (Lin, et al., 2010). This technology was used to quantify hESC colony attachment and growth in various smoke treatments. Data demonstrated that non-cytotoxic doses of conventional and harm reduction cigarette SS smoke impaired hESC colony attachment and growth significantly, but MS smoke did not. Moreover, hESC were more sensitive in most assays than mESC. Most significantly, for both species in all assays, SS smoke from harm reduction cigarettes was more potent than SS smoke from a conventional brand. This study clearly demonstrates the need to monitor harm reduction products carefully and to evaluate both MS and SS smoke emitted from them
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Toxicological Evaluation of Thirdhand Smoke Using In-vitro Models
Get PDFThirdhand smoke (THS), which recently emerged as a potential health hazard, is the residue left on surfaces when secondhand smoke clears. It contains volatile organic compounds (VOCs), nicotine and related alkaloids, and chemicals formed during aging such as tobacco specific nitrosamines. The purpose of this dissertation was to evaluate the cytotoxicity of THS using in vitro models. A protocol was optimized to extract THS from fabrics into cell culture medium at room temperature for 1 hour. This protocol was effective for evaluating cytotoxicity of the extracts using mouse neural stem cells (mNSC) in the MTT assay. Fresh THS extracts from terrycloth exposed to 133 cigarettes over 11 months killed mNSC, disrupted motility and the cytoskeleton, and caused fragmentation and vacuolization of cells. THS aged at room temperature for 5 months no longer killed mNSC, indicating that the above effects were caused by VOCs that were lost from THS during aging. Twenty-five VOCs in THS were screened for cytotoxicity to mNSC and adult lung cells, and acrolein was identified as the most toxic VOC. Acrolein killed mNSC and adult lung cells at 10-5 M and decreased proliferation at 10-6 M by affecting the expression of TFDP 1, CASP 3, ANAPC 2, and WEE1, genes involved in cell cycle regulation. A smoker’s car was then simulated, and car seat cover and carpet were exposed to low levels cigarette smoke for 1 month. THS from these fabrics induced single strand DNA breaks in mNSC and adult skin cells. Terrycloth exposed to THS in an indoor chamber became more cytotoxic with longer exposure and lost toxicity after aging in the absence of fresh smoke. Because proteins enhanced extraction of cytotoxic chemicals, infants mouthing THS contaminated objects may have elevated intake of THS toxicants due to proteins in their saliva. Batches of THS that did not affect cell survival/proliferation caused stress-induced mitochondrial hyperfusion, increased mitochondrial membrane potential, increased ATP production; increased oxidative stress; and decreased expression of genes involved in mitochondrial function. These data show that relatively low levels of THS can disrupt cellular functions and have the potential to adversely impact health
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A Vaper Paradox: Evaluation of the Relationship Between Electronic Cigarette Use and SARS-CoV-2 Infection
Get PDFThe 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
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Evaluation of Metal Emissions in Electronic Cigarettes
Get PDFElectronic cigarettes (EC) are tobacco-free nicotine delivery devices comprised of metal components that aerosolize fluid containing nicotine, a humectant, flavorings, and contaminants. The purpose of this project was to determine the concentration of elements, including metals, in the aerosol of cartomizer style electronic cigarettes (EC), disposable EC, electronic hookahs (EH), and tank style EC, how these concentrations change when puffing parameters are varied, and the source of the elements present in the aerosol. The metal analysis in fluids and aerosols was done using inductively coupled optical emission spectroscopy (ICP OES) and elemental analysis of atomizers was completed using scanning electron microscopy (SEM) coupled with and energy dispersive spectroscopy. There was considerable variation in performance of EC both across and within brands. Most EC tested required higher air flow rates to produce aerosol than conventional cigarettes require to produce smoke. The total concentrations of elements/metals present in EC aerosols varied by brand and style. For the disposable EC and electronic hookahs, the results were similar with each containing various metals in their aerosol. The total concentration of elements in the aerosol collected with a cold trap varied by brand and EC model (cartomizer EC - 487 to 5,861µg/L, disposable EC - 997 to 2.296 µg/L, tank-style - 617 to 4,382 µg/L), with silicon being the dominant element in the aerosol. These concentrations did change slightly, when more puffs were collected over time, when voltage was increased, and puff volume increased. The most common metals in the aerosol of all units were chromium, nickel, copper, zinc, silicon, tin, and lead, which originated from the wires, wicks, joints, and air tubes within the atomizing unit. There were design differences between different EC models, such as shifting from solder joints to brass clamps or welds, which reduced the concentration of metals in the aerosol. The main conclusions from this study are that metals were present in EC aerosols, and metals usually originated from the components of the atomizer unit
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Clinical Investigations on Human Health Responses to Thirdhand Smoke and Electronic Cigarette Exposure
Get PDFAbstract of the DissertationClinical Investigations on Human Health Responses to Thirdhand Smoke and Electronic Cigarette Exposure
by
Shane Sakamaki-Ching
Doctor of Philosophy, Graduate Program in Cell, Molecular, and Developmental Biology
University of California, Riverside, March 2022
Dr. Prue Talbot, ChairpersonBackground: Cigarette smoking causes diseases such as cancer, heart disease, and stroke. The diseases associated with electronic cigarettes (ECs) are not as well understood. Cigarette smoking leaves a residue, thirdhand smoke (THS), on indoor surfaces. The health effects of THS on humans is limited to one study. Our purposes were to: (1) identify urinary biomarkers of potential harm and exposure and correlate these to metal concentrations in urine from cigarette smokers and EC users; (2) determine the responses of humans dermally exposed to THS; and (3) identify molecular changes in keratinocytes exposed to THS extract.Methods: In Chapter 1, urine from non-smokers, EC users, and cigarette smokers was evaluated for biomarkers of exposure, effect, and potential harm. In Chapter 2, human participants were exposed dermally to clothing impregnated with filtered clean air or THS; their urine was analyzed for biomarkers of exposure and harm, and their plasma was analyzed using proteomics. In Chapter 3, molecular responses of keratinocytes to extracts from THS impregnated fabric were determined.Results: In Chapter #1, urinary biomarkers of exposure, effect, and potential harm were elevated in EC users and cigarette smokers; increased EC usage was correlated with elevated metal concentrations, which correlated with oxidative DNA damage. In Chapter #2, THS-exposure significantly elevated urinary 8-OHdG, 8-isoprostane, and protein carbonyls and activated pathways associated with inflammation, oxidative stress, and skin disease initiation. In Chapter #3, THS in keratinocytes caused proinflammation, and oxidative stress, mitochondrial damage, and elevated keratin 5 levels.Conclusion: Cigarette smoking and EC vaping elevated DNA damage by oxidization which was correlated with total metal concentrations in the urine of cigarette smokers and EC vapers, a change which may lead to disease. Dermal THS exposure significantly increased oxidative damage to DNA, lipids, and proteins in humans. Inflammation was elevated at 3 hours and persisted for 22 hours, which could in the long-term lead to disease. Keratinocytes exposed to THS extracts had elevated molecular risk factors for developing inflammatory skin diseases, including cancer. THS-induced responses in keratinocytes were similar to those in cigarette smokers, implicating THS as an environmental hazard that should be monitored
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Characterizing the Pathways That Initiate and Stop Dynamic Blebbing in Human Embryonic Stem Cells
Get PDFDynamic blebs are membrane protrusions on the surface of healthy cells that function in cell division and migration. The purpose of this dissertation was to discover better methods to control dynamic blebbing by identifying pathways that initiate and inhibit blebbing in human embryonic stem cells (hESC). Live cell imaging experiments demonstrated that dynamic and apoptotic blebbing were morphologically and temporally distinct during passaging of hESC. Dynamic blebs retracted faster than apoptotic blebs and had an intact cytoskeleton. Dynamic blebbing was prolonged by depolymerization of microtubules and stopped by drugs that disrupted actin microfilaments or inhibited myosin II. Plating on laminin-521 or Matrigel overcoated with laminin-111 efficiently stopped blebbing by activating an integrin-focal adhesion kinase pathway. To identify the pathway that initiates dynamic blebbing, we tested the hypothesis that the P2X7 calcium channel is activated by ATP released during passaging. Immunocytochemistry and PCR showed that the P2X7 receptor is expressed in hESC, but not in cells starting differentiation. P2X7 inhibitors and siRNA decreased dynamic blebbing. Extracellular ATP concentration increased during passaging. While apyrase, which degrades ATP, reduced the percentage of blebbing cells, addition of ATP to the culture medium prolonged blebbing. When Ca2+ was chelated by either EGTA or BAPTA, dynamic blebbing was inhibited. Rac activation was associated with decreased blebbing and cell attachment, while blebbing was activated though the ROCK pathway. These data support the idea that dynamic blebbing in hESC is initiated by extracellular ATP binding to P2X7, allowing Ca2+ influx which in turn initiates dynamic blebbing via the ROCK- myosin II pathway. Decreased extracellular ATP is accompanied by activation of Rac, cessation of blebbing, and attachment. These results introduce better ways to control dynamic blebbing and improve cell survival during passaging. The use of P2X7 inhibitors and laminin substrates enables attachment and improves single hESC plating efficiency, which will facilitate quantitative work in drug discovery and toxicology. These results also introduce: (1) P2X7 as a cell surface marker for pluripotency; (2) hESC as an excellent model for studying dynamic blebbing and (3) the possibility of using hESC, which are similar to epiblast cells, to study cell movements during gastrulation
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Search for the Holy Grail of Smoking: The Hidden Dangers of Electronic Nicotine Delivery Devices
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