Electronic cigarettes (ECs) are categorized into generations which differ in terms of design, aerosol production, and customizability. Current and former smokers prefer third-generation devices that satisfy tobacco cravings more effectively than older generations. Recent studies indicate that EC aerosols from firstand second-generation devices contain reactive carbonyls and free radicals and can cause in vitro cytotoxicity. Third-generation ECs have not been adequately studied. Further, previous studies have focused on cells from the respiratory tract, whereas those of the oral cavity, which is exposed to high levels of EC aerosols, have been understudied. We quantified the production of reactive carbonyls and free radicals by a thirdgeneration EC and investigated the induction of cytotoxicity and oxidative stress in normal and cancerous human oral cell lines using a panel of eight commercial EC liquids. We found that EC aerosols produced using a new atomizer contained formaldehyde, acetaldehyde, and acrolein, but did not contain detectable levels of free radicals. We found that EC aerosols generated from only one of the eight liquids tested using a new atomizer induced cytotoxicity against two human oral cells in vitro. Treatment of oral cells with the cytotoxic EC aerosol caused a concomitant increase in intracellular oxidative stress. As atomizer age increased with repeated use of the same atomizer, carbonyl production, radical emissions, and cytotoxicity increased. Overall, our results suggest that thirdgeneration ECs may cause adverse effects in the oral cavity and normal EC use, which involves repeated use of the same atomizer to generate aerosol, may enhance the potential toxic effects of third-generation ECs.

Graduate students have a lot on their plates. They are expected to do exceptional research, publish frequently, take classes, teach, train new students ... the list goes on and on. Eventually, the primary tasks become finding a job and graduating. An issue that can affect these fledgling chemists is the lack of information and support for those facing the decision between master’s and doctoral degrees. The availability of more information about this decision might eliminate the population of graduate students without clear career goals who stay in grad school because they see no other option. In my experience as a chemistry graduate student, a master’s degree is viewed with contempt even though there are a significant number of jobs in chemistry for applicants with a master’s degree. Yes, these are frequently “limited” to jobs in industry—another idea that is often viewed with contempt by those in academia—but that does not

Electronic cigarettes (ECs) have emerged as a popular alternative to conventional cigarettes. These devices operate by using resistive heating to vaporize a fluid consisting of a propylene glycol‐glycerol base, flavorings, and nicotine. Although considered safer than conventional cigarettes, recent studies have shown that EC vapor contains harmful compounds such as free radicals and carbonyls. A limited number of studies have shown that EC liquid and vapor can cause oxidative stress and cell death in vitro. While the majority of in vitro studies have focused on the toxicity in respiratory tract cell lines, the tissues in the oral cavity, which are exposed to high local concentrations of EC vapor, have been under‐studied. Our objective was to investigate the ability of EC vapor to induce cytotoxicity and oxidative stress in human oral cell lines. HGF‐1 human gingival fibroblasts and SCC‐25 human oral squamous cell carcinoma cells were exposed to a panel of commercially‐available fruit and tobacco‐flavored EC vapors using an air‐liquid interface exposure model, which mimics physiological exposures in vitro. Cytotoxicity and markers of oxidative stress were assessed after ecologically‐relevant exposure protocols. Cytotoxic EC vapors caused a greater than 30% reduction in cell viability. Nicotine was shown to ablate the effects of the cytotoxic EC vapors. There was no difference in the cytotoxic response to EC vapor between the cell lines tested. Treatment of SCC‐25 cells with a cytotoxic EC vapor caused a significant increase in intracellular ROS after a single 15 puff exposure. This increase occurred for both nicotine‐containing and nicotine‐free vapors. The differential effects of nicotine on cytotoxicity and oxidative stress suggest that either (1) cytotoxicity is not driven by ROS or (2) that nicotine protects cells by a mechanism that does not impact intracellular ROS levels. Ongoing studies are investigating the role of nicotine in this aerosolized system and the relationship between EC vapor constituents and cytotoxicity in human oral cells.Support or Funding InformationPenn State Tobacco Center of Regulatory Science; Penn State Department of Food ScienceThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An accurate depiction of protein structure and its mobility is necessary to understanding protein function and many methods exist to determine this. Obtaining protein structural characteristic by advanced electron paramagnetic resonance (EPR) spectroscopy techniques like double electron‐electron resonance (DEER) is emerging as a powerful technique that is complementary to other well established structural characterization tools. Such studies most commonly require the ligation of “spin labels” to different sites on the protein, often by a site‐specific reaction between a stable molecule containing a nitroxide radical and an amino acid residue. We seek to develop a robust set of tools for predicting structures of hard‐to‐characterize proteins by combining the distance information obtained from advanced EPR techniques with mobility information obtained from room temperature continuous wave EPR (CW EPR). Many methods of spin‐labeling a protein exist; however, the viability of these specific approaches for performing structural determination techniques like DEER has not yet been explored. The most common spin‐labeling methodology conjugates (1‐oxyl‐2,2,5,5‐tetramethylpyrroline‐3‐methyl) methanethiosulfonate (MTSSL) to a protein via a disulfide bond with a cysteine residue. To achieve this conjugation site‐specifically, all native surface‐located cysteine residues must be removed, which may not be desirable from a structural integrity standpoint. Utilizing unnatural amino acids can alleviate this issue and the spin‐label can be added to the protein via techniques such as click chemistry, a rapid cycloaddition that can occur at physiological conditions. In this work, we compare how different spin labeling techniques perform from an experimental point of view. We seek, overall, to determine a powerful and easy‐to‐use protocol for structural determination. Here, we use T4 Lysozyme as a well‐characterized protein model and compare 4‐alkyl TEMPO installed on the unnatural amino acid p‐‐azidophenylalanine (pAzF) with the aforementioned MTSSL/cysteine label. For this purpose, we use a cysteine‐free version of T4 Lysozyme and genetically encode either cysteine or pAzF to specific positions. The results presented focus on the structural rigidity of the spin labels, a key factor defining the flexibility of certain areas of the protein and the certainty of the distance determination. Moreover, as different spin probes display different spectroscopic characteristics, each spin label is evaluated in terms of DEER sensitivity and efficiency. Finally, we compare different advanced EPR techniques (DEER, DQC, relaxation enhancement) to determine the most robust in distance predictions.Support or Funding InformationThe work is funded by the Pennsylvania State University startup fund of Alexey Silakov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.