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In-depth analysis of e-cigarette filling solutions and their biological implications Project Narrative

This research focuses on the analysis of the components of e-cigarette filling solutions and their implications for biological systems. E-cigarettes are increasingly reported to cause adverse health effects and therefore may pose a significant public health risk.

In-depth analysis of e-cigarette filling solutions and their biological implications Project Summary

This proposal investigates electronic cigarette filling solutions. Although e-cigarettes are sold

as smoking cessation aids they also have been reported to cause adverse effects. Unregulated, e-cigarettes have been shown to contain varying levels of nicotine and other tobacco alkaloids. The FDA has indicated that the research of new and emerging tobacco products, including electronic cigarettes, is an important research priority. The specific goal of this research is to study the hypothesis that e-cigarette filling solutions are commonly mislabeled and contain unintended alkaloids (in addition to nicotine) which affect growth and viability of cells in culture. Additionally, we suggest that the selected tobacco alkaloids modify proteins. E-cigarette filling solutions will be analyzed by GC-MS and HPLC for identification and quantification of tobacco alkaloids. Lysozyme will be used as a model protein and subjected to the alkaloids to study protein modification. Cells will be grown in an incubator and subjected to individual tobacco alkaloids as well as the filling solutions to study the effects on growth and cell viability. The cells will also be lysed and compared to the lysozyme model protein. The sum of these studies will inform science-based policies regarding low concentrations of nicotine and its derivatives as to potential adverse effects on biological systems.

In-depth analysis of e-cigarette filling solutions and their biological implications Multiple PI Leadership Plan

Multiple PIs are necessary because the nature of the research requires strong leadership from

several very different scientific disciplines. Each PI brings vital skills that are necessary for the research proposal. This interdisciplinary blend of collaborators is required for the successful exploration of each specific aim. Potts provides the analytical expertise necessary for Specific Aim #1. She will also be responsible for providing alkaloid standards for the other PIs. Carver provides biological expertise which is necessary for Specific Aim #2; he has experience growing and studying cell cultures using undergraduate and high school students. Kim provides biochemical expertise and will focus on the protein chemistry explored in Specific Aim #3. Albu provides expertise in fluorescence spectroscopy needed in Specific Aim #3 and the computational needs that are required in all areas of the proposed research.

Potts will provide general oversight for the entire study, serve as contact PI and assume fiscal and administrative management. She will be responsible for communication with NIH and submission of financial reports. PIs will communicate weekly by email and meet as a team once a month to discuss key scientific issues and to make any decisions necessary for the progress of the research. Publication authorship will be based on the relative scientific contributions of the PIs.

Though not anticipated, conflicts will be addressed through a meeting of all PIs to discuss issues. If a resolution cannot be reached, appropriate departmental administrators will be asked to meet with the PIs to mediate a resolution. If more intervention for resolution is required, the UTC Senior Vice-Chancellor for Academic Affairs, Dr. Jerald Ainsworth, will have executive authority to settle the disagreement. Dr. Ainsworth has a scientific background in immunology and experience with NIH funding.

In-depth analysis of e-cigarette filling solutions and their biological implications Specific Aims

Electronic cigarettes (e-cigarettes), although sold as smoking cessation aids, are increasingly

reported to cause adverse effects. (Chen 2013) More consumers are responding to marketing of e-cigarettes as alternatives to traditional smoking products. In addition, this advertising reminds consumers that e-cigarettes are not covered by indoor smoking bans, and these ads also suggest, directly or indirectly, that e-cigarettes may be used to support smoking cessation. (DeNoon 2009) The Food and Drug Administration (FDA) has indicated that the research on new and emerging tobacco products, including e-cigarettes as tobacco products, is an important institutional priority.(Food and Drug Administration 2012) Preliminary analytical data have quantified the components in some e-cigarette filling solutions. The FDA found that not only did the filling solutions contain other tobacco alkaloids (in addition to nicotine) but also some solutions contained nicotine, even when labeled as nicotine-free.(Trehy, Ye et al. 2011) The overall goal of our research is to document the adverse effects of e-cigarette filling solutions under various laboratory conditions. The specific goal of this research is to determine whether filling solutions are commonly mislabeled and contain unintended alkaloids (in addition to nicotine) and which may affect growth and viability of cells in culture. Additionally, we suggest that selected tobacco alkaloids modify proteins. The sum of these studies will inform science-based policies regarding low concentrations of nicotine and its derivatives as to potential adverse effects on biological systems.

Specific Aim #1: Identify and quantify tobacco alkaloids in disposable e-cigarettes and associated filling solutions labeled as low or non-nicotine containing. We will analyze e-cigarette filling solutions labeled as low or non-nicotine containing by gas chromatography – mass spectrometry to identity any tobacco alkaloids present. Additionally, samples will be analyzed by high performance liquid chromatography for quantitative analysis. We will purchase supplies from at least five internet suppliers and determine the concentrations of several tobacco alkaloids, including nicotine, cotinine, myosmine, anatabine, anabasine and nicotyrine. The solutions to be analyzed will be labeled to contain 24 mg/mL of nicotine or less, or will be labeled nicotine-free.

Specific Aim #2: Document the effects of e-cigarette fluids on the growth and viability of cells in culture. We will study the effects of nicotine, cotinine, myosmine, anatabine, anabasine, and β-nicotyrine on three types of cultured mammalian cells commonly used in regular cigarette assays (human transformed lung cells, human skin fibroblast cells, and human lung fibroblast cells). We will assay the viability and proliferation of cell cultures to treatment with the previously mentioned alkaloids; both individually and in the combined e-cigarette products. Initially, cell growth curves and cell viability will be investigated using these chemicals as compared to non-treated cells. Cell proliferation rates will be compared to control cultures to determine if these chemicals alter normal mitotic rates. Cell assays will also determine if cells within the cultures are undergoing apoptosis at a variant rate compared to controls.

Specific Aim #3: Establish whether protein modifications are induced by selected tobacco alkaloids found in e-cigarettes. We hypothesize that nicotine and its analogs may induce modifications in a wide-range of non-nicotinic acetylcholine receptor (nAChR)-based proteins. In this context, we will investigate the nature of protein modifications induced by the selected tobacco alkaloids using model cellular proteins exposed to selected tobacco alkaloids. There are two sub-aims for this specific aim with the main analysis techniques being fluorescence spectroscopy and 2D-protein gel electrophoresis. The first sub-aim (3a) is to elucidate if selected tobacco alkaloids are able to determine any conformational changes in lysozyme utilizing fluorescence spectroscopy. The second sub-aim (3b) is to investigate if selected tobacco alkaloids are involved in protein expression at the cellular level. The cell study will be carried out by monitoring fluorescence behavior and 2D-gel electrophoretic features of cellular proteins by focusing on biomarkers representing protein expression. The two biomarkers for this sub-aim are cellular protein phosphorylation and the cellular behaviors affected by the intracellular level of Ca2+.

In-depth analysis of e-cigarette filling solutions and their biological implications Research Strategy

A Significance

In the last five years, electronic cigarettes (e-cigarette(s)) have become increasingly popular

among smokers.(Campagna, Caponnetto et al. 2012, McAuley, Hopke et al. 2012) Sold in both disposable and re-fillable forms, these devices are comprised of an atomizer and battery. Contained within the atomizer is a filling solution of glycol, water, flavorings and nicotine which is vaporized upon heating. Manufactures advertise these nicotine delivery devices as “safe” alternatives to traditional tobacco products. The Center for Tobacco Products of the Food and Drug Administration is concerned with the claims and has indicated the study of these forms of tobacco alternatives as one of their top research priorities.(Food and Drug Administration 2012)

In a letter to the open journal BMJ in 2010, Andreas Flouris and Dimitris Oikomou cite three major studies which focused on the analysis of nicotine in e-cigarette filling solutions.(Flouris and Oikomomou 2010) Two of the studies by government groups, the US-FDA and a Greek research group, found similar results: varying amounts of nicotine, even in samples labeled as non-nicotine containing. A private research group, HNZ of New Zealand found that samples were correctly labeled with nicotine content. Searches on SciFinder Scholar did not reveal if the Greek and HNZ studies were published in peer-review journals, but the FDA published their results in the 2011 in the Journal of Liquid Chromatography and Related Technologies.(Trehy, Ye et al. 2011) The conflicting results of these studies indicate that more research is needed in this area. Specific studies are needed that focus on samples labeled as containing low-nicotine or nicotine-free.

A large amount of research associates nicotine, cigarette tar, and other chemicals found in cigarette smoke with lung and cardiovascular disease is available. Studies have been performed that initially linked smoke inhalation with disease and mortality rates, and decades of further studies have discovered various chemicals within smoke, and their physiological effects.(Burke, Farb et al. 1997, Qiao, Tervahauta et al. 2000, Yashima, Ohara et al. 2000, Hoshino, Mio et al. 2001, Shields 2002, Frohlich M 2003) However, the relatively new e-cigarette has not been as well scrutinized. E-cigarettes are marketed as an alternative to regular cigarettes, and also as a potential cessation device.(Gornall 2012) Nicotine inhalation is found in e-cigarettes and is associated with a novel repertoire of chemicals.(Trehy, Ye et al. 2011) Overall, the potential adverse effects of e-cigarettes have not been thoroughly investigated and the interactions of the chemicals used or produced within e-cigarettes are not well understood.

Tobacco alkaloids such as nicotine were suspected to act on various nicotinic acetylcholine receptors (nAChRs). (Millar and Harkness 2008, Barik and Wonnacott 2009, Benowitz, Hukkanen et al. 2009)Consequently, many published studies have focused heavily on either the interaction of nicotine and the nAChR family or the mechanism of the nicotine-induced release of several neurotransmitters. (Millar and Harkness 2008, Barik and Wonnacott 2009, Benowitz, Hukkanen et al. 2009)However, recent studies revealed that nicotine targets or modifies a wide-range of non-nAChRs-based proteins in the brain tissues and regulates their expression level (Salomon, Marcinowski et al. 1996, Hejmad, Dajas-Bailador et al. 2003, Kane, Konu et al. 2004, Li and Wang 2007, Barik and Wonnacott 2009, Benowitz, Hukkanen et al. 2009) For example, Li and coworkers identified a number of proteins that are known to be modified upon exposure to nicotine, which are glutathione S-tranferases, peroxiredoxin, heat shock proteins, protein phosphatase, and several enzymes responsible for glycolysis and the citric acid cycle. (Li and Wang 2007) Kane and coworkers reported the up- or down-regulation of the genes for ubiquitin, ubiquitin-conjugating enzymes, and proteasomal subunits in different regions of the rat brains upon exposure to nicotine for 14 days.(Kane, Konu et al. 2004) Nicotine was also found to be involved in regulating the signaling pathway by altering the extracellular signal-regulated kinases and intracellular level of calcium ions.(Zhai, Li et al. 2008)It should be noted that the mentioned studies above focused mainly on several selected protein targets in brain tissues upon exposure to nicotine, therefore limiting our

understanding of the biological implications of tobacco products affecting whole proteomics. The biological functions of the non nAChRs-based proteins affected by the presence of nicotine can be categorized to into four types which are oxidative stress, protein modification/degradation, metabolism, and the cellular signaling pathways. (Salomon, Marcinowski et al. 1996, Li and Wang 2007) The suggested potential connection between tobacco alkaloids and protein modifications via oxidative stress is important since protein modifications linked to oxidative stress are known to be associated with many diseases such as Alzheimer’s disease (AD), Parkinson’s disease and hereditary renal amyloidosis.(Dobson 2003, Kane, Konu et al. 2004, Chiti and Dobson 2006, Li and Wang 2007, Ramierez-Alvarado 2008, Chiti and Dobson 2009)

A project overview for the proposed research is contained in Table 1. This table lays out the timeline for the project and clarifies the contributions of each of the collaborating groups. Though organized on a Fall – Summer schedule for each year, this could be altered based on the funding cycle.

Table 1: Project overview, including the contributions of collaborating groups

Project Activity 1st granted year 2nd granted year

Fall Spring Summer Fall Spring SummerInitialize the infrastructure: Student recruitment, purchase of equipment

Specific Aim #1: Potts’ group Develop methods using GC-MS and HPLC

Specific Aim #1: Potts’ group Analyze samples utilizing GC-MS and HPLC

Specific Aim #2: Carver’s group Optimize conditions for cell growth and viability assays

Specific Aim #2: Carver’s group Carry out cell growth and viability assays

Specific Aim #3: Kim and Albu’s groups Develop methods for fluorescent spectroscopy and 2D gel-electrophoresis-based approaches

Specific Aim #3: Kim and Albu’s groups Collect data utilizing fluorescent spectroscopy and 2D gel-electrophoresis

Dissemination: Conference presentations, Manuscript preparation/submission

B Innovation The proposed research is innovative because we will conduct a more thorough evaluation and analysis regarding which chemical additives are present in e-cigarettes and also determine if protein modification is possible due to the chemical additives. To our knowledge a study of this nature has not been done.

Specific Aim 1 focuses on the analytical determination of nicotine in possibly mislabeled filling solutions; those labeled as containing no or low levels of nicotine. Initial studies by the FDA have found this could be a significant issue with these unregulated products.(Trehy, Ye et al. 2011) More samples must be analyzed to determine how prolific mislabeling is across the e-cigarette

industry. Persistent mislabeling issues necessitate that legislation be developed to regulate these products. The presence of other tobacco alkaloids is of special concern because the unregulated e-cigarette company does not report the presence of these chemicals. The oxidation of nicotine (Sellergren, Zander et al. 1998) is a possible source for these other alkaloids, but impure starting materials could also lead to their existence in the solutions.

The effects of nicotine, tar, and other chemicals have been extensively studied using normal cigarettes. However, nicotine, cotinine, myosmine, anatabine, anabasine, and -nicotyrine, the major chemicals found in cartridges used in e-cigarettes, have not been well researched.(Trehy, Ye et al. 2011) Some initial surveys of refill fluids and their effects have been reported (Bahl, Lin et al. 2012), however, much more research is needed to better understand how these chemicals interact in different cell systems. Specific Aim 2 will establish initial datasets working with three different cell lines to study the chemical interactions as they are associated with e-cigarette refill solutions.

Specific Aim 3 focuses on the role of tobacco alkaloids toward protein modifications. To date, we are at the beginning stage of understanding the role of tobacco alkaloids in terms of possible proteome alteration. However the reported studies have been limited to the role of nicotine toward selected proteins in the brain tissues or cells isolated from the brain tissues. Furthermore, the role of nicotine and other tobacco products in terms of affecting the structural integrity of a protein requires a thorough investigation. For instance, many publications focused on the beneficial role of nicotine for AD by reducing aggregation of -amyloid(Salomon, Marcinowski et al. 1996, Zeng, Zhang et al. 2001, Liu and Zhao 2004, Zhang, Liu et al. 2006, Liu, Zhang et al. 2007), yet there is no clearly understood mechanism on how nicotine down-regulates amyloidosis leading to decreased neurotoxicity. Most importantly, major studies solely focused on the biological role of nicotine rather than including other tobacco products such as cotinine, anatabine, and myosmine.(Benowitz, Hukkanen et al. 2009, Trehy, Brown et al. 2012) Considering the fact that cotinine is a major metabolite of nicotine and smoking results in exposure to other tobacco alkaloids in addition to nicotine, it is logical to expand the proteomics study to other tobacco alkaloids.

In this background, we propose to investigate the effects of tobacco alkaloids toward protein modifications of the cellular proteins and an individual protein utilizing two effective approaches and the main analysis techniques are fluorescence spectroscopy and 2D-gel electrophoresis. While LC-MS is a more conventionally utilized technique for studying protein modifications, the proposed approaches offer advantages over LC-MS due to simple and less laborious work-up procedures, therefore suitable for this project involving undergraduate researchers. In addition, the Chemistry Department at UTC is not equipped with a proteomics-grade LC-MS. Furthermore, fluorescence techniques can be used to monitor conformational changes in proteins. The first approach is focused on investigation of the effects of tobacco alkaloids toward any conformational changes in lysozyme. Lysozyme was chosen for this study since its native form is relatively stable and it has been used in several studies focusing on protein modifications related to amyloidosis.(Booth, Sunde et al. 1997, Goda, Takano et al. 2000, Dumoulin, Jumita et al. 2006)

The second approach is to investigate if selected tobacco alkaloids are involved in protein regulation at the cellular level. We will focus on two biomarkers for this study, which are the level of cellular protein phosphorylation and the cellular features affected by the intracellular level of Ca2+. Protein phosphorylation or phosphoregulation of cellular proteins is the most commonly occurring type of covalent modification utilized in regulating the activities of many proteins especially cellular enzymes.(Tarrant and Cole 2009) Tumorous cells generally exhibit higher level of phosphorylated proteins compared to normal cells. Interestingly, the phosphorylation of certain proteins could trigger the ATP dependent ubiquitin/proteasome protein degradation pathway and result in apoptosis. Therefore, the level of phosphorylated proteins serves as an efficient biomarker for evaluating the effect of tobacco alkaloid on phosphoregulation. Calcium ions are involved in the calcium signaling pathway either through the membrane transport of Ca2+ via ion channels leading to membrane depolarization or through utilization of Ca2+ as a secondary messenger to activate protein kinase C. (Clapham 2007) Nicotine binding to some nAChRs results in activating the nAChRs and this further

leads to influx of Ca2+ as well as disruption in membrane potential. (Barik and Wonnacott 2009) In this context, there are two questions that remain to be answered. First, would other tobacco alkaloids result in an increase of the intracellular level of Ca2+? Second, would the Ca2+-dependent cellular processes such as apoptosis be affected by the presence of tobacco alkaloids? To answer these questions, we will utilize a bioluminescent Ca2+ indicator such as aequorin or coelenterazine to quantitate the intracellular level of Ca2+ upon exposure to the selected tobacco alkaloids. In terms of the Ca2+ signaling pathway affected by the presence of tobacco alkaloids, the main interest is apoptosis triggered by the activation of caspase-3 induced by the increased level of Ca2+ C Approach

Specific Aim #1: Identify and quantify tobacco alkaloids in disposable e-cigarettes and associated filling solutions labeled as low or non-nicotine containing. Gas chromatography – mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) will be used in a two-fold method for the determination of tobacco alkaloids in filling solutions labeled as nicotine-free or containing less than 24mg/mL of nicotine. Based on the results previously published by FDA researchers, we know that other tobacco alkaloids are present in the e-cigarette filing solutions.(Trehy, Ye et al. 2011) But also, we have previously quantified nicotine in filling solutions using both GC-MS and HPLC. Justice (2012)

We began this research in 2010, looking solely at nicotine concentrations. We initially chose GC-MS because we had been using this method for the determination of nicotine and other combustion products in cigarette butts. For the filling solutions that were analyzed, nicotine concentrations were determined to be at levels 85% of the reported value using GC-MS. We expanded the research using HPLC with UV detection in an effort to find a more economical method for quantitative analysis and to confirm our previous results. HPLC confirmed the GC-MS analysis as illustrated in Figure 1. Though both methods are ultimately suitable for quantitation, GC-MS analysis of nicotine and other tobacco alkaloids requires the addition of an internal standard.(Skoog, Holler et al. 2007) Cotinine is a commonly used internal standard for nicotine, but it has also been found in filling solutions as a contaminant.(Trehy, Ye et al. 2011) Additionally, the sample dilution with organic solvents that is required for GC analysis considerably changes the sample matrix. Therefore, GC-MS is a useful tool for identification, but not in determining concentration for this research.

After identification of the various alkaloids present in each filling solution by GC-MS, analysis will continue with quantification by HPLC with UV detection. Column separation of these compounds is facilitated with C18 stationary phase column. Previous studies by Trehy et al have found that the Phenomenex Gemini-NX 5mm C18 110A column improves resolution of the alkaloids so this column will be purchased and installed with our existing instrumentation – ThermoFinnigan Spectrosystem HPLC.(Trehy, Ye et al. 2011) At a buffered pH of 8.7, all of the compounds of interest achieve baseline resolution within 20 minutes. Prior to analysis of the filling solutions, the figures of merit (accuracy, reproducibility, selectivity and sensitivity) for the technique and specific instrument will be determined. These determinations include but are not restricted to limit of detection, limit of quantitation, linear dynamic range, number of theoretical plates, and column resolution.

Figure 1: Comparison of Nicotine Analysis by GC-MS and HPLC

Once alkaloid concentrations in the filling solutions have been determined, the data will be statistically analyzed to determine if the differences (if any) from the labeled concentrations are significant. A simple two-sided t-test can be used for basic comparison of the measured nicotine concentration to the product labels. As no reported values are available for the other alkaloids, we will compare the non-nicotinic concentrations by analyzing at least three filling solutions from the same manufacturer, labeled with the same concentration of nicotine. This relationship will also require a t-test but the comparison will be of two experimental means. Additionally, we will use multivariate analysis to determine if manufacturer identity plays a factor in the labeling differences by reducing all the possible variables (including labeled nicotine concentration, experimental nicotine and other detected tobacco alkaloid concentrations) through principal component analysis (PCA). The new resulting variables will reveal if there is a correlation between filling solutions from the same manufacturer. Low detection limits and contamination could be problematic and therefore it is crucial that sample preparation and storage be evaluated as possible sources of error.

Though we don’t anticipate any major problems with this analysis because previous research in our labs has proven it effective, alternate strategies for analysis might be necessary. Baseline resolution of the alkaloids by HPLC could be difficult, therefore a thorough study of eluent polarity and gradient adjustments maybe required. If resolution continues to be an issue, analysis by HPLC with a diode array detector would be explored as an alternative. Our current instrument cannot scan the UV spectrum as the compounds elute, but an HPLC with diode array detection would allow for full spectral analysis of each compound. A diode array HPLC is currently out for bid through the Chemistry Department and funds for the purchase have been encumbered.

Specific Aim #2: Document the effects of e-cigarette fluids on the growth and viability of cells in culture. To determine the growth and viability of cells, we will use cell cultures to assay the response of different cell types to treatment with the various chemicals, both individually and in the combined e-cigarette products. Cells, i.e., human transformed lung cells, human skin fibroblast cells, and human lung fibroblast cells, purchased from ATCC (American Type Culture Collection) will be cultured in a 5% CO2 incubator at 37°C and 95% relative humidity using standard methods already in place within the laboratory. This work has been initiated and the human transformed lung cell line CCL-185 has been established in the laboratory. Over the summer of 2013, we will start the initial screening with this cell culture. There are a number of cell cultures available through ATCC, so if we encounter any issues with basic cell culture protocols, we have the ability to purchase similar cultures for use in the project. Initially, established cultures will be grown with various concentrations of refill solution as well as isolated chemicals at a range of concentrations. These synchronic cultures will be used as a reagent for both Specific Aim 2 and Specific Aim 3.

Effects on growth and cell viability will be investigated as an initial screen of tobacco alkaloids. Cultures exposed to increasing chemical doses of alkaloids will be tested for proliferation and cytotoxicity using the MTT assay (Alley, Scudiere et al. 1988, van de Loosdrecht, Beelen et al. 1994). The MTT assay is a very robust assay that has been used extensively in cell cultures.(Mosmann 1983, Berridge, Herst et al. 2005) Data will be analyzed using a BioTek ELx800 microplate reader to compare control (non-chemically exposed) cultures to treated cultures. No observed adverse effect levels (NOAEL) will be determined by the dose–response curve. The treatments should provide a basic understanding of how cell cultures are affected by e-cigarette products, and provide a dataset that we can relate to the known effects of regular cigarette exposures. Specific Aim #3: Establish whether protein modifications are induced by selected tobacco alkaloids found in e-cigarettes. In order to investigate the nature of protein modifications induced by selected tobacco alkaloids, lysozyme will be utilized for sub-aim 3a as a model protein and the modifications of cellular proteins will be studied for sub-aim 3b. The compounds of interest use in Aim 3 will be nicotine, cotinine, myosmine and -nicotyrine.

Currently, we are in the process of optimizing conditions for sub-aim 3a. Lysozyme was chosen for this study since it is stable, has been used in several studies focusing on protein

modifications related to amyloidosis, and is fluorescent due to its 6 tryprophan and 3 tyrosine residues. (Goda et al. 2000, Protein Data Bank) Figure 2 shows a representative 1D-gel image of lysozyme after incubation with an amyloidosis-inducing toxin without the presence of tobacco alkaloids. As shown in the figure, we were able to induce oligomerization of lysozyme as early as 10 min of incubation, which is based on multiple bands appearing in the gel. Furthermore, the incubation of lysozyme with an amyloidosis-inducing toxin for 3 h or longer lead to amyloidal lysozyme which was insoluble, therefore the recovery was low as evident in L6 and L7 of the gel. It is our intent to investigate whether the addition of selected tobacco alkaloids to the same incubation conditions will reverse the induced amyloidosis.

Figure 3 shows a series of fluorescence emission spectra of lysozyme at pH 7.0 as the lysozyme concentration was increased from 5 M to 0.5 mM. The spectra were obtained at 37C using a Horiba Jobin Yvon Fluorolog-3 spectrophotometer with a full spectrum xenon pulse single source lamp. The samples were recorded in a 1 cm path-length quartz cuvette using an excitation wavelength of 280 nm. The emission spectra were recorded over the 290-800 nm range in increments of 1 nm with a band pass of 2 nm for both excitation and emission, and with an integration time of 0.5 s. We are currently in the process of optimizing the parameters such as band path and integration time in order to optimize the fluorescence spectral profile of lysozyme.

Strategy for sub-aim 3a: Lysozyme will be incubated with the compounds of interest (nicotine, anabasine, mysomine, -nicotyrine, and cotinine) at pH 7.0 and 37C in a time-dependent manner and the conformational changes in lysozyme will be detected by fluorescence spectroscopy before and after dialysis. In addition, amyloidal lysozyme will be incubated in the same manner as the non-amyloidal lysozyme. The comparison studies of conformational changes of amyloidal and non-amyloidal lysozyme upon exposure to the selected compounds will shed light on the role of tobacco alkaloids in terms of modulating protein structures focused on conformational changes.

Strategy for sub-aim 3b: Cells will be incubated with the compounds of interest as described in Specific Aim 2. After the incubation, the cells will be lysed and each aliquot of the cells will be subjected to further analysis such as 2D-gel electrophoresis and fluorescence analysis. For 2D-gel electrophoresis, an aliquot of the cell extract containing cellular proteins will be treated with staining dye to detect phosphorylated proteins after running gel electrophoresis. Then, the gels will be stained through multiple staining procedures and be submitted to image collection to overlay the images for in-combination analysis of phosphorylation and total-protein expression. For quantitating the level of phosphorylation, an aliquot of the same cell extract will be treated with a staining dye (either Pro-Q Diamond or fluorescein analogs) and submitted to fluorescence detection utilizing a Horiba fluorimeter available in Dr. Kim & Albu’s labs and a BioTek ELx800 microplate reader to be purchased through this support. For the Ca2+ -based biomarker study, an aliquot of the cell extract will be treated with a fluorescent Ca2+ indicator for monitoring the intracellular level of Ca2+ or with fluorescent caspase-3 antibody for monitoring the caspase-3-dependent apoptosis level prior to fluorescence detection.

Figure 2. Lysozyme amyloidosis-inducing toxin without the presence of tobacco alkaloids. MM, molecular marker; L1, unmodified lysozyme at time 0; L2, unmodified lysozyme at time 24 h; L3, lysozyme + toxin at 10 min; L4, lysozyme + toxin at 30 min; L5-L7, lysozyme + toxin at 1, 3, and 24 h, respectively.

Figure 3. Fluorescence emission spectra of lysozyme at various concentrations (5 M to 0.5 mM) at pH 7.0.

BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

Gretchen E. Potts POSITION TITLE

UC Foundation Associate Professor

eRA COMMONS USER NAME (credential, e.g., agency login)

GPOTTS EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable) MM/YY FIELD OF STUDY

Miami University, Oxford, OH B.S. 05/96 Chemistry University of Florida, Gainesville, FL Ph.D. 08/2000 Analytical Chemistry

Please refer to the application instructions in order to complete sections A, B, C, and D of the Biographical Sketch.

A. Personal Statement This research presented in this proposal focuses on the analysis and biological implications of electronic cigarette filling solutions. My analytical chemistry expertise is very necessary for this study. I will provide analytical analysis and determination for the filling solutions and I will also provide standards for the other researchers. During my Ph.D. education, I was trained in analytical analysis techniques as well as chemometrics. Both of these areas are crucial to the development of this project. For the last thirteen years at UTC, I have focused my research on environmental analytical analysis. Research in the area of cigarette litter led to the development of this project. For the last two years we have worked on the analysis of the filling solutions and for the last year, we have been studying the solutions using high performance liquid chromatography. I have given seven invited talks at universities and three invited talks at the Pittcon (the premier conference for analytical chemists). My students have given 14 research presentations at National ACS, Pittcon and regional ACS meetings. With my expertise and experience, I will be able to contribute meaningfully to the goals and specific aims of the proposed research.

B. Positions and Honors Positions: 2000-2002 Visiting Assistant Professor, College of Charleston, Charleston, SC

2002-2008 Assistant Professor, The University of Tennessee at Chattanooga, Chattanooga, TN 2008-present UC Fdn Associate Professor, The University of Tennessee at Chattanooga, Chattanooga, TN Honors

1996 Gamma Theta Phi, Chemistry Honor Society 1996 Mortar Board National Honor Society 2003 Sigma Xi Scientific Research Society 2002 – 2006 NSF – RSEC Program at UT-Knoxville, Faculty Mentor 2007 University of Chattanooga Foundation named professorship 2012 UTC Keep the Starts Shining Teaching and Other Experience: 2002 – present Instrumental Analysis, Methods of Environmental Analysis, Quantitative Analysis, research advisor on 8 departmental honors theses Affiliations American Chemical Society, Analytical Chemistry Division (Local Section Char, 2011) Member of the ACS Exam Institute Committee for the 2012 Quantitative Analysis Exam (2011-2012)

C. Selected peer-reviewed publications 1. Moerman, Jessica.W.; Potts, Gretchen E. Analysis of metals leached from smoked cigarette litter,

Tobacco Control, 2011, 20 (Suppl 1) i30-i35. 2. Easparam, Sarah; Potts, Gretchen E. Elemental Analysis of Shiitake Mushrooms by ICP-OES. J.

Underg. Chem. Res. 2006, 5, 83 – 87. 3. Brooks, Timothy; Bodkin, Tom E.; Potts, Gretchen E. and Stephanie A. Smullen. Elemental Analysis of

Human Cremains Using ICP-OES to Classify Legitimate and Contaminated Cremains. J. For. Sci. 2006, 51, 967 – 973.

4. I.B. Gornushkin, B.W. Smith, G.E. Potts, N. Omenetto and J.D. Winefordner, Some Considerations on the Correlation Between Signal and Background in LIBS using Single Shot Analysis, Anal. Chem. 1999, 71 (23) 5447 – 5449.

D. Research Support Completed Research Support University of Tennessee Research Foundation 01/09/2012 - 10/12/2012 Hassan Almoazen (PI), Transdermal Delivery of Trace Elements by Nanoemulsion Technology as an Alternative to Parenteral Delivery Role: CoPI UTC Faculty Summer Fellowship 05/01/2012 - 07/31/2012 Gretchen Potts (PI), Analysis of contaminants leached from cigarette litter into surface soils Role: PI ACS Division of Analytical Chemistry IYC Grant 08/24/2011 - 12/31/2011 Gretchen Potts (PI), Chemistry Concepts for Elementary Students: A Hands on Approach to Bring Science into the Classroom Role: PI UTC Faculty Summer Fellowship 05/01/2008 – 07/31/2008 Gretchen Potts (PI), Contaminants in the Little Sequatchie River: An environmental water analysis study, Role: PI UTC Faculty Summer Fellowship 05/01/2005 – 07/31/2005 Gretchen Potts (PI), Elemental Analysis of human cremains Role; PI UTC Grote Faculty Fellowship 05/01/2004 – 07/31/2004 Gretchen Potts (PI), Biomonitoring: A study of metal uptake in plants of the Chattanooga River Valley Role: PI UTC Provost Student Research Awards, 12 funded over the last 12 years, total funding of $10,998

Program Director/Principal Investigator ():

PHS 398/2590 (Rev. 06/09) Page 1 Biographical Sketch Format Page

BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2.


NAME

Ethan A. Carver POSITION TITLE

Associate Professor


ECARVER EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)



University of Tennessee at Chattanooga B.S. 05/91 Biology University of Tennessee at Knoxville Ph.D. 05/99 Biomedical Sciences The Jackson Laboratory Post-Doc 07/02 Developmental Biology

Please refer to the application instructions in order to complete sections A, B, C, and D of the Biographical Sketch. A. PERSONAL STATEMENT The goal of my research is to better understand the molecular processes involved and their relationships with each other during the process of muscle development and differentiation. I currently use zebrafish as a primary model for muscle development, but have an extensive background in murine genomics and development. I have a very broad background in murine genetics and genomics, with specific training at two historically significant mouse research facilities, Oak Ridge National Laboratory, and The Jackson Laboratory. During my doctoral work, my research centered on a positional cloning project in mice, as well as homology mapping between human and mouse genomes. I also became competent in advance microscopy through work on microscopes at the Laboratory and through the attendance of a course offered at Cold Spring Harbor Laboratory. As a postdoctoral fellow at The Jackson Laboratory, I carried out early developmental biology research on the Snail Family genes in mice. Work at Incyte Genomics and the National Center for Biotechnology Information, I learned bioinformatics and large dataset manipulation. As PI or co-Investigator on a number of university-, NSF- and NIH-funded grants, I have begun to establish my research laboratory and have developed collaborations between myself and researchers at the University of Memphis (Dr. Charles Lessman), and St. Jude Children's Research Hospital (Michael R. Taylor). Overall, I have initiated research projects and have started to disseminate results and regional meetings with the end goal to publish the findings in peer reviewed journals as I have previously done. B. POSITIONS AND HONORS Professional Experience: 1999-2002 Post-doctoral, The Jackson Laboratory, Developmental Biology 2001 Bioinformatics Consultant, Proteome/Incyte Genomics 2002-2005 Bioinformatics Scientist, ComputerCraft/NCBI contractor, National Center for Biotechnology Information, NIH 2005-11 Assistant Professor of Developmental Biology, University of Tennessee-Chattanooga, Department of Biological and Environmental Sciences 2011-present Associate Professor of Developmental Biology, University of Tennessee-Chattanooga, Department of Biological and Environmental Sciences Teaching and Other Experience: 2005-present Assistant Professor of Developmental Biology, UTC: Human Anatomy, Developmental Biology, Human Development and Disease, Environmental Genetics, and research electives


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Special Course Work: Advanced In Situ Hybridization & Immunocytochemisty. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. October 13-26, 1993. Professional Affiliations: Society for Developmental Biology National Association of Advisors for the Health Professions (NAAHP) Tennessee Academy of Sciences C. SELECTED PEER-REVIEWED PUBLICATIONS 1. Kim, J., Vaughn, AR., Cho, C., Albu, TV. and Carver, EA. (2012) Modifications of Ribonuclease A Induced by p-Benzoquinone. BioOrganic Chemistry 40:92–98. 2. Lessman, C.A., Taylor, M.R., Orisme, W., and Carver, E.A. (2010). Use of Flatbed Transparency Scanners in Zebrafish Research: Versatile and Economical Adjuncts to Traditional Imaging Tools for the Danio rerio Laboratory. Methods Cell Biol. 2010;100:295-322. 3. Murray, S.A., Carver, E.A., and Gridley, T. (2006). Generation of a Snail1 (Snai1) conditional null allele. Genesis. 44(1):7-11. 4. Savagner, P., Kusewitt, D.F., Carver, E.A., Magnino, F., Choi, C., Gridley, T., and Hudson, L.G. (2005). Developmental transcription factor slug is required for effective re-epithelialization by adult keratinocytes. J Cell Physiol. 202(3):858-866. 5. Oram, K., Carver, E. A.., and Gridley, T. (2003). Slug expression during organogenesis in mice. Anat Rec. 271A:189-91. 6. Carver, E. A., Oram, K., and Gridley, T. (2002). Craniosynostosis in Twist heterozygous mice: a model for Saethre-Chotzen syndrome. Anat Rec. 268:90-92. 7. Zhao, P., Iezzi, S., Carver, E., Dressman, D., Gridley, T., Sartorelli, V., and Hoffman E.P. (2002). Slug is a novel downstream target of MyoD. Temporal profiling in muscle regeneration. J Biol Chem. 277:30091-300101. 8. Carver, E. A., and Gridley, T. (2002). Epithelial-Mesenchymal Transitions and Cancer. Current Genomics Vol.3, No.4:355-361. 9. Carver, E. A., Jiang, R., Lan, Y., Oram, K. F., and Gridley, T. (2001). The Mouse Snail Gene encodes a Key Regulator of the Epithelial-Mesenchymal Transition. Moleclular and Cellular Biology 21:8184-8188. 10. Carver, E. A., Olsen, A., Hamann , J., and Stubbs, L. (1999). Physical Mapping of EMR1 and CD97 in Human Chromosome 19 and assignment of Cd97 to Mouse Chromosome 8 suggest an Ancient Genomic Duplication. Mammalian Genome 10:1039-1040. 11. Carver, E. A., Issel-Tarver, L., Rine, J., Olsen, A., and Stubbs, L. (1998). Location of Mouse and Human Genes Corresponding to Conserved Canine Olfactory Receptor Gene Subfamilies. Mammalian Genome 9:349-354. 12. Carver, E. A., and Stubbs, L. (1997). Zooming In on the Human-Mouse Comparative Map: Genome Conservation Re-examined on a High-Resolution Scale. Genome Research 7:1123-1137. 13. Kim, J., Carver, E. A., and Stubbs, L. (1997). Amplification and Sequencing of End Fragments from Bacterial Artificial Chromosome (BAC) clones by Single-Primer PCR. Analytical Biochemistry 253: 272-275. 14. Culiat, C., Carver, E. A., Walkowicz, M., Rinchik, E., Cacheiro, N., Russell, L., Generoso, W., and Stubbs, L. (1997). Induced Chromosomal Rearrangements as Tools for Identifying Critical Developmental Genes and Pathways in the Mouse. Reproductive Toxicology 11:345-351. 15. Stubbs, L, Carver, E. A., Cacheiro, N. L. A., Shelby, M., and Generoso, W. (1997). Generation and Characterization of Heritable Reciprocal Translocations in Mice. Methods: A Companion to Methods in Enzymology 13: 397-408. 16. Stubbs, L., Carver, E. A., Shannon, M., Kim, J., Geisler, J., Generoso, E., Stanford, B., Dunn,W., Mohrenweiser, H., Zimmermann, W., Watt, S., and Ashworth, L. (1996). Detailed Comparative Map of Human Chromosome 19q and Related Regions of the Mouse Genome. Genomics 35: 499-508. 17. Stubbs, L., Carver, E., Ashworth, L., and Lopez-Molina, L. (1996). Location of the DBP Transcription Factor Gene in Man and Mouse. Mammalian Genome 7:65-67.


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D. RESEARCH SUPPORT STEM Professional Development grant (Co-director/participant) 5/01/2013-12/31/2013 Learning Science through Writing: This grant is to provide teachers with new strategies to enhance learning in sciences. A series of exercises and workshop have been developed it illustrate how to implement these strategies into the working classroom. ($197,109) Role:Co-director NIH 1R15AR055798-01A1 Carver (PI) 9/01/2009-8/31/2013 The overall goal of this research is to gain a better understanding of the developmental processes involved in muscle development, while providing a dynamic learning environment for undergraduate students. In this proposal, we plan to identify and characterize a novel collection of zebrafish motility mutants and identify the defective gene(s) in these mutants. ($200,391) Role: PI NSF DBI-0922941 Carver (PI) 9/15/2009-8/31/2012 Acquisition of a Confocal Microscopy System for expansion of research and teaching capabilities within the Univeristy and surrounding region, and to increase collaborative studies. ($184,188) Role: PI NSF DBI-0821057 Carver (Co-PI) 9/01/2008-8/31/2011 Acquisition of a Microarray Scanner and Real-Time PCR System for Interdisciplinary Research and Teaching in an Undergraduate College Setting. ($87,250) Role: Co-Investigator Summer 2009 UC Foundation Faculty Summer Research Fellowship (PI) ($5000) Spring 2007 UC Foundation Faculty Research Grant (PI) #R04-1011-088 ($3000) Spring 2007 UC Foundation Instructional Excellence Award (PI) #R04-1011-082 ($1421) Fall 2005 UC Foundation Instructional Excellence Award (PI) #R04-1011-082 ($1465) 7/01-7/02 American Cancer Society grant recipient (PI) #PT-01-130-10-MGO 7/01 National Institute of Child Health and Human Development (PI) #1F32HD08714-01A1*** Declined to accept American Cancer Society grant 6/99-7/01 National Cancer Institute, Institutional Training Grant Recipient #CA09217



NAME

Kim, Jisook POSITION TITLE

Assistant Professor of Chemistry


JISOOK70 EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)



Chung-ang University, Korea B.S. 1993 Chemistry Chung-ang University, Korea M.S. 1995 Chemistry Case Western Reserve University Ph. D. 1996-2001 Chemistry University of Minnesota Post-doc 2001-2003 Medicinal Chemistry MIT Post-doc 2003-2004 Biol. Eng. & Toxicology

A. Personal Statement The role of Dr. Kim is to investigate the nature of protein modifications induced by selected tobacco alkaloids using a model protein and cellular proteins upon exposure to selected tobacco alkaloids utilizing 2D-gel electrophoresis. Dr. Kim’s research group developed analytical methodologies focused on non-amyloidal ribonuclease (RNase) which is similar to the target protein lysozyme. With minor modifications in the developed methodologies, Dr. Kim and coworkers will be able to study the induced modifications occurring in lysozyme and cellular proteins. Dr. Kim established a vigorous research program at the multidisciplinary biochemistry area in an effort to educate UTC undergraduate students. Dr. Kim has served as co-PI for four different NSF proposals, published 9 papers in biochemistry area-related journals and given 22 presentations at regional and national ACS meetings including 10 presentations given by her undergraduate research students.

B. Positions and Honors Positions: 1995-1996 Research Scientist, Daelim Petroleum Company, Korea 2004-2007 Non-tenure track assistant professor, Chemistry, Tennessee Tech University, TN 2007-Present Assistant Professor, Chemistry, University of Tennessee at Chattanooga, TN Teaching and Other Experience: 2004-2008 Non-tenure track assistant professor, Chemistry, Tennessee Tech University, TN 2007-Present Assistant Professor, Chemistry, University of Tennessee at Chattanooga, TN Professional Affiliations: American Chemical Society C. Selected peer-reviewed publications. 1. Kim J. (2013) “Biological implications of benzoquinones” (Book Chapter) in the book “Quinone: Occurrence,

Medicinal Uses and Physiological Importance”, Nova Science Publishers, Inc., ISBN 978-1-62618-323-0. 2. Vaughn AR, Redman, CB, Kang, SM, Kim J. (2013) Biological implications of 2-chlorocyclohexa-2,5-diene-

1,4-dione toward ribonuclease A. Advances in Bioscience and Biotechnology, 4: 22. 3. Kim J, Vaughn AR, Cho C, Albu TV, Carver EA. (2012) Modifications of ribonuclease A induced by p-

benzoquinone. Bioorganic Chemistry, 40: 92. 4. Kim J, Zhang Y, Ran C, Sayre LM. (2006) Inactivation of bovine plasma amine oxidase by haloallylamines.

Bioorganic & Medicinal Chemistry 14: 1444. 5. Kim J, Park SB, Tretyakova NY, Wagner CR. (2005) A method for quantitating the intracellular metabolism

of AZT amino acid phosphoramidate pronucleotides by capillary high-performance liquid chromatography-electrospray ionization mass spectrometry. Molecular Pharmaceutics 2: 233.

6. Kim J, Chou TF, Griesgraber GW, Wagner CR. (2004) Direct measurement of nucleoside monophosphate delivery from a phosphoramidate pronucleotide by stable isotope labeling and LC-ESI-MS/MS. Molecular Pharmaceutics 1: 102.

7. Kim J, Drontle DP, Wagner CR. (2004) Monitoring the intracellular metabolism of nucleoside phosphoramidate pronucleotides by 31P NMR, Nucleosides Nucleotides & Nucleic Acids 23: 48.

8. Kim J, Wagner CR. (2003) Studies of the intracellular metabolism of nucleoside amino acid phosphoramidates utilizing 31P NMR and LC/MS. Antiviral Research 57: 47.

9. Choo CKK, Kim KH, Lee YE, Kim J. (2002) A scientific study of Choson white ware: early porcelain from a royal kiln at Kwangju Usanni. Archaeometry 44: 199.

10. Ling K, Kim J, Sayre LM. (2001) Catalytic turnover of benzylamine by a model for the lysine tyrosylquinone (LTQ) cofactor of lysyl oxidase. Journal of American Chemical Society 123: 9606.

D. Research Support. Completed Research Support: NSF#0619476 09/01/06-08/31/09 Major Research Instrumentation grant Liquid Chromatography mass spectrometer for biochemical and environmental analysis Role: Co-PI NSF#0821057 09/01/08-08/31/11 Major Research Instrumentation grant Acquisition of a microarray scanner and real-time PCR system for interdisciplinary research and teaching in an undergraduate college setting. Role: Co-PI NSF Award #0922941 09/01/09-08/31/12 Major Research Instrumentation grant Acquisition of a Microscopy Core System Role: Co-PI NSF Award #CHEM-0951711 05/01/10-04/30/13 Major Research Instrumentation grant Acquisition of a Benchtop Single Crystal X-ray Diffractometer for the University of Tennessee at Chattanooga Role: Co-PI E. Advised students and student projects 1. Robert A. Patton “RNase modification by 1,4-benzoquinone and 1,4-hydroquinone.” 2. Steven W. Ledford “Electrophoretic analyses of RNase modification by 2-methyl-1,4-benzoquinone.” 3. Caitlin B. Redman “Kinetic analysis of RNase modification by a series of substituted 1,4-benzoquinones.” 4. Albert R. Vaughn “RNase modifications induced by selected PAH quinones.” 5. Albert R. Vaughn’s DHON Thesis “Modification of ribonuclease A induced by 2-chlorcyclohexa-2,5-diene-

1,4-dione.” 6. Min J. Kang “Kinetic analysis of RNase modification by a series of substituted 1,4-benzoquinones by UV-

Vis spectroscopy.” 7. George E. Campbell “Studies of biologically active molecules inducing plasmid DNA modification.” 8. Chris Cho "Kinetic analysis of RNase modification by a series of substituted 1,4-benzoquinones by UV-Vis

spectroscopy.” 9. Ashley Cardenal “Studies of biologically active [Rh9S3Cl3] inducing plasmid DNA modification.” 10. Anna Zyglewska “Lysozyme modifications induced by 2-chloro-1,4-benzoquinone.” 11. Jeffrey McDonald (Co-advisor with Dr. Albu) “UV-Vis spectroscopic analysis of lysozyme modifications

induced by selected quinones.” 12. Hendrik Greve “SDS-PAGE analysis of lysozyme modifications induced by selected quinones.”



NAME

TITUS V. ALBU POSITION TITLE

Assistant Professor of Chemistry


TIALBU EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)



University of Bucharest License 06/94 Chemistry University of Bucharest M.S. 06/95 Chemistry Case Western Reserve University University of Minnesota

Ph.D. Postdoctoral

08/00 07/02

Chemistry Chemistry

A. Personal Statement The present study focuses on the analysis and biological implications of e-cigarette filling solutions. Due to my vast experience in computational chemistry in particular and physical chemistry in general, I will be able to provide a distinct point of view to the study. After conducting research in thermal analysis of materials and kinetics of catalyzed reactions in my native country, I carried out computational studies as PhD student working on two different projects, one dealing to modeling the electrochemical oxygen reduction at the cathodes of fuel cells and the other dealing with modeling of properties of doped diamond. As a postdoctoral research associate, I worked on development and applications of direct dynamics methods in determining accurately rate constants and other dynamic parameters on a variety of gas-phase and solution reactions. As a faculty, I developed my independent research program focusing in application of computational chemistry to understand in more detail various molecular properties and reactivity. Identifying and characterizing pathways for hydrogen abstraction from hydrofluorocarbons, developing methods for accurate investigation of electrochemical oxygen reduction, analyzing association patterns in HF-H2O mixture, and characterizing molecular properties of thiosemicarbazones are few distinct studies I was conducting in the past few years. During my sabbatical leave two years ago, I began working on experimentally determining protein modifications induced by quinones using fluorescence spectroscopy. This project was a collaborative effort with two of current co-investigators (Drs. Kim and Carver). For family reasons, last summer I resigned my tenured position at Tennessee Tech and started a tenure-track position at UT Chattanooga. Since then, I continued both my computational and experimental research interests. In conclusion, I have been successfully involved in chemistry research for almost twenty years in a wide range of areas, and I will be able to significantly contribute at achieving to goals of the proposed study through participating as co-PI. B. Positions and Honors Employment 1994-1996 Researcher, Institute of Physical Chemistry of the Romanian Academy, Bucharest, Romania 2002-2007 Assistant Professor, Department of Chemistry, Tennessee Tech University, Cookeville, TN 2007-2012 Associate Professor, Department of Chemistry, Tennessee Tech University, Cookeville, TN 2012- Assistant Professor, Department of Chemistry, University of Tennessee at Chattanooga, TN Honors 1998 The Department of Chemistry Award for Excellence in Graduate Student Teaching, Case

Western Reserve University, OH 1998 Graduate Dean’s Instructional Excellence Award, Case Western Reserve University, OH 2001 Minnesota Supercomputing Institute Research Scholar Award, University of Minnesota, MN 2004 Ralph E. Powe Junior Faculty Enhancement Award, Oak Ridge Associated Universities

2010 Non-Instructional Faculty Assignment Award, Tennessee Tech University, Cookeville, TN C. Selected Peer-reviewed Publications (selected from 27 peer-reviewed publications) 1. Kim, J., Vaughn, A. R., Cho, C., Albu, T. V. & Carver, E. (2012). Modifications of Ribonuclease A Induced

by p-Benzoquinone. Bioorganic Chemistry, 40, 92-98. 2. Albu, T. V., Espinosa-Garcia, J. & Truhlar, D. G. (2007). Computational Chemistry of Polyatomic Reaction

Kinetics and Dynamics: The Quest for an Accurate CH5 Potential Energy Surface. Chemical Reviews, 107, 5101-5132.

3. Baburao, B., Visco, D. P. Jr. & Albu, T. V. (2007). Association Patterns in (HF)m(H2O)n (m + n = 2-8) Clusters. Journal of Physical Chemistry A, 111, 7940-7956.

4. Swaminathan, S. & Albu, T. V. (2007). Hybrid Density Functional Theory with a Specific Reaction Parameter: Hydrogen Abstraction Reaction of Trifluoromethane by the Hydroxyl Radical. Theoretical Chemistry Accounts, 117, 383-395.

5. De Silva, N. W. S. V. N. & Albu, T. V. (2007). A Theoretical Investigation on the Isomerism and the NMR Properties of Thiosemicarbazones. Central European Journal of Chemistry, 5, 396-419.

6. De Silva, N. W. S. V. N., Lisic, E. C. & Albu, T. V. (2006). Hybrid Density Functional Theory Investigation of a Series of Alloxan-Based Thiosemicarbazones and Semicarbazones. Central European Journal of Chemistry, 4, 646-665.

7. Swaminathan, S. & Albu, T. V. (2006). Hybrid Density Functional Theory with Specific Reaction Parameter: Hydrogen Abstraction Reaction of Fluoromethane by the Hydroxyl Radical. Journal of Physical Chemistry A, 110, 7663-7671.

8. Albu, T. V. (2006). Hybrid Density Functional Theory Study of Fragment Ions Generated during Mass Spectrometry of 1,3-Dioxane Derivatives. Rapid Communications in Mass Spectrometry, 20, 1871-1876.

9. Zuev, P. S., Sheridan, R. S., Albu, T. V., Truhlar, D. G., Hrovat, D. A. & Borden, W. T. (2003). Carbon Tunneling from a Single Quantum State. Science, 299, 867-870.

10. Albu, T. V., Lynch, B. J., Truhlar, D. G., Goren, A., Hrovat, D. A., Borden, W. T. & Moss, R. A. (2002) Dynamics of 1,2-Hydrogen Migration in Carbenes and Ring Expansion in Cyclopropylcarbenes. Journal of Physical Chemistry A, 106, 5323-5338.

11. Albu, T. V., Anderson, A. B. & Angus, J. C. (2002). Dopants in Diamond Nanoparticles and Bulk. Density Functional Study of Substitutional B, N, P, SB, S, PN, O, NN, and Interstitial H. Journal of Electrochemical Society, 149, E143-E147.

12. Albu, T. V., Corchado, J. C. & Truhlar, D. G. (2001). Molecular Mechanics for Chemical Reactions: A Standard Strategy for Using Multiconfiguration Molecular Mechanics for Variational Transition State Theory with Optimized Multidimensional Tunneling. Journal of Physical Chemistry A, 105, 8465-8487.

13. Albu, T. V. & Anderson, A. B. (2001). Improvements to an Ab initio Model for Electrochemical Processes: Application to the Outer-Sphere Oxygen Reduction. Electrochimica Acta, 46, 3001-3013.

14. Anderson, A. B. & Albu, T. V. (2000). Catalytic Effect of Platinum on Oxygen Reduction: An Ab Initio Model Including Dependence on the Electrode Potential. Journal of Electrochemical Society, 147, 4229-4238.

15. Anderson, A. B. & Albu, T. V. (1999). Ab initio Determination of Reversible Potentials and Activation Energies for Outer-Sphere Oxygen Reduction to Water and the Reverse Oxidation Reaction. Journal of American Chemical Society, 121, 11855-11863.

D. Research Support Ongoing Research Support Faculty Research Development Grant, Tennessee Tech University 07/01/2012-06/30/2013 Understanding biological activity of thiosemicarbazones using molecular modeling The goal of this project is to apply quantum mechanical methodologies to investigate molecular properties of two series of thiosemicarbazones and to correlate these properties with their observed biological activity. Completed Research Support Faculty Research Development Grant, Tennessee Tech University 07/01/2011-06/30/2012 A computational study on the nucleophilic attack on the C=C double bond of quinones The goal of this project is to carry out hybrid density functional theory computations of reactions, products, and transitions states for nucleophilic attacks at C=C bond of quinones by N-containing compounds.

Non-Instructional Faculty Assignment Grant, Tennessee Tech University 07/01/2010-06/30/2011 An investigation in the biological activity of quinones using fluorescence techniques The goal of this project is to investigate experimentally the modifications of model proteins (i.e., RNase) by benzoquinone and substituted quinones.

In-depth analysis of e-cigarette filling solutions and their biological implications Equipment and Environment

The Departments of Chemistry and Biology and Environmental Sciences are adequately

equipped in terms of the required instruments. Chemistry Laboratory and Major Equipment: The Chemistry Department at the

University of Tennessee at Chattanooga is housed in Grote Hall, a building renovated in 2010. Any sample preparation for this project will be performed in a lab that is 2040 sq ft in size, has 16 fume hoods and 11 lab benches. The HPLC analysis will be performed in an advanced instrumentation lab. It is 717 sq ft in size, contains one fume hood and three lab benches. Access to both of these labs is limited to authorized personnel only through the use of swipe card access.

The analytical determination described in Specific Aim #1 will be performed using existing equipment, including a ThermoFinnigan TraceGC PolarisQ and a ThermoFinnigan SpectraSystem HPLC purchased in 2002. A new HPLC with autsosampler and diode array detector is currently out for bid by the Chemistry Department (funds are already in place for the purchase) and it will also be used for the analysis once it is installed in August 2013. Drs. Kim and Albu’s labs are equipped well with most of the necessary instruments for Specific Aim #3 such as a Horiba Jobin-Yvon Fluorescence Spectrometer, a Shimadzu UV-Vis spectrophotometer, and several 1D-electrophoresis units. Repair and maintenance of all units is supported by a departmental endowment.

A 2D-gel electrophoretic unit and an imager that can visualize the protein gels stained by fluorescent dyes are needed to complete Specific Aim #3. Therefore, a small portion of the requested budget will be used to purchase a 2D-gel electrophoretic unit and an imager.

Biology Laboratory and Major Equipment: Located adjacent to Dr. Carver’s office in Holt Hall (next to Grote Hall) are research and teaching laboratories. The research laboratory is furnished with histological and molecular biology equipment. Within the Biology and Environmental Science Department, standard gas/air lines exist, as well as a Millipore distilled water system. Other appropriate equipment such as, microcentrifuges, two hybridization ovens, a UV transilluminator and photodocumentation system, UV/VIS spectrofluorometer, tissue homogenizer, Biotek Synergy HT spectrofluorometer, FMBio fluorescence imaging system, HPLC with UV Detector, Sorvall high-speed refrigerated centrifuge, -80°C freezer, liquid nitrogen storage tank, a nucleic acid vacuum concentration unit, a multi-system electroporation unit, digital scales and a Real-Time PCR System are present.

For tissue culture, a small NuAire, class II, tissue culture hood and a Fisher Scientific two layer growth chamber are available for use in another laboratory. A range of microscopy equipment is available. These include an Olympus BX51 phase contrast/fluorescent microscope with digital camera, an inverted microscope with a digital camera, an Olympus BX31 microscope and basic stereomicroscope equipped with a digital camera, and a separate Leica S8APO stereomicroscope with conventional and fluorescence light sources. There is also an Olympus Fluroview 1000 confocal microscope and a Joel NeoScope SEM. Repair and maintenance of hardware is contracted to local vendors.

in-depth analysis of e-cigarette filling solutions and ... · e-cigarette filling solutions will be...

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