Dr. Lori Hensley – Biology

Cannabinoids as Novel Therapeutics for Pediatric Cancers

 Ewing’s Sarcoma is a pediatric bone cancer that is highly aggressive, leading to a five-year survival rate of only 30% even with multi-modal treatment protocols.  Improved therapeutic options are desperately needed.  Our research has focused on the ability of non-psychoactive cannabinoids to induce death and inhibit metastases in cells from members of the Ewing’s sarcoma family of tumors and other solid pediatric cancers.  Our data demonstrate these compounds can successfully kill Ewing’s sarcoma cells and related tumor cells in vitro through the induction of apoptosis. Our data further suggest we can limit the migration of tumor cells and endothelial cells (required for new blood vessel formation to feed the tumors), potentially reducing their ability to spread throughout the body.  In order to test the efficacy of our drugs in a more realistic model of human cancer, we developed a novel bioluminescent mouse model of Ewing’s sarcoma in which engineered tumor cells are injected into the tibiae of mice, and the growth of  tumors in control and treated mice can be tracked using specific imaging techniques.  For summer research, we will be further investigating the effects of our compounds on metastatic potential and angiogenesis, and a larger mouse study is currently being planned. Also central to our summer research will be experiments to identify the receptor and subsequent cellular signaling pathways these cannabinoids are using to exert their effects. Students involved with this project will learn and use techniques such as tissue culture, cell viability assays, western blots, PCR, migration and invasion assays, and immunohistochemistry.

Dr. Marty Perry – Chemistry

Engineering cytochrome P450s for enantioselective oxidative reactions

 The overall aim of this study is to determine the molecular basis for enantioselective oxidations by CYP2C9 as a step toward engineering biocatalysts for environmentally friendly or “green” applications. CYP2C9 is a member of a large class of oxidative enzymes called cytochrome P450s (CYP for specific isoforms). The attractive properties of these enzymes has led to their use in the synthesis of bulk and specialty chemicals in industry and medicine, development of biosensors, and bioremediation of environmental pollutants. Despite these advances, the inherent high regio- and enantio-selectivity of P450 reactions have not been tapped for meeting the rising demand for chiral chemicals. It is hypothesized that the location ofspecific CYP2C9 residues in the active site are “hotspot” contacts in enantioselective binding and catalytic processes for the enzyme. CYP2C9 is one of the most enantioselective P450 enzymes, and thus the active site residues are likely optimized for chiral interactions. In following, those residues are sensitive to mutations of functional residues and make CYP2C9 an excellent target for identifying and validating enantioselective “hotspot” residues. In this study, a novel strategy is employed to identify contact residues that distinguish between enantiomeric substrates through computational studies and then assess their importance by mutating those residues and determining theeffects on binding and catalysis. Preliminary studies have already corroborated previous reports of critical residues and identified new leads for study. The completion of the project will ultimately provide a critical foundation for engineering improved or novel P450 biocatalysts that are capable of addressing demand for chiral chemicals using “green” chemistry. We will accomplish these efforts through a multi-institutional collaborative project involving computational (in silico) and biochemical (in vitro) approaches. Specifically, we will test our hypothesis through the following specific aims:

►Aim 1: Identify enantioselective “hotspot” residues involved in CYP2C9 complexes through computational docking studies.

►Aim 2: Validate enantioselective “hotspot” residues involved in CYP2C9 binding of molecules using mutant enzymes.

►Aim 3: Validate enantioselective “hotspot” residues involved in CYP2C9 catalytic reactions using mutant enzymes

Dr. Joe Bradshaw – Chemistry

Vitamin D deficiency has become a topic of interest in recent publications (1-7). Vitamin D, along with calcium, promotes proper bone growth in children and aids in the prevention of osteoporosis in older adults.  Vitamin D is present in two forms, Vitamin D3 and Vitamin D2 (Figure 1). D3 is produced after ultraviolet light-stimulated conversion of 7-dehydrocholesterol in the skin (3).  Vitamin D2 is derived from plant sources.  Both Vitamin D2 and D3 are metabolized in the liver to form 25-hydroxyvitamin D2 (25-OH D2) and 25-hydroxyvitamin D3 (25-OH D3) (Figure 2), respectively. In addition, biologically inactive 3-epi analogs of 25-OH D2 and 25-OH D3 have been reported, especially in young children (3). The levels of the 25-hydroxy metabolites are routinely measured for diagnostic assessment of vitamin D related diseases; however, recent studies have indicated that separation from the inactive 3-epi analogs may provide more accurate information for treatment and prevention.  Analytical methods that can accurately quantitate both of the 25-hydroxyvitamin D analytes in the presence of 3-epi analogs may become essential for diagnosis and monitoring of patients with vitamin D disorders.

 Vitamin D is derived in vivo primarily from UVB radiation impacting the skin where 7-dehydrocholesterol is converted to cholecalciferol (vitamin D3) through a photolytic, nonenzymatic reaction.  Smaller amounts of vitamin D are acquired through diet, including vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) from animal and plant sources, respectively. Vitamin D3 is metabolized to 25-hydroxyvitamin D3 in the liver and then to the active form 1,25-dihydroxyvitamin D3 in the kidney. Circulating levels of 1,25-dihydroxyvitamin D are tightly controlled and serve to modulate expression of specific genes through the vitamin D receptor.

Vitamin D acts to promote intestinal calcium and phosphate absorption and also controls their liberation from bone. In addition to rickets and osteomalacia, low serum 25-hydroxyvitamin D3 and D2, has been linked to hypertension, autoimmune diseases, and cancer (5,6).

The most abundant metabolite of vitamin D is 25-hydroxyvitamin D and it is considered one of the best indicators of vitamin D status. Accurately measuring 25-hydroxyvitamin D is necessary to adequately assess an individual’s vitamin D levels and to help determine the role of vitamin D in various diseases.

HPLC analysis of 25-OH D2 and 25-OH D3 is classically performed using C18 stationary phases. Under such conditions, the 3-epi analogs are not resolved and thus are included in the overall reported value. Recently Phinney, et al., reported the use of a cyano HPLC column for the effective separation of the 25-OH and the 3-epi forms for use in reference measurement procedures (1).   Although effective, the conditions necessitate a run time better than 40 minutes limiting its utility for routine highthroughput analyses.

Immunoassays suffer from high reagent cost, narrow dynamic range, and poor selectivity (inability to distinguish 25-hydroxyvitamin D3 from 25-hydroxyvitamin D2) (5). These inadequacies of immunoassays make them unattractive for vitamin D analysis in patient samples.  

Research will be conducted into development for a quick and efficient method for extraction of vitamin D from serum and analysis of vitamin D by HPLC.  The current generation of this technology is capable of reliably determining 25-hydroxyvitamin D concentrations across the entire clinical range.  Accurate and precise measurements of both 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 and the epi analoges should be routinely achieved using LC. The primary goal of this research is to develop a simple sample preparation step while accommodating high throughput for 25-hydroxyvitamin D runs, ~100 samples/day.

References

1. Tai, S. S.-C.; Bedner, M.; Phinney, K. W. Analytical Chemistry 2010, 82, 1942-1948.

2. Higashi, T.; Homma, S.; Iwata, H.; Shimada, K. Journal of Pharmaceutical and Biomedical Analysis 2002, 29, 947-955.

3. Higashi, T.; Shimada, K.; Toyo’oka, T. Journal of Chromatography B 2010, 878, 1654-1661.

4. Ginde AA, Liu MC, Camargo Jr CA, Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004, Arch Intern Med 2009;169:626-632

5. Borradale D, and Kimlin M, Vitamin D and disease: an insight into traditional functions and new roles for the ‘sunshine vitamin’, Nutr Res Rev 2009;22:118-136.

6. Adams, JS, Hewison M, Update in Vitamin D J Clin Endocrinol Metab 2010 95:471-478.

7. Lai JKC, Lucas RM, Clements MS, Harrison SL, Banks E, Assessing vitamin D status: Pitfalls for the unwary, Mol. Nutr. Food Res. 2010 54:1-10.

Dr. Detri Brech – Dietetics and Nutrition

Comparison of the Pre- and Post-Assessments of Height, Weight, BMI and Nutrition Knowledge of Children Participating in a Nutrition/Physical Activity Program to Children in a Control Group

The OBU student chosen to work with this project will assist with each phase of the research. Time spent in the research will be extremely rewarding. The following is a brief description of the project. The research project will consist of the student developing age-specific nutrition education materials to be presented weekly to the children ages 4 – 12 years in the treatment group. Prior to the beginning of the program, the student researcher will assist me in weighing and measuring each child, calculating the child’s BMI, and assessing nutrition knowledge of each child in the treatment and control groups. The months of June and July will be spent in running the nutrition/physical education program with the treatment group. Upon completion of the program, post-assessment measurements will be made of the treatment and control groups. The student researcher will then collate the data and calculate results comparing pre-test and post-test within and between each group. If you enjoy working with children, this project will impact your life.

Dr. Tim Hayes – Chemistry

 Phototoxicity of Modified Porphyrins: Potential Agents for Photodynamic Chemotherapy

Photodynamic therapy (PDT) was devised to circumvent many of the side effects of traditional chemotherapy.  In PDT, the agents that are used are non-toxic until exposed to certain wavelengths of light, which leads to the production of reactive oxygen species.  Porphyrins are heterocyclic compounds that are used as prosthetic groups in many enzymes; the best known is heme in red blood cells.  The first photosensitizer approved by the FDA for PDT was a porphyrin oligomer. 

One of the most common forms of cancer for which there is currently no satisfactory treatment is triple-negative breast cancer.  Many breast cancers can be treated with antagonists to estrogen or progesterone because these hormones drive proliferation of the tumor cells.  Brest cancers which express the cell surface protein HER2 can be targeted with an antibody against HER2.  However, cells which lack all 3 of these molecules and are ER(-)/PR(-_/HER2(-) (triple negative) are resistant to most treatments.  We would like to test the efficacy of our PDT agents against breast cancer cells that are triple negative.

There are at least 2 different subcellular targets for porphyrins.  Many dissolve in the cell membranes, targeted by the hydrophobic nature of the ring itself and constituents attached to the ring.  Others bind to DNA and RNA and induce photocleavage; these may be active in the metallated form.  In addition, some porphyrins are more active on some cell types than on others. 

Several series of modified porphyrins have been synthesized in the lab of Dr. Joe Bradshaw.  These were designed to enhance their water-solubility and, in one case, to add a side chain that may enhance transport across the blood-brain barrier.  My lab has been testing these porphyrin derivatives for phototoxicity.  The purpose of this project is to continue and extend testing of these compounds to characterize their utility as agents for photodynamic therapy, focusing specifically on triple-negative breast cancer. 

We will test the toxicity of these compounds with and without light exposure, the concentration dependence of phototoxicity, the best wavelengths for photoactivation and how well the compounds are taken up by cells.  Each porphyrin will be tested on several cell lines, including triple-negative and ER(+)/HER2(+) breast-cancer derived cell lines, to assess the variability of the phototoxicity between cell types.  We will use this data to correlate the modifications of the porphyrins with their phototoxicity and other properties to aid in design of the next generation of modifications to make them more water-soluble, less toxic in the dark and more phototoxic. 

Dr. Sara Hubbard – Chemistry

The analysis of bis-phenol A in water samples using solid-matrix fluorescence spectroscopy

 Bis-phenol A (BPA) is used in the production of epoxy resins and polycarbonate plastics and frequently remains in these materials.  In vitro and in vivo experiments show that BPA exhibits weak estrogenic activity by binding to and activating estrogen receptors.  Suspected effects of modified endocrine functions may be reduced fertility, altered development, and cancer in estrogen-sensitive tissues.  Infants and children are particularly at risk due to their still developing neurological and endocrine systems and the fact that their ability to detoxify and eliminate substances such as BPA is immature.  This concern led to a change in the plastic used for baby bottles and other containers, which removes BPA from the plastic, allowing manufacturer’s to claim their products are “BPA free”.  Despite this reduction of BPA in current food packaging, BPA is still present in our environment due to BPA-containing waste in landfills or ocean trash.  The environmental sources, though at low levels, are a particular concern for the exposure of infants and children and also for aquatic organisms.

 BPA is a fluorescent compound, which means following excitation, it will emit radiation at a longer wavelength than the exciting wavelength.  BPA is excited at ~280 nm and emits and ~310nm. This emitted light can be measured and correlated to the concentration of BPA present in a sample.  Fluorescence is a very sensitive and selective technique, which makes it possible to determine very low concentrations BPA with fewer concerns about outside interferences compared to absorption spectroscopy.  However, the fluorescence intensity can also be decreased (quenched) by collisions within a solution.  By preparing a solid sample, such as a sugar glass, containing the sample it is possible to detect lower concentrations of BPA compared to a sample in solution.

 The goals of this project are to obtain fluorescence data for BPA using sugar glasses as a solid matrix and to explore the effects of temperature and time on the leaching of BPA-containing materials into water samples.

Dr. Ruth Plymale- Biology

Sampling Fountain Beverage Machines for Biofilm-forming Bacteria

Biofilms are communities of microorganisms that are encased by a matrix composed of extracellular polymers and stuck to a surface, usually a moist or wet surface. Enclosed within the matrix, biofilm bacteria experience several benefits compared to planktonic (free-floating) bacteria. The biofilm matrix is adhesive, allowing biofilm microbes to firmly attach to the substrate and to each other. Thus, biofilm microbes have better access in aquatic environments to stationary environmental nutrients than do planktonic bacteria. Biofilm microbes are thus well-protected from various environmental stresses. In a previous study, researchers from Hollins University cultured bacteria from sodas, diet sodas and water dispensed from fountain beverage machines. The average number of bacteria cultured from each beverage type was not significantly different, despite significant differences in beverage pH. The findings that bacterial numbers were unaffected by low pH and were consistent across beverage types suggest that the bacteria were growing in the beverage machines as biofilms, although the Hollins University researchers did not determine the biofilm formation capacity of the bacteria they isolated. The goals of this project are to confirm whether bacteria may be cultured from beverages dispensed from fountain beverage machines and to quantify the biofilm-forming ability of microbes cultured from dispensed beverages.

Dr. Nathan Reyna – Biology

Identification of the expression patterns of 17 candidate genes in transgenic tobacco plants. 

Programmed cell death (PCD) is an essential cellular process characterized by cell shrinkage, nuclear condensation and fragmentation that leads to the selective elimination of cells and has been reported to play a major role in cystic fibrosis, oncogenesis and other disease occurrences.  There appears to be some evolutionarily conserved signaling pathways for an apoptotic-like programmed cell death (AL-PCD) in plants.  My research this summer will attempt to use a plant model, tobacco to identify new genes associated with oxidative stress and apoptosis.  In addition to new gene discovery we will examine the expression patterns of these genes (mRNA) using Quantitative Real Time PCR.  Students will learn multiple molecular biology related techniques. Students working on this project will be expected to present their work at both on and off campus venues. A portion of this project may be conducted at the University of Arkansas, Little Rock and will provide an opportunity for OBU students to make a connection with a graduate degree granting university. In addition this project is partially funded through the Arkansas Space Grant Consortium. Students will be expected to present their research off campus.

Dr. Jim Taylor – Biology

The Development of Arabidopsis in Lowered Atmospheric Pressure

The growth of plants in outer space will be dependent on how plants respond to altered atmospheric pressure, gravity, and modified light. The purpose of this study is to further investigate the development of plants at lower atmospheric pressure by including a hypobaric chamber. The experiment will include 50% of the earth normal 101 kPa using wildtype Arabidopsis and light and gravity sensitive and insensitive mutants. If time allows, altering the perception of gravity using a clinostat will be included in the experiments using the hypobaric chambers. These studies will give more insight to how plants develop at lower pressure and in altered gravity which will give a better understanding to how plants may develop in long term space travel.