Life Sciences

Jason Ray, Benjamin Bitner, Kyle Kener, and Brent Jackson, Brigham Young University Life Sciences Type 1 and Type 2 diabetes are increasing at an alarming rate. Both types of diabetes result in decreased functional β-cell mass, which is defined as the number of β-cells multiplied by their Glucose Stimulated Insulin Secretion rate. Decreased functional β-cell mass inhibits regulation of blood glucose levels. β-cells have an extremely low proliferation rate after adolescence, meaning the functional β-cell mass cannot naturally recover. Increasing functional β-cell mass could provide a cure for diabetes, either through pancreatic islet transplants or through enhancement of the endogenous β-cell population. Nkx6.1 has been shown to increase β-cell proliferation by inducing the nuclear receptors Nr4a.1 and Nr4a.3. We have shown that Nkx6.1 increases expression of the gene c-Fos, and that c-Fos induces expression of Nr4a1 and Nr4a3. Furthermore, we have shown that c-Fos is sufficient to induce proliferation of β-cells in the INS-1 832/3 cell line and in primary rat islets. Finally, using lenti-sh-c-Fos to create a stable c-Fos deficient stable cell line, we have demonstrated that Nkx6.1 mediated proliferation is modified by the lack of c-Fos. We propose a model by which c-Fos is a critical link between Nkx6.1 and Nr4a mediated β-cell proliferation.

Kari Norman, Utah State University Life Sciences The identification of important areas for biodiversity is essential for affective allocation of limited conservation resources. Since Myers’ seminal biodiversity hotspot paper in 2000, great strides have been made in more accurate global mapping. While global mapping identifies important patterns in large-scale biodiversity, conservation management rarely if ever occurs on the same scale. Finer scale mapping is therefore essential to make research applicable for on the ground decision-making. This project focuses on North America, a continent that holds no priority areas when included in a global analysis. Using citizen scientist data of multiple vertebrate and plant taxa, we created maps of species richness and significantly rare species. In addition, we created similar maps based on range map data and compared to determine if the two data types produced different biodiversity priority areas. The results of this study provide informative maps about the locations of species, their vulnerability, and how human action may impact them, as well as underline the importance of understanding the data behind their construction.

Tyler White, Kyle Jensen, Jacob Hatch, Trevor Wienclaw, Brian Hair, and Aaron Trent, Brigham Young University Life Sciences The bacterium Staphylococcus Aureus (SA) is a common commensal organism of the human nose and skin that can lead to diseases such as pneumonia, endocarditis, and meningitis. These SA infections are usually remedied via antibiotic treatment with methicillin. However, over the course of frequent exposure to various antibiotics, the bacteria have evolved resistance to methicillin to create resistant strains (MRSA) that is completely resistant to this drug and many others (leaving vancomycin as the last viable option). As a result, the need for an antibiotic alternative treatment for this infection is becoming increasingly crucial especially in hospitals where nosocomial transmission of the bacteria is prevalent. Phage are bacterial-specific viruses that have shown promise as anti- bacterial agents for human bacterial pathogens. Thus far, we have isolated 18 different samples of phage that lyse MRSA and 51 strains of S. Aureus (over 20 of which are MRSA). We are currently testing all phage samples against these bacterial strains to determine which phage possess a broad tropism to kill many SA/MRSA isolates. We are also conducting experiments to determine the relative lytic ability of each phage. Lytic phage with broad tropism and/or strong lytic ability will have their genomes sequenced in order to verify their respective novelty. Novel phage will be aggregated to form a highly virulent cocktail that can be used to treat a broad spectrum of SA and MRSA infections.

Michael Hinckley and Cayden Westwood, Dixie State University Life Sciences Clinical laboratory standards state that urine samples should be tested within 2 hours of collection. If testing is delayed beyond that time frame the sample needs to be refrigerated to inhibit urea conversion to ammonia and an increase in urine pH. Here medical lab science students investigated whether urine samples left at room temperature for 48 hours resulted in significant increases in urine pH with concomitant microbial growth. Five clean-catch urine specimens were obtained and promptly tested for pH levels using a sterile pH meter (EcoTestr, Oakton Instruments). Samples were then aliquoted into capped and uncapped containers that were left at room temperature. Samples were tested at 0, 8, 20, 24, 32 and 48 hours. The pH values from the uncapped and capped samples from these time periods were analyzed using a paired two tailed t-test. Results indicated one sample out of five was significant (P = .05). Initial samples were gram stained, then read to determine the presence of bacteria. Specimens were cultured on 5% Sheep Blood and MacConkey agar plates. Plates were read for bacterial growth at 24 hours incubation and growth was identified on two samples. At 48 hours, both uncapped and capped urine cultures were plated again and three samples demonstrated bacterial growth the following day. Microbial testing identified normal urogenital flora and pathogenic bacteria. Urine with pathogenic bacteria demonstrated significant increases in pH, while additional cultures with bacterial growth also increased but not with significance. Future studies could employ a larger sample size from both healthy and diseased individuals. Furthermore, identification of microbes that will thrive in acidic and alkaline pH would be of interest.

Joshua Peterson, Brigham Young University Life Sciences Cancer kills millions of people every year. Cancer occurs when cells proliferate at an excessive rate and do not die as regularly functioning cells do. In cancer cells, the mechanism that initiates apoptosis (cell death) is inhibited. These cells eventually multiply to the point where they interfere with physiological function and cause death. Therefore, one of the aims of cancer research is to find treatments that initiate apoptosis in cancer cells. Many current chemotherapeutic (anti- cancer) treatments are toxic to all mitotic cells, rather than to cancer cells alone. Studies have shown that resveratrol, which is found in grapes, peanuts, and berries, facilitates apoptosis in cancer cells without causing apoptosis in regular cells. The apoptotic activity of resveratrol in tumor cells is dependent on a large, sustained increase in cytoplasmic calcium ion concentration. In properly functioning cells, plasma membrane Ca2+- ATPase (PMCA) pumps excess calcium from the cytosol to the extracellular space. PMCA prevents toxically high levels of calcium and maintains cytoplasmic calcium homeostasis. Using live cell microscopy to monitor intracellular calcium ion concentration, we explore the direct and indirect effects of resveratrol on PMCA activity in MDA-MB-231 (a breast cancer cell line).

Coral Gardner, Southern Utah University Life Sciences Sea spiders (pycnogonids) are a small group of exclusively marine arthropods which resemble terrestrial spiders. One family, the Colossendeidae can grow to a very large adult size, much larger than any other pycnogonid. Colossendeis colossea, the largest known species, has a leg span of up to 70 cm and is found in both very deep ocean waters and shallower water in the Antarctic. Since it has been very difficult to obtain and study live specimens of this species, very little is known of their biology and natural history. We have obtained a number of preserved specimens, all labeled C. colossea, from the United States National Museum and are asking the question, are all of these specimens actually C. colossea? To answer this question, the specimens are being compared to syntypes from the Museum of Comparative Zoology and also to the original type description (Wilson, 1881). Based on previous scientific papers on C. colossea and closely related species, the following are used to separate species within this genus: relative proportions of terminal segments of walking leg and of pedipalp; location and number of eyes and shape of eye tubercle; proboscis shape and orientation; number of spine rows on terminal segments of oviger and shape of oviger terminal claw; abdomen size and orientation. Based on these, our preliminary results demonstrate that some of the United States National Museum specimens are not C. colossea since they differ from the syntypes and type description in several of the above characteristics.

Morgan Christiansen, Taylor Hoybjerg, and Ryan Cook, Brigham Young University Life Sciences Asthma is a chronic allergic disorder manifest by airway restriction due to inflammation, bronchoconstriction, and increased respiratory mucous secretion. As many as 300 million people worldwide are affected by asthma. It is becoming increasingly prevalent, especially in countries experiencing urbanization and Westernization. Asthma is currently the most common chronic illness among children in the U.S. and the third leading cause of hospitalization for children aged 0 – 15 yrs. Reservoir dust collection and area air sampling are the two primary methods of measuring allergen levels in house dust. Allergen sensitization leading to asthma is thought to occur prior to age six while the immune system is still naïve. In the case of the dust mite allergen Der p1, the exposure window may be as early as age two. However, little evidence is available to establish a dose–response relationship between inhalation exposure and early immunological sensitization to allergens. Temperature and relative humidity play a major role in dust mite survival and proliferation—indoor humidity above 50-60% in arid environments has been shown to support dust mite populations. Evaporative “swamp” coolers add moisture to cool the air and this increased humidity can create favorable environments for dust mite survival. The purpose of this research is to quantify the levels of dust mite antigen in homes with air conditioners and homes with swamp coolers in Utah Valley to determine the role humidity plays in the abundance of dust mite antigens. We are testing the hypothesis that homes with swamp coolers exhibit higher levels of antigen due to heightened humidity levels.

Morgan Hughes, Utah State University Life Sciences Elk feeding stations are used throughout the Western US as a means to prevent depredation on private lands (Smith,2001). Many of the unintended effects of such artificial congregation remain unexamined. In many species, increased densities result in increased parasite loads (Dietz, 1988) adding physiological stress to individual animals and reducing the economic value of the animal to sportsmen (Choquette, 1956). Through laboratory analysis of fecal float samples, I will monitor changes in the number of parasite eggs for elk at Hardware Ranch feeding station over the winter season. This is to discover if there may be negative implications of feeding stations which should be further examined. Increased prevalence of parasites could also indicate an increased danger for transmission of other diseases which are a threat to domestic livestock (Williams, 2002).

Amanda Hobson, Carrie Draney, Andrew Stratford, Thomas Becker, Danhong Lu, Michelle Arlotto,

Matthew Durrant and Dennis Eggett, Brigham Young University Life Sciences The drugs amantandine and rimantadine were previously used to treat influenza A infections in humans. In 2005, a mutation in the Influenza A M2 channel conferred resistance to these drugs, rendering them obsolete against treating influenza. The S31N amantadine-resistance mutation in the influenza A M2 sequence currently occurs more frequently in nature than the S31 wild type. Overcoming this mutation with new drug compounds is the focus of Influenza A M2 channel researchers. However, there are several other identified mutations that have also been shown to confer resistance to these drugs. These other mutations are thought to occur only in small frequencies in nature. A statistical analysis of 1,007 unique M2 protein sequences shows an enhanced frequency for the S31N/V27A dual amantidine resistant mutation in recent years, especially in swine, compared to expected frequencies based on the occurrence rates of individual mutations in wild type (S31) M2. The development of the S31N/V27A variant in the Midwestern US swine may be a harbinger of novel human strain development. At the same time, the different propensities for the V27A as compared to the V27T dual mutant may reflect differences in viral fitness or protein energetics, and this information could be exploited to focus drug development so as to reduce further drug insensitivity. V27A/S31N is a possible path forward for the evolution of M2, which may convey a new level of drug resistance and should receive attention in drug design.

Brent Knoblauch and Marty Larsen, Dixie State University Life Sciences Chytrid fungus (Batrachochytrium dendrobatidis) is one of the major hypothesized theories behind global amphibian decline. Canyon Tree frogs (Hyla arenicolor) found in Zion National Park have been found to contain infected individuals, however population sizes have grown despite infection with the usually deadly fungus. The present study sought to demonstrate a correlation between skin temperatures and fungal proliferation, and investigated what enabled Hyla arenicolor to survive and reproduce with chytrid present. Ten frogs were sampled with sterile swabs from each of seven different study areas found in Zion National Park. Along with each swab a temperature was taken via infrared thermometer and recorded. DNA was also extracted from the swabs, and Batrachochytrium dendrobatidis- specific primers were used in a touchdown PCR protocol to determine infection rates. Swabs from specimens with higher temperatures were expected to display lower instances of infection. This would establish for the first time a clear demonstration that high skin temperatures were destroying the fungus in infected individuals in the wild.

Matz Indergard, Paul Spruell, and Fredrich Govedich, Southern Utah University Life Sciences Dispersal of organisms allows them to colonize new habitats and may buffer against extirpation due to localized catastrophic events. For organisms inhabiting ephemeral environments, dispersal can be challenging, as suitable habitat is often small, isolated spatially, and unpredictable temporally. Fairy shrimp are small invertebrates that occupy ephemeral freshwater pools filled by rainfall. Fairy shrimp produce resting (resistant) eggs when conditions are not favorable. Upon the return of supportive conditions, these eggs will then hatch. Little is known about the methods of dispersal used by fairy shrimp. However, it has been suggested that the resting eggs are the most likely life stage to provide dispersal opportunities. We examined two proposed methods of egg dispersal for fairy shrimp (Brachinecta sp) from a series of ephemeral pools just north of Three Peaks near Cedar City, Utah. We hypothesized that dispersal could be attributed to prevailing winds or water runoff, which would disperse eggs in a direction corresponding to prevailing wind patterns or to outflow following the surface gradient. To conduct our test we established three reference points equally spaced in the linearly arrayed series of pools. We then collected soil samples at randomly determined locations around these center points to form an overlapping radial grid. We then added purified water to each of our samples and hatched any shrimp eggs that were present in the sediment. We then mapped the overall concentrations of shrimp in our sampling area. Results to date do not reveal an obvious pattern with prevailing wind direction and dispersal.

Bryce Anderson, Kemais Ehlers, Deborah Johnson, and Stephen Persaud, Brigham Young University Life Sciences Antigen presenting cells digest and display foreign proteins from infected cells on the major histocompatibility complex (MHC) that is recognized by T cells via their T cell receptor (TCR). LLO56 and LLO118 are CD4+ helper T cells with TCRs specific for the same Listeria monocytogenes epitope. Despite differing by only 15 amino acids, these TCRs have dramatically different primary and secondary responses to infection. TCRs have very low affinity for peptide MHC. We determined to generate high affinity T cell receptors to test if T cell activation would be improved. We reasoned that the single chain LLO118 and LLO56 TCRs (Vβ2-linker-Vα2) could be subjected to directed evolution to generate mutants that are more stable and then used as a template for engineering high affinity T cell receptors. Single chain LLO118 and LLO56 were fused to the yeast surface protein Aga-2 and error prone PCR was used to generate mutagenic libraries. Stabilized single chain TCRs (scTCRs) were selected for using biotinylated Vβ2 and Vα2 antibodies and anti-biotin beads. First generation clones with increased stability compared to wild type were isolated for both LLO118 and LLO56. A second mutagenic library using the first generation mutants as templates was produced and the most stable clones were selected after temperature denaturation, permitting isolation of clones with increased stability. We are currently engineering high affinity T cell receptors by generating affinity libraries using site directed mutagenesis of the CDR3 regions. These libraries are sorted for their ability to bind to MHC tetramers and individual clones are tested using flow cytometry. Generation of pathogen specific high affinity TCRs will increase our understanding of how T cells are activated and could also provide infection specific diagnostics and therapeutics.

Benjamin Bitner, Jason Ray, Kyle Kener, and Brent Wright, Brigham Young University Life Sciences β-cell mass is lost in both major forms of diabetes. Mature β-cell have restricted proliferative capacity. Studies aimed at increasing β-cell mass frequently have the unwanted side effects of decreased glucose stimulated insulin secretion (GSIS) or increased apoptotic rates. Without functional β-cells, the body is unable to reduce elevated blood glucose, which results in hyperglycemia induced maladies. Enhanaced GSIS could be used as a treatment for diabetes. Overexpression of the β-cell transcription factor Nkx6.1 induces β-cell proliferation, enhances GSIS and protects against apoptosis. Nkx6.1 induces expression of VGF, which is necessary for enhanced GSIS. Microarray analysis of Nkx6.1 expressing primary rat islets demonstrates upregulation of c-Fos at 24 and 48 hours after adenoviral transduction. We have shown that c-Fos upregulates expression of VGF. Finally, preliminary data suggest that c-Fos modulates GSIS as demonstrated in INS-1 β-cell line, stable INS-1 knockdown cells transduced with lenti-sh-c-Fos and in primary islets. We present a model by which c-Fos is necessary for Nkx6.1 mediated enhanced GSIS by inducing expression of VGF.

Coral Gardner and Don Long, Southern Utah University Life Sciences Irrigation of lawns and gardens in Cedar City, Utah is accomplished in two fundamentally different manners. In older neighborhoods, water is diverted from a natural stream (Coal Creek) into a series of canals and ditches for residential flood irrigation, compared to newer neighborhoods, which use sprinklers or similar devices from well water. The overall objective of this project is to better understand the chemical and biological changes that occur in irrigation and runoff waters in Cedar City. We are addressing the following three hypotheses. 1) Changes will be observed in water chemistry as surface water moves from Coal Creek through Cedar City. 2) Irrigation strategies influence water chemistry during periods of high precipitation. 3) Microbial community changes will be associated with differences in water chemistry. Water chemistry data including dissolved oxygen, conductivity, pH, alkalinity, dissolved organic matter and nitrate/nitrite were collected weekly at eight sites and during high precipitation events. Over the course of four months, there was a reduction in alkalinity levels among all sites. During high precipitation periods, nitrate was detected in newer neighborhoods. Dissolved oxygen and pH were at higher levels, while salinity and conductivity were lower in a reservoir site relative to irrigation canals. Future work will investigate bacterial community composition in Cedar City waters. We will isolate bacterial DNA from water samples and amplify the 16sRNA segment of DNA using the polymerase chain reaction. We will then correlate bacterial community composition to the water chemistry results described above.

Brianne Palmer, Utah State University Life Sciences Quaking aspen (Populus tremuloides) are declining in the interior west. Aspen are critical for the maintenance of wildlife habitat and are one of the few broadleaf trees in the western forest ecosystem. In western landscapes, it has recently been determined that a large proportion of aspen trees are triploid (three copies of each chromosome) and the remaining trees are diploid (two copies of each chromosomes). In this study we attempted to find differences in the physiology between the two cytotypes to determine future management strategies The size and density of stomata trees is likely to influence the survival of the species in water- and heat-stressed environments, since stomata control both photosynthesis rates and rates of water loss. Individuals with larger stomata or greater stomatal density may be efficient photosynthesizers but may be at risk for water loss during transpiration in environments with low precipitation and hot temperatures, such as those often seen during summers in the intermountain west. To determine if there is physiological differences between the cytotypes we measured the variation between stomatal sizes and densities between the cytotypes using cellulose acetate leaf impressions and microscope imagery. We collected leaves from twelve aspen stands (eight diploid and four triploid) representing the two cytotypes in Swan Flats and Fish Lake, Utah. From these analyses, we deduced that the variation in stomatal size and density is primarily among clones rather than among cytotypes. Further data collection and analyses will occur in the spring of 2015.

Austin Thompson and David Walton, Brigham Young University Life Sciences When we sustain a traumatic injury to the peripheral nervous system (PNS), our bodies elicit a series of responses to try to heal the acquired damage, including inflammation and repair processes. One of these responses is the increased expression of nerve growth factor receptor (NGFR), which helps to stimulate regeneration of the nerve. In a normal, healthy PNS, NGFR is rarely found. Following damage to the nerve, NGFR can be found in high levels around the damaged area. In our study, we are simulating traumatic injury to the sciatic nerve of rats in order to study the effects of regeneration after a local application of nerve growth factor (NGF). We are using both a physical crush model and a focal demyelination model to simulate the nerve injury. In the crush model, we are examining the effect of a crushed extracellular matrix (ECM) on degeneration and subsequent regeneration. In the focal demyelination model, we are investigating the effects of local demyelination with an intact ECM on degeneration and regeneration. In two additional experimental groups, we will perform an intraneural injection of NGF into the damaged sciatic nerve one week after the crush or a lysolecithin injection at the damaged site. We are examining the nerve both qualitatively using SEM and immunohistochemistry and quantitatively using electrophysiology. This allows us to understand the role of the ECM in regeneration, and its effect on the rate of regeneration. We hypothesize that the addition of NGF in combination with the increase of NGFR after injury will increase the rate of nerve regeneration. We expect regeneration to be faster in the focal demyelination model due to the presence of intact ECM than in the crush model where the ECM is damaged.

Westen Archibald, Trevor Smart, and Emily Forsyth, Brigham Young University Life Sciences The Beaver Dam Wash National Conservation Area is considered an important biodiversity hotspot for the Western United States. The wash is an ecotone on the Northeastern edge of the Mojave Desert and is directly adjacent to both the Colorado Plateau and the Great Basin. Thus, this area contains various species of flora and fauna from each of the surrounding biomes. Prior research of the Beaver Dam Wash has documented high aboveground biodiversity for a desert ecosystem however no research has been conducted on belowground diversity. For this reason we chose to characterize the diversity and distribution of entomophilic nematodes, as they are commonly used as a biological indicator to the surrounding ecosystems. We hypothesized that because plant and insect biodiversity is high here, we would also find a diversity of entomophilic nematodes that is higher than the surrounding areas. To test this hypothesis we collected soil samples from 15 sample sites representative of the diversity of the different ecological communities. We tested for patterns of codistribution between entomophilic nematodes and environmental variables, such as plant cover, proximity to water, presence of organic matter, elevation, ecosystem classification, and soil chemistry. We extracted nematodes from the soil samples and sequenced the 28s rDNA region of representative individuals from each sample. Nematode diversity was low; one species was broadly distributed, X others were more patchily distributed. No correlation was found between above ground factors or soil chemical properties (ppm phosphorous, percent organic matter, and percent soil moisture). We conclude that the distribution of entomophilic nematodes in this ecological confluence is to some degree stochastic and uncoupled from aboveground diversity or belowground soil conditions.

Simranvir Kaur, University of Utah Life Sciences The placenta is the major source of fetal serotonin during pregnancy, which is essential for fetal brain development. In Utah, approximately 13% of pregnant women take Selective Serotonin Reuptake Inhibitors (SSRIs) to treat depression, the use of which has been correlated to significantly lower serotonin levels in cord blood for newborns. Studies suggest association between maternal SSRI use and adverse outcomes such as preterm birth, cognitive deficit, and disruption of serotonergic systems. However the effect of SSRIs on placental gene expression, serotonin synthesis and transport in the placenta, is not known. This study evaluates the impact of maternal SSRI use on placental gene expression and levels of serotonin in the cord blood using a nested case-control observational study model. Biological samples will be collected until 20 cases (women taking SSRIs during pregnancy) and 20 appropriately matched controls have been enrolled into the study. Participants also complete an online questionnaire to measure depression and anxiety levels as well as document any medication they have taken during pregnancy. Data and sample collection for this study is still in progress. Once enough samples have been collected for batch analysis, we will complete RT-PCR and ELISA, expected in Spring 2015. Upon complete data analysis, we expect this study will help in targeting mothers who are at risk for adverse pregnancy outcomes and further provide suggestions for intervention.

Matthew Durrant and Shuqing Xu, Brigham Young University Life Sciences Researchers at the Max Planck Institute for Chemical Ecology have recently sequenced the Nicotiana attenuata (wild tobacco) genome. This genome affords researchers new opportunities to understand the evolution of this organism. One method for analyzing the evolution of specific genes in a given genome is referred to as phylostratigraphy, which makes use of large-scale BLAST sequence similarity searches. I designed a pipeline using the python programming language that implements a phylostratigraphic analysis to estimate the evolutionary age of all ~35,000 genes belonging to N. attenuata. By analyzing the large amounts of data produced by this BLAST search, each gene was assigned an estimated age through comparing the taxonomies of all other organisms that share similar protein sequences. This effectively answers the question “How old is this gene?” for each gene in the entire N. attenuata genome. Previous studies have produced microarray data that tracks the transcriptomic response of N. attenuata to an herbivore attack. By comparing the newly gathered gene evolutionary age information with this previously gathered microarray data, several new insights into the molecular signaling pathways of N. attenuata were made. It was found that at 1 hour following an herbivore attack, the transcriptome of N. attenuata is evolutionarily young, suggesting that the initial response to an herbivore attack recruits genes that have evolved more recently in the organism’s evolutionary history. At 5 hours after attack, however, there is a distinct decrease in the overall age of the N. attenuata transcriptome, suggesting that the organism is recruiting more ancient genes that are used to reconfigure the transcriptome of the organism. Beyond 5 hours, the transcriptome is once again relatively young, and it is clear that it has indeed been reconfigured to provide a more herbivore-specific defense response. This demonstrates a novel, evolutionary approach to analyzing signaling pathways in plants.

Stephen Tahan, Westminster College Life Sciences

Taylor Woodward, Brigham Young University Life Sciences The prevailing view is that enhancement of dopamine (DA) transmission in the mesocorticolimbic system, consisting of DA neurons in the ventral tegmental area (VTA) that innervate the nucleus accumbens (NAc), underlies the rewarding properties of alcohol and nicotine (NIC). Dopamine neurotransmission is regulated by inhibitory VTA GABA neurons. We have shown previously that VTA GABA neurons are excited by low-dose ethanol, but inhibited by moderate to high-dose ethanol. The aim of this study was to evaluate the role of []6 nicotinic cholinergic receptors (nAChRs) in ethanol effects on VTA GABA neurons as well as DA release in the NAc. In electrophysiology studies, superfusion of ethanol enhanced the frequency, but not amplitude, of mIPSCs recorded in acutely dissociated VTA GABA neurons from GAD GFP mice. The []6 nAChR antagonist []-conotoxin P1A did not affect mIPSCs, but prevented the ethanol (30 mM)-induced increase in mIPSC frequency. While microdialysis studies show that ethanol enhances DA release in the NAc, we and others have found that ethanol decreases DA release at terminals using fast scan cyclic voltammetry (FSCV). We have reported that ethanol inhibition of DA release at terminals in the NAc of ethanol-naïve animals is mediated by GABA. Using FSCV in the slice preparation, ethanol inhibited DA release in the NAc. Superfusion of the []6 nAChR antagonist []-conotoxin MII did not affect DA release, but prevented ethanol inhibition of DA release. Taken together, these findings suggest that ethanol enhancement of GABA inhibition of VTA GABA neurons is mediated by []6 nAChRs located on GABA terminals to other VTA neurons, affecting DA release in the NAc. Results from this study could provide a pharmacologic rationale for considering drugs that act selectively on nAChRs as therapeutic agents for the treatment of alcohol dependence and alcohol and NIC co-dependence.

Corey Wolf, Westminster College Life Sciences The yeast species Saccharomyces cerevisiae is used in numerous industries including brewing, baking, and winemaking. In recent years an increased demand for low ethanol beers has pushed breweries to develop a beer that is both rich in flavor and low in alcohol content. The current strategies employed to decrease the ethanol concentration in beer are costly, time intensive, and result in less flavorful beers. In this study, a method for decreasing alcohol content in the brewing process through the use of metabolic inhibitors of Alcohol Dehydrogenase is tested. Alcohol Dehydrogenase is the enzyme responsible for the conversion of acetaldehyde, an intermediate in the metabolic pathway of yeast, into ethanol. Two competitive inhibitors of Alcohol Dehydrogenase, Pyrazole and Fomepizole, were applied to small scale fermentations in varying concentrations in an attempt to decrease the overall ethanol yield of the beer. The application of either inhibitor resulted in an initial decreased rate of fermentation overall, measured as a change of sugar and ethanol concentration. In the later stages of fermentation, the rate of ethanol production returned to normal, suggesting the inhibitors were either catalyzed or removed by the cell. While this method for manipulating the products of fermentation did not yield the desired effects, this study did highlight the importance of the fermentation pathway for maintaing healthy Saccharomyces cerevisiae cultures. This research may be helpful in understanding the complete metabolic pathway of brewer’s yeast, with further application to organisms with conserved pathways.

Merrill Christensen, Brigham Young University Life Sciences Selenium (Se) has established chemopreventive efficacy against prostate cancer, the second leading cause of cancer death among men in the US. Currently, most studies only employ one single chemical form of Se, even though different forms of Se act through varied mechanisms to achieve their anticancer effects. In this study, we propose that systematically combining multiple forms of Se will produce an optimal combination of Se compounds to inhibit in vitro prostate cancer cell growth. As proof of principle, we (1) synthesized Se nanoparticles (nano Se), (2) determined the IC50s of methylseleninic acid (MSA), sodium selenite, and nano Se in PC-3 cells, (3) utilized mixture designs and response surface methodology to direct our combination experiments with these three compounds, (4) tested the fifteen combinations determined in the previous step, and (5) constructed a polynomial model to derive the optimum combination of MSA, sodium selenite, and nano Se to inhibit PC-3 cell growth. Our results showed that combining different forms of Se compounds enhanced its chemopreventive effect. Future studies will expand the use of three Se-containing compounds to four and demonstrate similar effects in xenograft mice.

Chelsea Moody, Dixie State University Life Sciences A relatively new pathogen, chytrid fungus, has been a major cause of decline in amphibians worldwide. This pathogen has been found on Canyon Tree Frogs in Zion National Park. Earlier data suggested that chytrid did not affect populations of Canyon Tree Frogs in Zion National Park. We predicted that since the populations weren’t impacted by the presence of the fungus, there must exist a mechanism by which the frogs rid themselves of the infective agent. To test this prediction, we captured and swabbed ten frogs in each of nine canyons in Zion National Park during the summer of 2013. Swabs were sent to the San Diego Zoo to be analyzed for the presence of chytrid DNA. Chytrid infection rates and population sizes were compared with data from previous years. As expected, the data showed that two of the three infected canyons experienced declines in the percentage of frogs testing positive for the fungus. Thus, these frogs must possess a mechanism to survive this pathogen, whereas most amphibians do not. Our results may prove helpful to other biologists seeking to understand how amphibians can survive this pathogen.

Eric Swenson, Dixie State University Life Sciences Underwater invasive species such as mussels and algae attach to submersible substrates that are advantageous for growth, and often include recreational boats that are frequently moved from one waterway to another. This can lead to rapid and uncontrolled spread of the invasive species. Through the use of newly developed ionic surfactants, growth and attachment of species on submersible surfaces can be inhibited. New surfactants have been incorporated into the final coating of typical product sealants to prevent the attachment of various marine organisms. By integrating the surfactant into the coating, an increase in the usefulness and longevity of effective inhibition is expected, as compared to commercially available alkali anti-fowling agents that are applied after the sealing process. It is hypothesized that the adherence properties possessed by marine organisms will be compromised by the introduction of ionic surfactants through changes in the chemical makeup of the exposed surfaces. The ultimate goal is to develop a substrate surface in which there is no affinity for attachment and/or causes fatality to the organism. If the desired outcome is reached, the potential benefits that ensue could create drastic improvements in areas of invasive species prevention and management in the desert southwest fresh-water lakes. Additionally, extension of the surfactant application to metal surfaces could have significant impacts on efficiency in water treatment systems, as well as cargo shipping in saltwater environments.

David Baird, Utah Sate University Life Sciences We obtained from the Bureau of Land Management (BLM) 30 years of monthly waterfowl population surveys completed at Pariette Wetlands in the Uintah Basin, Utah between 1980 and 2010. The Pariette Wetlands are the largest wetlands managed by the BLM within the lower-48 states and are comprised of 4,033 acres of land. Pariette Wetlands is surrounded by about 6,000 square miles of land where oil and gas production is the major activity. This waterfowl refuge is a significant location for migrating waterfowl species within the Pacific Flyway and provides important summer habitat for resident waterfowl. Our objectives were to determine what the trends were for waterfowl population abundance, occupancy, and species richness.

Sharisa Nay, Brigham Young University Life Sciences Gene therapy is a growing field of science with the potential to improve thousands of lives. With an eye toward improving the effectiveness and longevity of gene therapies, my project examines the preferential binding tendencies of the histone protein variants Htz1 and Htas1. Htz1 is the Ceanorhabditis elegans homolog of H2AZ, an important variant of the H2A histone. This protein has been shown as necessary for survival and as playing a role in the prevention of ectopic heterochromatin spread. Htas1 is another variant of H2A that plays a role in the increased transcription of sperm-producing genes. The preferred positioning of these variants on naked DNA is not yet known. Through a DNA extraction, nucleosome reconstitution, and DNA digest and sequencing, we will take these variants and examine where they are prone to localize within the N2 Bristol strain of c. elegans. This will allow us to include DNA sequences on our gene insertions possessing a high binding-affinity for these transcription-promoting histones. Thus, if we can identify the locations at which these variants will localize within DNA, we will be able to insert these preferred constructs into the genes used for gene therapy and thereby increase the effectiveness of gene therapies.

Shwan Javdan, University of Utah Life Sciences Each year in the United States, over 22,000 new cases of ovarian carcinoma are diagnosed and 14,000 women die from its progressive cancer stages. Anthracyclines, a class of common chemotherapy drugs, have long been the primary treatment for this and many other cancers, but they often leave patients with cardiotoxicity, hepatotoxicity, and other adverse effects. Polymer-drug conjugates using poly[N-(2-hydroxypropyl)methacrylamide] (poly-HPMA) are nanosized, water-soluble constructs that accumulate passively in solid tumors by the enhanced permeability and retention effect as well as actively by cell surface targeting methods. As such, they have exhibited reduced toxicity in the body. The goal of this study was to develop a novel, targeted HPMA copolymer-drug conjugate for the treatment of ovarian carcinoma. To accomplish this, an antibody fragment called Fab’, targeted to ovarian carcinoma cell surface antigen OA3, was bound to an HPMA copolymer-gemcitabine conjugate. Gemcitabine is a nucleoside analog used in chemotherapy that has demonstrated considerable effectiveness in recent years. The copolymer-gemcitabine conjugate was successfully developed following the synthesis of all the requisite components, including Fab’, diblock chain-transfer agent (di-CTA), N-(2-(2-pyridyldithio)ethyl)methacrylamide (PDTEMA), and polymerizable backbone-degradable gemcitabine derivative. Syntheses of di-CTA and PDTEMA were optimized and then characterized by high-performance liquid chromatography, H-NMR, and mass spectrometry. Monomers were combined by reversible addition-fragmentation chain-transfer polymerization, and then the Fab’ fragment was bound to the polymer backbone via a disulfide exchange reaction with PDTEMA. The targeted copolymer-drug conjugate was then verified by fast-protein liquid chromatography as well as ultraviolet-visible spectroscopy. A number of in vitro tests have demonstrated the properties of an efficient drug delivery system. Flow cytometry displayed active accumulation of the copolymer-gemcitabine conjugate to OVCAR3 cell surfaces. Furthermore, IC50 measurement found promising drug efficacy comparable to modern anthracyclines. Future work will involve in vivo evaluation of the conjugate’s therapeutic efficacy in nude mice bearing OVCAR3-xenografts.

Whitney Badal, Brigham Young University Life Sciences Light therapy has been utilized to treat alcoholism and opiate-dependent rats as well as ameliorating symptoms of Parkinson’s disease. As both addiction and Parkinson’s Disease (PD) are often associated with decreased dopamine transmission in the striatum, it is likely that light therapy is able to increase dopamine release. A similar technique called near-infrared light treatment has also been shown to be effective in mice in restoring the function of dopamine cells in the substantia nigra pars compacta (an area associated with PD). A possible explanation for this is that light catalyzes the formation of neuromelanin. It is likely that neuromelanin is a neuroprotective cellular agent that is able to reduce damage caused by reactive oxygen species. Using UV-IR spectrophotometry, we show that in the presence of photostimulation, dopamine (0.3-30 uM) oxidizes and polymerizes into neuromelanin. Since hydrogen peroxide catalyzes this formation of neuromelanin, it is likely that this is a radical-polymerization reaction, suggesting that neuromelanin may be a radical scavenger. Additionally, the presence of the selective iron chelator desferrioxamine, the calcium chelator EGTA, or lack of calcium in the artificial cerebral spinal fluid markedly reduces the formation of neuromelanin. Using fast scan cyclic voltammetry in mouse horizontal and/or coronal brain slices, dopamine release in the nucleus accumbens core was enhanced by light exposure, in particular UV and short-wavelength visible light. These findings indicate that both iron and calcium are necessary for melanization in neural tissues and that light-induced melanization enhances dopamine release, suggesting a physiological role for melanization in synaptic transmission.

Chase Barker, Utah Valley University Life Sciences Central Research Question: Phylogenetic relationships of mayflies are still not very well known, however molecular and morphological data have begun to shed light on the relationships of these insects (Ogden et al. 2009). Our central question is to elucidate the phylogenetic relationships within the mayfly family Baetidae.

Julia Hull, Weber State University Life Sciences Endophytes are fungi that live within aerial portions of plants for most or all of their life cycle without causing visible signs of disease. Gall forming aphids, Pemphigus betae, are highly competitive over gall site selection (Moran 1993), forming galls on the leaves of narrowleaf cottonwood and their hybrids. The favored gall location overlaps with areas of highest endophyte probability. I hypothesized that a negative correlation would exist between endophyte infection and aphid galling on leaves of backcross hybrid cottonwood trees.

Brianne Palmer, Utah State University Life Sciences Inversions have plagued the valleys in Utah resulting in built up pollution carpeting the cities and spreading into surrounding ecosystems. The ecological impact of these inversions is unknown. Inversion-based pollution events deposit nutrients and pollutants in the ecosystems. The purpose of this study is to determine if lichens, due to their unique physiology, preserve a record of inversion-based nitrogen deposition in order to assess the ecological impact of the inversions. In June and July of 2013 we collected 111 samples of lichens from sites exposed to the inversions, along Red Butte Creek and on an elevation gradient on Grandeur Peak in Salt Lake City. The collection focused on two species of lichens (Xanthomendoza montana and Xanthomendoza fallax) because they are easily identifiable and live in all the sampling sites. The samples were ground, run through a stable-isotope mass spectrometer and analyzed for the %N and δ15N. If lichens hold a record of inversion-based depositions there should be more nitrogen in lichens from the sampling sites closer to the pollution source (Salt Lake City). Once the data was complied, the resulting graphs showed no correlation between %N and distance from Salt Lake City between δ15N and the pollutant source. However, the isotopic variability within the samples was greater than expected and requires further research.

Preston Manwill, Southern Utah University Life Sciences Plants produce compounds, secondary metabolites, which can be harnessed for medicinal uses. Caryophyllene, a secondary metabolite produced by a variety of higher plants, has shown promise as an analgesic and anti-inflammatory. Caryophyllene synthase is the final enzyme in the biosynthetic pathway that produces caryophyllene. The gene and enzyme responsible for caryophyllene production have been observed in crop plants and model organisms, but few studies have investigated caryophyllene production in indigenous plants. We investigated the production of caryophyllene in Ericameria linearifolia (Narrowleaf goldenbush) because it is a shrub native to southwest North America. The first portion of this research examined sequence similarities between caryophyllene synthase gene orthologs in E. linearifolia and other plant genera. Genomic DNA was isolated from E. linearifolia leaves collected from plants growing in southern Utah. Literature reviews and database inquiries have provided DNA sequences for similar synthases, from which primers will be designed to copy and sequence the E. linearifolia caryophyllene synthase gene, confirming the orthologs presence. Following confirmation of the gene, mRNA will be extracted from leaf material and oligo(dT) primers used to synthesize cDNA. Subsequent cDNA amplification and cloning of products into a vector will allow for transformation into a protein-producing bacterium. These final steps make the downstream procedures of protein production, structure determination, and functional characterization of the protein possible. Comparison with enzymes that produce similar organic compounds may result as well, and would provide insight that could improve the biosynthesis of medicinal compounds.

Preston Manwill, Sourthern Utah University Life Sciences Plants produce myriad secondary metabolites (essential oil) that play a role in ecological interactions. Species evolve unique mixtures of organic compounds due to strong selective pressures that act on metabolic pathways. Few studies have investigated the antimicrobial properties of secondary metabolites that evolved against soil microbes associated with native plants. The essential oil of Mojave goldenbush (Ericameria linearifolia), a shrub native to the Intermountain West, was characterized and its bacteriostatic abilities against root associated and non-root associated bacteria morphs were examined in this study. We hypothesized E. linearifolia oil would act as a greater growth inhibitor against native soil bacteria with which it has evolved than against bacteria from non-associated soils. Secondary metabolites were extracted using steam distillation and analyzed with gas chromatography mass spectroscopy. A Kirby-Bauer assay tested presence, size and retention of inhibition zones (IZ) produced against soil bacteria. The essential oil of E. linearifolia contained twenty secondary metabolites, two of which, limonene and sabinene, constituted greater than fifty percent of the oil. Presence and retention of IZ produced by E. linearifolia metabolites varied significantly by community association and bacteria morph. The hypothesis was supported by IZ that were largest against bacteria cultures isolated from soil that surrounded Mojave goldenbush roots and least effective against cultures isolated from non-root associated soil. Our findings suggest that secondary metabolites of E. linearifolia have evolved to specifically prevent negative interactions with bacteria most associated with the plant.

Cameron Sargent, Brigham Young University Life Sciences Although viruses present a potential solution for treating and studying many harmful diseases, they have yet to be utilized successfully, largely due to an overall lack of understanding of the interactions between viruses and their hosts. This study, however, demonstrates the efficacious usage of comparative genomics in further elucidating viral reproduction mechanisms, a step in overcoming this hurdle. Following the sequencing and annotation of genomes of the B4 subcluster of mycobacteriophage, a previously unstudied selection of viruses, comparative genomics analysis identified regions of proteins from these viruses that are highly conserved, or highly similar. One of these B4 phage proteins was then compared on the basis of its predicted folded structure and its amino acid composition to a protein with the same function from another phage, whose structure and DNA binding mechanism was determined via NMR spectrometry. Gene sequence alignments identified the region of highest conservation in the B4 phage. Protein folding then revealed that this region created a structure that was analogous to the functional region of the protein studied elsewhere, indicating that it too recognizes, binds to, and modifies DNA in the same manner. Furthermore, another DNA-binding motif with high conservation among B4 phage was observed in both the folded B4 proteins and the protein analyzed via NMR spectrometry. This comparison procedure not only further elucidated the function of an important gene in B4 phage but also displayed that comparative genomics, a resource-effective and computer-based approach, can successfully identify the critical functional regions of a protein. This study therefore provides a novel procedure for further understanding the mechanisms of virus-host interactions, which in turn facilitates the successful application of viruses in treating and studying diseases.

Eliza Warren, Brigham Young University Life Sciences We have previously demonstrated that ethanol inhibition of medium spiny neurons (MSNs) in the nucleus accumbens (NAc), and γ-aminobutyric acid (GABA) neurons in other ethanol-sensitive brain areas, is mediated by GABA(B) receptors. The aim of this study was to evaluate the involvement of GABA and glutamate (GLU), and in particular GABA(A), GABA(B) and NMDA receptors, in mediating ethanol inhibition of dopamine (DA) release in the NAc. Using fast scan cyclic voltammetry (FSCV), we evaluated the effects of ethanol on DA release in the NAc core of C57/BL6 and CD-1 mice. In the slice preparation, local stimulation evoked robust, frequency-dependent DA release in the NAc, with maximal release at 20 Hz. Ethanol decreased DA release with an IC50 of 60 mM in C57Bl6 mice. In anesthetized C57BL6 mice, ethanol decreased DA release with an IC50 of 2.0 g/kg. Superfusion of the GABA(B) receptor antagonist CGP55845 enhanced DA release 85%, while neither the GABA(A) receptor antagonist bicuculline, the GABA(A) receptor agonist muscimol, the GABA(B) receptor agonist baclofen, nor the NMDA GLU antagonist APV significantly affected DA release. CGP55845 blocked ethanol inhibition of DA release, while neither bicuculline nor APV altered ethanol’s effects. GABA inhibited DA release at 0.5-10 uM but not at 0.1-1 mM. Compared to ethanol effects on DA release in C57BL6 mice, CD-1 GAD GFP knock-in mice, which only express GAD at 50% levels, were significantly less sensitive to ethanol. As DA release was sensitive to low concentrations of GABA, CGP55845 may act as a GABA(A) rho receptor antagonist, and neither bicuculline nor baclofen had any effect on DA release, ethanol may be acting on extrasynaptic GABA(A) rho receptors on DA terminals to inhibit DA release in the NAc.

Tamara Fox, Weber State University Life Sciences Clostridium botulinum has been implicated in cases of infant botulism across the United States. It is recommended that infants under the age of one year not be fed honey because of the presence of C.botulinum spores. The goal of this project is to determine whether honey produced in small and large apiaries in Utah contain varying amounts of toxin producing C. botulinum. Honey samples will be collected from hives maintained in Utah and tested for the presence of toxin producing strains of C. botulinum. Samples will be dissolved and centrifuged to isolate the spores and then superheated to release the DNA. Testing will then be done through a multiplex polymerase chain reactions (PCR) using primers specific for 16s rRNA, Clostridia species, and toxins A, B, E, and F. The presence and type of toxin producing Clostridia species will be compared with a Chi-Squared Test of Independence. Research will be completed by February of 2014 and we expect small apiaries will have a lower frequency of toxin producing C. botulinum strains than large apiaries and that toxin phenotype will vary between the two groups. The results will increase understanding on the variance of C. botulinum in Utah honey and will contribute to further research on this topic.

Kevin Nay, Southern Utah University Life Sciences Leech taxonomy has traditionally been based on morphological characters, but with new developments in DNA technology many taxonomists are starting to use genetic information in descriptions of new species. Leeches in southern Utah are poorly inventoried with respect to many other aquatic animals. There have been few morphological inventories of leeches and even fewer descriptions of the genetic diversity within leeches. Landscape genetics is a powerful tool used to understand geographic patterns of genetic diversity. Southern Utah has many naturally isolated bodies of water due to the climate and the dramatic changes in elevation in this part of the country. The landscape genetic study of leeches in southern Utah will provide us with a better understanding of genetic differentiation within southern Utah leeches. The mitochondrial DNA (CO I region) will be used to estimate genetic diversity and examine the relationships among individuals in two populations of leeches. I hypothesize that leeches in southern Utah will have greater genetic diversity then historically recognized from morphological studies suggesting a new species of leech. The study will lead to better understanding of the taxonomy and identification of southern Utah leeches.

Preston Manwill, Southern Utah University Life Sciences Members of Anthoxanthum (sweetgrass) have a history of ceremonial and medicinal use. Coumarin, a secondary metabolite produced by the grass, is an anticoagulant and antimicrobial agent. Antimicrobial properties of commercially available European A. odoratum metabolites have been nominally investigated, but no North American species have been studied. Additionally, European sweetgrass is purported to be available in both diploid and polyploid strains. Our research objectives were to: 1) determine if ploidy level strains do exist, and 2) investigate secondary metabolite production and evolution in commercial strains and A. hirtum, a Great Basin native. Using epidermal casts and fuschin staining, differences in guard cells and nuclei were surveyed. Secondary metabolites from the A. odoratum strains and A. hirtum were obtained through steam distillation and a vacufugation protocol that concentrated hydrosols. GC/MS analyses characterized and quantified secondary metabolites. The secondary metabolites coumarin, dihydrobenzofuron, and dihydroactinidiolide were identified, with the first and latter greatest in A. hirtum. Significant guard cell differences between strains, as well as species, were observed. Two ploidy strains of A. odoratum were suggested. A Kirby-Bauer assay tested presence, size and retention of inhibition zones (IZ) produced against soil bacteria. Gram-staining initially characterized bacterial morphs. Secondary metabolites from the diploid strain were most effective against all bacterial morphs, but polyploid metabolites also generated and retained IZ against diploid root associated and non-root associated morphs. The native sweetgrass produced IZ only against root associated bacteria with which it had evolved. Future work will include similar studies of other North American Anthoxanthum species.

Justen Despain, Brigham Young University Life Sciences Mycobacterium ulcerans is an acid fast intracellular bacteria that is the causative agent of Buruli Ulcer, a disease endemic in tropical regions of Africa. The disease starts as a nodule that progresses into a necrotizing skin lesion. Treatment ranges from antibiotic therapy to surgical removal of surrounding skin to stop necrotization of the skin. Owing to the intracellular nature of M. ulcerans, antibiotic therapy can be an ineffective treatment option. It has been previously shown that mycobacteriophage can reduce extracellular bacterial load in mice infected with M. ulcerans. In this study, we propose a method of killing intracellular M. ulcerans by mycobacteriophage using avirulent Mycobacterium smegmaits as a vector.

Lee Leavitt, University of Utah Life Sciences The ventral respiratory column (VRC) is a region in the brainstem shown to control breathing patterns in mammals. Using activation and inhibition of neurons in this region, classes have been assigned based on response-combinations. Using a mouse model, cells from this region are dissociated, plated and incubated with a dye that indicates changes in cytoplasmic calcium levels. Hundreds of cells are measured while varieties of pharmacological agents are applied. Response-combinations provide a profile of the receptors found on these neurons. Previously, varieties of cell classes were shown to contain NMDA receptors (receptors linked to learning and memory). However, specific compositions of subunits within these receptors are not known. These receptors are ligand gated ion channels composed of four non-covalently bound proteins. Each subunit has a different activation profile determined by interactions of agonist and antagonists. Conantokins (peptides isolated from snail venom) and other compounds further afford understanding of the architecture the assigned cell-classes. This project has continued to classify the subunit compositions of NMDA receptors with the ultimate goal of understanding which NMDA receptor subunits are present in each class. This will provide valuable information on the VRC’s function, and will allow for pharmacological innervations to change behavior in this region.

Dasom Kim, Brigham Young University Life Sciences The main purpose of the project is to compare the phylogeography of a species of stonefly (Klapopteryx kuschelli) and a species of dragonfly (Rhionaeshna variegata) in Patagonia. Specifically, the project will study how geography and behavior (i.e., their dispersal abilities) have affected their evolutionary histories. Of all the varied climates and geography in South America, comparatively few phylogeographic studies have been conducted with insects, especially in Patagonia. This study will provide an important foundation for a comparative phlyogeographic study of two insect groups inhabiting the same regions of Patagonia. Also, if funded, this project will give me an exclusive opportunity to interact with international research institutions in South America as well as their scientists.

Derrick Crawford, Brigham Young University Life Sciences Members of the FGF family of signaling factors are key components in distal outgrowth and patterning of the vertebrate limb. These factors are expressed and secreted by the apical ectodermal ridge (AER) on the distal margin of the limb. Blocking their function is known to truncate the limb skeleton. Conversely, replacing the AER with beads soaked in Fgf protein can rescue limb outgrowth and patterning. Our lab has demonstrated that one of the mechanisms whereby the Fgf/AER functions is to mediate directed outgrowth of the adjacent mesenchyme. As the AER regulates growth of mesenchyme toward itself, it would be predicted that the AER’s dimensions would be important for shaping the mesenchyme that it recruits. We have found that the shape of the AER changes over time in a manner that corresponds to the shape of limb elements as they form along the proximal distal axis. Further, mutants that exhibit defects in the dimensions of the AER show corresponding anomalies in the limb skeleton. Given these observations it would be predicted that an Fgf soaked bead being of fixed spherical dimensions would only be capable of forming a cylindrical, rod-shaped limb. A bead placed posteriorly fulfills this expectation whereas a bead placed apically does not. We provide a molecular explanation for this discrepancy. We have also manipulated the shape of the AER surgically and find that similar to beads the shape and the AP position of the AER dictates the shape of the forming limb skeleton.

Jake Gamboa, Brigham Young University Life Sciences It is estimated that approximately 350,000 people in the United Stated die annually from post-myocardial infarction arrhythmias. A majority of these people will undergo a surgery that results in partial or complete removal of the stellate ganglion and other nerve fibers of the pharyngeal space in an attempt to prevent over stimulation from the neurons to the area of dead heart tissue and, therefore, future arrhythmias. However, without a somatomototopy, it is unclear what physiological effects partial or full sympathectomies may have. We will create a three-dimensional map of the pharyngeal space nerve plexus which will, in turn, allow for a more accurate and precise surgery.

Mark Soffe, Utah State University Life Sciences SSBs are DNA binding proteins that are essential components of cells and play key roles in DNA replication, repair, and recombination. Here we utilize two biochemical properties associated with the E. coli SSB protein to develop a novel procedure to purify proteins using a resin-free strategy. 1. E. coli SSB binds to single stranded DNA (ssDNA) with extremely high affinity (K = 1013 M-1), indicating very tight binding. 2. It is also a unique protein with respect to its purification – it is possible to obtain greater than 95% pure SSB from the total cell lysate without using any sort of column or resin, utilizing polyethyleneimine (PEI) and ammonium sulfate precipitation. Our design uses SSB as an affinity/solubility tag to enhance the solubility and expression of difficult-to-purify proteins, and allows for the simple, resin-free purification using PEI and ammonium sulfate precipitation. There also may be a possibility to co-express protein dimers and possibly tetramers using this method. Constructs have been made that include the SSB gene, along with the ability to fuse any gene of interest, as well as a TEV Protease cleavage sequence allowing for proteolytic cleavage after gene expression. Two genes of interest have been cloned in thus far—TEV protease and Rad51. In this proposal I outline experiments to develop this strategy further and test our proof of principle concept and its application to a broader set of target proteins.

Teressa Paulsen, University of Utah Life Sciences Breast cancer is still the most common cancer among women regardless of race or ethnicity. The focus of our research is to uncover the mechanism breast cancer cells use to escape the inherent limitations of the telomere and obtain immortality. The protective end of a chromosome, the telomere, degrades with each cellular division. The cellular response to telomere dysfunction is to activate programmed cell death. Therefore, this type of damage normally limits the proliferative potential of the cell and subsequently carcinogenesis.

Josh Hall, Weber State University Life Sciences Reproduction in birds is extremely conservative with the vast majority of the birds adopting bird-egg contact incubation to maintain an appropriate microclimate for embryonic development (Deeming, 2004). The Great Salt Lake is a vital nesting site for American Avocets (Recurvirostra Americana) that shows extreme temperatures and hostile environments where nest success can be as low as 1 -14% (Cavitt, 2008). Constancy of incubation, i.e. the time that the eggs are in contact with an adult, is a major indicator of nest success and environmental conditions. Our goals were to examine some of the costs natural selection places on embryos and parents to maintain a constant embryo temperature. We hypothesized that incubation attentiveness would increase across the nesting cycle. Over 200 AMAV nests were surveyed. Thermal probes were used to record various nest microclimates at every minute. A pseudonest with painted chicken eggs was also created and a thermal probe was placed to measure the ambient temperature without any adult incubation. A motion sensitive camera was placed over nests to examine differences in parental care. Nests will be divided into three phases: early, mid, and late incubation. Thermal data will be analyzed using descriptive statistics and mean variance values to calculate how incubation constancy varied throughout these phases. We expect this data to tell us more on how natural selection is working on these populations and some possible theories of how this developed.

Xin Wan, University of Utah Life Sciences Endothelial dysfunction exists in individuals with diet-induced obesity (DIO) and type 2 diabetes (T2DM). Markers of endothelial dysfunction include reduced phosphorylation (p) of endothelial nitric oxide (NO) synthase (eNOS) to total eNOS (p-eNOS:eNOS), and attenuated endothelium-dependent vasorelaxation. Free fatty acids (FFAs) are elevated in individuals with DIO and T2DM. Our laboratory has shown that when: (i) endothelial cells are incubated with saturated FFA palmitate; (ii) mice are infused with lard-oil; and/or (iii) when mice are fed with high-fat diet, protein phosphatase 2A (PP2A) binds directly with eNOS. When this occurs, the association among Akt-Hsp90-eNOS is disrupted, p-eNOS:eNOS is impaired, and endothelium-dependent dysfunction occurs. This is prevented using pharmacological and genetic approaches that limit production of FFA metabolite ceramide. It is unknown whether PP2A inhibition per se is protective. We hypothesized that arterial dysfunction in obese vs. lean mice is prevented by PP2A inhibition. Seven-week-old, male, C57B16 mice consumed standard (CON, n=20) or high-fat (HF, n=20) chow for 12-weeks. Subgroups (n=10) of CON and HF mice received IP injections of saline (vehicle; V) or Lixte Biotechnology 100 (LB1, 1 mg/kg/day) for the last 14-days. Preliminary experiments verified that LB1-treatment for 3 and 21 days decreases (p<0.05) arterial PP2A activity. HF mice gained weight and developed peripheral glucose intolerance vs. CON mice regardless of LB1 treatment. Endothelium-dependent vasorelaxation was impaired (p<0.05) in HF-V vs. CON-V mice, but dysfunction was less severe (p<0.05) in HF-LB1 mice. p-eNOS:eNOS was reduced (p<0.05) in arteries from HF-V vs. CON-V mice, but p-eNOS:eNOS was similar in arteries from HF-LB1 and CON-LB1 mice. Akt and Hsp90 co-immunoprecipitation with eNOS was impaired (p<0.05) in HF-V vs. HF-CON mice, but this was not observed in arteries from HF-LB1 and CON-LB1 mice. These findings suggest that PP2A activity suppression in vivo is sufficient to preserve endothelial function in obese mice.

Molly Van Engelenhoven, Utah State University Life Sciences For thousands of years farmers have known that soil organisms can destroy crops but only in the past ten years have researchers begun to understand that complex interactions among soil organisms and plants can determine plant diversity and productivity. Much of the research in this young field of study has come from qualitative greenhouse experiments and separately from simple mathematical models. Here, we report findings from a six-year plant-soil feedback (PSF) field experiment that assessed the effects of soil organisms on the growth of twelve plant species common to the intermountain west. Initially, twelve native and exotic species were randomly assigned to 1550 experimental plots in a field. After four years of growth, the plants were killed. Next, the same plant species were randomly assigned to the 1550 plots again to test how each plant species responded to the soil microbial communities ‘cultivated’ by each other plant species. Two years later plant growth responses to different soil types were measured. Most plants showed large growth differences on different soil types. For example, Bromus tectorum grew twice as much on Pseudoroegneria spicata-cultivated soils than on self-cultivated soils. When the plant growth responses observed in this experiment were used in a mathematical model, PSFs were found to play a critical role in determining which plants were likely to be dominant in a community and how productive these different plant communities would be. In summary, this large-scale experiment provides strong evidence for the role of PSFs in plant productivity and diversity. Results have implications for natural methods of effective native plant restoration, biofuel production and weed control.