Life Sciences

Ashley Calder, Utah Valley University Life Sciences An estimated 50-80% of dementia patients suffer from Alzheimer’s disease (AD). Currently there is no test to diagnose AD except post mortem. Recent papers indicate that AD affects the cytoskeleton and cellular structure through mutations that alter structural proteins, and that dysfunction of the cytoskeleton may play a pivotal role in AD and other neurodegenerative diseases. In particular, specific genetic components of AD affect microtubule and actin filaments that control endocytosis, exocytosis, the shape and size of the neuron, vesicular transport along neurites (dendrites and axons), and fibril formation. The goal of our research is to determine if breast cancer molecular subtypes can be used as a model for AD. Breast cancer is comprised of five molecular subtypes that contain different molecular structures depending on mutations specific to each subtype and the proteins being synthesized. These mutations and their expressed proteins change the characteristics of the cytoskeleton and resulting properties of the cell such as size, shape and stiffness. Both computer simulation and experiment have demonstrated that high-frequency ultrasound in the 10-100 MHz range is sensitive to these properties. For this study, ultrasonic tests were conducted on monolayer cell cultures of breast cancer cell lines of different subtypes. Ultrasonic waveforms were analyzed by transforming them into their corresponding spectra. The positions, widths, and shapes of the spectral peaks were compared and correlated to model results using a pattern recognition algorithm. Preliminary results indicate that cell stiffness and size can be determined from the measurements. Further analyses of these and additional data will determine if ultrasound is sufficiently sensitive to differentiate between the molecular subtypes of breast cancer. Results from these analyses, future studies with neuron cell cultures, and application of the results to the development of a minimally invasive, in vivo method for accurately diagnosing AD will be discussed.

Ashley Nelson, Brigham Young University Life Sciences The motivational effects of opiates and ethanol switch from a dopamine (DA)-independent to a DA-dependent pathway when the animal is in a drug-dependent state. A corresponding change occurs in ventral tegmental area (VTA) GABA(A) receptors in opiate-dependent animals, which switch from a GABA-induced hyperpolarization of VTA GABA neurons to a GABA-induced depolarization. The aim of this study was to evaluate VTA GABA neuron excitability, GABA synaptic transmission to VTA GABA neurons and GABA-mediated DA release in the nucleus accumbens (NAc) under ethanol-naïve and dependent conditions. To accomplish these studies, we used standard whole-cell and attached-cell mode electrophysiological techniques to evaluate acute and chronic ethanol effects on VTA GABA neurons in GAD GFP mice, which enabled the visual identification of GABA neurons in slice preparation. In naïve animals, superfusion of ethanol (IC50 = 30 mM) and GABA(A) receptor agonist muscimol (IC50 = 100 nM) decreased VTA GABA neuron firing rate in a dose-dependent manner. Compared to saline-injected controls, in animals made dependent on ethanol by twice daily injections of 2.0 g/kg ethanol, neither ethanol nor muscimol significantly affected VTA GABA neuron firing rate on average. We and others have found that ethanol decreases DA release at terminals, as measured by fast scan cyclic voltammetry. We have recently reported that ethanol inhibition of DA release at terminals in the NAc of ethanol-naïve animals is mediated by GABA, possibly from VTA GABA neurons that project to the NAc. We evaluated the effects of ethanol on DA release in the same ethanol-dependent animals. Compared to controls, superfusion of ethanol did not significantly affect DA release. Together, these findings suggest that VTA GABA neurons undergo a switch in GABA(A) receptor function with chronic ethanol, which results in a corresponding switch in DA release, perhaps resulting from adaptations in VTA GABA neuron input to the NAc.

Bradley Prince, Brigham Young University Life Sciences Learning and memory occur due to adaptive brain changes in response to our environment. These changes are mediated by synaptic plasticity, particularly within the hippocampus, where spatial and declarative memories occur. Plasticity can either strengthen or weaken synapses, known as long-term potentiation (LTP) or long-term depression respectively. While many forms of synaptic plasticity are N-methyl-D-Aspartate receptor-dependent, recently endocannabinoids were identified to mediate several new forms of hippocampal synaptic plasticity. Endocannabinoids bind to receptors such as cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1), and mediate several forms of plasticity, including in the hippocampus. However, new research has demonstrated a non-CB1/TRPV1-dependent endocannabinoid synaptic plasticity in the hippocampus. While the receptor(s) involved is currently unknown, several potential candidate receptors that bind the endocannabinoid anandamide have been identified. These are orphan G-protein coupled receptors (GPRs) whose distribution in the brain and/or function is unknown. GPR55 is of particular interest as it activates second message systems, including increasing intracellular calcium. Using quantitative RT-PCR, electrophysiological and memory behavioral tasks we examined hippocampal GPR55 expression and function. GPR55 is indeed expressed in hippocampus of both rats and mice. Cellular expression is currently being examined and appears to be rare in interneurons and more likely expressed by pyramidal cells. Interestingly, application of the GPR55 agonist LPI (2 µM) to wild-type mice demonstrates a decrease of LTD in brain slices. This LPI effect was not noted in GPR55 knock-out mice in the presence of LPI. This data suggest GPR55 is physiologically relevant in the hippocampus. This is the first direct evidence we are aware of that a novel endocannabinoid receptor directly effects hippocampal LTD. Because neurodegeneration that affects memory is typically associated with an increase in LTD, this provides a potential target to slow the advance of diseases such as Alzheimer’s.

Audrey Butler, Utah Valley University Life Sciences High-frequency (HF) ultrasound has been shown to be sensitive to a range of breast pathologies, and is being explored for the intra-operative assessment of lumpectomy margins. This sensitivity is believed to arise from microstructure-dependent interactions of ultrasound in the tissue. The objectives of this study were to develop breast tissue phantoms with microstructures that accurately mimic the histology of normal and malignant tissue, and to determine the effects of these microstructures on HF ultrasonic spectra (10-100 MHz). Phantoms were created from a mixture of water, gelatin, and soluble fiber. To simulate various breast tissue histologies, polyethylene beads, polyethylene fibers, and nylon fibers with a range of diameters were embedded into phantoms. Microstructures ranging from randomly dispersed beads to bead-fiber constructs resembling terminal ductal lobular units (TDLUs) were modeled and tested. Pitch-catch and pulse-echo measurements were acquired using 50-MHz transducers, a HF pulser-receiver, and a 1-GHz digital oscilloscope. Spectra were derived from the data and peak densities were determined from the spectra. Peak density, which is the number of peaks and valleys in a specified spectral range, has been shown to correlate with tissue complexity. Preliminary results from dispersed beads (58-925 µm diameter) of constant volume concentration (0.8%) indicated that the smaller beads produced higher peak densities than the larger beads with a consistent and statistically significant trend. These results substantially improve upon previous phantom studies and upon results from original breast cancer studies, demonstrating the strength of the HF ultrasound response to tissue microstructure. The higher peak densities can be attributed to either the higher number of scatterers for small beads or the size of scatterer in relation to the ultrasonic wavelength. These and other results from more advanced histologically accurate microstructures modeling TDLUs will be discussed.

Lance Deeter, University of Utah Life Sciences Continuous-flow left ventricular assist devices (LVADs) are used in advanced heart failure patients either to bridge them to transplantation or as a permanent-destination therapy. We determined whether chronic exposure to non-pulsatile blood flow and acute increases in coronary perfusion pressure associated with LVAD implantation would influence arterial function. Arteries from a transmural biopsy of the left-ventricle were obtained from ten male patients (54±4 years old) at the time of LVAD implant (n=17, 184±25 µm i.d.) and 239±51 days later upon LVAD explant (n=21, 281±22 µm i.d.). Lmax tension was determined and dose-response curves to potassium chloride (KCl, 10-100 mM) were performed using isometric tension techniques. Next, bradykinin (BK, 10-6 to 10-10 M) and sodium nitroprusside (SNP, 10-4 to 10-9 M) concentration-response curves were completed on vessels precontracted to ~65% of maximal tension development. Maximal BK-induced vasorelaxation was greater (p<0.05) at explant (85±5%) vs. implant (59±9%), while SNP evoked responses (~90%) were similar between time-points. These findings suggest coronary endothelial function is improved by LVAD implantation. Heart failure was precipitated by a myocardial infarction in six of the ten patients. These are referred to as “ischemic” patients whereas the remaining four are “non-ischemic” patients. We hypothesized that coronary vascular responses would be improved by LVAD implantation to a greater extent in ischemic vs. non-ischemic patients. In ischemic patients maximal BK-induced vasorelaxation was greater (p<0.05) in coronary arteries obtained at explant (87±6%, n=14, 305±30 μm i.d.) vs. implant (53±11%, n=12, 204±33 μm i.d.). In non-ischemic patients maximal BK-induced vasorelaxation was similar in arteries obtained at explant (79±9%, n=7, 232±21 μm) and implant (72±17%, n=5, 135±13 μm). SNP responses were similar (~90%) between groups at implant and explant. Collectively, our data suggest that LVAD implantation improves endothelium-dependent vasorelaxation in ischemic but not in non-ischemic patients.

Dusting Day, Brigham Young University Life Science Atherosclerotic vascular disease is the leading cause of morbidity and death in the United States. Approximately 1.4 million surgical procedures are required every year for treatment of vascular disease and its subsequent issues. While saphenous vein and internal mammary artery grafts are most commonly chosen by physicians, many patients who are in need of arterial grafts have vessels that are not ideal for grafting because of damage to the vessels or disease. This introduces the necessity for synthetic blood vessel grafts that function precisely as natural vessels in vivo. Our blood vessel research team has entered the tissue engineering field in its most exciting effort: the scalable rendering of cell-seeded vascular constructs with rapid prototyping machines or 3D printers. We have built and are modifying a 3D printer to deposit living endothelial and smooth muscle cells into vascular structures. Using agar, alginate, or collagen gels as placement media, cells can be arranged in shapes resembling multilayered artery tubules and proliferate to form functional arteries. The endothelial layer and smooth muscle layer of cells interact to secrete a natural extracellular matrix (ECM) between them. We have successfully cultured endothelial cells and are perfecting our technique of harvesting aortic smooth muscle cells for culture. These cells will be encapsulated in a gel we have optimized for cell adhesion and proliferation and will then be printed with our rapid prototyping machine into the shape of a blood vessel. After proper cell growth and secretion of the ECM we will subject our synthetic graft to tensile strength testing, thrombosis tests, and eventually implantation into an animal for observation of any immunogenic effects. Our project’s success would bring an array of new treatment options through biomedical engineering that would save many lives of those who suffer from cardiovascular disease.

Joseph Linzey, Brigham Young University Life Sciences Dopamine (DA) D2 receptor expression parallels DA levels in the brain and these autoreceptors on DA neurons been shown to be modulated by long-term ethanol exposure. We have previously demonstrated that VTA GABA neurons also express D2 receptors, and that DA and D2 receptor agonists markedly enhance the excitability of VTA GABA neurons, opposite to their well-known inhibition of DA neurons. Most importantly, D2 receptor antagonists block ethanol inhibition of VTA GABA neurons and D2 receptor expression in VTA GABA neurons down-regulates with chronic ethanol. This study evaluates short-term D2 receptor adaptation in VTA GABA neurons and in DA release in the nucleus accumbens (NAc) by acute ethanol. In electrophysiology studies in anesthetized rats, periodic iontophoretic application of DA, or the D2 agonist quinpirole, markedly enhanced VTA GABA neuron firing rate, which was initially inhibited by ethanol, but resulted in latent and marked rebound excitation 30-60 min following injection. Using fast scan cyclic voltammetry (FSCV), we evoked DA signals in the core of the NAc by electrical stimulation of the medial forebrain bundle at the level of the lateral hypothalamus (60 Hz, 24 pulses). Intraperitoneal (IP) administration of ethanol (1.0-3.0 g/kg) dose-dependently decreased the amplitude of the MFB-evoked NAc DA signal. IP administration of the D2 antagonist eticlopride (1 mg/kg) markedly increased (250%) the amplitude of the evoked DA signal. When ethanol was administered after eticlopride it increased the amplitude of the DA signal an additional 42%. These findings suggest that ethanol induced decreases in evoked DA release may be due to autoreceptor feedback. Work is in progress to evaluate the short-term expression of D2 receptors in VTA GABA neurons following acute ethanol and to evaluate the effects of ethanol-induced short and long-term adaptations in VTA GABA neuron D2 expression in mediating ethanol effects on DA release in the NAc.

Brittany Hammontree, Dixie State University Life Sciences Depending on metabolic conditions or dietary preferences, people can often become deficient in critical vitamins and minerals. For example, a number of people are deciding to become vegetarians, and vitamin B12 deficiencies could become a huge epidemic, as this essential vitamin is only obtained through meat products. This issue was the driving force to look deeper into new ionic liquid materials and how they could be used as a transport agent for vitamin B12, along with other vitamins. Ionic liquids are organic salts that are currently being explored in many scientific fields due to their unique properties. However, using ionic liquids as a transporter in transdermal applications has yet to be explored Developing new mechanisms of administering nutrients via transdermal processes can increase the bioavailability and effectiveness of vitamins and minerals that often cannot survive oral administering due to the acidity and molecular absorption via the stomach. This research focused on finding the right ionic liquid with high solubility of the individual vitamins. Several ionic liquids were developed, and the different vitamins were tested for solubility levels. This greater solubility allows for maximum exposure of the vitamin during transdermal delivery. In particular, two different vitamins were tested – vitamin K and Protoporphyrin, a chemical analog to vitamin B12. Additionally, the effect of these ionic liquids on the physiology of the blood and plasma as it enters the body past the skin layers is critical to understand. In addition to transdermal applications of vitamins, transport of these vitamins to other countries and remote locations could have tremendous implications. Ionic liquids tend to increase shelf life of solutes, and the availability to provide these materials during medical missions or service trips would be increased substantially, particularly in more remote settings.

Anindita Ravindran, University of Utah Exercise and Sport Science Obesity predisposes individuals with Type II Diabetes to cardiovascular complications such as impaired blood vessel function. Due to the elevation of free fatty acids (FFAs) in obese individuals, ceramide, a lipid metabolite, accumulates and might contribute to the inability of a blood vessel to constrict or relax appropriately. Vessel dysfunction is partly caused by the inability of the endothelium, the innermost protective lining of blood vessels, to synthesize and release nitric oxide (NO). Our data indicate that ceramide impairs endothelial NO synthase (eNOS), the enzyme that synthesizes NO. In order to study mechanisms by which ceramide might impair eNOS, it is important to measure cellular ceramide production in response to pharmacological and genetic manipulations. Previously we used P-32 radioactive assays to measure ceramide accumulation. However, the use of radioactivity is expensive, potentially hazardous, and waste disposal is an environmental concern. Therefore, I sought to import a less harmful, more cost effective, yet accurate technique of measuring ceramide production by immunofluorescence (IF). IF allows ceramide to be tagged with a primary antibody which can be detected by a secondary antibody conjugated with a fluorescent dye. I have observed that 250, 500, and 750 uM palmitate (pal) incubation for 3 h increases (p<0.05) endothelial cell ceramide accrual in a dose-dependent manner. Further, a FFA-independent method to alter ceramide accrual i.e., 3 h incubation of cells with N-oleoylethanolamine, also elevates (p<0.05) ceramide production. Importantly, I have shown that 500 uM palmitate-induced ceramide accrual can be prevented (p<0.05) by two structurally dissimilar inhibitors (10 uM myriocin, 1mM L-cycloserine) of the rate-limiting enzyme responsible for ceramide biosynthesis i.e., serine palmitoyl transferase (SPT). None of these inhibitors impairs cell viability. These data indicate that IF is an accurate and reproducible method whereby ceramide accrual can be quantified in endothelial cell systems.

Monica Cervantes, Utah Valley University The central research question of this project was to determine if high-frequency ultrasound is sensitive to tissue pathology at the microscopic level. Previous studies on surgical specimens have shown that high-frequency ultrasound may be sensitive to a range of breast pathologies including fibroadenomas, atypical ductal hyperplasia, fibrocystic changes, and carcinomas. The ultrasonic parameters that were sensitive to pathology were the number of peaks (the peak density) of the first-order spectra of the waveforms (one forward Fourier transform), and the slope of the second-order spectra of the waveforms (two consecutive forward Fourier transforms). The ability to determine pathology rapidly and with minimal specimen preparation would make high-frequency ultrasound particularly well-suited for real-time use during cancer surgery to ensure all of the malignant tissue has been removed. The purpose of this research was to determine the sensitivity of the peak density and spectral slope to tissue microstructures other than those found in breast cancer. The results of this study would not only support the results from the breast cancer studies, but also extend those results to the detection of cancer and other diseases in a range of organs and tissues. The research methodology included the following steps. (1) Freshly excised bovine organs were obtained from a meat packaging facility, including the heart, liver, and kidney. (2) Specimens approximately 3x3x1 cm in size were dissected from the organs and tested immediately with ultrasound. (3) Both pitch-catch and pulse-echo waveforms were acquired from the samples. (4) The data were analyzed by determining the peak densities and spectral slopes. The results showed that the more heterogeneous tissues of the heart, the vascular structures (aorta, vena cava, etc.), displayed significantly higher peak densities than the muscle tissues. Similarly, the ureter, which has greater heterogeneities in its structure (larger and more varied), displayed significantly higher peak densities than the cortex and medulla tissues. No significant trends were observed for the liver tissue, or for the spectral slopes except for kidney medulla tissue. Heterogeneity and peak density in high-frequency ultrasonic spectra that may be useful for performing real-time pathology during cancer surgery.

Robert Bradford, Utah Valley University Biology Most organisms exhibit latitudinal gradients in diversity (i.e., taxonomic richness decreases as latitude increases). Few studies have sought latitudinal gradients in lichens, especially in the midlatitudes. Our primary questions were: 1) do lichens along the west coast of the United States show latitudinal gradients? 2) If so, what is the rate of change and does the level of taxonomic richness affect this rate? We hypothesized that lichens would show a reverse latitudinal gradient in the region, as has been documented for lichens elsewhere in the northern hemisphere, but at a considerably smaller scale. This study fills in the gap in our understanding of lichen latitudinal gradients over large areas of North America. It also functions as a baseline for future climate change and conservation efforts. Our study area is bound at the south by the California-Mexico border (32.331° N) and at the north by the Washington-Canada border (47.178° N), and extends inland from the coastline to the crest of the Sierra Nevada Mountains (116.083° W, at its eastern -most point). We divided the region into 218 roughly equal-area (cite) grid cells using GIS, each bordered with latitudinal and longitudinal lines. We derived a list of all vouchered lichen specimens in each grid cell using Consortium of North American Lichen Herbaria, an online database. The data were synonymized, and species, generic, and familial richness were calculated for each grid cell. We found no correlation (R2 = 0.2306) between latitude and species richness, using the raw vouchered data. What we did find was a strong correlation (R2=0.9069) between sample density and species richness. These results are biased by sample density and do not reflect what is naturally occurring. We hypothesize that we can get an unbiased estimate of richness with MaxEnt models. Using the georeferenced lichen distributions and related climate data, we constructed species distribution models of all species with five or more occurrences (990 species). In GIS, we projected all 990 distribution models and our 218 grid cells together to calculate species richness for each cell at various thresholds (i.e. likelihood of occurrence at 10%, 20%, 30%, etc.).

Vance Almquist, Utah State University Plants, Soils, and Climate Fire induced soil water repellency has been characterized across a variety of soils and landscapes as being a cause of watershed degradation and surface water pollution. The repellency is due to the condensation of volatile polyaromatic hydrocarbons onto soil particles. Although repellency is known to reverse, in some locations the reversal takes months; whereas, in other locations it may take years. Little is known about the reversal mechanisms and how they lead to such a large range of reversal times. Access to untreated fired-affected sites, can be unpredictable and samples vary greatly from site to site. Therefore, a model compound that mimics fire-induced soil hydrophobicity is needed to be able to systematically investigate soil hydrophobilicty reversal mechanisms. Fluorene is a relatively non-toxic, hydrophobic polyaromatic hydrocarbon. The behavior of fluorene coated sand grains was investigated under laboratory conditions using quartz sand. Moreover, its fluorescent properties could be used to non-destructively monitor its degradation over time. In this context fluorene was studied as a possible model compound for the study of mechanisms involved in the reversal of fire-induced soil hydrophobilicy. The compound was subjected to conditions known to degrade or reverse water repellency including temperature, UV-light, and moisture content. Changes in hydrophobicity were monitored using the ethanol drop test and fluorescent imaging. Digital image processing techniques with the public domain software, ImageJ, produced by the National Institute of Health, were used to analyze the images and generate spatial maps of treatment effects on fluorene degradation and hyrdophobicity reversal. Our results indicate that the hydrophobic fluorene-coated sand layers were degraded by treatments such UV light known to reverse hydrophobicity in fire-affected soils, thus suggesting that fluorene may serve as a suitable model compound for producing hydrophobic layers on course grained material.

Jesse Cobell, Brigham Young University Biology Alzheimer’s disease (AD) is the sixth major cause of death in the U.S. However, at present, no diagnostically useful serum markers for AD have been identified. Hence, we used a novel serum proteomic approach to interrogate the low molecular weight proteome for serum biomarkers. This allowed for survey of around 5000 low MW species. To reduce ion suppression, an acetonitrile precipitation step was used to remove high abundance serum proteins. Protein-depleted sera from 58 cases and 55 controls were analyzed by cLC-ESI-QTOFMS/MS using reverse phase chromatography. Data were reviewed using Applied Biosystem’s Analyst-QS software to compile spectra. Differentially expressed peptides (cases vs. controls) were analyzed statistically using the Student’s t-test. This led to discovery of 36 candidate biomarkers. Additionally, we compared AD subjects with more severe disease (Clinical Dementia Rating (CDR) =3) with non-demented individuals (CDR=0) and found 23 biomarkers. Furthermore, on comparison of mild and moderate stage AD individuals (CDR = 0.5, 1, 2) with those with severe disease (CDR = 3), we found 24 biomarkers. Some of these biomarkers appeared more prominent in one gender. We then fragmented several of these biomarkers on an LTQ-Orbitrap XL hybrid mass spectrometer and cLC-ESI-QTOF-MS/ MS system using collision-induced dissociation to determine amino acid sequence analysis. We have identified 5 biomarkers and are in the process of identifying the remaining biomarker species. This serum proteomics approach found statistically different peptide abundances in subjects with AD. Additional biostatistical evaluations are underway to determine sensitivity and specificity of individual biomarkers and their combinations. Future studies will assess biomarkers according to disease stage and validate current biomarkers in blinded comparisons of other AD sera. This serum proteomics approach appears promising in locating and identifying clinically useful serum biomarkers of AD.