Human SKIP (skeletal muscle and kidney-enriched inositol phosphatase) is a 51 kDa inositol polyphosphate 5’-phosphatase that is found in many tissues including brain, eye, and abundantly in the heart, kidney and skeletal muscle.(1-2) SKIP translocates from the endoplasmic reticulum at rest to the cell membrane upon insulin signaling which stimulates protein complex formation involving the insulin receptor.(3) SKIP exhibits affinity towards the fatty acid phosphatidylinositol 4,5-bisphosphate diC16 (PIP2 or PI(4,5)P2)(4) and it is thought that SKIP hydrolyzes the D-5 position of PIP2.(2) PIP2 is a phospholipid component of cell membranes where it is involved in insulin signaling.(5) The purpose of this experiment was to obtain recombinant, human, enzymatically active SKIP to be used as a tool to study signaling pathways associated with SKIP enzymatic activity. For this purpose, a HIS-SKIP chimera was produced in T7 Shuffle cells. Cultures containing T7 Shuffle cells transformed with pLATE31 SKIP were grown to log phase and protein expression was induced with IPTG (1 mM). Fast protein liquid chromatography (FPLC) using a nickel chromatography column was used to purify HIS-SKIP. HIS-SKIP was detected at 51 kDa using a Western blot using an anti-HIS probe. HIS-SKIP was more pronounced on the Western blot as concentration increased. The total concentration of the non-pure protein mixture which included HIS-SKIP obtained was 1186 µg/ml, as determined by a Bradford assay with a BSA standard curve. The total contribution of HIS-SKIP to the total protein concentration was determined to be ~30% or , ~356 µg/ml as assessed by SDS-PAGE. SKIP enzymatic activity was not detected using the Malachite Green Standard Curve in the partially pure preparation. There may be various reasons for not detecting enzymatic activity.
Proteases are a class of enzymes whose function is to cleave peptides into smaller amino acid chains or free amino acids. Two such proteases, Acyl Peptide Enzyme Hydrolase (APEH) and acylase (ACY), function in unison to cleave N-acylated amino acids from the N-terminal end of peptides so that these peptides can become further metabolized in the cell. In this pathway, APEH will first cleave an N-acylated amino acid from the end of a peptide. Then, ACY will remove the acetyl group from the amino acid so it can be recycled or further metabolized. While the enzymatic activity of APEH and ACY have been studied extensively alone, little has been done to study flux through the pathway. Understanding enzyme flux is useful since enzymes rarely work independently of one another in vivo but rather work in a coordinated fashion to perform their respective functions. This research illustrates a novel method to observe how both enzymes work together using Gas Chromatography Mass Spectrometry (GCMS). Using a selected N-acetylated dipeptide, the release of 2 free amino acids-Ala and Met- is detected by GC-MS and correlates to APEH and ACY activity, respectively. Using standard curves and time courses, the rate through APEH and ACY was determined. This method serves as a relatively simple way to study enzymes in vitro in an attempt to better understand the pathways function in vivo. In addition to measuring the flux of more than one enzyme in metabolic pathway, the use of this GCMS method has revealed that the enzymatic activity of the proteases in question do not function universally across different cell lysates. For example, it was determined that the rate of flux through APEH/ACY was 5 times faster in A549 cancer cell lysates than in normal blood lysates. The GCMS method described herein allows comparative studies of APEH/ACY flux and has potential to deepen the knowledge of how other these enzymes work together in both normal and disease states.
Nanomaterials are becoming increasingly common in the environment as they are engineered for technical purposes. Because nanotechnology is a relatively new technology, studies of nanomaterial toxicity effects are ongoing. For this study, Escherichia coli (E. coli) was used to determine the toxicity of coated zinc oxide, uncoated zinc oxide, iron oxide, and copper nanomaterials. Control conditions included no nanomaterials and zinc macromaterial. ATP fluorescence was used to monitor the growth curve of E. coli with and without nanomaterials, and culture methods were used to examine E. coli growth with varying concentrations of nanomaterials. The results so far show that as the concentration of nanomaterials increased, E. coli growth or ATP levels decreased and then increased again. These results were compared to E. coli growth without nanomaterials and E. coli growth with zinc macromaterial. Some nanomaterials may have had a greater impact on E. coli growth than other nanomaterials. These ATP fluorescence tests and culture methods will lead to future chemotaxis studies on the effects of nanomaterials as an attractant, repellent, or inert ingredient on E. coli movement.
Do fish have personality? Repeatability of behavioral traits between sexes in the live-bearing fish Brachyrhaphis roseni
Several recent studies suggest that individual animals have what could be described as 'personality'. My research will address whether fish have personality and the important implications if they do. Specifically, my project addresses whether personality is consistent between sexes. I also look at whether personality will be the same among individuals regardless of the type of environment, or in this case test, it is set in. These are important questions, as there is little understanding of how personality is differentially expressed between sexes and what the best methods to measure personality with consistency are. Personality traits can affect a variety of biological processes, including dispersal, competition, predation risk, mate choice, cognition and decision making, and even speciation. Previous work in my lab suggests that there could be differences in boldness between some Brachyrhaphis species (Ingley et al. 2014), but there was no evidence for differences among sexes. However, my preliminary work in B. roseni suggests that males and females differ in the expression of their boldness behaviors over time. This research will also extend our understanding of the origins of personality by focusing on fishes.
In the ongoing effort to improve clinical diagnosis and treatment for breast cancer patients, understanding tumor heterogeneity remains a focal point of medical research. While individual gene aberrations or mutations have been identified as indicators of breast cancer prognostic outcomes, research in head and neck squamous cell carcinoma has revealed that paired genetic mutation events can be associated with decreased patient survival. We set out to identify such paired genetic mutation events associated with prognostic outcomes in breast cancer patients. Using somatic mutation, chromosomal aberration, and clinical patient data available online via The Cancer Genome Atlas, we performed Cox proportional hazards-ratio analysis on 50 prognostic event pairs of single nucleotide polymorphisms (SNP) and copy number variants (CNV) across 897 patients. Resulting hazards ratio scores indicate the effect of variables on patient life expectancy, with values greater than 1 indicating a decrease in patient life expectancy. Preliminary work has revealed a meaningful decrease in survival probability in patients with both the PIK3CA SNP and BRIP1 CNV, with a hazard ratio score of 1.85. PIK3 pathway mutations are frequently associated with breast cancer tumorigenesis, and BRIP1 aberrations may interfere with BRCA1-interacting helicase, suggesting a possible role in breast cancer tumorigenesis. Interestingly, CNV events paired with mutation events in the tumor-suppressing TP53 gene do not appear to experience the same decrease in survival probability. We continue to investigate these prognostic indicators, possible recorded mechanisms in the literature, and present our findings.
One of the most pressing needs within the medical community is the demand for transplantable organs. On average, a name is added to the waiting list for an organ every 10 minutes in the United States, and 22 people per day on average die waiting for a transplant. Even if a patient receives an organ, the risks associated with the transplant are high -- from the immunosuppressant drugs required to the risk of graft-versus-host disease (GvHD). If organs could be engineered using a patient’s own cellular material, transplants could take place without the risk of GvHD or the need for immunosuppressants. One of the keys to creating any organ is first building a vascular network that could feed the surrounding tissue. This is where our research comes in. Our group is working to create fully functional and patient-specific blood vessels in vitro using a 3D printer. Our 3D-printed blood vessels could be used as the foundation for further tissue engineering or for transplants themselves. In this research, we use the freeform reversible embedding of suspended hydrogels (FRESH) printing method developed by TJ Hinton at Carnegie Mellon University. With the FRESH method, we will 3D print vascular networks of collagen. Successfully implanting said vascularized networks in patients will be made much more achievable by our being able to create a FRESH slurry which is sterile, providing an environment in which human umbilical vein cells (HUVECs) pervading the collagen can grow. In this presentation, I will discuss the progress my team has made in reaching the aforementioned goal. The vitality of the HUVECs will be reported on as a metric of our ability to produce a sterile slurry.
Alouatta palliata (the mantled howler monkey) and Ateles geoffroyi (the black handed spider monkey) are both found in the rainforests of Central and South America. The two species can coexist because they have different dietary needs, A. palliata relies on leaves (foliverous), which are abundant, while A. geoffroyi eats fruits and roots (frugivorous) which are less abundant and more patchily distributed. This difference in diet reduces competition between the species for resources. A. palliata will often form closed multimale-multifemale groups, with up to 40 individuals, and spend most of their day eating or sleeping. A. geoffroyi will typically spend their days traveling, foraging, resting, or engaged in social activities. A. geoffroyi typically lives in multimale-multifemale groups, and form a fission-fusion group structure, this means that their groups are more fluid and change often. This makes A. geoffroyi less territorial and eliminates competition for resources. Both species have an obvious male dominance hierarchy, but there is a difference in the activity pattern of the two species, with A. palliata being less active than A geoffroyi. Due to the social structures of both species, I hypothesize that I will see more affiliative behaviors in both species, than I will aggressive/agnostic. Due to the limited feeding competition in the folivorous A. palliata, I predict that the A. palliata will maintain closer intraspecific proximities than the A. geoffroyi. Data were collected at the La Selva Biological Station in Sarapiquí, Costa Rica, From May 11th to May 22nd, 2017. I conducted instantaneous scan samples in five-minute increments. At each interval, I recorded their behavior and their proximity to the nearest individual. The behaviors were divided into agnostic (fight/grab, bite, and grunt/growl) and affiliative (foraging, playing, rest, traveling, and grooming), and proximities were estimated in meters. It was found that A. geoffroyi and A. palliata showed higher rates of affiliative behaviors than they did aggressive, only 1% of recorded behaviors were agnostic supporting my hypothesis. It was also found that A. palliata maintained an overall proximity of 3.45 meters and A. geoffroyi 4.2 meters. A. palliata who relies on the abundance of leaves, experienced less feeding competition, and maintained closer proximities, supporting the hypothesis. A. geoffroyi experiences greater competition due to less abundance of food, which was reflected in the farther proximities in A. palliata, which also supports the hypothesis.
The orphan G protein-coupled receptors GPR18 and GPR119 are expressed in hippocampal pyramidal cells.
The hippocampus, a location in the brain associated with learning and memory, has been shown to be a site for synaptic plasticity (the strengthening and weakening of synapses over time based on activity levels). Synaptic plasticity mechanisms such as hippocampal long-term potentiation (LTP) and long-term depression appear to be essential in memory formation. While most classical plasticity studies examined the NMDA receptor, the first receptor identified to mediate LTP, other receptors including cannabinoid receptor CB1 are also involved in various types of hippocampal plasticity. As plasticity is so critical to memory, it is necessary to understand these additional mediators of plasticity in order to provide tools to mitigate memory disorders such as Alzheimer’s disease and dementia, which still lack effective treatments. We previously identified plasticity modification by endocannabinoid pathways that were independent of canonical CB1. Therefore, we examined novel orphan G protein-coupled receptors, GPR18 and GPR119. Both GPR18 and GPR119 can be activated by the endocannabinoid anandamide; GPR18 is also activated by THC and endogenous lipid N-arachidonyl-glycine. Using reverse transcription real-time PCR we identified GPR18 and GPR119 expression in mouse hippocampus. PCR products were isolated by gel electrophoresis to demonstrate the appropriate sized amplicon, which was cut from the gel and sequenced to validate them as GPR18 or GPR 119. To confirm PCR data, we used immunohistochemistry to examine GPR18 and GPR119 protein expression and location. We note antibody labeling of GPR18 in cell bodies of CA1, CA3 and dentate gyrus pyramidal cells. Collectively, our data indicate that GPR18 and GPR119 are expressed in the hippocampus. To determine the potential physiological roles of hippocampal GPR18 and GPR119, we performed field electrophysiology experiments. We found no differences in baseline transmission or in LTP. However, we did find significant differences in post-tetanic potentiation (PTP). We have yet to perform field electrophysiology experiments on GPR119, and also hope to look at CA3 interneuron transmission for both proteins. We then intend to use hippocampal slices from GPR18 and GPR119 knockout mice for field electrophysiology experiments. We believe our research will give important insight as to the location and possible functions of GPR18 and GPR119 in hippocampus function and memory.
Biofilm induction in mucormycosis-causing fungi and the synergistic antifungal activity of amphotericin B and thyme oil
Fungal infections caused by opportunistic pathogens have gained clinical importance in the last decade, with a significant increase in infections due to the Zygomycetes, Mucor, Rhizopus and Absidia. These serious and sometimes fatal infections are often associated with biofilm formation. The formation of biofilms often increases resistance to antifungal agents when compared to free living colonies. This study investigates both the biofilm formation and the antifungal susceptibility of two species known to cause mucormycosis infections namely: Rhizopus oryzae and Absidia corymbifera. Upon successful biofilm formation, the synergistic effects between thyme oil and amphotericin B were tested. Results indicate that both R. oryzae and A. corymbifera form biofilms under specific conditions, and that these biofilms were significantly inhibited by thyme oil. The MIC50 of thyme oil on Absidia corymbifera and Rhizopus oryzae was 0.0005 μL/mL and 0.0001 μL/mL respectively. Results also indicate a strong synergistic relationship between amphotericin B and thyme oil when used in combination against fungal biofilms.
The central dogma of biological processes is the conversion of information stored in DNA into proteins, through the mechanisms of transcription and translation. For the proper functioning and survival of an organism, these processes must be performed with great fidelity. Nonsense-mediated mRNA decay (NMD) is a process occurring in conjunction with translation that surveilles mRNAs containing nonsense mutations, also called premature termination codons (PTCs), and causes their rapid degradation. In the absence of NMD, mRNAs containing nonsense mutations are translated into truncated proteins. In this way, the cell prevents the translation of truncated proteins, which can lead to lethality within the organism due to misfolding or the buildup of unusable proteins lacking critical domains. To assess how the loss of NMD affects the viability of individuals carrying naturally occurring genetic variants in this experiment, we crossed Drosophila melanogaster (fruit fly) mutants that lack NMD pathway function to a series of lines that contain naturally occurring variants. The source of these variants is the well characterized Drosophila Genome Reference Panel (DGRP). Of the 168 DGRP lines tested, we discovered that 11 lines showed significant viability difference when NMD is inactivated. Of these 11 lines, 9 showed a significantly decreased viability, while the other 2 lines demonstrated a significantly increased viability. Our major conclusion is that the majority of naturally occurring variants have no significant influence on the viability of the fly when NMD is absent. However, a small number of variants do produce a strong effect on viability, presumably because they are detrimental to the organism if not broken down by the NMD pathway.
Background: Mayflies are a very fascinating and primitive lineage of insect, being one of the first to develop flight and the only taxonomic order that has a subimago stage (a winged stage before sexual maturity). As nymphs, mayflies can go through as many as 50 molts, with the exact number being dependant on the species. Some species of Ephemeroptera are parthenogenetic, meaning that female mayflies can reproduce viable offspring without a male present. However, parthenogenetic mayflies can reproduce via sexual reproduction when males are present. Purpose and essential question: We will be constructing an artificial stream mesocosm for the purpose of rearing parthenogenetic species of mayflies in order to examine their transcriptome data throughout the species lifecycle. Methods: We have constructed three recirculating stream systems consisting of two chambers for a total of 6 chambers holding a combined 50 gallons of water. We will run the water first through a temperature controlled cooler to keep the water at the same temperature of the stream the mayfly nymphs were collected from, and a basic aquarium filter will be used to keep the water as clear as possible. We will periodically add newly collected stream water to provide fresh algae and microbes for the mayfly nymphs to eat. The sides of our tank will be lined with mesh to simulate vegetation along the stream. We will take nymphs out at various stages of development and collect transcriptome data from them. Once the subimago and adults emerge they will be removed from the stream and their eggs will be collected to support the next generation. Conclusion: This project is expected to produce a population of genetically identical mayflies and to have extracted multiple sets of transcriptome data from this population. We plan to continue to cultivate further generations from this colony and undertake more transcriptome data analysis from these populations.
Diabetes mellitus reduces and eventually depletes a person’s ability to regulate his or her blood glucose levels due to either pancreatic beta cell death or reduced sensitivity to insulin throughout the body. Preliminary cell death and insulin insensitivity necessitate increased insulin production by the remaining beta cells. This increased demand on the cells eventually leads to the death of additional beta cells perpetuating the cycle and exacerbating the problem. Type II Diabetes (T2D) is characterized by increased levels of circulating Free Fatty Acids (FFA), particularly Palmitate (PA)—the primary fatty acid synthesized by the liver. In order to better understand the degenerative effects of increasing FFA levels on pancreatic beta cells, rat beta cell insulinoma (INS-1) cells were incubated in progressively increasing concentrations of PA (0.15 mM, 0.3 mM, and 0.5 mM). The cells cultured in higher concentrations were step-wise increased from culture in lower PA concentrations to simulate the progression of T2D hyperlipidemia. In order to more fully explore and understand how beta cell functionality changes during progressively higher concentrations of FFA, cells from each PA concentration level along with a corresponding control were stimulated with glucose and the secreted insulin was measured. All cells were measured against a control cell line that has received no treatment, which served as a model for a standard, non-diabetic individual. Glucose stimulation, insulin secretion, and total insulin content were measured to provide insight into how we can best prevent and treat diabetic symptoms and causes.
Modeling Decidual NK cell transfer of Granulysin to Trophoblast Cells using stimulated Peripheral Blood NK cells
Natural killer (NK) cells are the most abundant immune cell type in the human first trimester placental interface (over 70%). Named decidual natural killer cells (dNK), they must protect the maternal-fetal interface from infection while maintaining tolerance to the semi-allogeneic fetus. dNK are less cytotoxic than peripheral blood NK cells (pNK), even though they express high levels of cytotoxic granules, particularly granulysin (GNLY), which is expressed at higher levels in dNK than pNK. GNLY is a pore-forming antimicrobial peptide produced by cytotoxic lymphocytes (CD8+ T cells and NK cells)4. It selectively permeabilizes bacterial and parasitic membranes, allowing granzymes (serine proteases) to enter and trigger oxidative microbial programmed cell death. While bacterial killing usually involves killing the infected cell, dNK and pNK are able to kill intracellular bacteria in primary trophoblast (placental cells) and trophoblast cell lines (JEG-3) without killing the host cells. The killing mechanism is dependent on transfer of granulysin from NK cells to trophoblasts, without degranulation of granzymes and perforin. The transfer may occur through direct contact between NK and JEG-3 by cytoplasmic projections, or by constitutive secretion of GNLY by dNK and uptake by JEG-3, which occurs by clathrin-mediated endocytosis. While GNLY is detected in JEG-3 (by fluorescent staining and flow cytometry) after co-culture with freshly isolated dNK, the levels of GNLY transferred to JEG-3 by freshly isolated pNK are too low for detection, albeit sufficient to kill bacteria. I waned to know whether pNK also transfer GNLY to trophoblasts like dNK and the mechanism responsible for transfer. To address this question, I activated pNK to increase their expression of GNLY to levels that would allow me to detect GNLY transfer to JEG-3.
Aaron Leifer, Jasmine Banner, Collin Christensen, Trevor Lloyd, Kenneth Call, Brigham Young University Diabetes Mellitus has become a worldwide epidemic affecting over 400 million people. Both type 1 and type 2 diabetes result from the body’s inability to produce or respond to insulin in order to regulate blood sugar. In both cases, the insulin secreting ë_-cells in the pancreatic Islets of Langerhans have become endangered and in many cases non-functional. The function of these ë_-cells is defined by their ability to multiply and maintain a steady number, to defend against induced cell death and ultimately to secrete insulin. Since ë_-cell production reaches its peak during fetal development, this would suggest that diabetics have an inactive pathway to produce functional ë_-cells. However, recent studies have identified key transcription factors that aid pancreatic progenitors in becoming functional ë_-cells. Pdx1 is a transcription factor that is active throughout the ë_-cell pathway and found in mature ë_-cells. Research has identified Pdx1 as a key component in helping both ë±-cells and ë_-cells proliferate and even in reprogramming ë±-cells to become functional ë_-cells. Additionally, Pdx1 has been identified to help ë_-cells effectively secrete insulin. We present data demonstrating the effect of Pdx1 adenoviral over-expression on three independent markers of functional ë_-cell mass: 1) cell proliferation, 2) cell survival, and 3) insulin content and secretion. Defining the effect of Pdx1 on each of these parameters will provide further data to explore therapeutic interventions for diabetic patients.
Epithelia encase most of our internal organs and, indeed, the whole body. To serve as an effective barrier against pathogens, epithelia must regulate cell numbers even as cells continuously divide, grow, and die. Cell extrusion happens when a cell fated to die signals neighboring cells to squeeze it out of the epithelium using an actomyosin ring. The extruded cell may undergo apoptosis, a highly regulated process of programmed cell death, or be a live cell that later dies through anoikis, or cell death through loss of contact with the basement membrane and survival signals. Typically, cells extrude apically, away from the basal membrane, and are removed from the lumen of an organ or gland. However, some cells are able to extrude basally and survive post-extrusion, a strategy that an oncogene-bearing tumor cell might use to achieve metastasis. KRAS is one such oncogene that is often mutated in human cancer. The wildtype gene governs multiple pro-growth and survival pathways in the cell. A mutation in one amino acid, called KRasV12, converts it to an oncogene and allows the protein to be constitutively active, inappropriately enhancing its function. This often leads to runaway cell proliferation and tumorigenesis. In prior tissue culture studies we observed that KRasV12 cells are significantly more likely to extrude basally than apically, and could possibly migrate and invade. We therefore hypothesized that KRasV12-driven basal extrusion leads to metastasis, a novel idea that has never been demonstrated. We sought to test our hypothesis in a zebrafish model of epithelial cancer. We injected one-cell embryos with EGFP-labeled KRasV12 or CAAX control, sorted for embryos positive for transgenes at 24 hpf, and fixed the embryos at 48 hpf. We performed immunostaining for EGFP, E-cadherin (periderm marker) or p63 (basal cell marker), and the nucleus (DAPI). We used the expression of caspase-3, a protein activated during apoptosis, and nuclear morphology to determine cell death. Using E-cadherin or p63 expression to mark the borders of the developing epidermis, we quantified live or dying cells either on the epidermal surface or internalized into the fish body. Indeed we find that embryos with the KRasV12 oncogene have a significantly higher percentage of live internalized cells compared to control, suggesting that these may later give rise to metastases. We are currently performing live imaging studies to ascertain if KRasV12 cells spread via basal extrusion and make it to the bloodstream.
The prevalence of diabetes, a disease that affects all ages, continues to increase at a rapid rate. Type 1 diabetes results from the autoimmune destruction of pancreatic β-cells while type 2 diabetes results from insulin resistance, which leads to eventual β-cell apoptosis. In both diseases, preventing the death of insulin producing β-cells, or regenerating functional β-cell mass, would be an essential step for finding a cure. The orphan nuclear receptor Nr4a1 has been shown to play an important role in β-cell growth. In vitro, overexpression of Nr4a1 leads to increased β-cell proliferation, and lentiviral knockdown of Nr4a1 leads to decreased β-cell proliferation. Lentiviral knockdown of Nr4a1 also reduces glucose induced mitochondrial respiration, insulin secretion, and the expression of some genes associated with glucose metabolism. To better understand the role Nr4a1 plays in the progression of diabetes, we examined a full-body knockout mouse model under conditions of a normal or high fat diet. We measured blood glucose weekly, weight monthly, and glucose tolerance monthly until 20 weeks of age. When harvested at 20 weeks, we measured mitochondrial respiration of various tissues and the levels of B-cells, T-cells, and neutrophils in the blood. Our data demonstrate systemic effects of Nr4a1 in terms of fuel utilization and diabetic progression.
Investigation of the synergistic effect of Origanum vulgareÛø(Oregano) Oil and Amphotericin B to inhibit fungal biofilm in species known to causing Mucormycosis
Karaleen Anderson, Mariel Hatch, Caeleb Harris, Anastasiia Matkovska, Kendrick Kiggins, Levi Neely, Utah Valley University Mucormycosis is a life-threatening disease that occurs in immunocompromised individuals, such as burn, cancer and diabetic patients. Amphotericin B is the current line of treatment for the disease, however it is known to have many adverse side effects including cell toxicity. Due to the high mortality and morbidity associated with the disease even when treated with Amphotericin B, it is vital that new combination therapeutic techniques be investigated in order to more effectively treat the disease. Mucromycosis is most often caused by a filamentous, opportunistic fungi called Rhizopus oryzae. This species causes up to 80% of infections and is the most common species isolated from confirmed Mucormycosis sites. Origanum vulgare (oregano) oil has been shown to have broad anti-microbial properties in various studies. This study investigates the ability of oregano oil to lower the concentration of Amphotericin B needed to successfully inhibit R. oryzae biofilms. Various concentrations of oregano oil and Amphotericin B are tested to determine the optimal concentration ratio that maximizes biofilm inhibition. Synergistic activity of oregano oil and Amp B could be used to decrease the amount of Amphotericin B needed to treat Mucormycosis infections while still utilizing the antifungal properties of Oregano oil.
Matt Austin, Brooke Smyth, Lauren Manwaring, Moroni Lopez, Brigham Young University Type 2 diabetes is characterized by the inability of pancreatic ë_-cells, which secrete insulin, to regulate blood glucose levels. The glucose-regulating mechanisms of these dysfunctional ë_-cells exhibit a gradual insensitivity to insulin, caused by prolonged hyperglycemia. Treatment for individuals suffering from Type 2 diabetes is limited to supplementary insulin injections. However, recent studies have revealed that powerful anti-oxidants called flavanols, which are found in cocoa, affect insulin secretion and glucose tolerance of ë_-cells. We isolated three fractions from the whole cocoa extract: monomeric catechin-rich, oligomeric procyandin-rich and polymeric procyandin-rich flavanols. Because cellular respiration is closely related to insulin secretion, we hypothesize that these fractions may exert their anti-diabetic effects by enhancing cellular respiration. To determine the effects of cocoa flavanols on ë_-cell respiration, we performed respiration assays on INS-1 ë_-cell lines incubated with increasing concentrations of whole cocoa extract, monomeric, polymeric and oligomeric catechin fractions or a control. We present data demonstrating the effect of these compounds on ë_-cell respiration. Advancements based on our research could provide an innovative therapeutic alternative to current diabetes treatment and new insight into the respiratory pathways of ë_-cells, affording new targets for a multitude of potential gene therapies.
The alkene functional group is commonly found in many important organic molecules. The restriction to rotation of a carbon-carbon double bond leads to isomeric molecules that can have similar physical properties and can be difficult to separate by conventional techniques. Therefore, it is important that synthetic reactions produce one alkene isomer in excess to avoid a potentially difficult separation. Here we present our study of a stereospecific palladium-catalyzed cross-coupling reaction for the production of alkyl-substituted trans-alkenes.
Multivalent Human Serum Albumin ÛÒ Anti-CD20 Fab’ Conjugates for Induction of Apoptosis in Lymphoma Cells
Christian Kodele, Lian Li, Jane Yang, University of Utah Non-Hodgkin lymphoma (NHL) is an immune disease mostly of B-cell origin (eighty-five percent of the time) as well as the ninth leading cause of cancer death in the United States. Although treatments for NHLs greatly improved following the FDA approval of Rituximab (RTX), refractive malignancies still occur that are nonresponsive and/or resistance to current therapies in at least a third of all patients. This has been attributed both to the inability of immune effector cells (eg., macrophages, natural killer cells) to hypercrosslink ligated monoclonal antibodies (mAbs), and to Fc receptor (FcR)-mediated endocytosis or ÛÏtrogocytosisÛ of CD20 antigens. In order to address these clinical obstacles, we designed a novel paradigm in macromolecular therapeutics that can specifically kill cancer cells without a drug. This paradigm is based on the use of anti-CD20 Fab’ fragments in a multivalent system. Crosslinking of CD20 receptors leads to receptor clustering, transfer to lipid rafts, opening of a calcium channel, and ultimately apoptosis. Additionally, the removal of the Fc fragment resulted enticingly in both the rendering of the system to be immune dependent and in decreasing the numerous adverse effects. In this study, we have used human serum albumin (HSA) as the multivalent carrier of RTX based Fab’ fragments. We have covalently attached multiple Fab’ fragments to HSA, characterized the nanoconjugate’s physiochemical properties, and evaluated its efficacy to induce apoptosis of Raji B cells in vitro. The efficacy of the nanoconjugate to induce apoptosis was determined with Annexin V assay and flow cytometry. The interaction of the nanoconstruct with Raji cells was characterized using confocal microscopy of Cy5 labeled conjugates. As predicted, the HSA-(Fab’)x conjugate was able to induce cell death in vitro. The results of the Annexin V apoptosis assay showed that 38.9 percent of the cell population treated with the conjugate became apoptotic, while 13.6 and 15.7 percent of the cell populations untreated and treated with whole RTX mAb became apoptotic respectively. Furthermore, images recorded by use of confocal microscopy suggest that the attachment of HSA-(Fab’)x conjugate to the cell membrane is CD20 specific. While not conclusive, the combination of these results suggest that the mechanism of action involves cross-linking of the CD20 receptor, which subsequently induces apoptosis. We believe these results warrant further investigation of the mechanism of action of HSA-(Fab’)x, as well as the treatment potential of this nanoconjugate.
Dipodomys microps, the chisel-toothed kangaroo rat, is a small desert rodent occurring in a region that has undergone substantial ecological changes due to rangeland expansion. Although generally considered a saltbush specialist, the natural diet of D. microps consists of saltbush (Atriplex confertifolia) a C4 plant and blackbush (Coleogyne ramosissima) a C3 plant. To determine if human-driven environmental change has impacted the natural diet of D. microps we took advantage of natural differences of carbon stable isotopes between C4 and C3 plants. We predicted that increased abundances of blackbush due to rangeland expansion and corresponding decreases of saltbush will have led to diet shifts in D. microps. To test this prediction we sampled fur of kangaroo rats from Inyo County, California housed in the Museum of Vertebrate Zoology from three time periods: before the landscape change (1917, N=10), during the disturbance (1973-1978, N=10), and recent samples (2008-2009, N=8). We observed minor shifts in percent of saltbush in diets of D. microps. In 1917 saltbrush consumption averaged 48%, in the mid 1970s saltbrush ingestion averaged 47i%, and by the late 2000s the average consumption dropped to 37%. These observations are consistent with my prediction that environmental factors caused kangaroo rats to favor blackbush. However, the low percentage of C4 reliance suggests this species may not be a true Atriplex specialist as previously thought.
In many species, including humans, a decline in reproductive ability is associated with many deleterious health conditions. Some species maintain reproductive ability throughout life, and reproductive decline is often associated with the end of life. Ovary function in particular seems to be correlated in longevity. Previous studies suggest that preserved ovarian function is correlated with increased mean lifespan; however, the role of the germ cells within the ovary is not well understood To investigate the relationship between germ cells and somatic cells and their role in expanding longevity, this project measured the life span of 17-month-old mice that received either germ cell-containing ovaries or germ cell-depleted (somatic-only) ovaries. In this experiment, young, germ cell-containing and germ cell-depleted ovaries from 60-day-old mice were transplanted into 17-month-old mice. Life span was then observed and contrasted with the mean life span in this line of female mice (644 days). In the recipient mice that possessed germ cell-containing ovaries, life span increased by 10%. This result was similar to previous experiments where mice received transplants at 11 months of age and life span was increased by 11%, suggesting the age at transplant had little influence on remaining life span. However, when germ cells were depleted from the young ovaries prior to transplantation, life span was increased by 21%. These results suggest that somatic cells have a major role in longevity and that the germ cells were not essential for reproduction-directed extension of life span. While germ cell-depleted ovaries lacked germ cells, the somatic cells remained intact and functional. This may indicate that somatic cell function is necessary within ovaries to produce and increase in life span as seen with other studies. From these results, germ cell depletion can be concluded to significantly increase lifespan within our mice subjects. Germ cell-depleted ovaries lead to increased lifespan and health span. Further experiments will be done in order to analyze ovary effect on longevity. Current experiments are being conducted to analyze in what ways ovaries influence longevity and overall health. This question is the basis for experiments currently being conducted.
Nanostructures and the manufacturing of them are being researched for applications in areas such as controlled drug release, bio-sensors, solar cells, and data storage. The nanostructure known as the gyroid is particularly promising for application in these areas because of the continuous, fully connected channels that spiral through it periodically and uniformly. The gyroid can be manufactured through self-assembling block-copolymers or surfactants, but the mechanism for the assembly is not well understood. A better understanding of the parameters that control the formation of this structure will tell us how we can better control the formation of the gyroid structure. Our goal is to use molecular dynamics simulations to find the mechanism behind the formation of the gyroid in a model system. We were able to simulate its spontaneous formation and tested the efficiency of a number of order parameters, something that can distinguish between the gyroid and the surrounding mixture. As the gyroid forms, it passes through a transition state: the point where it has a 50% probability of forming or melting. The complexity of the gyroid structure makes it difficult to find an order parameter that can capture the structure at the transition state. However, by using committor analysis methods we were able to identify the transition state. We developed several order parameters that can distinguish gyroid from the isotropic mixture and, using Aimless Shooting and Maximum Likelihood Optimization, we ranked these parameters according to their effectiveness.
Late gas defects in aging cheese result in significant losses to the manufacturer. Lactobacillus wasatchensis, a novel non-starter lactic acid bacteria (NSLAB), has recently been identified as an important cause of late gas defect. Controlling growth of this unwanted NSLAB may be done by incorporation of bio-protective LAB cultures (BP-LAB) into the cheese during manufacture, which could inhibit its growth during cheese aging. Previous research has shown that several BP-LAB cultures inhibit Lb. wasatchensis to varying degrees but the exact mode of inhibition has not been determined. Quantification of inhibition between BP-LAB cultures and Lb. wasatchensis was done using the spot test with the agar-flip method then measuring inhibition zones in comparison to time incubated. MRS agar with 1% ribose (MRS-R) was inoculated with each BP-LAB and incubated anaerobically at 35°C for 48 h to form a spot colony. Inoculated agar was flipped over and a Lb. wasatchensis strain swabbed on the exposed surface then incubated anaerobically at 25°C for up to 72 h. In addition, potential synergistic quantification of inhibition by co-BP-LAB strains was tested by taking 1 mL each of two different BP-LAB strains growing them together, and then following the previous described protocol. The five most inhibitory BP-LAB cultures were Lactobacillus rhamnosus LB3, Lactobacillus paracasei P-210, Lactobacillus brevis 13648, Lactobacillus casei F19, and Lactobacillus paracasei Lila. Four different co-cultures were tested LB3/ P-210, LB3/P-220, P-200/P-210, and P-200/P-220. No significant increases in the inhibition zones were observed when BP-LAB cultures were paired versus individual strains. Results confirmed that selected BP-LAB strains can inhibit growth of Lb. wasatchensis. Currently, we are trying to isolate the bacteriocins produced by these BP-LAB.
Lactobacillus wasatchensis (WDC04) is a novel, slow growing, non-starter lactic acid bacterium (NSLAB) causing late gas formation in aging cheese, which results in significant economic losses to the producer. During cheese aging, organic acids can be produced by other NSLAB organisms or purposefully added to the cheese during manufacture. Organic acids are often used as preservatives. They are often found naturally in foods, generally don’t affect flavor or product quality, and under acidic conditions, are able to enter bacterial cells effectively altering the cell’s proton motive force. The effect of selected organic acids in their natural concentration range (2.5-560 mM) in Cheddar cheese was investigated for their ability to inhibit Lb. wasatchensis. Five organic acids (lactic, formic, propionic, citric and acetic) that can be produced by NSLAB organisms were selected and added at minimum, median, and maximum concentrations to individual wells of a 48 well plate containing MRS broth with 1% ribose (MRS + R) inoculated with WDC04. Growth rates were determined on a Tecan Infinite 200 PRO plate reader over 40 hours and results graphed on Excel. Both formic and citric acid showed inhibition of Lb. wasatchensis. As formic acid concentrations increased, the inhibitory effect also increased. The maximum concentration (100 mM) showed the most inhibition, the median concentration (63.15 mM) had an inhibitory effect between maximum and minimum, and the minimum concentration (26.3 mM) caused minimal inhibition. The addition of citric acid at the minimum (12 mM) and median (13.5 mM) concentrations showed similar inhibition. The use of these acids at concentrations normally found in Cheddar cheese is a potential antimicrobial measure to prevent or reduce late blowing in aging cheeses.
Examination of proteins bound to nascent DNA in mammalian cells using BrdU-ChIP-Slot-Western technique
Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples
Reactive oxygen species (ROS) are highly regulated in the body by antioxidants. When the regulation of ROS in the body is hindered, it can lead to oxidative stress and cell damage. The Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme plays a part in reducing ROS and damage due to oxidative stress. It has been shown that the hyperglycemic conditions present in type 1 and type 2 diabetes mellitus impairs the activity of the G6PD enzyme, leading to oxidative stress and damage in endothelial cells1. Here, however, the activity of G6PD in red blood cells was specifically analyzed and determined to be lower in red blood cells treated with high glucose compared to controls. It is anticipated that upon addition of an anti-oxidant to the red blood cells treated with high glucose, the G6PD activity level will normalize. References 1. Zang, Z., Apse, K., Pang, J., & Stanton, R. C. (2000). High glucose inhibits glucose-6-phosphate dehydrogenase via cAMP in aortic endothelial cells. The Journal of Biological Chemistry, 275(51), 40042-40047. Doi: 10.1074/jbc.M007505200
Research has rarely been conducted past 2003 on comparing behaviors of zoo gorillas in different environments. The few relative studies suggest that, of course, naturalistic behavior is more common in naturalistic environments; ie. outside enclosures. "Exhibition of gorilla social groups in naturalistic habitats has been shown to...facilitate expression of species-typical behavior and enhance reproduction" (Lukas, K. ). How does this species-typical behavior differ from an outdoor enclosure to an indoor enclosure? With the Hogle Zoo's upcoming closure of the outside exhibit, during the winter months, I intend to draw a conclusion as to what differences the gorillas might display.
A long-standing hypothesis in the field of neuroethology has been that organisms performing similar behaviors use similar underlying neuronal circuitry. Recently, though, studies focused on locomotion have shown that this may not necessarily be true. Work in sea slugs has demonstrated that homologous behaviors can be generated by a variety of neuronal connections. Our work looks at reproductive behaviors and asks whether those behaviors are mediated by the same neuronal connections in two species of leech. Research in Hirudo verbana has shown that reproduction is mediated by a neurohormone, hirudotocin, that is stored in and is released from a circuit of characterized neurons called Leydig Cells. A second leech, Macrobdella decora, has the same reproductive strategy as H. verbana, including a similar progression of behaviors. Using immunohistochemistry, we have identified Leydig Cell homologs in M. decora. Additionally, when we inject either M. decora or H. verbana with a hormone similar to hirudotocin, we observe reproductive-behavior output that is nearly identical. We intend to compare these species further by fully characterizing the Leydig Cell network using electrophysiology. These results will clarify the relationship between reproductive behavior and neuronal circuitry in leeches and give insight as to how neural circuits evolve across species.
Objectives We evaluated the effects of different levels of stored iron (ferritin) within normal ranges on immune cell group production and inflammation markers in cross-country athletes. Research Methodology Forty-one NCAA division 1 cross-country athletes, ages 18 to 25 years old (Male: 19; Female; 22), had blood drawn at the beginning of the cross country season. Blood was collected from subjects and analyzed by complete blood count (CBC) at McKay Dee Hospital in Ogden, Utah. Enzymatic spectrophotometry was used to determine participants ferritin levels. Cytokines IL-1β IL-6, IL10, GM-CSF, IL-5, and IL-4 were measured at baseline by the magnetic multiplex panel for Luminex TM platform (at University of Connecticut). Based on ferritin levels, participants were divided into two groups, high ferritin, and low ferritin. Student’s t-test was used to compare cytokines and CBC mean difference between low and high ferritin groups. Pearson correlations were used to determine associations between cells groups and cytokines under low or high ferritin conditions. IBM SPSS statistics 22 software for Windows was used to analyze the collected data. Results Participants in the high ferritin group had higher levels of IL-1β (p=0.04) and IL-5 (p=0.05) and eosinophils (p=0.02) when compared to the low ferritin counterparts. In contrast, IL-4 (p=0.04) was significantly higher in the low ferritin group. Moderate-strong correlations were found between eosinophils and cytokines; IL-1β (r=0.64) and IL-5 (r=0.59) in the high ferritin group. Conversely, eosinophils from the low ferritin group correlated strongly with IL-4 (r=0.86) Conclusions In this study, it was observed that eosinophils correlated with different cytokines depending on the iron storage status. Our results are in accordance with previous studies showing that IL-1β down regulates ferroportin (FPN) by increasing hepcidin levels which in turn decrease iron absorption. As iron storage in the form of ferritin increases, the need to absorb iron decreases inversely to IL-1β. Additionally, we observed increases in IL-4 in the low ferritin group. IL-4 has been observed to signal an increase in iron uptake (absorption) and mobilization. This is accomplished through transferrin (Tfn) expression. Furthermore, eosinophils and IL-5 were higher in the high ferritin group. IL-5 is a hematopoietic cytokine produced by eosinophils, it is possible that the increases in this biomarker were linked to the increases in red blood cell production observed on the high ferritin athlete group. Our results indicate that Iron storage status measured by ferritin levels may modulate cytokine release and immune cell profile.
Parker Booren, Nathanael Jensen, Talon Aitken, Samuel Grover, Jackie Crabree, Brigham Young University Diabetes continues to grow at a rapid rate, affecting the lives of both young and old. Both Type 1 and Type 2 diabetes lead to eventual ë_ cell depletion (and subsequent decrease in insulin secretion). This can be treated through ë_ cell transplantation from the pancreata of cadavers. Currently, collecting sufficient ë_ cells for one diabetic patient requires pancreata from multiple cadavers. If proliferation can be induced in a donor’s aged ë_ cells, transplantation would become more effective as one donor now becomes sufficient to serve one or two patients. Nkx6.1 is a transcription factor that increases insulin secretion and induces proliferation of young rat ë_ cells (5 weeks) through the upregulation of its target genes: VGF, Nr4a1 and Nr4a3. Aged rat ë_ cells (5+ months) fail to proliferate after overexpression of Nkx6.1. We have also shown that upregulation of Nkx6.1’s target genes is disrupted in these aged ë_ cells. This may be due to changes in expression of a binding partner necessary for Nkx6.1’s upregulation of these target genes or to changes in Nkx6.1 posttranslational modifications that impede binding partner interactions in aged ë_ cells. We present data from co-immunoprecipitation and mass spectrometry experiments that reveal the presence or absence of Nkx6.1’s binding partner in young and aged ë_ cells. Furthermore, we present mass spectrometry results of Nkx6.1 posttranslational modification from young and old ë_ cells. This data will increase understanding on the ability of Nkx6.1 to upregulate its target genes in an aged ë_ cell.
Diabetes affects over 30 million Americans and 185,000 Utahn’s. Type 1 and Type 2 diabetes are characterized by decreased functional β-cell mass and insulin production. Diabetes also results in increased circulating glucose and fatty acid levels, which damage and destroy β-cells over time. Our study will shed further light on the effects of palmitate, the most commonly made fatty acid in the liver, on hyperlipidemia. In this study we test the specific effects of chronic palmitate exposure on various cell lines acclimated to 0.15 mM, 0.3 mM, and 0.5 mM concentrations of palmitate. We demonstrate the effects of progressive long-term exposure to palmitate on β-cell proliferation and resistance to apoptosis. We demonstrate mechanistic changes that result in the observed phenotypes. Our goal in this study is to explore how β-cells adapt to exposure to hyperlipidemia, and to define interventions to protect β-cells from the harmful effects of hyperlipidemia.
Aphthous stomatitis, more commonly known as canker sores, are painful ulcers that form in the epithelial tissues the mouth. Those affected by these sores typically develop a canker multiple times per year, with the sores lasting a duration ranging between a few days to a few weeks. Approximately 40% of college age adults report experiencing canker sores at least 3-6 times within a one-year period. For how prominent the sores can be, relatively little is known about what causes canker sores and why some people are affected, and others are not. Although the cause of canker sores is unknown, evidence is beginning to suggest that bacteria within the oral cavity could play a role in forming canker sores. The DEFB1 gene is an antimicrobial peptide. Within humans, one DEFB1 allele has been shown to correlate with increased bacterial infections including increases in cavities. This study seeks to determine if carriers of the affected DEFB1 allele are at an increased risk for canker sores. We genotyped subjects unaffected by canker sores for the DEFB1 gene, and compared them to subjects who report as regularly affected by canker sores in order to determine is the different DEFB1 alleles correlate to canker sore infection rates.
A number of genes have been researched to determine their role in athletic performance. Some genes have been discovered to give a performance advantage in either power or endurance events. In order to find genetic association with athletic performance, we collected DNA from 200 Division III track athletes and determined their genotype for the following genes: ACE, ACTN3, NOS3, BMP2, PPARA, ACSL1, ACVR1B, GABPB1, BDKRB2. The genotypes of these athletes were analyzed to determine if one version of the gene was advantageous over another in both power and endurance events. Additionally, the genotypes were analyzed to see if combinations of the advantageous genotypes result in a further advantage over other combinations. By accomplishing these analyses, we look to take another step toward predicting the success of an athlete based off the individual’s genotype. We also aim to identify which genes will predispose an athlete to be most successful. By understanding how these genes coincide, athletes could then know which events they would be genetically predisposed for and thereby determine which events would give them the most success.
Aaron Leifer, Jasmine Banner, Collin Christensen, Trevor Lloyd, Kenneth Call, Brigham Young University Approximately 9.4 percent of the United States is affected by type 1 or type 2 diabetes. Diabetes results from the body’s inability to maintain healthy blood glucose levels due to the loss of pancreatic ë_-cells (insulin secreting cells) or from the body’s insulin sensitive cells becoming insulin resistant. Both type 1 and type 2 diabetes results in a loss of functional ë_-cells. The current treatments for diabetes are insulin injections or transplants, many times requiring up to three donors per transplant. Neither option is an optimal cure: insulin injections do not cure the disease, and transplants are not available to the majority of people. We propose that being able to replicate ë_-cells in-vivo would allow us to provide a cure to diabetes. ë_-cells stop reproducing (proliferating) soon after birth except in a few occasions such as obesity and pregnancy, leading us to believe that there are key gene(s) that induce cell proliferation when activated. Finding these gene(s) would present a viable cure, being able to grow ë_-cells in-vivo for transplantation or even injection. The gene MafA is present in mature ë_-cells and previous research has revealed its vital role in the pancreas. MafA is turned on around embryonic day 15.5 and steadily increases expression up until the cell becomes a mature ë_-cell. The time period when MafA is turned on corresponds with when a ë_-cell is proliferating and developing leading us to believe that MafA is crucial to finding a cure. Here we show the effect of MafA overexpression on INS1 832/13 ë_-cell proliferation, survival, and insulin secretion.
The ACE gene plays a key role in regulating blood pressure through the Renin-angiotensin-aldosterone system. Within the ACE gene, there are two alleles that have various phenotypic effects: the insertion (I) and deletion (D) alleles. The I allele contains a 287 base pair transposon inserted within an intron, while the D allele lacks this transposon. Carriers of both D alleles have increased ACE activity, which is associated with an increased risk of coronary heart disease and other cardiovascular diseases, while carriers of both I alleles have decreased ACE activity. Currently, the mechanism behind both actions is not understood. As transposons are often targets for DNA methylation, methylation status of the ACE gene may influence the activity of the gene and may contribute to ACE activity levels. In this study, we evaluated the methylation status of carriers of the I and D allele to determine if the I allele is targeted for DNA methylation. The results of this study may have implications in the transcriptional activity of other genes where transposons are present. If methylation spreads from the transposon to other portions of the gene body in ACE, then it can be inferred that similar effects would occur in other genes containing transposons.
Quantifying Minimum Banding Size and Survival in fledgling American White Pelicans from Gunnison Island
Every year, nearly 20,000 American White Pelicans (Pelecanus erythrorhynchos) raise young on Gunnison Island in the North arm of the Great Salt Lake. The Gunnison Island pelican colony is the second largest in the Western United States, and is crucial to the maintenance and viability of the species. Pelicans in Utah are increasing in number, which is a conservation success story, but larger numbers of the birds means a higher likelihood of range extension in search of resources. As a result, serious concerns have been raised about pelican resource use, including eating game fish from reservoirs and colliding with airplanes. To specifically address some of these concerns and to understand basic population ecology, The Utah Division of Wildlife Resources (UDWR) outfits adult pelicans with solar-powered transmitter backpacks and bands around 500 juvenile pelicans annually. In general, only large-bodied fledgling birds, which are chosen based on sight alone, are selected to be banded, with smaller birds being released. The primary goal of this research project was to correlate and make recommendations for reliable morphometrics that could quickly and quantitatively be used to select pelicans for banding. To do so we collected morphometric data from both banded and nonbanded fledgling pelicans during the annual UDWR event. Measurements included beak length, weight, tarsal-metatarsal diameter, tarsal-phalanges diameter, and parasite load. Our findings suggested that beak length correlated most strongly with pelican weight, and would therefore be the most reliable morphometric to use. Our secondary goal was to understand the effects of banding on fledgling survival. This process included a tag sweep of Gunnison Island prior to pelican arrival and right after departure. Later research will determine survival rates for each size group. This research will help answer the question of whether or not the current banding system impacts pelican survival. Additionally, increasing our understanding of population dynamics will help conservation managers construct plans that ease tensions between the natural resource (pelicans) and other land-use shareholders.
In evolutionary/systematic biology, phylogenetic reconstruction relies on hypotheses gleaned from historical (usually fossil/ paleontological), morphological, or molecular data—compounded and analyzed by various statistical or logical methods that are intended to yield the most accurate evolutionary tree based on the data entered. Phylogenetic discordance usually occurs when different genetic data generate competing evolutionary hypotheses; or it is uncertain which, of all espoused, is the true phylogeny. Discordance is philosophically interesting because it can, in certain cases, pull apart justificatory sources concerning alternative analytical methods—especially in cases where the same data and evolutionary parameters are assumed, but discordant phylogenies nonetheless result. In other cases, discordance can allow us to understand genetic and lineage persistence, divergence, and manifestation over time—especially in cases where isolated molecular data (e.g. mitochondrial DNA versus nuclear DNA) generate discordant phylogenies for the same groups of organisms. Discordance can often be the foundation for discussion about the ontological status of species, about what should count as phylogenetic data, about what kinds of assumptions we should make regarding the nature of evolution, and so on. Understanding discordance provides a framework for understanding the nature of the scientific method and knowledge production therein; and specifically, in this case the precarious nature of phylogenetic reconstruction. Discordance shows us that even today, systematics is a dynamic and malleable process, in which the only thing really set in stone might be the paleontological data. In partnership with the GSLC, we began with a simple case: Divergent histories of the Polar Bear/Brown Bear complex, analyzed using both mitochondrial and nuclear DNA. Though both are ‘molecular data’, each convey a slightly different genetic history. Nonetheless, it turns out, contributing valuable evidence about the truly unique and intertwined histories of the two bear lineages. For our research we attended the Evolution Meetings, hosted annually by ASN, SSB, and SSE (this year in Portland, Oregon). We sought out similar cases of contemporary discordance presented by current evolutionary biologists from around the world. We have been working with the GSLC to provide these concrete cases in hopes of creating a module that would simply and clearly demonstrate modern attempts by real biologists to grapple with discordance. Our poster displays some of the most tractable cases, which illuminate Discordance engendered both by conflicting data, and by conflicting analytical methods. We have also outlined our current research project into the justificatory purchase of Parsimony, stemming from this endeavor.
Assessing the Role of Temperature in Dengue Fever Outbreak Dynamics with Wolbachia Transinfection Control Methods.
Dengue fever is one of the most common mosquito borne viral infections in the world; its spread is most prominent in tropical climates. The infection is caused by bites from mosquitoes carrying the virus. Recently, biologists have discovered that if you infect mosquitoes with bacteria called Wolbachia (a process called transinfection), and release them into the wild, they will spread this bacteria to the local mosquito population. Wolbachia bacteria causes an inhibited ability for the mosquitoes to transmit the Dengue virus, as well as a decreased mosquito lifespan. Several studies have shown that the Wolbachia bacteria can be used as a control method to mitigate the severity of Dengue fever outbreaks. We developed a mathematical model using a system of nonlinear differential equations to investigate how Wolbachia transinfections could diminish the spread of the Dengue virus in human populations. We then used MATLAB computational software to solve our model numerically, so we could simulate the spread of Dengue fever with a wide variety of environmental and human factors. With this information, we can determine how quickly and how far the Dengue virus can spread under variable factors such as environmental temperature, size of mosquito population, (both local and Wolbachia carrying), what time of year the Wolbachia mosquitoes are released and human population size. The numerical results of our model show that use of the Wolbachia bacteria can significantly reduce the severity of Dengue fever outbreaks, and that the environment temperature can play a large role in mitigating the outbreak. Our model can be used by mosquito abatement centers, and adjusted for local factors to determine optimal strategies to control disease outbreaks most efficiently.
Our laboratory is determining the contribution from endothelial cell (EC) autophagy to aging-associated endothelial dysfunction. Autophagy is a process whereby damaged cellular cargo is delivered to the lysosome for degradation and eventual recycling or removal. Data from our laboratory indicate that: (i) autophagy occurs in ECs and arteries; (ii) autophagy-related (Atg) mRNA in ECs and protein expression in arteries is lower in old (22 months) vs. adult (7 months) mice; (iii) endothelial dysfunction displayed by arteries from old vs. adult mice is rescued by pharmacological activation of autophagy; and (iv) exercise-training upregulates vascular autophagy in adult mice. Thus, robust rationale is provided to test the hypothesis that exercise-training lessens endothelial dysfunction displayed by old mice by increasing vascular autophagy. 21-month old male C57Bl6 mice were familiarized with treadmill running for 2 days. On days 3 and 4, all mice completed a body composition assessment (nuclear magnetic resonance;NMR) and a maximal exercise test wherein total work performed was calculated. Next, animals ran on a motorized treadmill 6 x per week x 15-60-min per session @ 10-20% grade x 11.5 weeks (ETR, n=12) or remained sedentary (SED, n=6). SED mice maintained familiarity with treadmill-running by completing 1 x 15-min bout per week. After 11.5 weeks NMR and maximal exercise tests were repeated on all mice. Additionally, myocardial function was evaluated using echocardiography, indices of autophagy-related EC mRNA (RT-PCR) and arterial protein (immunoblotting) were estimated, and arterial function was quantified using isobaric and isometric procedures. Total work (kgm) performed during the maximal exercise test at 11.5 weeks was greater (p
Oxygen levels in water environments affect many aspects of the world around us. Fish require diffused oxygen to live, our pipes will corrode more easily if oxygen levels are too high, etc. For this reason, we have focused our research on fabricating a microfluidic device that is sensitive enough to measure small amounts of oxygen, is durable enough to be reused, and is cost effective. Our device can measure oxygen in anoxic environments (water that contains very low levels of oxygen) and is made out of PDMS, which is an extremely durable substance. We have based our design off of the STOX electrode which uses a double membrane set-up to measure oxygen levels. The STOX electrode is very expensive to make and is extremely fragile. We have successfully made a device and performed field tests.
are used to distinguish meaning in a second language (L2). For example, Spanish learners must acquire the /i/-/ ɪ/ contrast to distinguish the words ‘sheep’ and ‘ship’. However, much less work has investigated the acquisition of L2 allophones (variant pronunciations of a contrastive sound which do not distinguish meaning in a language). For example, an English-speaking learner of Spanish must learn that [b] does not sound the same at the beginning of a word as it does between vowels. In the present study, we investigate how L2 allophones are acquired. In particular, we examine whether adult L2 learners can make use of lexical cues from sound-meaning pairings. Participants were exposed to 16 words from an artificial language (8 differing in the sounds [m]-[l] and 8 differing in [b]-[β]). Each word was presented auditorily at the same time as an image depicting its meaning appeared on the computer screen. Participants were randomly assigned to one of two exposure conditions (Same Image and Different Image). Participants in the Different Image group heard auditorily words differing only in the sounds [b] and [β] paired with two distinct images (e.g. [bati] ‘apple’, [βati] ‘penguin’), whereas the Same Image group were exposed to the same auditory words but saw a single image (e.g. [bati] ‘penguin’, [βati] ‘penguin’). At test, participants were shown images and heard an auditory form and had to determine if they matched or mismatched. Taking advantage of the fact that sound contrasts that are used to distinguish word meaning are perceived more readily than sound differences that do not, participants in the Different Image group should perform more accurately at test then those in the Same image group for the [b-β] contrast. A second test examined whether participants’ extend their knowledge about the sounds to a new set of words. Participants were exposed to 6 new words (3 [b] and 3 [β]), but not their similar sounding pair (e.g. they were exposed to [βanu] ‘elephant’, but never heard [banu]). At test, they again decided whether learned auditory stimuli matched pictures. If adult L2 learners do utilize the lexical cues to learn about L2 sounds, then the Different Image group should more readily detect mismatches that contain the [b-β] contrast than the Same Image group.
Sequencing and Annotation of 12 Bacteriophage Genomes To Aid In Discovering a Treatment For Methicillin-Resistant Staphylococcus aureus
Methicillin-Resistant Staphylococcus aureus (MRSA) is a widespread pathogen, present largely in hospital, community, and livestock agriculture settings. MRSA may also adapt into Vancomycin Resistant Staphylococcus aureus (VRSA) which is immune to all common antibiotics, and is largely untreatable. Due to MRSA’s ability to generate biofilms and adapt to antibiotics, a new form of treatment will soon become necessary, especially if VRSA begins to become prevalent in hospital or community settings. The purpose of this research is to sequence and annotate the genomes of 12 bacteriophage found by my lab through community and livestock based MRSA strains, and to use that information to gain understanding of what characteristics make a lysin or a polysacharidase the most effective at destroying MRSA or MRSA biofilms. To do this, I extracted the DNA from a high titer lysate using the phenol chloroform isoamyl alcohol method, and then purified the DNA by running the whole genome DNA through agarose gel electrophoresis, and then eluting the DNA out of the agarose. The genomes are then subjected to library preparation in order to go through Next generation sequencing on an Illumina HiSeq 2500. This phase of the project is still on-going, but once several phage genomes have been sequenced, I will annotate that data, and use it to find patterns as to what creates the most effective lysin proteins, and polysaccharidase proteins. This data can then be used by biochemists to design a drug modeled after the optimum lysin or polysaccharidase, or it can be used to select the phage itself which will be best suited for fomite decontamination or treatment of MRSA infection. Future studies could include intentionally inducing mutation of a phage to grow in a strain of MRSA outside of it’s natural host range, and observing the change in the coding for the lysin protein in the wild type and mutant strains.
Courtney Smith, Brigham Young University
Evaluation of cultivatable rhizosphere-associated bacteria isolated from Manzanita (Arctostaphylos patula) in Southern Utah for plant growth promoters
Manzanita (Arctostaphylos), an evergreen shrub common to desert regions of the western United States, are of interest for their ecological role as an important food source for wildlife, for erosion control on dry, rocky, slopes, and, for their potential use in xeriscaping due to their drought tolerance, showy flowers, and attractiveness to birds and butterflies. Very little information is available on beneficial microbes associated with manzanita despite growing evidence that rhizospheric, endospheric, and phyllospheric microbes play important roles in promoting plant growth, strengthening resistance to plant pathogens, controlling frost injury, and in controlling plant diversity within local ecosystems. Mechanisms by which plant-associated microbes influence host fitness include plant hormone production, protection of the plant from pathogen colonization, and nutrient cycling, including nitrogen fixation and phosphate solubilization. In this study, cultivation-based techniques were used to isolate bacteria from the rhizospheres of the greenleaf manzanita (Arctostaphylos patula), to characterize the isolates for evidence of plant growth promoting activities, and to identify the isolated species using 16S rRNA gene sequencing. A total of 14 manzanita plants were sampled from three distinct populations in the Cedar City region of Southern Utah. From these samples, 47 rhizospheric bacterial isolates were identified through 16S DNA sequencing. Species common to all three locals included Clavibacter michiganensis, Caulobacter sp., Pseudomonas koreensis, Pseudomonas fluorescens, and Bacillus simplex. Several of these species have been reported to promote plant growth and to contribute to pathogen resistance in other plant species, suggesting a possibility of similar beneficial associations with Manzanita.
Medical and biological research depends on successful protein detection. One method of protein detection is by mass spectrometry, where the proteins are detected according to their mass and charge. Mass and charge result in a mass to charge ratio (m/z). This method is simple and effective because it requires extremely small amounts of sample and can run hundreds of experiments over the course of an hour. One limitation in using mass spectrometry for protein research is caused by the large molecular mass of proteins. Protiens have large molecular weights, such as ~12,000 grams per mole for cytochrome c, in comparison to other ions, such as 85 grams per mole for rubidium ion. Mass spectrometers have less accurate measurements at when the m/z ratio is large. Increasing the charge, however, allows protein ions to be detected at lower m/z ratios and can result in more accurate measurements. Our project focuses on using additives in the solvent sprayed to increase the charge state on the protein when it is sprayed, also called supercharging. This will allow the protein to obtain greater resolution and signal in mass spectrometers. This would increase the effectiveness of research on proteins that have medical and biologically important applications, such as curing Alzheimer’s, fighting cancer, and combating diabetes. In our experiments, we will be using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) coupled with Electrospray Ionization (ESI). Solutions containing proteins, potential supercharging additives, and a supporting solvent will be sprayed from a small capillary against a charged plate, which will generate protein ions in the gas phase. This is ESI. The ions will then enter the mass spectrometer and spin within the electromagnetic field generated by a cyromagnet, and then the observed m/z ratios will be Fourier transformed. This is FTICR. The types and concentrations of proteins and additives will be to test the effectiveness of different additives on different proteins. In a lab in California, it was observed that using smaller glass tips for electrospray resulted in higher charge states for the proteins sprayed. This leads us to expect that adding silicate to the solutions will have a similar effect. We also anticipate that other potential additives will be found and can then be used as well.
The association of the serotonin transporter and oxytocin receptor genes on affiliative behaviors in rhesus macaques
Studies show that social relationships play a role in physical and mental health and that an individual’s genetic variation may influence their sociability. It is widely believed that the serotonin and oxytocin systems influence social behavior. The two variants of the serotonin transporter (SERT) gene code for efficiency of the serotonin system, with the long (L) allele associated with greater efficiency and the short (s) allele associated with lower efficiency. The two variants of the oxytocin receptor (OXTR) gene code for availability of oxytocin, with the ancestral allele (G) associated with increased oxytocin availability and the mutant allele (A) leading to lower availability. While these genes are present in humans, orthologous SERT and OXTR genes are present among rhesus macaques (Macaca mulatta), a nonhuman primate species commonly used in translational research assessing genetic contributions to behavior due to their genetic, social, and physiological similarities to humans. To assess the contributions of SERT and OXTR genotypes to social behavior, at three-to-four months of age, blood samples were obtained from 127 rhesus monkeys (55 males, 72 females) housed in large social groups of 100 or more members. DNA was extracted and genotyped for the SERT genotype for all subjects and OXTR genotype for 33 subjects. As adults (M=6.23 years), subjects’ affiliative behaviors (e.g., number of social partners, rates of grooming, initiating social affiliation, and their frequency of social proximity) were recorded by trained observers during four 300-second observations. One-way ANOVA with genotype as the independent variable and social behaviors as the dependent variables were used to determine the relationship between genotype and affiliative behaviors. An association between SERT genotype and subjects’ average number of social partners neared traditional statistical significance (F(2,117)=2.749, p = 0.068), with a posteriori analyses showing that subjects homozygous for the L allele exhibited significantly more social partners when compared to subjects homozygous for the s-allele subjects. Further, OXTR genotype was associated with the frequency of receiving groom from social group members (F(2,26)=8.058, p = 0.002), with a posteriori analyses showing that subjects that were homozygous for the A allele showed increased rates of being groomed by others when compared to subjects with other genotypes. These findings suggest that subjects homozygous for the L allele for the SERT gene and subjects homozygous for the A allele for the OXTR genotype showed evidence for greater affiliation than do subjects with other genotypes.
We are using GigaPan Units and game camera/trail camera traps to identify the return of different plant types (i.e. grasses, shrubs, trees), the rate of regrowth (how quickly the different plant types return), and animal movement patterns. There are three locations: A) Control Site - near the burn, but not directly affected by the fire; B) High Intensity Site – north of Brian Head; C) Moderate-Low Intensity Site– east of Brian Head. At sites B and C we are taking two photos with the GigaPan to really capture the green sprouting in the midst of the black burn. We are anticipating that by using the image J and descriptive statistics we will be able to see a moderate growth rate change and return of species into the area that was burned. The pictures at our control site will help contrast our findings at the other sites.