Science Poster Day Abstracts

University of California,
Los Angeles
Seventeenth Annual
SCIENCE POSTER DAY
May 13, 2014
12:00 – 3:40 p.m.
Ackerman Grand Ballroom
UCLA Science Poster Day wishes to thank the following for their generous support in making this event possible:
Scott Waugh, UCLA Executive Vice Chancellor and Provost James Economou, UCLA Vice Chancellor for Research Patricia E. Turner, Dean and Vice Provost of Undergraduate Education Victoria Sork, Dean of Life Sciences Joseph Rudnick, Dean of Physical Sciences Vijay Dhir, Dean of Engineering Eugene Washington, Vice Chancellor, UCLA Health Sciences Dean, David Geffen School of Medicine Alan Robinson, Associate Vice Chancellor, Medical Sciences Executive Associate Dean, David Geffen School of Medicine The Clinical and Translational Science Institute This project received support from the NIH/NCRR/NCATS UCLA CTSI Grant Number UL1TR000124 Abstracts were received directly from the authors by electronic submission. Every effort has been made to reproduce the content of the abstracts according to the electronic version submitted, except in certain circumstances where changes were made to comply with Science Poster Day standardized format or upon request by the author. Editors do not assume any responsibility for proofreading or correcting any scientific, grammatical, or typographical errors. Photos and video footage taken by UCLA during Science Poster Day may be used for promotional purposes on behalf of the university.
SCIENCE POSTER DAY Table of Contents Welcome Letter from Dean Turner………………………………………………….……………………………………………..............i Schedule of the Day.......................................................................................................................................ii Abstracts ………………………………………………………………………………………….…………………………….………… 1 ‐ 128 Undergraduate Research Awards (2013‐2014) ………………………………………………………………………….………….129 
Undergraduate Research Fellows…………………………………………………………………………….………….129 
Undergraduate Research Scholars….………………….………………………………………………….……….…..130 Boyer Research Scholars Ehrisman Research Scholars Gottlieb Research Scholars Hilton Research Scholars Lau Research Scholars Litton Research Scholars MacDowell Research Scholars Miller Research Scholars Norton Rodman Scholar Oppenheimer Scholars Silva Research Scholars Sparks Research Scholars Van Trees Research Scholars Wasserman Research Scholars

CARE Scholars …………..…………………………………………………………………………….…………….……………132 
MARC U*STAR Program Scholars...…………………………………………………………..…………………………132 
UC LEADS Scholars………………..………………………………………………………………….……………….………..132 
MSD Scholars ……………………………………………………….………………………………..……………………………132 
Beckman Scholars...…………….…………………………………………………………………..……………..…………..132 
Howard Hughes Research Scholars…………..…………………………………………..……..……………………..133 
UCLA Amgen Scholars…………………..........................................................................................133 
Science Poster Day 2013 Dean’s Prize Winners…………………………………………………..………….……134 
SACNAS Conference Award Winners…………………………………………………….………….……………..….134 
ABRCMS Conference Award Winners ………………………………………………….…………….…..……………134 
CAMP Conference Award Winners ………………………………………………..…….………….………...……….134 
ERN Conference Award Winners.………………………………………………………….………..…….…………….134 Student Group Sponsors UCLA Undergraduate Science Journal Staff………………................................................................................135 UCLA SACNAS & CityLab Staff………………………………………………………………………..…….…………..…………….........136 Indices Alphabetical Index of Students in the Biomedical Research Minor……………………………………………..………....137 Course Research Presenters……………………………….……………………………………………..…………………………………..138 Alphabetical Index of Student Presenters…………..………………………………………………………………………..139 ‐ 144 Alphabetical Index of Mentors…………………………………………………………………………..…………………………145 ‐ 148 URC‐Sciences Staff………………………………………………………………………….………………………………….…………………..149 URC‐Sciences Faculty Advisory Board………………………………………………………………………………….……………….…149 UCLA Annual Undergraduate Research Poster Sessions……………………………………………...…..Inside Back Cover UCLA
Division of Undergraduate Education
Office of the Deans and Vice Provost
2300 Murphy Hall
143801
May 13, 2014
Welcome to the Seventeenth Annual UCLA Science Poster Day! UCLA has a long and proud history of
encouraging students to participate in research. Our undergraduates engage in pioneering research with
distinguished teacher-scholars. As a testament to this tradition, 385 posters will be presented today as part of
UCLA’s 17th annual Science Poster Day. The explosion of student research on campus is notable and is in
keeping with our mission to promote undergraduate research and other capstone experiences as part of a
UCLA undergraduate education.
UCLA provides a rich array of academic research opportunities, from the Student Research Program to
Honors Thesis courses. Undergraduates have opportunities to work with faculty in the College and in the
professional schools, including the Schools of Dentistry, Medicine, Nursing and Public Health. Today posters
will be presented by students working with faculty in the following departments: Anesthesiology,
Atmospheric and Oceanic Sciences, Bioengineering, Biological Chemistry, Chemical Engineering, Chemistry
& Biochemistry, Civil & Environmental Engineering, Cognitive Science, Dentistry, Earth, Planetary & Space
Sciences, Ecology & Evolutionary Biology, Electrical Engineering, Gastroenterology, Hematology &
Oncology, Human Genetics, Infectious Diseases, Institute of the Environment & Sustainability, Integrative
Biology & Physiology, Material Science & Engineering, Mechanical & Aerospace Engineering, Medicine,
Microbiology, Immunology & Molecular Genetics, Molecular & Medical Pharmacology, Molecular, Cell &
Developmental Biology, Nephrology, Neurobiology, Neurology, the Neuropsychiatric Institute,
Neurosurgery, Obstetrics & Gynecology, Ophthalmology, Orthopedic Surgery, Pathology, Pediatrics,
Pulmonary Disease, Physics & Astronomy, Physiology, Psychiatry& Biobehavioral Science, Psychology,
Radiological Sciences, Nursing and Surgery. I invite you to browse the Undergraduate Research Center –
Sciences site (http://www.ugresearchsci.ucla.edu) for information about more opportunities for undergraduate
research.
I thank the Undergraduate Research Center - Sciences office for organizing this event. I am also indebted to
the faculty who direct research programs administered by the Undergraduate Research Center - Sciences
office. This event is sponsored by the UCLA Undergraduate Science Journal, and supported by the Clinical
and Translational Science Institute (CTSI) and the School of Medicine as well as the Division of
Undergraduate Education.
One hundred and nineteen students have been selected to be judged during Science Poster Day. At the end of
the poster presentations, we will honor students that have done an exceptional job sharing their research
findings. The Dean’s Prize Award Ceremony will include a talk by Dr. James Economou. This awards
ceremony will celebrate their successes and the successes of all the students who presented at Science Poster
Day.
All participants in Science Poster Day are deserving of recognition. They have challenged themselves to
extend their learning beyond their classrooms. Good luck to the prize nominees and thank you all for sharing
your exciting scientific discoveries.
Sincerely,
Patricia Turner
Dean and Vice Provost for Undergraduate Education
i SCIENCE POSTER DAY
Schedule of the Day 11:00–12:00 p.m. Registration for SESSION ONE presenters ONLY! Please check in at the registration desk before setting up your poster. 12:00–1:10 p.m. 12:15–1:15 p.m. 1:15–2:25 p.m. 1:30‐2:30 p.m. 2:30‐3:40 p.m. 3:40‐4:00 p.m. 4:00–4:10 p.m. 4:10–4:30 p.m. 4:30–4:45 p.m. Presentation of Posters – SESSION ONE Students with numbered posters 001‐150 will present during this session. Registration for SESSION TWO Presentation of Posters – SESSION TWO Students with numbered posters 151‐299 will present during this session. Registration for SESSION THREE Presentation of Posters – SESSION THREE Students with numbered posters 300‐450 will present during this session. Remove Posters Welcome and Introduction of Speaker Presiding: Tama Hasson, Assistant Vice Provost, Undergraduate Research Keynote Speaker Dr. James Economou, Vice Chancellor for Research, “Team Science” Dean’s Prize Awards Ceremony Presiding: Tama Hasson, Assistant Vice Provost, Undergraduate Research ii SPD 2014 SESSION ONE 1 The Effects Of 31 Knockdown In Dopaminergic Cells And Its Relation To Parkinsons Disease KHADIJ ASSANI, Ciara Martin, and David Krantz Parkinson's disease (PD) is a neurodegenerative disorder characterized primarily by the progressive loss of dopaminergic neurons in the substantia nigra. Epidemiological studies suggest that gene‐environment interactions may play a significant role in the etiology of sporadic PD. Specifically, studies have shown that exposure to pesticides increase the risk of contracting PD, however, the neurotoxic mechanisms of action are still unknown. Dr. David Krantz's lab uses the fruit fly, Drosophila melanogaster, to study the gene‐environment interaction involved in PD with particular focus on the role of neurotransmitter transporters and synaptic transmission. The PD‐linked fungicide ziram inhibits E1 ligase, the essential first step in the UPS, which is essential in regulating the degradation of proteins via the proteasome. We hypothesize that it is ziram's ability to inhibit E1 ligase, and not other molecular mechanisms, that are responsible for its link to PD. To test this hypothesis in the fly, we will specifically mimic the effects of ziram on E1 ligase through genetic constructs. The genetic construct, UAS‐E1‐RNAi, inhibits the E1 protein in the protein degradation pathway. Expression of the RNAi construct in dopaminergic cells is pupal lethal, due to the critical role of dopamine in cuticle development. Thus, in order to obtain viable adults, the GAL4‐GAL80 temperature sensitive system will be used. Finally, we will determine if more robust knockdown of RNAi is sufficient to cause dopaminergic cell loss. 2 Effects of Irreversible Electroporation (IRE) on Peri‐pancreatic Vessels and Bile Ducts NAOMI A. SO, Stephen T. Kee, Edward W. Lee Irreversible Electroporation is a minimally invasive treatment that causes cell death via apoptosis through high frequency electrical currents to the target tissue. IRE has been shown to have the potential of preserving vital structures in a clearly delineated manner and can be monitored with real‐time imaging. Here, we evaluated the effects of IRE on swine pancreatic tissue, monitoring its effects on peri‐pancreatic vessels and bile ducts. 18 Yorkshire pigs underwent contrast‐enhanced CT angiogram examination of the SMA, SMV, common bile duct, and duodenum before and after ablation of the pancreatic parenchyma abutting major vessels with follow‐up imaging and sacrifice at 24 hours, 7 days, and 4 weeks. Weekly CT angiography studies and lab studies were performed and analyzed for changes. In acute studies, there was extensive tissue damage mostly due to coagulative necrosis of the pancreas as well as infiltration of immune cells. In the chronic studies, there was ductal regeneration but no pancreatic acinar tissue and glandular function was lost. In all samples, the bowel was preserved except for minimal injury to the muscularly layer of the bowel in 2 animals. Otherwise, IRE did not cause any other complications. Pancreatic tumors that are in advanced stages often cannot be resected due to invasion of surrounding vessels. Therefore, IRE has great potential in its ability to effectively ablate pancreas while keeping neighboring vessels intact and thus improve treatment outcome. 3 Characterizing a Model of Idiopathic Pulmonary Fibrosis from Induced Pluripotent Stem Cell Fibroblasts KELLY DARMAWAN, Preethi Vijayaraj, Kathy Chung, Mehrsa Mehrabi, Brigitte Gomperts Idiopathic pulmonary fibrosis (IPF) is a fatal disease that usually occurs in the lungs of older adults. It is characterized by failure of alveolar re‐epithelization after chronic lung injury, persistence of fibroblasts in the form of fibrotic foci, deposition of extracellular matrix, and distortion of lung structure ultimately resulting in respiratory failure and death. The cause of idiopathic pulmonary fibrosis is unknown, with no known treatment except for lung transplant. The fibrotic foci in the lungs are associated with the progression of the disease. These are areas of cellular matrix with collections of actively proliferating myofibroblasts that produce collagen. However, very little is known about IPF because no good scarring model has been established yet. Through the study of cells derived from induced pluripotent stem cells (iPSCs), my lab has found that iPSCs derived from idiopathic pulmonary fibrosis patients creates an accurate scarring model for IPF. We have confirmed this by verifying known characteristics of IPF cells in iPSC derived cells, such as resistance to apoptosis and the role of TGFb as a profibrotic mediator. Through an apoptosis assay where apoptosis was induced and the results were analyzed using FACs staining, we found that like IPF cells, the iPSC derived cells are also resistance to apoptosis. In addition, a Luciferase assay determined that the iPSC derived cells secreted their own TGFb, similar to IPF cells. These and similar assays suggest that the iPSCs are a good model for IPF. 1
SPD 2014 SESSION ONE 4 Exploring Gene‐Environment Interactions Implicated in Parkinson's Disease Using a Parquat and Maneb Model In Drosophila VANESSA NUNEZ, Ciara A. Martin, Angel Barajas, and David E. Krantz Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of dopaminergic (DA) neurons resulting in motor dysfunction and other deficits. Overexpression and mutations of the α‐synuclein gene in humans has been linked to familial forms of PD. However, disease progression likely relies on an individual's genetic background and/or environmental exposures. In particular, prolonged exposure to the pesticides paraquat and maneb has been shown to increase the risk of PD up almost two fold. Individuals with variants in the a‐synuclein gene were shown to be at increased risk of PD when they were exposed to pesticides. Our lab has developed a paraquat and maneb model of PD in the fruit fly, Drosophila melanogaster. We report that survival and motor ability significantly decreased upon exposure to paraquat alone or with paraquat and maneb combined. Interestingly, it was only combined exposure to both paraquat and maneb that led to significant DA cell loss. We assessed the DA cell counts and survival curves of our model in the presence of over‐expression of human mutant a‐ synuclein. We observed a significant decrease in longevity in flies with an overexpression of α‐ synuclein that were exposed to paraquat plus maneb and at only two weeks of exposure found a significant loss of DA neurons. Thus, our work confirms epidemiological data linking paraquat and maneb exposure to PD in addition to further implicating α‐synuclein in gene‐environment interactions. 5 Neutrophil Extracellular Traps(NETS) down regulate the activity of Invariant Natural Killer Cells (iNKTcells) Darshan Randhawa, Isela Valera, Ramesh Halder, and Ram Raj Singh Neutrophilic granulocytes (neutrophils) are the most abundant innate immune cells circulating in the blood at any given moment. iNKT cells are a conserved T cell population that can rapidly produce a large amount of cytokines in response to a foreign pathogen. iNKT cells are essential in innate immune responses against pathogens. They also can play a regulatory role in the development of autoimmune disease, asthma, and cancers. Both neutrophils and iNKT cells are part of the initial innate response. Neutrophils activated by TLR ligands and/or Phospholipids suppresses iNKT cell activity and/or responsiveness, through pro‐inflammatory cytokines. The experiment is conducted simultaneously in human and mice models. Blood from human patients is collected and iNKT cells are isolated and cultured. Flow cytometry for neutrophil activity markers and for iNKT cells are measured to establish baseline activity level. NETs are then cultured with iNKT cells and the subsequent activity levels are measured using immunoflourense and flow cytometry. Incubation of NET's with iNKT cells derived from same patient has resulted in down regulation of iNKT cells. iNKT cells cultured in the presence of NETs down regulated the expression of CD69. In addition, the soup from NETosis, had only minimal down regulation of iNKT CD69 expression. This data suggests that physical NET and iNKT cell interaction results in the down regulation of iNKT activity. 6 Lineage‐based Wiring Properties in the Drosophila Brain DANIEL TRAN, Jennifer Lovick, and Volker Hartenstein The motivation for this study was to better understand how neurons form functional circuits because this information is largely unknown. Drosophila were used because they provide a valuable model to study neural circuitry given the myriad genetic tools available to them and the fact that their neurons are grouped into clonal units called lineages. This study aimed to identify a certain primary neuronal cluster with branches terminating in the mushroom body medial lobe of the Drosophila brain. It was hypothesized that the cluster contains neurons from both the DALv2 and DALv3 lineages (DALv being short for dorso‐anterior‐lateral position). In order to identify the neurons in this cluster, we determined the spatial expression patterns of two genetic sequences specific to the DALv2/3 lineage: GMR48B12 and engrailed. A line of flies containing the genetic construct, GMR48B12‐GFP, were produced to visualize GMR48B12 expression. Their brains were dissected, stained with anti‐en to visualize engrailed expression, and mounted for confocal microscopy. It was determined that there is a possible subset of DALv3 primary neurons within the cluster. Evidence also suggests that the cluster associates with both the DALv2/3 lineages due to its close spatial position to DALv2/3 secondary neurons. These results open the door for future experiments to better characterize these neurons and to elucidate their identity. 2
SPD 2014 SESSION ONE 7 Stress May Promote Productivity in College Students ANA I. ETCHISON, Meredith S. Sears, and Rena L. Repetti Individuals who procrastinate experience higher overall levels of stress; however, the way individuals respond to stress on a short‐term basis is less clear. College students (N = 137) rated their mood, daily stressors (e.g., assignments due), procrastination and work‐approach coping behavior four times a day for five days. The sum of each individual's work or academic stressors reported over the course of five days was correlated with average levels of procrastination (r = .23, p < .01), indicating that students experiencing higher work demands were also more likely to procrastinate. Daily negative mood, procrastination and work‐approach behavior were examined using a logistic multilevel model. Feelings of stress actually predicted a decrease in procrastination (B = ‐.03, p < .05), and a corresponding increase in work‐
approach behavior (B = .10, p < .001), at the next measurement a few hours later. This suggests that while procrastination increases with the number of stressful work‐related demands, in the short‐term students appear to respond proactively to feelings of stress by approaching work demands rather than avoiding them. Understanding both short‐ and long‐term associations between procrastination and stress will allow us to create more effective learning environments. Future studies should examine the role of different types of stressors in predicting stress and procrastination behavior. 8 Bone Formation in Osteoporotic Mice is Induced by Systemic Administration of NELL‐1 LESLIE CHANG, Greg Asatrian, Aaron W. James, Choon G. Chung, Omar Velasco, Xinli Zhang, Chia Soo and Kang Ting Osteoporosis is a major health concern that plagues elderly and post‐menopausal patients. This disease leads to progressive deterioration of bone microarchitecture, resulting in an increased risk of bone fracture. Therefore, new therapies for the prevention and treatment of osteoporotic fractures are necessary. NELL‐1 is a novel osteoinductive protein has been found to promote endochondral and intramembranous ossification and is a potential future therapy for osteoporosis. NELL‐1 is highly specific and promotes osteoblastogenesis while reducing osteoclast formation, which reduces bone formation. Localized delivery of NELL‐1 has been found to produce bone regeneration, but a systemic delivery of NELL‐1 would prevent osteoporotic fracture. Using a murine tail injection model, we studied the capacity for recombinant NELL‐1 to prevent ovariectomy (OVX)‐induced osteoporosis. Changes in bone mineral density were evaluated using Dual‐energy X‐ray absorptiometry (DEXA) and high‐resolution microtomography (microCT) imaging. Cellular differentiation was analyzed through histological and immunohistochemical staining. In both SHAM and OVX groups, NELL‐1 demonstrated its osteogenic potential as it mediated significant increases in bone mineral density. Histological samples also correlated this osteogenesis through increased osteoblast and decreased osteoclast numbers in the NELL‐1 treated groups. These findings hold compelling implications for the use of NELL‐1 as an anti‐
osteoporotic therapy for the aging population. 9 Development of an in vitro Model for Premalignancy of Lung Squamous Cell Carcinoma through Signaling Pathway Modulation YEON SUN KIM, Manash K. Paul, Bharti Bisht, and Brigitte N. Gomperts Lung cancer is the most lethal type of cancer, killing more people around the world than any other cancer. A particular subtype of lung cancer, lung squamous cell carcinoma (LSCC) is thought to progress through distinct premalignant stages. Knowledge of the progression of LSCC is vital for its early diagnosis, treatment, and prevention; however, the genetic alterations specific to each stage of this premalignant progression have yet to be clarified. Previous literature has revealed pathways that are significantly implicated in LSCC. By chemically modulating some of these known pathways in airway basal stem cells, the presumed cells of origin for LSCC, we have built an in vitro mouse model of LSCC which demonstrates hyperplasia, metaplasia, and dysplasia the progressive stages of LSCC premalignancy. This model has substantial clinical bearing: using this in vitro model, a high‐throughput‐assay can be developed to screen for compounds to build a targeted chemoprevention strategy, which may ultimately stop the premalignant progression of LSCC. 3
SPD 2014 SESSION ONE 10 In Situ Hybridization of 3‐Hydroxysteroid Dehydrogenase Mutation Demonstrates Loss of Androgen Receptor CLARK CHEN, XuFeng Chen, and Jiaoti Huang 3‐β‐Hydroxysteroid Dehydrogenase (3‐β‐HSD) is an important enzyme in the synthesis pathway of steroid hormones. 3‐β‐HSD mutation in prostate cancer (PC) has been hypothesized as the defining change that makes prostate cancer androgen independent. Researchers have been concerned with androgen independence since the primary treatment of PC is with hormonal therapy. Past research has indicated the correlation between androgen independence and metastatic rates. Cell lines were first used to prove the concept, LNCaP and VCaP both express androgen receptors and they were compared against PC3 and DU145 cells which do not express androgen dependence. Then all lines were tested using Rolling Circle Amplification (RCA) to demonstrate the percentage efficiency for application in human models available in tissue block samples. Tissue micro array (TMA) blocks held 120 different samples for the RCA. Multiplex assays highlighting wild‐type and mutant 3‐β‐HSD were done with specifically targeting padlock probes and varying fluorescence. The power of this project, if successful, opens up the field of oncology to a wider catalog of patient genomic DNA to correlate in the advancement of cancer by increasing assaying quantity a hundred fold. 11 Dentate Gyrus Contributions to Auditory Fear Generalization MOLLY S. HODUL, Vanessa Rodriguez Barrera and Michael S. Fanselow There are multiple factors affecting memory maintenance and storage, and numerous disorders associated with impaired memory. When a fear memory is stored, some innate responses are generalized to new experiences, allowing for preparation to novel events that may be threatening. The inappropriate generalization of fear is a hallmark symptom of fear and anxiety disorders. Acetylcholine (ACh) is an important neurotransmitter for the storage of memory and recently, the dentate gyrus has been implicated in the generalization of fear. The dentate gyrus is a structure within the hippocampus that undergoes neurogenesis (NG), or the birth of new cells throughout a lifespan; this area contains many cholinergic neurons. We set out to see whether administration of scopolamine (ACh antagonist) will result in a change in the amount of fear generalization using an animal model of fear conditioning. Rats will either receive saline or scopolamine and undergo fear conditioning to a specific tone. Following conditioning, we will present novel tones that were never paired with shock and measure how much rats generalize their fear responses to safe tones. To assess whether neurogenesis affects fear generalization we will use a transgenic mouse in which all post‐natal NG is absent. Transgenic and control mice will undergo the same behavioral task. We hypothesize that interfering with the cholinergic system or eliminating NG will result in a deficit of fear generalization. 12 Testing Novel Compounds to Prevent Alpha‐synuclein Aggregation in Mice BENJAMIN BOODAIE, Garima Dutta, Chunni Zhu, Hanan Baker, Tianze Yi, Diana L. Price, Eliezer Masliah, and Marie‐
Francoise Chesselet Abnormal accumulation of the presynaptic protein α‐synuclein in the brain is thought to underlie neurodegeneration in Parkinson's disease and Lewy Body Dementia. A thorough study of α‐synuclein using computer‐based molecular modeling showed that the protein combines to forms pore‐like oligomeric structures that alter ion conductivity of affected neurons. These studies thus point to a soluble, oligomeric form of α‐synuclein (as opposed to the insoluble form associated with Lewy bodies) as the root of neuronal cell death in these diseases. Accordingly, a novel class of bicyclic peptidomimetic compounds was designed using structure‐activity relations simulations to specifically block the formation of this toxic oligomeric species. In the current study, we are using an α‐synuclein overexpressing mouse model to test the efficacy of two of these compounds in reducing the formation of toxic α‐synuclein aggregates. We will also examine the effects of these drugs on neuropathology as well as associated motor and cognitive deficits with the hopes of shedding light on mechanism of action. 4
SPD 2014 SESSION ONE 14 Use of Hygromycin Phosphotransferase to Develop Retroviral Replicating Vectors with Expanded Transgene Capacity REBECCA M. TALKIN, Kip Hermann, and Noriyuki Kasahara Retroviral Replicating Vectors (RRVs) are highly efficient gene therapy vectors capable of delivering a transgene throughout a tumor mass. The size of the transgene is currently limited to ~1.2kb, in part due to the high mutation rate of Reverse Transcriptase, which results in 'drop‐out’ of DNA that is unnecessary for virus survival. We hypothesized that conferring a selective advantage to RRVs that retained a large transgene would result in evolution of a strain with expanded transgene capacity. To test this, we inserted the antibiotic resistance gene, hygromycin phosphotransferase (Hph), which is 1.5kb, into the RRV. The virus (RRV‐Hph) was passaged through PC3 cells that were treated with or without hygromycin. RRV given hygromycin selective pressure retained the transgene for more passages than those not exposed to hygromycin. However, after selective pressure was removed, the transgene dropped out in fewer passages than from the original, unpassaged virus. This may be due to co‐transduction of the cells by two viruses: intact RRV‐Hph, which confers hygromycin resistance to the cells, and a kinetically advantaged 'drop‐out’ virus that outcompetes RRV‐Hph once selective pressure is removed. We plan to isolate RRV‐Hph to determine if it is more stable than the original RRV, and sequence the virus to identify mutations that may contribute to improved genomic stability. If a more stable version of the virus has evolved, it could be used to develop more effective gene therapy vectors for cancer and other diseases. 15 Visualizing Assembly of Differently Oriented Dipole Moments within Carboranethiols on Metal Substrates BRANDON M. MATTHEWS, John C. Thomas, Harsharn S. Auluck, Logan A. Stewart, Chad A. Mirkin, and Paul S. Weiss
Self‐assembly, at the molecular level, is governed by interactions between the substrate and deposited molecules, and among neighboring and nearby molecules. Synthetic approaches in nanomaterials exploit these interactions at the single‐molecule level, giving way to materials whose size, shape, and functionality are regulated by nanoscale interactions which can be tuned. Eutectic gallium‐indium (EGaIn) was chosen to help determine the roles of these interactions at the nanoscale as supramolecular assembly can direct surface morphology in the liquid state. EGaIn was subjected to shear forces in solution via ultrasonication, with cage molecules m‐1‐carboranethiol or m‐9‐
carboranethiol added as ligands to create self‐assembled monolayers on the subsequently formed nanoparticles. Scanning electron microscopy and transmission electron microscopy were used to examine the synthesized nanoparticles, including determinations of their shape, size, and oxide coverage. Preliminary results reveal correlations between particle shape and dipole orientation, as m‐1‐carboranethiol produced faceted particles and m‐9‐
carboranethiol produced spherical particles. Carboranethiols enable the formation of stable monolayers that appear to dictate the resulting shape, size, and oxide coverage of EGaIn nanoparticles based on the individual dipole orientation. Polarization modulation infrared reflection absorption spectroscopy was also used to monitor varied dual codeposited carboranethiols on Au{111}, determining the molecule that dominated surface co 16 Basolateral Amygdala AMPA, Not NMDA, Glutamate Receptor Activation Is Necessary for the Ability of Reward‐Paired Cues to Bias Action Selection ALICE S. WANG, Venuz Y. Greenfield, and Kate M. Wassum Environmental reward‐paired cues influence reward‐seeking decisions by biasing one towards actions that earn the same reward predicted by the cue, an effect that requires the basolateral amygdala (BLA). The chemical or receptor systems in the BLA that regulate this ability are unknown. Glutamate receptor function is important for memory formation and recall as well as the ability of reward‐paired cues to bias action selection. Hence, we evaluated the role of two glutamate receptors N‐methyl‐D‐aspartate (NMDA) and a‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) in BLA on cue‐biased action selection and Pavlovian conditioned responding using the Pavlovian‐to‐
instrumental transfer (PIT) paradigm. In this paradigm, rats are trained in Pavlovian and instrumental conditionings and tested on the effect of reward‐paired cues on decision‐making. Prior to test, rats were infused with either saline, 1ug or 3ug of APV‐D, a selective NMDA receptor antagonist, or the same dose of NBQX, a selective AMPA receptor antagonist, into the BLA. We found that inactivation of AMPA, not NMDA, glutamate receptors abolished the ability of the reward‐paired cues to bias the selection of actions towards that earning the same outcome as predicted by the cue. Inactivation of neither glutamate receptor subtype affected conditioned food port approach responding induced by the reward‐paired cue. Together, these results show that BLA AMPA, not NMDA, glutamate receptors bias action selection by recalling sensory‐specific representation of a reward. 5
SPD 2014 SESSION ONE 17 Effects of Gender and Types of Emotions on the Judgment of Emotional Emergence JINGQI YU, Hanul Shin, Devan D. Sheth and Elizabeth L. Bjork The study assessed the effects of the expressers' gender (male vs. female) and their emotions (happiness vs. anger) on perceptions of emotional expressions. We had several hypotheses, the most important of which was that a male's expression of anger would be identified earlier, while a female's expression of happiness would be identified earlier. Seventeen participants were presented with gradients showing a male and a female model's facial expressions ranging from neutral to extreme and asked to decide the earliest point (threshold) along gradients at which they noticed a change from a neutral to an identifiable emotion, or the emergence of an emotional expression. The hypothesis was supported by the result (p < .001); that is, a male's expression of anger was noticed earlier, whereas a female's expression of happiness was noticed earlier. These findings further the understanding of human communication, which has the potential to improve emotional perception. 18 Retinoic Acid in Treating Glioma Patients HUYTRAM N. NGUYEN, Arthur P. Chou, Tie Li, Fei Liu, Weidong Chen, Reema R. Mody, Desiree Sanchez, Bowen Wei, Gregory M. Lucey, Jennifer Quan, Reshmi Chowdhury, William Yong, Phioanh Nghiemphu, Timothy Mutation in isocitrate dehydrogenase 1 (IDH1) is present in a large percentage of gliomas and is associated with improved patient survival. Retinoic acid treatment with isotretinoin may delay recurrence in malignant gliomas; however, its survival benefit remains controversial. This study aims to evaluate the effects of isotretinoin (ITR) in patient with diffuse gliomas. We retrospectively identified glioma patients treated at UCLA and collaborating institutions. Patients' charts and molecular biomarker studies were reviewed. Survival data was evaluated by Kaplan‐
Meier analysis and Cox proportional hazard model. Result: Our cohorts included 128 grade II, 153 grade III, and 549 grade IV glioma patients. Patients with IDH1 mutation showed improved overall survival (OS); Grade II: 135.7 months vs. 69.5 months (mutant vs. wildtype, n=115 and n=13, p=.002); Grade III: 114 months vs. 31 months (n=112 and n=41, p=.002); and Grade IV: 36.1 months vs. 17.9 months (n=69 and n=480, p<.001). Treatment with ITR upfront is associated with increased median OS in grade IV IDH1 wildtype patients, who received concurrent radiation/temozolomide upfront and bevacizumab at recurrence; median OS is 30.0 months (n=25, ITR) vs. 21.5 months (n=116, non‐ITR), p=.006; Cox model HR=2.13, p=.006. IDH1 mutation is associated with longer OS in three tumor grades. Grade IV patients with IDH1 wildtype appears to receive survival benefit from upfront treatment with retinoic acid. The etiology of this association is unclear. 19 Striatal Adenosine Blockade and Conservation‐Withdrawal Behaviors BRANDY A. BRIONES, Traci Plumb and Thomas R. Minor Stress and anxiety disorders are the most common mental health disorders amongst the human population, affecting about 40 million American adults. High on‐going levels of blood cortisol and activation of adenosine A2A receptors (A2ARs) both play an important role in the development of behavioral depression in a rat model of Post‐traumatic Stress Disorder (PTSD) comorbid with depression. Adenosine binding in A2ARs disrupts dopamine signaled motivation, preventing adaptive stress coping, thus resulting in behavioral depression. Blockade of A2ARs in the dorsal medial striatum (DMS) of the brain is proposed to decrease the negative effects of traumatic stress (i.e., helplessness in animal models). Rats were implanted with bilateral cannulae into the DMS (bregma: AP ‐0.4, ML +2.6, DV ‐5.0) during stereotaxic surgery. Following recovery from surgery, rats were exposed to traumatic inescapable shock or simple restraint. Rats were tested for their ability to learn to escape administered shocks in a shuttle‐escape apparatus, 24‐
hours after traumatic stress. Ten minutes prior to testing, rats' cannulae were infused with A2AR antagonist CSC (8‐(3‐
chloro‐styrl) caffeine) or vehicle. Observation of shuttle‐escape testing revealed a greater deficit in learned‐escape performance of shock‐control rats, exhibiting learned helplessness, compared to restraint‐control, restraint‐
experimental, and shock‐experimental rats, exhibiting minor learning deficits. These findings may be significant in treating stress disorders such as PTSD. 6
SPD 2014 SESSION ONE 20 Effects of Overexpression of FoxP2 in Area X on Male Zebra Finch Song CHAE Y. KIM, Nancy F. Day, and Stephanie A. White A gene mutation in FOXP2, a transcription factor, results in severe language deficits, and results in structural abnormalities of the basal ganglia. To alleviate neurogenetic speech and language deficits, the mechanisms for learned communication must be understood. Though language is a uniquely human trait, songbirds have a subcomponent of language, vocal learning, that can be studied in a lab. In songbirds, FoxP2 expression in Area X, an avian basal ganglia brain region dedicated to singing, changes based on social context: FoxP2 is low when the bird sings alone and high when he sings to a female. To determine if overexpression of FoxP2 affects adult zebra finch song, male birds were injected with an adeno‐associated virus designed to overexpress FoxP2. Songs were recorded during three social conditions (directed, undirected, and 2 hours after non singing). Overexpression of FoxP2 is predicted to remove differences between the three states because the virus maintains high FoxP2 levels. Syllabic changes in adult male zebra finch songs following FoxP2 overexpression are analyzed to identify differences. Our preliminary data suggest that, as expected, syllables sung during undirected song have greater variability than those sung during the directed and non‐singing conditions. However, there were no conclusive results that suggest that overexpression of FoxP2 reduces variability between social contexts. Studying how FoxP2 affects singing behavior in birds allows us to investigate genes critical for human speech acquisition. 21 Investigating the Metabolic Regulation of Stem Cell Differentiation through Uncoupling Protein 2 ANDREA CHAIKOVSKY, Jason Hong, and Michael Teitell Human pluripotent stem cells (hPSCs) exhibit a distinct metabolic profile. While differentiated cells rely mainly on oxidative phosphorylation (OXPHOS) for energy, hPSCs rely mainly on glycolysis to support their rapid proliferation. Upon differentiation, the metabolic machinery within hPSCs shifts from a highly glycolytic to a highly oxidative pattern of carbon trafficking. Although it has been thought that differentiation drives this metabolic shift, recent studies like those involving uncoupling protein 2 (UCP2) indicate that this metabolic shift actually precedes and may regulate or enable differentiation. UCP2 helps elevate glycolytic flux in hPSCs and is repressed upon differentiation, coinciding with the transition to OXPHOS as the main cell energy source. A recent study has found that inhibiting UCP2 repression prevents hPSCs from differentiating properly by forcing the retention of pluripotent gene expression and reducing differentiated gene expression despite exposure to differentiating stimuli. To further investigate the metabolic regulation of hPSC differentiation, we used bioinformatics to identify other genes responsible for shuttling and modifying carbon substrates, such as genes involved in glycolysis, the pentose phosphate pathway, and lipid biosynthesis that are repressed upon differentiation, similar to UCP2. We are continuing to analyze the regulatory regions of these genes in order to identify common regulatory mechanisms for these metabolic genes, and possibly for driving stem cell differentiation. 22 Characterization of Fibril and Oligomer Forming Segments of TAR DNA Binding Protein 43, TDP‐43 HAMILTON TRINH, Elizabeth Guenther and David Eisenberg TAR‐DNA binding protein 43 (TDP‐43) has been shown to be associated with the neurodegenerative disorder,Amyotrophic Lateral Sclerosis (ALS), through the formation of amyloid‐like deposits in motor neurons. The structure of these deposits is not yet known. Using Rosetta energy predictions, amino acid segments of TDP‐43 (304‐
333 and 333‐362) were targeted as potential oligomer forming regions. These segments were recombinantly expressed and purified using a combination of affinity chromatography and high performance liquid chromatography. Electron microscopy and antibody binding were used to characterize segments that had been shaken for various timepoints. Results from these studies show that segments display fibrillar structures within 24 hours of shaking at a concentration of 500 microM. These fibrils exhibit toxicity towards motor neurons at concentrations as low as 50microM. Continued characterization of these segments will enable an understanding of how TDP‐43 can aggregate in ALS patients. Future objectives will include continued attempts at crystallization of these segmentsand optimization of oligomer forming conditions in order to gain insight into the regions of TDP‐43 that are essential for oligomer formation. 7
SPD 2014 SESSION ONE 23 Genetic Interactions that Regulates Layer Specificity through Modulation of RhoGTPase Cdc42 Activity in the Drosophila Visual System CY. KIMBERLY TSUI, Matthew Y. Pecot, Yi Chen and S. Lawrence Zipursky In the Drosophila visual system, synaptic connections are organized into discrete layers for precise neuronal network formation. Lamina neuron L3 reaches its target medulla layer M3 in two steps: First, L3 target to a common domain in the outer medulla by N‐Cadherin (CadN) adhesion and Semaphorin‐1α (Sema‐1α)/Plexin A repulsion. Next, L3 segregates into M3 partially by Sema‐1α signaling. Using mosaic analysis with a repressible cell marker (MARCM), we demonstrated Cdc42 acts cell‐autonomously to regulate the second step of L3 targeting. In a RNA interference screen, Cdc42's reported activator dPix was identified to also be involved in L3 targeting. We hypothesize that CadN and Sema‐1α regulate specific steps in L3 targeting by precisely modulating cytoskeletal dynamics through interaction with different downstream effectors, Cdc42 and dPix. We developed a genetic system to cell‐autonomously study these interactions at different stages of L3 targeting. By MARCM, individual L3s, rendered homozygous for null‐mutations in our genes‐of‐interest by mitotic recombination in multiple chromosomes, are specifically labeled. Labeling of heterozygous cells is prevented by a repression system. We first examined L3s with null‐mutations in Cdc42 and Sema‐
1α. Mutant L3s showing appropriate mis‐targeting phenotypes were generated. However, wild‐type cells were labeled due to ineffectiveness of the repression system, giving false‐positive results. We are currently repeating the study with a repressor that has been tested to be effective. 24 Horizontal Gene Transfer of a Diversity‐Generating Retroelement between Bacteroides Species KAYVAN C. SASANINIA, Yanling Wang and Jeffery F. Miller Diversity‐generating retroelements (DGRs) are a novel family of genes that facilitate rapid sequence diversification of targeted protein‐encoding genes. Bacteroides fragilis 638R and B. thetaiotamicron VPI 5482, commensal members of the gut microbiota and opportunistic pathogens, were newly identified to contain a potentially adaptive DGR in their genomes. Recent bioinformatic analysis suggests these DGRs are carried on mobile genetic elements including conjugative transposons. We aimed to detect if DGRs are capable of transferring between Bacteroides species via horizontal gene transfer. Previous attempts to detect in vitro transfer of the DGR‐containing conjugative transposon were unsuccessful, possibly due to lack of host‐derived signals. Here we attempted to detect DGR transfer in vivo using a gnotobiotic mouse model. Germ‐free mice were inoculated with donor and recipient Bacteroides species with respective antibiotic resistance markers. Fecal samples were collected and putative transconjugants were selected on medium plates with double antibiotics. The genetic background of putative transconjugants was further determined using a colony PCR assay. Although distinct colonies were seen on double antibiotic plates, none matched the profile of a true transconjugant. These results suggested that DGRs are not being transferred under the current experimental conditions. Understanding mechanisms governing conjugative transposon transfer can elucidate how DGRs are disseminated and the potential roles they play for host fitness 25 One Byte at a Time: Memory for the Apple Logo MEENELY NAZARIAN, Adam B. Blake and Alan D. Castel A logo is a simple representation of a company that is designed to be memorable. Prior research suggests that frequent interaction with an object does not necessarily predict accurate recall of its features or location (e.g. coins, fire extinguisher); however, the Apple logo was designed to be aesthetically pleasing, relatively simple, and to promote positive consumer associations with the product. This study examines whether people can accurately recall and recognize the Apple logo. We found that participants show surprisingly poor memory for such a pervasive logo, as measured through recall (drawings) and forced‐choice recognition (among an array of similar Apple logos with altered features). While Apple users perform as poorly as PC or hybrid users, they show higher confidence in their ability to recognize the logo. This study suggests that even frequent exposure to simple items with the central purpose of increasing memorability does not always lead to accurate recall. This study has implications for market applications, but also lends important insight into research on the interactions between attention and memory in real‐world settings. 8
SPD 2014 SESSION ONE 26 Rhabdomyolysis in Obese Trauma Patients JONATHAN B. LEE, Galinos Barmparas, Joshua Chan, Taryne Imai, Nicolas Melo, Matthew Bloom, and Eric Ley Patients who sustain traumatic injuries are at risk for development of rhabdomyolysis. The effect of obesity on this risk is largely unknown. This study aims to characterize the role of obesity in the development of rhabdomyolysis after trauma. This was a retrospective review of trauma patients admitted to the surgical intensive care unit. Patients with available creatine kinase (CK) levels were selected. Admission and highest creatinine, highest CK levels and the need for hemodialysis were recorded. Patients were divided into two groups based on their Body Mass Index (BMI): The overweight/obese group with BMI>25 and the non‐overweight/obese group with BMI<25. The Primary outcome was CK>10,000 U/L. Results: There were 96 patients (49%) with BMI>25 and 102 (51%) with BMI<25. There was no difference in the admission or highest creatinine levels between the two groups (p=0.79 and p=0.83). After adjusting for confounding factors, BMI>25 patients were at a significantly higher risk of having a CK>10,000 U/L (15% vs. 5%; adjusted p=0.02). In a forward logistic regression model including all factors potentially associated with development of rhabdomolysis, BMI>25 was independently associated a CK>10,000 U/L (AOR: 5.2; 95% CI: 1.3, 20.9; adjusted p=0.02). Overweight patients are at a significantly higher risk for developing rhabdomyolysis after trauma. Based on the findings of this study, aggressive monitoring of CK levels to prevent rhabdomyolysis in this patient population is highly encouraged. 27 Utilizing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐Cas to Model Tom70 Mitochondrial Morphology Defects in Zebrafish GARY M. SHMORGON, Jisoo Han and Carla M. Koehler The mitochondrion is an essential organelle that is involved in a variety of processes. The mitochondria synthesize the required energy for the cell in the form of ATP. They also participate in different signaling pathways. Defects in mitochondrial morphology have been shown to be linked to several diseases such as optic neuropathy, Charcot‐Marie‐
Tooth disease and Parkinson's disease. In order to observe both the phenotypic and genotypic defects easily, zebrafish (Danio rerio) knockout models of different genes will be created utilizing the CRISPR‐Cas system. This system of genomic editing will used to create a knockout model of the translocase of outer membrane 70 gene (Tom 70), which encodes for an outer membrane protein that acts as an import receptor and is part of a complex of proteins. At this point, we have obtained a PCR digest product that suggests that the CRISPR‐Cas system is working at a high efficiency rate. This research project will allow us to determine what morphological defects occur as a result of a mutation to such a crucial outer membrane mitochondrial protein. 28 Potential Biphonation in Australian Amphibians Visualized by Spectrograms and Close‐Returns Plots PAULINA M. YOUNG and Peter M. Narins Because a larynx consists of a single unit, animals that possess larynges, including amphibians, should be unable to produce multiple sounds concurrently. However, audio recordings of Littoria pearsoniana and Cophixalus australis, both Australian tree frogs, show evidence of overlapping harmonic stacks in their call frequencies, called biphonation. Recordings were made in the forests of eastern and northeastern Queensland, Australia, for L. pearsoniana and C. australis, respectively. The calls were analyzed with SELENA, an audio analysis program that displayed calls in a time versus frequency spectrogram. A majority of calls of both species contained probable biphonation. Six exemplary calls were cross‐analyzed with the Close‐Returns plots method; no clear overlap was found. Since the FFT intervals used for Close‐Returns were half of that in the SELENA spectrograms, a new approach reduced the FFT on the SELENA spectrograms of both species. The presence of biphonation in these new spectrograms was debatable. It may be that a higher FFT interval, though instrumental in improving resolution, elongates the edges of the harmonic stacks, causing misleading overlap. Though L. pearsoniana and C. australis vocalizations did not show significant overlap, those of other animals may. The results are an indication to others that adjusting FFT on spectrograms may distort the appearance of a call. When investigating potential biphonation, it would be more reliable to employ an audio analysis method that does not depend on FFT. 9
SPD 2014 SESSION ONE 29 Synthesis of Indium Complex with Mono‐Substituted Tridentate Ferrocene Ligand JUN GAO, XINKEWang, Stephanie M. Quan and Paula L. Diaconescu Polymers are useful in a variety of applications such as plastics and drug delivering agents. Indium complexes have shown catalytic properties in polymerization reactions. Ferrocene chelating ligands provide good stability of the resulting metal complexes. In comparison to traditional di‐substituted ferrocene tetradentate ligands, mono‐
substituted tridentate ferrocene ligands may form metal complexes with a more open coordination sphere around the metal center. Therefore, we attempted to synthesize an indium complex supported by a mono‐substituted ferrocene ligand. 30 Newborn Neurons' Role in Contextual Learning and Recall TAM T. TRAN, Sarah Hersman, Lili Hodgins and Michael S. Fanselow The hippocampus is important in establishing contextual fear conditioning. However, the role of newborn neurons in the dentate gyrus is unknown. Our study will determine if these newborn neurons are incorporated into circuits active during spatial recall, and what contributions these neurons make to context discrimination. To probe this, mouse subjects were trained and tested over four‐days' time in two distinct contexts, after which subjects' brain tissue was extracted and immunolabeled for c‐Fos (an immediate early gene transiently expressed by activated neurons) and 5‐
bromo‐2'‐deoxyuridine (BrdU, incorporated into newborn neurons). During testing, subjects demonstrated increased freezing response to the shock‐paired context, but not the non‐shocked context, indicating differential fear acquisition and context discrimination. Immunohistochemical labeling showed that during testing, newborn neurons were activated at high levels, but labeling revealed no correlation between the activity of newborn neurons and the subjects' level of context discrimination. Also, newborn neuron activity was not predicted by exposure to either context. These results show that newborn neurons are highly involved in spatial recall, and future experiments will be necessary in order to continue to investigate the role of newborn neurons in context discrimination. 31 Identification of Key Regions and Residues Controlling Abeta Folding and Assembly KIMBERLY K. HOI, Margaret M. Condron and David B. Teplow Neurotoxic oligomers formed by the amyloid beta‐protein (Abeta) in the brain may play a key role in the etiology Alzheimer's disease (AD), and are therefore attractive therapeutic targets. For knowledge‐based drug development, the biophysical and structural characteristics of Abeta must be well understood. Here, the physical biochemistry of Abeta monomer folding and assembly was studied using scanning D‐amino acid substitution. Using Photo Induced Cross‐Linking of Unmodified Proteins (PICUP) and subsequent SDS‐PAGE, we identified specific amino acids important in controlling Abeta40 and Abeta42 oligomerization. Further, the fibril formation kinetics of Abeta variants was quantified using Thioflavin T. Preliminary circular dichroism results demonstrated that single D‐amino acid substitutions can alter the rate of Abeta folding into stable secondary structures. The two predominant forms of Abeta in humans contain 40 or 42 amino acids, termed Abeta40 and Abeta42, respectively. We observed that the same substitution in these two peptides produced differences in oligomerization and fibril formation, consistent with previous studies suggesting that the two isoforms form oligomers through distinct pathways. In Abeta42, amino acids at the C‐terminus seemed to play an important role in oligomer formation. In Abeta40, D‐[K16] through D‐[D23] formed Thioflavin T‐positive amyloid fibers. The structure‐assembly relationships established in here have the potential to provide specific targets for the design of therapeutic agents for AD. 10
SPD 2014 SESSION ONE 32 Shape Based Detection of G. lamblia and C. parvum Using MATLAB Image Processing ANDREW P. GIFFORD, Hatice Ceylan and Aydogan Ozcan When performing cell phone microscopy using the camera of a standard smart phone, the means to detect and quantify the cells in the sample was absent. Multiple cell types can be distinguished from each other by their physical properties. MATLAB was utilized in the development of a shape detection algorithm, which could be used to identify G. lamblia cysts and C. parvum oocysts within a sample. Using image segmentation, this algorithm could successfully distinguish between these two cell types based on their shape and size; however, the algorithm failed when cells were densely packed together. This requires further analysis and experimentation in order to develop a consistently successful algorithm, which will allow for accurate detection and recognition of bacteria within a sample. 33 Investigating the Osteogenic Potential of Skin Cell Derivatives and their Response to a Bone Inducing Protein BEN SCHOENBERG, Agustin Vega‐Crespo, and James Byrne Our lab is currently researching a subpopulation of osteogenic stem cells found in adult human skin cells. We are trying to isolate and genetically modify these cells by introducing a protein responsible for bone growth and repair, known as Bone Morphogenetic Protein‐2 (BMP2). The long‐term goal for this project is to help treat patients experiencing significant back pain through spinal fusion. Our hypothesis is that human skin cell derivatives respond to and elaborate BMP2, which could represent an important target in manipulating the osteogenic potential of human skin cell derivatives. Human skin cell derivatives were differentiated for 10 days in osteogenic differentiation media alone or supplemented with recombinant human BMP2. Alizarin red staining (showing extracellular matrix) was quantified with spectrophotometry. Gene expression analyses were performed using quantitative polymerase chain reaction. BMP2 levels in conditioned media were measured by enzyme‐linked immunosorbent assay during osteogenic differentiation. We are currently still in the process of collecting data, but we expect to see that exogenous rhBMP2 significantly increases the in vitro osteogenic potential of human skin cell derivatives. Furthermore, we expect to see increased levels of downstream transcript levels, including RUNX2 and osteocalcin. Our preliminary results suggest we can induce human skin cell derivatives to become bone cells. 34 Potential Translational ER‐Beta Ligand Shows Protective Effect on Experimental Autoimmune Encephalomyelitis MAVIS S. PENG, Noriko Itoh, and Rhonda R. Voskuhl Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system that is characterized by inflammation and neurodegeneration. Currently only anti‐inflammatory drugs are present in the market. Although inflammation may be suppressed, the neurodegenerative process continues, causing an accumulation of disability in MS patients. Thus there is a need for neuroprotective drugs. Previously our lab has studied an ER‐Beta ligand, Diarylpropylnitrile (DPN) that has shown to be neuroprotective. However, with 70‐fold selectivity for ER‐Beta over ER‐
Alpha, a relatively low selectivity, there is concern that DPN may exert proliferative effects that are known to be mediated through ER‐Alpha. This study focuses on another ER‐Beta ligand that has over 1000‐fold selectivity of ER‐
Beta over ER‐Alpha, greatly reducing the chances of binding to ER‐Alpha. As our subjects, we used a mouse model of MS, experimental autoimmune encephalomyelitis (EAE) and performed in two strains with different disease courses, wildtype C57 Black/6 (C57BL/6) and wildtype non‐obese diabetic (NOD). Using EAE clinical scoring and analysis of rotarod treading times, we determined that ER‐Beta ligand treatment reduced clinical EAE severity in both male and female C57BL/6 and NOD mice. The ER‐Beta ligand shows promising translational potential because of its prospective ability to act as a safe neuroprotective drug. We will further assess the ligand's neuroprotective effects through neuropathology. 11
SPD 2014 SESSION ONE 35 Investigating the Role of the Contra‐lesional Cortex in Forelimb Recovery After Experimental Traumatic Brain Injury BRITTANY A. HESSELL, Derek R. Verley, Daniel Torolira, Annabel Klees, Richard L. Sutton and Neil G. Harris Being a leading cause of death and injury, traumatic brain injury (TBI) has become increasingly prevalent in the United States. In patients that sustain TBI's, recovery is very slow, with maximal recovery occurring much later after injury. Patients with TBI rarely reach full recovery status, and usually never completely regain their lost functions. In our study, we strive to investigate the role of the contra‐lesional cortex in the recovery of motor deficits, specifically in forelimb reaching abilities. We hypothesize that the contra‐lesional cortex acts to inhibit functional recovery of the ipsi‐lesional cortex through callosal connections. Our results indicate that through silencing the contra‐lesional cortex with muscimol, a GABA‐A receptor channel agonist, reaching success and accuracy increase in the impaired forelimb, manifesting in an overall improvement in functional recovery of the affected forelimb. 36 Structural Biology of Two Mutations in Amyloid ß Protein ELISABETH HODARA, Robin Roychaudhuri, Margaret M. Condron and David B. Teplow. Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder associated with aging. It has been shown to be associated with the aberrant folding and assembly of the amyloid beta protein (Abeta), a cleaved translational product of the amyloid precursor protein (APP) gene. A recent study reported the effect of two naturally occurring mutations at position 673 of the APP gene, equivalent to Ala2 of Abeta. The first mutation, resulting in an Ala to Thr (A2T) substitution, protected against cognitive decline and AD. The second mutation, resulting in an Ala to Val (A2V) substitution, was protective in the heterozygous state but caused AD in the homozygous state. The fact that these two mutations occur at the same site, Ala2, but cause different phenotypes indicates that position 2 within Abeta may play a pivotal role in determining its folding and assembly characteristics. We performed biophysical and biochemical studies to elucidate the structural basis for the published phenotypic effects of the two mutations. We conducted experiments using Photo‐Induced Cross‐Linking of Unmodified Proteins (PICUP). followed by SDS‐PAGE. to analyze oligomer size distribution of the most pathogenic form of Abeta, Abeta42 (containing 42 amino acids). We determined the peptide backbone dynamics using CD spectroscopy and we investigated the rates of formation of beta‐sheet structure by Thioflavin T fluorescence. We determined the time‐dependent evolution of morphologies of the assemblies using electron microscopy. 37 Characterization of Cytospin‐B as a Novel Mitotic Protein EMMANUELLE J. HODARA, Ely G. Contreras, Ankur A. Gholkar and Jorge Z. Torres Cancer is a deregulation of genes involved in tumor suppression, genomic integrity, cell differentiation, survival and apoptosis resulting in an imbalance between cell division and cell death. Thus, investigating the role of novel mitotic proteins can be instrumental in understanding new signal transduction pathways that lead to tumorigenesis, and finding new therapeutic targets. Cytospin‐B is a novel mitotic protein. It is overly expressed in a number of cancer cell lines but not in regular tissues, and this over‐expression has been correlated with high induction apoptosis. Cytospin‐B is known to interact with NPM/B23, a phosphoprotein which plays multiple roles in cell growth and proliferation. In order to elucidate the role of Cytospin‐B in the mitotic process, we are investigating the nature of its interaction with B23 and other mitotic proteins. We purified Cytospin‐B via a tandem affinity purification system (LAP/TAP) with both Enhanced Green Fluorescent Protein tag and S‐tag, and performed proteomic analysis by mass spectrometry. We expect to find B23 as well as other key mitotic proteins such as alpha‐tubulin, Mad2, crest, and p50. Based on the results, we will proceed to localize the protein at various stages in the cell cycle by immunofluorescence, and analyze its physiological activity by siRNA assays. In understanding the role of Cytospin‐B in cancer‐causing pathways, we hope to reveal it as a target for inhibition and find molecular inhibitors that could be instrumental in cancer therapies. 12
SPD 2014 SESSION ONE 38 Deletion of Autophagy Leads to Diabetes Development in Heterozygous Human‐Islet Amyloid Polypeptide Transgenic Mice RASIKA DESHPANDE, Jacqueline Rivera, Tatyana Gurlo and Peter C. Butler Type 2 Diabetes (T2DM) is characterized by a deficit in beta‐cells due to increased apoptosis and the formation of intracellular toxic oligomers of islet amyloid polypeptide (IAPP). Mouse models indicate that heterozygous expression of human IAPP (h‐IAPP) in beta‐cells does not lead to diabetes development. The autophagy pathway was shown to be important for the survival and function of beta‐cells. We hypothesize that transgenic mice heterozygous for h‐IAPP and deficient in the autophagy pathway would develop diabetes because the autophagy pathway may be involved in the degradation of aggregate‐prone h‐IAPP. We first generated our groups of mice: control, heterozygous h‐IAPP and Atg7 knockout (KO) were the controls for Atg7 KO: heterozygous h‐IAPP mice. Fasting blood glucose measured for 15 weeks showed that only the Atg7 KO: heterozygous h‐IAPP mice developed diabetes between the ages of 10‐14 weeks. Pancreatic sections were analyzed for beta‐cell mass and apoptosis (TUNEL). Beta‐cell mass was significantly reduced in Atg7 KO: heterozygous h‐IAPP mice while the TUNEL positive beta‐cells were increased compared to control groups. In addition, Atg7 KO: heterozygous h‐IAPP mice and Atg7 KO mice showed significantly increased nitrotyrosinated inclusions. These results show that deletion of autophagy in the presence of heterozygous expression of h‐IAPP results in diabetes development associated with a deficit in beta‐cell mass due to increased apoptosis and oxidative stress. 39 Identification of Retinal Degeneration Genes: Amalgam, Cell‐Cell Adhesion, Phagocytosis ARASH GHAFFARI‐RAFI, Tiffany Mao, Alex Tonthat, Samuel Bounds, Sampat Sindhar and Frank Laski Genomic screens provide an exciting avenue for accelerating the discovery of loci contributing to specific diseases, including age‐related retinal degeneration (RD), the leading cause of blindness in the United States and worldwide. Via a screen exploiting the model organism Drosophila melanogaster, our study bids to identify new RD loci and their functions. Although past research in D. melanogaster has focused on recessive viable genes, ours examines recessive lethal, which have yet to be studied and are more difficult to study for RD mutant phenotypes hence requiring nontraditional genetic techniques to identify these genes. We used an RNAi approach, in which double stranded RNA (dsRNA) is expressed only in the D. melanogaster visual system to knockdown the gene of interest. Then these transgenic flies are aged in light and histologically analyzed for any signs of RD. In preliminary experiments, my screen of 130 phagocytosis and cell adhesion genes identified 11 loci with potential RD mutant phenotypes. In particular, expression of dsRNA of the cell membrane protein Amalgam resulted in a severe age‐dependent RD. I also report the potential of Amalgam playing a role in light dependent RD, in which proper cell adhesion may be paramount for inhibiting photoreceptor atrophy. This report presents a summary of the newly identified genes contributing to RD susceptibility, and proposes a future course of action for elucidating the non‐phototransduction molecular processes contributing to visual impairments. 40 Determining Temperatures Associated With the Evolution of Metazoan Life by Applying 'Clumped' Isotope Thermometry to Precambrian and Cambrian Soil Nodules From South Australia To Screen For Diagenesis STEVE FLORES, Robert Eagle, Gregory Retallack and Aradhna Tripati During the Precambrian, life on Earth was primarily unicellular consisting of Bacteria and Archae; it wasn't until the 'Cambrian Explosion’ where living conditions on Earth changed allowing evolution to accelerate and making life on Earth more diverse and complex. To investigate conditions associated with these transitions, we are using a new tool, clumped isotope thermometry. Clumped Isotope thermometry is a thermodynamic proxy, and can be used to determine temperatures of carbonate formation and study paleoclimate using 13C‐18O isotope abundances in carbonate minerals. While there is still room for improvement using clumped isotopes, as it is a new tool, it represents the best method for determining temperatures of formation in carbonate minerals. I will be investigating the temperature of formation for soil nodules with calcite from South Australia for four separate time periods: Cryogenian, Ediacaran, Cambrian and Ordovician. Powdered samples will be measured for clumped isotopes on a mass spectrometer. Cap‐47 values will be reported, from which temperature of formation will be derived. The temperature of formation that is obtained will then be investigated to see if samples underwent any alteration through diagenesis (i.e., burial heating and recrystallization). Unaltered samples will be investigated further to assess temperatures associated with critical transitions during the evolution of metazoan life. 13
SPD 2014 SESSION ONE 41 Development of Cortical Features Associated with Visuospatial Functioning in 22q11.2 Deletion Syndrome ERIC JUNG, Wen‐Ching Tran, Ami Mehta, Carolyn Chow, Rachel Jonas and Carrie Bearden 22q11.2 Deletion Syndrome (22qDS) is a neurogenetic syndrome caused by a hemizygous deletion on chromosome 22. Among other cognitive and physiological anomalies, a marked deficit in visuospatial ability is exhibited in 22qDS (Bearden et al. 2001). This study examined whether brain structural features, such as cortical thickness and surface area, correlated to visuospatial functioning as measured by the Rey Complex Figure Test (RCFT) in 22qDS. Trajectories of age were examined to uncover any effects of 22q on brain development and visuospatial functioning. RCFT performance was found to be significantly lower in the 22qDS group than the control group. In studying whether RCFT scores appear reflective of cortical features, we hope to suggest an age‐sensitive neuroanatomical basis for the visuospatial deficit seen in 22qDS. 42 Functional Characterization of a Novel Toxoplasma gondii Rhoptry Transporter Protein ANDREW H. LIN and Peter J. Bradley Apicomplexa are a phylum of parasitic protozoa that infect a wide range of vertebrates and are responsible for a large number of diseases in warm‐blooded animals, including malaria and toxoplasmosis. The apicomplexan T. gondii, which chronically infects a third of the world's population, uses a sophisticated strategy for infection involving a unique rhoptry organelle that is still incompletely understood. This study looks specifically at a novel rhoptry protein that has been bioinformatically determined to be a major facilitator protein and transporter in T. gondii. We hypothesize that this protein is essential in T. gondii's invasion pathways. To functionally characterize this novel rhoptry protein, we used a protein tagging method that utilizes a hemagglutinin tag, followed by a knockdown of our protein of interest. We have verified that gene model TgME49_316280 indeed codes for a novel rhoptry protein that localizes specifically to the bulbous ROP portion of the rhoptry. Additionally, an attempt to knockout our protein failed, suggesting that our novel rhoptry protein is crucial in the invasion pathway of T. gondii. Understanding the mechanisms by which T.gondii invades host cells is the first step in designing better therapies for this potentially deadly parasite. 43 Selective Plane Illumination Microscopy Enables Visualization of Calcium Ion Flux in the Zebrafish Heart. PETER PELLIONISZ, Bryce Jacobson, Ann Cavanaugh, Laurent Bentolila, Jau‐nian Chen and Katsushi Arisaka. Defects in calcium efflux and reuptake in cardiomyocytes are associated with heart failure and cardiac arrhythmias. In Zebrafish tremblor mutants, uncoordinated irregular cardiac contractions are due to a calcium extrusion defect. The synthetic compound efsevin is reported to restore persistent, coordinated, and rhythmic contractions in tremblor mutant cardiomyocytes. In order to study calcium gradients in mutant Zebrafish hearts and investigate the role of efsevin regarding calcium handling, a selective plane illumination microscope (SPIM) is constructed. The SPIM system utilizes a light‐sheet based orthogonal objective arrangement to provide optical sectioning while reducing phototoxic damage and photobleaching in comparison to conventional confocal techniques. Furthermore, use of a scientific complementary metal‐oxide detector (sCMOS) permits high frame rate image acquisition at native resolution. The SPIM system is utilized with fluorescent calcium indicators in genetically engineered Zebrafish to investigate the clearing of calcium from cardiomyocytes following muscle contraction. A significant application of this technique is the fast, high resolution imaging of both live and large‐volume biological samples. 14
SPD 2014 SESSION ONE 44 Biodistribution in Tissue of Novel Drug Molecules via Matrix‐Assisted Laser Desorption/Ionization Imaging Mass Spectrometry JOSEPH M. FOWLER, Jonathan F. Waxer, Carly N. Ferguson, Richard A. Gatti and Joseph A. Loo Premature termination codons (PTC) are the result of mutations within an organism's genome that can cause a wide variety of genetic disorders. One such genetic disorder is ataxia‐telangiectasia (AT), which causes decreased coordination, mental retardation, and premature death in patients due to a PTC‐generated AT‐mutated protein. Nonaminoglycoside have been shown to allow read through of PTC's, which allows proper translation of genes. While nonaminoglycosides have been found to treat PTC genetic disorders in vitro, they are untested in vivo. Two nonaminoglycosidic read‐through compounds of interest (RTC 6 and 16) should theoretically be able to pass the blood‐
brain barrier to treat neural or brain PTC disorders because of their small size and non‐polar characteristics. A total of forty‐eight mice were tested following an intraperitoneal injection: sixteen untreated; sixteen RTC 6 treated; sixteen RTC 16 treated. Imaging Mass Spectrometry (MALDI‐IMS) was used to identify the presence of RTC 6 and 16 in the liver, heart, kidneys, lungs, and brain of dosed mice. As expected, no masses of RTC 6 or 16 were also found in the organs of control mice. Metabolites and adducts of RTC 6 and 16 localized in the brains of treated mice, revealing their ability to cross the blood‐brain barrier. Traces of the drugs were found in the heart and lungs of treated mice, pointing towards possible side effects. After complete tissue testing, the next step will be to test AT‐mouse models to determine if the compounds are efficacious. 45 Characterization of a Mycobacterium Tuberculosis System for Iron Acquisition from Heme NEDA BIONGHI, Michael V. Tullius and Marcus A. Horwitz Mycobacterium tuberculosis (Mtb) is the primary etiological agent of tuberculosis, one of the world's leading causes of death. Understanding the biology and methods of survival for this pathogen are vital to combating the disease. Like most bacteria, Mtb requires iron to survive. Our laboratory recently described a novel Mtb heme iron acquisition system, and the genomic region responsible for heme uptake was identified. To extract the iron contained in the hemin structure, Mtb is believed to use a heme‐degrading enzyme (Rv3592) to break down the molecule intracellularly and release free iron. However, an Mtb mutant defective in siderophore‐mediated iron acquisition (mbtB deletion) and lacking Rv3592 is not completely defective in heme iron acquisition, suggesting an alternative mechanism for acquiring iron from heme transported into the cell. To identify potential genes functioning in heme acquisition, we are creating a genomic library from the siderophore‐deficient Mtb mutant and carrying out complementation studies in a siderophore‐deficient BCG strain. Unlike Mtb mbtB, BCG mbtB is incapable of growth in low concentrations of hemin and complementation with the Mtb mbtB genomic library is expected to allow for the growth of BCG mbtB in these concentrations of hemin. As a complementary approach, we have isolated hemin‐
adapted clones of BCG mbtB that grow with reduced concentrations of hemin and intend to conduct RNA seq analysis to characterize the functional difference between adapted and unadapted strains. 46 Transmucosal Delivery of Celecoxib into Oral Cancer Lesions from Multifunctional Nanodiamond Constructs ALBERT T. YEN, Kangyi Zhang, Ho‐Joong Kim, Giulia Daneshgaran and Dean Ho Celecoxib (Cxb) is an insoluble drug with the potential to treat oral cancers. However, Cxb administration via ingestion results in low on‐site bioavailability and high systemic toxicity. Direct injections are traumatic and inconvenient. To overcome these challenges, sustained and localized delivery of Cxb into oral cancer lesions is required. However, Cxb molecules are unstable in the aqueous oral cavity. Another challenge to overcome is the protective mucus layer that covers the oral mucosa and traps most particulates. To facilitate the release of Cxb into oral cancer lesions, we propose an adhesive, biodegradable patch embedded with mucopenetrating nanodiamond‐Cxb complexes. Nanodiamonds (NDs) are carbon nanoparticles that act as effective drug carriers due to their functionality, biocompatibility, and drug sequestration capability. Our NDs are polymer‐functionalized to allow for mucosal penetration. We are currently investigating the mucopenetrative efficiency of our multifunctional ND construct with Franz diffusion cell experiments and in vitro cell models. Furthermore, we are generating Cxb loading and release profiles for our ND construct with UV spectroscopy. Preliminary cell viability assays conducted by treating oral cancer cells lines with ND‐Cxb complexes have also shown that ND‐Cxb may be better at killing tumor cells than free Cxb. Overall, our mucopenetrating ND‐Cxb platform can overcome the barrier nature of mucous membranes, serving as a model for nanoparticle‐mediated transmucosal drug delivery. 15
SPD 2014 SESSION ONE 47 Nicotine Withdrawal Enhances Neuronal Activity Within the Interpeduncular Nucleus Via a Mechanism Dependent on Alpha(a)2‐Containing Nicotinic Receptors MONTANA UPTON Tobacco use is the leading cause of preventable death in the United States, and is highly addictive in nature. An important mediator of continued tobacco use relates to the unpleasant withdrawal symptoms induced by smoking cessation. Thus, by understanding the mechanisms mediating tobacco withdrawal symptoms, more effective therapeutic interventions could be provided. Nicotine, a primary psychoactive constituent in tobacco smoke, binds to neuronal nicotinic acetylcholine receptors (nAChRs). Neuronal nAChRs are proteins with distinct pharmacology and expression patterns within brain regions associated with nicotine reward and withdrawal. A nAChR subunit with high expression patterns within the IPN, an area that has been associated with nicotine addiction and reward, is the α2 subunit. To better clarify the neuronal mechanisms involved in nicotine addition, our lab studies the necessity and sufficiency of α2 nAChR subunits in modulating withdrawal behavior in mice. My studies over the course of the last year have assessed whether the absence of nicotine withdrawal in the α2 nAChR subunit null mutant mouse is influenced by a lack of neuronal activation in these mice within the IPN. Using immunohistochemistry, my preliminary results in wildtype mice demonstrate that nicotine withdrawal can enhance the expression of the immediate early gene cfos, a marker for neuronal activation, within the IPN. These effects were absent in the α2 nAChR subunit null mutant mouse. Mice were provided by Dr. Shardhad Loftipour. 48 Women Overestimate Past Penis Model Girth, Yet Prefer Larger Penis in Short‐Term Partners SHANNON LEUNG, JINKYUNG (JAYMIE) PARK and Nicole Prause Women's preferences for penis size in different mate types may reveal the nature of size preferences. Studies have not examined the relationship context for penis size preference, and methods to date have relied on 2D, flaccid penis models. In this study, 3D plastic penile models were constructed across a range of normative human penis sizes were used to assess women's ability to recall penile size for both immediate and delayed recall. Women tended to recall the model as slightly smaller than it was immediately after viewing it, yet overestimated the size, especially the circumference, after a delay. In the second part of the study, participants were also asked to report their desired penis size for a short term versus a long‐term sexual partner through a questionnaire by using the penile models as a reference. Women also reported a preference for a partner with a larger circumference for a short‐term sexual relationship, whereas they did not have a preference with respect to length. These results are consistent with higher pleasure motives active during short‐term relationships, but also raise questions about women's ability to accurately recall their past experiences with different penis sizes. 49 Differential Mitochondrial Gene Expression in Patients Undergoing Mechanical Circulatory Support Device Implantation ELEANOR CHANG, Martin Cadeiras, C.Y. X'avia Chan, Galyna Bondar, Nicholas Wisniewski, Maral Bakir, Jay Chittoor, Tam Khuu, and Mario Deng A main cause of death in Advanced Heart Failure (AdHF) patients is the immune related Multiple Organ Dysfunction syndrome (MOD). Previous studies have indicated that mitochondrial dysfunction in the context of severe energy depletion and detrimental organ damage via oxidative stress has emerged to be a key contributor to the pathogenesis of MOD. We hypothesize that mitochondrial dysfunction develops in the setting of AdHF before and after MCSD implantation. We collected PBMC samples from 11 AdHF patients undergoing MCSD implantation and 3 healthy controls. Samples were collected 1 day before surgery and at days 1, 3, 5 and 8 postoperatively. Purified mRNA was subjected to whole‐genome mRNA sequencing analysis. Two‐way ANOVA was used to identify significant differentially expressed genes. Biological significance of clusters was assessed by gene ontology (GO) enrichment. 44 out of 290 mitochondrial genes were observed to be differentially expressed between the control, low, and high risk groups. The genes in the high risk group were expressed higher in magnitude in comparison to the other two groups. Those selected genes were found to be linked to major inflammatory response mechanisms and programmed cell death. Differential mitochondrial gene expression was observed in patients is a strong predictor of AdHF‐associated MOD risk. Further examination of the temporal alterations in the mitochondrial gene expression in conjunction with whole PBMC gene expression profiling may benefit the daily clinical management of AdHF patient 16
SPD 2014 SESSION ONE 50 Discs Large Homolog 1 (Dlgh1) Splice Variant Specific Expression Changes in Response to T Cell Receptor (TCR) Stimulation MICHELLE D. TIBBS, Jillian A. Crocetti, Oscar Silva and M. Carrie Miceli CD8+ cytotoxic T lymphocytes (CTLs) play a vital role in the immune response to viral infections through direct lysis of infected cells and production of proinflammatory cytokines. Although these responses are both initiated through T cell receptor (TCR) engagement of antigen, their activities are not always paired. Scaffolding protein Discs large homolog I (Dlgh1) plays a critical role in facilitating these responses, suggesting that Dlgh1 might regulate these two functions through independent mechanisms. Dlgh1 has two main splice variants in CTLs: Dlgh1 AB and Dlgh1 B. While Dlgh1 AB directs both granule‐dependent cytotoxicity and proinflammatory cytokine production, Dlgh1 B only mediates granule‐dependent cytotoxicity. We hypothesize that changes in splice variant expression ratio impacts functional output of the CTL. In order to test this hypothesis we examined the expression levels of total Dlgh1, Dlgh1 AB and Dlgh1 B transcripts in mouse CD8+ CTLs. Though our results had some variation over many experiments, much of the data show that total Dlgh1 mRNA, and perhaps protein levels, decrease at early time points and recover at later time points in response to stimulation. Interestingly, Dlgh1 AB, but not Dlgh1 B, mRNA decreased in response to stimulation, leading to an increased Dlgh1 B/Dlgh1 AB ratio. These results, supported by a preliminary functional output experiment, suggest that T cells might selectively regulate the expression of Dlgh1 splice variants in order to uncouple various TCR‐dependent functions 51 25‐Hydroxyvitamin D Dose‐Dependently Regulates Expression of Cathelicidin in TLR‐Stimulated Macrophages SIMA AMIN, Kathryn Zavala, John S. Adams and Martin Hewison Macrophages utilize toll‐like receptors (TLR) to recognize foreign pathogens leading to phagocytosis and bacterial killing. TLR activation stimulates expression of the vitamin D receptor (VDR) and the enzyme, 1alpha‐hydoxylase, which converts precursor 25‐hydroxyvitamin D (25D) into biologically active 1,25‐dihydroxyvitamin D (1,25D). 1,25D binds VDR and acts a transcription factor upregulating expression of the antimicrobial peptide, cathelicidin (CAMP). This suggests an intracrine vitamin D system for regulation of the antibacterial response. In this study, we assessed the 25D dose response curve to determine the concentration of 25D needed to restore cathelicidin expression following TLR activation. Monocytes were collected from healthy blood donors and stimulated with TLR ligand, Pam3CSK4, on Day 0 or Day 1. Cultures were supplemented with vehicle, 10nM 1,25D, or 25D (20nM, 100nM, 250nM, or 500nM). After 24 or 48 hours, RNA was harvested and cDNA synthesized. Quantitative PCR was used to measure gene expression of three vitamin D system genes: 1alpha‐hydroxylase (CYP27B1), vitamin D receptor (VDR), and 24‐hydroxylase (CYP24A1); and the antimicrobial peptide: cathelicidin. Our results demonstrate that supplementation with 500nM 25D upregulates cathelicidin expression to a level comparable to 10nM 1,25D. More broadly, the significance of this study is to support the hypothesis that vitamin D sufficiency is important for maintaining the appropriate response to infection. 52 Applying Optogenetic Techniques in a Huntington's Disease model to Inhibit or Excite Somatostatin Positive Interneurons to See the Effect in Inhibitory Activity in Cortical or Striatal Neurons KRISTA N. RUDBERG, Sandra M. Holley and Michael S. Levine In Huntington's disease, an increase in GABA may be affecting the medium spiny neurons, which leads to lack of motor control. The source of this increased GABA is unknown, but persistent low‐threshold spiking interneurons are suspected. Using channelrhodopsin‐2 and halorhodopsin to respectively excite and inhibit somatostatin‐positive persistent low‐threshold spiking interneurons, we found that evoked GABA did not contribute to the overall GABA increase in medium spiny neurons and cholinergic neurons, but found an indication of a small contribution from spontaneous GABA. Therefore, persistent low‐threshold spiking interneurons probably make up part of the previously shown increase in GABA in Huntington's disease. In addition, there is promising data that interneurons in the striatum are affected differently than the same class of interneurons in the cortex of a Huntington's disease model, suggesting that they play different roles in the disease. 17
SPD 2014 SESSION ONE 53 Spatial and Temporal Expression of Notch and Fibropellin Variants TAMER GHESH, MATTHEW ARGUELLO, TREVOR RAFFERTY, DANIEL TAYLOR, Lydia Ann and Pei Yun Lee Strongylocentrotus purpuratus is a model organism of deuterostome development, a group which includes humans and other chordates. This experiment focuses on the characterization of putative Notch variants using BLAST analyses and phylogenetic trees. Notch is vital in the process of cell differentiation during neurogenesis. The temporal and spatial expression of these genes were determined through reverse transcription polymerase chain reaction (RT‐PCR) and whole mount in situ hybridization (WMISH). Only the following RT‐PCRs showed expression: Gene 1 at 0 and 24 hours, Gene 2 at 24 hours, Gene 3 at 48 hours, and Gene 4 at 0 and 48 hours. However, the results of the WMISH do not support the RT‐PCR results. These results are significant because they help contribute to our understanding of the expression of putative Notch genes. 54 Sustained Local Delivery of the Chemotherapeutic Taxol to Treat Glioblastoma via Tunable, Amphiphilic Diblock Copolypeptide Hydrogels VISHWAJITH RAMESH, Matthew Garrett, Shanshan Zhang, Timothy J. Deming and Harley I. Kornblum Glioblastoma is the most common primary malignancy of the central nervous system. The current standard of therapy involves surgical resection, chemotherapy, and radiation. However, these have been shown to have only a modest effect on survival and recurrence is nearly universal. One limitation of chemotherapy is the restricted ability of most drugs to cross the blood‐brain barrier. Diblock copolypeptide hydrogels (DCH) are biocompatible materials that can be modified to integrate well with brain tissue, carry a variety of molecules, and degrade gradually over time, making them strong candidates for site‐specific, sustained drug delivery. In this study, we constructed DCH loaded with 0%, 0.5%, or 0.8% taxol and tested the feasibility of the DCH delivery vehicle using an in vitro assay with cells suspended in media. We used a well‐established model of glioblastoma (U87). We found that both taxol‐loaded constructs were able to reduce U87 cell viability. Based on these results, with further optimization to maximize effective drug delivery as well as injectability (for in vivo studies), DCH proves to be a promising replacement for the current therapies for glioblastoma. 55 Treatment of Yeast with Polyunsaturated Fatty Acids Decreases Coenzyme Q Content MINHHAN N. PHAM, Hui S. Tsui, Chris Allan, and Catherine F. Clarke The coenzyme Q‐deficient Saccharomyces cerevisiae coq mutants are highly sensitive to treatment with polyunsaturated fatty acids (PUFAs). PUFAs undergo lipid peroxidation due to the presence of bis‐allylic H atoms which are abstracted quickly in the presence of reactive oxygen species (ROS). Such lipid peroxidation produces toxic products that are presumed to kill the coq mutants. Previous studies suggest that the reduced form of Q (QH2) is protective because it acts as a chain terminating antioxidant. Thus lipid peroxidation stress may lead to oxidized Q in response to oxidative stress, and such perturbations of the redox state of Q/QH2 may directly affect mitochondrial function and lead to cell death. In this study wild‐type yeast were treated with different concentrations of linolenic acid (Lnn) to study the impact on the content of Q in response to lipid peroxidation. We observed that the yeast Q?content decreased with increasing concentrations of Lnn. The content of reduced QH2 decreased more dramatically than that of oxidized Q. We are currently assessing whether treatment with isotope‐reinforced PUFAs (D‐PUFAs) may protect the cellular Q content from degradation due to lipid peroxidation. In addition, study of Q autoxidation products may help to determine the process by which the content of total Q decreases after treatments with PUFAs. 18
SPD 2014 SESSION ONE 56 Stathmin (Oncoprotein 18) Deregulation Induces Differential Apoptosis in Liver Cancer Cells Infected with Hepatitis C Virus (HCV) NATALIE M. LIU, James Vu, Nu Lu, Darshil Patel, and Samuel French Patients with chronic HCV risk developing aggressive liver cancer. Using mass spectrometry, we previously identified the cytoskeleton protein stathmin as differentially up‐regulated in liver cancer cells when in the presence of HCV. Stathmin over‐expression is associated with breast, prostate, and lung cancer. Stathmin promotes microtubule destabilization and is deactivated when phosphorylated at four different serine residues. We aim to identify how HCV takes advantage of stathmin deregulation to activate liver cancer pathways. If HCV helps to inhibit apoptosis by increasing the level of activated stathmin, then cytoskeleton destabilization is the potential mechanism through which HCV induces liver cancer. We used transient transfection to over‐express four different GFP tagged stathmin mutants into two liver cancer cell lines (with and without HCV). Utilizing flow cytometry to analyze the rate of apoptosis within only the transfected population, we find that there is no definitive trend affecting apoptosis when single phospho‐site stathmin mutants are over‐expressed. Our results suggest that a stathmin mutant lacking all four phosphorylation sites, and therefore cannot be deactivated, may be necessary to sustain a clear effect on apoptosis. In conclusion, cytoskeletal destabilization is a mechanism through which HCV can induce cancer. Thus, stathmin could be a future target for therapy to halt the progression of liver cancer in patients with hepatitis C. 57 Transcriptome Analysis of the Human Corneal Endothelium: Insights into Gene Regulation of the Corneal Endothelial Dystrophies CYNTHIA WANG, Ricardo F. Frausto, Tina M. Bui, and Anthony J. Aldave Purpose: To comprehensively characterize human corneal endothelial cell (HCEnC) gene expression, age‐dependent differential gene expression and to identify expressed genes mapped to chromosomal loci associated with the corneal endothelial dystrophies PPCD1, FECD4 and XECD. Methods: Total RNA was isolated from ex vivo corneal endothelium obtained from four pediatric (=11 years old) and five adult (=53 years old) healthy donor corneas. The RNA samples were processed and hybridized to the Affymetrix GeneChip 1.1ST array. Data analysis was performed using the gene expression module within the Partek Genomics Suite software. Differentially expressed genes were validated by quantitative‐PCR (qPCR) and nanoString technology. Results: Transcripts were identified corresponding to 13,697 genes in HCEnC. Nine genes displayed the most significant differential gene expression between pediatric and adult HCEnC. Analysis of the PPCD1, FECD4 and XECD loci demonstrated transcription of 53/95, 27/40, and 35/69 protein coding genes respectively. Conclusions: An analysis of the corneal endothelial transcriptome reveals the expression of almost 14,000 genes, with less than 1% mapped to chromosomal loci associated with PPCD1, FECD4 and XECD and less than 0.1% demonstrating significant differential expression between pediatric and adult HCEnC. This data will serve as a resource for scientists investigating HCEnC gene expression to focus the search for the genetic basis of several corneal endothelial dystrophies with unidentified genetic bases. 58 Identification of the Antigenic Epitope in Phospholipase A2 Receptor Mediating Idiopathic Membranous Nephropathy VINSON LAM, Liyo Kao, and Quansheng Zhu ABSTRACT WITHHELD 19
SPD 2014 SESSION ONE 59 ABSTRACT RETRACTED 60 Importance of Kaposi's Sarcoma‐Associated Herpesvirus Open Reading Frame 54 and Ubiquitin‐
Associated Protein 1 Interaction in Evading Host Interferon Response SARA M. SHU, Harding Luan, Danyang Gong, Ronika Sitapara Leang, Shaoying Lee, Laura Liu, Emily Duong, Justina Leo, Leming Tong, Ren Sun and Ting‐Ting Wu Human gamma‐herpesviruses, including Epstein‐Barr virus and Kaposi's Sarcoma‐associated herpesvirus, are known for establishing lifelong persistent infections and are associated with several maligancies. Understanding how gamma‐
herpesviruses evade the host immune response is crucial for developing an effective vaccination strategy. Type I interferons (IFNs) represent the first line and critical component of host antiviral response. Gamma‐herpesviruses counteract this host type I IFN response, facilitating their life‐time persistence. Open Reading Frame 54 (ORF54) is one of the viral gene products that blocks type I IFN induced signaling and was found to down‐regulate type I interferon receptor 1 (IFNAR1). However, the underlying molecular mechanism of IFNAR1 down‐regulation remains to be elucidated. Previous protein‐protein interaction studies identified Ubiquitin Associated Protein 1 (UBAP1) as an interacting protein candidate of ORF54. To determine the functional significance of the ORF54 interaction with UBAP1 in down‐regulating IFNAR1, we will map the residues on ORF54 that are essential for interacting with UBAP1 and examine the effects of abolishing the interaction with UBAP1 on ORF54 function through mutating the binding residues. Successful characterization of this interaction will reveal a novel mechanism by which gamma‐herpesviruses inhibit the host immune responses and contribute to improving the efficacy of interferon therapies for virus‐
associated cancers. 61 Thermally Responsive Trehalose Glycopolymer as a Readily Removable Smart Excipient for Protein Stabilization. MALTISH M. LORENZO, Muhammet U. Kahveci, and Heather D. Maynard Synthesis of block copolymers of N‐isopropylacrylamide (NIPAAm) and trehalose based monomers by atom transfer radical polymerization (ATRP) and their potentials as a smart stabilization agent for proteins is described. N‐
isopropylacrylamide monomer was polymerized via atom transfer radical polymerization (ATRP) using an alkyl halide radical initiator and a copper catalyst. A modified disaccharide, styrenyl trehalose, was also polymerized by ATRP to yield a known protein stabilizing agent. Poly(NIPAAm) and Poly(styrenyl trehalose) will be used as macroinitiators to polymerize styrenyl trehalose and NIPAAm, respectively, to yield block copolymers. Poly(NIPAAm) has been shown to have a drastic changes in solubility in aqueous solution when exposed to elevations in temperature above its lower critical solution temperature (LCST). This behavior will be utilized for the preparation of 'smart†polymer‐protein systems to separate the protein from the stabilizer, namely poly(NIPAAm)‐b‐poly(styrenyl trehalose) or poly(styrenyl trehalose)‐b‐polyNIPAAm. After purification, the block copolymers will be incubated at various concentrations with a selected protein and exposed to multiple lyophlization cycles to elucidate the effects of the polymer on protein stability. We aim to create a thermo‐responsive polymer, which, upon changing environment temperature, should dissociate from the protein, precipitate and be easily filtered out to obtain a native active protein. 20
SPD 2014 SESSION ONE 62 High Expression of EMT Markers as Early Detection for Triple Negative Breast Cancer WENDY SILVA and Gustavo Miranda‐Carboni Breast cancer represents the second most commonly diagnosed cancer type amongst women in the United States. Triple‐negative breast cancers (TNBC) are aggressive and frequently metastasize to the lung and brain leading to a poor prognosis. Here we aim to identify properties of non‐malignant breast lesions that predict progression to metastatic TNBC. We hypothesize that TNBC with metastatic potential arise in response to activation of Wnt/beta‐
catenin signaling. Secretable Wnt‐ligands from activated basal‐epithelial stem cells elicit either autocrine and or paracrine cellular responses and thereby promote epithelial‐to‐mesenchymal‐transitions (EMT), invasion, and metastasis. To investigate the role of Wnt/beta‐catenin signaling we have collected samples of the earliest identifiable breast lesions (atypical hyperplasia) from African‐American women at high‐risk to develop TNBC. We determined the expression pattern for known markers of the Wnt/beta‐catenin signaling pathway (BMI‐1, HMGA2, and WNT10B) in normal, atypical hyperplasia, and TNBC samples by immunohistochemistry. Preliminary results revealed a higher expression of BMI‐1, HMGA2, and WNT10B in atypical hyperplasia and TNBC than observed in normal tissue. These results are promising, and reveal that the presence of these markers may lead to earlier detection of TNBC in women who are at high risk to develop TNBC. Innovative therapeutics to prevent metastasis for TNBC patients based on these EMT markers can be developed, and increase the survival rate of patients with TNBC. 63 Generating a Zebrafish Model of Deafness‐Dystonia Syndrome Using Transcription Activator‐Like Effector Nuclease (TALEN) Technology CHRISTINA K. JAYSON, Meghan Johnson, Jisoo Han and Carla M. Koehler Defects in mitochondrial assembly lead to a broad spectrum of diseases. However, suitable animal models to facilitate understanding the underlying basis for these diseases are limited. Here, zebrafish models are developed for the neurodegenerative, mitochondrial deafness‐dystonia syndrome caused by mutations in components of the mitochondrial protein import pathway, TIMM8A and TIMM8B. Zebrafish lines expressing a fluorescently labeled protein that resides in the mitochondria in different tissues, including primary motor neurons, the brain, heart, and muscle were generated to permit phenotypic characterization. These tissues are typically affected by mitochondrial disease due to their high energetic requirement. Using the transcription activator‐like effector nuclease (TALEN) technology, TIMM8A and TIMM8B knockout zebrafish lines are being generated. Preliminary results indicate that the TALENs have successfully targeted the locus for each construct. Defects in mitochondrial function and organ development, including the neural system, will be characterized in embryos and adult fish. Mitochondrial morphology and movement in the axons and axonal development in primary motor neurons will be imaged. Impairment in mitochondrial trafficking in the neurons as well as affected development of the cardiovascular system is expected. Modeling deafness‐dystonia syndrome in zebrafish will enable studies of the mechanics of mitochondrial diseases, allowing further investigation of the consequences of a compromised protein import system. 64 Resistive Switching in Percolative Nanoparticle Thin Films ERIC J. SANDOUK, Henry O. Sillin, Adam Z. Stieg and James K. Gimzewski Recently, memristive devices have been the subject of significant consideration for their potential use in nanoelectronics and non‐volatile memory applications. Here we investigate the nonlinear current‐voltage properties of a relatively new memristive system, thin films of silver nanoparticle ‐ organic polymer composites. Silver nanoparticles (d = 80‐100 nm) were synthesized via the polyol process in the presence of polyvinylpyrrolidone, a protective agent, and deposited onto silicon oxide substrates after the addition of excess organic polymer. A forming step was required to functionalize the devices and was achieved by several applications of 7‐9V voltage sweeps. Once functionalized, the thin film devices exhibited controllable resistive switching between discrete resistance states that depended on parameters such as temperature and bias voltage. Because of their tunable resistance states, highly increased spatial density, ease of fabrication and low power consumption, nanoparticle thin films may soon find a place in electronics alongside current CMOS technology. 21
SPD 2014 SESSION ONE 65 Temporal and Spatial Expression of Strongylocentrotus purpuratus mist and deltex‐4 not Detected Within 72 Hours of Fertilization KYLE BUDROVIC, JASON BUNN, TYLER ICE, LYDIA ANN and Pei‐Yun Lee Studying gene expression in Strongylocentrotus purpuratus at the larval stage provides insight into homologous developmental genes in a deuterostome model to aid in the understanding of human development. This paper attempts to localize, both temporally and spatially, Strongylocentrotus purpuratus unknown genes mist and deltex‐4 II. The genes were subjected to bioinformatic analysis, followed by vector cloning and colony analysis. RNA probes were hybridized to embryos fixated at the 24, 48 and 72‐hour stages of embryonic development to determine spatial expression. Reverse transcription PCR results showed no bands within the first 72 hours of fertilization, positive controls excepted. In situ hybridization results showed no staining for either mist or deltex‐4 II probes, confirming negative RT‐PCR results. Both experiments confirm that these genes, whose likely products are transcription factors and Notch signal pathway receptors, are not expressed within the early Strongylocentrotus purpuratus development. 66 Optimization of an Orally Viable Read‐Through Compound (RTC) for the Treatment of Duchenne Muscular Dystrophy (DMD) DAVID B. CHU, Thomas Gintjee, Alvin Magh, Refik Kayali, Matthew Alschuler, Yinhan Wang and Carmen Bertoni Duchenne muscular dystrophy (DMD) is a severe neurodegenerative disorder that results in overall muscle degeneration and eventual death. Approximately 13% of the mutations causing DMD are nonsense mutations that generate a premature stop codon and result in a truncated and nonfunctional dystrophin protein. RTC13 is a read through compound that has been previously shown to restore dystrophin expression in mdx mice after intraperitoneal administration. RTC13Y, an orally viable prodrug of RTC13, was developed to increase bioavailability of RTC13. We have now tested the viability and efficacy of chronic systemic administration of RTC13Y in mdx mice alongside PTC124, a preexisting read through compound in the late stages of clinical trials. We administered RTC13Y and PTC124 via oral gavage in mdx mice 3 times a day at intervals of 4 hour between administrations, at doses of 30 mg/kg and 60 mg/kg. Functional analyses using the grip and wire tests showed a significant improvement in mdx mice treated with RTC13Y compared to untreated mdx mice or mdx mice that received PTC124 at the same dose range. To further determine the functional activity of our lead RTC, we have analyzed muscle isolated from mdx mice treated with RTC13Y and PTC124 for dystrophin expression and their ability to resist to eccentric force contractions. Our results demonstrated a significant improvement in mice treated with RTC13Y compared to untreated mdx controls, suggesting that RTC13Y is a promising candidate for the treatment of DMD. 67 Ultra‐high Throughput Isolation of Circulating Tumor Cells Using Vortex‐aided Inertial Microfluidic Device VICTOR SAN HOU YU, James Che, Manjima Dhar, Derek E. Go, Melissa Matsumoto, Edward B. Garon, Jonathan Goldman, Rajan P. Kulkarni, Elodie Sollier, and Dino Di Carlo ABSTRACT WITHHELD 22
SPD 2014 SESSION ONE 68 Lentiviral Delivered PPARg shRNA Improves Bone Microarchitecture by Altering the Balance of Osteogenesis and Adipogenesis JUNG KIM, Jia Shen, Aaron W. James, Kevork Khadarian, Shen Pang, Choon G. Chung, Raghav Goyal, Greg Asatrian, Xinli Zhang, Kang Ting, and Chia Soo Skeletal aging is associated with alterations in bone formation and breakdown and results in increased fat in bone marrow. An increase in fat cells contributes to low bone mineral density. Ppargamma is a transcriptional regulator of fat cell differentiation and a regulator of bone formation and breakdown. The down‐regulator of fat cell differentiation through the inhibition of Ppargamma may have a therapeutic role in the promotion of bone maintenance and prevention of age‐related bone loss. We used immunodeficient mice to knock out Ppargamma in the femoral bone marrow. We analyzed microcomputed topography for bone density, bone volume, and bone thickness. Femoral samples were analyzed by immunostaining for markers of bone formation, bone breakdown and fat cell differentiation. Femurs treated with Ppargamma knockout in vivo exhibited a significant absence of fat cells in limbs and low fat contents in marrow. Additionally, Ppargamma knockout samples showed increases in trabecular bone mineral density, bone volume and bone thickness which can be caused by increased bone growth or decreased bone breakdown. These outcomes suggest that the deprivation of Ppargamma leads to the proliferation of the trabecular bone by reducing fat cells in marrow and altering bone formation and breakdown. These results lead to a better understanding of the impact of Ppargamma regulation on bone health, and therapeutic studies will be focused on the efficacy of Ppargamma inhibition for improving bone growth and maintenance.
69 Introduction of Multi‐ and Single Worm Trackers, and Modifications Made for Behavioral Analyses of C. elegans SHIJIA W. LU, Shirley Cheng, Steve Mendoza, Katsushi Arisaka C. elegans is a convenient animal to use in behavioral experiments. In order to characterize behavior, it is necessary to track the motion of these worms over time. We describe a worm tracker that we have used for our experiment to show speed, velocity, and angular position. The worm tracker we show first is a multi‐worm tracker, calculating the center of mass for each worm. However, there is a need for higher resolution studies of subtle motions, such as head movement or bending, which may be relevant for the behavioral study of C. elegans. As well as exploring higher definition worm trackers, we have worked in developing single worm trackers. We will describe our progress in modifying our worm tracker for experiments such as detailing the response curve when C. elegans reverses from a harmful stimulus (such as ultraviolet light or touch). We hope that the general worm trackers can further help in the behavioral analysis of C. elegans. 70 Spatial and Temporal Gene Expression of Jagged‐1b, Fibrillin, Notch, and Fibropellin at Successive Developing Embryonic Stages of Strongylocentrotus purpuratus JAMIE HO, HARRISON HO, KRISTIE TAGUMA, Lydia Ann, and Pei Yun Lee In this study, the identities of four unknown genes were determined, as well as where and when they are expressed during development. The purple sea urchin, Strongylocentrotus purpuratus, was used as the model organism since echinoderms are evolutionarily close to chordates and are relatively simple organisms during embryogenesis. The genes were determined to be Jagged‐1b, Fibrillin, Notch, and Fibropellin using BLAST and phylogenetic analysis. Whole mount in situ hybridization (WMISH) and reverse transcription PCR in conjunction were utilized to see both temporal and spatial expression. RNA probes for WMISH were obtained through gene cloning, bacteria transformation, plasmid isolation, and DNA sequencing. Jagged‐1b is not expressed within 72 hours after development based on in situ and RT‐
PCR results. RT‐PCR data suggests Fibrillin is expressed at 48 and 72 hours after development, however WMISH only confirms low expression at 48 hours at the apical region. RT‐PCR suggests that Notch is expressed at 48 and 72 hours after development but in situ only confirmed expression at 48 hours in the hind gut. Fibropellin is expressed 24 hours after development according to the RT‐PCR, but in situ suggests that it is not expressed at all. Since the sea urchin genome encodes for many vertebrate‐related genes, studying S. purpuratus' genome is a convenient model system to help elucidate the function and expression of human genes. 23
SPD 2014 SESSION ONE 71 Examining Insular Cortices' Projections To the Hypothalamus and Other Brain Regions. NAYER S. TOMA, Rajesh Kumar and Ronald M. Harper ABSTRACT WITHHELD 72 Studies of the BBSome in Trypanosome brucei SELENA ZHOU, Michelle Shimogawa, and Kent Hill Trypanosoma brucei are protozoan parasites responsible for African trypanosomiasis, a disease of high medical and economic significance in sub‐Saharan Africa. There are currently no efficient drugs available for this lethal illness, so studying the biological mechanisms of the parasite is essential in order to elucidate a treatment. One area of interest is the BBSome, a conserved complex of seven Bardet‐Biedl syndrome (BBS) proteins (BBS1, 2, 4, 5, 7, 8, 9) that mediates protein trafficking to and from the flagellum in a variety of organisms. All seven subunits are conserved in trypanosomes, but their specific functions are unknown. To better understand BBS protein function, localization of epitope‐tagged BBS1 and BBS4 was determined by immunofluorescence. Both subunits showed localization to the flagellum base, consistent with presence of a BBSome complex and a role for the trypanosome BBSome in regulating the flagellum targeting of proteins. Generation of antibodies against specific BBS proteins provides a second approach to studying the BBSome and is currently in progress. Protein fragments from BBS1 and 9 were cloned, recombinantly expressed in bacteria, purified, and used to produce antibodies in rabbits. Antibodies will be used for immunofluorescence and other techniques such as immunoprecipitation and Western blotting to study the native BBSome complex. Characterizing the role of the BBSome in trypanosomes may shed light on African trypanosomiasis and other primary cilia diseases. 73 Elucidation of the Embryonic Temporal Expression Patterns of Caspase‐6‐Like, Caspase‐7‐Like, BAX Inhibitor 1‐Like, and CRADD‐Like in Strongylocentrotus purpuratus DANIELLE MIHORA, BRIAN KHOA NGUYEN, Danielle Ryba, ANNABELLE SOARES, Marco Morselli and Pei‐Yun Lee Model organisms, such as the purple sea urchin (Strongylocentrotus purpuratus), allow researchers to elucidate gene function, cellular pathways, and developmental processes, all of which can then be extrapolated to similar processes in other organisms or be used for further study of genes, as they pertain to disease. The purposes of this study were to identify four S. purpuratus genes using BLAST and phylogenetic analysis and to determine their temporal and spatial expression via reverse transcription PCR (RT‐PCR) and whole mount in situ hybridization (WMISH), respectively. The gene sequences were identified as caspase‐6‐like, caspase‐7‐like, CRADD‐like, and BAX inhibitor‐1‐like (BI‐1‐like). Caspase‐6‐like and BI‐1‐like displayed temporal expression at 0, 24, 48, and 72 hours post‐fertilization. Caspase‐7‐like showed expression during the first three time points; CRADD‐like did so during the last three time points. Caspase‐6‐
like, caspase‐7‐like, and BI‐1‐like showed endodermal expression at the 48 hour time point. This suggests that apoptotic pathways play at least a minimal role in sea urchin development. Such apoptotic pathways draw significant parallels with those that form the basis of human diseases such as cancer, thereby inspiring areas of interest in future biomedical research. 24
SPD 2014 SESSION ONE 74 Conventional Cytogenetic Characterization of Pancreatic Carcinoma Reveals Highly Complex Karyotypic Abnormalities DAVID S. SHABSOVICH, Nagesh Rao, and Carlos A. Tirado Pancreatic cancer is the fourth leading cause of cancer death worldwide, with an average 5‐year survival rate of approximately 6 percent. Cytogenetic analysis has allowed for the elucidation of clinically significant chromosomal abnormalities in both solid tumors and hematological malignancies, put pancreatic cancer still does not have any FDA‐
approved cytogenetic assays. In the present study, conventional cytogenetic analysis of 4 pancreatic carcinoma cell lines was completed using Giemsa banding (G‐banding). Cells were cultured, harvested, and dropped on slides, and chromosomes were stained using Wright‐Giemsa stain coupled with preceding pancreatin treatment. 20 sets of metaphase chromosomes were analyzed per cell line. G‐banding analysis revealed highly complex karyotypic abnormalities in all cell lines analyzed, including multiple numerical and structural aberrations. Structural aberrations included deletions, duplications, and balanced and unbalanced rearrangements. Further conventional and molecular cytogenetic (fluorescence in situ hybridization and array comparative genomic hybridization) analysis is currently being conducted on cell lines and primary tumor specimens in order to comprehensively characterize pancreatic carcinoma cytogenetically. Ultimately, phenotypic correlation of cytogenetic abnormalities will provide a framework for the development of diagnostic and prognostic cytogenetic assays as well as targeted therapies to combat the malignancy.
75 Melanopsin, an Intrinsically Photosensitive Retinal Protein, is Expressed in a Novel Cell Type in the Pain‐Sensitive Trigeminal Ganglion EILEEN K. NGUYEN, Anna Matynia, Sachin Parikh and Michael Gorin In the clinical condition called photoallodynia, normal levels of light can cause severe pain in individuals suffering from ocular diseases, migraines, and traumatic brain injury. Currently, no treatment exists for photoallodynia other than light avoidance. Photoreceptors called intrinsically photosensitive retinal ganglion cells participate in non‐image‐
forming vision through a photopigment called melanopsin. Melanopsin is involved in innate light aversion, but the connection between light and pain remains unclear. Melanopsin expression in the pain‐sensing trigeminal nerve, which innervates the cornea and facial regions, could delineate a mechanism for light‐dependent ocular pain. To identify this novel connection, melanopsin expression was analyzed in mice using RT‐PCR and immunohistochemistry in various tissues. In the trigeminal root ganglion, a region that receives direct peripheral pain input and projects to the trigeminal nucleus, melanopsin colocalized with a neuronal marker, identifying a novel cell type. Melanopsin transcription was also detected in the choroid, cornea, and spinal trigeminal nucleus, most likely from projections of these neurons. Melanopsin expression in the trigeminal nerve highlights a novel connection between light and pain that occurs independently of retinal activation. Future aims include testing for intrinsic light sensitivity in the trigeminal system and convergent signaling from light and pain in this new cell type, which may have implications for topical, rather than invasive, treatment. 76 Investigating autophagy as a regulator of growth, metabolism and survival in LKB1 mutant non‐small cell lung cancer. SARAH SIMKO, Rob McMickle, and David Shackelford In 2014 an estimated 220,000 people in the US alone will be diagnosed with lung cancer with few personalized or targeted therapies for these patients. My research studies the LKB1 gene, which is mutationally inactivated in ~30% of non‐small cell lung cancer cells (NSCLC), constituting a large patient population. LKB1 is a tumor suppressor and upstream kinase that regulates the AMP regulated kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1). Through regulation of AMPK and mTORC1, LKB1 controls tumor cell growth, metabolism and autophagy. The purpose of this study was to study autophagy in LKB1 mutant lung tumor cells to understand how this cellular process regulates cell death or survival. Autophagy is a conserved cellular process that degrades proteins and organelles to free up metabolites during cellular stress. Due to its involvement in times of metabolic stress, autophagy may have a key role in dictating the survival or death of cancer cells. We developed an assay allowing us to monitor autophagy lung tumor cells in real time using a fluorescent‐tagged reporter protein LC3. LKB1 mutant lung tumor cells are highly sensitive to the metabolic therapeutic phenformin and we discovered that under physiologically relevant glucose levels, phenformin in combination with an autophagy inhibitor selectively killed LKB1 mutant NSCLC cell by apoptosis. Importantly, by understanding the molecular regulation of autophagy in lung cancer we aim to design better therapeutic strategies for the treatment LKB1 mutant NSCLC. 25
SPD 2014 SESSION ONE 77 Dysfunction in Circadian Behavior in a Mouse Model of Rett Syndrome MATTHEW I. DERAKHSHESH, Dawn H. Loh, Quan Li, Danny Truong, and Christopher S. Colwell Rett Syndrome (RTT), an X‐linked disorder, is progressive in nature and can manifest itself in the forms of autism, ataxia, dementia and loss of purposeful hand use. Molecularly, RTT is caused by a mutation in the gene encoding Methyl‐CpG‐Binding Protein 2 (MeCP2), a transcriptional regulator. MeCP2 mutant mice serve as models for identifying the symptoms of RTT and the role of MeCP2 in causing those deficits, but little has been uncovered about the circadian aspect of the disorder. We hypothesized that like other progressive neurological disorders such as Huntington's and Parkinson's diseases, MeCP2 mutant mice exhibit circadian deficits and that further destabilization of their circadian rhythms can result in a shorter life span. We exposed Mecp2 male knockout (KO) mice to various lighting conditions to examine dysfunction in the circadian system through wheel running activity. Our findings demonstrate deficits in circadian rhythms related to locomotor activity that include decreased overall activity, greater fragmentation, as well as decreased power and precision. Next, we conducted a weekly 6‐hour phase advance on the KO mice, the results of which indicate that the phase advance induced faster death. Finally, when exposed to a dark pulse at ZT 2 and a light pulse at ZT 14, the KO mice also show deficits in masking. Taken together, the data demonstrates a unique role of circadian dysfunction in RTT, and that potential therapies targeting the entrainment of circadian rhythms reduce the manifestation of their symptoms. 78 RAP‐011 Efficiently Rescues Erythropoiesis in Zebrafish Models of Diamond Blackfan Anemia TIANNA WILSON, Jason Ear, Haigen Huang, Zahra Tehrani, Victoria Sung, Thomas O. Daniel, Rajesh Chopra and Shuo Lin Diamond Blackfan Anemia (DBA) is a rare blood disorder characterized by a lack of red blood cell production in the bone marrow causing severe macrocytic anemia in affected individuals. Steroid treatments remain as the primary mode for treating DBA. However, patient responses vary and there are undesirable side effects. Currently, bone marrow transplants are the only curative option for DBA patients, but can lead to severe complications. This raises the need to better understand the pathology of the disease for the development of novel treatments and an effective cure. Although the direct cause of DBA is not definitive, it is widely accepted that malfunctioning ribosomal proteins cause blood progenitor cells to undergo premature apoptosis. Approximately 50% of DBA cases have been shown to be associated with a mutation in ribosomal proteins, including RPS19 and RPL11. The zebrafish model system is particularly useful for studying blood development because their development occurs externally, the embryos are transparent, and they can progress through the early stages of development without blood cells. Using zebrafish models of ribosome stress, we have identified a novel role of the Activin/TGF‐beta signaling pathway in the pathogenesis of ribosome stress. Furthermore, the function of this pathway appears to be independent of p53. 79 Computer Vision Aided Analysis of the Complex Bacterial Signaling at Early Stage Previews to Biofilm Formation JAIME DE ANDA, Kun Zhao, and Gerard C. L. Wong The secondary messenger cyclic di‐GMP is used by bacteria as a signal for motility and biofilm formation. This molecule plays a major role in bacteria interactions for further growth to the development of bacterial colonies. Since biofilms are implicated in chronic infections of the host, the study of motility at early biofilm formation can give insight towards the development of novel anti‐infective treatments. Previews studies have developed methods to measure the production of such cdiGMP, by using the cyclic di‐GMP‐responsive cdrA promoter to encode green fluorescent protein (GFP) genes. For this study, we have observed under a microscope, the ability of bacteria to attach and populate the surface of a flow cell. The presented data is on an overproducing bacterial strain and a wild type cyclic di‐
GMP producing Pseudomonas Aeruginosa. We collected images at early stages of attachment with an EMCCD camera on an Olympus IX81 microscope. Images were collected in the bright‐field every 3s, for 300 images, with a 1 frame of GFP data taken under UV light during a period of 20 h. Each data set was processed using novel computer aided tracking algorithms. From the individual tracking of each bacterium, we are able to retrieve characteristic morphological distributions of the bacterial colonization of the surface upon its progression toward biofilm formation. We focus on the complex interactions between the overproducers and the under/non‐producers of cyclic di‐GMP. 26
SPD 2014 SESSION ONE 80 Functional Characterization of Porcine Intrinsic Cardiac Nervous System KRISHAN PATEL, CHRISTOPHER KANAAN, Pradeep Rajendran, Jeffrey Ardell, Andrew Armour and Kalyanam Shivkumar The intrinsic cardiac nervous system (ICN) is a network of interconnected neuronal aggregates called ganglionated plexi (GPs), which are composed of parasympathetic efferent, sympathetic efferent, sensory afferent and local circuit neurons. There is increasing evidence that GPs play an active role in control of cardiac function. The aim of this study was to functionally characterize the neuronal subpopulations that exist within the left atrial ganglionated plexi (LAGP). Activity from multiple intrinsic cardiac (IC) neurons in the LAGP was recorded in five anaesthetized pigs using a 16‐
channel linear microelectrode array. In total, 72 neurons were identified based on their specific amplitudes and waveforms. Induced changes in IC neuronal firing rate were evaluated in response to the following interventions: (1) focal cardiac mechanical distortion (afferent stimulus); (2) electrical activation of vagus nerves or stellate ganglia (efferent stimuli); and (3) occlusion of the inferior vena cava or thoracic aorta (afferent stimuli). In addition to firing rate, activation recovery interval, a surrogate marker of local action potential duration, was measured during each intervention. Overall, 72% of IC neurons responded to afferent stimuli, 47% to efferent stimuli, 38% to both afferent and efferent stimuli, and 15% were non‐responsive. We hope to gain insight into the neural remodeling that occurs at the level of the ICN in healthy versus diseased states by comparing this data with similar data collected from infarcted animals. 81 Genetic Dissection of Orbitofrontal Sulcogyral Morphology in Families with Bipolar Disorder LORRAINE U. ALIRE, NEDA GHASSEMI, POYI WU, Noor B. Al‐Sharif, and Scott C. Fears Bipolar I Disorder (BP) is a psychiatric disorder characterized by recurring and alternating episodes of mania and depression. Mapping the genetic basis of BP disorder has presented many challenges. Research suggests that the emotional and behavioral symptoms of BP disorder are caused by abnormalities in a brain circuitry consisting of the ventral prefrontal cortex and the limbic system. The orbitofrontal cortex (OFC) has a central role in this brain network, as it is involved in cognitive decision‐making and emotional regulation and processing. However, information on the heritability of and the association between OFC sulcogyral morphology and BP disorder remains scarce. Previous literature suggests that the OFC exhibits three varying H‐shaped sulcogyral patterns based on the contiguity of the lateral and medial sulci of the cortex. Therefore, using structural magnetic resonance imaging data, the present study aims to classify the sulcogyral patterns (Types I, II, III) of the OFC and determine the heritability of this endophenotype in families with heavy genetic loading for BP disorder from two closely related populations in Costa Rica (n=285) and Columbia (n=242). The large sample size and use of multi‐generational pedigrees provide the present study with statistical power to estimate heritability (the proportion of variance due to genetic factors) and association with BP disorder. Future genetic linkage studies can then be performed to identify genes leading to OFC morphology associated with bipolar disorder. 82 X‐AFm Stabilization as a Mechanism of Preventing Conversion Phenomena in Calcium Aluminate Cements GABRIEL FALZONE, Magdalena Balonis, and Gaurav Sant Phase conversion phenomena are often observed in calcium aluminate cements (CACs), when the water‐rich (CAH10, C2AH8) hydrates formed at early ages, in time, expel water to form more compact, less‐water rich structures (C3AH6). These phase conversions follow a pathway regulated by the thermodynamic stabilities of the phases. Based on this premise, it is proposed that by provoking the precipitation of phases more stable than those encountered along the conversion pathway, conversion phenomena can be prevented in CACs. Based on this insight, X‐AFm formation (in this case, X = NO3‐), provoked by the sequential addition of calcium nitrate (CN) additives is identified as a means of preventing conversion. A multi‐method approach comprising x‐ray diffraction, thermal analytics, and evaluations of the mechanical properties is used to characterize solid phase balances and evolutions of strength for systems cured at 25°C and 45°C. The results highlight the absence of the C3AH6 (hydrogarnet) phase across all systems and curing conditions considered, with no decrease in strength being noted when CN additives are used. The experimental outcomes are supported by insights gained from thermodynamic simulations which highlight thermodynamic selectivity as means of regulating and controlling the evolutions of phase balances: using inorganic salts in CACs, and more generally in cementitious material systems. 27
SPD 2014 SESSION ONE 83 Acetylcholine Mediates Cue Stimulated Reward Seeking Action in Nucleus Accumbens Core YIQIONG XU, Anne Collins and Kate Wassum External cues that signal rewards can trigger reward‐seeking actions. For instance, needles invigorate an addicts' drug seeking actions because they associate needles with pleasure. Neurotransmitters such as dopamine and acetylcholine (ACh) are involved in decision making and reward seeking behavior. Understanding the neural mechanisms of these will aid the development of new therapeutic methods for compulsive addiction disorders. We hypothesize that ACh in the nucleus accumbens core mediates Pavlovian to instrument transfer (PIT), the ability of reward‐paired cues to invigorate a reward‐seeking action response. Rats were trained in behavioral operant chambers equipped with a generator that produces either a tone or white noise, and a photo beam that detects when the rat enters for food. The rats were then given a series of PIT tests to evaluate their cue‐triggered responses. Prior to the PIT test, the animals were injected with a cholinergic receptor antagonist. The data suggests that after the muscarinic receptor was antagonized, the cue invigorated actions decreased, while nicotinic receptor antagonists did not change PIT significantly in the nucleus accumbens. The results have revealed that in the nucleus accumbens core, acetylcholine mediates PIT through muscarinic receptors. Future experiments will be done to show that acetylcholine antagonists can be beneficial to counteract addictive behavior and may be used as a therapeutic method to alleviate addiction problems. 84 Alternative Precursors to Q Biosynthesis in Escherichia coli EMILY WENG, Letian Xie, and Catherine F. Clarke Coenzyme Q is an electron carrier that transports electrons from complexes I and II, to complex III, in the electron transport chain. In addition to this essential role, coenzyme Q (also known as ubiquinone or Q) is also an antioxidant. Consequently, it has been a subject of interest as a nutritional supplement. However, Q is also involved in the generation of reactive oxygen species, therefore characterization of the polypeptides involved in Q biosynthesis may aid the development of Q supplements for human use. The pathway of Q biosynthesis (which requires at least eleven genes, COQ1‐9, ARH1 and YAH1) is not completely known, and it has been found that distinct intermediates accumulate in Saccharomyce cerevisiae when compared to Escherichia coli. The proposed pathways in S. cerevisiae and E. coli diverge after the formation of 3‐polyprenyl‐4‐hydroxybenzoic acid and are thought to converge after 2‐
polyprenyl‐6‐methoxy‐phenol. More recently, it has been shown that para‐amino benzoic acid (pABA) acts as a precursor to Q biosynthesis in S. cerevisiae. To investigate pABA's function in Q biosynthesis in E. coli, we labeled various strains of E. coli using 12C‐pABA, 13C6‐pABA, and 99% 13C6‐pABA, 4 hydroxy benzoic acid (4HB), resveratrol, and analyzed using LC/MS/MS. We postulated that in wild type E. coli, 13C6‐pABA can be incorporated into Q. Experimental results yielded a range of results, revealing novel intermediates and elucidating information on intermediate conversion between the 4 HB and pABA pathway to Q. 85 Progress Toward the Synthesis of Bicyclo[3.1.0] Fluorinated Thymidine Analogues DAVID W. SHIA and Michael E. Jung Nucleoside analogues are a promising class of antiviral and anticancer agents that have recently been considered as candidates in antisense oligonucleotide therapy. The bicyclo[3.1.0]hexane thymidine analogue has been shown to adopt a conformation that closely mimics that of naturally occurring thymidine. Furthermore, incorporation of the analogue in oligonucleotide strands has been shown to increase the melting temperature of the heteroduplex, suggesting an increased thermodynamic stability. However, the properties of a fluorinated bicyclo[3.1.0] nucleoside analogue have not yet been explored. Fluorination at the C3 position on the bicycle presents an interesting challenge that involves the selective protection of one secondary alcohol over the other. Although certain silyl compounds have been shown to give selective protection of the 3' and 5' hydroxyl groups, the subsequent fluorination proves difficult due to the fluoride mediated silyl deprotection that has been found to occur over the desired fluorination. Thus, to achieve the desired fluorinated product, either a fluoride resistant selective protection or the selective activation of the C3 hydroxyl group must be achieved. Here, we present the work that has been done toward the synthesis of the bicyclo[3.1.0] fluorinated thymidine analogue from the commercially available starting material, D‐ribose, through a key Grubb's ring closing metathesis followed by an alcohol directed Simmons‐Smith cyclopropanation. 28
SPD 2014 SESSION ONE 86 Beta Adrenergic Receptors in a Diffuse Axonal Injury Model of Head Trauma BRANDON K. NGUYEN, Alex W. Lamb, and Eric J. Ley Beta adrenergic receptor (BAR) activity alters the related immune deficiency that occurs after traumatic brain injury (TBI). How BAR affects neuronal cells that are exposed to environments that mimic TBI, including oxygen glucose deprivation (OGD) or mechanical stretch that mimics diffuse axonal injury (DAI) is unknown. The aim of this study was to expose a neuronal cell culture to BAR agonist Isoproterenol (ISO) and/or antagonist Propranolol (PROP) before and after OGD and stretch injury to determine how BAR alter neuronal cell cytotoxicity in TBI. Immortalized mouse hippocampal cell line, HT‐22, and primary mouse cortical cultures underwent OGD and were given hypoxic media and immediately placed in an anoxic incubator for 2 hours. DAI was performed by injecting 7PSI into the wells to provide stretch injury. Cytotoxicity was assessed using a Lactate Dehydrogenase assay. We found that the beta adrenergic receptor is seen to be highly involved in the secondary injury in both OGD and cellular stretch models of traumatic brain injury. The use of beta adrenergic receptor agonists and antagonists can modulate different cellular responses that are seen in each injury model. Interestingly, ISO is seen to have variable impact after DAI, which is not reversible by addition of PROP. Further studies on the mechanism behind adrenergic agonist and antagonist behavior in these models are warranted based on these findings. 87 Characterization of BarH‐like, SIX3, ASC‐like, and Lox in Strongylocentrotus pururatus Embryogenesis ZACK COHEN, KATHERINE LI, ALEX MACEDO, TIEN PHAN, Daniel Malkin, and Pei Yun Lee Genomic conservation between Strongylocentrotus purpuratus (purple sea urchin) and other deuterostomes may provide insights into early development in humans. Using S. purpuratus as a model organism, we combined bioinformatical analysis, gene cloning, RT‐PCR, and whole mount in situ hybridization to identify and characterize the temporal and spatial expression patterns of BarH‐like, SIX3, Achaete‐scute‐like, and Lox at up to 72 hours post‐
fertilization. BarH‐like and Achaete‐Scute‐like were not expressed in developing embryos at the time points examined. SIX3 expression localized to the periphery of the animal pole, the tip of the gut, and the mouth in the blastula, gastrula, and pluteus larva stages, respectively. Lox was restricted to the hindgut in the gastrula and pluteus stages. These data suggested that SIX3 and Lox play roles in the development of the sea urchin gut. The expression profiles of the genes provide essential preliminary information required for an understanding of their roles in development. Studies of S. purpuratus gene functions during embryogenesis will have implications for mechanistic studies of cell specification during early development in humans. 88 Isolation of Amyloid Beta‐Protein Oligomers JOSEPH L. CONOVALOFF, Eric Y. Hayden and David B. Teplow Amyloid beta‐protein (Abeta) forms fibrils in the brain that were believed to be the direct cause of Alzheimer's disease (AD). However, evidence supports the 'oligomer cascade hypothesis," which posits that oligomers may be the proximate pathologic agents. We have used rapid, zero‐length, in situ chemical cross‐linking (PICUP; photo‐induced cross‐linking of unmodified proteins) to stabilize the oligomer states, enabling the isolation and study of pure Abeta40 oligomers of specific size (number of monomers). However oligomers of the more neurotoxic isoform of Abeta, Abeta42, have been difficult to purify using the same procedures used for Abeta40. We reported that Abeta42 containing the amino acid substitutions Phe10 and Tyr42 behaves similarly to wild‐type Abeta42. We now have isolated [F10,Y42]Abeta42 oligomers that remain stable for characterization studies. In this method, the cross‐linked protein was fractionated by two sequential SDS‐PAGE (gel electrophoresis) steps, and the resulting oligomer bands were excised and electro‐eluted. We report that the addition of 25% (v/v) dimethyl sulfoxide to the cross‐linked protein, as well as 6M urea to the second SDS‐PAGE, improved the purity of the isolated oligomer populations. The pure oligomer populations were dialyzed against 0.1% TFA and lyophilized. We now will use these pure lyophilizates for biophysical and biochemical study. Importantly, our procedure for isolating oligomers could be of use in other diseases, e.g., Huntington's, Parkinson's, and Lou Gehrig's diseases. 29
SPD 2014 SESSION ONE 89 Changes in Craving, Withdrawal and Affective State During a Laboratory Smoking Lapse Task REJOYCE GREEN, Spencer Bujarksi, Jenessa Shapiro, and Lara A. Ray Smoking is a leading source of death and disability. Previous studies have shown craving and withdrawal, characterized by negative affect, to maintain smoking behavior. Less is known about the direct effects of smoking on positive mood. We examined changes in craving, withdrawal, and positive and negative mood in participants who completed a laboratory smoking lapse task with incentives to delay and reduce smoking following twelve hours of abstinence (n=77). We hypothesized that participants who smoke more cigarettes during the task will have greater reductions in craving, withdrawal, and negative mood and greater increases in positive mood as compared to baseline. Consistent with our hypotheses, regression analyses revealed the number of cigarettes smoked during the task to be associated with greater reductions in craving (p < 0.001), withdrawal (p < .001), and negative mood (p = 0.05). No significant effect on positive mood (p = .44) was observed. This study suggests that acute reductions in craving, withdrawal and negative mood during a quit attempt may serve to perpetuate smoking lapse/relapse whereas changes in positive mood may be less influential. 90 Bipolar and Substance Use Disorder Comorbidity: Impact on Trait Impulsivity and Reinforcement Learning LAP‐WOON KEUNG, Eliza Congdon, Terri Teshiba, Dara Ghahremani, Joseph Ventura, Edythe London, Russell Poldrack, and Nelson Freimer Substance use disorders are highly prevalent in patients with Bipolar Disorder (BP), and while this comorbidity predicts poor outcome, the mechanisms underlying this comorbidity are unknown. Some previous studies have shown increased trait impulsivity and poorer neurocognitive function in BP patients with a history of substance use disorder (SUD) comorbidity, but others have failed to demonstrate a difference between BP with and without history of SUD. To address this discrepancy, we analyzed an existing dataset, including 56 BP patients and 1137 healthy controls with and without a past diagnosis of SUD. Our hypothesis was that trait impulsivity and performance on a probabilistic reversal learning task (PRLT), which measures the ability to learn from positive and negative feedback, would be sensitive to BP‐SUD comorbidity. In terms of trait impulsivity, there was an effect of both BP and SUD diagnoses, such that individuals with either diagnosis had higher trait impulsivity than individuals without diagnoses. In terms of PRLT performance, there was an interaction between BP and SUD, such that BP patients with a past diagnosis of SUD performed worse than all other participants. Although limited in sample size, our findings lend resolution to which aspects of neurocognitive function are sensitive to BP‐SUD comorbidity. Elucidation of the mechanisms that underlie this comorbidity may help to identify risk factors, and identify a subgroup of patients that is more homogenous and therefore more suitable for genetic investigation.
91 Conditional Knockout of CCL2 from Astrocytes Ameliorates Experimental Autoimmune Encephalomyelitis in Mice ALEXANDRIA HOFFMAN, Roy Kim, Noriko Itoh, Rory Spence, Micheal Sofroniew, and Rhonda Voskuhl Multiple sclerosis (MS) is a degenerative auto‐immune disorder estimated to affect 2.3 million people worldwide. The disease is characterized by inflammation and the loss of protective myelin sheaths in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is the animal model for (MS). Chemokine (C‐C motif) ligand (CCL2) is associated with the recruitment of immune cells to the CNS during EAE. Previous studies using universal CCL2 knockouts have demonstrated its importance in the initial and effector phase of the disease. In an effort to better understand the role of CCL2 we generated a conditional knockout using CCL2 floxed mice and astrocyte specific Cre mice. These mice, along with their wild‐type littermates were induced with EAE and monitored for clinical disease symptoms and further examined for the degree of demyelination in the CNS. In the conditional CCL2 knockout we saw a significant amelioration of symptoms late in EAE compared to their littermate controls. Additionally, we found a markedly reduced loss of myelin and axons in the CNS of the conditional knockouts. We also saw a less diffuse activation of reactive astrocytes and microglial cells in white and gray matter of the CNS. The lack of CCL2 from astrocytes protects the CNS by reducing the infiltration of immune cells and reducing diffuse activation of astrocytes and microglia cells in the CNS which leads to protection of myelin axons. In conclusion, the expression of CCL2 from astrocytes plays a key role in ameliorating late EAE. 30
SPD 2014 SESSION ONE 92 The Search for Self‐Avoidance: Identifying the Role of DSCAMB in Mediating Self‐Avoidance in Zebrafish GERARDO A. TELLEZ, Donald P. Julien and Alvaro Sagasti The somatosensory system in zebrafish is an elaborate system of sensory neurons that innervate the skin with highly branched arbors. The non‐overlap among these neurons is termed self‐avoidance, and it optimizes covering of a defined territory. This phenomenon has been studied extensively in Drosophila, where cell adhesion molecules have been found to be mediators of this process. Specifically, the Down Syndrome Cell Adhesion Molecule (DSCAM) has been implicated in playing a key role in mediating self‐avoidance in Drosophila. This project will investigate the role of DSCAMB by looking at the phenotype of DSCAMB null mutants and analyzing whether it does play a role in sensory neuron self‐avoidance. 93 Optic Nerve Crush Dramatically Reduces Behavioral Light Aversion but Light Perception is Retained in Mice JASON KESSLER, Anna Matynia, Sachin Parikh, Allen Rodriguez, Nicholas Brecha, and Michael Gorin People suffering from traumatic brain injuries or migraines, even visually blind patients, can experience photoallodynia, an abnormal, painful hypersensitivity to low light intensities. In severe cases, these patients often live in extreme social isolation to minimize light‐induced chronic pain. Previous results show that melanopsin‐expressing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) preferentially survive optic nerve crush and mediate innate light aversion. Here, the relationship between ipRGCs and light aversion was investigated using optic nerve crush in mice. Firm bilateral crush was performed to block light transmission to downstream visual brain regions, resulting in blindness. Immediately following optic nerve crush, mice were subjected to a behavioral assay, revealing dramatically reduced light aversion. Over the following four weeks, mice demonstrated a complete lack of light aversion, indicative of the loss of visual function. However, a non optic nerve, light‐detecting pathway is implicated by significantly retained light aversion in mice treated with morphine or nitroglycerin, both of which are known to sensitize trigeminal ganglion neurons. Recently identified melanopsin‐expressing trigeminal ganglion cells may be an alternative light‐
sensing neurocircuit mediating this behavior. Ongoing experiments will assess functional activation of melanopsinergic trigeminal ganglion cells to investigate this hypothesis. 94 Generation of Paramyxovirus Matrix Interactomes by Comparative Proteomics to Interrogate the Molecular Basis and Significance of Matrix Nuclear‐Cytoplasmic Trafficking TIM VOROS, Mickey Pentecost, Ajay Vashisht, Arnold Park, James A. Wohlschlegel, and Benhur Lee Paramyxoviruses (PVs) are (‐) sense RNA viruses that replicate in the cytoplasm and bud from the plasma membrane. Matrix (M) is the major structural protein of PVs involved in viral morphogenesis and budding at the plasma membrane. Curiously, several PV matrix proteins are reported to localize to the nucleus, although the biological significance of matrix nuclear localization is unknown. We previously showed that the nuclear localization of Nipah virus M is regulated by its monoubiquitination. To shed light on the biological function of matrix nuclear localization, we performed a comparative proteomics screen to identify the cellular interacting partners of 7 M proteins representing PVs from all five genera. We generated inducible cell lines expressing 3X‐Flag‐tagged M from Nipah, Hendra, Ghana (GH‐M74a), Sendai, hPIV2, NDV, and Measles virus. Lysates were subjected to anti‐Flag immunoprecipitation and M‐interactomes were generated by 2 dimensional liquid chromatography followed by tandem mass spectrometry. Within the interactomes we identified numerous enzymes involved in ubiquitin conjugation (e.g. Cullin ring ligases), nuclear import/export factors (Importin‐A, B, CRM1, and Nuclear pore components). Unexpectedly, matrix interactomes were also enriched in factors that regulate mRNA transport, processing, and stability. Based on bioinformatics and preliminary functional analysis, we hypothesize that M nuclear trafficking is a virulence mechanism that promotes viral replication by interfering with host gene expression. 31
SPD 2014 SESSION ONE 95 Antibody Conjugated Vault Nanoparticles Show Increased Incorporation in Mouse Lung Epithelium KUSH V. BHATT, John S. Tran, Benjamin Lopez, Valerie A. Kickhoefer, Leonard H. Rome, and Vedang A. Londhe Vault nanoparticles were discovered in 1986 by Leonard Rome of UCLA. Nanoparticles are described as endogenous ribonucleoprotein particles with unknown function. Moreover, vault nanoparticles have drawn attention for potential delivery of synthetic molecules. Our study investigated the ability of nanoparticles to enter Mouse Lung Epithelium (MLE 15) cells and whether conjugating nanoparticles with Anti‐EGFR antibody could affect incorporation. We hypothesized that conjugating nanoparticles with antibody would enhance incorporation. MLE 15 cells were grown in DMEM media at 37 degrees. Cells were exposed to anti‐EGFR tagged with GFP to confirm expression of EGFR, then exposed to nanoparticles with and without anti‐EGFR. Image analysis for quantification was done using immunofluorescence microscopy and ImageJ software. Total RNA was extracted from cells and used to measure CXCL1 mRNA expression levels. EGF receptor expression was confirmed on MLE 15 cells. Recombinant vault nanoparticle (CP‐
MVP‐z/INT‐m‐Cherry) uptake was noted in MLE 15 cells and nanoparticles conjugated with Anti‐EGFR incorporated at higher rate than nanoparticles without antibody (p < 0.01). Moreover, vault nanoparticles elicited robust expression of CXCL1 (23 fold), which was attenuated when nanoparticles were coupled with Anti‐EGFR antibody (<2 fold). Nanoparticles conjugated with anti‐EGFR antibody show higher levels of incorporation in epithelial cells. 96 RNA Sequencing of Adipose Tissue Identifies Genome‐Wide Regulation of Gene Expression in Metabolic Disease MARCUS ALVAREZ, Linga Reddy MV Prasad, Rita M Cantor, Arthur Ko, Elina Nikkola, Johanna Kuusisto, Aldons J. Lusis, Markku Laakso, and Paivi Pajukanta Cardiovascular disease (CVD) is a major cause of death worldwide. One major risk factor for CVD and related metabolic disorders is triglyceride (TG) levels, which is regulated by adipose tissue. We hypothesize that investigating genome‐
wide genetic and transcriptome signatures of adipose tissue will reveal novel variant‐specific mechanisms contributing to TG levels in human. To investigate our hypothesis, we analyzed genome, transcriptome, and phenotype data from 598 Finnish males from the metabolic syndrome in men (METSIM) cohort. We obtained about 8 million DNA variants at a threshold of 5 % minor allele frequency (MAF). Adipose RNA was sequenced and reads were mapped to the human reference genome. Exons, transcripts, and genes were quantified from read counts and normalized. TG levels, body mass index (BMI), and insulin levels were regressed on normalized gene expression data, and significantly associated genes. Then linear regression was used to look for DNA variants affecting expression levels of significantly associated genes. To increase our power to find regulatory variants, we also implemented allele specific expression (ASE) analysis. We predict to find several novel variants and genes involved in CVD and metabolic disease that will help elucidate new mechanisms in adipose tissue that contribute to the development of risk factors for disease. Our future studies are targeted to validate these findings and identify the specific pathways and mechanisms by which these variants and genes act in cardiometabolic disease.
97 TSLP and CSF‐1 Regulate Post‐Stroke Neurogenesis ANANT S. RANDHAWA, Andrew J. Brumm, and Thomas S. Carmichael Ischemic stroke causes tissue damage in the brain. This induces the proliferation of neural progenitor cells (NPCs) in the subventricular zone (SVZ) and signal for them to migrate out to a site adjacent to the site of stroke, commonly referred to as the peri‐infarct cortex. By using a mouse model of distal middle cerebral artery occlusion, we have shown that SVZ‐derived neuroblasts (immature neurons) preferentially migrate to angiogenic vasculature in the peri‐
infarct cortex. We developed a set of candidate vessel‐neuroblast signaling interactions through cell‐specific whole genome expression profiling at 7d, post stroke. Data from these interactions identified TSLP and it's receptor (TSLPR/IL‐7R‐alpha) and CSF‐1 and it's receptor (CSF‐1R) as novel ligand‐receptor systems that are upregulated after stroke. TSLP and CSF‐1 ligands are upregulated in peri‐infarct angiogenic blood vessels and their respective receptors are upregulated in stroke‐responsive neuroblasts. In vivo studies of TSLP and CSF‐1 knockdown (loss‐of‐function) assays show a decrease in the number of stroke‐responsive neuroblasts within the peri‐infarct cortex vs. control at 14d after middle cerebral artery occlusion (MCAO). We used a non‐cell‐specific EF1 promoter to drive micro RNA (miR) constructs to knockdown the expression of TSLP and CSF‐1 in peri‐infarct tissue. These results tell us that TSLP and CSF‐1 are involved in driving the proliferation and differentiation of SVZ‐derived NPCs by modulating neurogenesis after stroke. 32
SPD 2014 SESSION ONE 98 Modulation of Dopamine Receptors Induces Behavioral Changes in Zebrafish BINH K. NGUYEN, Lisa Barnhill, and Jeff Bronstein Parkinson's disease is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra of the basal ganglia, which leads to impairments in motor function and eventual cognitive and memory deficits. The primary neurotransmitter of the basal ganglia is dopamine. Dopamine is therefore important in motor function and we are attempting to utilize a zebrafish model system to screen chemicals for their ability to modulate the dopamine pathway. To establish a screen for dopamine behavioral effects in zebrafish, we are using apomorphine, a fast‐acting dopamine receptor agonist, and haloperidol, a dopamine receptor inhibitor, as proof‐of‐concept controls. These drugs were administered to 7‐day old zebrafish (n = 12 per condition) at various incubation times, and subsequent behavioral changes, such as swimming distance and speed, were monitored. Apomorphine, at 1 hour post‐treatment, significantly increased large distance movements (> 2 mm) at a concentration of 1 uM relative to DMSO controls, whereas haloperidol, at 4 hours post‐treatment, significantly decreased large distance movements at a dose of 5 uM. These results suggest that modulating dopamine receptor activity can impact zebrafish behavior. In the future, we hope to use this method to screen zebrafish treated with chemicals that may interfere with the dopamine pathway. These control treatments can then be used to try and rescue behavioral changes in treated fish. 99 Identifying Key Components of Mycobacterial Cell Wall That Promote NOD2 Activation PHUONGANH M. LE, Mirjam Schenk and Robert L. Modlin Nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2) is an intracellular pattern recognition receptor for muramyl dipeptide (MDP), which is part of the mycobacterial cell wall that confers adjuvant activity for inducing B and T cell responses. We recently identified that in leprosy, NOD2 activation induced an IL‐32‐dependent dendritic cell (DC) differentiation program, crucial for host defense against microbial infection. Our goal is to identify fractions of M. leprae MDP that induce a strong NOD2 response. In order to address this, we cultured HEK‐Blue NOD2 cells, which is a human cell line co‐transfected with the NOD2 gene and the secreted alkaline phosphatase (SEAP) reporter gene under the control of the NF‐kB promoter. We measured activation of NOD2 in these cells by the addition of HEK‐blue SEAP substrate medium. Collaborators provided us with fractionated MDP that we tested for the amount of NOD2 activation using the HEK‐Blue NOD2 reporter as well as cytokine induction in human monocytes. We identified two candidate fractions (F17 and F18) that showed significant activation of NOD2, which will be further investigated. These experiments should contribute to potential therapeutic strategies against infectious disease by targeting the activation of NOD2, which is crucial for DC differentiation and adjuvant activity. 100 Small Intestine is a Major Organ Affecting Systemic Inflammation in Models of Atherosclerosis and Vascular Dysfunction LEYLA GHAFFARI, Sepideh Shakeri, Zarina Barseghyan, Maryam Shabihkhani, Nika Karimi, Roshanak Alialy, Zohreh Takallou, M. Reza Rostami, Nasim Pourtabatabaei, Greg Hough, and Navab, Mohamad Introduction. Being the site of digestion, modifications, reassembly and absorption of hundreds of vital compounds, the intestine plays a pivotal role in the organism’s survival. In addition the excretion and elimination of the toxic waste from peripheral tissues that arrives through the liver and bile is left to intestine. Objective. Our group sought to determine the role of HDL mimetic peptides with high affinity for lipid mediators of inflammation in modulating systemic inflammation. Methods. An animal model of inflammation and atherosclerosis, LDL receptor deficient mouse was maintained on a high fat‐high cholesterol diet for two weeks. One group received a) the laboratory rodent chow diet b) a second group was provided with the HDL mimetic peptide 5F in the diet at 200 ug per 4 gr food, c) the third group was administered 50 ug per day of the peptide by subcutaneous injection. After two weeks the mice were fasted overnight, they were anesthetized, blood was removed from retro orbital sinus, plasma was prepared and cryopreserved at ‐80 degrees centigrade. Using liquid chromatography and electrospray ionization mass spectrometry we determined the levels of 5F and the proinflammatory HETEs & HODEs and oxidized phospholipids in plasma. Results. Our group observed that administering the HDL mimetic peptide reduced the level of proinflammatory molecules (p<0.015) regardless of the route of administration despite that the circulation concentration of the peptide was orders of magnitude higher (p<0.0001) when injected. Conclusion. Our group found that the intestine has a major l
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SPD 2014 SESSION ONE 101 Non‐cell Autonomous Inhibitory Effect of Cystatin E/M SANDHIYA RAVICHANDRAN, Natarajan Venkatesan, and Eri S. Srivatsan Cervical cancer is the second most common cancer among women around the world. Although human papilloma virus (HPV) has been associated with cervical cancer, the infection alone is insufficient for tumor development. The inactivation of cystatin E/M, a protease inhibitor, has been observed in many cancers and has been showed to be related to the development of cancer stem cells. Our study focuses on both the non‐cell autonomous and intracellular mechanism of tumor cell growth inhibition. Although we have observed non‐cell autonomous growth inhibition with cystatin E/M containing supernatant, we did not observe growth inhibition with the purified protein. It is likely that the protein is inactivated during the fractionation procedure. Our studies have also shown that re‐expression of cystatin E/M results in the downregulation of NFKB‐driven cytokines, IL‐10 and MMP‐9. It is also known that cisplatin resistance leads to NFKB activation. We have created cisplatin resistant cells from the parental sensitive CST6 cells, a cervical cancer cell line. We confirmed cisplatin resistance with increased presence of cyclin D1, by western blotting. Induction of cystatin E/M resulted in cell growth inhibition so we hypothesize that cystatin E/M is binding to NFKB and decreasing its transcriptional activity. Both cytoplasmic and nuclear fractions will be investigated to identify the molecular mechanism of cystatin E/M‐driven growth suppression. 102 Characterizing the Mitochondrial Proteome in Relation to Genome, Function, and Disease JESSICA LEE, Amanda J. Lin, Caitlin M. Black, Derrick Huang, Natalie Hernandez, Jeong H. Choi, Jun Zhang, and Peipei Ping Mitochondria are responsible for crucial life‐sustaining processes, such as energy production, metabolic signaling regulations, and apoptotic initiation. Mitochondrial dysfunction has been implicated in numerous complex diseases, from Alzheimer's and Parkinson's diseases to cancers and cardiomyopathies. To understand the mechanisms driving mitochondrial function and dysfunction, our lab had previously examined four model systems ”mouse heart, mouse liver, human heart, and drosophila”to characterize relationships between mitochondrial proteome features, biological function, and disease. Our present study expands the scope of our research by combining proteomic and genomic perspectives and employing bioinformatics analysis to investigate whether correlations also exist between chromosome origin, biological function, and disease, and how they differ between different organs and organisms. We expect patterns in protein expression and proteins abundances to implicate candidate chromosomes as 'hotspots’ for specific functional clusters and diseases. Comparison of mouse liver and heart mitochondrial proteomes reveals significant variability in protein abundance, indicating the importance of regulatory and environmental involvement in organ function. These findings can then inform experimental model selection for studying and treating complex diseases. 103 GILZ Regulates Hematopoietic Stem Cell Self‐renewal TONATIUH MONTOYA, Vincenzo Calvanese and Hanna K.A. Mikkola. Hematopoietic stem cells (HSC) establish, maintain and reconstitute the entire blood system upon transplantation. Attempts to generate functional HSCs from pluripotent cells have yielded cells of limited self‐renewal ability, with no therapeutic use. Understanding basic HSC biology will bring us closer to generating functional HSC. To identify potential self‐renewal regulators, we used microarray analysis comparing the transcriptomes of CD34+CD38‐
CD90+GPI80+ human fetal liver cells, a self‐renewing HSC enriched population, to CD34+CD38‐CD90+GPI80‐ cells, composed of closely related, but non‐self‐renewing progenitors. Glucocorticoid‐induced leucine zipper (GILZ) was among the most differentially expressed genes uncovered by this analysis. As GILZ has been shown to regulate apoptosis in other stem cells, and since apoptosis regulation is key for self‐renewal, we hypothesized a role for GILZ in maintaining self‐renewal by regulating HSC stress. To test this hypothesis, we overexpressed GILZ in HSC and observed a higher proportion of HSC‐enriched CD34+CD38‐CD90+ cells after culture, suggesting that GILZ can enhance self‐
renewal. Furthermore, shRNA knockdown of GILZ in HSC resulted in loss of CD34+CD38‐CD90+GPI80+ cells, revealing an essential role for GILZ in self‐renewal. These findings call for further investigation into the mechanism by which GILZ regulates HSC function, and will enable us to improve strategies to generate functional HSC. 34
SPD 2014 SESSION ONE 104 Elucidating a Novel Vitamin C Synthesis Pathway in Caenorhabditis Elegans MARIA E. PEDRAZA, Alexander N. Patananan and Steven G. Clarke Vitamin C is an important antioxidant that neutralizes free radicals in cells, participates in a variety of enzymatic reactions, and protects against human diseases. The three main vitamers of vitamin C are dehydroascorbate, D‐
isoascorbate, and the most common L‐ascorbate. Much is known about the important roles ascorbate plays in vertebrates, yet little is known about its presence in invertebrates. Caenorhabditis elegans, a common invertebrate model organism, has several enzymes whose homologs in vertebrate organisms require ascorbate. Therefore, we investigated if ascorbate is present in C. elegans by normal and reverse phase high‐performance liquid chromatography (HPLC) and gas chromatography‐mass spectrometry (GC‐MS). Interestingly, we detected the presence of ascorbate in eggs, L1 larvae, mixed worms, and gravid adults despite C. elegans lacking many of the enzymes plants and animals require for ascorbate biosynthesis. Furthermore, we incubated intact worms with various precursors known to be necessary for ascorbate biosynthesis in other organisms, but none increased the amount of ascorbate in C. elegans, suggesting a novel pathway. The presence of ascorbate in C. elegans is a first for invertebrate organisms, and we are currently elucidating the biochemical pathways for the biosynthesis of this molecule. Finding a novel pathway in C. elegans will broaden our understanding of vitamin C synthesis and its importance to organisms, as well as answering questions about the evolutionary patterns of ascorbate synthesis. 105 The Role of Climate Change in Mass Extinctions: Using Stromatolites to Constrain Temperatures During Times of Biotic Crisis ROBERT GAMMERIELLO, Yadira Ibarra, Carlie Pietsch, Victoria Petryshyn, and Aradhna Tripati Stromatolites are laminated sedimentary structures that are commonly thought to be created by cyanobacteria, either through the trapping and binding of sediment, or through metabolically‐induced precipitation. Stromatolites first appear in the fossil record ~3.8 Ga. The occurrence of stromatolites in the fossil record decreases before the Precambrian‐Cambrian boundary, and strikingly they subsequently only re‐emerge in abundance at times of biotic crisis, such as the Permian‐Triassic and Triassic‐Jurassic extinctions. Both of these mass extinctions are coincident with large scale volcanism. Associated increases in atmospheric CO2 caused increases in ocean acidity and increases in global temperatures, and these environmental perturbations are hypothesized to have been key drivers of mass extinction, along with anoxia and euxinia. In order to understand the role of climate change in mass extinctions, we are estimating temperatures across both boundaries using clumped isotope analyses of stromatolites. Samples were collected from the Lower Triassic Virgin Limestone (Nevada) and the Cotham Marble (Triassic‐Jurassic boundary United Kingdom). Subsequent to petrographic analysis, samples were micro‐drilled to obtain primary phases. Our analysis has yielded plausible temperatures for the Triassic‐Jurassic stromatolites (24.6 to 44.2 °C) suggesting warmer ocean waters. Temperatures for the Permian‐Triassic stromatolites range from 87.9 to 122.4 °C, indicating diagenetic mixing with the primary, biogenic carbonate. 106 K‐Selected Ungulate Species Are Less Likely to Suddenly Die in Stressful Situations SAJAN SHAH, SHANE PATEL, Barbara Natterson‐Horowitz, and Daniel T. Blumstein Animals (including humans) may suddenly die of heart failure when in stressful conditions. To gain insights into human sudden cardiac death, we studied capture myopathy in ungulates. Of the 306 species of ungulates, 58 species have had reports of sudden cardiac death associated with escape, capture, or handling. We conducted a series of phylogenetic analyses to identify life history correlates of sudden death. We identified an r/K syndrome whereby K‐
selected species were less likely to suddenly die in stressful situations. This result provides the basis for future studies in humans that directly examines the effect of r/K life‐course trajectories on sudden cardiac death. 35
SPD 2014 SESSION ONE 107 Creating a High Yield Method for Quantification of Lipid Metabolism in Rapidly Dividing Cells AMY K. YU, ERIC S. WANG, Joseph P. Argus, Joo Hee Sohn, Kevin J. Williams, Moses Q. Wilks and Steven J. Bensinger
Rapidly‐dividing cells require higher quantities of lipids to sustain growth and proliferation. Understanding the amount and source of cellular lipids could shed light on ways to support or hinder division. However, our previous methods of analyzing and quantifying lipids in cellular samples were impure and limited in their accuracy, scope, and efficiency. In particular, these methods were unable to quantify the lipids in primary cells. Here, we developed a new high‐yield, efficient method of quantifying and analyzing lipids in cells and tissue using GCMS. This method effectively analyzes sterols and 12‐24 carbon fatty acids derived from a wide range of lipid classes. It uses an efficient acid methanolysis procedure that displays significantly higher yields than previous methods. Furthermore, it is compatible with the use of 13C to analyze lipogenesis pathways. Our new high‐yield method allows for analysis of lipid metabolism in a variety of normal and pathologic states. 108 Social Environmental Influences on Infants' Face Preferences YOSELIN GUTIERREZ, and YOSELIN CORDON Preference for own race faces begins to emerge around 3 months and may be shaped by social environments. The mechanisms driving development of face preferences can provide insight into the influence of social environments on the development of face processing and stereotyping. We are extending the findings of a previous study that presented upright faces to infants using the same method as the present study. Latino and Caucasian infants 9‐ to 12‐
months old are presented with side‐by‐side inverted female Caucasian, Latina, and African American faces. Eye movements are recorded by an eye tracker, and longer looking suggests visual preference. Current results show significantly more looking to African American than Latino (t=2.19, p=.043) and Caucasian faces (t=3.23, p=.005) and significantly more looking to Latino than Caucasian (t=3.04, p=.007) faces regardless of infant ethnicity. These results are consistent with results of looking to upright faces, and suggests that infants might process faces less holistically than adults. We are testing more infants and conducting eye movement scan path analyses to explore mechanisms of development of same and other race face perception. 109 Mechanism of Transthyretin Stabilization JOSHUA A. CHOU, Lorena Saelices, and David S. Eisenberg Transthyretin (TTR) is a tetrameric protein that when dissociated, aggregates causing Transthyretin Amyloidosis. A TTR variant with a methionine residue at the 119 position (T119M) has been shown by previous studies to be nonpathogenic compared to wild‐type TTR. T119M TTR when combined in cis or trans with the pathogenic TTR V30M is also protective. Residue T119 is located in the central TTR hydrophobic pocket, shown to be the target site for stabilizing drugs. In order to uncover the mechanism of prevention of TTR aggregation, we created various TTR mutants. Aggregation assays were performed on wild‐type and mutant TTR, quantifying TTR aggregation by measuring turbidity and following protein concentration of the insoluble fraction of the samples. Mutations to methionine, tryptophan, or tyrosine at position 119 showed no signs of aggregation. We also mutated various residues of the hydrophobic pocket and found that 108W and 15W mutants also do not aggregate. Our results indicate that these bulky, hydrophobic amino acids stabilize the hydrophobic pocket, preventing TTR from dissociation and aggregation. Further experiments will design small peptides to target this pocket to prevent TTR aggregation, as potential drugs against TTR Amyloidosis. 36
SPD 2014 SESSION ONE 110 Identification of Morgana Protein Interactors Important in Cell Division ELY CONTRERAS, Ankur A. Golkar, and Jorge Torres Cancer is a disease caused by abnormal cells with the capacity to undergo many cycles of cell division. Understanding cell division and more specifically how different proteins coordinate the formation of the mitotic spindle, is necessary because inhibition of spindle function may be a potential way to stop the proliferation of cancer cells. Morgana is a protein involved in the assembly of the mitotic spindle and has been linked in centrosome amplification. Centrosome amplification increases errors in chromosome segregation and can potentially give rise to malignant cells. Morgana has been shown to have protein‐protein interactions with Hsp90, ROCKI and ROCKII, yet very little is known about their role during mitosis. This study aims to further characterize Morgana by identifying the proteins that interact with it during mitosis and to determine their role and Morgana's in cell division. To identify Morgana interactors, a stable cell line expressing LAP‐tagged (Localization and Affinity Purification tagged) Morgana was generated. The LAP‐tagged protein complexes were purified by performing tandem‐affinity purifications. The identity of the protein complexes were determined by mass spectrometry. From the mass spectrometry analysis I identified Morgana's known interactor Hsp90, along with new interacting proteins BUB3 and MAP 1B. The new interactors are involved in the assembly of the mitotic spindle and suggest Morgana has an essential role in promoting proper cell division, important for understanding cancer progression. 111 Decoupling Subjective Heaviness and Density in Humans' Judgments of Object Value JEANETTE ZHU, Megan A. K. Peters, and Ladan Shams, Ph.D. An object's weight can heavily influence humans' judgments of its value, with a positive correlation between weight and value evident in various areas of decision making. Previous studies use stimuli in which density and weight are perfectly correlated: to increase weight, density is increased with volume held constant. However, it was recently demonstrated that weight and density perception are dissociable, and that humans possess biases about objects' density based on their size. Therefore, it is necessary to decouple density and weight to understand their individual effects on value judgments. Participants view two sets of differently‐sized objects, and are asked to lift them and judge their relative value; in one set, objects possess constant density, while in the other they possess constant perceived weight. We hypothesize that density provides the primary influence on value judgments, over and above the effect of weight. Our findings will provide insight into the underlying factors driving value judgments, informing future studies of heaviness perception as well as value‐based decision making. 112 Engineering Budding Yeast for High Production of Geraniol WAI MAN (EMILY) YU, Anthony DeNicola, and Yi Tang Vinblastine is one of the front‐line anti‐cancer drugs approved by FDA; however, it is one of the most costly drugs to produce because isolating the compound from the native plant host Catharanthus roseus is expensive. Therefore, our lab is developing yeast as an alternative host to produce the metabolite intermediates needed for vinblastine. Our lab aims to increase the titer of geraniol, the first committed step of the iridoid alkaloid pathway by engineering the native mevalonate pathway of the yeast strain. First, the enzyme encoding geraniol, geraniol synthase (GES), was integrated into the yeast chromosome. To increase the low production of geraniol, upstream genes in the mevalonate pathway, such as HMG‐CoA reductase (HMG1) and farnesyl pyrophosphate synthetase (ERG20), were mutated and integrated into the yeast strain BY4741. Yeast transformation using homologous recombination was performed for the integration of these genes. Three strains with the integration of different upstream genes were created. The three strains are BY4741 with GES, with GES and a truncated copy of HMG1 (tHMG1), and with GES and plasmid of mutated ERG20. The production of geraniol from these yeast strains was monitored and quantified using gas chromatography‐
mass spectrometry (GC‐MS) with a calibration curve. The geraniol concentration of BY4741 with GES and a truncated copy of tHMG1 was 0424mg/L, and that of BY4741 with GES and plasmid of mutated ERG20 was 0.396mg/L. We hope to develop a stable pathway of geraniol for Vinblastine production. 37
SPD 2014 SESSION ONE 113 The Role of Hematopoietic Cells in Coronary Artery Development VINCENT HUANG, Gentian Lluri and Atsushi Nakano Coronary artery formation is a complex process that involves crosstalk between the epicardium and myocardium that promotes epithelial‐mesenchymal transition (EMT) to generate the coronary arteries. Our recent data suggest that the developing heart tube serves as a de novo source of definitive hematopoiesis, highlighting the interdependence of the heart and blood during embryogenesis. We hypothesize that the hematopoietic system is crucial for the development of coronary arteries by affecting epicardial EMT. The lack of hematopoiesis in Runx‐1 knockout mice lead to a reduction in coronary artery coverage, a thin myocardium and atrioventricular septum defects. We also utilized ex‐
vivo heart culture and whole mount staining to determine that hematopoietic ablation in Vav1‐DTA mice resulted in a reduction in the coronary microvasculature. Vimentin and Snail2 were downregulated while E‐Cadherin and Snail1 did not show statistically significant changes in mutants, indicating reduced epicardial EMT in Runx‐1 and Vav1‐DTA mutants. We also employed immunofluorescence and determined that the number of cardiac tissue macrophages, a major hematopoietic cell residing in the heart, temporally and spatially correlated with coronary formation. Furthering knowledge of coronary artery formation may potentially lead to a treatment involving the de novo formation of coronary collateral to treat the millions of patients afflicted with coronary artery disease and myocardial infarction and greatly impact the field of cardiac regeneration. 114 Electromyographic Evaluation of Reaching and Grasping Performance in Rats with Cervical Spinal Cord Injury BENITA JIN, Guillermo Garcia‐Alias, Sharon Zdunowski, Hui Zhong, Roland R. Roy, and V. Reggie Edgerton Cervical spinal cord injury (SCI) can severely impair the ability to reach and grasp by damaging the corticospinal tract, a descending system that has been implicated in the control of skilled hand movements. Many studies employ rodent models to assess SCI treatment and recovery by studying behavioral and anatomical measurements. However, up to date, there are few studies that have analyzed muscle activity with electromyography (EMG) to better understand SCI impairment and plasticity in reaching and grasping. The aims of this study were to (1) characterize the temporal recruitment of the forelimb muscles in control animals during reaching and grasping, and (2) in the same animals analyze the EMG once the corticospinal tract was damaged in the medullar pyramids or in the C4 spinal cord segment. Rats were trained in the single pellet reaching task. EMG was recorded in the proximal and distal muscles of the upper forelimb during reaching and grasping. We show that deviation in the timing and activity of muscle recruitment from that of the standardized EMG sets significantly lowered the success rate of a reach. These deviations were exaggerated in the SCI rats: rats with pyramidotomy terminated reach movement at the grasp stage. SCI rats with strychnine treatment demonstrated over‐activation of early reaching stages and re‐compensation. EMG is able to reveal changes in muscle recruitment patterns that may underlie changes in reaching behavior after SCI, and offers a window into functional mechanisms of recovery. 115 Lack of BLOC‐1 and Brain Development. SONIA ZAHEER, Frank Y. Lee, Linh L. Mikutowicz, Diana Nguyen, Esteban C. Dell’ Angelica, Cristina A. Ghiani and Christopher S. Colwell. Recent evidence is suggesting a neurodevelopmental origin for psychiatric disorders such as schizophrenia (SCZ). Several genes have been associated to this disorder, among them is DNTBP1 which encodes Dysbindin, one of the subunit of the Biogenesis of Lysosome‐related Organelle Complex‐1 (BLOC‐1). The expression levels of this complex are high in the developing brain and drastically reduced in mice deficient in any of the 8 subunits forming this complex, such as the Pallid mouse. Thus, the Pallid mice provide the opportunity to study the impact of BLOC‐1 deficiency on brain function and in psychiatric disorders such as SCZ. In our present work, we examined the cytoarchitecture of neurons in the Dentate Gyrus of the hippocampus. In the mutant mice, these neurons displayed fewer dendrites as compared to wild‐type controls. Additional abnormalities in the hippocampal structure are seen in the Pallid mice. These observations indicate that BLOC‐1 function is important for hippocampal neuronal maturation and are consistent with a link between mutations in the genes coding for this complex and neurodevelopmental disorders. 38
SPD 2014 SESSION ONE 116 The Impact of Cocaine on Macrophage Function and Susceptibility to HIV Infection JEFFREY Z. SHEN, Irene Kim, Dhaval Dixit, Andrew Cross, Jerome A. Zack, and Dimitrios N. Vatakis HIV is the causative agent of AIDS, a debilitating disease affecting more than 35 million people worldwide. Highly active antiretroviral therapy (HAART), the mainstay of current treatment regimes for AIDS, effectively suppresses symptoms but does not fully halt HIV pathogenesis. Epidemiological studies suggest that many HIV‐positive individuals use stimulants such as cocaine and methamphetamine, and the use of such substances is correlated with increased disease progression. However, the mechanisms by which these substances act have not been fully elucidated. We hypothesized that cocaine suppresses macrophage function because macrophages have a central role in the immune response and express sigma‐1 receptors, which have been shown to be mediate the stimulant and toxic properties of cocaine. Through flow cytometry analysis we show that physiologically relevant concentrations of cocaine activate monocyte‐derived macrophages to significantly increase expression of surface activation markers such as CD16, and possibly CD68 as well. We also show that this activation is mediated through the sigma‐1 receptor. In future studies we will examine whether macrophage function is impaired through phagocytosis assays and whether cocaine facilitates HIV infection of macrophages. This research is significant because it seeks to clarify how cocaine, a commonly used substance among AIDS stricken individuals, modifies the immune system, which may lead to development of novel and effective treatments methods tailored to these individuals. 117 Characterization of Spinal Interneurons Responsible for Standing and Stepping in Spinally Transected Mice MELINDA W. NG, Erica Dale, Hui Zhong, Niranjala Tillakaratne, and Victor R. Edgerton The lumbosacral spinal circuitry is responsible for standing and stepping after a complete mid‐thoracic spinal cord lesion. The goal of the current study is to identify the interneurons that contribute to these movements which occur independent of supraspinal control. We hypothesize that there is a unique group of interneurons that respond to sensory inputs and are responsible for standing and stepping. Mice were spinally transected and step‐trained for 10 weeks. At the end of the training, mice were injected with pseudorabies virus (PRV) to label the neurons associated with stepping circuitry. Mice were subsequently walked on a treadmill to allow for the reporter protein, cfos, activation; spinal cords were sectioned via cryostat and labeled using immunohistochemistry. Fluorescent microscopy showed neuron activation in the lower thoracic and lumbar regions. These data, as well as identification of cholinergic interneuronal networks, will allow an initial characterization of anatomical and physiological maps of the neurons responsible for bipedal stepping and standing after spinal transection in vivo. If the specific pattern of related circuitry can be identified, therapeutics geared towards activation of these networks can be simplified. 118 Functional Role of Cajal‐Retzius Neurons in the Postnatal Mouse Neocortex SAHANA KRIBAKARAN, Amaya Miquelajauregui, Mate Marosi, and Carlos Portera‐Cailliau Cajal Retzius (CR) neurons found in layer one of the neocortex are cells that play a vital role in neuronal migration and cortical lamination during development, but the functional role of these cells in the postnatal neocortex is still poorly understood. Here, we perform in vivo electrophysiology with optogenetics, where channelrhodopsin 2 (ChR2) coupled with yellow fluorescent protein (YFP) is expressed in Ebf2‐Cre mice to selectively stimulate CR neurons at postnatal ages (P) 6 to 10, while simultaneously recording local field potentials (LFP) and single‐cell patch‐clamp recordings from layer (L) 2/3 of the mouse neocortex. Preliminary results show that optogenetic stimulation at P10 leads to distinct LFP responses in L2/3 as well as in deeper cortical layers. To confirm the specificity of the response, we will perform further experiments at younger ages (P6), a time point when more CR neurons are present and the ChR2‐YFP labeling is more specific. We expect that optogenetic activation of CR neurons will trigger post‐synaptic currents in pyramidal neurons in L2/3, and possibly early network oscillations. If we directly record excitatory LFPs that are time‐locked with the light stimulation, this will give strong evidence that the CR neurons of L1 are synapsing onto the L2/3 pyramidal cells. By understanding this neocortical circuit, we hope to further elucidate underlying mechanisms for neurodevelopmental disorders such as autism spectrum disorders, epilepsy, schizophrenia, and bipolar disorder in future studies. 39
SPD 2014 SESSION ONE 119 New Findings related to the Progression of Alzheimer's disease Biomarkers in people with Down syndrome JODI C. HWANG, SAMANTHA M. SARWAR, CAITLYN Y. LEW, and Linda D. Nelson This is a first‐ever longitudinal study in which in vivo changes in pathological load were examined in people with Down syndrome. A cohort of 15 individuals with Down syndrome (mean age = 39.0 years, SD = 10.9 at baseline) was examined at two points in time (mean span = 2.87 years, SD = 0.83). To measure pathological progression, we used a radiolabeled probe, [18F]FDDNP, that binds to beta‐amyloid senile plaques and neurofibrillary tangles (tau) in the brain, two primary biomarkers of Alzheimer's disease. Our aim was to identify which region(s) of the brain were most affected by this disease and which area(s) of the brain showed the greatest increase in pathological load. The efficacy of the radiolabeled probe, [18F]FDDNP, in identifying physical signs of Alzheimer's disease in the brain was demonstrated. The [18F]FDDNP results revealed that frontal lobe was most affected in this cohort. From a clinical standpoint, this sample underwent statistically significant emotional changes that included increased levels of depression and indifference, or unresponsiveness to a person's environment. This pattern of frontal involvement was upheld in terms of concomitant statistically significant decline in neurocognitive areas of emotional and behavioral function. Increased emotional instability would be expected in cases where frontal lobe involvement was present. These findings provide strong support for detection of Alzheimer's disease progression in the frontal lobe. 120 The Effects of Sleep Quality on Virtual Reality Learning and Overnight Forgetting DANIEL LIN, Joey K.‐Y. Essoe, Joseph Tran, Jenny Zhou, James Mutter, Dana Frostig, Jonathan Yang, Nicco Reggente, and Jesse Rissman. As virtual reality (VR) becomes increasingly used as research and education tool, understanding what factors modulate its effectiveness has become a pressingly necessary, yet often neglected, area of research. Research shows that sleep is important for memory consolidation and thus retention. The current study examines the effects of sleep quality on VR learning—particularly, overnight forgetting. While immersed in VR, participants learned the pronunciations and meanings of 42 Swahili nouns in three learning sessions, each followed by cued recall as retrieval practice. They were then tested again the next day. The difference in memory recall performance between the final test of Day 1 and the initial test of Day 2 was defined as an overnight forgetting score. Sleep quality was measured on Day 2 using the Pittsburgh Sleep Quality Index (Buyess, 1998). We hypothesize that sleep quality will negatively correlate with overnight forgetting, such that the better a one sleeps, the less one will forget the learned material (i.e., better retention). If confirmed, our results might inform user‐end adjustments to make the most of VR‐based education. 121 HIV Reservoir Cells are Established Early On During Acute Infection JOSE A. ORTIZ, Lorelei Bornfleth, and Martha L. Blum HIV causes an incurable infection. Although infection can be suppressed indefinitely with combination anti‐retroviral therapy (cART), viral replication eventually resumes when therapy is stopped. A DNA copy of the viral genome, called a provirus, remains permanently integrated in the host cell genome and serves as a template for viral replication when therapy is stopped. The small population of resting cells with integrated provirus, the reservoir, is the main barrier to full eradication of HIV. It is not known how soon after acute infection this reservoir is formed nor how stable it is over time. This research project investigates the time point at which HIV reservoir cells become established by analyzing the sequence evolution of HIV. We hypothesize that the viral reservoir might be composed of homogenous sequences that arise from viral copies predominantly established during acute infection. Samples from three subjects from the Acute Infection Early Disease Progression Program (AIEDRP) in California were collected at three different time points. Viral RNA was extracted from serum samples and converted to cDNA using RT‐PCR. Full length nef genes from cDNA were amplified using nested PCR and cloned into a proviral vector. Individual nef clones were then sequenced using Sanger cycle sequencing to develop a phylogenetic tree for genetic analyses. This research reveals the time point when HIV reservoir cells become established and could potentially influence treatment strategies to help HIV‐infected individuals. 40
SPD 2014 SESSION ONE 12‐1:10pm 122 Timing and Localization of Olig, Nkx2.4, FoxQ2 and NFE2 Gene Expression in Embryonic Development of Strongylocentrotus Purpuratus JESSICA FERNANDEZ, VARSHINI SUNDARAM, JONATHAN TAN, KAZUTO URIU, Daniel Malkin, Pei Yun Lee Echinoderms such as Strongylocentrotus purpuratus are great model organism for studying gene function, developmental processes and cellular pathways. Sea urchin studies provide knowledge that is applicable to other complex organisms including human. Four uncharacterized genes of Strongylocentrotus purpuratus were identified via BLAST search and phylogenetic analysis, and their expression patterns were determined by RT‐PCR and Whole Mount In Situ Hybridization (WMISH). The four genes were identified as oligodendrocyte transcription factor (Olig), homeobox protein Nkx2.1 (Nkx2.1), forkhead box Q2 (FoxQ2) and nuclear factor erythroid derived 2 (Nfe2). Through RT‐PCR, Olig was found not to be expressed anytime during the sea urchin development. In situ hybridization results show no signs of expression of Olig at 24h or 48h but there may be either ubiquitous or nonspecific staining at 72h. RT‐PCR results show expression of Nkx2.4 at 24h and 48h; however, the control had incorrect band size. In situ hybridization results show light staining at 24h, strong staining at 48h, and light staining at 72h. For NFE2L1, RT‐PCR results suggest there is expression at 24h, 48h, and 72h; however, the control had incorrect band size. In situ hybridization could not be completed for NFE2L1. The study provides a further understanding of both S.purpuratus and human embryonic development, and it opens the door for future experiments to test the function and cellular pathways of these genes. 123 ABSTRACT RETRACTED 124 ndi1 Expression in Intestinal Stem Cells Extends Lifespan in Drosophila Melanogaster JACQUELINE V. GRANIEL, Jae H. Hur, David W. Walker As organisms age, mitochondrial activity is reduced and this may be a major cause of aging. Previous studies have shown that the alternative internal NADH‐ubiquinone oxidoreductase (ndi1), a yeast gene that can functionally substitute for some complex I functions of the electron transport chain in metazoans, can increase mitochondrial activity in Drosophila melanogaster and extend life when expressed in neurons. Dietary restriction (DR) and reduced fertility have been correlated with lifespan extension and we set out to determine if the mechanisms involved in ndi1‐
mediated lifespan extension overlap with these paradigms. We examined the influence of tissue‐specific ndi1 expression on lifespan, feeding behavior and fertility. We show that ndi1 flies live longer and eat more, suggesting ndi1‐mediated lifespan extension is not a result of DR. Furthermore, we observed an increase in feeding frequency and an increase in sugar consumption in the long‐lived flies. Similarly, decreased fertility that has been associated with some long‐lived flies was not observed in ndi1 flies. Overall, tissue‐specific ndi1 expression does in fact extend lifespan without any obvious physiological trade‐offs. More assays must be done to uncover the mechanism behind ndi1‐
mediated lifespan extension to better understand aging and protect against age‐related diseases. 41
SPD 2014 SESSION ONE 125 Neuropathological Data on Two Adult Subjects with Down Syndrome Offer Surprising Results Concerning Cerebellar Involvement in Alzheimer's Disease PAMELA J. LANGMAIER, SAMANTHA M. SARWAR, SAMMIE W. MUI, Spencer Tung, and Linda D. Nelson People with Down syndrome are at a significantly higher genetic risk for Alzheimer's disease as compared to the general population. This is a retrospective, descriptive case study of two adults with Down syndrome who were part of a larger cohort (N equals 15) followed over a four year period of time. Results of post‐mortem analyses for these two subjects offer new evidence of biomarker buildup in a region of the brain that was previously not considered to be affected by this disease: the cerebellum. Another important structure was the amygdala, an area deep within the temporal lobe, known to control emotional functioning. Biomarkers, such as beta‐amyloid and tau, usually appear later in the Alzheimer's disease state, and tau was found in abundance in the amygdala region in both of these subjects with Down syndrome compared to sporadic Alzheimer's disease controls. Clinically, these two subjects showed significant decline in areas of emotional functioning, in accordance with the pathological findings. In addition, a radiolabeled probe identified in vivo levels of the beta‐amyloid and tau in these two subjects. This research offers preliminary support for the need to further investigate why the cerebellum is affected in people with Down syndrome and Alzheimer's disease. It also offers support for the need to consider temporal lobe structures in addition to those that are typically known in the general population to be affected by Alzheimer's disease (e.g., hippocampal region). 126 Lyve1 Distinguishes the Divergence of Hemogenic Precursors during Embryonic Development ASHLEY YEON JOO KIM, Lydia K. Lee, and Hanna K. A. Mikkola Hematopoietic stem cells (HSCs) are self‐renewing multipotent cells are capable of differentiating into progenitors of all blood cell types. Embryonic hematopoiesis occurs in three waves that overlap spatially and temporally: Primitive (red blood cells and macrophages), transient‐definitive (erythromyeloid progenitors, EMP), and definitive (multipotent, self‐renewing HSCs). Understanding the divergence in regulation of hemogenic precursors is critical in developing in vitro transplantable HSCs. However, current research is hindered by inability to purify the diverse precursor populations. Through conditional targeting, we identify the lymphatic vascular endothelial receptor 1 (LYVE1) as a differential marker of the transient‐definitive and definitive waves. To characterize LYVE1 expression, we developed a lineage tracing model (Lyve1‐eGFP‐hCre; Rosa26‐YFP) and cultured the hemogenic organ explants in OP9 and OP9DL1. Differentiation into lymphoids, erythroids, and myeloids, shown by flow cytometry and colony forming assays, demonstrates that EMPs and subset of HSCs express LYVE1. In contrast, LYVE1 lineage does not label primitive erythropoiesis, as confirmed by immunostaining and absence of erythroid defect in Lyve1‐eGFP‐hCre; Scl‐/‐ embryos. Our data indicate that the divergence of the two waves from mesoderm must occur prior to formation of LYVE1+ hemogenic endothelium. These findings provide a new model to selectively study the yolk sac EMPs that ultimately seed the fetal liver and give rise to self‐renewing HSCs. 127 ABSTRACT RETRACTED 42
SPD 2014 SESSION ONE 128 Characterization of Embryonic Temporal and Spatial Expression Patterns of Notch‐a, Notch‐b, Notch‐c, and Delta 3 in Strongylocentrotus Purpuratus KELLY Y. CHEN, JESSICA GONZALEZ, ANNA KOLENTSOVA, MINDY TRUONG, Lydia Ann, and Pei Y. Lee Research on model organisms can lead to a better understanding of the developmental processes of chordates, such as modern humans. This study attempts to characterize the spatial and temporal expression of four genes in Strongylocentrotus purpuratus embryos. First, BLAST and phylogenetic analysis was performed in order to predict the identity of the unknown genes. Next, genes were cloned using PCR, ligation, and bacterial transformation. RNA probes for whole mount in situ hybridization (W‐MISH) were synthesized for determination of spatial gene expression. Sea urchins embryos were collected for use in reverse‐transcription PCR (RT‐PCR) in order to determine temporal gene expression. Based on these results, it was concluded that notch‐a, notch‐c, and delta 3 are expressed maternally. Notch‐a, notch‐b, and notch‐c are expressed 24 to 72 hours post‐fertilization, while delta 3 is not. Notch‐a is expressed throughout the ectoderm and primary mesenchyme cells at 24 hours post‐fertilization. These findings help lead to a better understanding of the S. purpuratus genome and widen the potential for its use in further research that is relevant to humans. 129 Treatment of a Mouse Model of Multiple Sclerosis with Highly Specific Estrogen Receptor‐β Ligands, WAY‐202041 and WAY‐200070 Ervin Herrera, Masuma Syed, Duc Nguyen, Spencer Moore, Timothy Yoo, Anahit Poturyan1, Shalini Kumar and Seema Tiwari‐Woodruff Multiple sclerosis (MS) is an autoimmune neurodegenerative disease which affects the central nervous system (CNS) and is characterized by motor, sensory, and cognitive deficits. Current therapies for MS come mostly in the form of immunomodulatory drugs, which are not directly neuroprotective. Our lab has shown that the estrogen receptor β (ERβ) ligand DPN has a direct neuroprotective effect in mouse models of MS. WAY‐202041 has a >200‐fold selectivity for ERβ over ERα, compared to the 70‐fold selectivity of DPN. Our aim was to investigate the potential neuroprotective effects of prophylactic and therapeutic treatment with the WAY compounds in a chronic mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Treatment with WAY both prior to and after disease onset improved EAE clinical scores. Immunohistochemistry of brain and spinal cord sections from WAY‐202041‐treated mice showed improved myelin density, and increased oligodendrocytes compared to vehicle‐treated group. Together, these findings strongly support a neuroprotective effect of WAY treatment in a chronic mouse model of MS. Therefore, the WAY compounds should be further investigated as potential treatment options for MS. 130 Posterior Polymorphous Corneal Dystrophy 3 is Associated with Agenesis and Hypoplasia of the Corpus Callosum MICHELLE JANG, Ashley Roldan, Ricardo Frausto, Anthony Aldave The corpus callosum (CC) is a sheet of nerve fiber bundles derived from neural crest tissue that connect the right and left hemispheres of the brain. Posterior polymorphous corneal dystrophy (PPCD) is a dominantly inherited disorder of the neural crest‐derived corneal endothelium. In approximately 1/3 of affected probands screened to date, mutations have been identified in the zinc‐finger E‐box binding homeobox 1 (ZEB1) gene. Both ZEB1 and ZEB2 have been previously associated with developmental abnormalities of the brain. While a variety of ocular abnormalities have been previously reported with an abnormally developed CC, this is the first report that an inherited disorder of the cornea is associated with this type of CNS abnormality. Slit lamp examination and DNA collection for screening of the ZEB1 gene were performed for members of two families with individuals previously diagnosed with posterior polymorphous corneal dystrophy. The proband of the first family was diagnosed shortly after birth with ACC. She was subsequently diagnosed with PPCD, prompting screening of the ZEB1 gene, which identified a novel deletion (c.449delG; (Gly150Alafs*36)). The proband of the second family was also diagnosed with ACC and PPCD. ZEB1 sequencing identified a novel deletion (c.1913‐1914delCA; p.(Ser638Cysfs*5)) present in the heterozygous state, which was also identified in the proband's affected mother. We report the first association of PPCD3 with malformations of the CC. 43
SPD 2014 SESSION ONE 131 Beta‐Arrestin‐1 regulates estrogen membrane signaling in hypothalamic neurons MATTHEW C. ABRAMS, Angela M. Wong and Paul Micevych Estradiol membrane‐initiated signaling (EMS) mediates a wide variety of the rapid neural events. Estradiol stimulation induces the trafficking and internalization of membrane estrogen receptor‐alpha (ERalpha). In neurons, ERalpha trafficking to the membrane is dependent on activation of PKCtheta. Internalization occurs with the sequential phosphorylation of the receptor by G‐protein‐coupled receptor kinases (GRKs), binding of an arrestin, and sequestration into early endosomes. GPCRs furthermore can associate with the signaling molecule beta‐arrestins because of the inherent scaffolding structure and function. In the present study, we initially used an immortalized neuronal cell line N‐38s derived from the ARH to examine whether beta‐arrestins mediated cell signaling and internalization. This cell line was partly chosen because the same beta‐arrestin isoform (Arrb1) is expressed in the rat's ARH and in N‐38 neurons. The effect of Arrb1 knockdown on membrane ERalpha physiology was then assessed in vitro. In N‐38 neurons, estradiol induced MAPK phosphorylation and internalization of membrane ERalpha. However, Arrb1 siRNA mediated Arrb1 knockdown abrogated the estradiol‐induced MAPK phosphorylation and membrane ERalpha internalization. These results indicate that both EMS and internalization of membrane ERalpha are dependent on Arrb1 and that in the whole animal, estradiol action that induces sexual receptivity requires a functional Arrb1. 132 Smad4 is Regulated by Phosphorylations that Integrate TGF‐beta, FGF and Wnt Signaling Pathways TATSUYA ARAKI, Hadrien Demagny and Edward M. De Robertis Smad4 is an intracellular component of Transforming Growth Factor Beta (TGF‐beta) signaling that controls the differentiation of endoderm and mesoderm during early development. Smad4 is currently thought to function constitutively. A bioinformatic screen uncovered putative phosphorylation sites in the linker region of Smad4. The sequence information suggested the hypothesis that Smad4 is phosphorylated first by Mitogen Activated Protein Kinase (MAPK) (activated by Fibroblast Growth Factor FGF), and then by Glycogen Synthase Kinase 3 (GSK3) (inhibited by Wnt). In this study, we discovered a cross‐talk between TGF‐beta, FGF, and Wnt signaling at the level of Smad4 liker phosphorylation. To demonstrate the involvement of FGF and Wnt in the regulation of TGF‐beta signaling, reporter gene and RT‐PCR assays were conducted. Wnt was shown to increase TGF‐beta activity but interestingly, only in the presence of FGF. In situ hybridization of Xenopus embryos showed that the phosphorylation‐resistant mutations of putative MAPK or GSK3 sites in Smad4 linker region were sufficient to significantly alter the differentiation and developmental fates. Using a novel luciferase reporter for Smad4 activity (from the mouse Chordin promoter), we showed that Wnt greatly increases TGF‐beta signaling in Xenopus animal cap explants. The GSK3 sites in Smad4 were required for this potentiation. Our data suggest that Smad4 is an important center for integrating the FGF, Wnt, and TGF‐beta signaling pathways to regulate early development. 133 Nomogram of the Immune Response After Heart Transplantation MARAL BAKIR, Tam Khuu, Martin Cadeiras, Galyna Bondar, Nicholas Wisniewski, Victoria Groysberg, Eugene Depasquale, Joselina Fuentes, Linda Rangel, Jennifer Zhang, Elaine Reed and Mario Deng Rejection and infection are common complications after Heart Transplantation and a major challenge is to assess the immune status of each patient to minimize these risks. We hypothesize that a nomogram of the ideal immune response could be developed for the management of heart transplantation and to develop a nomogram of the response to immunosuppression. Research was conducted to create a nomogram of the ideal immune function against which individual patients' immune status can be compared to predict their risk for developing rejection, infection or other major outcomes. We retrospectively analyzed, prospectively collected data from 109 adult heart transplant recipients between January 2010 and May 2013 who had been followed with brain natriuretic peptide, anti‐
HLA Class I and II antibodies, Allomap molecular expression testing, and Cylex immune function test assay. This data was then normalized and evaluated in the context of immunosuppression management over time along with rejection and allograft function parameters. To visualize the information, a HeatMap, was conducted and a clustering algorithm was used to sort the variables according to their correlation. A pattern emerged which resembled the clinical practice and showed that as immunosuppressant levels decreased antibody, Cylex and Allopmap levels increased. These initial results suggest that development of a nomogram will allow us to individualize immunosuppression management as well as predict patient outcome based on individual patient's response.
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SPD 2014 SESSION ONE 134 The Role of Inhibitory Smads in Cardiac Cushion Development RANA BESADA, Andrew W. Harmon, and Atsushi Nakano Aortic valve disease afflicts 2% of the U.S. population, and congenitally malformed valves are predisposed to calcification among the elderly. Previous studies revealed that bone morphogenetic protein (BMP) signaling is essential for proper valve formation, so we sought to further investigate is pathway's role. In mice, valve formation begins at embryonic day 9.0 (E9.0) when endocardial cells of the outflow tract and atrioventricular canal begin to undergo epithelial to mesenchymal transition (EMT) in response to signals from nearby myocardium. This transition initially results in the formation of cushions, which proliferate and are then remodeled into mature valves by E15.5. Inhibitory Smad6 and Smad7 attenuate the BMP pathway. Previous Smad6 knockout studies reported hyperplastic valve. To determine the mechanism behind hese large valves, we performed immunohistochemistry to test for proliferation and EMT markers in cardiac cushions at multiple developmental time points in Smad6 knockout mice and controls. In early stages, there was no difference in the number of phosphohistone‐H3‐positive cells or Vimentin‐
positive cells, suggesting that cell proliferation and EMT are not affected in mutants. However, in later stages, proliferation was greater in the Smad6 knockout mice. These results suggest that the Smad6 may be involved in valve cell proliferation after EMT process. Understanding this process will give greater insight on mechanisms underlying congenital diseases, which afflict 19 to 75 of every 1000 live births. 45
SPD 2014 SESSION TWO 151 Microreaction Catalyzed by Multivariate Metal‐Organic Framework TRENTON OTTO, Nanette N. Jarenwattananon, Stefan Giaggler, Jonathan W. Brown, Arek Melkonian and Louis‐S. Bouchard We investigated the performance of zinc‐based metal‐organic framework (MOF) catalysts that were post‐synthetically modified with the homogeneous palladium catalyst Pd(CH3CN)2Cl2 for the hydrogenation of propylene in a packed‐
bed, tubular microreactor. The catalytic conversion was analyzed across a range of metal loadings, reactant flow rates, feed concentrations, and reactor temperatures. The conversion was optimal at moderate metal loadings, stoichiometric excess of hydrogen, and relatively mild temperatures. While the activation energy varied with metal content, the predominant factor for the reaction rate was the Arrhenius prefactor. The catalytic activity depended strongly on reactant feed composition but showed no dependence on total flow rate, indicating a diffusion‐limited process. CO poisoning of the MOF catalyst was found to be reversible, suggesting a weak binding mechanism. 152 Inflammation and Oxidative Stress Pathways in BACHD Mice DANH T. LE, Analyne M. Schroeder and Christopher S. Colwell Huntington's Disease (HD) is a neurodegenerative condition that leads to overall brain atrophy as a result of a genetic mutation caused by an expansion of a CAG repeat region in the gene encoding huntingtin. The typical characteristics of this disease include motor, cognitive, and psychiatric disorders, often described as irrepressible abnormal movements. The second leading cause of death within the HD population is cardiovascular disease suggesting that although the disease causes the degeneration of neurons in the striatum that the peripheral tissues such as the heart may be impaired as well. RNA from the hearts of young and old wild‐type and BACHD mice were extracted to measure the changes in expression levels of inflammatory and oxidative stress genes through real‐time PCR. We found differences in the CT threshold levels of actb, Catalase, and Cyclophillin A as well as the inflammatory marker IL‐10 between the various groups. Overall, based on the genes tested so far, we found that aging plays an important factor in altering the gene expression levels of inflammation and oxidative stress markers. Future experiments will continue to examine other inflammatory and oxidative markers to look for changes in gene expression within the hearts of wild‐
type and BACHD model mice. The purpose of this study is to determine if disruption in inflammation and oxidative stress pathways may be the mechanism that accelerates the risk of cardiovascular dysfunction leading to disease. 153 The Use of Stimulating Images to Assess Visual Perception of the Terrestrial Hermit Crab JI SUN LEE, Xiaoge Ping, Dennis Garlick and Aaron P. Blaisdell There is an ongoing research on how well various species can perceive objects visually, and hermit crabs are one species that has been the focus of research. In our study, hermit crabs were presented with various stimulating images on a computer screen in three independent experiments with three different conditions: inverted, grey‐scale, and color. The hiding behavior was recorded by a program to determine whether they discriminated the stimuli. Experiment 1 and 2 utilized habituation and unhabituation. Once the subjects in control and experimental groups habituated to a control image, the experimental group was given the new stimuli: an inverted hawk for Experiment 1 and grey‐scale for 2. Latency in seconds to hide from the stimulus and the number of crabs that hid on the test trials did not differ among the two groups in both experiments. In Experiment 3, red, blue, and green colored circles were stimuli, whose order was counterbalanced. The number of crabs that hid and the average time of hiding were different among the three colors, and the color blue produced the strongest response. Our research provides more insights into hermit crab species and also crustaceans. 46
SPD 2014 SESSION TWO 154 Early Empathic Responsiveness as a Predictor of Later Language Ability in Infants at High or Low Risk for Autism Spectrum Disorder ANDREA E. BRARD and Ted Hutman Earlier research has found a relationship between empathy and language. For example, infants who showed higher levels of empathy at 12 months demonstrated higher language ability at 36 months (Hutman et al., 2012)‐‐ suggesting that infants' sensitivity to changes in the affective state of another person supports the development of language skills. We will investigate the possibility that responsiveness to another person's distress predicts language ability better than language ability predicts responsiveness to distress. One hundred and fifty eight infants at high or low risk for Autism Spectrum Disorders (ASD) were evaluated on both their distress response and on language ability at 12, 18, and 24 months. Responsiveness to distress was measured using previously published coding scales and language was measured using a standardized instrument. We predict that infants with higher distress response scores at 12 months will exhibit higher language scores at 18 and 24 months. Such findings may suggest that infant's level of empathic ability may better predict language skills than do language skills better predict empathic ability. Earlier research has even suggested that empathy might occur very early developmentally (Geangu et al., 2010). As such, targeting early empathic responsiveness with appropriate intervention may improve language development. 155 Synthesis of Mesoporous Thin Films of CdS Nanocrystals by Cross Linking Ligand Stripped Polymer Templated Nanocrystals JUSTIN C. ONDRY, Shauna Robbennolt and Sarah H. Tolbert Block copolymer templating of ligand stripped nanocrystals is a robust method that has been applied to making a variety of mesoporous noble metal, metal oxide, and metal selenide nanocrystal films. However the use of thermal processing to remove the polymer template can have detrimental effects on other material properties such as grain size and crystal structure. Due to the size dependence of the band gap in semiconductor nanocrystals, this grain growth changes the optical and electronic properties of polymer templated films. Here we present a new method to form mesoporous films of CdS nanocrystals that avoids thermal processing. In this method, ligand stripped CdS nanocrystals assemble with a diblock‐copolymer template to form an organic inorganic composite. The resulting film is then soaked in a solution of a small molecule cross linking agents. After soaking, the nanocrystals form a robust network. Next the polymer template was gently dissolved out of the film leaving behind a porous film of CdS nanocrystals. These films show disordered but homogeneous porosity as observed by SEM, and maintain the initial nanocrystal size, as determined by UV‐Vis spectroscopy and X‐ray diffraction. The high surface area of these materials and the well defined nanocrystal size make them interesting for a range of applications. 156 Bilingual Imitation Study CRYSTAL KUMTONG, Natsuki Atagi, Elizabeth Goldenberg and Catherine Sandhofer Children learn many new things about their environment and language through imitating and interacting with adults. How do children know who to imitate? This study aims to understand whether monolingual and bilingual children differ in their selective imitation process when learning to play with novel toys. Preschool‐aged monolingual and bilingual children were observed for the likelihood of imitating a monolingual actor over a bilingual actor. Three‐ to five‐year‐old monolingual and bilingual children were shown video clips of two actors playing with novel toys in different ways. After the children watched the video, they were handed the toys and observed for how they play with the toys. Preliminary results show monolingual children to not show a preference for an actor when imitating an action. However, bilingual children tend to prefer to imitate the bilingual actor. These results suggest monolingual and bilingual children may have cognitive differences when dealing with social cues from the environment. As the population becomes more diverse, academic programs such as the dual‐language programs and having bilingual tutors may have great benefits for bilingual children. 47
SPD 2014 SESSION TWO 157 Expression of p16 in Colon Cancer and Cyclin D1 in Gastric Cancer Predicts Response to CDK4/6 Inhibition In Vitro ANN R. YUFA, Zev A. Wainberg, Adrian Anghel, Shariar Adhami, Amy M. Rogers, Tin Manivong, Habib Hamidi, Dylan Conklin, Richard S. Finn and Dennis J. Slamon Cyclin‐dependent kinases (CDKs) play a significant role in regulating cell cycle progression through association with cyclins. CDK4 and CDK6 interact with Cyclin D1 to mediate hyperphosphorylation of retinoblastoma (Rb) during early G1 phase. Palbociclib is a highly selective inhibitor of CDK4 and CDK6 which functions by blocking Rb phosphorylation resulting in G1 arrest in sensitive cell lines. Panels of 17 gastric and 27 colon cancer cell lines were exposed in vitro to palbociclib over various concentrations to generate dose response curves. Analysis of Variance was used to identify differentially expressed genes between sensitive and resistant cell lines. Using gene amplification and expression data, lines were selected for further analysis via western blot and flow cytometry. Palbociclib demonstrated anti‐
proliferative activity for 14 gastric and colon cancer cell lines tested. Cell lines with either Cyclin D1 or HER2 amplification expressed greater sensitivity to the compound, while Cyclin E‐amplified cells emerged as resistant. In contrast to other cancers, expression levels of p16 (CDK4 inhibitor) and p21 (CDK2 inhibitor) in colon cancer indicated that p16 loss and p21 gain predict for resistance rather than sensitivity to CDK4/CDK6 inhibition. Palbociclib demonstrates anti‐proliferative activity in several gastric and colon cancer cell lines. Molecular markers found to predict for sensitivity to this agent enhance patient selection for future clinical studies of palbociclib. 158 The Impact of Impaired Maternal Treg Functionality on Trans‐Generational in Utero Growth Restriction THALIA M. NGUYEN, Maria N. Yesayan and Daniel A. Khan. Pregnancy is an immunologic mystery. The fetus is 50% paternal, a difference which should cause maternal immune rejection. The maternal immune system invokes FoxP3+ T regulatory cells (Tregs) to mediate immune tolerance. We used a transgenic mouse that co‐expresses human Diphtheria Toxin Receptor (DTR) in FoxP3 expressing cells, which when treated with Diphtheria Toxin (DT) Tregs are deleted in vivo. We noted that DTR pups are small compared with wild‐type (B6) mice. We hypothesized that the DTR transgene causes a subtle break in Treg function leading to Intra Uterine Growth Restriction (IUGR). To exclude a genetic cause, we did a cross breeding (DTR x B6) and found that pups of DTR (F) x B6 (M) were significantly smaller (~525.7g) than pups of B6xB6 (~733.5g) or pups of B6 (F) x DTR (M) (~745.5g). Mating the Heterozygous IUGR females (DTR (F) x B6 (M)) with B6 males also resulted in IUGR pups (~580.6g), reflecting a trans‐generational effect. After birth, IUGR pups grew faster than wild type and at 18 months were heavier and had higher random blood glucose. IUGR fetuses expressed higher levels of IGF‐1 than normal growth fetuses. Tregs from pregnant DTR mice failed to acquire specificity for fetal antigen. The results in mice suggest that human IUGR, which usually defies explanation, may be related to a subtle break in immune tolerance during pregnancy leading to the known risks (obesity, diabetes) associated with human IUGR and that also has a trans‐
generational effect. 159 The Analysis of Diffusion Model and ROF Model in Image Denoising SHUYI WANG and Jing Qin Image processing based on mathematical modeling has been well developed and still has attracted much attention due to broad applications. With the increase of image pixels per unit, the sensitivity of modern devices to noise increases as well. However, one of the biggest challenges of image denoising model is to remove noise while preserving various image features such as textures and edges. Therefore, in our work, we examined two of the most popular nonlinear image denoising models, including the diffusion model, and the total variation based one, i.e., ROF model, proposed by Rudin, Osher and Fatemi. By converting the respective minimization problems into partial differential equations, we are able to apply the finite difference techniques to solve those equations. Peak signal‐to‐
noise ratio is the ratio between the maximum possible power of a signal and the power of noise. Numerical experiments and parameter adjustments on a variety of images have shown that the ROF model has consistent outstanding performance over the diffusion model in terms of PSNR. 48
SPD 2014 SESSION TWO 160 Characterization of Dmbx1B, Atoh8‐like, RXR, and LMX1 and Determination of Their Temporal and Spatial Expression in Early Development of Strongylocentrotus purpuratus JOONHEE KIM, Siddharth Pandey, Amit Randhawa, Dylan West, Marco Morselli and Peiyun Lee The purple sea urchin is a deuterostome which produces optically clear embryos that undergo rapid cleavage and gastrulation and are easy to manipulate for various experiments. In this study, four previously uncharacterized genes of purple sea urchin were identified through BLAST and phylogenetic analysis, and their temporal and spatial expression patterns were determined by RT‐PCR and Whole Mount In Situ Hybridization (WMISH). Putative identities of the genes were found to be Diencephalon/Mesencephalon Homeobox 1B (Dmbx1B), Atonal Homolog 8‐like (Atoh8‐
like), Retinoic X Receptor (RXR), and LIM Homeobox 1 (LMX1). These genes are involved in early development during neurogenesis or kidney development. Based on WMISH and RT‐PCR data, Dmbx1B was expressed in the mouth at 48 and 72 hours. Atoh8‐like was expressed around the vegetal plate and surrounding ectoderm at 24 and 48 hours and in the gut at 72 hours. RXR expression was found around the vegetal pole at 24 and 48 hours. There was no expression of LMX1 during early embryonic development. Characterization of these genes and determination of their gene expressions are essential for studying gene regulatory networks (GRNs). This study may elucidate the evolution of these genes, allow for further understanding of homologous genes, and reveal potential implications in the origin of certain human diseases or disorders. 161 Testosterone Prevents Cortical Atrophy in the Mouse Model for Multiple Sclerosis SHANNON H. WAILES, Chandler R. L. Mongerson, Rory D. Spence and Allan Mackenzie‐Graham Previous research has demonstrated via magnetic resonance imaging (MRI) that testosterone treatment was able to restore cortical atrophy in men with Multiple Sclerosis (MS) . However, the neuropathology behind this restoration is currently unknown. Therefore, we used experimental autoimmune encephalomyelitis (EAE), the most common mouse model of MS, to elucidate the neuropathology behind testosterone's neuroprotection. We post‐treated gonadally intact male EAE mice with either a placebo or testosterone pellet. Following 25 days of EAE, mice were imaged using ex vivo MRI. Our results indicated that testosterone treatment was able to restore cortical atrophy. Furthermore, immunohistochemisty showed that testosterone treatment was able to prevent demyelination, inflammation, axonal loss and synaptic loss in the cortex. Future studies can now focus on the cellular target and mechanisms of testosterone treatment on the cortex in EAE. 162 The Pro‐Angiogenic Effects of Fibromodulin in Wound Healing KERMIT S. ZHANG, Jia Jian, Ching Yun Hsu, Xinli Zhang, Kang Ting, Chia Soo and Zhong Zheng Fibromodulin (FMOD) is a 59 kD extracellular matrix (ECM) proteoglycan with critical roles in scarless fetal skin repair, transforming growth factor‐beta (TGF) modulation, and collagen fibrillogenesis. Previous studies have recently elucidated exogenous FMOD significantly reduced adult scar formation, improved collagen formation, and had greater wound tensile strength. Our research focused on further elucidating the role of FMOD's ability to promote blood vessel formation (angiogenesis) during cutaneous wound healing. In vitro assessments were evaluated by using tube formation assays of Human Umbilical Vein Endothelial Cells (HUVEC) on Geltrex (purified basement membrane extract). FMOD significantly increased capillary‐like tube formation in vitro as assessed by dimensional (total length of cellular network per field) and topological (number of junctions, branches, and meshes per field) parameters. Angiogenic effects of FMOD were also evaluated by in ovo chick chorioallantoic membrance (CAM) assay. FMOD markedly induced blood vessel formation on the chorioallantoic membrane of chicken embryos. Importnatly, FMOD promoted capillary formation along with upregulated expression of angiogenic growth factors in rat primary closure cutaneous wounds, as assessed by immunohistochemistry and quantitative RT‐PCR. The promising effects of FMOD as an angiogenesis promoter can serve as a novel, and effective agent in healing poorly vascularized wound areas such as chronic diabetic wounds. 49
SPD 2014 SESSION TWO 163 Rapid Access to Composite Peptide Macrocycles by Friedel‐Crafts Alkylation Towards Solid‐
Supported Synthesis of a Pin1 Targeted Library HAYOUNG YOUN, Tristan E. Rose and Patrick G. Harran Composite peptide macrocycles hold potential for binding therapeutic targets owing to their large, complex topology and restricted conformation. A designed template harboring a latent cinnamyl carbocation can be readily appended to synthetic peptides. Protic acid activates the template, initiating rapid and efficient macrocyclization by Friedel‐Crafts alkylation of nucleophilic aromatic side chains. Here, we examine an extension of this methodology to on‐resin reactions wherein macrocyclization and release from the solid support are achieved simultaneously. Thus, structurally distinct macrocycles can be prepared by incorporating two simple steps into conventional Fmoc peptide synthesis. Towards construction of a pilot screening library, we have examined six model peptides containing the ‐Glu‐Pro‐ motif known to bind the active site of the mitotic regulatory enzyme Pin1. C‐terminal ethanolamide and glycine spacers were tested as attachments to the chlorotrityl polystyrene resin. We have studied macrocyclization under twelve different reaction conditions, and have identified and examined a persistent low purity observed in reactions of tryptophan‐containing peptides. These studies provide important advances towards construction of a Pin1‐targeted macrocycle library. 164 Migration and Proliferation of Fibroblasts and Keratinocytes in 1‐,2‐,3‐ Dimensional In Vitro Wound Healing Models VIRATA YINDEEYOUNGYEON and Bill J. Tawil. Chronic wounds are harmful to a patient's health and quality of life because they do not observe the expected healing stages in a normal time span. To successfully close the wound, fibroblasts and keratinocytes must be present and functional. These specialized cells aid the proliferation phase by facilitating cell migration and proliferation to initiate the extra cellular matrix and tissue genesis. Most chronic wounds remain in the inflammatory phase and rarely transition to the proliferation phase. To create the wound‐healing model, we seeded fibroblasts and keratinocytes on 1‐, 2‐, and 3‐dimensional models with fibrin microbeads. We then quantified the migration and proliferation of these cell types using bead circumference and the alamarBlue assay in order to understand the cell profiles during the proliferation phase. Preliminary results revealed that fibroblasts migrated fastest on a 2D fibrin construct while keratinocytes migrated best on a 1D plastic surface. The proliferation assay showed that both of the cells proliferated highest on the 1D plastic surface. These experiments will model the differences in cell migration and proliferation in various microenvironments in order to optimize the viability and performance of each cell type, constructing better wound healing models. 165 Isolation of Representatives of Proteobacteria and Actinobacteria in a Soil Sample of Kona Coffee Plant XUAN DANG, ANGEL KONG, AN NGUYEN, Sabrina Solouki, Jordan Moberg‐Parker, Kris Reddi and Giorgia Pirino Kona coffee (Coffea arabica) is a top gourmet coffee grown almost exclusively on histosols soil in Hawaii. Salgado et al. found Coffea arabica rhizosphere to be the host for various nitrogen fixers in a study in 1997. Preliminary results, on the other hand, suggest that Actinomyces may be present in the sample soil. Collectively, we hypothesized that the soil sample of a Kona coffee tree would include nitrogen fixers of phylum Proteobacteria and antibiotic producers of phylum Actinobacteria. Two approaches were taken—cultivation dependent and cultivation independent. In the cultivation dependent approach, nitrogen fixation assay and antibiotic producing assay were employed. In the cultivation independent approach, metagenomic DNA of all bacterial members in the sampled community was extracted and isolated. Taxa were assigned based on 16S rRNA sequence alignment (BLASTn) and structural alignment (RPD‐II). 88% of tested were able to fix nitrogen while 45% were able to produce antibiotics. Phylogenetic trees support both hypotheses. This suggests that histosol soil making up the rhizosphere of Kona coffee possesses a diversity of nitrogen fixers and antibiotic producers. Studies on these bacteria can help engineer more eco‐friendly nutrient rich soil while reducing greenhouse emission by histosols soil. Furthermore, exploring antibiotic producers may result in the discovery of novel antibiotics that can target parasitic bacterial species while being utilized as a tool to combat the antibiotic resistance war. 50
SPD 2014 SESSION TWO 166 Orphan ABC Transporter Abcc6 Contributes to Catecholamine‐Induced Cardiac Fibrosis DORIT T. STEIN, Christoph D. Rau, Milagros Romay, Rozeta Avetisyan, Jessica Wang, Yibin Wang and Aldons J. Lusis Cardiac fibrosis, a complex phenotype driven by multiple genes and pathways, results in a loss of elasticity and a degradation of regular heart function and is seen in patients with heart failure. Prior studies have correlated levels of cardiac fibrosis to the severity of heart failure but few systems‐level genetic studies of fibrosis in heart failure have been reported. The Hybrid Mouse Diversity Panel (HMDP) is a unique resource of inbred mouse strains that allows for high‐resolution genome wide association studies. The Heart Failure HMDP Project aimed to identify loci contributing to the cardiac fibrosis phenotype. Eight‐week old mice from 80 HMDP strains were treated with 30 ug/g of body weight/day of Isoproterenol (ISO) for 3 weeks, sacrificed, and fibrosis was assessed with Masson's Trichrome staining. Genome wide mapping of ISO‐induced cardiac fibrosis mice revealed Abcc6, an orphan ABC transporter linked to the human disease pseudoxanthoma elasticum, as a possible candidate for ISO‐induced fibrosis. Nineteen HMDP strains contain an Abcc6 splice‐site mutation which was linked to cardiac fibrosis in the ISO‐treated animals but not the control animals. Abcc6 knockout and knock‐in mice confirmed the contribution of Abcc6 to cardiac fibrosis, with the addition of Abcc6 having a protective effect compared to mice which lacked Abcc6. Possible pathways through which Abcc6 may be acting must be identified such that its role in heart failure may be further understood. 167 Differential Regulation of the Host Type I Interferon Immune Response by the Adaptor Molecule, TRAF3 SHIVAM ZAVER, Anurupa Dev, Kislay Parvatiyar, Roghiyh Aliyari, Jose Pindado, Yaya Wang, Amir A. Ghaffari, Brian J. Zarnegar, Jeannie Q. He and Genhong Cheng The innate arm of the immune system provides the first line of defense against invading pathogens. In the context of innate antiviral immunity, the induction of the type I interferon (IFN) pathway, characterized by activation of the transcription factors IRF3 and NFkB, is critical in limiting viral replication and spread. Much attention has been shifted towards the identification of key upstream sentinel proteins that recognize viral components to active IRF3, but little is known on the downstream regulation of the IRF3‐‐>IFN signaling axis. While the adaptor protein TRAF3 is known to be a positive regulator of the IFN response to RNA, here provide evidence for a differential function for TRAF3 in the IFN response to DNA and RNA virus infection, indicating that cellular host defense pathways are divergent at the level of TRAF3. Traf3‐/‐ MEFs produced elevated levels of type I IFNs in response to HSV‐1 or B‐DNA, and exhibited less HSV‐1 viral replication compared to wildtype cells. Cells lacking TRAF2 or cIAP1/2 components of the TRAF3‐containing complex also exhibited a heightened type I IFN response to DNA. A clearer understanding of how pathogenic DNA is recognized at the molecular level should aid in the ongoing development of antiviral therapeutics. 168 A Potential Antioxidative Role for Humanin in Hypoxic Human Trophoblastic Cells IL SEOK JEONG, Gina C. Capodanno and Kuk‐Wha Lee Interuterine growth restriction (IUGR) is a clinical condition in which the fetus fails to receive proper oxygen and nutrition delivery from the placenta, leading to various pregnancy complications such as preterm birth. Hypoxia plays a vital role in inducing oxidative stress, a biological state in which output of reactive oxygen species (ROS) surpasses intrinsic antioxidant defenses, one of which we propose is humanin (HN). This 24 amino acid peptide has already exhibited cytoprotective effects in various oxidant‐induced disease models such as amyloid‐beta‐induced Alzeheimer's disease and atherosclerosis. We hypothesize that HN will be induced endogenously in hypoxic human trophoblast cells and that exogenous delivery of HN to the cells will antagonize oxidative stressors. Previous studies denote the effective chemical achievement of hypoxia with cobalt chloride. Dose‐dependence and time‐course experiments will be conducted on the hypoxic cells, followed by immunoblot analysis, to assess HN peptide induction. Further studies are required to study the exact role of HN in human trophoblasts and a potential therapeutic role for HN in the treatment of oxidative stress inducing disease states. 51
SPD 2014 SESSION TWO 169 Electroencephalogram Analysis Using the Empirical Wavelet Transform KATHRYN HEAL and Jerome Gilles Wavelet decomposition methods have proven quite useful in the analysis of signals. They are regularly implemented in fields like communications, physics, and biomedical applications. A weakness of classical wavelets is that they are built independently of the analyzed signal; this can lead to some limitations of the extracted information. An extension, called the "empirical wavelet transform" (EWT), was recently proposed as a data‐driven approach to this decomposition problem. Although it has been established that this new method provides greater resolution for signal analysis, it still relies on one parameter: the number of desired frequency sub‐bands. The removal of this parameter would make this method more robust and user‐friendly. Here we propose a method to fully automate the EWT. The problem we address can be reduced to finding a parameterless domain‐segmentation method based on the particular signal. Our proposed technique is based on a scale‐space representation of the spectrum of the considered signal to detect the "optimal" number of sub‐bands. In collaboration with the Neuroscience department, we investigate the use of this tool to analyze brain signals. Our aim is to detect key rhythms in EEG signals. The ability to extract such information is of great importance to better understand and/or predict the origin of epileptic seizures or brain malfunctions in the case of Parkinson's disease. We will present some preliminary results of such analysis and will introduce some challenging questions for future work. 170 The Effect of Human Presence and Human Activity on Risk Assessment and Flight Initiation Distance in Skinks MARILYN M. MCGOWAN, PRIYA D. PATEL, JOLIE D. STROH and Daniel T. Blumstein Antipredator behavior and risk assessment of many species is affected by the presence of humans and their activities. Previous studies have largely been conducted on birds and mammals and relatively less is known about human impacts on reptiles. We looked at flight initiation distance (FID) as a measure of risk assessment in inland blue‐tailed skinks (Emoia impar), and tested the direct and indirect effects of humans on risk assessment. We first examined the effects of varying levels and types of human disturbance and activity on skink FID. We found that skinks flushed at significantly longer distances in areas with the least human activity. We then tested the degree to which skinks are able to discriminate different numbers of humans by comparing FID across three different types of approaches. Skinks did not significantly differentiate between a single approacher and a single approacher coming from a group of two other people, but did flush at greater distances when approached by three people simultaneously. Despite skinks not being directly harvested or harassed by humans, their human discrimination abilities are refined. Overall, skinks habituate to a variety of human activities and perceive a larger threat when the number of human approachers is greater. 171 PI3K/AKT and RAS/MAPK Co‐Activation Induce an Epithelial‐Mesenchymal Transition (EMT) and Cellular Plasticity in Prostate Cancer Cells BILL QUACH, Marcus Ruscetti, Zhixiang Lu, David Mulholland, Yi Xing and Hong Wu Prostate cancer is the second leading cause of cancer‐related death in American men. While localized prostate cancer is treatable, metastatic, castration‐resistant prostate cancer usually leads to death. The epithelial‐mesenchymal transition (EMT) is one explanation for the development of metastatic disease and proposes that epithelial tumor cells acquire mesenchymal characteristics in order to become motile, invade through the surrounding tumor microenvironment, travel through blood vessels, and seed metastases at distant sites. To assess a direct role for EMT in prostate tumor progression and metastasis, our lab has recently developed the Pb‐Cre+; PtenL/L; KrasG12D/+; Vim‐
GFP model that recapitulates metastatic human prostate cancer and exhibits EMT at the primary tumor site. By using GFP to mark Vimentin, a mesenchymal marker, and EpCAM as an epithelial marker, we are able to isolate epithelial, mesenchymal and EMT‐like tumor cells from mutant mice by FACS. Interestingly, sorted populations have the capacity to trans‐differentiate into epithelial and mesenchymal lineages without additional stimuli in vitro. While epithelial cells are dependent on the PI3K/AKT and RAS/MAPK pathways for growth and survival, mesenchymal cells are resistant to PI3K and MAPK inhibition in vitro. However, mesenchymal cell growth and survival is inhibited by treatment with the HDAC inhibitor LBH589 both in vitro and in vivo, suggesting that epigenetic alterations may regulate cellular plasticity between mesenchymal and epithelial states. 52
SPD 2014 SESSION TWO 172 Assessing the Effect of Cortical Electrical Stimulation On Animals' Spontaneous Behaviors and Their Motor Electrophysiology JONATHAN KEYES, Guillermo G. Alias and Reggie Edgerton Damage to the corticospinal tract impairs one's ability to perform skilled movement. Current clinical methods include non‐invasive methods such as the transcranial magnetic stimulation (TMS) to evaluate the integrity of the corticospinal tract, but do not provide effective therapy due to its mobility limitations. In the present study, we have implanted electrodes on the motor cortex and evoked corticospinal motor potentials while the animals were freely moving inside a cage. We have categorized the animals' behaviors and studied the cortical electrical stimulation (CES) responses under different stimulation parameters. These behaviors are then synchronized with the animals' motor electrophysiology to determine the optimal stimulation and the proper behaviors needed to induce an evoked potential in the muscle. The result of this study can provide an avenue to novel therapeutic method to test spinal cord connectivity and to promote spinal cord regeneration as it serves as a basis to how CES can affect an animal's spontaneous behavior and muscle electrophysiology. 173 Generation of T and NK Cells from CAR Expressing Human Embryonic Stem Cells KATIE C. PANNELL, Deirdre D. Scripture‐Adams, Emily Lowe, Nixian Zheng, Hongying Chen, Masakazu Kamata, Bao Nguyen, Irvin Chen, Zoran Galic and Jerome A. Zack Successful lymphocyte development from human embryonic stem cells (hESCs) would result in a variety of new research avenues with direct clinical implications, especially in the treatment of HIV and other diseases of the blood. Our lab has published successful T cell generation protocols from the H1 line hESCs using both feeder‐dependent and feeder‐free methods of hematopoietic precursor production. We have also used previously published methods to generate natural killer (NK) cells from hESCs and have shown they express a variety of protein receptors typical of mature NK cells. We now seek to use these in vitro methods of lymphocyte development to generate T and NK cells from hESCs that have been modified by transduction with a chimeric antigen receptor (CAR) lentiviral vector. We expect that lymphocytes containing sequence from this multi‐component vector will have both increased cytotoxic effects against HIV infected cells, as well as resistance to HIV‐1 through the activity of two anti‐HIV small hairpin RNA sequences. This derivation of genetically modified lymphocytes could lead to a more efficient immune response in future NK and T cell based therapies for HIV. 174 Clonal Analysis of Adipose‐Derived Adult Tissue Regenerative Cells for Autologous Cell Therapies and Tissue Engineering MARCO MRAVIC, W. Reef Hardy and Bruno Peault When isolated in aggregate, perivascular stem cells (PSCs) from human adipose tissue exhibit multipotency in vitro and the ability to repair several adult musculoskeletal tissues upon implantation. However, the regenerative properties of these PSCs, including pericytes from the microvasculature and adventitial cells from larger vessels, have not been investigated at the single cell / clonal level. We believe only a portion of isolated PSCs are multipotent and can self‐
renew, and that cell surface molecules can distinguish such fractions. None of 192 single CD31‐ CD45‐ CD146+ CD34‐ pericytes nor 384 single CD31‐ CD45‐ CD146‐ CD34+ adventitial cells purified by flow cytometry (N=2 human lipoaspirates) produced clonal cultures. However, high levels of intracellular aldehyde dehydrogenase (ALDH) 1A1, a putative stem cell marker, enhanced the clonogenic potential to 39% of 480 adventitial cells (N = 5 donors). None of 384 ALDH+ pericytes (N = 4 donors) could self‐renew. Twenty‐one ALDH+ adventitial cell clonal cultures were expanded for differentiation assays, proliferation assays, and cell surface marker analysis. Of clonal cultures assayed thus far, 17/21 and 4/19 differentiated to adipocytes and osteoblasts in vitro, respectively (p<0.01). Within 150 hours, 12/15 cultures doubled (p<0.05) while 3/15 doubled rapidly (<36 hrs). Cell surface marker profiles of these clones show immense heterogeneity. We aim to discover and validate novel molecular criteria to isolate enriched fractions of PSCs for regenerative therapies... 53
SPD 2014 SESSION TWO 175 Comparison of Alloplastic Materials for Extraction Socket Augmentation in the Mini Pig EDWIN ESHAGHZADEH, Armita Fartash, Armand Keuroghlian, Joan Pi‐Anfruns, Olga Bezouglaia, , Sotirios Tetradis, Peter Moy and Tara Aghaloo Socket augmentation is performed using a bone graft substitute to minimize remodeling following tooth extraction. Presently, the literature lacks comparison among alloplastic bone graft materials for socket augmentation. The purpose of this research study was to compare the effectiveness of different biomaterials for socket augmentation in the mini pig. Tooth extraction of two molars on the right and left side of the mandible and maxilla of six Yucatan mini‐
pigs was performed. Socket augmentation was performed with seven alloplastic bone graft materials: T1‐HA‐BTCP, ABB, BCP, HA‐BTCP, BTCP, HA‐c, and HA‐cc. The biomaterials were randomly assigned and utilized to graft the sockets in the mandible and maxilla of each pig. One socket was used as control in each pig, where no bone graft was placed. Following micro‐CT analysis after four months of healing, 3‐D reconstructed images were made to analyze bone volume to tissue volume measurements, buccal‐lingual bone width, buccal‐lingual bone loss, and vertical bone loss. This study found that application of bone graft substitutes via socket augmentation should be considered optimal treatment at the time of extraction. ABB, BCP, HA‐BTCP, HA‐c, and HA‐cc all displayed significant difference in vertical or horizontal bone measurements when compared to extraction without bone grafting. Within all measurements analyzed, HA‐BTCP was consistently found to be more effective than healing without grafting. Socket augmentation may improve bony contours and facilitate implant placement. 176 Targeted Block Copolypeptide Vesicles for the Enhanced Delivery of Chemotherapeutics ALLISON T. YIP, Brian S. Lee, Alison V. Thach, April R. Rodriguez, Timothy J. Deming and Daniel T. Kamei Cancer is the second leading cause of death worldwide. Although chemotherapy remains one of the most common cancer treatment options, current chemotherapeutic drugs are nonspecific, eliciting negative side effects by killing healthy cells along with cancer cells. The development of nanoscale drug delivery vehicles is exciting because of their potential to encapsulate and protect the drug as it circulates throughout the body. In the current study, we prepare negatively charged block copolypeptide vesicles from poly(L‐glutamate)60‐poly(L‐leucine)20 (E60L20) that are decorated with polyethylene glycol (PEG), loaded with the chemotherapeutic doxorubicin (DOX), and conjugated with a targeting protein transferrin (Tf). These Tf‐conjugated, DOX‐loaded, PEGylated E60L20 vesicles (Tf‐DPEL) demonstrated extended drug release and a 1.5‐fold decrease in the IC50, suggesting that the use of the Tf‐DPEL vesicles can improve drug carrier efficacy in comparison to the non‐targeted vesicle construct. These block copolypeptide vesicles therefore have the potential to become effective nanosized drug‐delivery vehicles for targeted chemotherapy treatment. 177 Individual differences in working memory performance as a function of the local integrity and regional connectivity of the hippocampus CODY KOMMERS, OMRI RACCAH, Nicco Reggente and Jesse Rissman Although the hippocampus is well known to contribute to the storage and retrieval of long‐term memories, emerging data suggests that the hippocampus may also contribute to the online maintenance of task‐relevant representations in some tests of working memory. To the degree that hippocampal mechanisms serve to facilitate performance on short delay memory tasks, individual differences in hippocampal microstructure could contribute to across‐subject variance in working memory performance. To examine the relationship between hippocampal structure and function, we obtained the diffusion‐weighted images (DWI) of a large cohort of subjects from the Human Connectome Project MRI dataset. We used the DWI to compute diffusion tensor images (DTI), which in turn were used to generate whole‐brain mean‐diffusivity (MD) maps. MD in deep gray matter has been construed as an indirect measurement of local microstructural deficits (Kim et al., 2013). Thereby, we aimed to assess the underlying integrity of each subject's hippocampal gray matter and use examine whether these measures can account for variance in memory performance across subjects. Average MD within the left hippocampus was found to be significantly correlated with performance on a Working Memory List Sorting Task. This current study extends previous findings and contributes to the debate surrounding the role of the hippocampus in working memory. We plan to conduct further analyses aimed at characterizing the role of fronto‐hippocampal connectivity in working memory performance. 54
SPD 2014 SESSION TWO 178 Cholesterogenesis Inhibitor Lovastatin Increases Transduction Efficiency of Lentiviral Vectors in a Hematopoietic Cell Line KG1a KYOKO YAMAGUCHI, Zulema Romero Garcia, Fabrizia Urbinati and Donald B. Kohn Gene therapy has emerged as an alternative treatment for monogenic diseases and many other non‐hereditary disorders. Lentiviral vectors (LV) have been successfully used in several clinical trials in the last two decades. The vector particles have been pseudotyped to express a surface glycoprotein called VSV‐G that increases tropism and particle stability, promoting the use of these LVs for gene therapy. The LV being studied expresses a Beta‐globin transgene with anti‐sickling activity and carries a large, complex cassette (Beta‐AS3) and has a low transduction efficiency compared to simpler LVs. Previous studies showed that VSV‐G binds to the low density lipoprotein receptor (LDL‐R) to enter the cell. Our hypothesis was that the addition of Lovastatin, a cholesterogenesis inhibitor, to human cells will cause an up‐regulation of the LDL‐R; and due to the increased expression of the VSV‐G receptor will also induce an increase in the transduction efficiency of the LV. When transduction experiments with Beta‐AS3 LV were carried out in a human hematopoietic cell line (KG1a), we observed that the treatment of these cells with Lovastatin caused an increase in (1) LDL‐R expression, as determined by FACS, and (2) transduction efficiency, i.e. VCN, as analyzed by qPCR. These data support the correlation between LV receptor expression and transduction efficiency, and suggest that Lovastatin treatment could sensitize hematopoietic cells to transduction. Current studies are being performed using primary human CD34+ hematopoietic cells. 179 Characterizing a Phr Peptide Quorum Sensing Cassette in Streptococcus Pneumoniae that May Influence Pathogenicity DAVID H. CHAN, Sharon E. Hoover and Beth A. Lazazzera Quorum sensing is a form of cell‐cell communication in bacteria which regulates gene expression according to cell density, eliciting a response when the local concentration of a small molecule secreted by the cells is high enough. We have identified a novel quorum sensing gene cassette in Streptococcus pneumoniae that contains genes encoding for a secreted peptide signaling molecule (phrB) and its probable transcription factor target (tprB). This cassette is an appealing target for analysis due to its location on pneumococcal pathogenicity island 1 (PPI‐1), a horizontally acquired genomic region implicated in virulence. Introduction of a construct to delete tprB resulted in the desired deletion of tprB, but also resulted in undesired disruption of genomic regions immediately surrounding tprB. However, deleting phrB did not cause any anomalies in the surrounding genomic region. Future experiments will insert tprB under the control of a fucose‐inducible promoter at another locus and delete tprB at its original locus. Successfully deleting the endogenous copy of tprB when a second copy is provided in trans would suggest that tprB is essential to the cell, while the inability to do so would suggest that there may be genetic features preventing recombination at tprB. In the future, we will assess the phrB and tprB deletion strains for effects on the expression of genes in PPI‐1. Results from this study will help expand our knowledge of peptide signaling molecules in Gram‐positive bacteria. 180 Cell‐type Specific Polysynaptic Viral Tracing Reveals How Chronic Morphine Alters the Striatal Connectome MAXWELLL ROTH, Daniel Nachun, Ani Minasyan, Christopher J. Evans, Hongwei Dong and Wendy M. Walwyn Chronic administration of morphine, a mu opioid receptor agonist, has been shown to reduce dendritic spine density in striatal medium spiny neurons (MSNs). We proposed that these opiate‐induced structural adaptations could alter the functional connectivity of these MSNs. To assess this adaptation we used a retrogradely transported polysynaptic pseudo‐rabies virus (PRV263) that expresses a floxed Brianbow cassette. We microinjected this virus after 10 days of chronic morphine or saline exposure into the major efferent targets of D1 and D2 MSNs, the substantia nigra pars reticulata and the ventral pallidum respectively. When exposed to Cre‐Recombinase expressing D1 and D2 MSNs, the viral dTomato gene is excised enabling the expression of Green Fluorescent Protein (GFP). These GFP expressing virions are retrogradely transported across the synapses of these target D1 and D2 expressing striatal MSNs effectively labeling the direct and indirect connections to these MSNs. Analyzing the distribution and number of GFP labeled cells in different brain regions, we found that chronic morphine exposure altered the afferent connectome of both these MSN subtypes. Specifically, in D2‐Cre mice, chronic morphine was associated with fewer GFP‐labeled cells in the insular cortex suggesting an opiate‐induced region and cell‐type specific re‐organization of the cortico‐striatal network, a change that may be important in mediating the adaptations associated with chronic drug exposure. 55
SPD 2014 SESSION TWO 181 Downregulation of NF‐kB Activation and Adhesion Molecule Expression in the Tumor Endothelium: A Potential Immune Escape Mechanism Implemented by Pancreatic Cancer Cell Lines RYAN D. FRESHMAN, Georg Hilfenhaus and Luisa Iruela‐Arispe Cancer cells have the capacity to diminish infiltration of circulating leukocytes, thereby evading the immune response and enabling tumor progression. In order for leukocytes to infiltrate tumors, they must bind to adhesion molecules expressed by the endothelium of tumor‐associated blood vessels. Here we assess the effects of pancreatic cancer cells on adhesion molecule expression in the endothelium and aim to identify targets that can promote immune cell infiltration. As the nuclear factor kappa B (NF‐kB) pathway controls the expression of several adhesion molecules, we established a human umbilical vein endothelial cell (HUVEC) reporter line expressing GFP under an NF‐kB promoter. We co‐cultured these HUVECs with a panel of human pancreatic cancer cells using a transwell assay in the presence of tumor necrosis factor alpha (TNF‐a), and NF‐kB activation and adhesion molecule expression were assessed. Most pancreatic cancer cells decreased NF‐kB activation in co‐cultured HUVECs. This correlated with a transcriptional downregulation of several adhesion molecules, including E‐selectin, ICAM‐1, and VCAM, and was partly mediated by non‐contact effects. Downregulation of TNF‐a receptors was observed for one pancreatic cancer cell line. Thus far, a number of kinase inhibitors were unable to restore NF‐kB activation in co‐cultured HUVECs. Our work provides evidence for the potential of tumors to evade the immune response via NF‐kB mediated downregulation of adhesion molecule expression. 182 Body Composition Analysis of Ischemic Heart Failure Patients SIMA AMIN and Tamara Horwich Ischemic heart failure is correlated with higher mortality when compared to non‐ischemic HF. This study investigates the relationship between adiposity, lipid serum levels and lean body mass (LBM) in HF patients with and without ischemic etiology. Advanced HF patients (n=316, mean age 55+/‐14 years, left ventricular ejection fraction 36.5+/‐
15.7) had BIA measured with InBody 520 (Biospace Inc. CA), an 8‐point tactile electrode system employing 5, 50, and 500 kHz frequencies at right and left arms and legs, and trunk segments. Both groups had similar severities of HF. Other variables were gathered from patient charts. Ischemic HF patients were predominantly male and significantly older than non‐ischemic HF patients. Total cholesterol and LDL were significantly lower in the ischemic group. Ischemic patients were more likely to have diabetes. Significantly more ischemic patients were prescribed beta‐blockers and statins. LBM, measures of adiposity (waist circumference, percent body fat, BMI), and water levels did not significantly differ between the two groups. More aggressive cholesterol lowering methods may have been used to lower total cholesterol and LDL in the ischemic HF group. Further investigation of the link between ischemia, HF, and outcomes in HF is needed. 183 Molybdenum Bi‐ and Tri‐Boride Systems: Synthesis and Analysis AVALON H. DISMUKES and Richard Kaner Refractory metal borides have recently generated intense interest in materials chemistry. These compounds have been shown to possess many advantageous properties, such as exceptionally high hardness, electrical conductivity, and even superconductivity. Previous research was directed at tungsten tetraboride in conjunction with Chris Turner's (current graduate student) project but new research has been directed at molybdenum‐boride systems. Higher molybdenum borides (i.e. MoB2, MoB3, Mo0.91B3, and MoB4) are currently under exploration as compounds of interest in this category of materials. However, the complex phase relationships in the molybdenum‐boron system complicate the preparation of phase‐pure samples. Here we expand upon the phase relationships in arc melted MoB2 and MoB3. Systematically varying the ratio of boron to molybdenum in sub‐ to super‐stoichiometric amounts has yielded samples approaching phase purity. System compositions are examined by X‐ray diffraction (XRD) and their grain structure analyzed by scanning electron microscopy (SEM). We also demonstrate preferential phase formation of the MoB2 structure in both binary and ternary solid solutions despite wide stoichiometric variation, as suspected from previous reports (and literature). This work enables further exploration of the properties of molybdenum borides such as through hardness measurements, thermogravimetric stability testing, and crystallographic phase design. 56
SPD 2014 SESSION TWO 184 Delivery of Bone Morphogenetic Protein 2 and Phenamil to Induce Osteogenesis of Adipose‐
Derived Stem Cells CHOONG SUNG IM, Jiabing Fan, Zhongkai Cui and Min Lee Bone morphogenetic protein 2 (BMP2) is a widely used osteoinductive factor to stimulate bone regeneration in clinic, but the high therapeutic dosage causes adverse effects such as ectopic bone formation. Thus, there is a need to develop alternative strategies that can effectively complement BMP activity. Recently a small molecule phenamil was shown to enhance osteoblast differentiation by stimulating BMP2 signaling. We hypothesized that co‐delivery of BMP2 + phenamil will enhance osteogenesis of adipose‐derived stem cells (ASC). To test this, we fabricated poly lactic‐
co‐glycolic acid (PLGA) into 3D porous scaffolds, which were further modified with apatite coatings for enhanced cellular responses and efficient delivery of BMP2 and phenamil. We studied the release kinetics of BMP2 and phenamil from the scaffolds using ELISA and UV spectrometry, respectively. ASC viability and osteogenic differentiation were examined with live/dead cell staining and alkaline phosphatase (ALP) activity, an early osteogenic marker. Both BMP2 and phenamil showed sustained release profiles up to 3 weeks. Delivery of BMP2 + phenamil resulted in significantly increased ASC proliferation and ALP expression on the scaffolds compared to the BMP2 or phenamil alone, or unloaded control. This study suggests that combination of BMP2 + phenamil can enhance BMP2‐induced osteogenesis and potentially reduce total BMP2 dose requirement without compromising osteogenic efficacy. Currently, this approach is being tested in an in vivo calvarial defect model. 185 Exploring the Biosynthetic Potential of the Marine‐derived Filamentous Fungus Penicillium Oxalicum
FRANK SUN, Jaclyn M. Winter and Yi Tang Natural products are specialized metabolites that are made by living organisms in nature. A large number of these metabolites belong to the polyketide class of natural products and are responsible for the biosynthesis of blockbuster drugs. Filamentous fungi are prolific producers of these polyketides and serve as great resources for new pharmaceutical discoveries. Fungal polyketides are produced by iterative polyketide synthases (IPKSs), which are megasynthases that use different combinations of a single set of catalytic domains to create different metabolites. Unfortunately, the programming rules of IPKSs is still poorly understood and limits what we can bioengineer with these enzymes; however, with continuous characterization of IPKSs, we will be able to define these elusive mechanisms and harvest the metabolites for testing. Specifically, we will focus on the genomic mining and characterization of IPKSs from the marine‐derived fungal strain, Penicillium oxalicum that we isolated from the Sea of Cortez. Based on its unique chemical profile, the genome of P. oxalicum was sequenced, annotated, and mined for IPKS‐containing clusters (656 and 1688) in order to characterize them and identify the natural products they are associated with through analysis of different gene knockouts. Understanding how these enzymes synthesize natural products will provide a foundation for designing therapeutic agents with increased biological activity to deal with deadly microbial pathogens and diseases. 186 The Effect of Lipid Composition on the Conformational Equilibria of Rhodopsin MICHELLE LIN, Ned Van Eps and Wayne Hubbell Rhodopsin is the primary photoreceptor of rod cells and is essential for visual signal transduction. A study of the effect of different lipid compositions on the conformational equilibria of rhodopsin was performed to better understand protein‐lipid interactions in membranes. Rhodopsin was purified from bovine retinas and incorporated into nanodiscs, which consist of a lipid bilayer and a membrane scaffold protein. By assembling nanodiscs containing various lipid components such as palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC), palmitoyl‐2‐oleoyl‐glycero‐3‐phosphoserine (POPS), 1,2‐dioleoyl‐glycero‐3‐phosphocholine (DOPC), and 1,2‐dioleoyl‐glycero‐3‐phosphoserine (DOPS), the lipid's influence on rhodopsin's activity was examined. Measurements were made with UV‐visible spectroscopy to probe the Schiff base attached chromophore of the receptor. While the POPC/POPS membrane strongly favored the rhodopsin intermediate, metarhodopsin‐II, with decreasing pH, the DOPC and DOPC/DOPS membranes appeared to stabilize the protein as metarhodopsin‐II in a pH‐independent fashion. DOPC/DOPS had a stronger effect than DOPC alone on metarhodopsin‐II stabilization. Thus, the lipid composition influences the conformational equilibrium near the chromophore, possibly due to differences in acyl chain saturation of the lipids, headgroup charge, or hydrocarbon chain length. The rhodopsin study is significant as the conformational changes in response to lipid environments can be extended to the rest of the G‐protein‐coupled receptor family. 57
SPD 2014 SESSION TWO 187 Luminescence Characteristics of Laser‐Created Bubbles in Pressurized Water at Low Temperatures BRANDON R. BERG, Royce E. Coleman, Daniel Yeung, Eli Van Cleve and Gary A. Williams A pulse at 1064 nm from a Nd:YAG laser can create bubbles in water that collapse and emit light at the point of maximum collapse. After formation, the bubbles increase to a size of order 1 mm, and then quickly collapse. The adiabatic compression of the gas in the bubble heats the interior of the bubble to over 8000 K and forms a plasma, which at the maximum collapse point emits a pulse of light of duration approximately 5‐10 nanoseconds. This luminescence pulse, detected with photomultipliers, can be described by two parameters: the pulse duration, and the pulse area, which can be used to roughly calculate the photon flux from the bubble. Of interest in this experiment is the behavior of the luminescence pulse as the temperature of the water is lowered from 20 C to 1 C, and the pressure increased from 1 bar to 5 bars. An initial hypothesis was that the luminescence might be affected by either the increasing viscosity of the water, or by the decrease of the water vapor in the bubble at low temperatures. The preliminary data shows that as temperature decreases, the duration of the pulse remains constant, while the total flux of light increases, by nearly a factor of 1.5 at 1 C. This indicates that the changing viscosity does not play a major role, and that the brightness increase can be attributed to the decreasing water vapor in the bubble. Modeling the luminescence as blackbody emission shows that the temperature in the interior of the bubble has increased by nearly 1000 K when the water temperature is lowered to 1 C. 188 Measurement of Ion Temperature in a Laboratory Plasma JIACHEN LIU, Seth Dorfman, Walter Gekelman, Patrick Pribyl, Anton Bondarenko and Troy A. Carter. Alfven wave is a low‐frequency oscillating wave in a magnetized plasma. Understanding Alfven wave is important because it may play a significant role in chromosphere heating, solar wind turbulence, fast ions in a tokomak and other modern unsolved plasma physics problems. This research focuses on the measurement of plasma ion temperature using the width of the ion spectral line of the plasma. A monochromator is first used to measure cold (‐0.1ev) spectral lines of a mercury lamp to account for instrumental broadening. A convolution of the instrumental broadening and plasma simulation (PrismSPECT) is compared with the actual plasma ion line. As the temperature of the plasma rises, the width of the spectral line is expected to increases as well. Through this relationship, we are able to measure plasma ion temperature from the width of the ion line. This measurement will be crucial in future experiments aimed at studying Alfven wave behavior in hot ion plasma. Results may shed light on one or more of the plasma physics problems mentioned above. 189 Sarcospan as a Therapeutic Target in Treating Duchenne Muscular Dystrophy EVA MA, Elizabeth M. Gibbs, Thien Nguyen and Rachelle H. Crosbie‐Watson Duchenne muscular dystrophy (DMD) is a progressive skeletal muscle wasting disorder that inevitably leads to death due to cardiorespiratory failure. DMD results in the loss of the dystrophin‐glycoprotein complex (DGC) from the muscle membrane, which renders the sarcolemma susceptible to contraction‐induced damage. Over‐expression of the utrophin‐glycoprotein complex (UGC) and alpha‐7beta‐1 integrin compensates for the loss of the DGC and rescues the dystrophic pathology in the mdx mouse model of Duchenne. Sarcospan (SSPN), which is a core component of both the DGC and the UGC, is the main focus of this study. Previously, three‐fold levels of human SSPN (hSSPN) over‐expression in mdx mice increased expression of the UGC and alpha‐7beta‐1 integrin at the sarcolemma membrane and demonstrated amelioration of dystrophic pathology. However, ten‐fold levels of hSSPN over‐expression resulted in early lethality and toxicity in wild‐type mice. Here, we clarify the role of species‐specific SSPN (mSSPN) in skeletal muscle by investigating mSSPN‐Tg:mdx mice for signs of toxicity. Hematoxylin and eosin staining, immunofluorescence assay, and Evan's blue dye assay of quadriceps taken from 6 week old mice were used to determine mSSPN effects on sarcolemma integrity and myofiber regeneration. Physiological tests such as grip strength and wire hang analysis were performed on mdx and mSSPN‐Tg:mdx mice to evaluate muscular strength and mechanical improvement. This information will be necessary to move forward with clinical trials utilizing hSSP 58
SPD 2014 SESSION TWO 190 Characterizing the Significance of ORF31 in Gamma‐Herpesviruses' Late Gene Regulation LAURA B. LIU, Shaoying Lee, Harding Luan, Sara Shu, Emily Duong, Justina Leo, Ren Sun and Ting‐Ting Wu Kaposi's Sarcoma‐associated Herpesvirus (KSHV) and Epstein‐Barr Virus (EBV) are two human gamma‐herpesviruses associated with cancers, especially in infected immuno‐compromised patients. Like all herpesviruses, progression of gene expression during lytic replication consists first of immediate early genes, followed by early genes, and finally late genes. The late genes encode structural components of virions and their expression is essential for the completion of the viral lytic cycles. Because KSHV and EBV do not have an effective cell culture model to study viral lytic replication, we utilize a closely related virus, murine gammaherpesvirus‐68 (MHV‐68) as a model. Previously it was found that in MHV‐68, five open reading frames (ORF)18, 24, 30, 31, and 34 are highly conserved among gamma‐herpesviruses and are required for late gene expression. The expression of late genes is tightly regulated by viral DNA replication but how these two processes are linked together is not clear. ORF31 was selected for further analysis for this project because KSHV ORF31 was previously shown to interact with ORF41, one of the components in the viral DNA replication complex. This result provides a clue to a possible link between late gene expression and viral DNA replication. We will first confirm that the same interaction exists between MHV‐68 ORF31 and ORF40/41 and then determine significance of this interaction in regulating late gene expression. The study will enhance our understanding of the gamma‐
herpesvirus lytic replication cycle. 191 Precise Control of Gene Expression during Hematopoietic Differentiation from Human Pluripotent Stem Cells by Electroporation Mediated RNA Delivery CHONG BIN HE, William Kim and Gay M. Crooks Transplantation of hematopoietic stem cells (HSCs) has been used to treat patients with blood disorders. Unfortunately, the number of appropriate donors is insufficient to accommodate the high demand for transplantation. One potential solution is the generation of HSCs from human pluripotent stem cells (hPSCs), which include embryonic stem cells (hESCs) and induced pluripotent stem cells. Although many studies have shown that it is possible to generate hematopoietic progenitor cells (HPCs) from hESCs, the resulting HPCs do not resemble the HSCs found in vivo. One possible way to improve the hematopoietic differentiation from hESCs is by controlling the gene expression of key master regulatory transcription factors that are important for the onset of definitive hematopoiesis. The purpose of our project is to adapt electroporation mediated RNA transfection for use during hematopoiesis from hESCs and assess its effectiveness as a means to precisely and transiently manipulate gene expression. Our preliminary data show that we can introduce both mRNA and small interfering RNA (siRNA) into cells at key checkpoints in hESC‐
derived hematopoiesis. We show that the inhibition of adaptor protein LNK by introduction of siRNA through electroporation resulted in a three‐fold increase of CD 34 positive cells after hESC‐derived hematopoietic differentiation. The ultimate goal is to utilize transient RNA delivery to precisely control gene express toward the generation of transplantable HSCs from hPSCs. 192 Afferent Vagal Nerve Fibers Impact Efferent Vagal Nerve Response and Promote Heterogeneity in Epicardial and Endocardial Repolarization NUPUR MAKKAR, Kentaro Yamakawa, Eileen So, Pradeep Rajendran, Jonathan Hoang, Aman Mahajan, Kalyanam Shivkumar and Marmar Vaseghi The vagus nerve (VN) is known to modulate cardiac repolarization, however the specific role of afferent fibers in the VN is not well understood. The purpose of this study was to evaluate the effects of deafferentation of the VN on ventricular repolarization, at baseline and during vagal nerve stimulation (VNS). In 6 Yorkshire pigs, the heart and bilateral cervical vago‐sympathetic trunks were exposed, and the VN was isolated. A 56‐electrode sock was placed on the ventricles, and a 64‐electrode basket catheter was placed in the left ventricle in order to obtain epicardial and endocardial activation recovery interval (ARI) recordings. ARI, a measure of refractory period changes, was recorded during baseline and VNS with an intact VN as well as after unilateral and bilateral decentralization of the VN. After unilateral decentralization, ventricular ARI shortened compared to the intact condition. After bilateral decentralization, ventricular ARI shortened even further. Following decentralization, a reduced VNS current was required to bring about a similar change in heart rate and ARI. Dispersion of repolarization (DOR) decreased after unilateral decentralization and bilateral decentralization. Results supported that afferent fibers of the vagal nerve play a role in modulating the efferent vagal input to the heart. Deafferentation increases the effectiveness of vagal nerve stimulation while also decreasing heterogeneity. 59
SPD 2014 SESSION TWO 193 The Search for Dark Matter: Two Strong Candidates DAVID CHOI, Alexey Lyashenko and Katsushi Arisaka 95% of the entire Universe is still unaccounted, and this is labeled dark matter and energy. Axion and Weakly Interacting Massive Particle (WIMP) are believed to be two strong candidates to be dark matter particles. A high Axio‐
electric coupling constant coupled with a low background noise provides a likely probability to detect an Axion. Furthermore, interactions between WIMPs and normal matter produce scintillation, which can be converted to energy. In this poster, I present an ideal Axio‐electric coupling constant that outweighs the XENON100 detector background noise and compare it to a realistic constant. I also present an updated conversion for determining energy with nuclear recoil calibration data from the XENON100 detector. For Axions however, in order to overcome the XENON100 background noise, the ideal Axio‐electric coupling constant requires being unrealistically large. For WIMPs, the new energy conversion provides a more reliable approach in targeting dark matter. With promising results, both candidates show the great potential for improvements in our search for dark matter. 194 Determination of the Antibiotic Resistance and Production Properties of Microbes in the Rhizosphere of Acokanthera Oblongifolia KRISTINE HUYNH, NITYA REDDY, Jessica Tsui, Gregory Harper, Jared Liu, Kris Reddi and Giorgia Pirino Acokanthera oblongifolia can be used to treat snakebites, itches, and internal worms in Africa; however, the fruit, bark, and root are highly toxic to human. As a result, this plant is not well studied. Characterizing microbes of A. oblongifolia can lead to the discovery of novel bacteria with antibiotic production or resistance properties and aid the development of novel antibiotics for therapeutic treatments. The toxin‐producing property of A. oblongifolia suggests that a large percentage of antibiotic resistant and producing microbes should be present in the rhizosphere. In the following study, bacteria were isolated from the plant soil and antibiotic resistance and production assays were performed to assess antibiotic resistance and production properties, respectively. Results indicate that within the 30 isolates tested, none were able to produce Vancomycin while over 50% were resistant to at least one of the following antibiotics: Erythromycin, Tetracycline, Rifampin, Cefuroxime and Sulfamethoxazole with Trimethoprim. 16S rDNA analysis of the individual isolates and metagenomic analysis of the microbial community confirmed that isolates with antibacterial resistance belong to phylotypes with microbes that produce antibiotics. These results suggest that the antibiotic resistant property of each isolate was developed in response to the toxicity of A. oblongifolia, rather than competing against the other microbes in the community. 195 Lamina Neuron Targeting in the Visual System of Drosophila MIRIAM BEYDER, Wael Tadros and S. Lawrence Zipursky The molecular mechanisms underlying the formation of synaptic connections are still poorly understood. Few genes responsible for directing these processes have been identified. The Drosophila visual system is an ideal system in which to address this question due to its amenability to genetic and molecular techniques. Within the fly medulla, second order lamina neurons target to five (M1‐M5) distinct synaptic layers. In an effort to identify novel factors involved in wiring, we conducted a genetic screen addressing the targeting of lamina neurons L1 and L2, which normally target to the M1/M5 and M2 layers respectively. We employed mosaic analysis with a repressible cell marker (MARCM) to assess the effects of large chromosomal deletions, or deficiencies on the targeting of these two neurons. This approach permits the generation and visualization of individual homozygous mutant lamina neurons surrounded by heterozygous tissue. Mutant lamina neurons were identified by variations in morphology, projections to inappropriate layers, and expansion into surrounding columns of the medulla. To date, we have identified four deficiencies, Df(3R)Exel6204, Df(3R)Exel6183, Df(3R)Exel6193, and Df(3R)Exel6167, which result in distinct morphological and targeting phenotypes. Ultimately, we hope to identify the genes responsible for these and other phenotypes found in this screen. Overall, this research will further the understanding of existing pathways involved in neuronal wiring through the identification of novel factors. 60
SPD 2014 SESSION TWO 196 Prolonging the Lifespan of Fibrin Hydrogels Through the Use of Methacrylated Glycol Chitosan and Its Effect on the Proliferation and Differentiation of Mouse Bone Marrow Stromal Cells AUSTIN M. COPP, Soyon Kim and Min Lee The development of an injectable, biocompatible, and biodegradable hydrogel system has long been of interest for the field of tissue engineering. To achieve this aim, several studies have focused on the use of proteins that are used as scaffolds in natural biological processes. One such protein, Fibrin, a protein found in blood that is a key component of hemostasis, has been extensively studied with varying degrees of success. Using fibrin hydrogels as a tissue engineering scaffold has been shown to induce proliferation and differentiation of mouse bone marrow stromal cells in prior studies. Fibrin hydrogels, however, degrade very quickly, limiting their viability for long term therapeutic treatments. The focus of this study was to increase the lifespan of fibrin hydrogels through the use of glycol chitosan, a water soluble biopolymer that has been modified to contain methacrylate groups. This Methacrylated Glycol Chitosan (MeGC) has the additional benefit of being photopolymerizable, allowing the gels to be cured by blue light , an application that would be useful for in‐vivo administration, as blue light is less cytotoxic then UV light. To further reduce the cytotoxic effects of this system, Riboflavin, or Vitamin D, was chosen as the photo‐initiator for its biocompatibility as well. The results of this study have shown that MeGC‐Fibrin composite hydrogels increase the lifetime of the fibrin hydrogels while still retaining some of its proliferation and differentiation capabilities. 197 Transferrin Variants for Enhancing the Drug Delivery Efficacy of Nanoparticles STEPHANIE J. WANG, Ricky Y.T. Chiu, Takuma Tsuji, Juntian Wang, Christina T. Liu and Daniel T. Kamei Current chemotherapies are effective at killing cancer cells, but also demonstrate similar toxicities towards healthy cells, resulting in severe side effects for patients. To address this issue, researchers have investigated transferrin (Tf), an iron carrier glycoprotein, as a targeting ligand since its receptors are often overexpressed on the surface of cancer cells. However, Tf is limited in its efficacy as a drug carrier due to its low cellular association, or short residence time inside the cell. Based on predictions generated by our mathematical model, our group previously engineered Tf variants, which exhibited increased cellular association due to lowered iron release rates and were able to more effectively deliver a conjugated protein toxin compared to native Tf. Here, we investigated the effects of our Tf variants on nanoparticles by first extending our mathematical model to accurately represent the events of a Tf‐
conjugated nanoparticle trafficking pathway. In vitro radiolabeled trafficking studies with the PC3 prostate cancer and A549 lung cancer cell lines validated the model's predictions. Subsequently, cytotoxicity studies performed in PC3 and A549 cells and also in a mouse PC3 xenograft model demonstrated that this increase in cellular association further translates into an enhanced ability to deliver the chemotherapeutic doxorubicin. Our findings illustrate the benefits of using a systems analysis in a forward engineering approach to design novel targeting ligands. 198 The Role of Murine gammaherpesvirus 68 Open Reading Frames 40 and M1 in Modulating the Unfolded Protein Response Jane Wang, Jiaying Feng and Ren Sun The Gammaherpesvirinae subfamily of Herpesviridae is known for its inclusion of the cancer‐implicated Epstein‐Barr virus (EBV) and Kaposi's sarcoma‐associated herpesvirus (KSHV). A genomic screen of MHV‐68, a model for EBV and KSHV, identified the ORF40 and M1 genes to be regulators of the cellular unfolded protein response (UPR). UPR activates in response to the accumulation of unfolded or misfolded proteins in the host ER lumen and aims to restore normal cell function by halting protein translation and increasing the production of protein chaperones to unclog host machinery. Because viruses exploit host cellular machinery, viral synthesis often leads to ER stress and consequentially triggers the UPR. In vitro studies suggest that ORF40 represses while M1 induces activation of the response, and we hypothesize that ORF40 is expressed earlier in the viral replication cycle to suppress induction of UPR while M1 is expressed later to stimulate production of chaperone proteins that aid in viral protein folding. To investigate the order of ORF40 and M1 expression kinetics, a dual‐tagged ORF40‐FLAG, M1‐HA virus was constructed and will be infected in 293T human embryonic kidney cells; mRNA and protein will be collected every 4 hours on a 24‐hour scale and analyzed through qPCR and Western blotting. This study of the MHV‐68 and its host may further elucidate the viral‐
host interactions and potentially lead to the development of novel therapeutic treatments. 61
SPD 2014 SESSION TWO 199 Proteomic Characterization of the Nexin‐Dynein Regulatory Complex in Trypanosoma brucei. MARSHA R. CHENG, Gerasimos Langousis and Kent Hill The Nexin‐Dynein Regulatory Complex (N‐DRC) is a massive protein complex that is an integral part of virtually all eukaryotic flagella (also known as cilia). N‐DRC consists of approximately ten subunits, yet its exact composition remains enigmatic. The function of N‐DRC is unclear; it is thought to regulate axonemal dyneins and hence proper flagellar beating, an essential physiological function for diverse eukaryotic cells. It was shown recently that mutations in DRC1, a subunit of N‐DRC, lead to primary ciliary dyskinesia, a disorder characterized by dysfunctional motile cilia in humans. Moreover, studies in green algae have shown that absence of DRC1 leads to disruption of N‐DRC. Flagella assembly and motility is paramount in the growth and pathogenesis of Trypanosoma brucei, the causative agent of lethal sleeping sickness. In particular, N‐DRC proteins have been shown to be essential for normal parasite motility. Immunofluorescence of epitope‐tagged DRC1 allows for confirmation of its location in the axoneme. Assuming that DRC1 is a subunit of N‐DRC (based on previous research in Chlamydomonas and humans), proteins in N‐DRC that depend on DRC1 for assembly are determined through SILAC proteomics using DRC1 mutants. 200 Characterization of Insoluble Protein Aggregation in C9ORF72 TANYA S. KIM, Mochtar Pribadi and Giovanni Coppola A hexanucleotide repeat expansion in C9ORF72 is the leading cause of familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This intronic expansion mutation is translated through a mechanism known as Repeat Associated Non‐ATG Translation (RANT), producing protein deposits in the brains of affected patients. We hypothesize these deposits are toxic insoluble aggregates, capable of spreading through a prion‐like propagation. These aggregates may include one or more of the possible RANT products. To determine the toxicity of these aggregates, each possible product of C9ORF72 RANT was introduced to 293 cells and analyzed for potential protein aggregation and propagation. We found that only the Glycine‐Alanine (GA) RANT product forms high molecular weighted fibrillary aggregates. However, co‐transfection of the GA frame with the other five possible frames showed no co‐localization with the other RANT products. This study suggests that the GA expansion frame possesses a greater potential for forming aggregating RANT product of the non‐canonical C9ORF72 hexanucleotide repeat translation. However preliminary results do not sufficiently support a prion‐like propagation. 201 Developing Liver Specific Vector for Gene Therapy Application JANE KANG, Weidong Xiong and Vaithi Arumugaswami FAH (fumarylacetoacetate hydrolase) is needed to break down the amino acid tyrosine in the liver and kidneys. Without FAH, a tyrosine product called fumarylacetoacetate accumulates, causing liver and kidney problems characteristic of type I tyrosinemia, which affects 1 person out of 100,000 worldwide. Genetic material can be delivered into hosts to treat such genetic disorders via viral vectors. Furthermore, our goal is to develop a vector for liver specific expression of target genes. Reporter analysis of the promoter of microRNA‐122 (mir‐122), which is abundantly expressed in liver tissue, showed that a 280 base pair minimum promoter element is critical for liver specificity. My preliminary results show that I have successfully cloned the mir‐122 promoter upstream of the FAH gene in an expression vector. I included promoters, such as CMV (cytomegalovirus) and AAT (alpha 1‐antitrypsin) as positive controls. The mir‐122, AAT, and CMV promoters were PCR amplified and digested with Xba1 and Age1 restriction enzymes. Subsequently, these fragments were subcloned upstream of the FAH gene in an adenoviral shuttle vector. Currently, I am evaluating the specific expression of the FAH gene using a liver cell line and HeLa cell line. In conclusion, I have cloned and verified various promoters in an adeno‐shuttle vector. In the future, I will generate adenoviral vectors using the shuttle clones. Subsequently, these vectors will be evaluated for rescuing FAH gene deficiency in a knockout animal model. 62
SPD 2014 SESSION TWO 202 The Role of Autophagy and MicroRNAs in Regulating Tissue Growth in Drosophila Melanogaster SINA FAMENINI and Julian A. Martinez‐Agosto Autophagy is a tightly regulated intracellular catabolic process involving the lysosomal degradation of cytoplasmic organelles and proteins and essential for maintaining cellular homeostasis. RNA interference mediated for miRNAs, is another cellular regulatory mechanism responsible for the post‐transcriptional silencing of a wide array of genes. Dicer1 is an enzyme responsible for silencing genes by cleaving double‐stranded hairpin RNA molecules and forming the RNA induced silencing complex (RISC). While autophagy and miRNA pathways have been extensively studied individually, many questions about their interactions remain unanswered. Most interestingly, patients have been identified to carry mutations in Dicer1 and in known autophagy genes. In order to study the relationship between autophagy and miRNAs, tissue specific UAS/Gal4 drivers were used to induce the expression of RNAi constructs targeting the pathway in Drosophila Melanogaster. Analysis of the larval and adult progeny revealed aberrant tissue growth conditions. Downregulation of specific ATG genes and Dicer1/2 revealed undergrowth phenotypes associated with apoptosis without affecting PI3K and TOR pathways. Downregulation of autophagy in a background of reduced Dicer1 activity caused distinct combinatorial undergrowth phenotypes in the wing imaginal discs of third instar larva and in adult flies. These findings demonstrate a specific requirement for autophagy and miRNA signaling in tissue growth. 203 Antimicrobial Peptide Cathelicidin Inhibits Salmonella Mediated Intestinal Inflammation and Fibrosis
BOWEI SU, Samantha Ho, Kyriaki Bakirtzi, Richard E. Isaacson, Charalabos Pothoulakis and Hon‐Wai Koon Cathelicidin is a family of endogenous anti‐microbial peptides with anti‐inflammatory effects. We determined whether exogenous cathelicidin can modulate intestinal fibrosis in an animal model of intestinal inflammation. 129SvJ mice (n=6 per group) were pretreated with streptomycin orally followed by Salmonella typhimurium (strain SL1344) 3x10(8) cfu/100ul using oral gavage; selected mice received cathelicidin expressing lentivirus (Camp‐LV) 1x10(7) infectious units per mouse intravenously on day 11. Cecal tissue, the main location of Salmonella induced inflammation and fibrosis, was obtained on day 21 for analyses. Chronic Salmonella infection induced cecal inflammation with fibrosis and stricture (7x increase in TNF mRNA p=0.0001 and 3.6x increase in collagen Col1a2 mRNA p=0.0005). Cecal inflammation and fibrosis were significantly reduced by mouse cathelicidin expressing lentivirus (40% reduction in TNF mRNA p=0.01). Salmonella induced cecal endogenous cathelicidin expression (140 fold p=0.0001). Cathelicidin expressing lentivirus further increased its expression to 268 fold (p=0.03). Cathelicidin treatment reduced Salmonella induced cecal histological damages (60% reduction in histology score, p=0.0001) and collagen deposition (50% reduction in fibrosis score, p=0.0001) as observed by H&E staining and Masson Trichrome staining. Lentiviral administration of mouse cathelicidin modulates Salmonella induced intestinal inflammation and fibrosis in mice. 204 The Effects of Predation on Germination Potential in the Mojave Desert Shrub Catclaw Acacia, Acacia (Senegalia) greggii A Gray THEA PERCIVAL, CASSIE HUYNH and Keith Gaddis Seed herbivory is a dominant limiting factor to reproduction in plant species. Total destruction of a seed reduces the number of offspring a plant contributes to the next generation. Some seeds are capable of sustaining minor amounts of damage, and still germinating. We investigate the effects of herbivory damage to seeds of the California native desert shrub Cat Claw Acacia, (Senegalia greggii), by comparing germination among seeds mechanically damaged. We observed seeds for 34 days, in a controlled laboratory setting, recording the dates of first growth and mortality. We found that minor amounts of herbivory act to increase germination success in seeds, but also leads to a higher likelihood of mortality. This study indicates a level of resilience in desert plant species, and informs predictions of dispersal in this and similar species. Information about desert species in particular is incredibly valuable as the debate popularizes of using the desert to harness renewable energy, which will inevitably alter what has remained until now a relatively untouched environment. 63
SPD 2014 SESSION TWO 205 Affinofile Profiling of Infant and Maternal HIV‐1 Envelopes Demonstrates That Increased CCR5 Usage Efficiency is Selectively Favored in Mother‐to‐Child HIV‐1 Transmission AMIT SUMAL, Kelechi Chikere, Angelique Cercillieux and Benhur Lee Although millions of children have been perinatally infected with HIV‐1, various aspects behind this mode of transmission are still unclear. Previous studies have found that only a limited subpopulation of maternal virus is typically transmitted to an infant, and suggest that these viruses possess selective advantages to transmission. In particular, HIV‐1's envelope glycoprotein has been seen as a target of this selective pressure, but current studies have only addressed its role in evading the maternal immune response. The envelope also plays an essential role during infection, and its relative use of CD4 and CCR5 during cell entry is known to influence infectivity. To determine which receptor's usage efficiency is selected for in perinatal transmission, we investigated the CD4 and CCR5 usage efficiencies of fourteen maternal and infant envelopes. These envelopes were analyzed using GGR Affinofile cells, which can be simultaneously induced to express CD4 and CCR5. The infectivity data of each envelope was then processed by Viral Entry Receptor Sensitivity Analysis (VERSA), which quantitatively describes an envelope's relative usage of CD4 and CCR5 as a VERSA angle. Comparing VERSA metrics for each envelope found that every transmitted envelope used CCR5 more efficiently than its corresponding maternal envelope. This result proposes that perinatal transmission effectively selects for increased CCR5 usage efficiency and implicates mucosal macrophages and CD4+ memory T cells as primary targets of perinatal transmission. 206 Characterization of Acetohydroxyacid Synthase in Clostridium thermocellum KANE NANIA and James C. Liao Acetohydroxyacid synthase (AHAS) is an enzyme that catalyzes the first step of the biosynthesis of branched chain amino acids. Preliminary results have indicated that in Clostridium thermocellum, this enzyme is also involved in a key step in the production of isobutanol, acting as a 2‐ketoisovalerate decarboxylase (KIVD). C. thermocellum is an attractive organism for its potential to produce biofuels like isobutanol as it can degrade lignocellulosic biomass at high temperatures. In this work, the AHAS subunits of C. thermocellum have been cloned, purified, and characterized for both their expected AHAS activity as well as their KIVD activity. In addition, we investigate the effects of a point mutation in the gene encoding the regulatory subunit aimed at decreasing end‐product inhibition. 207 Frames of Reference in Sensory Integration: How eye position influences audiovisual computations JASON M. CARPENTER, Brian Odegaard and Ladan Shams How does the position of our eyes influence what we see and hear? When the brain encodes sensory input, each sensory modality encodes information in its natural reference frame: visual information is encoded with respect to the retina, and auditory information with respect to head position (Pouget et al., 2002). When we receive simultaneous audiovisual information, the brain could encode combined signals in an eye‐centered reference frame, head‐centered reference frame, or some combination of the two. In our study, subjects localized synchronous flashes of light and bursts of sound, with eyes fixated centrally (aligned reference frames) or peripherally (misaligned reference frames). Data were analyzed using a Bayesian Model, which can identify subjects' prior localization biases that depend on eye and head position. We hypothesized that during peripheral fixation, localization responses would reflect both eye and head‐dependent biases, indicating the brain uses a hybrid reference frame when encoding stimuli. Results will provide novel insights into how the brain computationally processes audiovisual information, and the role that eye position plays in these calculations. 64
SPD 2014 SESSION TWO 208 Protein Kinase M Maintains Long‐Term Sensitization and Long‐Term Facilitation in Aplysia RASHI SINGH, Kaycey Pearce and David L. Glanzman Understanding how the brain maintains long‐term memories is one of the major outstanding unknowns in modern neuroscience. Evidence from mammalian studies indicates that activity of a protein kinase C (PKC) isoform, protein kinase M zeta (PKMzeta), plays a critical role in the maintenance of long‐term memory. Recently, a PKM isoform, known as PKM Apl III, was cloned from the nervous system of Aplysia. We examined the role of PKM Apl III in long‐
term memory maintenance in Aplysia. Intrahemocoel injections of the pseudosubstrate inhibitory peptide ZIP (zeta inhibitory peptide) or the PKC inhibitor chelerythrine erased the memory for long‐term sensitization (LTS) of the siphon‐withdrawal reflex (SWR) as late as 7 d after training. In addition, both PKM inhibitors disrupted the maintenance of long‐term (>24 h) facilitation (LTF) of the sensorimotor synapse, a form of synaptic plasticity previously shown to mediate LTS of the SWR. Our results support the idea that long‐term memory in Aplysia is maintained via a positive‐feedback loop involving PKM Apl III‐dependent protein phosphorylation. Furthermore, the demonstration that PKM activity underlies the persistence of LTF of the Aplysia sensorimotor synapse, a form of synaptic plasticity amenable to rigorous cellular and molecular analyses, should facilitate efforts to understand how PKM activity maintains memory. 209 An investigation Into the Functional Role(s) of the lncRNA Xist Repeat Domains During Initiation of X‐
Chromosome Inactivation WALTER MANCIA, Amy Pandya‐Jones and Kathrin Plath One function of the rapidly growing class of non‐coding RNAs is to effect changes in chromatin state. However, the mechanism by which these lncRNAs exert their function remains unclear. Here we use X chromosome Inactivation (XCI) in mouse Embryonic Stem Cells (ESC) to study how the long non coding RNA Xist regulates chromosome‐wide gene silencing. In mammalian female cells, one of the two X‐chromosomes is transcriptionally silenced during development to compensate for the dosage imbalance of X‐linked genes between males(XY) and females(XX). The crucial RNA molecule required for initiation of XCI is Xist. Upon differentiation of female ESCs, Xist is rapidly upregulated and coats the future Xi in cis, leading to a transcriptionally silenced state of the chromosome that is maintained throughout all subsequent somatic cell divisions. Recent work has revealed that during the initiation of XCI, Xist exploits the three‐dimensional architecture of the chromosome to spread in a sequence independent manner across the chromosome. This finding has prompted us to define the function of six repeat‐regions of Xist in mediating Xist RNA function. To this end, we are engineering six targeting vectors that upon homologous recombination on the X chromosome in mouse ESCs will each generate a version of the Xist transcript that is deleted of a single repeat. We will test the ability of each Xist mutant to coat the X‐chromosome and induce silencing by Fluorescence In Situ Hybridization for the Xist RNA and X‐linked genes. 210 Not So Hot Spots? High Speciation Rates Do Not Explain Reef Fish Species Richness in the Coral Triangle MERICIEN VENZON, Andrew Noonan, Peter F. Cowman, Daniel L. Rabosky and Michael E. Alfaro Amongst all marine habitats, coral reefs have historically been recognized as areas of highly concentrated diversity, with a maximum amount of fish diversity occurring in the Indo‐Australian Archipelago (IAA) a collection of tens of thousands of islands recognized for its diverse biotas and high numbers of endemic species. In the last decade the IAA has been recognized as an important engine of marine fish biodiversity through three possible mechanisms: the IAA as a center of origin, a center of refuge or survival, and as a center of accumulation. More recently, a dynamic role for the IAA has been suggested with the region acting during different geologic intervals as a center of origin or refuge. We tested these hypotheses using a suite of comparative phylogenetic methods across eight major lineages of reef fishes. Contrary to the center of origin hypothesis, we find little evidence for exceptional speciation rates within the IAA. Similarly, we found no evidence for time‐dependence of diversification rates for most families. Analysis of diversification rates across additional marine hotspots also showed that speciation rates within these regions are not exceptionally rapid. Elevated rates of dispersal into the IAA detected across all families support the center of accumulation hypothesis, and suggest that the connectivity between regions is the main factor driving extant global biodiversity patterns. 65
SPD 2014 SESSION TWO 211 Hypomethylating Agent Decitabine Increases Expression of Immunogenic Antigen in Glioblastoma Multiforme PRADHAN U. BHAT, Joseph P. Antonios, Richard G. Everson, Horacio Soto, Linda M. Liau and Robert M. Prins Once considered an immune‐privileged site, it is now known that the immune system is capable of mounting responses to foreign antigen in the central nervous system. Advances in immunotherapy have utilized this knowledge in targeting glioblastoma multiforme (GBM), an astrocytic tumor with poor clinical outcomes. However, GBM remains resistant to immunotherapeutic approaches due to its low immunogenicity. Recent studies have revealed expression of cancer‐testis antigens (CTA) by some human malignancies. While CTAs are immunogenic and may therefore elicit an immune response, they are not endogenously expressed by glioma. We hypothesize that the induced expression of CTA may provide a novel immunotherapeutic target on GBM. Our study investigated the use of decitabine, a hypomethylating agent, to induce expression of the CTA NY‐ESO‐1 in glioblastoma. Primary GBM lines were treated with the drug, and compared to primary lines, which endogenously express NY‐ESO‐1. Results of quantitative polymerase chain reaction were analyzed to determine differences in relative NY‐ESO‐1 expression among cell lines. The antigen's expression in GBM following decitabine treatment was found to be upregulated. Our findings suggest the therapeutic use of decitabine increases expression of immunogenic CTA NY‐ESO‐1 and may enhance immunotherapy outcomes. Future investigations will utilize exCELLigence assays to monitor recognition and killing of decitabine treated GBM by NY‐ESO‐1 transduced T‐cells. 212 Anatomical Impact of Reelin Haploinsufficiency and Prentatal Organophosphate Pesticide Exposure on Mouse Corpus Callosum and Hippocampus DAE WOONG KIM, Brian R. Mullen and Ellen M. Carpenter Autism Spectrum Disorders (ASD) are neuropsychiatric disorders that exhibit symptoms of impaired communication, reduced emotional response, poor social skills, and compulsive repetitious behaviors. While the exact etiology of ASD has not been identified, it is believed that a combination of genetic and environmental factors contributes to the development of ASD. This study further explores the interaction between genetic and environmental factors by examining the combinatorial effect of reelin deficiency and prenatal exposure to organophosphate pesticides. Previous experiments have shown that mice with the reelin deficiency or the exposure to pesticide result in some behaviors seen in ASD patients, but the combination of the two variables shows a paradoxical mitigation of ASD‐like behavior. This study is aimed towards observing neuroanatomical changes, specifically the white matter tracts of the corpus callosum and the cell morphologies of the hippocampus. The data from this study shows changes in sagittal cross sectional areas of the corpus callosum in mice affected by both reelin haploinsufficiency and prenatal chorpyrifos (CPO) exposure. There were no changes seen with animals that had either reelin heterozygocity or CPO treatment alone in cross sectional sagittal areas of white matter. In addition, we looked for morphological changes within granular and pyramidal cells of the hippocampus. Prenatal CPO increased spine density and reelin haploinsufficiency showed decreased spine density. 213 Dopamine‐Resistant (DRD2 A1) Receptor Polymorphism May Lead to Food Addiction AMY TRANG, Joanna Yeh, Zhaoping Li and Susanne M. Henning Obesity is a large public health concern that continues to be studied today. Food addiction may be relevant because it activates dopaminergic reward regions in the brain. Those with the Taq1A polymorphism of the A1 allele may be addicted to certain foods and overeat to counteract reduced dopamine receptor density. To evaluate associations between DRD2 genotype, food cravings, overeating, and body mass index (BMI), we conducted questionnaires (Power Food Scale/PFS, Food Craving Inventory/FCI), drew blood, and recorded body measurements. 12 healthy obese adolescent patients participated, with mean BMI 37.3 (±4), mean FCI score 6.96 (±3), and mean PFS score 39.4 (±18.7). FCI and PFS were positively correlated with BMI, (r‐value 0.18‐0.37). Adolescent patient blood samples have not been genotyped yet. 132 healthy college students participated, with 68 samples genotyped up to date. Those with at least one A1 allele had mean BMI 23.2 (±4), mean FCI score 1.2 (±0.5), and mean PFS score 2.9 (±0.9). Those lacking the A1 allele had mean BMI 23.5 (± 3.4), mean FCI score 1.1 (±0.6), and mean PFS score 2.7 (±0.8). After analyzing 68 data sets, no difference was observed between average scores of FCI (p‐value: 0.711), PFS (p‐value: 0.361), or BMI (p‐value: 0.744) of A1/A1 and A1/A2 compared to A2/A2 genotypes. Final data analyses will be performed after all genotyping will be completed. Food cravings and food addiction may contribute to obese phenotypes through the DRD2 A1 polymorphism. 66
SPD 2014 SESSION TWO 214 Functional Characterization of Novel Rhoptry Effector Proteins in Toxoplasma gondii JENNIFER NGO and Peter J. Bradley The phylum Apicomplexa are obligate intracellular parasites that invade their mammalian host cells using specialized secretory organelles. Within this phylum are Plasmodium falciparum, the causative agent of malaria, and Toxoplasma gondii, an important pathogen of immune‐compromised persons. These parasites are able to actively invade host cells with two apical secretory organelles, the micronemes and rhoptries. The protein constituent of the rhoptries includes rhoptry neck proteins (RONs) and rhoptry body proteins (ROPs). The RONs help the parasite to pass through the host cell membrane by forming a moving junction; whereas the ROPs are injected into the host cytosol to further hijack the host cell. Previous analyses have shown that secreted ROP proteins consist of kinases, protein phosphatases and novel rhoptry proteins. ROP9 has been identified as a possible novel effector protein to target the host cell and a putative virulence determinant. In this project, we investigate ROP9 whose characteristics make it a good candidate to be a regulatory element during host infection. The effector protein is predicted to be a soluble effector protein that is also phosphorylated. We confirmed that ROP9 may play a role in host hijacking systems with an exogenously expressed green florescent protein chimera. To assess the function of ROP9, we knockout the gene with a drug resistance marker through homologous recombination. The knockout was successful which suggests ROP9 is not an essential protein in the parasite’s lytic cycle, but is likely an effector that modulates host functions. We are now generating tandem‐affinity fusion proteins for immunoprecipitation and complementing the wild‐type gene to get a clearer understanding of both in vivo and in vitro phenotypes. 215 Multi‐level Immune Cell Flow Cytometry and Gene Expression Characterization of the Immune Response of Patients with Advanced Heart Failure Undergoing Mechanical Circulatory Support Therapy JAY R CHITTOOR, Yael Korin, Martin Cadeiras, Johanna .M Schaenman, Murray H. Kwon, Tiffany Sidwell, Fadi Kandarian, Galyna Bondar, Mario C. Deng and Elaine F. Reed A main cause of death in Advanced Heart Failure (AdHF) after Mechanical Circulatory Support Device (MCSD) implantation is the Multiorgan Dysfunction Syndrome (MOD). We aimed to incorporate multi‐level immune cell flow cytometry and genome wide transcriptome analysis to improve phenotype characterization. PBMC from 11 patients were obtained at days ‐1, 1, 3, 5 and 7 before and after MCS surgery, and from 3 healthy volunteers at similar timepoints. Immunophenotyping (IP) was performed with multi‐color monoclonal antibody T cell panel on LSR Fortessa. Whole‐genome transcriptome profiling was assessed by Next‐gen mRNA Sequencing. AdHF risk was assessed using the InterMACS classification and MOD was quantified by organ function parameters. Bioinformatics analysis was performed. Heat maps were constructed for visualization. Compared to lower risk patients (InterMACS level 3, n=5) high risk patients (levels 1 and 2, n=6) showed IP profile of chronically activated CD57+PD‐1+ CD8 T cell subsets with high frequencies of memory and terminally differentiated (TEMRA) cells before MCSD surgery across all 5 time‐points. Genes involved in the establishment of these IP showed differential gene expression (q< 0.05). Combination of gene expression and IP‐ supervised markers predicted high risk MOD patients after MCSD surgery. Simultaneous assessment of IP surface marker and gene expression are useful to improve characterization and risk prediction in patients with AdHF who undergo MCSD surgery. 216 Analysis of Interview Data from IBS Patients Exposed to Physical Activity Shows Reduced Stress and Improved Mood SUN YOUNG PARK, Leila Shahabi, Bruce D. Naliboff and David Shapiro Irritable Bowel Syndrome (IBS) is a gastrointestinal disorder that presents with symptoms of abdominal bloating, pain from bowel movements, and altered bowel habits. Previous studies have shown that there is a link between stress, the brain and symptoms of IBS. Physical activity, such as yoga and walking, is thought to impact both physical and psychological well‐being, and, some postures are specifically designed to reduce pain and stress. This study is designed to assess the effectiveness of the movement‐based approaches to alleviate IBS symptoms. Here we analyze qualitative, semi‐structured interview data of 26 IBS patients undergoing two types of physical activities. Patients were randomly assigned to either lyengar yoga or structured walking groups and the interviews were conducted afterwards. The data was divided into three groups: reduction of IBS symptoms, mood alleviation and treatment efficacy. The post‐treatment interviews suggest that yoga is associated with alleviating IBS symptoms in 64 percent of interviewed patients, though many still considered it as a complementary to other standard treatments. The results also show that walking is associated with reduced stress and improved mood in 89 percent of interviewed patients and there was a preference for walking over medications. The study suggests that yoga and walking are effective in alleviating IBS symptoms, and this highlights the importance of physical activity in IBS patients. 67
SPD 2014 SESSION TWO 217 Induction of Human Germ Cell Fate by Overexpression of Transcription Factors In Vitro TIASHA AYUMI SHAFIQ , Sofia Gkountela and Amander T. Clark Correct germ cell development is required for the generation of male and female gametes so that genetic and epigenetic information can be carried into the next generation. Abnormalities in germ cell development can cause germ cell tumors as well as infertility problems, which affect about 10‐15% of men and women in the US. Human Primordial Germ cells (PGCs) can be spontaneously differentiated from Human Embryonic Stem cells (hESCs) in vitro, but these immature PGCs are very rare and do not develop into mature PGCs that could be clinically relevant in the future. To increase the yield of PGCs and to overcome the differentiation bottleneck, the aim is to induce expression of BLIMP1, PRDM14 and NANOS3, three critical genes in germ cell specification during the differentiation process. Using Zinc Finger Nucleases, the construct containing PRDM14 and NANOS3 is being inserted into the AAVS1 safe harbor locus in hESCs and expression induced using the TET‐ON system. Moreover, BLIMP1, PRDM14 and NANOS3 are separately cloned into pCDH vectors to produce lentiviruses and transduced into hESCs. The protein and gene expression of the plasmids have been verified using western blot and qRT‐PCR respectively. These tools will be used to observe an increase in PGC numbers, survival and mature traits after hESCs differentiation. Therefore, developing mature PGCs in vitro from human pluripotent stem cells could be of importance in understanding human germ cell development and possibly treating infertility problems.
218 High Fat, High Cholesterol Diet Induces Systemic Inflammation and Vascular Complications Through Increasing Lysophosphatidic Acid in the Intestine GABRIELLA R. MARVIZI, Sabere Dejbakhsh, Victor Fung, Nika Karimi, Farhad Sedigh, Roshanak Alialy, Nasim Golchin, Maryam Shabihkhani, Greg Hough and Mohamad Navab Apolipoprotein A‐I mimetics showed promise in animal models of disease and improved HDL function in humans when given orally at high doses despite achieving low plasma peptide levels. However when high plasma levels were achieved with low doses of peptide given intravenously or by subcutaneous injection, improvement in HDL function was not seen. This raised the possibility that the site of the peptide's anti‐inflammatory action is the intestine. Lysophosphatidic acid is a factor shown to be involved in inflammation. LDL R‐/‐ mice were fed a high fat, high cholesterol diet for two weeks. Another group received HDL mimetic peptide in the diet containing high fat and cholesterol. Mice were then fasted, blood was removed, and plasma prepared. The animals were perfused with cold saline and the small intestine removed. Lysophosphatidic acid levels in plasma and in intestine were determined using liquid chromatography electrospray ionization mass spectrometry. The high fat diet resulted in a significant rise in plasma and intestine lysophosphatidic acid compared to the values for the mice on the chow diet (p<0.01. p<0.001 respectively). The arteries of the experimental mice showed fatty streaks and lipid accumulation. However, the group that received the HDL mimetic peptide did not show significantly elevated levels of lysophosphatidic acid compared to the chow group. Feeding a high fat, high cholesterol diet to animal models results in inflammation and vascular dysfunction, and HDL mimetic peptide reduces this effect.
219 Understanding Transcriptional Changes in Retinal Degeneration in Drosophila Melanogaster SAMPAT SINDHAR, Alex Tonthat, Amy Alayari, Sam Bounds, Zafar Gill, Stephanie Jensen, Tiffany Mao, Jackie Mao, Arash Rafi, Shanelle Shahery and Frank Laski Retinal degeneration (RD) diseases, such as retinitis pigmentosa, are the leading cause of irreversible blindness and have been shown to have significant genetic contributions. To identify genes involved in RD, the lab has been using the UAS/Gal4 system coupled to RNAi to create eye‐specific knockdowns in Drosophila melanogaster. The screen identified importin genes, which transport molecules to the nucleus, as having an RD phenotype. Given the role of importin genes, we hypothesized that damage to the photoreceptor cells would cause a signal molecule, bound by an importin, to travel to the nucleus and upregulate the expression of rhabdomeric proteins After white‐eyed Drosophila were aged in the light to induce damage to the photoreceptor cells, RT‐PCR was used to determine the relative gene expression of NinaE, the major rhodopsin protein, and NinaA, a chaperone protein. When compared to Drosophila aged in the dark, Drosophila aged in light exhibited a two‐fold decrease in NinaE expression and about a three‐fold increase in NinaA expression. Downregulation of NinaE in light may be a mechanism to detect less light and reduce apoptosis in photoreceptor cells, as ß‐arrestin/rhodopsin complexes have been shown to lead to cell death. Other experiments showed that an action potential was likely needed to mediate the decrease in NinaE expression and prior exposure to light likely insulated the eyes from changes in transcription. The insights gained from this project may be useful in understanding retinal degeneration diseases.
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SPD 2014 SESSION TWO 220 The Role of PACAP in Cortical Spreading Depression IRVIN C. LIEN, Serapio M. Baca and Andrew Charles Pituitary adenylate cyclase‐activating peptide (PACAP) is a polypeptide that stimulates adenylate cyclase and subsequently increases the cAMP level in target cells, thereby affecting signaling cascades in the cell important for physiological processes. PACAP is a potent dilator of blood vessels that is believed to be an important mediator of neurovascular function. Administration of PACAP evokes an attack of migraine in susceptible individuals, and PACAP is a promising therapeutic target for migraine. In our study, we investigated the potential role of PACAP in basic mechanisms of migraine. We used optical intrinsic signal (OIS) imaging and local field potential (LFP) recordings to study the phenomenon of cortical spreading depression (CSD), a slowly propagated wave of brain excitation followed by inhibition that is believed to underlie the migraine aura. We compared the neural and vascular patterns of CSD in knock‐out mice lacking PACAP with those of wild‐type mice. Our preliminary data indicate that CSD propagates with different rates in mice lacking PACAP as compared with controls, and that there is a larger and sustained constriction of blood vessels following CSD in the PACAP knock‐out mice. These results indicate a potentially important role of PACAP in basic mechanisms of migraine, and support further investigation of therapies for migraine and other neurological disorders that target PACAP and its receptors. 221 Metaheuristics of Agent Based Swarm and Contagion DANIEL MOYER and Andrea Bertozzi Swarming is a natural phenomenon observed in numerous animal species, such birds, fish, and humans. Groups of individuals move cohesively and exhibit complex group behavior despite the lack of obvious leadership or communication. Previous mathematical literature has explored particle based models for reproducing such swarms, with more recent work introducing an emotional contagion along with the dependence on space and velocity. In the present work we explore a contagion model that allows parameters to respond to selection pressure. In particular, we present numerical results of simulated evolutionary selection on a variety of parameters. Two separate genetic algorithms were implemented and run for a large number of generations on the same numerical framework. The framework itself implemented a multi‐threaded linked cell scheme. Both algorithms show apparent convergence to the same biologically interpretable values. 222 The Potent Growth Promoter Lysophosphatidic Acid Induces Systemic Inflammation That Is Reduced By HDL Mimetic Peptides NEGAR ARAM, Sepideh Shakeri, Nasim Golchin, Zarina Barseghyan, Katayoun Abtin, Soode Nili, Loris Orbelians, Victor Fung, Shila Safar, Susan Hama, Reza Peyman, Yalda Behbahanian,Greg Hough and M. Navab Introduction. Oxidized lipids can act as lipid mediators of inflammation. Lysophosphatidic acid (LPA) has been known as a potent tumor promoter acting in many ways to contribute to uncontrolled growth and metastasis. In addition (LPA) can contribute to atherogenesis and cardiovascular dysfunction. Objective. In the present study our group sought to determine the effect of lysophosphatidic acid in a mouse model of atherosclerosis on the induction of proinflammatory molecules and on systemic inflammation. Methods. LDL receptor null mice were maintained on a laboratory rodent chow for two weeks. One group received in addition, 1 ug per gram diet of LPA. After two weeks the mice were fasted overnight, anesthetized, blood was removed from the retro orbital sinus, plasma prepared. Using LC ESI‐MS we determined circulating lysophosphatidic acid levels. The plasma levels of SAA, was determined using an ELISA kit. Results. The plasma levels of LPA in the animals that received this lipid in their diet was higher than those on the rodent chow alone (p less than 0.012). Plasma SAA also showed a significant increase in the group that received LPA (p less than 0.011). These increases correlated with the lesion scores in the mice (r2= 0.65). Conclusion. These findings indicate that oral LPA can induce systemic inflammation and contribute to vascular complications. 69
SPD 2014 SESSION TWO 223 Achievement Goals and Metacomprehension Accuracy CHARLIE HALL, Kenji Ikeda, Kou Murayama, Mo Ally, Carole L. Yue and Alan D. Castel Accurate metacomprehension (MCP) requires both object‐level processing (e.g., basic reading comprehension) and meta‐level processing (e.g., self‐monitoring of comprehension) and promotes effective learning. Inaccurate MCP often leads to unwise study choices and ineffective learning. The present study examined the potential interaction between two factors that may influence MCP accuracy: working memory capacity (WMC) and achievement goals. High WMC allows learners to engage in dual‐level processing, leading to greater accuracy. Mastery achievement goals (i.e., intent to master a skill) are associated with both object‐ and meta‐level processing, whereas performance achievement goals (i.e., intent to perform normatively) are associated with only object‐level processing. Therefore, we predicted that mastery goals would improve MCP accuracy and learning, especially for high‐WMC participants. Undergraduates completed a WMC task before reading comprehension task, composed of unimportant, important, and inference questions about six different passages, with mastery or performance goals introduced at encoding. Though mastery goals led to numerically higher accuracy, this effect did not reach significance (p = .23), with no interaction between goal type and WMC. Interestingly, however, performance goals led to better performance on important questions (p < .05), but also greater overconfidence than did mastery goals. These results indicate that achievement goals may have unexpected effects on MCP accuracy and, subsequently, learning. 224 Hedgehog Signaling in the Postnatal Mouse Retina Development LILLIAN LAI, Kiyo Sakagami, Xiangmei Zhang and Xian‐Jie Yang The vertebrate retina consists of seven specific neuronal cells types that are responsible for sensing and processing visual information. Elucidating the mechanisms underlying retinal development will provide insights into retinal degenerative disease processes and treatments. Previous studies have shown that Hedgehog (Hh) signaling profoundly influences progenitor cell proliferation and cell fate decision in the embryonic mouse retina. However, its role in postnatal stages remains uncharacterized. To study the requirement of Hh signaling in postnatal mouse retina, the cellular response to Hh signals was eliminated by conditionally ablating the Smoothened (Smo) gene, a critical component of the Hh signaling pathway, with the Cre‐lox recombinase system. Smo was deleted in retinal progenitor cells at embryonic day 11.5 and retinas were analyzed at early postnatal stages and at maturation. At postnatal day 0 (P0), the mutant retina revealed an overproduction of retinal ganglion cells at the expense of progenitor cells. Surprisingly, mutant retina displayed phenotypes that were largely similar to wildtype retinas at P30. Here, we provide evidence suggesting that the Smo deficient retina auto‐corrected its excess of retinal ganglion cells and compensated for the reduced progenitor cells during early postnatal development. The adaptive changes observed in postnatal mouse retina offer broad implications for retinal plasticity and the capability in coordinating a balanced production of neuronal cell types. 225 Correction of the Dystrophin Gene Defect Mediated by PNA‐ssODNS Conjugated to Cell Penetrating Peptides in a Mouse Model for Duchenne Muscular Dystrophy YU TANG MEI, Fiona Bhondoekhan, Alvin Magh, Farnoosh Nik‐Ahd and Carmen Bertoni Duchenne Muscular Dystrophy (DMD), a severe recessive X‐linked muscle degenerative disease characterized by mutations in the dystrophin gene, results in progressive muscle weakness and deterioration. Our laboratory seeks to develop effective therapies permanently restoring dystrophin expression in DMD. We recently demonstrated that targeting Satellite Cells (SCs), muscle stem cells, repaired the dystrophin gene defect. We also showed that single‐
stranded oligodeoxynucleotides (PNA‐ssODNs) can induce single‐point mutations in the dystrophin gene and correct the dystrophin gene defect in a DMD mouse model. To increase the efficacy of PNA‐ssODNs distribution into SCs, we tested the ability of cell penetrating peptides (CPPs) to assist PNA‐ssODNs in crossing the plasma membrane and target SCs. Mdx5cv DMD mice models were injected with different dosages of CPP‐PNA‐ssODNs and Tibialis Anterior muscles were injured 2‐8 weeks prior to sacrifice to activate SCs. Restoration of dystrophin protein expression in skeletal muscles was then assessed. Dystrophin expression analysis in muscle fibers revealed improvement for CPP‐
PNA‐ssODNs and PNA‐ssODNs treated mdx5cv mice. Quadriceps, Gastrocnemius, and Tibialis Anterior muscles exhibited higher dystrophin expression compared to Diaphragm, and sustained expression of dystrophin in muscles suggested gene correction occurred in SCs and contributed to muscle repair. These studies have important clinical implications in the developments of an effective treatment for Duchenne Muscular Dystrophy. 70
SPD 2014 SESSION TWO 226 Phytoplankton Community of the Ballona Wetlands Ecological Reserve ANTHONY NOREN, JACKIE LAM, KRISTINE LEON, Ellisa Soberon, Jessie Jaeger, Mahsa Ostowari, Vanessa De Anda, and Rebecca Shipe An intensive 24‐hour field study was conducted to evaluate tidal and physical environmental conditions associated with the phytoplankton community in the intertidal channels of the Ballona Wetlands. One of the few remaining wetlands in Southern California, the Ballona Wetlands consist of two main channels that connect to the Ballona Creek where it empties into the coastal ocean. Temperature, salinity, pH, dissolved oxygen and phytoplankton community composition were sampled hourly on March 6‐7, 2014 from the east and west channels AND the adjacent creek. In addition to a daily cycle of lower temperatures at night, we found that there was a higher temperature variability in the west channel relative to the other sample sites. Preliminary data shows that the west channel has the highest species richness of the three sites. This richness is possibly due to the east channel having both inflow and outflow tidal gates whereas the west channel only allows outflow. All sample sites contain a phytoplankton community dominated by diatoms of the genera Cylindrotheca, Nitzchia, Navicula, and unidentified pennate diatoms. In addition, the west channel contains the diatom genera Pleurosigma, Stephanopyxis, Haslea, Fragilariopsis, and Skeletonema. Interestingly macroalgae spores and the harmful algal bloom taxa Prorocentrum were also abundant in the west channel. This baseline study of the phytoplankton community of Ballona Wetlands suggests a possible role of wetlands as a source or sink of ecologically unique and potentially harmful algae. 227 Mice with two X chromosomes exhibit increased development of obesity EMILIO M. RONQUILLO, Jenny C. Link, Xuqi Chen, Arthur P. Arnold and Karen Reue Obesity is an epidemic associated with heart disease, the leading cause of death in the United States. While marked variability in fat proportion and distribution has been observed between men and women, the mechanisms governing sex differences in obesity remain largely unexplored. Our project utilizes the four core genotype (FCG) mouse model, which generates XX and XY males as well as XX and XY females, to examine hormonal and genetic determinants contributing to the development of obesity. We previously found that XX mice, compared to XY mice, had increased fat accumulation in the absence of gonads. We hypothesized that we would see a similar trend within a gonadally intact cohort. When FCG mice were fed a high fat diet, we observed significant increases in body weight, proportional fat mass and respiratory ratios of XX mice compared to XY counterparts. Increased liver expression of four genes (Ddx3x, Kdm6a, Eif2s3x and Kdm5c) thought to escape X‐inactivation suggests a potential mechanism for sex chromosome complement differences. Further studies on mechanisms involved in metabolic sexual dimorphism will contribute to a growing body of research that will help to inform better diagnosis, and possibly treatment, of both men and women suffering from or at enhanced risk for metabolic dysfunction. 228 Diagnosing Seizure Disorder by Understanding Patterns of Comorbidities and Pharmaceutical Management EMILY A. JANIO, KAAVYA R. RAMAN, JUSTINE M. LE, Wesley T. Kerr, Jessica M. Hori, Sarah E. Barritt, Akash B. Patel, Chelsea T. Braesch, Eric S. Hwang, Emily C. Davis, David Torres‐Barba and John M. Ste Seizures occur for a multitude of reasons in patients with complex medical histories. As such, distinguishing between epileptic (ES) and non‐epileptic seizures (NES) is a challenge. We examined if the pattern of comorbidities and pharmaceutical treatments that a patient was using, could help distinguish epileptic from non‐epileptic seizures. To find these patterns, we studied epileptologists' outpatient clinical notes concerning 298 patients with medication resistant seizures (218 ES, 80 NES). These patients later were diagnosed with the gold standard diagnostic assessment: 72+ hour in‐patient closed circuit video‐encephalography monitoring. Based on the findings in these notes, our C4.5 decision tree achieved 79% leave‐one‐out cross‐validation accuracy (95% confidence interval 74‐84%: better than chance, permutation test, p<0.001). While our method had high sensitivity (90%), it was limited by low specificity (51%). This suggests that it was more effective in ruling‐in epilepsy than ruling‐out non‐epileptic seizures. The structure of the learned decision tree provided intriguing insight that could be interpreted to better illuminate the medical and psychiatric causes, effects and associations of seizures. This work could be used to better identify which patients may benefit from anti‐epileptic medications, as well as identify key non‐seizure comorbidities that, if left untreated, could reduce patients' quality of life even if their seizures are controlled. 71
SPD 2014 SESSION TWO 229 Identifying a Novel Disease Locus for Multiple Epiphyseal Dysplasia PATRIC J. HO, Karthika Balasubramanian, Bing Li and Daniel H. Cohn Multiple Epiphyseal Dysplasia (MED) is a rare inherited skeletal dysplasia characterized by childhood joint pain and mild short stature, ultimately leading to early‐onset osteoarthritis. Dominant mutations in COMP, MATN3, COL9A1, COL9A2, and COL9A3, all resulting in structural abnormalities in cartilage extracellular matrix proteins, and recessive mutations in SLC26A2, resulting in a defect in post‐translational sulfation of cartilage extracellular matrix proteoglycans, have all been found to produce MED. These mutations account for MED in approximately 85% of the documented cases; therefore, the molecular basis of MED remains unknown in about 15% of the diagnosed cases. Several families negative for mutations in the known MED genes were selected for exome sequencing studies. As a result, dominant de novo variants in PRICKLE1 and PTH2R were identified in one MED case. A mutation screen of 32 additional MED patients revealed that none of the patients had any significant disease‐causing variants in PTH2R or PRICKLE1, suggesting that mutations in PTH2R or PRICKLE1 are not common causes of MED. Gene expression studies revealed that, unlike PTH2R, PRICKLE1 is expressed at high levels in cartilage, making it a more promising candidate associated with MED. Furthermore, homozygosity for a nonsense mutation in mouse Prickle1 results in under‐
mineralized, shorter long bones, providing evidence of PRICKLE1's role in cartilage development. Studies are currently underway to determine how the mutation in PRICKLE1 might cause MED. 230 Sense of Safety of Multi‐Ethnic Youth: Does the Ethnic Composition of the School Matter? STEPHANIE GARCIA, Jessica Morales‐Chicas and Jaana Juvonen Despite the growing number of the biracial individuals within the overall population, little is known about what school factors that protect this specific population's well‐being. The present study assessed the relationship between percentage of school diversity (ranging from 50% to 78% ) and perceived school composition and its impact on multi‐
ethnic students' feelings in school . We hypothesized that multi‐ethnic students feel less vulnerable in school settings that are diverse (they are less likely to stand out) and where they perceive more students like them (i.e. multi‐ethnic). A total of 800 multi‐ethnic students from 20 public middle schools in California completed self‐reported measures of safety and perceived victimization. Results demonstrate that more diversity in schools is associated with higher levels of safety and less victimization, but amount of perceived same‐ethnic peers was not significant. Findings highlight the overall ethnic diversity of the schools as a protective factor of multi‐ethnic youth. 231 Expanding the Search for Water Ice in Lunar Craters Using Albedo‐Temperature Correlation SZILARD GYALAY and David A. Paige Currently orbiting the Moon is NASA's Lunar Reconnaissance Orbiter (LRO). On board this spacecraft are several instruments‐‐among them the Diviner Lunar Radiometer Experiment (DLRE) and the Lunar Orbiter Laser Altimeter (LOLA). The former measures thermal emission of the lunar surface, while the latter measures the topography of the Moon by reflecting 5 beams at 28 Hz off of the surface. Because LOLA also measures the energy and width of these returning laser pulses, a map of the albedo across the lunar surface at a wavelength of 1064 nm can be created. While water would normally evaporate off the surface of the moon the instance the sun shines on it‐‐the tilt of the moon and the lips of craters create permanently shadowed regions at the poles, which would theoretically allow water ice to persist. High reflectance in cold regions could suggest the presence of this water ice, Previous work has shown this negative correlation between albedo and temperature within craters poleward of 70 degrees latitude. To see if this correlation continues, craters from 40 to 70 degrees from the equator have had their reflectance and temperature data (normalized to 315K) examined for this correlation. The inverse correlation appears to continue, though whether this is truly due to the presence of volatiles such as water or just space weathering remains to be seen. The presence of water on the moon would have implications on the possibility of extraterrestrial life or the feasibility of establishing a permanent base on the Moon. 72
SPD 2014 SESSION TWO 232 Defining the Role of Oxidative Stress‐Induced Genomic Instability and Energy Metabolism Genes in Tumorigenesis. ZIJUN SOPHIE ZHAO, Anastasia Lomova, Aspram Minasyan, Johanna Scott, Nicholas Graham and Thomas Graeber Normal cells in the presence of sufficient oxygen perform cellular respiration. However, most cancer cells in the presence of sufficient oxygen rely heavily on glycolysis, a phenomenon known as the Warburg effect or aerobic glycolysis. Another 'hallmark†of cancer is genomic instability resulting in tumors with aberrant copy numbers of genes. In our research, we hypothesize that metabolic gene copy number changes drive tumorigenesis. To test our hypothesis, we use mouse embryonic fibroblasts (MEFs), an accepted system for studying cell transformations in vitro. MEFs, which are prone to become genomically unstable in culture, are exposed to different stressors including genetic manipulations and oxidative stress. Depending on the stress factor, the MEFs are predicted to respond with less or more copy number alterations. Western blot and real‐time polymerase chain reaction (qPCR) are employed to measure the protein expression levels and gene copy number changes in MEF cells. Our preliminary data suggests that amplification of enolase2 and hexokinase2 gene copies are potential drivers responsible for the metabolic switch from cellular respiration to aerobic glycolysis, and hence, tumorigenesis. Our lab has identified the amplification and deletion of metabolic genes as a driving force underlying the recurrent pattern of DNA copy number alterations seen in cancer patients. Future results have the potential to further elucidate the genetic basis of cancer metabolism and development. 233 Modifications in Striatal and Cortical Inhibition from Parvalbumin‐Expressing Interneurons in the Q175 Knock‐In Mouse Model of Huntington's Disease ALINA Z. NAQVI, Laurie Galvan and Michael S. Levine Huntington's Disease (HD) is a progressive, adult‐onset neurodegenerative disorder that is characterized by movement, cognitive, and psychiatric disturbances. There is degeneration of striatal medium‐sized spiny neurons (MSNs) followed by loss of cortical pyramidal neurons (CPNs). Differences in MSN and CPN spontaneous inhibitory postsynaptic currents (IPSCs) were observed in symptomatic R6/2 mice, a rapidly progressing model of HD. We investigated if the source of this GABAergic (inhibitory) increase is due to parvalbumin (PV)‐expressing interneurons. MSNs and CPNs receive GABAergic inputs from PV interneurons. Previously, using optogenetics, the PV‐MSN and PV‐
CPN GABAergic synaptic transmission was increased in symptomatic R6/2 mice. Now, we are investigating if this modification could be observed in a slowly progressing HD mouse model, the Q175 knock‐in model. Experiments were conducted at two stages: pre‐symptomatic (2 months) and symptomatic (12 months). We are examining spontaneous IPSCs in both stages of wild‐type (WT) and Q175 mice. By optically activating PV interneurons expressing channelrhodopsin (ChR2), a cation channel, we observed GABAergic responses in MSNs and CPNs in WT and Q175 mice. Activation of PV interneurons in pre‐symptomatic Q175 mice showed no (or mild) GABAergic response modification. At the symptomatic age, preliminary data shows interesting findings. These results may further confirm findings in R6/2 mice indicating a potential GABA dysfunction in the striatum and cortex of Q175 mice. 234 Reliability of Diffusion Tensor Imaging Tractography for Facial Nerve Imaging in Patients with Vestibular Schwannoma MONICA MATHUR, Nicole Cremer and Isaac Yang The ability to visualize the facial nerve (FN) in relation to the location of vestibular schwannoma (VS) is crucial for pre‐
operative surgical planning and may increase the safety of surgery. Diffusion tensor imaging (DTI) tractography has enabled pre‐operative, in vivo, mapping of the FN. We conducted a meta‐analysis to assess the accuracy of DTI tractography for locating the FN in VS resection planning. A PubMed search on 'vestibular schwannoma facial nerve tractography was done. Inclusion criteria were gross total resection of VS, pre‐operative identification, and intraoperative localization, of the FN. An overall reliability rate was calculated by comparing the intraoperative location of the FN, as described by the surgeon, with the pre‐operative location by DTI tractography in order to quantify the accuracy of DTI tractography. 38 cases of VS fit our inclusion criteria. In 89.5% of these cases, surgical findings agreed with the location of the FN as mapped by DTI tractography. Of these cases, all had post‐operative HB grade I or II compared to HB grade II to IV when imaging was not possible. DTI tractography is a reliable method for imaging the FN in relation to VS, and may decrease FN injury during surgery. However, DTI tractography has limitations, and further studies are needed in order to better understand what factors correlate with successful location of the facial nerve and DTI tractography in patients with VS. 73
SPD 2014 SESSION TWO 235 Computer‐Aided Diagnosis of Epilepsy Using Clinical Information AKASH B. PATEL, SARAH E. BARRITT, JESSICA M. HORI, Wesley T. Kerr, Emily A. Janio, Kaavya R. Raman, Justine M. Le, Chelsea T. Braesch, Eric S. Hwang, Emily C. Davis, David Torres‐Barba, John M. Stern, Noriko Salamon and Mark S. Cohen Distinguishing between epileptic and non‐epileptic seizures is a challenge. On average, the time from the first seizure to the diagnosis of non‐epileptic seizures is 7 years; during which, a majority of patients often are misdiagnosed with epilepsy and treated inappropriately with anti‐epileptic medications. This exposes patients to the serious side effects of these medications. One of our laboratory's goals is to create an automated system that can aid physicians in distinguishing patients with epileptic and non‐epileptic seizures. We accomplish this by inspecting the out‐patient clinical notes from patients with medication‐resistant seizure disorder that were later diagnosed with the gold standard diagnostic assessment: 72+ hour in‐patient closed circuit video‐electroencephalography (VEEG) monitoring. Using a combination of the known risk factors for epilepsy and non‐epileptic seizures reported in 228 clinical notes, our decision tree achieved an accuracy of 65%. While this appears low, it is comparable to the accuracy of neurologists prior to VEEG monitoring. The structure of our decision tree also provided meaningful information about the interpretation of each risk factor in each patient. This work may help diagnose, and thereby more effectively treat, these patients that are in need. In addition to serving patients with seizure disorder, the computer‐aided diagnostic methods we develop here may be applicable to the diagnosis of other maladies in the future. 236 Molecular Epidemiology Shows Low Prevalence of Hepatitis C ‐ HIV Co‐infection in a Los Angeles Cohort LAUREL CLARE, Lorelei Bornfleth, Kara Chew, Pamina Gorbach, Marjan Bavanbakht and Martha Lewis Blum Hepatitis C Virus (HCV) is the fastest rising co‐infection with HIV in U.S. urban populations and a main contributor to mortality among HIV‐infected persons via liver‐related disease. There is evidence of increasing rates of sexual transmission of HCV associated with non‐injection drug use in HIV‐infected men who have sex with men (MSM). Here, HCV prevalence was investigated in an HIV‐infected MSM cohort of non‐injection drug users, Metromates (N=186), from Los Angeles where rates of co‐infection have not been fully described. Prevalence was hypothesized to be 10%. The association of transmitted HIV and HCV drug resistance was also examined to identify risk factors. Plasma samples were screened by qPCR designed to detect all HCV genotypes. Multiple regions of HCV NS3 protease, NS5B polymerase, and structural genes E1 E2 and Core, were amplified and sequenced. HIV protease and reverse transcriptase were also sequenced. Phylogenetic analysis was used to determine relatedness. HIV and HCV amino acid sequences of the protease and polymerase were examined for drug resistance‐associated mutations. In total, 3 co‐
infected MSM were found, two genotype 1a and one genotype 3a, resulting in a 1.6 % prevalence. Phylogenetic analysis showed the sequences were unrelated. They all lacked known drug resistance mutations. Therefore, MSM in the Metromates cohort from Los Angeles exhibit low prevalence of HCV‐HIV co‐infection. Overall, improved understanding of HCV and HIV co‐infection can provide data to help improve prevention efforts. 237 The Effects of Familism on Psychological Distress in Latina Women in the U.S. AZUCENA VILLALOBOS, Christine Guardino and Christine Dunkel Schetter Familism, a cultural value prominent among Latinos, is characterized by a strong sense of interdependence between individuals and their families. It emphasizes maintaining close family relationships, prioritizing family over self, and contributing to the well‐being of the family (Sabogal, Marin, Otero‐Sabogal, VanOss Marin, & Perez‐Stable, 1987). Previous research has linked familism to better psychological health among college students (Campos, Ullman, Aguilera, & Dunkel Schetter, in press). This study will examine the correlates and consequences of familism in a sample of 420 U.S.‐ and foreign‐born Latina mothers recruited in a large network study in five regions of the U.S. Analyses will examine whether familism varies by socioeconomic status and acculturation, and test associations between familism and indicators of psychological distress. We predict that education, income, and years in the U.S. will be negatively associated with familism. Based on the psychological benefits of familism demonstrated in previous studies, we expect that higher familism will be associated with lower postpartum depressive symptoms and lower perceived stress, especially among foreign‐born Latinas. 74
SPD 2014 SESSION TWO 238 Refinery Fenceline Monitoring Using Multi‐Axis Optical Absorption Spectroscopy to Determine Facility‐Averaged Emission Fluxes ELLYN GRAY, Olga Pikelnaya and Jochen Stutz Urban air pollution, one of the most critical environmental problems in today's world, is caused by anthropogenic emissions of hydrocarbons and nitrogen oxides. To develop and monitor effective air pollution mitigation strategies, it is essential to provide accurate measurements of emissions. Large industrial facilities are particularly difficult to monitor, as many components of these facilities can contribute to the overall emissions. Consequently new strategies to monitor these emissions are needed. Multi‐Axis Differential Optical Absorption Spectroscopy (MAX‐DOAS) is an emerging remote sensing technique which uses narrow‐band molecular absorption features to identify trace gases and determine their atmospheric concentration. Here we present a novel application of MAX‐DOAS to monitor NO2 and HCHO emissions from a local refinery. These observations are necessary for validating reported emission fluxes and understanding local ozone production. We discuss the deployment strategy for determining the facility‐averaged emission fluxes using a dual MAX‐DOAS technique that is based on instruments both upwind and downwind of the refinery to find the net flux divergence. Our technique hinges on the simultaneous operation of two identical and highly comparable instruments, making long‐term instrumentation stability an essential factor in the design. We present the initial characterization of this design for NO2 and HCHO measurements, as well as outline its implementation for future emission inventory calculations. 239 Preference of Caenorhabditis Elegans for Isothermal Movement MOJDEH TABIBIAN, Evan Yang and Katsushi Arisaka Temperature affects nematode Caenorhabditis elegans navigation behavior, which can be observed through thermotaxis. We examined C. elegans' development of memory for the temperature of cultivation by placing them on a thermal gradient and observing their movements. We expected that starved C. elegans placed on a thermostatically regulated gradient would accumulate around the cultivation temperature in search for food. When we analyzed the movement of C. elegans by taking images, we found that the majority of them were centered on the temperature of cultivation as expected. The C. elegans left a trend traveling along the same temperature, showing preference for that particular temperature. Contrary to our expectation, a few populations of C. elegans were found to prefer accumulating at temperatures other than temperature of cultivation. This variety in navigation behavior may be an indication of individual differences due to genetic activity. 240 Optimizing Stellar Data From Adaptive Optics Images of the Galactic Center Using Empirical Noisemaps SAUNDRA ALBERS For over two decades, the Galactic Center Group has studied the center of the Milky Way Galaxy. This encompasses everything from the supermassive black hole at its very center to the various stellar populations that surround it. The most prohibitive obstacle in these studies involves the tremendous blurring effect that occurs when light from distant objects is obstructed by the turbulence of the earth's atmosphere. In 2004, the introduction of Adaptive Optics, which directly correct for atmospheric distortion using deformable mirrors, delivered views of the galactic center with unprecedented resolution. We use a program called Starfinder to examine stellar orbits in high resolution, allowing us to study a fascinating array of phenomena such as black holes, stellar dynamics, evolution, and even experimental verification of general relativity. My work optimizing this data extraction from Adaptive Optics images improves our ability to learn more about all of these regimes. I will create tools such as empirical noise maps to better analyze the Adaptive Optics data collection. My optimization techniques allowed Starfinder to detect fewer false sources and more real stars with greater accuracy. This yielded more definite orbital statistics, giving us more powerful tools for studying stellar dynamics and evolution in the region, along with testing general relativity. 75
SPD 2014 SESSION TWO 241 Gene Expression of HLF, RFX3, BHLHE23, and E2F6 in Strongylocentrotus purpuratus Embryo Development SANDRA CORDOVA, ANNA MAYOR, TOM NGO, RAQUEL TORRES, Daniel Malkin and Pei Yun Lee Developmental biology studies the mechanisms leading to cell differentiation and organism growth and is essential to understanding multiple diseases including birth defects and cancer. Strongylocentrotus purpuratus, or the purple sea urchin, is ideal for studying developmental biology due to its accessibility, transparent embryos, and its similarities to human development. In this study, four S. purpuratus genes were identified: Hepatic Leukemia Factor (HLF), Transcription Factor RFX3 (RFX3), Basic helix loop helix transcription factor E23 (BHLHE23), and Transcription Factor E2F6 (E2F6). First, the BLAST sequencing program was used to identify these genes. Afterwards, a phylogenetic analysis was conducted to confirm their identities. Next, gene expression levels and localities were determined by RT‐
PCR and whole mount in situ hybridization. Gene cloning techniques were conducted to amplify target genes then colony PCR verified the presence of gene inserts in the transformed bacterial colonies. After successful gene amplification, RNA probes were synthesized and used in whole mount in situ hybridization. RNA probes were hybridized to S. purpuratus embryos at different developmental stages (24 hours, 48 hours, 72 hours) and gene expressions were observed. Results reveal that HLF, RFX3, and E2F6 are expressed at all three developmental stages of purple sea urchin embryo development. On the other hand, BHLHE23 is not expressed or has limited expression during the first 72 hours of purple sea urchin development. 242 Electrophysiological Alterations In Medium‐Sized Spiny Neurons Of a Knock‐In 175 CAG Repeat Mouse Model Of Huntington's Disease CONNY H. TRAN, Tim Indersmitten and Michael S. Levine The identification of the gene responsible for Huntington's disease (HD), caused by a CAG repeat expansion in the huntingtin gene, allowed for the creation of many genetic mouse models of HD. Each was generated using a different approach in delivery of the mutated gene. There is evidence that knock‐in models are more faithful representations of human HD in terms of genetic context and in recapitulating the slow progression and neuropathology of HD. We describe the electrophysiological changes in medium‐sized spiny neurons (MSNs) of a knock‐in mouse model, termed Q175, during disease progression. Voltage‐clamp whole‐cell recordings from striatal MSNs were used to characterize alterations in basic membrane properties and synaptic transmission in homozygous and heterozygous Q175 mice at 2, 7, and 12 months of age. Relative to age‐matched wild‐type littermates, changes in membrane properties included increased cell input resistance and decreased cell holding current. Alterations in neuronal communication were largely absent in Q175 mice at 2 months, but marked by 7 months. These included increased frequency of spontaneous inhibitory postsynaptic currents (sIPSC) and reduced frequencies of spontaneous and miniature excitatory postsynaptic currents (sEPSC and mEPSC). Our results indicate that the Q175 model exhibits similar electrophysiological alterations compared with other HD mouse models and support the hypothesis that Q175 mice manifest a slow progressing form of HD. 243 Melanopsin Localization in Peripheral and Central Pain Pathways in Photoallodynia HALINA YEE, Anna Matynia and Michael B. Gorin Photoallodynia, also known as photophobia, is exacerbated ocular pain in response to normal levels of light. The molecular mechanisms involved in its central and peripheral pain pathways are unknown. This research studies whether melanopsin‐containing intrinsically photosensitive retinal ganglion cells interact with corneal nociceptors to mediate pain, and localizes melanopsin expression in the brain. Dry‐eye mouse models were used to elucidate photoallodynia's peripheral pathways. Neuronal degeneration was observed with peroxidase‐catalyzed amplification systems in benzalkonium chloride‐treated corneas. Colocalization studies of melanopsin and beta‐tubulin in the brain using green‐fluorescent‐protein transgenic mice examined photoallodynia's central pathways. The amplification systems were unsuccessful, possibly due to the cornea's fibrousness. In colocalization studies of melanopsin in the trigeminal ganglion, goat anti‐rabbit and donkey anti‐rabbit secondary antibodies double‐stained a rabbit anti‐
melanopsin primary antibody. Immunofluorescence yielded melanopsin‐containing fibers that travel as a fiber tract and diverge deep into the medulla, as well as in the trigeminal ganglion's periphery. The results indicate melanopsin's role in the trigeminal ganglion's innervation of the cornea, and its subsequent role in photoallodynia's central pathways. This identifies potential therapeutic targets for those suffering from photophobia. 76
SPD 2014 SESSION TWO 244 The Effect of Sarcospan Loss in Cardiac Function REGINALD T. NGUYEN, Michelle Parvatiyar and Rachelle H. Crosbie‐Watson Sarcospan (SSPN), a small tetraspanin sarcolemmal protein, promotes cell adhesion and stabilizes the dystrophin and utrophin glycoprotein complexes. SSPN loss leads to the destabilization of skeletal muscle adhesion complexes and reduces dystrophin localization. The loss of dystrophin underlies the disease, Duchenne muscular dystrophy (DMD), which leads to severe muscle wasting and cardiac dysfunction. Although our lab has focused on the role SSPN plays in skeletal muscle, the role of SSPN in the cardiac system is unknown. We examined WT and SSPN‐null mice under saline and isoproterenol challenge. SSPN‐null mice displayed hypertrophy evidenced as increased heart/body weight ratio and systolic/diastolic ventricular dimensions, and decreased left ventricular ejection fraction. Upon isoproterenol challenge, SSPN‐null hearts displayed an increased E/A ratio, indicative of restrictive ventricular filling and decreased fractional shortening. Immunofluorescence data from SSPN‐null mice showed decreased levels of dystrophin and sarcoglycans, with increased Beta‐1D integrin, compensating for loss of stabilizing adhesion complexes, relative to WT. SSPN‐null hearts displayed increased fibrosis compared to WT, with the difference being enhanced by isoproterenol challenge. SSPN loss also contributes to diminished Akt signaling, illustrated by a reduction in active P‐Akt levels. SSPN plays an important role in cardiac infrastructure, maintenance, and function, while its removal promotes cardiac hypertrophy and tissue damage. 245 Characterization of a Novel Small Molecule Inhibitor of Anthrax Lethal Toxin RENAE LOPEZ CRUZ, Charlie Chi‐Lee Ho and Kenneth Bradley Anthrax is an infectious disease caused by the spore‐forming bacterium Bacillus anthracis, which has been classified by the Center for Disease Control as a Tier 1 bioterrorism agent. The bacterium secretes a binary toxin called lethal toxin (LT), comprised of two protein subunits: protective antigen (PA) and lethal factor (LF). PA mediates the cellular entry of LF via endocytosis. Acidification of the late endosome induces the formation of a pore in the endosomal membrane allowing LF to enter the cytosol and exert its virulent effects upon the host cell. Our goal is to further elucidate the intoxication pathway by investigating the mechanism by which a novel compound, RC1, prevents LT‐induced cell death in murine macrophages. Key events underlying cellular toxin entry were assessed through cell‐based viability assays and western blots to determine the stage of intoxication blocked by RC1. We found that RC1 protects host cells when added up to 30 minutes post‐intoxication and that it also protects cells from a diphtheria toxin chimera that employs the same entry mechanism as LT. Additionally, our results show that RC1 acts downstream of cellular toxin entry but prior to toxin activity in the cytosol. Based on these data, we hypothesize that the molecular target of RC1 is necessary for the translocation of LF from the lumen of the endosome into the cytosol. Identifying the molecular target of RC1 will further our understanding of anthrax pathogenesis and similar toxin‐mediated diseases, which may lead to new therapeutics. 246 Effect of Anti Inflammatory Peptide J on Circulating Markers of Inflammation in a Mouse Model of Atherosclerosis SHERWIN TOLUIE, Loris Orbelian, Persia Bakhtiari, Katyoun Abtin BSc, Zarina Barseghyan, Kamran Toluie, Greg Hough and Mohamad Navab In populations there is a strong inverse relation between HDL levels and coronary heart disease (CHD). Over the past three decades, the use of statins has significantly reduced the incidence of CHD. A large percentage of patients however, still present with CHD and need treatments reducing the coronary artery disease risk in them. Several peptides have been designed that sequester inflammatory molecules and reduce vascular complications in preclinical studies. Anti inflammatory peptide J (AIPJ) is one example. Objective. In the current investigation we sought to study the effect of the AIPJ on serum amyloid A (SAA). Methods. LDL receptor deficient mice, were maintained on a Western type Diet for 8 weeks. Group 1 received normal chow , Group 2, WD alone, Group 3, WD supplemented with AIPJ. After 8 weeks, fasting blood was removed, plasma separated and SAA was determined using a commercial kit. Results. AIPJ reduced the circulating SAA levels (p< 0.01) as compared with the WD alone. Conclusion. In animals that received the peptide J, the removal of oxidized lipid by the peptide likely affected several oxidative reaction cascades and covered pathways that are involved in the induction of inflammatory molecules such as SAA. It would be useful to determine if the effect is on the liver, via the macrophages or on other tissues and cell types. The current observation might have therapeutic implications and be useful in reducing vascular inflammation in hyperlipemia. 77
SPD 2014 SESSION TWO 247 Creating an shRNA Library from the TIM‐3 cDNA PHILIP S. SYED, Jonathan Rick, Scott Kitchen and Anjie Zhen Human Immunodeficiency Virus (HIV) is a virus which is able to escape the body's adaptive immune response, mediated by T‐cells. HIV mutates too quickly for T‐cells to recognize epitopes and clear the infection. As the disease continues, T‐cells remain activated too long and express exhaustion markers such as TIM‐3. A hypothesized solution to avoiding this exhaustion is to lower expression of TIM‐3, which may be accomplished by small hair‐pin RNA (shRNA) specific to the TIM‐3 mRNA; the only caveat is identifying an effective shRNA. Creation of shRNA's from a source cDNA is possible by a technique known in scientific literature as Enzymatic Production of an RNAi Library (EPRIL). In this technique, the target cDNA is digested non‐specifically into small fragments, which are ligated to specific probes, digested with restriction enzymes, and processed to yield vectors with shRNAs for different sequences in the cDNA. So far, a TIM‐3 cDNA was cloned into the TOPO vector and extracted by PCR of the established vector with primers specific to the ends of the TIM‐3 sequence, and the resulting cDNA has been digested by DNase I and processed into short, blunt, double‐stranded fragments. These may further be subjected to the enzymatic procedure of EPRIL and high‐throughput screening. Ultimately, the generated TIM‐3 shRNA's which will be identified as effective in high‐
throughput screening will be co‐expressed in a cell along‐side the chimeric antigen receptor, CD4‐, where the effects of TIM‐3 knockdown can be examined in vitro. 248 Distinct Roles of CCN1 and CCN2 in Skeletal Development TIEN PHAN, Jie Jiang and Karen Lyons The CCN proteins regulate various biological functions such as cell adhesion, migration, proliferation, chondrogenesis, and skeletal development. In the bone tissue of vertebrate species, the expression of most CCN family members has been observed in both osteoblasts and chondrocytes; however, little is known about the roles of CCN1 and CCN2 in embryonic limb development. To evaluate the roles of CCN1 and CCN2 during endochondral bone formation, phenotypic and molecular changes in cartilage‐specific conditional knockout mice were investigated. Histological and immunohistochemical methods were used to quantify proliferation, apoptosis, and senescence in mutant embryos at embryonic day 16.5. Preliminary data revealed that compared to wild type embryos, CCN1 mutants had 19.6% and 24.9% less proliferating cells in the growth plate's resting and proliferative zones, respectively. CCN2 mutants had 26.7% and 33.0% less proliferating cells in the same areas of growing tissues. The double mutants showed similar patterns to those of CCN2 mutants, although less pronounced. The data demonstrated impaired chondrocyte proliferation and hypertrophy as a result of CCN1 and CCN2 deletions, which corresponded to skeletal dimorphism as seen in previous studies. A better understanding of CCN1's and CCN2's functions in chondrocytes during development holds promise for prevention and therapeutic treatment of diseased conditions such as osteoarthritis. 249 Administering 15‐HETE, a Lipid Mediator of Inflammation Orally or by Subcutaneous Route Induces Systemic Inflammation to a Similar Degree. ZANIAR MORADIAN, Katayoun Abtin, Sepideh Shakeri, Nasim Golchin, Amir Dadgostar, Bita Khorram, Samra Vazirian, Greg Hough and Mohamad Navab Arachidonic acid and linoleic acid undergo oxidative modification and a series of lipid mediators of inflammation are generated in our organism continuously. Hydroperoxides such as HPODES and HPETEs that are generated this way are highly toxic and we convert them to less toxic species including HETEs and HODEs. Our group focuses on the effects of 15‐HETE in systemic inflammation and the role that the intestine plays. LAL R‐/‐ mice were maintained on laboratory chow. Mice groups received a) chow alone, b) chow containing 1 ug per gram diet of 15‐HETE, or c) subcutaneous injections of 15‐HETE at 0.5 ug twice a week. Following two weeks the mice were fasted overnight, blood was removed from retro orbital sinus. Using LC‐ESI‐MS/MS the levels of oxidized fatty acids and using ELISA levels of SAA was determined. The plasma levels of HETEs and HODEs and that of SAA was similar in mice that received the 15‐HETE orally or by subcutaneous route. Thus the oral 15‐HETE in the intestine is capable of inducing reactions leading to the induction of hepatic SAA which is an important acute phase reactant. The fact that oral 15‐HETE was able to induce hepatic SAA as effectively as injected 15‐HETE indicates the importance of the intestine as a site contributing to systemic inflammation. It would be useful to demonstrate the differential role of the intestinal cells (enterocytes) vs the microbiota of the intestine in this phenomena. 78
SPD 2014 SESSION TWO 250 Optimization of Bessel Beam for Sheet Illumination Microscopy PAOKUAN CHIN, Blake Madruga and Katsushi Arisaka Bessel Beam is praised as the self‐reconstructing beam. Its virtue lies in its invariant thickness along the propagation axis and the ability to reconstruct itself when obstructed. Sheet microscopy can utilize Bessel beam by scanning it across a plane to create a light volume of remarkable surface area and minimal volume. This advantage results in an especially thin sheet, in order to optically slice samples in question along the previously defined plane. A Bessel Beam can be created by an axicon, a cone shaped lens, or by an annular slit. The former one allows much higher photon efficiency. In the latter method, two parameters that govern the length and thickness of the Bessel Beam have already been mathematically calculated. But that is not the case for Bessel Beam constructed by the former method. In this paper, we report the two parameters of that case. We found them empirically by simulation in Zemax, a program for optical design. With these two parameters in hand, one can shape a Bessel Beam specific for the need of imaging certain samples. However, the Bessel Beam is often condemned for the high percentage of photons residing in its surrounding rings. We verify this property and propose two solutions to this issue. 251 Interleukin‐10 Administration to Dystrophic Mice Modulates Muscle Inflammation and Increases Muscle Differentiation IVAN FLORES, Chiara Rinaldi and James G. Tidball Duchenne muscular dystrophy (DMD) is a fatal disease characterized by muscle necrosis, inflammation, and variability in muscle fiber size. Pro‐inflammatory M1 macrophages increase muscle damage by releasing cytolytic free radicals. Subsequently, anti‐inflammatory M2 macrophages promote muscle regeneration. Recent findings show that interleukin‐10 (IL‐10) can shift muscle macrophages to an M2 phenotype, a change that coincides with muscle differentiation. Ablation of IL‐10 from mdx mice increased muscle damage and weakness suggesting that increased delivery of IL‐10 to dystrophic muscle will decrease pathology.We are testing that possibility by injecting mdx mice with IL‐10 and then assaying pathology. Our findings show M1 macrophages decrease, reflected by reduction in inducible nitric oxide synthase, while M2 macrophages increase, indicated by elevated arginase expression. Additionally, elevated levels of muscle regulatory factor‐4 (MRF4) in treated muscles indicate an increase in muscle differentiation. Muscle cell expression of MRF4 is elevated when they are co‐cultured with IL‐10 stimulated macrophages. Collectively, these findings indicate that IL‐10 can promote muscle differentiation through a macrophage mediated process. Further investigation will test whether IL‐10 also has a direct effect on muscles. Establishing a relationship between muscle differentiation and IL‐10 levels may lead to the development of therapeutic strategies for attenuating the pathology of DMD in human patients. 252 High Throughput, Label Free Cell Viability Assay Using Deformability Cytometry DEWAL GUPTA, Mahdokht Masaeli and Dino Di Carlo Cell viability assays are a critical component of many studies yet many still rely on randomized single cell analysis to form conclusions on a sample population. Deformability cytometry has the ability to bring large population analysis to eliminate bias that can result from single cell analysis. This study determines the efficacy of a microfluidic device that allows for the detection of live and dead cells in a randomized population without the need for staining or labeling. Deformability cytometry uses the deformation of cells under high, controlled pressure to pick up on the differences between the mechanical properties of live and dead cells. To test this, chromatin modifying drugs were used on Jurkat cells, a t‐cell leukemia cell line, in order to induce a controlled, dose dependent cellular apoptosis. The cells were analyzed using deformability cytometry for three continuous days, along with a live/dead staining assay to determine population viability. Using statistical algorithms, the cells could be clustered into two distinct populations (live and dead) using only the deformation and size of the cells. The algorithm was able to distinguish the amount of live cells within the population successfully and as accurately as the staining assay. These results show that our device is a functional, high throughput viability assay that is much cheaper, faster, and reliable than conventional tools. 79
SPD 2014 SESSION TWO 253 Single Motor Unit Adaptation After Primate Spinal Cord Injury STEVE GUZMAN, Sharon Zdunowski, Roland Roy, Hui Zhong and V. Reggie Edgerton Considerable upper limb function can be recovered in a non‐human primate following a severe cervical spinal cord injury (SCI). However, the neural reorganization strategies underlying this recovery remain unclear. We hypothesize that one mechanism that contributes to this recovery can be attributed to selective muscle reinnervation and even denervation of the muscle fibers within a given of motor pool. In our study, six rhesus monkeys underwent C7 hemisection and EMG electrode implantation while video and EMG recordings were assessed during a trained hand task, both pre and post‐lesion. Following the injury, we observed an immediate loss of the ability to activate most muscles of the upper limb as required for reaching, grasping, and retrieving food. However, about 6‐24 weeks after injury we found varying degrees of muscle reactivation of different motor pools among different upper limb muscles. Based on the EMG burst patterns during SCI recovery there seems to be a proliferation of aberrant synaptic connections, which results in elevated levels of activation and a higher instances of co‐contraction of selected pairs of muscles. In the more advanced stages of recovery it appears that reinnervation of more muscle fibers and/or retraction of connections to some muscle fibers within a unit of orphaned muscle fibers is seen. These changes in motor unit properties may contribute to the progressive improvement in motor function following cervical spinal cord injury. 254 Investigation Into Early Theropod Physiology Using CO2‐47 Mass Spectrometry on Eggshell Fragments William M. Henrikson and Robert A. Eagle Carbonate clumped isotope thermometry is a relatively recent breakthrough in paleothermometry that improves over the Epstein‐Urey Carbonate‐Water thermometer by including measurement of the rare isotopologue 13C18O16O. The rare heavy isotopes 13C and 18O are thermodynamically favored to form bonds together (clump) in an inverse relationship with temperature of the precipitate which allows us to back out temperature independent of the isotopic content of the solution (as was needed before but impossible for vertebrates). Analysis was done on early Jurassic Eggshells from Torvosaurus, an early Theropod, as well as soil carbonates found nearby using this method. Measurements around 39 degrees celsius suggest that Torvosaurus may have had body temperatures similiar to those of large modern mammals or birds. 255 The Effects of Osmotic Stress on Nuclear Physical Properties in Arabidopsis Thaliana SAM YANG and Amy C. Rowat Nuclear organization plays a critical role in regulating gene expression. In metazoan nuclei, nuclear physical properties are strongly regulated by osmolarity: extracellular osmolarity decreases nuclear volume and alters chromatin condensation. While plants are constantly subjected to osmotic stresses, the effects of osmotic stress on the physical properties of their nuclei remain unexplored. The aim of the present study is to determine the extent to which osmotic stress in A. thaliana alters nuclear physical properties. To investigate the effects of hypo‐osmotic stress on nuclear volume in vivo, root tissue from A. thaliana is incubated in distilled water for varied time scales. Thereafter, nuclei are labeled with a DNA intercalating dye and laser scanning confocal microscopy is used to measure nuclear volume. Additionally, nuclei are isolated from leaf tissue and incubated in solutions of varying osmolarities in vitro. Subsequent image analysis reveals the relationship between osmotic stress and nuclear physical properties. A deeper understanding of the mechanisms by which osmotic stress alters nuclear physical properties changes may provide additional insight to how plants respond to environmental stresses. 80
SPD 2014 SESSION TWO 256 Immunophenotypic Identification of Pericyte Markers in Human Perivascular Soft Tissue Tumors YU‐HSIN YEN, Marco Mravic, Sarah M. Dry, and Bruno Peault and Aaron W. James Several perivascular‐ and pericytes‐specific antigens have been validated for both ex vivo isolation and in vivo identification of perivascular stem cells (PSCs) in several human tissues. These PSCs are capable of regenerating and developing into connective and musculoskeletal tissues. Same antigens were first to be investigated in several perivascular and mesenchymal soft tissue tumors. Immunochemical staining was performed on paraffin embedded slides of human perivascular tumors, including glomus tumor (n=11, ages 25‐73) and PEComa (n=16, ages 25‐86). Glomus tumors have diffused positive expressions against pericyte markers CD146, PDGFR‐beta, and alpha‐SMA in all 11 patients. Non‐tumor vessels nearby are used as an internal control, and endothelial marker CD31 was used as a negative control for lesional tissue. Also, because PSC are putative mesenchymal steam cells (MSCs) expression of MSC markers CD44, CD73, CD90, and CD105 in these tumors were also examined. Patchy to diffused co‐expression of MSC markers CD90 (10/11 patients) and CD105 (4/11 patients) was found. A range of expression patterns was identified in PEComa samples, including a distinctly perivascular distribution of pericyte marker positivity in tumor cells, to a more widespread sheet‐like positivity. In summary, perivascular soft tissue tumors showed characteristic and reproducible pericyte marker expression, which may indicate a perivascular cell origin. Further immunophenotyping and characterizing may improve clinical diagnosis and treatment options. 257 Neuronal Control of Blood Progenitor Maintenance SUNJONG JI, Tina Mukherjee and Utpal Banerjee Maintenance of hematopoietic progenitors in Drosophila involves both intrinsic and extrinsic factors. Recent studies have shown that GABA from Kurs6‐positive neurosecretory cells is secreted into the circulating hemolymph upon olfactory stimulation and binds to metabotropic GABAB receptors expressed on blood progenitors, which is necessary and sufficient for progenitor maintenance. This study investigates the role of other neurosecretory cells in blood progenitor maintenance. Neurosecretory cells were genetically ablated and their effects on blood progenitor maintenance were assessed via immunohistochemistry. It was found that Okt30‐ and Jan204‐positive neurosecretory cells are indeed also involved in blood progenitor maintenance. This study links the actions of peptidergic neurosecretory cells and the deprivation of their secreted neuropeptides to the integrity of the hematopoietic and innate immune systems in Drosophila. 258 The Role of Rabex‐5 in Tissue Growth Maintenance in Drosophila melanogaster NAVNEET K. RAMESH and Julian A. Martinez‐Agosto Endocytosis is the process by which molecules, such as growth factors, are internalized by a cell. This process has been implicated in the regulation of growth in Drosophila melanogaster, primarily through vesicular fusion mediated by members of the Ras family of GTPases. One main component of the endocytic pathway is Rab5, which is activated by a guanine exchange factor known as Rabex‐5. We hypothesized that Rabex‐5, through its role in endocytosis, plays a pivotal role in regulating the fusion and recycling of growth factors in Drosophila. We observed that loss of Rabex‐5 conferred abnormal proliferation in the imaginal wing disc. We then sought to determine if Rabex‐5 remotely controlled growth signaling in the wing disc from another part of the fly. Rabex‐5 downregulation in the brain led to a smaller wing disc, suggesting that Rabex‐5's role in growth regulation may be linked to insulin production and secretion. Immunohistochemical analysis revealed that the Ras, Wnt, and AKT pathways were elevated in the Rabex‐5 mutant, along with the previously reported increase in JNK and STAT. This increase in signaling could all be facilitated by an increase in PVR signaling. Based on our findings, we propose a model by which elevated JNK leads to an upregulation of PVR, which subsequently activates another pathway to generate the abnormal growth in the Rabex‐5 mutant. Ultimately, this study suggests that Rabex‐5 is a crucial negative regulator of growth, but may serve as both a tumor suppressor and oncogene in the cancer context. 81
SPD 2014 SESSION TWO 259 Temporal and Spatial Expression of Norepinephrine Gene, E23 Gene, Netrin 3 Gene, and Netrin 1 Gene in Strongylocentrotus purpuratus Embryo Development FELIX V. CHEN, KEVIN TRAN, DAN RAGUSANO, EDWARD HUI, Daniel Malkin, Lydia Ann and Pei Yun Lee The temporal and spatial expression patterns of Norepinephrine gene, E23 gene, Netrin 3 gene, and Netrin 1 gene in Strongylocentrotus purpuratus during embryonic development and the functions of the genes are reported here. The functions of the genes were first identified using a BLAST analysis. A phylogenetic analysis using MEGA6.0 was conducted to confirm the gene identities identified from BLAST analysis.. The genes were tested in RT‐PCR for temporal expression during the first 72 hours of embryonic development and were followed up by whole mount in situ hybridization (WMISH) to further analyze spatial expression of the gene during embryonic development. The results of the WMISH showed no expression of Norepinephrine at any stage, the expression of E23 was only present at 24 hours, Netrin 3 was expressed later at 48 hours and 72 hours, and Netrin 1 was also expressed only at 48 hours and 72 hours. The results indicate that Norepinephrine is not expressed during embryo development and is likely turned on after gastrulation to regulate energy metabolism. E23 is a transcriptional regulator that turns on early in embryo development to initiate cell division and is turned off soon after. During gastrulation Netrin 3 and Netrin 1 are expressed in the endoderm and at the mid gut at the archenteron. 260 Mechanisms of Ventricular Arrhythmias Induced by Fibrosis and Stress Shankar Iyer and Thao P. Nguyen Fibrosis is known to contribute passively to sudden cardiac death, in the form of ventricular tachycardia and fibrillation (VT/VF), by creating structural barriers that interrupt electrical conduction. However, recent evidence of direct coupling between myocytes and fibroblasts via gap junctions suggests a more active role. We hypothesize that under hypokalemic stress, fibroblast‐myocyte coupling promotes actively the formation of arrhythmogenic triggers, such as early afterdepolarizations (EADs) and triggered activity (TA). Optical voltage and calcium imaging techniques were used to observe wave propagation across cocultures of neonatal ventricular myocytes (NRVMs) and fibroblasts paced at 2‐4Hz and exposed to hypokalemic stress (2.7mM K). In control NRVM monocultures, pacing produced uniform electrical wave propagation. Introducing an empty space acting as a physical barrier in the monolayer impeded wave conduction and resulted in reentrant wave activity. Filling the empty space with fibroblasts resulted in more severe wave conduction defects, seen as wavebreaks. Under hypokalemic stress, EADs and TAs originated from border zones of fibroblast‐coupled myocytes in these cocultures. Our results suggest that fibroblast‐coupled myocytes actively promote arrhythmias at the tissue level by producing EADs and TAs under hypokalemic stress. These findings shed light on a novel mechanism by which fibrosis promotes arrhythmias in the whole heart. 261 Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Plays a Role in Regulating Embryoid Bodies (EBs) Differentiation under Hypoxic Conditions YING LIN, Yuan Xie, Jessica Cinkorpumin, Xiangzhi Meng and William Lowry Hypoxic conditions have been indicated as key factors in regulating human development. Previous studies in Lowry lab have shown that hypoxia (2% O2), which is closer to the physiological level (5% O2) compared to normoxia (21% O2), could facilitate the generation of glia cells and promote neural progenitor cell (NPCs) maturation in vitro. Due to the important effects of hypoxia, it is worthwhile to conduct further studies on the mechanism of how hypoxia affects the maturation of human embryonic stem cells (hESCs) in vitro. Hypoxia Inducible Factors (HIFs) have been suggested to response to oxygen tension change, but the mechanism of how they regulate cell differentiation is still unclear. We hypothesized that lowering the oxygen tension will facilitate the transcriptional and functional maturity of human pluripotent stem cell by mimicking conditions in vivo. ARNT has been discovered to stabilize other HIF members in hypoxia. EBs derived from XFiPS‐scramble/shARNT ES cells was used as a model to study the gene regulation mechanism. By conducting reverse transcriptase PCR, we found that the ectoderm genes and mesoderm genes were regulated in different directions for shARNT EBs in hypoxia compared to scramble controls. The results indicate the important role that ARNT play in germ layer formation in hypoxia. Our studies of oxygen tension in EBs differentiation not only provide a potential method to facilitate hESCs maturation in vitro, but also is vital to gain a better understanding of early human fetal development. 82
SPD 2014 SESSION TWO 262 Spatial Dynamics of Species Invasions in Diamond Food Webs CALEB BECKETT and Priyanga Amarasekare The diamond food web module, in which two consumer species share both a basal resource and a predator in common, occurs quite commonly in known trophic communities. Many examples exist of this community resulting from a species invasion by an exotic consumer species into a native community consisting of a resource, consumer, and predator. In a single community, this manner of coexistence must be mediated by a tradeoff in competitive ability and susceptibility to the predator between the resource species, but not all empirical cases are consistent with this. In these cases, it is likely that spatial mechanisms involving metacommunities are involved. We develop a mathematical model consisting of multiple habitat patches in an uninhabitable matrix. All patches are linked by dispersal, and each is inhabited by a local community. Through model analysis and numerical simulation, we show that dispersal in the presence of spatial heterogeneity in resource productivity leads to qualitatively different outcomes from a local community regarding invasibility and coexistence. The outcome of an invasion is contingent on the level of variation in resource productivity across space, the mode of dispersal, and the nature of the invading species' competition‐
predation tradeoff relative to its competitor. These results suggest landscape and species characteristics which may aid in management decisions and biocontrol measures. 263 Effects of Exercise and Antidepressants on Levels of Brain‐Derived Neurotrophic Factor and Glucocorticoid Receptors in the Hippocampus after Traumatic Brain Injury JASPREET K. GARCHA, Shyama Nair and Grace Griesbach Traumatic Brain Injury (TBI) is an increasing health concern that afflicts more than 10 million people annually and can have social, physical, and behavioral effects. Studies show that exercise and antidepressants increase neuroplasticity after TBI through increased brain‐derived neurotrophic factor (BDNF) after a two‐week post injury period. Increases in BDNF are absent during this post‐injury period when there is a heightening of the hypothalamic‐pituitary‐adrenal axis response. BDNF help support the survival and growth of neurons and thus are important in our understanding of TBI. To identify the levels of BDNF, rats with a sham or fluid percussion injury (FPI) were placed in different conditions including fRW (forced running wheel) or sedentary and were injected with either the antidepressant desipramine or a vehicle. The levels of BDNF were analyzed using the offline setting of a stereoinvestigator program to understand the influences of exercise on this key molecular marker of neuroplasticity. We had anticipated that antidepressants would lower the stress response and increase BDNF production in the dentate gyrus of the hippocampus. However, our results indicate that there is no statistically significant difference between the groups in number of BDNF. This may be due to the delayed exercise at 5‐6 weeks showing that early desipramine administration does not lead to increase in BDNF. It is critical to understand when and how to treat TBI through analyzing BDNF levels in order to further our understanding of the effects of exercise and antidepressants and how to incorporate them into better treating traumatic brain injury. 264 Knockdown of Smooth Muscle Beta‐1 Integrin Reveals Matrix Assembly Deficits via Tissue Transglutaminase MICHAEL J. RALE, Onika D.V. Noel, Julia Mack and M. Luisa Iruela‐Arispe Integrins, as cell adhesion molecules, are crucial in the development, maintenance, and continued remodeling of resilient, blood vessel networks. In a recent in vivo study, beta‐1 integrin knockout in murine smooth muscle cells resulted in an embryonic lethal phenotype, with site‐specific formation of aneurysms in the ascending aorta, highlighting the importance of this particular integrin in extracellular matrix (ECM) assembly and vessel morphogenesis. To better understand beta‐1 integrin's role in in vitro ECM production and assembly, we utilized a CRE‐LOX knockout system to produce mouse immortalized smooth muscle cells (IMSMCs) that lacked beta‐1 integrin. We hypothesized that the loss of this integrin would result in differential transcript production of ECM related proteins (e.g. fibronectin, collagen, crosslinkers) and aberrant ECM morphology. RT‐PCR of knockout IMSMCs revealed reduced production of fibronectin transcript that declined over seven weeks. ECM crosslinkers, lysyl oxidase and tissue transglutaminase (tTG2), also showed reduced transcript production. Deoxycholate assay for insoluble fibronectin coupled with detection of incorporated biotin‐cadaverine showed reduced tTG2 crosslinking activity. Immunofluorescence microscopy further reveals the impact of beta‐1 integrin knockout on ECM, which resulted in loss of mature matrix assembly and a desensitized response to the addition of 200 ng/ml tTG2. Our results highlight the importance of beta‐1 integrin and tTG2 in the development of vessel morphology. 83
SPD 2014 SESSION TWO 265 Selection and Screening for Efficient Methanol Bioconversion in Escherichia coli CHRISTOPHER M. WONG, Igor W. Bogorad and James C. Liao With the continual demand for clean renewable sources of energy, biofuel production has proven to be an attractive alternative to traditional petroleum synthesis. Novel metabolic pathways such as non‐oxidative glycolysis (NOG) provide 100% theoretical efficiency in the conversion of accessible and inexpensive carbon sources into higher biofuels. Methanol dehydrogenase (Mdh) can convert methanol into formaldehyde, which can be synthesized with ribulose‐5‐phosphate to produce fructose‐6‐phosphate, a precursor in the NOG cycle. However, the Mdh gene in E. coli has yielded low expression and activity. Here, we design a scheme to screen for potential Mdh mutants with higher expression and activity than the current form. Effective forms of the enzymes 3‐hexulose‐6‐phosphate synthase (Hps) and 6‐phospho‐3‐hexuloisomerase have been identified and transformation into E. coli has previously shown high activity in the assimilation of formaldehyde to form fructose‐6‐phosphate. A strain of E. coli with knocked out ribulose‐5‐phosphate epimerase (Rpe) and transformed Mdh‐Hps‐Phi allows for selection of an efficient Mdh mutant when grown on ribose media. Selection for an efficient mutant of Mdh opens up potential metabolic pathways for the production of higher biofuels from a readily available fuel source. 266 Paleozoic temperatures from trilobites and brachiopods TYLER VOLLMER, YUNG WING CHEAH, Uwe Brand, Robert Eagle and Aradhna Tripati The Paleozoic is the first Era in the Phanerozoic, and is associated with major transitions in the evolution of multicellular life. During the Paleozoic, three mass extinctions occurred: the End Ordovician (443 Ma), Late Devonian (359 Ma) and the Permo‐Triassic (251 Ma) extinction that marks the end of the Paleozoic. I will be looking at trilobite and brachiopod samples from Mid and Upper Ordovician and Mid Devonian, the time period before mass extinction to see if there is evidence for any changes in temperature that might have contributed to the extinction. Articulated brachiopod shells and trilobite carapaces are composed of low‐Mg calcite, which is thought to be relative well‐preserved. Previous studies have used these fossiliferous remains as an archive of ocean water chemistry, arguing that some specimens preserve the oxygen, carbon and strontium isotope composition of seawater from when they were alive. I will using the clumped isotope method, which is useful for determining paleo‐
temperatures of carbonate samples. This proxy is sensitive to diagenesis, and I will identify specimens that may be unaltered and should be used for paleoclimate reconstruction. We have trilobite and coeval brachiopod remains spanning the Ordovician to Devonian that were previously screened for diagenesis in Brand, 2004. Samples will be analyzed by mass spectrometry to determine their isotopic composition, and carbonate formation temperatures calculated. Non‐altered samples may help us determined if changes 267 Towards An Understanding Of Herpesvirus RRM2 and Host Cell Metabolism TAYLOR M. BROWN, William J. Sullivan and Heather R. Christofk Differences between human proteins and their viral homologs have not been fully characterized, and thorough understanding of these differences may provide key insights into viral replication and infection. Ribonucleotide reductase small subunit (RRM2) is a component of the enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which is essential for DNA replication. Human RRM2 can undergo phosphorylation, while a viral homolog of RRM2 maintains an aspartic acid conservation throughout the herpesvirus family. The aspartic acid may lie in the same pocket and mimic the human phosphorylation event, resulting in constitutive activity of viral RRM2. To test this hypothesis, human breast epithelial cells were engineered to stably overexpress RRM2 or mutant versions of the protein, (S376D and S377D). Low levels of RRM2 expression were detected via immunoprecipitation of wildtype and S376D cells, and S377D cells resulted in apoptosis, suggesting that constitutive RRM2 expression is not tolerated. To overcome the observed repression of constitutive RRM2 expression, the wild‐type and mutant RRM2 constructs were sub‐cloned into doxycycline‐inducible vectors. The cellular effects of elevated wild‐type and mutant RRM2 expression will be investigated and compared via proliferation assays, cell cycle analysis, and metabolic profiling. Understanding the mechanism of viral RRM2 in promoting replication could be important for development of therapeutic strategies for viral infections. 84
SPD 2014 SESSION TWO 268 Using Change Blindness to Study the Effect of Visual Attention in Visual Area V4 DANIEL J. FOSTER, Fabrice Arcizet, Koorosh Mirpour and James W. Bisley Visual attention is necessary to perceive and react to our world; when we do not attend something, we are often completely unaware of it. This lack of perception is exemplified in change blindness tasks in which we are unable to detect a difference between two scenes of objects separated by a blank screen, even though the change may be huge. Past studies have suggested that any behavioral effects of attention, including change blindness, are due to a change in neural activity in visual area V4; the current study seeks to test this hypothesis. Subjects were trained in a simplified change blindness task comprised of one, two, four, or eight objects on a screen. Subjects had to pay attention to all objects presented and determine if one of the objects rotated. While attention can be focused when only one object is presented on the screen, attention must be spread with more than one object presented. Given the results of past studies we expected to see the neural activity of V4 correlate with the degree to which attention is spread; high activity correlating with focused attention and lower activity with spread attention. Using extracellular electrodes to measure spiking activity from single neurons, we see no correlation of V4 activity with the number of objects. Since the V4 activity is unchanging and different behaviors based on object number is seen, these results suggest that V4 is not responsible for the behavior associated with attention, as has been suggested in the past. 269 Olfactory Plasticity in Entomopathogenic Nematodes JOON HA LEE and Elissa A. Hallem Many parasites, including entomopathogenic nematodes (EPNs), use host‐emitted olfactory cues to locate hosts. However, how parasitic nematodes respond to host‐emitted odors remains poorly understood. In particular, little is known about how parasitic nematodes integrate host odor cues with environmental cues such as temperature and intrinsic cues such as age to mediate context‐appropriate host‐seeking behaviors. To address this question, we are investigating the olfactory behavior of EPNs from the genera Steinernema and Heterorhabditis. We find that EPNs are attracted to the general host cue carbon dioxide under all conditions tested. However, responses to many odorants exhibit extreme olfactory plasticity as a function of IJ cultivation temperature and/or age. For example, in Steinernema carpocapsae, many odorants that are strongly attractive at lower temperatures are strongly repulsive at higher temperatures and vice versa. In other species, we find type and extent of olfactory plasticity varies among EPNs. In addition, we find that foraging strategy can also vary with temperature. For example, Steinernema carpocapsae behaves more like an ambusher at higher temperatures and more like a cruiser at lower temperatures. Some EPNs are found in geographical regions that undergo substantial seasonal temperature variation, and we hypothesize that plasticity of olfactory behavior and foraging strategy may enable EPNs to optimize host seeking under changing environmental conditions. 270 Maladaptive Natural Reward Seeking During Opiate Withdrawal KAY E. LINKER, Venuz Y. Greenfield, Nigel T. Maidment, Sean B. Ostlund and Kate M. Wassum A significant aspect of drug addiction is that addicted individuals will seek out the drug at the expense of other rewards, despite negative consequences of use and in the face of a decline in drug‐induced euphoria or 'high', perhaps indicating the value of the drug is inflated. Opiate withdrawal has previously been characterized as a motivational state that will enhance opiate‐seeking actions upon experience with the drug during the withdrawal state. We attempted to investigate the effect of opiate withdrawal on natural reward seeking. Rats were trained to receive sucrose or water reward through a reward‐seeking chain of instrumental actions and then were given chronic morphine exposure on a regimen previously shown to induce dependence. Following the morphine treatment rats were tested in withdrawal for their instrumental incentive learning behavior. We found that rats showed enhanced sucrose or water reward‐seeking actions, but only after the reward was experienced in the withdrawal state. Interestingly, this enhanced reward seeking had no correlation with the emotional experience of the reward, highlighting the maladaptive nature of this state. Elevated reward seeking was blocked by inactivation of mu‐opioid receptors of the basolateral amygdala. 85
SPD 2014 SESSION TWO 271 Carbon Dioxide Gustatory Receptor Involvement in Drosophila Hematopoiesis REBECCA M. BARBER, Jiwon Shim and Utpal Banerjee Drosophila hematopoiesis involves not only a localized development pathway in the lymph gland of flies, but also a more systemic pathway in which various biological systems seem to play a crucial role in regulating hematopoiesis. Previous research has shown that neural systems such as, olfaction, can have a profound effect on blood differentiation. The present study looks at the neural sensation of taste, and investigates the role of carbon dioxide gustatory sensation in regulating hematopoiesis. Drosophila carbon dioxide detection works by way of two gustatory receptors, Gr63a and Gr21a. Using genetic manipulations and confocal microscopy, results have shown that depletion of these two receptors significantly increases the number of crystal cells, a blood cell type that deals with wound healing, within the lymph glands of flies. The downstream projection neurons from these receptors also cause an increase in crystal cells upon depletion. To further investigate the mechanism by which these receptors regulate crystal cell differentiation additional experiments examining factors such as, insulin signaling, glutamate signaling, and normoxic and hypoxic signaling will be performed. This project aims to elucidate the exact mechanisms that allow for gustatory regulation of blood development. The overall findings of this study will provide novel insights into blood development, demonstrating a unique sensory involvement in Drosophila hematopoiesis that may be conserved in other organisms. 272 Directing Differentiation of Transplanted Neural Progenitor Cells with a Hyaluronan Biopolymer Hydrogel Matrix in Stroke ANDREW R. BERG, Pouria Moshayedi, Lina Nih, Jonathan Lam, Tatiana Segura and S. Thomas Carmichael Stroke is the leading cause of long‐term adult disability in the United States with more than 795,000 cases per year. Brain self‐repair processes do not efficiently restore lost neurological functions, therefore it is important to find a way to reconstruct the neural circuitry. Transplanting neural progenitor cells into the brain may accomplish this, but cell survival is poor due to the hostile infarct microenvironment. High molecular weight hyaluronic acid (HA) hydrogels can 'conceal†transplanted cells from local inflammatory signals to improve their survival, but also prohibits cell differentiation and tissue interaction. To overcome this limitation, the HA biopolymer backbone can be engineered with synthetic RGD, YIGSR, and IKVAV motifs from extracellular matrix proteins, heparin, bone morphogenetic protein 4, and brain derived neurotropic factor. In the current study, neural progenitor cells (NPCs) generated from human induced pluripotent stem cells were encapsulated within HA hydrogels containing these bioactive components and implanted into NOD scid gamma mice one week after cortical ischemic stroke. Concentrations of bioactive components were varied to study their effects on NPCs in vivo. Preliminary results suggest that the bioactive components attached to the HA hydrogel influence survival and proliferation of the transplanted NPCs. Continuing investigation of angiogenesis, neurogenesis, and NPC differentiation will provide further insight for designing a suitable transplantable matrix for stroke cell therapies. 273 Prkag2 is a Mediator of Catecholamine Induced Cardiac Hypertrophy YI CHANG, Christoph Daniel Rau, Jessica Wang, Aldons J. Lusis and Yibin Wang Cardiovascular disease is one of the leading causes of death or chronic illness in the United States and the rest of the world. Many forms of cardiovasular disease involve cardiac hypertrophy, in which the mass of the heart maladaptively increases due to a stressful environment. Through careful analysis of the Hybrid Mouse Diversity, Panel using genome‐
wide association and network analysis, a gene known as Prkag2 has been identified as a possible candidate for contributing to cardiac hypertrophy. Prkag2 is a member of the AMP Kinase complex and acts to regulate the catalytic subunit of the complex. Through siRNA‐mediated knockdown in neonatal rat ventrical cardiomycocytes (NRVMs), we have deduced that knockdown of Prkag2 does in fact induce cell proliferation and increase in cell‐size after the addition of isoproterenol(ISO), a catecholamine which stimulates the beta‐adrenergic receptors of the NRVMs and induces hypertrophy. It also results in increase expression in known hypertrophic markers, when preforming qPCR. These in vitro experiments are promising and will be further confirmed using in vivo knockout mice. If proven to be a contributing cause of cardiac hypertrophy, knowledge of the Prkag2 gene will be a great foundation for future clinical trials 86
SPD 2014 SESSION THREE 300 Characterizing the Role of headcase in Drosophila Stem and Progenitor Cells MELISSA E. TRUONG, Pedro Resende, D. Leanne Jones Intestinal stem cells (ISCs) are adult stem cells in the Drosophila intestine that are important for maintaining tissue homeostasis. ISCs give rise to enteroblasts (EBs), which further differentiate into the absorptive and secretory cells of the intestine. Previous work in the lab identified the Drosophila headcase gene as a factor expressed throughout the testis stem cell niche, which appears to prevent programmed cell death of somatic support cells. However, neither its expression nor role has been explored in other stem cell niches, such as the intestine. We show that headcase is expressed and required for maintenance of the proliferative ISCs within the intestine. Loss of headcase through RNA interference results in complete to near‐complete loss of ISC and EBs. This loss is rescued by blocking apoptosis by expression of the anti‐apoptotic baculovirus protein p35. Surprisingly, flies are able to survive for over three weeks after headcase knockdown, which is contradictory to the existing model that stem cell proliferation is absolutely essential for tissue turnover. This result suggests that either stem cell‐mediated tissue turnover is not absolutely necessary or supports the existence of compensatory mechanisms to maintain homeostasis after stem cell loss. These two possibilities will be explored further in future studies, using the DNA‐damaging agent bleomycin to force tissue turnover in intestines depleted of stem and progenitor cells. 301 Axin2 and Pleiotrophin Expression Following White Matter Ischemic Stroke in Mice MICHAEL E. REITMAN, Shira Rosenzweig and S. Thomas Carmichael Stroke is a debilitating neurological disorder which affects approximately fifteen million people yearly world‐wide and is the leading cause of adult long‐term disability. Cortical ischemic stroke and the mechanisms by which neuronal death and repair occurs has been a key area of neuroscientific research. However, subcortical ischemic stroke in white matter (WMS), which accounts for approximately 25% of all stroke subtypes, and the mechanisms underlying repair and recovery in white matter ischemia have yet to be thoroughly investigated. Recently, our lab devised a unique mouse model of white matter stroke involving the microinjection of an endothelial NOS inhibitor N5‐(1‐iminoethyl)‐L‐
ornithine along the white matter ventral to the forelimb motor cortex and using laser capture microdissection and RNAseq a transcriptome was generated for cells surrounding the infarct core following the use of this model which revealed differential gene expression. Utilizing immunohistochemistry, we have explored the expression levels of two identified proteins, Axin2 and Pleiotrophin (PTN) at critical time‐points following WMS. We report that Axin2 is up‐
regulated outside the infarct core at both 7 days and 2 months post‐stroke in a population of fibrous astrocytes while PTN expression increases in a population of astrocytes inhabiting the sub‐ventricular zone. We conclude that Axin2 and PTN show differential expression following WMS indicating possible involvement in repair following white matter ischemia and potential as therapeutic targets 302 Identifying Small Molecule Activators of GDP Bound RAB5 JOSHUA WEINREB and John Colicelli RAS is a GTPase and also one of the first oncogenes identified. The human genome encodes more than 170 RAS‐
related GTPases that participate in virtually all cell functions. When bound to GTP, these proteins adopt an 'active’ conformation with high affinity for downstream effector molecules. Upon GTP hydrolysis, GTPases convert to an 'inactive’ conformation with low affinity for downstream effectors. We hypothesize that some small molecules can activate a GDP‐bound GTPase, not by releasing GDP but by allosterically inducing a conformational change that mimics the high affinity for downstream effectors normally associated only with a GTP‐bound GTPase. I am testing this hypothesis by developing an assay to identify small molecule activators of RAB5, a GTPase that regulates receptor endocytosis in mammalian cells. Constitutively activated RAB5 may lead to increased degradation of receptor tyrosine kinases (RTKs) and thereby reduce RAS activity, which could be therapeutic for solid tumors of epithelial origin. 87
SPD 2014 SESSION THREE 303 Heterospecific Alarm Call Eavesdropping in Non‐Vocal White‐Bellied Copper‐Striped Skinks (Emoia cyanura) HOLLY FUONG, KATHRYN N. KEELEY, YASEMIN BULUT, and Daniel T. Blumstein Many species benefit from listening to the vocalizations of their predators as well as the alarm calls of other species. This eavesdropping is an important way to acquire information of predator location and threat magnitude. Eavesdropping is commonly studied in mammals and birds, and it has only recently been reported to occur in reptiles. Studies that examine lizard responses to playbacks of both predatory calls and heterospecific alarm calls are absent, even though eavesdropping may be especially important in non‐vocal species. On a field research quarter in French Polynesia, we broadcast predator vocalizations, alarm calls from a non‐predatory bird (red‐vented bulbul, Pycnonotus cafer), and social calls from red‐vented bulbuls to determine if non‐vocal white‐bellied copper‐striped skinks (Emoia cyanura) could discriminate between them. Upon hearing red‐vented bulbul alarm calls, skinks reduced their rate of looking compared to a baseline period. However, they did not respond significantly to red‐vented bulbul social calls or potential predators' calls. Upon hearing red‐vented bulbul alarm calls, skinks reacted significantly differently than when they heard social calls or predatory calls. Our study is the first to look at the responses of both predator and heterospecific alarm call playbacks in lizards. White‐bellied copper‐striped skinks most likely depend on heterospecific vocalizations for predator information because they are non‐vocal and found low on the forest floor, making it harder to identify predators. 304 Exploring Linker Length as a Method to Increase Protein‐Poly(poly(ethylene glycol methyl ether) acrylate) Conjugation Yield PETER C. NAUKA, Juneyoung Lee, Heather D. Maynard There has been a growing interest in exploring the use of protein‐polymer conjugates to improve the pharmacokinetic properties of various therapeutic proteins in vivo. In particular, conjugating poly(ethylene glycol) (PEG) to proteins has yielded twelve FDA‐approved products currently on the market. PEG is known to improve the properties of proteins by decreasing immunogenicity and increasing half‐life in vivo and reduce dosage frequency. Due to these successes, there is much research interest in examining PEG derivatives, particularly branched polymers, for additional benefits while minimizing individual branch size. This would allow for the use of larger PEG's, while minimizing accumulation in vivo. One such candidate is poly(poly(ethylene glycol methyl ether) acrylate) (pPEGA), but the conjugation has been observed to be relatively low compare to linear PEGs. In this study, our aim is to improve the yield of conjugation by varying the length of hydrophilic oligo ethylene glycol linker between the polymer side chains and the protein‐reactive end group of chain transfer agent (CTA) The CTA's were synthesized by a four‐step synthesis and verified by NMR, MS, UV/Vis and IR. The polymers used for this project are being synthesized via reversible addition‐fragmentation chain transfer (RAFT) polymerization and verified using NMR and gel permeation chromatography (GPC). Following polymer synthesis, impact of linker length will be described by conjugating various proteins. 305 Oxytocin Reduces Social Working Memory Performance in Individuals with Higher Levels of Social Anxiety SPENCER UEMURA, Meghan L. Meyer, Benjamin A. Tabak, Michael R. Irwin, Matthew D. Lieberman, and Naomi I. Eisenberger The neuropeptide oxytocin (OT) has become popular in psychological research, touted for its broad range of social cognitive and anxiolytic effects (Bartz et al., 2011). However, more recent studies of oxytocin's effects have produced mixed results (Bartz et al., 2011). One possibility for these mixed results is that the tasks used to study oxytocin's effects on social cognition vary in difficulty. Thus, we examined how oxytocin influences social cognitive performance (the ability to keep track of multiple people's traits) on a social working memory (SWM) task with a high difficulty level. In a randomized, double‐blind, placebo‐controlled, between‐subjects study, we examined the effects of OT vs. placebo on reaction time and accuracy for a SWM task (considering friends' traits) as well as a cognitive working memory (CWM; alphabetizing friends' names) task. No main effects of drug condition were found for SWM or CWM. Among subjects with higher levels of social anxiety, OT was found to significantly decrease accuracy for SWM tasks (but no relationship was found with CWM). These findings contribute to developing more cautionary views of oxytocin's effects on anxiety and social cognition (MacDonald & Feifal, in press). 88
SPD 2014 SESSION THREE 306 BCG Vaccine‐Mediated Has No Apparent Neuroprotection in the SOD1G93A Murine Model of Amyotrophic Lateral Sclerosis AN V. NGUYEN, Jesse Li, Zheying Chen, Hoa Dang, and Daniel Kaufman Approximately 5,600 people in the U.S. are diagnosed with Amyotrophic Lateral Sclerosis (ALS), a progressive neurodegenerative disease that affects motor neurons, annually. The disease progresses quickly, and patients' survival is usually 2‐5 years after diagnosis. Thus, there is a need to develop new therapies to slow the progression of ALS, using methods of with widespread accessibility such as vaccination. It has been established that ALS exhibit neuroinflammatory reactions by certain activated microglia, astrocytes and T‐cells. Thus, modulating these neuroinflammatory reactions via the modulation of Tregs, whose action limits inflammatory responses, might be neuroprotective. Fortunately, it has been shown that Bacille Calmette‐Guerin (BCG), an attenuated live bovine bacillus used worldwide against tuberculosis meningitis, can induce Tregs response during persistent infection. We hypothesized that BCG vaccination, by inducing Tregs responses, can indirectly reduce the number of particularly harmful microglia activated during the neuroinflammation, thus slowing down disease progression and prolonging survival in the SOD1G93A mouse model of ALS. We were interested in two time points for BCG vaccination, before and late after disease onset, to see whether or not they affected the course of treatment. So far, preliminary results suggest that there is no difference between ALS transgenic mice treated with saline or BCG. However, more consideration is needed to establish an optimal BCG dosage and potentially necessary boosting. 307 Luciferase Assisted Proteome Detection Platform for Plant Systems Biology BENJAMIN LEE, Ziqiang Zhu, and Chentao Lin The biological phenomena observed in plants are the results of the integration of different environmental and internal cues into the signaling pathways that converge to regulate the expression of key regulatory genes. Furthermore, before convergence different pathways interact both synergistically and antagonistically to regulate the activity of the various pathways. Though these pathways in the Arabidopsis have been extensively studied, the underlying molecular mechanisms remain largely a puzzle. This lack of understanding is natural because in plant biology research to date there is no scientific methodology that allows for highly sensitive, inexpensive, in vivo, and real‐time kinetic analyses of proteomic behavior. Though mass‐spectrometry based proteomic studies have been done, it is not a suitable platform to systematically study individual proteins and determine their biological functions. Given the above, we have undertaken the effort to develop the Luciferase Assisted Proteome Detection (LAPD) library of transgenic Arabidopsis so that we can acquire a systems understanding of the Arabidopsis. Here, the LAPD project is reviewed. 308 Genetic Moderators of Treatment Response to Dexmethylphenidate in Children and Adolescents with ADHD SUSAN N. CHANG, Christopher P. Laughlin, James T. McCracken, and Erika L. Nurmi Background: The stimulant dexmethylphenidate (d‐MPH) is an effective treatment for hyperactive and inattentive symptoms associated with pediatric attention deficit hyperactivity disorder (ADHD). Treatment‐induced growth slowing, however, is a common adverse effect associated with morbidity and poor treatment adherence. Substantial variation in response and growth effects is common and may be explained by genetic factors. This study investigated whether genetic variation in drug targets (SLC6A3/DAT1, SLC18A2/VMAT2, TAAR1) and metabolic enzymes and transporters impacting drug disposition (ABCB1, NR1I2) could help explain differential outcomes in treatment response to d‐MPH. Methods: Height was measured at baseline and at Weeks 4 and 8 in 202 children participating in the NIMH TRECC Project 1 Study. Treatment response was determined by scores on the Clinical Global Improvement Scale. Variants previously shown to impact protein function or to have significant associations were genotyped using the TaqMan platform for allelic discrimination. Results: In our dataset, treatment‐induced growth changes were influenced by genetic variation in SLC6A3 (rs27072, p=4.2E‐07), TAAR1 (rs5008782, p=1.49E‐09), and SLC18A2 (rs363227, p=3.05E‐24) while treatment response was influenced by genetic variation in SLC18A2 (rs363227, p=0.01). Understanding the pharmacogenomic factors moderating treatment response and adverse effects could lead to strategies to individualize treatment matching and achieve more effective therapeutics in the future. 89
SPD 2014 SESSION THREE 309 Effect of Fructose Metabolism on Cue‐based Incentive Motivation LINDA TSAN, LAURA A. DUONG, ANNIE HUYNH, Alisa R Kosheleff, Nigel T Maidment, and Niall P Murphy Hypersensitivity to drug‐paired cues often leads to relapse in former drug addicts. While often attributed to mesolimbic dopamine sensitization, where a hyper‐reactivity to dopamine transmission is to blame, the molecular mechanisms are not well understood. This study tested the hypothesis that this increase in sensitivity to drug‐paired cues results from a disruption in insulin signaling in dopamine neurons, which negatively impacts synaptic dopamine clearance via dopamine transporters. Tested in the context of repeated cocaine exposure in rats, this disruption was predicted to heighten dopamine transmission for cocaine‐paired cues, thus increasing cocaine‐seeking behavior. Additionally, because exposure to fructose has been shown to promote insulin resistance in the the brain by disrupting insulin signaling, we predicted that a high fructose diet would exacerbate increases in dopamine transmission for cocaine‐paired cues, resulting in even more cocaine‐seeking behavior. A Pavlovian‐instrumental‐
transfer paradigm, in which rats were trained to associate cocaine administration with auditory cues and lever presses, was implemented to evaluate this hypothesis. The increase in cocaine‐seeking behavior by fructose‐exposed rats in the extinction phase of PIT, when compared to rats consuming a normal diet in our experiment adds to growing evidence that hypersensitivity to drug‐paired cues is tied to disruptions in insulin signaling in dopamine neurons, and that that these disruptions are exacerbated by a high‐fructose diet. 310 Heterospecific Non‐alarm Vocalisations Influence Risk Assessment and Antipredator Behaviour in Common Mynas LILAH HUBBARD, WILLIAM KING, Anmy Vu and Daniel T. Blumstein Animals rely on their acoustic environments to gain information regarding predator threats and social opportunities. However, because individuals have limited attention, focusing on a particular aspect of their acoustic environment may affect their ability to allocate attention elsewhere. The distracted prey hypothesis suggests that animals may be distracted by certain stimuli, inhibiting their ability to detect approaching predators. Similarly, the distraction‐conflict hypothesis suggests that social stimuli may also distract individuals. We tested these hypotheses in common mynas (Acridotheres tristis) by playing back three different stimuli: motorcycle noise, social red‐vented bulbul (Pycnonotus cafer) calls, and social common myna calls. We examined their response to each stimulus by measuring time allocation to various behaviours immediately before and during broadcasting the three playbacks. We then studied how these stimuli affected their antipredator behaviour by measuring the distance at which they fled from an approaching predator (flight initiation distance; FID). We found that mynas responded to all three stimuli by delaying their return to relaxed behaviour following the playbacks compared to a silent treatment. In contrast to the distracted prey hypothesis, however, we found that mynas fled at greater distances when hearing red‐vented bulbul social calls than during our silent treatment. This suggests that rather than distracting, some social vocalisations may enhance prey vigilance and lead to earlier flight. 311 Fibromodulin Reduces Scar Formation in Rodent and Porcine Cutaneous Wound Models ANDREW T. NGO, Janette N. Zara, Xinli Zhang Kevin S. Lee, Chen‐Shuang Li, Weiming Li, Grace X. Chang, Kang Ting, Chia Soo and Zhong Zheng The annual cost of treating cutaneous scarring and similar wound healing complications approaches three billion dollars. Unfortunately, current methods to reduce scar formation are ineffective or have undesirable side‐effects. Therefore, there is a pressing need for the development of alternative therapies to reduce scar formation. Fetal cutaneous wounds heal by forming normal tissue, rather than scar tissue as in adult cutaneous wounds. Previously, we found high levels of a small leucine‐rich proteoglycan called fibromodulin (FMOD) in the fetal extracellular matrix. FMOD's expression drops during the transition from fetal‐type scarless repair to adult‐type scarring repair. By using FMOD loss‐ and gain‐of‐function rodent models, we demonstrated that FMOD is necessary and sufficient for fetal‐type scarless healing. In this study, we found that application of FMOD to adult rodent and porcine wounds significantly reduced scar formation. Furthermore, we determined that FMOD operates by stimulating fetal‐type fibroblast behavior in adult fibroblasts, as shown by enhanced fibroblast migration and myofibroblast differentiation and contraction, which caused more rapid wound closure, and accelerated myofibroblast clearance after re‐
epithelialization, which reduced scar formation. FMOD therefore seems promising for human use. Further investigation could elucidate the cellular signaling pathways by which FMOD acts. With a better understanding of the mechanism, more effective therapies for scarless wound healing could be developed.
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SPD 2014 SESSION THREE 312 Simultaneous Cryogenic Anchoring and Radiofrequency Ablation for Cardiac Arrhythmia Treatment JASON LOW, RICHARD SEO, KE ZENG, THEODORE KEE, and Warren Grundfest Cardiac arrhythmia is a disease characterized by abnormal electrical conduction in the heart that results in ineffective pumping. Dysfunctional nodes in the conduction pathway or in the cardiac muscles lead to irregular heartbeat patterns that can potentially induce severe complications such as cardiac arrest. Radiofrequency ablation, the current gold‐standard cardiac arrhythmia treatment procedure, has proven effective but suffers from shortcomings due to instability between the RF tip and the target site. We tested a cryogenic anchoring system that can be combined with RF ablation to create a stable ablation tip that physically attaches to the cardiac surface. A prototype anchor that incorporates RF ablation with a cryogenic adhesion system was shown to anchor to cardiac tissue with enough adhesion strength to maintain attachment during cardiac contractions. The thermal interference between heat generated by RF ablation and the cryogenic temperatures of cryogenic anchoring was not significant, demonstrating the feasibility of utilizing both technologies simultaneously. These results suggest that combining RF ablation and a cryogenic anchor into one catheter enables a physician to treat cardiac arrhythmia with improved stability and may be utilized for anchored tissue ablation of other organs where instability is an issue. 313 Fear the Spear: Spearfishing Induced Behavioral Changes of an Un‐harvested Species Inside and Outside a Marine Protected Area KATHARINE A. LANGEL, MADISON J. THOMAS, DIEM SAMANTHA C. TRAN, Daniel T. Blumstein By prohibiting fishing, marine protected areas (MPAs) provide a refuge for harvested species. Humans are often perceived as predators by prey, and animals often respond fearfully to humans. Thus, fish response to humans on and off an MPA can provide insights into their perception of humans as a predatory threat. Previous studies have found differences in the distance that harvested species of fish initiate flight (flight initiation distance ‐ FID) from humans inside and outside an MPA, but less is known about un‐harvested species. This study focused on whether lined bristletooth (Ctenochaetus striatus), an un‐harvested surgeonfish, can discriminate between a snorkeler and a snorkeler with a spear gun inside and outside of an MPA in Mo'orea, French Polynesia. Lined bristletooth FID was significantly greater in the presence of a spear gun and varied depending on if the spear gun encounter was inside or outside of the MPA. These results imply a level of sophistication of fish antipredator behavior, generates questions as to how a non‐targeted species of fish could acquire fear of humans, and demonstrates that behavioral surveys can provide insights about antipredator behavior that may have consequences for conservation and management. 314 Improving the Time to Detection of a Micellar Aqueous Two‐Phase System Integrated with the Lateral‐Flow Immunoassay ALISON V. THACH, David Y. Pereira, and Daniel T. Kamei A protein detection assay that requires no power, lab equipment, or trained personnel would enable diagnostic testing at home, in resource‐poor settings, and in war zones. One such test that satisfies these criteria is the lateral‐flow immunoassay (LFA). However, LFA typically exhibits low sensitivities that prevent its use in these applications. Our lab previously improved the sensitivity of LFA by using colloidal gold nanoparticles (GNPs) decorated with antibodies to capture the target protein and concentrate it in one of the two phases of a micellar aqueous two‐phase system (ATPS). The concentrated protein sample was then extracted and detected on an LFA test strip used in the competition assay format. Although this approach significantly lowered the detection limit, the micellar ATPS phase separates in a test tube on the order of hours, prohibiting its use in a point‐of‐need application. We therefore integrated the micellar ATPS on the LFA paper membrane, and discovered that the duration for macroscopic phase separation was reduced to minutes. To better understand this process, we performed fundamental studies to examine the effects of size and surface properties of the GNPs on their ability to flow through the paper membrane. This understanding will allow us to better engineer a device to be used at the point‐of‐need for a variety of applications. 91
SPD 2014 SESSION THREE 315 Pitted Collagen Hydrogels as a Biomimetic Scaffold for Intestinal Tissue Engineering TIFFANY YEH, Garrett Brinkley, Francisco Lei, and James C.Y. Dunn Treatment of many diseases today requires surgical interventions that are significantly invasive to patients. Methods of treatment for intestinal diseases are no different. These treatment methods are not only highly invasive but many times are ineffective and even result in total loss of intestinal function. Attempting to address this problem, tissue engineering provides minimally invasive methods of treating these intestinal diseases. Specifically, implantable biomimetic scaffolds are used in an effort to regenerate the intestinal epithelium. To further this investigation of regenerating the epithelium, pitted collagen scaffolds were fabricated using by laser cutting pits into the hydrogels directly after gellation. Analysis of these scaffolds was done using fluorescent markers to monitor the growth and proliferation of mouse and human myofibroblasts that when proliferated delivers growth factors and support crypt cell growth. Further observations were done on co‐cultures of both types of myofibroblasts and crypt cells using similar fluorescent markers and confocal fluorescent microscopy. This project will aid in the design of future intestinal implants and provide a more patient‐friendly treatment option for inflammatory intestinal diseases. 316 Morphogenesis of Basal Skin Cells during Sensory Axon Ensheathment MARCI F. ROSENBERG, Jeffrey Rasmussen and Alvaro Sagasti Zebrafish touch‐sensing neurons innervate the skin with elaborate peripheral arbors. These arbors are free endings, devoid of associated glia which normally ensheath axons to provide mechanical and metabolic support. Previously, our lab found that basal skin cells ensheath these sensory endings. Similar ensheathment channels are seen in the skin of a wide variety of other organisms, suggesting they perform essential functions; however, neither the mechanisms leading to channel formation nor the impact they have on sensory function is known. Using transgenic zebrafish and confocal microscopy, we measured the appearance of cytoskeletal markers associated with axon sheaths. We found that two membrane reporters (GFP‐CAAX and GFP‐PH‐PLC) first localize to ensheathment channels, then a reporter of filamentous actin (Lifeact‐GFP), then finally junctional reporters (plakoglobin‐YFP and alpha‐catenin‐YFP). Blocking peripheral sensory axon development by injection of a neurogenin1 morpholino prevented the formation of ensheathment channels and recruitment of these reporters. We thus propose a model whereby a sensory axon‐
derived signal polarizes the basal skin cell, leading to remodeling of the actin cytoskeleton and formation of a channel for the extending axon. Adherens junctions then seal the opening over the axon to stabilize the sheath. We hope that by blocking ensheathment channel formation we will gain insights into how the skin modulates sensory axon function and/or survival. 317 Identification of Candidate Genes for a Corneal Dystrophy of Bowman Layer Not Associated with a Mutation in the Transforming Growth Factor Beta‐Induced Gene DEREK J. LE, Ricardo F. Frausto, Ashley N. Roldan, Lydia B. Ann, and Anthony J. Aldave Corneal dystrophies are a group of disorders that are characterized by impaired vision due to loss of clarity in the cornea. Most corneal dystrophies exhibit a dominant Mendelian inheritance pattern and have been linked to mutations in genes expressed in the cornea. Corneal dystrophies of the Bowman layer (CDB) have been previously linked to dominant mutations in the exons of the transforming growth factor beta‐induced gene (TGFBI). However, a three‐generation Argentinian family was diagnosed with CDB and lacked mutations in TGFBI, suggesting a different, causative gene that has not been identified. Using whole‐exome sequencing of affected and non‐affected family members, variants in affected individuals and absent in unaffected individuals were identified. Human genetic variation databases, corneal gene expression data, and mutation pathogenicity prediction tools were used to further filter causative variants. After filtering, ten variants in ten genes were identified. Mutation prediction tools identified three deleterious variants in three genes: BAZ2A, a component of the nucleolar remodeling complex; CYP26A1, an enzyme involved in retinoic acid metabolism; and PAPLN, an extracellular matrix glycoprotein. In addition, CYP26A1 is located 1 megabase from 10q23‐24, a region on chromosome 10 previously linked to a family with similar form of CDB. While mutations in all ten genes should be screened for in other affected families, a mutation in BAZ2A, CYP26A1, and PAPLN likely represent the genetic basis for this disease. 92
SPD 2014 SESSION THREE 318 The Effects of Relative Humidity on Tropical Lake Temperatures AKSHAT MAHAJAN and Aradhna K. Tripati One of the most useful tools in climate modelling and simulation is the fact (reproduced independently by general circulation models) that relative humidity remains constant at progressively higher elevations, even as water vapour pressure and saturation vapour pressure fall in accordance with the lapse rate. Historically, modeling attempts have assumed that surface relative humidity remains constant spatially and temporally ‐ however, recent data reveal this is not the case. In particular, temporal trends in surface relative humidity are revealed to be most pronounced in the Tropics. Our understanding of water vapour interactions in the atmosphere relies on our understanding of relative humidity distribution and behaviour. We outline a plan to apply a model of relative humidity change to paleoclimatic records on Earth in order to assess the model's validity in describing climate reconstructions and future simulations. We present preliminary Pleistocene and Holocene lake temperature reconstructions to aid us in our work, derived from clumped isotope analysis of microbialities, sediment and gastropod shells. 319 Design of Line Confocal Microscope High‐Speed 3D Imaging TIMOTHY SHERRY and Katsushi Arisaka This project seeks to utilize a scientific CMOS camera and sheet illumination to try to capture a 3D image at high speeds relative to other confocal microscope systems. I have utilized a cylindrical lens to create a narrow sheet alongside a galvanometer to scan the beam across the sample quickly to generate the 3D image on the sCMOS sensor. By using the newly developed rolling shutter mode on the sCMOS camera that goes line by line, I am able to achieve confocality without the normal use of a slit. The system utilizes a 470‐nanometer laser that can be swapped with a 488‐nanometer laser to excite green fluorescent protein to allow neural network imaging of C. Elegans. This system can also be attached to current wide‐field microscopes to convert them into line confocal systems allowing for higher resolution at much cheaper prices than a regular confocal microscope as well. The system has performed well in trials, and should present itself as a viable and economical alternative to current confocal microscope systems. 320 Dorsal Hippocampus Modulates Behavioral Sensitivity to Ambiguous Situations in Rats ESTHER W. YANG, PATRICIA L. STAN, Cynthia D. Fast, M. Melissa Flesher, Nathaniel A. Nocera, Michael S. Fanselow, and Aaron P. Blaisdell We often encounter ambiguous situations where information needed for decision‐making is unavailable, yet we still make decisions by drawing on past experiences. Neural processes mediating such inferential reasoning remain poorly characterized. We investigated the neural mechanisms for how rats apply inferential reasoning strategies to make decisions in ambiguous situations. In Experiment 1, we investigated the role of acetylcholine (ACh) in the dorsal hippocampus (DH) during tests with ambiguously or explicitly absent cues. Micro‐infusions of the ACh antagonist, scopolamine, in the DH disrupted performance in the ambiguous situation. In Experiment 2, we examined c‐Fos (an early gene product indicative of neural activity) expression of rats engaged in a reasoning task versus rats engaged in a task that did not recruit higher‐level reasoning processes. C‐Fos expression patterns in the DH depended on the rat engaging in a reasoning task when it was faced with an ambiguous test condition. Collectively, these results offer insight into neural mechanisms involved in complex instrumental discriminations of ambiguous situations. 93
SPD 2014 SESSION THREE 321 The Effect of FOXP2 Overexpression in the Avian Basal Ganglia on the Time Course of Deafening‐
Induced Deterioration in Song Quality ROZI AULAKH, Nancy F. Day, and Stephanie A. White Mutations of the transcription factor FOXP2 in humans causes problems with speech and language learning and production. Zebra finch songbirds have been used as a model to study FOXP2, as they learn their song during a critical period and rely on auditory feedback to learn and maintain song. FoxP2 knockdown within the song control region of the avian basal ganglia interferes with vocal learning in juvenile finches. To explore the possible role of FoxP2 in adult song maintenance as a means for studying the gene in humans, we investigated the time course of deafening‐induced deterioration of song. We hypothesized that overexpression FoxP2 alters the time course of deafening in adult birds. For this experiment, some birds received an adeno‐associated virus that causes overexpression of Foxp2, and some received an adeno‐associated virus that transduced green‐fluorescent protein instead. Thereafter, birds were either deafened or received a sham surgery. Song motifs from before and after deafening were analyzed. We determined that the deafened birds that received FoxP2 virus underwent an accelerated rate of song deterioration compared to the sham birds. 322 Hypoxia‐induced Insulin Resistance Inhibits Oxygen‐dependent Growth in Drosophila melanogaster DANIEL M. WONG, Kristin E. Owyang and Julian A. Martinez‐Agosto In Drosophila, hypoxia systemically restricts larval growth, but the molecular mechanism through which it does so remains poorly characterized. We show that Sima, the alpha subunit of the Drosophila hypoxia‐inducible factor, accumulates in nuclei of larval fat body cells during hypoxia and inhibits systemic growth when overexpressed in this same tissue. Additionally, hypoxia and Sima overexpression in the fat body cause the retention of Drosophila insulin‐
like peptides (Dilps) in the larval brain. Ubiquitous overexpression of the insulin receptor rescues hypoxic growth restriction, suggesting that hypoxia‐induced insulin resistance in peripheral tissues serves to inhibit systemic growth. A survey of tissue‐specific insulin receptor overexpression under hypoxic conditions identified the trachea as sufficient to rescue growth inhibition. Activation of insulin signaling in the larval trachea under hypoxic conditions results in increased tortuosity, phenocopying that observed in Warts (LATS1/2 in mammals) mutant trachea. Similar to trachea‐
specific activation of insulin signaling during hypoxia, downregulation of Warts in the trachea also rescues hypoxic growth restriction. Collectively, our results demonstrate that hypoxia induces insulin resistance in peripheral tissues and that insulin‐ and Warts‐mediated growth of the tracheal system is sufficient to rescue hypoxic growth inhibition. These findings may be applicable to other instances in which an adaptive response to hypoxia is required. 323 Innate Immune Response of Drosophila to Entomopathogenic Nematode Infection JENNIFER PENA, Mayra Carrillo, and Elissa Hallem Entomopathogenic nematodes (EPNs) are lethal parasites of insects that are of interest as biocontrol agents for insect pests and as models for harmful parasitic nematodes of animals and plants. EPNs have symbiotic bacteria that colonize their gut and that aid them in killing their host. The goal of this study was to establish a model system for investigating the immune response to nematode infection and to characterize the insect immune response to nematode infection. To do this, we investigated the ability of the EPN Steinernema carpocapsae to infect Drosophila melanogaster. Using a natural infection assay, we found that S. carpocapsae can infect and kill D. melanogaster, that infection induces expression of a subset of antimicrobial peptides (AMPs), and that AMP expression is a response to the bacterial symbiont rather than the nematode. In addition, we found that infection causes melanization, an insect immune response used to encapsulate pathogens. A higher rate of melanization was observed following infection with axenic EPNs, suggesting that the bacterial symbiont may suppress melanization. Infection of D. melanogaster with seven different EPN species revealed that EPNs vary in their virulence, and infection of five different Drosophila species with S. carpocapsae revealed that Drosophila species vary in their susceptibility to infection. Our results establish a model system for investigating the immune response to parasitic nematode infection, and may be useful in enhancing the efficacy of EPNs as biocontrol agents. 94
SPD 2014 SESSION THREE 324 From Bench to Bedside: Cloning and Functional Testing of HIV‐1‐Specific TCRs from CD8+ T Cells for Adoptive Therapy PRIYA K. PATEL, Hwee Ng, Sangeun Park, Christian Hofmann and Otto O. Yang HIV‐specific cytotoxic T lymphocytes (CTLs) maintain the health of infected persons temporarily but eventually fail. A main reason is the high mutation rate of HIV. CTLs kill infected cells by recognizing viral sequences presented by HLA class I as epitopes on the cell surface, but the virus can mutate and no longer be recognized by CTLs. However, combinations of different CTLs, through T cell receptor (TCR) gene therapy might combat this problem. The aim is to clone and functionally test HIV‐specific TCRs for this purpose. Genes for TCRs recognizing HIV epitopes were isolated by PCR from infected persons and cloned into a lentiviral vector. TCR functionality was determined in a Jurkat‐T cell assay. To characterize TCR activity against epitope variants we will challenge TCR‐transduced primary CD8+ T cells with target cells infected with HIV with epitope mutations. We generated HIV clones with epitope variants via site‐directed mutagenesis, and vectors to transduce the appropriate HLAs into target cells. We cloned and tested six TCRs, one against RQANFLGKI (B*1302), three against KRWILLGLNK (B*2705), and two against GLNKIVRMY (B*1501). T1 and H9 target cells were transduced with B*1302, B*1501, and B*2705. Combining several TCRs could cause pressure on HIV and prevent escape from the immune system. Ultimately this could be used for TCR gene therapy to treat HIV. 325 Improved Delivery of Diphtheria Toxin to Cancer Cells Using Transferrin Variant‐Conjugated Nanoparticles KATHRYN R. DERN, Kristine M. Mayle, Ricky Y. T. Chiu, Stephanie J. Wang, and Daniel T. Kamei Due to the nonspecific toxicity of current standard‐of‐care cancer treatments, there is a demand for a drug carrier that exhibits specificity toward cancer cells while leaving healthy tissues unharmed. One commonly used targeting ligand for cancer therapy is the iron‐carrying protein transferrin (Tf), whose receptor is overexpressed by cancer cells relative to healthy cells. However, rapid recycling by cells, or low cellular association, limits the time frame in which Tf resides in the endosome and can release its cytotoxic payload. Previously, our lab determined that one way to increase the cumulative residence time of Tf within a cell is by decreasing its rate of iron release. Our lab hypothesized that a nanoparticle decorated with diphtheria toxin (DT) and a variant Tf exhibiting a lower iron‐release rate would demonstrate increased drug carrier efficacy compared to the wild‐type Tf‐conjugated counterpart. We performed in vitro cytotoxicity studies of nanoparticles decorated with DT and Tf in human prostate cancer cells. We found that the variant Tf‐conjugated nanoparticles displayed enhanced cytotoxicity compared to the wild‐type Tf‐conjugated nanoparticles, indicating that the variant Tf is a superior targeting ligand for the treatment of cancer. Our variant Tf has the potential to improve cancer therapy in the future by increasing the drug carrier efficacy of nanosystems and reducing toxic side effects to cancer patients undergoing treatment. 326 Dissecting Social Motility in African Trypanosomes through RNA‐Seq Transcriptomics and RNAi Functional Analysis RINA KIM, Michael Oberholzer, Sean Gallaher, Artur Jaroszewicz, Matteo Pellegrini, HoangKim Nguyen and Kent Hill
Cell‐cell communication and social behavior are widespread in microbes, such as fungi or bacteria. Social behavior was recently discovered in protozoan parasites. However, its contribution to disease pathogenesis and transmission are poorly understood. African trypanosomes (Trypanosoma brucei) are flagellated protozoan parasites that cause sleeping sickness in humans and related diseases in livestock in Sub‐Saharan Africa. T. brucei parasites engage in social behavior or 'social motility†when cultivated on semisolid agarose surfaces. This behavior is characterized by trypanosomes assembling into multicellular communities that engage in polarized migrations across the agarose surface and cooperate to divert their movements in response to external stimuli. Developmental transformation from single cell to multicellular life‐style in T. brucei is expected to be regulated by changes in gene expression. To identify the mechanisms underlying social motility we applied a RNA‐Seq transcriptome comparison of trypanosomes cultivated in suspension culture versus surface conditions. The results indicate that a set of genes is differentially regulated between the two culture conditions. Current effort is focused on dissecting the contribution of identified genes to social motility using RNA interference (RNAi) knockdown approaches. The study of social behavior in microbes is expected to open new paradigms for considering host‐parasite interaction and cell‐cell communication, giving way to new strategies for therapeutic intervention. 95
SPD 2014 SESSION THREE 327 Circadian Clock Rescue: Scheduled Feeding in BACHD Mice RICHARD FLORES, Dawn H Loh, Danny Truong, Analyne Schroeder, Yu Tahara, and Christopher Colwell Many suffering from neurodegenerative diseases such as Huntington's disease (HD) experience a wide variety of ailments. Prior to the appearance of motor dysfunction in HD patients is the prevalence of poor sleep, suggesting a problem with the circadian clock. BACHD mice, a mouse model of HD, exhibit similar symptoms both in poor motor skills and circadian rhythms. The suprachiasmatic nucleus (SCN) is the master clock of the body that synchronizes biological processes throughout the body including digestion. This study targets this feedback to the SCN by allotting BACHD and wildtype mice a six hour window per day in which they can feed. Preliminary results indicate that scheduled meals are able to ameliorate their poor rhythms as demonstrated by them staying asleep for longer throughout the night and being more active during the day. Even more intriguingly, the BACHD mice's progressive motor coordination decline slowed and in some cases improved compared to the control group. Further studies will determine whether a protocol of scheduled mealtimes can be used as a treatment for failing circadian clocks in HD and other neurodegenerative diseases. 328 Superfluid Helium‐4 Transition on Multi‐walled Carbon Nanotubes VITO M. IAIA, Emin Menachekanian and Gary A. Williams A technique for generating third sound, a thickness and temperature wave in a superfluid film, has been developed for superfluid helium‐4 films that coat the surface of multi‐walled carbon nanotubes. The temperature oscillations are detected with carbon resistance bolometers. The nanotubes are packed in an annular resonator that is vibrated with a mechanical shaker assembly, consisting of a permanent magnet mounted on springs, and surrounded by a superconducting coil. Driving the coil with an oscillating current and sweeping the drive frequency between 100‐200 Hz vibrates the cell and excites the resonant third sound mode of the helium film. This is seen as a sharp resonance in the Fast Fourier Transform analysis of the bolometer signal. A problem with the original setup was that the cell was rigidly coupled to the mixing chamber of the refrigerator that provided the cooling to low temperatures, below one Kelvin. The vibrational heating warmed up the refrigerator, preventing measurements on the thinnest helium films below 4 atomic layers, which require cooling below 0.2 K. A new configuration suspended the cell as a pendulum on a string, with thermal contact made by copper wires. Piezo sensor measurements showed this reduced the vibration to the mixing chamber by nearly two orders of magnitude. This experiment allows a better understanding of unique superfluid properties, like the strong oscillations found in the third sound velocity at integer helium atomic layers. 329 Function of Opioid Kappa Receptor (OPRK)/Dynorphin signaling pathway in Human Chondrocytes SAUMYA SHAH, Ling Wu and Denis A. Evseenko Osteoarthritis, which involves articular cartilage degeneration, is characterized by a loss of extracellular matrix proteins and glycosaminoglycans (GAG), and an increase in pro‐inflammatory cytokines such as TNF alpha. Our previous studies have shown that human fetal chondrocytes highly express opioid receptor kappa (OPRK), whose presence and function during cartilage development has not been reported. Our prior experiments have indicated that activation of OPRK increases GAG production while down‐regulating expression of genes such as MMP13 (involved in collagen and extra‐cellular matrix degradation). Current experiments study the mechanism underlying the increased GAG production upon stimulation of OPRK agonist. We treated pig chondrocytes, pig and adult human cartilage explants with TNF alpha (a cytokine that induces degradation of cartilage matrix by inducing catabolic genes) and with/without OPRK agonist dynorphin. Results of flow cytometry indicated that increased apoptosis due to TNF alpha was mitigated in presence of dynorphin. Additional experiments will be conducted involving the measurement of changes in ECAR (Extracellular Acidification Rate) in the presence and absence of dynorphin in adult pig chondrocytes as well as differences in dynorphin concentration in synovial fluid of patients with/without osteoarthritis. Ongoing work suggests that OPRK agonists such as dynorphin may be helpful in osteoarthritis therapy via retention of GAG in articular cartilage and protection of chondrocytes from apoptotic stimuli. 96
SPD 2014 SESSION THREE 330 Photodegradable Polyacrylamide Gels for Protein Recovery After SDS‐PAGE VALERIE A. WINTERS, Jason Kerr and Andrea M. Kasko Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) is an extremely useful analytical method for determining protein molecular weight via size separation. Many researchers wish to take advantage of the high resolution of the gels to purify small quantities of protein. Though some methods exist for extracting proteins, they either require expensive chromatographic elution equipment or very harsh conditions to degrade the stable and chemically inert matrix of the polyacrylamide gel. These conditions irreversibly damage all proteins. To address this limitation, we have incorporated photodegradable crosslinkers into our polyacrylamide gels for inexpensive and facile protein recovery. Application of UV light to a specific protein band cleaves local photodegradable bonds and increase the network mesh size, allowing for release of the desired protein. 331 Characterization of Yeast Prion Ure2 Amyloid Fibril Structure TIFFANY Y. LIN and Zhefeng Guo Prions are infectious proteins that underlie fatal neurodegenerative disorders called prion diseases, such as Creutzfeldt‐Jakob disease in humans and mad cow disease in cattle. Prion protein forms long unbranched protein fibrils called amyloids. The fibril structure is essential for understanding the role of fibrils in these diseases. A unique feature of prion diseases is the strain phenomenon, in which distinct strains display consistent characteristics such as incubation period, distinct patterns of PrP‐Scrapie distribution, and relative severity of spongiform changes in the brain. The molecular basis of strains is elusive. One hypothesis is that different amyloid structure underlies different prion strains. To investigate the molecular basis of prion strains, we used yeast prion protein Ure2 as a model system. We prepared Ure2 fibrils under two temperature conditions: 4 degrees C and 37 degrees C. We performed repetitive seeding to obtain a homogenous fibril population for analysis. Transmission electron microscopy reveals differences in fibril morphology, and thermodenaturation of the fibrils shows differences in fibril stability. The difference in biochemical properties suggests that these fibrils have different molecular structures. Future work to transform these fibrils into yeast cells will determine if they can lead to different yeast prion strains. Detailed studies of fibril structures will further reveal the structural basis of the strain phenomenon. 332 Electrophysiological Effects of Right and Left Vagal Nerve Stimulation on Ventricular Myocardium Kentaro Yamakawa, Eileen L So, Pradeep S Rajendran, JONATHAN HOANG, Nupur Makkar, Aman Mahajan, Kalyanam Shivkumar and Marmar Vaseghi Vagal nerve stimulation (VNS) is generally regarded to be cardioprotective. However, the regional ventricular electrophysiological effects of right and left VNS remain unclear. The purpose of this study was to assess the effects of right and left VNS on the electrophysiological properties of the ventricular myocardium. In 12 Yorkshire pigs, the heart and bilateral cervical vago‐sympathetic trunks were exposed and the vagal nerve isolated. Activation recovery interval (ARI), a surrogate for action potential duration, was analyzed at baseline and during right and left VNS. A 56‐electrode sock was placed on the ventricles (n=12) for epicardial and a 64‐electrode basket catheter was placed in the left ventricle (LV) for endocardial ARIs (n=9). A Millar conductance catheter was used to acquire hemodynamic data. Right and left VNS decreased heart rate, LV pressure, dp/dt max and comparably prolonged ARI. Changes in dp/dt max had the strongest correlation with ventricular ARI effects. There were no anterior‐posterior‐lateral regional differences during VNS. However, greater prolongation was demonstrated at the apex than the base and at the endocardium than the epicardium. Unlike sympathetic stimulation, right and left VNS have comparable effects on ventricular repolarization. The functional effects of VNS are greater on the endocardium than the epicardium, and on the apex than the base. Changes in dp/dt max demonstrated a stronger correlation with the electrophysiological effects of VNS than either heart rate or blood pressure. 97
SPD 2014 SESSION THREE 333 Screening for Suppressors of an Ancient Atypical Kinase in Coenzyme Q Biosynthesis HEI TONG LAM, Theresa P.T. Nguyen and Catherine F. Clarke Coenzyme Q (ubiquinone or Q) is a lipid electron carrier in the electron transport chain. COQ8 is identified as one of the eleven yeast Saccharomyces cerevisiae genes essential for Q biosynthesis. COQ8 encodes a putative kinase responsible for the stability of the CoQ synthome, a high molecular mass Coq polypeptide biosynthetic complex. Previous studies have characterized seven coq8 yeast point mutants unable to synthesize Q. In this study, we isolated six revertants that correct the Q biosynthetic defect present in the coq8‐3 point mutant. Each of the six revertants retain the original mutations present in the parent strain, but can grow in medium containing nonfermentable carbon source and produce readily detectable amounts of Q6 as assayed by LC/MS‐MS. We are investigating the nature of the suppression in these revertants. We hope to identify via molecular genetic approaches the synthetic interaction between the suppressors and the Coq8 polypeptide. 334 Small Molecules that Increase Life Expectation (SMILEs) Extend Life Span Through Targeting Mitochondria and Reactive Oxygen Species Induction JOSE J. TORRES, Xudong Fu and Jing Huang Much effort has been emphasized on pharmacological perturbation of biochemical pathways that extend life span of model organisms. In previous studies in our lab, we successfully screened several Small Molecules that Increase Life Expectancy (SMILEs) which extend the lifespan of Caenorhabditis elegans through targeting bioenergetics. To add, pathways induced by oxidative stress, which is tightly linked to bioenergetics, have been implicated to increase lifespan of Caenorhabditis elegans. For this reason, we wanted to determine if there were potential links between SMILEs and reactive oxygen species (ROS) that induce oxidative stress in mammalian cell lines. To do this, we treated various mammalian cell lines with SMILEs implicated in increasing lifespan of Caenorhabditis elegans to observe whether any of these small molecule drugs affected ROS levels indicated by the oxyblot method and using the 2’,7’ dichlorofluorescein diacetate dye. We found the SMILEs our lab had previously discovered to increase lifespan of Caenorhabditis elegans increased ROS levels. This suggests that these SMILEs induce moderate amount of ROS and potentially activate downstream protective mechanisms that cause cells to be resistant to oxidative stress. The results also indicate ROS and downstream pathways may play an important role in the anti‐aging effect of SMILEs, and other small molecules drugs that can induce moderate amounts of ROS might also be able to increase lifespan of model organisms. 335 The Role of Pou3f1 and Bcl11b in Respiratory Motor Circuit Formation ERIC Y. WANG, Albert Y. Han, and Bennett G. Novitch Breathing is a critical voluntary and involuntary muscular movement necessary for survival. Muscles that are involved in breathing are controlled by a dedicated neural circuit in the central nervous system. The respiratory motor neurons (MNs) directly responsible for the movement of the diaphragm and other muscles involved in breathing are part of the hypaxial motor column (HMC) of the spinal cord. Two genes, Pou3f1 and Bcl11b, have been found to be expressed in distinct sub regions of the HMC. We have generated DNA constructs that we are currently using in gain‐of‐function experiments to test our hypothesis that Pou3f1 and Bcl11b are involved in the differentiation of HMC MNs. Our initial results reveal that there is reciprocal inhibition of Pou3f1 and Bcl11b expression in the developing spinal cord. We hope to elucidate how these genes affect MN fate specification, columnar organization, cell body migration, and axon trajectory. 98
SPD 2014 SESSION THREE 336 Enhancing the Lateral‐Flow Immunoassay Using Gold‐Coated Magnetic Nanoparticles for Rapid Detection of Proteins PHUONG T. NGUYEN, Alison V. Thach, Ricky Y.T. Chiu, and Daniel T. Kamei A rapid, sensitive, and affordable point‐of‐care diagnostic device is pivotal in preventing the spread of diseases caused by infectious agents. Here, we have investigated the combination of gold‐coated magnetic particles (GMPs) with aqueous two‐phase systems (ATPSs) to concentrate the biomarkers prior to detection with the lateral‐flow immunoassay (LFA). Using the model protein transferrin (Tf), the GMPs conjugated with antibodies against Tf rapidly captured and concentrated Tf molecules in solution using an external magnet and an ATPS comprised of polyethylene glycol and potassium phosphate. Our preliminary results indicate that, the extracted GMPs are compatible with the subsequent LFA test. For future work, the GMPs and different ATPSs will be optimized to achieve at least a 10‐fold improvement in the detection limit of LFA for Tf. With this improved sensitivity, we envision that this device can be used to detect many biological agents. 337 Induction of Artificial Blinking for Super‐Resolution Optical Fluctuation Imaging (SOFI) Analysis JASON L. CHANG, Jianmin Xu, and Shimon Weiss With the application of advanced mathematics in Super‐resolution Optical Fluctuation Imaging (SOFI), the diffraction limit of light can be surpassed enabling the visualization of live cancer cells on the nanoscopic level. In order to apply SOFI analysis, fluorescent probes need to blink stochastically in the millisecond time range, but these probes are limited and intense excitation light are required to induce blinking. Thus, a nucleotide‐quencher system was created to allow for the artificial induction of blinking and fine control over blinking rates. This system involves nucleotide chains attached to the target of interest and a fluorophores at the other end. Quenchers conjugated to complimentary nucleotide chains are introduced to the sample. The binding and unbinding of nucleotide chains through base pairing result in dark and bright states of the fluorescent probes. Altering the length of complimentarity allows for the fine‐
tuning of blinking rates. The longer the complimentary sequence, the slower the blinking rate, and vice versa. Through this system, millisecond blinking on surfaces and fixed heLa cells were achieved without the requirement of powerful excitation lights, which are harmful to cells. The application of this technique to live cells coupled with SOFI analysis will enable real‐time, live cancer cell imaging of dynamic states on the nanoscopic level. 338 Organization of Actin Filaments by Cardiomyopathy Associated Metavinculin Mutants REBECCA M. MCGILLIVARY, Zeynep A. Durer, and Margot E. Quinlan Dilated cardiomyopathy is a heritable heart disease characterized by enlarged and weakened ventricles. This impairs the heart's ability to pump blood to the body, and often results in heart failure. Mutations in the protein metavinculin have been found in patients with dilated cardiomyopathy. Vinculin is a ubiquitously expressed protein that anchors actin filaments to membranes. Metavinculin is a larger isoform of vinculin that anchors actin filaments to intercalated discs, the sites of force transmission between cardiomyocytes. Metavinculin is known to sever actin filaments and create actin meshworks, while vinculin is known to bundle actin filaments. We are investigating structural alterations to actin filament networks that are caused by mutations in metavinculin. Using bulk assays and fluorescent microscopy, we are studying the actin filament binding, bundling, and severing activities of metavinculin and the cardiomyopathy mutants. Our data show that the cardiomyopathy mutants bind to actin filaments with higher affinities than wild‐type metavinculin and that neither wild‐type metavinculin nor the mutants bundle actin filaments. We are currently investigating the actin filament severing capabilities of the cardiomyopathy mutants using total internal reflection fluorescence microscopy. Changes in actin filament organization may affect the ability of intercalated discs to transmit force and withstand mechanical stress, leading to the development of cardiomyopathy. 99
SPD 2014 SESSION THREE 339 The Selective Pressures Maintaining Phenylalanyl‐tRNA Synthetase Editing Function in Escherichia coli KELLY P. VAN, Nathaniel S. Howitz, and Beth A. Lazazzera Aminoacyl‐tRNA synthetases attach the correct amino acid onto the corresponding tRNA during translation. Sometimes these aminoacyl‐tRNA synthetases (aaRSs) mischarge tRNAs with similar amino acids. Such error‐prone aaRSs have an editing domain that hydrolyzes the incorrect amino acid. Under standard lab growth conditions, there is no observable difference between wild type Escherichia coli and phenylalanyl‐tRNA synthetase (PheRS) editing‐
defective mutant E. coli during exponential growth. To understand the role PheRS editing has for E. coli and know what conditions require translational accuracy, we observed the growth and death rates of wild type and PheRS editing‐defective E. coli under various conditions. Through comparing colony‐forming units (CFU) of both strains when grown in different conditions, it was observed that editing function does not play a role in surviving short‐term desiccation or exposure to UV radiation, but may play a role in surviving long‐term stationary phase. The next question to investigate is whether PheRS editing‐proficient strains have a competitive advantage over PheRS editing‐deficient strains during long‐term stationary phase. To do this, we will put neutral markers into both the wild type and editing‐
defective strains, create a mixed inoculum, and perform the long‐term stationary phase survival assay, while monitoring the CFU of each strain. These experiments will reveal the selective pressures that have led to the evolution and maintenance of aaRS editing function in bacteria. 340 A Micro‐Precision 3D Electrophysiology Recording System ISAAC CASSAR, TREVOR DAVIS, and Wentai Liu As neural devices seek to achieve greater stimulation resolution by increasing the amount of electrodes in a device, they cause a decrease in the pitch of these electrodes leading to electrode 'cross talk’. Our research of electrode cross‐
talk and waveform generation has sought to address this problem with a novel stimulation/recording system. A 3‐
dimensional micro‐motor is used to scan microelectrode arrays to map electric fields. Based on cable theory, with external stimulation neurons respond to the second derivative of the electrical field, so data is analyzed and displayed as the second derivative to view the resolution of this waveform instead of the raw waveform. The results found are strongly suggestive that high density microelectrode arrays alone will not be enough to drastically improve electric field resolution. Using our novel system, we can continue to explore various stimulation patterns in order to provide greater resolution. 341 Customized Biomimetic Scaffolds Created by Indirect Three‐Dimensional Printing for Tissue Engineering JU‐YEON LEE, Bogyu Chio, Benjamin Wu, and Min Lee Three‐dimensional printing (3DP) is a rapid prototyping technique that can create complex 3D structures by inkjet printing of a liquid binder onto powder biomaterials for tissue engineering scaffolds. Direct fabrication of scaffolds from 3DP, however, imposes a limitation on material choices by manufacturing processes. In this study, we report an indirect 3DP approach wherein a positive replica of desired shapes was printed using gelatin particles, and the final scaffold was directly produced from the printed mold. To create patient‐specific scaffolds that match precisely to a patient's external contours, we integrated our indirect 3DP technique with imaging technologies and successfully created custom scaffolds mimicking human mandibular condyle using polycaprolactone and chitosan for potential osteochondral tissue engineering. To test the ability of the technique to precisely control the internal morphology of the scaffolds, we created orthogonal interconnected channels within the scaffolds using computer‐aided‐design models. Because very few biomaterials are truly osteoinductive, we modified inert 3D printed materials with bioactive apatite coating. The feasibility of these scaffolds to support cell growth was investigated using bone marrow stromal cells (BMSC). The BMSCs showed good viability in the scaffolds, and the apatite coating further enhanced cellular spreading and proliferation. This technique may be valuable for complex scaffold fabrication. 100
SPD 2014 SESSION THREE 342 Determining Antibiotic Synergies with Vancomycin in Escherichia coli ALICE ZHOU, CASEY BEPPLER, CAROLINE NGUYEN, Minh Quan Nguyen, Jack Mao, Tina Kang, and Jeffrey H. Miller Antibiotic resistance is a growing and urgent issue within the healthcare industry. Potentiators of existing antibiotics and combinatorial drug therapies provide valuable tools for treatment of multi‐drug resistant pathogens. This study examines drug interactions with vancomycin, a powerful antibiotic used for Gram‐positive bacterial infections. However, because it is incapable of penetrating the outer membrane of Gram‐negative bacteria except in extremely small amounts, vancomycin is clinically ineffective against Gram‐negative bacteria, including Escherichia coli and related pathogens. We propose that drugs with highly synergistic interactions with vancomycin can potentiate its effects in Gram‐negative bacteria and provide new treatment possibilities against infections. In order to study vancomycin synergies in E. coli, we use deoxycytidine deaminase‐deficient and peptidyl‐prolyl cis‐trans isomerase‐
deficient strains, which show increased sensitivity to vancomycin and other antibiotics. Synergies detected in these backgrounds through endpoint optical density analysis were then examined in wild‐type E. coli in order to determine their clinical potential. Of the antibiotics tested, vancomycin was found to exhibit strong synergistic behavior when combined with nitrofurantoin and trimethoprim in all backgrounds. These findings suggest that combinatorial drug therapies with vancomycin could provide effective treatment for multi‐drug resistant Gram‐negative bacterial infections. 343 Portable Spectrometer Attachment for Smartphones ALBERT TAO, Qingshan Wei, and Aydogan Ozcan Optical sensors have become indispensable tools for chemical and biosensing applications. The development of portable and cost‐effective sensing platforms is essential to move advanced sensing techniques from the laboratory to the point of use. We demonstrate here a portable spectroscopic sensor based a smartphone with a spectral resolution of 0.2 nm per pixel (nm/px). The smartphone's camera is integrated with an optical attachment device to create a high resolution spectrometer. The attachment is composed of the following: a light source, a cuvette‐held sample, a diffraction grating, an external optical lens, and a frame for to hold the components together. A white LED is used as the light source, which provides a full visible spectrum. The light will then pass through the sample and the diffraction grating. When the light passes through the sample, the sample will absorb certain wavelengths due to its absorption properties. This transmitted light will be diffracted as it passes the grating to form a visible spectrum, and an optical lens will enlarge the resulting spectrum to be viewed on the smartphone. To obtain accurate spectral readouts, three monochromic laser beams in red, green, and blue color with known wavelengths were used as references for the three‐point spectral calibration. A possible application for the spectrometer is to combine it with colormetric chemical assay for field use, such as monitoring heavy metal ions in water. 344 Type I Interferon Inhibition of Vitamin D Dependent Antimicrobial Pathway by Suppression of Interleukin 32 HARRIS CHOE, Marcos Munoz, Poorva Vaidya, Dennis Montoya, and Robert Modlin Latent tuberculosis (TB) infects one third of the world's population and ten percent of that population transforms into active TB. TB primarily infects the macrophages in the lungs, while the main source of the infection stems from the infectious bacteria, Mycobacterium tuberculosis. Active TB has been shown to have a type I interferon signature, which can inhibit the interferon gamma (IFN‐gamma) induced responses. Our preliminary data show that IFN‐gamma induces interleukin‐32 (IL‐32), which can induce a vitamin D dependent antimicrobial response against TB, in which IL‐
32 proceeds to up‐regulates CYP27b1 from an the enzyme that metabolizes vitamin D from its inactive 25D form to the bioactive 1,25D form, then activates vitamin D receptor (VDR), and then initiates antimicrobial peptide production. Here we show that interferon beta (IFN‐beta) can inhibit IL‐32 production and induction of the vitamin D antimicrobial activity by measuring IL‐32 induction of the vitamin D antimicrobial pathway using quantitative real‐time PCR after pretreatment with IFN‐beta. Our preliminary data show that IL‐32 serves a vital role in the antimicrobial activity against TB, but that this response may be inhibited by type‐I interferon induction during active TB. These findings suggest that inhibition of IFN‐beta may lead to successful treatment of TB. 101
SPD 2014 SESSION THREE 345 Overweight Girls and Emotional Adjustment: Do Some Friendships Help More Than Others? LUISANA SUCHILT, Leslie Echols and Jaana Juvonen While all overweight adolescents are at risk for negative outcomes, overweight females are at heightened risk for poor mental health (Needham and Crosnoe, 2005). White overweight females‐‐who defy the body weight norm for their ethnic group‐‐may be especially vulnerable to emotional maladjustment (Lanza, Echols, and Graham, 2013). In this study we examine whether having friends from other ethnic groups (with a variety of body weight norms) may serve as a protective factor for White adolescent females who are overweight. Using multilevel modeling with a sample of over 500 White females in 6th grade, our findings indicate that having cross‐ethnic friends buffers the negative effect of body mass index (BMI) on self‐worth and body image. In subsequent analyses we will investigate whether friends from ethnic groups with a higher BMI norm (e.g., Latino) are more protective than friends from ethnic groups with a lower BMI norm (e.g., Asian). This study highlights an important advantage of school ethnic diversity, as the initial findings suggest that friendships with peers from ethnic groups with different body weight norms may be essential for youth who don't fit the norm for their own group. 346 In Vivo Evaluation of Electrospun Polycaprolactone Graft for Anterior Cruciate Ligament Engineering
AZADEH N. NAZEMI, Natalie L. Leong, Nima Kabir, Armin Arshi, Frank Petrigliano, David R. McAllister, and Benjamin Wu The anterior cruciate ligament (ACL) is a band of anisotropic, dense connective tissue that connects the femur and tibia and is critical for the knee's structural stability. ACL rupture is a common ligamentous injury that often necessitates surgical intervention and accounts for over $1 billion in annual costs in the United States. The current reconstructive methods use autologous or allogeneic tissue but suffer from drawbacks including donor site morbidity and limited supply, leaving opportunity for the exploration of a tissue‐engineered substitute. We examine the use of a biomaterial, polycaprolactone, to serve as a platform for ligament reconstruction in a rat model. Electrospun PCL grafts were fabricated, laser cut, and coated with either collagen or heparin along with basic fibroblast growth factor, human foreskin fibroblasts, or both. Immunohistochemistry specific for macrophage infiltration indicates minimal inflammatory responses at 8 and 16 weeks postoperatively. Histological analysis of collagen reveals noticeable collagen deposition by week 8, and scaffold degradation followed by aligned scar tissue deposition by week 16. Mechanical testing data shows a trend of increasing stiffness and peak load to failure from day 0 to week 16, indicating that the polymer approaches native ACL properties. An overall trend of decreased inflammatory response, increased collagen deposition, and increased scaffold strength with time is observed, showing the potential for a ligament engineered approach to treat ACL rupture.
347 Similarities of Phototaxis and Mechanotaxis Escape Response in C. Elegans SHIRLEY CHENG, Shijia Lu, Timothy Sherry, and Katsushi Arisaka Many organisms induce nociception to avoid danger and stressful conditions. A unique and complex nociceptive response has been characterized in C. elegans when stimulated with mechanical and thermal stimuli. More recently, it has also been found that blue and UV light provoked a similar negative phototactic response. However, the connection between light response and nociceptive behavior in C. elegans is unknown. The behavioral response of C. elegans to blue laser light was observed first. Using an AmScope‐camera and laser setup, it was found that stimulation of the nose and anterior part of the body elicited a specific escape response that is also observed in the touch response. Upon stimulation, C. elegans moves backwards and then executes a sharp ventral omega turn, changing its direction of locomotion. Previous research on mechanosensation of C. elegans has mapped out the neural mechanisms of that response, which include nociceptors. Future studies will include using a worm tracker program that can compare the dynamic motion of C. elegans to light and touch stimuli. If the data is consistent between the two responses, it can provide convincing evidence that the neural network involved in the different stimuli are related. This study can further our understanding of how sensory transduction is conserved in C. elegans and how this behavior promotes survival by avoiding threatening stimuli. 102
SPD 2014 SESSION THREE 348 Investigating the Role of Nucleotide Deficiency and Oxidative Stress in Maintaining Genomic Instability in Human Induced Pluripotent Stem Cells BRIAN TRUONG, Patrick C. Lee, Agustin Vega Crespo, Jonathan Tang, Alexandra Ciminera, and James A. Byrne Human induced pluripotent stem cells (hiPSCs) hold significant promise for personalized medicine, however, genetic aberrations are observed following in vitro culture of hiPSCs. Recent studies have shown that nucleotide deficiency promotes genomic instability in cancer cells. Our first hypothesis is that: 1) insufficient production of deoxyribonucleotide triphosphate (dNTP) pools is an important cause of hiPSC genomic instability, and 2) enabling the nucleoside salvage pathway by predefined deoxyribonucleoside (dN)‐supplemented culture medium will increase dNTP pools and alleviate genomic instability. Our second hypothesis is that: 1) oxidative stress is a major cause of genomic instability in hiPSCs, and 2) decreasing reactive oxygen species levels via the antioxidant ascorbic acid (AA) will alleviate genomic instability. In this study, we found that dNTP levels in hiPSCs were smaller than in the fibroblasts of origin despite similar nucleotide biosynthesis gene expression. We also found that dN supplementation in hiPSCs led to a recovery of two of the four, which indicated a promising method of recovering nucleotide pools. We ultimately found that DNA damage levels in hiPSCs were greater than in fibroblasts, which could also be partially rescued by dN and AA supplementation. These data supported our hypothesis that dN and AA supplementation may provide a viable method of alleviating genomic instability caused by nucleotide deficiency and oxidative stress in hiPSCs. 349 The Effects of Chronic Neuropathic Pain on Neuronal Connectivity of the Dopamine Pathway ANI MINASYAN, and Wendy M. Walwyn While most studies focus on the physical effects of chronic neuropathic pain, what can be equally salient and long lasting are the affective disorders that follow. In this study, my goal was three‐fold: ensure the cuff model is appropriate and affective in inducing neuropathic pain, track the presence of anxiety and or depression as a result of said pain, and finally, link these affective changes to specific changes in striatal connectivity. Neuropathic pain was induced by wrapping a 2mm plastic, hallow cuff around the sciatic nerve of the right leg in D1‐Cre and D2‐Cre mice. A slew of behavioral tests were then conducted to test for the presence of both anxiety‐like and depression‐like phenotypes. In addition, a von Frey assay was conducted bi‐weekly to ensure the presence of allodynia, or hypersensitivity to previously innocuous stimuli. Finally, a pseudorabies virus was injected in order to trace changes in striatal connectivity. Thus far, data indicates that the model is in fact an effective one. Results from successive allodynia measures indicate that the neuropathic pain persists for approximately 8 weeks before waneing off. Results from the novelty suppressed feeding test indicate that both anxiety and depression ensue as a result of the cuffing. The marble burying test further supports the presence of anxiety. Preliminary data suggests that differences in striatal connectivity are subtle but present. Control animals display labeling in the amygdala and entorhinal cortex ipsilateral to the injection site. 350 Identification of Personalized Therapies for LKB1 Mutant Non‐small Cell Lung Cancers using a High Throughput Screen of FDA Approved Compounds ALEX YANG, Robert L. McMickle, and David B. Shackelford The LKB1 gene, which regulates cell growth, metabolism, and energy balance, is mutated in ~30% of non‐small cell lung cancer (NSCLC). Lung tumor cells with LKB1 mutations accumulate numerous defective mitochondria and are sensitive to energy stress induced by mitochondrial inhibitors (MIs). We hypothesize that this Achilles' heel of LKB1 mutant (LKB1MUT) lung tumor cells may be therapeutically exploited using MIs. In a hypothesis driven drug screen of FDA approved MIs, we treated two isogenic mouse lung tumor cell lines expressing either functional or mutant LKB1. To perform this screen we developed a 96 well high throughput assay that measures both mitochondrial membrane potential and apoptosis. Results of my screen demonstrated that the LKB1 mutant lung tumor line was significantly more sensitive to a mitochondrial inhibitor MI007 compared to those with the LKB1 gene. We further confirmed that MI007 selectively induced both energy and mitochondrial stress as well as apoptosis in LKB1MUT cell lines. These results lend support to the idea that LKB1MUT lung tumors may be susceptible to MIs due to their high concentrations of defective mitochondria and their inability to regulate energy stress. We are currently screening MI007 against panels of LKB1MUT human NSCLC tumor lines as well as initiating pre‐clinical studies mouse models of NSCLC. Identifying FDA approved compounds that selectively target LKB1 mutant lung tumors will allow us to repurpose and develop better targeted therapies for patients with LKB1 mutant NSCLC. 103
SPD 2014 SESSION THREE 351 Determination of Mineral‐Specific Clumped Isotope Acid Digestion Fractionation Factors Using Heating Experiments and Mass Spectrometry DREW HENRY, Jianwu Tang, Jed Mosenfelder, Robert Eagle, and Aradhna Tripati Clumped isotope thermometry involves the determination of formation temperatures of carbonates from the fraction of isotopologues containing multiple rare isotopes (oxygen‐18 and carbon‐13). At high temperatures, the abundance of these isotopologues should be stochastic. At lower temperatures, there is a tendency for heavy isotopes to form bonds with each other. However, spectroscopic determination of isotope ratios with high precision is difficult in solids, and so 13C‐18O bond abundance is not measured in the solid phase. Instead, analysis of carbonates is performed using gas source mass spectrometry, by reacting the carbonate samples with phosphoric acid and measuring the evolved CO2 gas. As an oxygen atom is lost during the conversion of CO32‐ groups to CO2, this reaction is hypothesized to result in mineral and acid digestion temperature‐dependent fractionation. In order to quantify this fractionation between CO32‐ and CO2, this experiment seeks to determine acid fractionation factors for carbonate samples of varying composition by randomizing samples through intense heating and comparing analyte CO2 measured composition to the expected composition for a stochastically distributed sample. From this analysis, future carbonate measurements can be calibrated to account for acid digestion fractionation. 352 Emotion Recognition from the Biological Motion of Walking in Point‐Light Displays GRACE LEE, Junzhu Su, and Hongjing Lu The human visual system has the ability to detect and analyze different patterns of limb movements. To investigate this ability, we used point‐light displays, which represent bodily motion with bright lights located at each main joint of the human body. In this study, we focused on the biological motion of walking as a means by which humans recognize emotions in their most natural state of movement. We utilized the Continuous Flash Suppression (CFS) paradigm by presenting the point‐light displays to only one eye at a time while suppressing the other eye with flashing images. Many studies have used CFS as a technique to investigate perceptual dominance, but few have employed it to investigate the relationship between perception and emotion. In this experiment, three different point‐light walkers portraying three emotions (happiness, sadness, and neutral) were presented in different orientations (upright, inverted, and scrambled). Preliminary results show shortest detection time for the upright orientation of walkers expressing happiness and sadness, but not for neutral. These results indicate that eliciting an emotion triggers an observer to notice limb movement faster. Furthermore, the walker evoking happiness provided the shortest detection time, due to the increased amplitude of shoulder movement and fast walking speed. This relationship between emotion and orientation of bodily joints provides implications as to how humans detect other people's body movements in social contexts. 353 Using Aqueous Two‐Phase Systems to Rapidly Concentrate Viruses for Improving the Detection Limit of the Lateral‐Flow Immunoassay ERIK JUE, Ricky Y.T. Chiu, Cameron D. Yamanishi, Benjamin M. Wu, and Daniel T. Kamei The development of point‐of‐need (PON) diagnostics has the potential to prevent pandemics and protect against biological warfare threats. The lateral‐flow immunoassay (LFA) is suitable for PON detection because it does not require power or equipment, is portable and cheap, is easy to use, and produces a rapid time‐to‐result. However, LFA has low sensitivity compared to its laboratory‐based counterparts. To improve LFA sensitivity, a polymer‐salt aqueous two phase system (ATPS) was used to concentrate biomolecules prior to the detection step. In these studies, it was found that the target phase containing the concentrated biomolecule can be extracted prior to phase separation equilibrium, reducing the time of the extraction step. A model virus bacteriophage M13 was then predictively concentrated between approximately 2‐ and 10‐fold by altering the volumes of the two phases of the polyethylene glycol (PEG)‐potassium phosphate ATPS. The ATPS was combined with LFA, and the colloidal gold indicator used for LFA was decorated with PEG to provide steric stabilization when exposed to the salt‐rich environment of the target phase. These decorated gold nanoprobes were used to demonstrate a 10‐fold improvement in the LFA detection limit, which was confirmed through quantitative analysis with a custom MATLAB script. This approach of combining a rapid ATPS with LFA has great potential for PON applications, especially since greater concentration‐fold improvements can be achieved by further varying the volume ratio. 104
SPD 2014 SESSION THREE 354 Spatiotemporal Imaging Spectroscopy ‐ A New Method of Tissue Characterization PRATIK A. MISHRA, SIDDHARTH BHARGAVA, Adria J. Sherman,Asael Papour,Zachary Taylor and Warren S. Grundfest
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer in the world. Approximately 350,000 of the 650,000 people diagnosed with HNSCC die every year. This is in part due to the difficulties in achieving complete surgical resection. HNSCC spreads in the aerodigestive tract and the oral cavity. Prognoses could be improved if surgeons could completely excise the tumor at the time of initial surgery. Fewer than 50% of patients with oral cancer will survive past 5 years. The extent of surgical resection is limited in part due to difficulty in accurately visualizing differences in normal and malignant tissue. Current post‐surgical treatment options include reoperation, radiotherapy, and chemotherapy, but these do not affect overall patients' survival rates. To address these problems, our research group has developed a novel spatiotemporal imaging spectroscopy (STIS) system which uses nanosecond pulsed ultraviolet LEDs to excite tissue fluorescence and a gated iCCD camera for detection. Decay times are directly converted into image contrast by a unique normalization algorithm developed by our group at UCLA. Image results of 23 surgical biopsy tissues at different spectral bandwidths clearly displayed a difference between malignant and benign regions of the tissues without the need for exogenous fluorescent dyes. If tissue characterization can be performed in situ at the time of surgery, surgeons may be able to perform more accurate oncological resections with improved margins. 355 The American Association for the Surgery of Trauma: A Multicenter Prospective Observational Study of Trauma Patients on Novel Oral Anticoagulants BEATRICE SUN, Galinos Barmparas and Eric Ley Anticoagulants are blood‐thinning drugs that prevent the formation of blood clots, although they also carry risks of bleeding complications. Trauma is the 5th leading cause of death in the US and the 9th among people over 65 years. The number of elderly and patients on anticoagulants presenting to trauma centers has been increasing. Patients on Warfarin have been found to have worsened outcomes following injury; however, the effect of newer anticoagulants such as Dabigatran or Rivaroxaban on the outcomes of trauma patients is unknown. We hypothesize that patients taking novel oral anticoagulants will have worsened outcomes following trauma compared to patients on Warfarin. We further hypothesize that these outcomes will occur independent of derangements in traditional anticoagulation tests such as PT, INR and aPTT, but may correlate with derangements in their admission thromboelastography (TEG) scores. This is an ongoing prospective multicenter study of patients on novel oral anticoagulants admitted to 6 Level I trauma centers, such as Cedars‐Sinai. Over the 2‐year study period, we expect to include over 500 patients, comprising the largest study to date specifically examining this problem. Data and outcomes are observational and are collected through the AAST MIT online data entry system. The overall objective is to identify injury patterns, reversal strategies, and outcomes among patients taking novel oral anticoagulants. The data collected will help provide guidelines for future trauma care of patients on anticoagulants. 356 Investigating the Diverse Mechanisms of Osteogenesis Imperfecta KATRINA H. LEE, Lisette Nevarez, Wenjuan Zhang, and Daniel H. Cohn Osteogenesis imperfecta (OI) is an inherited skeletal dysplasia often characterized by deformed skeletal structure, increased susceptibility to fractures, and shortened stature. OI was previously believed to be inherited in an exclusively autosomal dominant fashion, with variants in genes that encode type I collagen, COL1A1 and COL1A2. However, recent studies have identified additional recessively inherited mutations in various other genes involved in the type I collagen biosynthesis pathway, leading to a paradigm shift in the scientific community. It is currently known that these rare recessive mutations account for approximately 10% of OI cases. The type 1 collagen biosynthesis pathway has yet to be fully understood, leaving possibility for unidentified novel genes that may also be associated with OI. In the following study, an attempt to identify novel OI associated genes was conducted by the use of PCR, HaloPlex Sequencing, and Whole Exome Sequencing. Genomic analysis revealed that 54 cases had causative variants in already known OI associated genes. However, 31 of the cases showed no variants in any of the currently known OI associated genes. The data suggests that these 31 negative cases may be due to variants in genes not currently known to be associated with OI. Further investigation into these 31 negative cases may uncover novel OI associated genes. The study provides a platform that may serve as a basis to a better understanding of the genetic and mechanistic pathways leading to OI. 105
SPD 2014 SESSION THREE 357 Mapping Primary Lineages in the Early Drosophila Larval Brain PUJA MEHTA, ANGEL KONG, Jaison Omoto, and Volker Hartenstein The Drosophila larval brain is a model to study brain development and its mature circuitry. Our laboratory employs a genetic/anatomic analysis of the Drosophila neuronal lineages; genetically and structurally distinct clonal units of neuronal progeny which derive from individual neural stem cells (neuroblasts). Neuroblasts in the embryo generate the primary lineages which represent the functional neurons of the larval stage. Secondary lineages which are born in the larva (by the same neuroblasts) generate the neurons which will ultimately comprise the adult brain. Previous studies from the Hartenstein Lab have identified and mapped the neuroblast location and projection pattern of all secondary lineages at the late larval stage. The axon tracts of secondary lineages at this stage represent the immature scaffold of adult circuitry. However, a similar map does not exist for the axon tracts of the primary lineages. Hence, the goal of this project is to map the pattern of primary axon tracts in the early larva. This will generate a landmark system; a foundation for future studies analyzing the structure of individual primary neurons. In addition, we aim to document the spatial relationship between primary axon tracts and the emerging secondary neuronal lineages in the early/mid larval brain. From the information gathered, one can ultimately generate a complete map of the Drosophila lineages throughout development. 358 Identification of Novel Toxoplasma gondii Proteins via Vicia Villosa Lectin Affinity Chromatography KEVIN WANG, Eric Peng, Jonathan M. Wastling, Mae Huynh, Vern Carruthers and Peter J. Bradley Toxoplasma gondii maintain its intracellular life cycle using an extraordinary arsenal of parasite specific organelles. These include the inner membrane complex (IMC), the rhoptries, the micronemes, and the dense granules. While these unique compartments play a central role in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia Villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL‐binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in localized proteins from the different organelle compartments. The first of these, TGME49_018240 localizes to the IMC (denoted IMC18). To assess the function of IMC18, we disrupted its gene via homologous recombination. The knockout was successful, demonstrating that IMC18 is not essential. We have also shown that IMC18 undergoes substantial proteolytic processing that separates the C‐terminal domain from the predicted glycosylation site. We are performing pull‐down experiments coupled to mass spectrometry to identify IMC18's interacting partners to obtain a clearer understanding of Toxoplasma's IMC. We are similarly exploring the rhoptry neck protein, RON11, which was identified in these analyses. To access the function of RON11, we conditionally knockdowned the protein using a protein destabilizing method. The method will assist us in understanding RON11 and rhoptry function in general. 359 Immunohistochemical Analysis of Neuropeptides in the Suprachiasmatic Nucleus of the BACHD Mouse Model of Huntington's Disease ZOE MACDOWELL KASWAN and Christopher S. Colwell Huntington's Disease (HD) is a genetic neurodegenerative disease characterized by degeneration of the basal ganglia. However, many non‐motor symptoms including cognitive and emotional deficits are present as well, and there is growing awareness that these symptoms occur early in disease progression. HD patients suffer from poor sleep and altered melatonin rhythms for years before the onset of motor systems, consistent with the disruption of circadian rhythms. The circadian system is anatomically centered in the suprachiasmatic nucleus (SCN). Neurons in this brain region are defined by the expression of two neuropeptides: vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP). Previous work with the R6/2 mouse model of HD and post‐mortem HD patients has shown a decrease in neuropeptides in the SCN late in disease progression. My goal is to use immunohistochemical approaches to examine the expression of these peptides in the SCN of the BACHD mouse model of HD at age of symptom onset in an attempt to determine the cause of circadian disruption. We hypothesize that neuropeptide levels will be decreased in the HD mice as compared to littermate controls. It is our hope that by understanding the cause of circadian dysfunction, we can develop novel treatments for HD patients that will improve their quality of life and ameliorate symptoms. 106
SPD 2014 SESSION THREE 360 The Involvement of Estrogen in the Facilitation of Spatial Memory in the Western Scrub‐Jay (Aphelocoma californica) BRIGIT D. HARVEY, Michelle A. Rensel, and Barney A. Schlinger Estrogens are known to influence spatial memory and learning in the hippocampus of the vertebrate brain. Studies on mammalian models suggest that estrogens influence the structure and function of the hippocampus by increasing the number of dendritic spines on neurons. The avian hippocampus has been shown to contain aromatase as well, an enzyme that converts testosterone to estradiol (E2). Food‐caching species have relatively larger hippocampi than non‐
food‐caching species and therefore serve as a good model for examining the role of E2 on spatial memory. Therefore, in this experiment, we examined the effect of E2 and an aromatase inhibitor, fadrozole (FAD), on the performance of a spatial task in the Western Scrub‐Jay. Female scrub‐jays tended to cache fewer items during the test than male subjects. Overall cache retrieval accuracy was unaffected by treatment with E2 or FAD. However, subjects given E2 prior to the cache test showed an increased latency to find food during retrieval, despite there being no differences in overall searching behavior. These data suggest that elevated E2 during memory formation is detrimental, while E2 manipulation during retrieval has no impact on spatial memory function. These results are consistent with other studies in songbirds and suggest that regulation of estradiol is critical for the maintenance of appropriate spatial memory capabilities. 361 Brain Volumetric Analysis of Body Dysmorphic Disorder ALEX ZAI, Sarah Madsen, Teena Moody, and Jamie Feusner Body dysmorphic disorder (BDD) is a severe psychiatric condition in which individuals are preoccupied with perceived defects in their appearance. Little is known of the pathophysiology of BDD, and previous morphometric studies have produced discrepant results. To investigate morphometric abnormalities in the largest sample to date, we compared brain volumes from high‐resolution T1 magnetic resonance images of 49 unmedicated BDD subjects and 44 healthy controls. We examined total gray matter (GM), total white matter, and regions of interest including the bilateral thalamus, bilateral anterior cingulate, bilateral medial orbitofrontal cortex, left inferior frontal gyrus, and caudate laterality. We also examined correlations with clinical variables. Statistical analysis revealed no significant volumetric difference between groups, supporting a previous study. However, Voxel‐Based Morphometry analysis revealed a significant positive correlation between HAMA scores and GM volume in the dorsal head of the right caudate nucleus (p<0.05), suggesting that BDD sufferers with higher GM volume in that region are more anxious. These results complement previous BDD studies that found functional abnormalities in the caudate, and prior studies of related disorders such as OCD and anxiety. These findings may assist in identifying salient regions for further investigation of the pathophysiology underlying the clinical symptoms, and could be incorporated into biomarker algorithms to diagnose and treat those afflicted. 362 An Examination of Parent and Child Antagonism among Children with and without Developmental Delay PATRICK G. HENTSCHEL, STEPHEN P. PEREZ, Willa A. Marquis, and Bruce L. Baker Rationale: Antagonism [AN], marked by overt verbal (e.g., blame) and non‐verbal (e.g., sarcastic tone) displays of negativity, has been linked to psychological distress in children with typical development (Harold et al., 1997). Little is known about the impact of AN among children with delay, despite their higher risk of maladjustment. Aim: We explored status (children with typical development [TD], intellectual disability [ID], or Autism Spectrum Disorders [ASD]) group differences in observed child and parent AN and will examine how AN relates to child (e.g., IQ, problem behavior) and parent (e.g., stress) factors. Methods: Participants were 182 (95 TD; 34 DD; 53 ASD) 13‐year‐old children and their parents. Child and parent AN were coded from the Parent‐Child Conflict interactive lab task. Other measures include the Child Behavior Checklist (CBCL; Achenbach, 2001) and the Alabama Parenting Questionnaire (Elgar et al., 2007). Results: Chi‐square tests showed significant group differences in parent (X2(2)=11.90,p<.01) but not child AN (X2(2)=4.38,p>.05). Parent prevalence was 24.2% [TD], 41.2% [ID] and 9.4% [ASD]. Future analyses will explore how AN relates to child and parent factors differentially by status. 107
SPD 2014 SESSION THREE 363 The Hematopoietic Microenvironment in Oncogene Expressing Mice Exhibits an Impaired Potential to Support Blood Cell Formation ERIK WOODRUFF, Michael Fice, and Kenneth Dorshkind In order to define the cell of origin of pediatric B‐acute Lymphoblastic leukemia (B‐ALL), we utilized a BCR‐ABL transgenic mouse (BCR‐ABL Tg) to model disease initiation and progression. We observed that marrow cellularity in BCR‐ABL Tg that had not yet developed B‐ALL was significantly lower than in control animals. This result led us to propose that stromal cells that form the hemtaopoietic microenvironment had a decreased capacity to support hematopoiesis. The objective of this study is to test this hypothesis. To investigate the hypothesis that stromal cells from BCR‐Tg mice do not support hematopoiesis, we replicated the bone marrow environment using in vitro conditions described by Dexter. Bone marrow from BCR‐ABL Tg mice was used to establish adherent layers of stromal cells. Cultures from control, wild type mice were initiated in parallel. Following treatment of the stromal cell cultures with mycophenolic acid to remove hematopoietic cells, the stromal cell cultures were recharged with wild type bone marrow cells. Cell counts and FACS analysis were performed over a 5‐week period to compare the potential of BCR‐
ABL Tg and wild type stroma to support hematopoiesis. The frequency of myeloid cells in cultures established with BCR‐ABL Tg and wild type stroma was similar. However, the total number of myeloid cells in the BCR‐ABL Tg cultures was significantly lower at all time points. These data suggest that oncogene expression in the hematopoietic microenvironment impairs blood cell development. 364 Timing and Localization of RFX3, E2F3, and Hlf Transcription Factors in Embryonic Development of Strongylocentrotus purpuratus ERFAN FARIDMOAYER, CYRUS KHOYLIAR, Nicholas Loporchio, Sneh Patel, and Pei Yun Lee Evolutionary proximity of echinoderms to chordates makes Strongylocentrotus purpuratus a valuable model organism in the study of genetic developmental patterns with the potential of possessing similar pathways to the chordate phylum. Along with their prompt development and availability, the study of gene expression in embryonic developmental stages can build the foundation for further research in genomic regulation. This project focuses on determining the spatial and temporal expression of four S. purpuratus genes: Hepatic leukemia factor, basic Helix‐
Loop‐Helix, Regulatory Factor X‐3, and E2F3 transcription factors. These genes were initially identified by BLAST and phylogenetic analyses. Reverse Transcription PCR at 0 hr, 24 hr, 48 hr, and 72 hr developmental stages were used to check for maternal and zygotic gene expression. Whole Mount in situ Hybridization was also performed, and staining was analyzed to determine localization of gene expression at 24 hr, 48 hr, and 72 hr developmental stages in S. purpuratus. Analysis of RT‐PCR results revealed maternal and continued expression of all genes until the 72 hr pluteus stage in S. purpuratus. Whole Mount in situ Hybridization data suggested a transition in the expression of Hepatic leukemia factor, E2F3, and Regulatory Factor X‐3 from the vegetal plate at the 24 hr stage to the endoderm at the 72 hr stage. In addition, expression of Hepatic leukemia factor, E2F3, and regulatory factor X3 was also observed at the apical ectoderm at the 24 hr and 48 hr developmental stages. 365 Controlling Ice Crystallization for Tissue Engineering Scaffold Microarchitecture SHANNON WONGVIBULSIN and Benjamin Wu The control of ice formation can serve as a powerful tool in tissue engineering scaffold fabrication. With ice‐
templating, scaffolds with varying architectures can be fabricated. As the polymer solution freezes in ice‐templating, the growing ice crystals push aside the solid phase, which consists of polymer chains. The sublimation of the ice crystals through lyophilization leaves voids in the scaffold. These voids mirror the ice structure and serve as the scaffold's pores, which play an important role in the overall microarchitecture of the scaffold. Because architectural cues impact cellular processes, it is important to understand the factors that impact scaffold architecture in order to utilize these parameters in designing successful scaffolds for tissue engineering applications. In this study, we analyze the role of freezing time, undercooling, and pre‐cooling in ice crystal formation using an aqueous 4% w/v chitosan‐
alginate solution as a model solution. We demonstrate that larger pore sizes can be achieved with decreased undercooling and increased freezing time. Furthermore, cooling the polymer solution (pre‐cooling) prior to freezing allows us to reduce gradients in pore sizes and achieve more homogeneity in pore size distribution within the scaffold. Employing the understanding of how freezing time, undercooling, and pre‐cooling influence ice‐templating can allow for the fabrication of tissue engineering scaffolds with specific microarchitectures conducive for various tissue engineering applications. 108
SPD 2014 SESSION THREE 366 Elucidating the Role of ORF34 in Gamma‐herpesvirus Infection HARDING LUAN, Yong Hoon Kim, Tiffany Hsu, Ronika Sitapara Leang, Stefanie Jaeger, Nevan Krogan, Ren Sun, and Ting‐Ting Wu The two human gamma‐herpesviruses, Kaposi's Sarcoma‐associated herpesvirus (KSHV or HHV‐8) and Epstein‐Barr Virus (EBV or HHV‐4) are associated with several cancers totaling more than 200,000 cases per year. Like all other herpesviruses, progression of gene expression during lytic infection of the viruses is temporally regulated. Genes are expressed in three phases: immediate‐early and early genes together encode proteins necessary for viral replication, and late genes encode structural proteins. To date, regulation of late gene transcription is poorly understood. Previously, our lab showed that five early genes, open reading frames (ORFs) 18, 24, 30, 31, and 34, are essential for late gene expression. To begin elucidating the role of each of these proteins, we selected ORF34 to perform immunoprecipitation and subsequent mass spectrometry to identify interacting proteins. We confirmed by co‐
immunoprecipitation that ORF34 interacts with RNA Polymerase II (Pol II). Using site‐directed mutagenesis, we generated eight mutant ORF34 expression plasmids with mutations at evolutionarily conserved residues. In complementation assays, a mutation from aspartic acid to alanine at position 154 abrogates late gene expression. This phenotype appears to be caused by decreased binding to Pol II. Exploiting this essential association may lead to a novel therapeutic strategy for treatment of chronic gamma‐herpesvirus infection. 367 Loss of Reelin in the Tumor Environment Promotes Metastasis of Primary Breast Cancer Cells JOAN W. CHOU, Alec Chiu, Elvira Khialeeva, and Ellen M. Carpenter Reelin is an extracellular matrix glycoprotein originally observed to play a significant role in cell migration during brain development. However, recent studies have demonstrated that reelin signaling is also present in other tissues such as the mammary glands and has been shown to affect the formation of the mammary ductal network and to inhibit the migration of mammary epithelial cells lining the lumen of the ducts. Recent unpublished findings from our lab have shown that loss of reelin signaling also affects breast cancer metastasis. In these studies, 4T1 mouse mammary tumor cells implanted into mammary fat pads formed a primary tumor and metastasized to the lungs. In mice carrying mutations for reelin or Dab1 genes, a decrease in number of metastatic nodules was seen in the lungs. Here, we set out to determine what differentiates tumors raised in wild type mice from those raised in reeler or Dab1 mutant mice using histology and immunohistochemistry. We found that tumors from wild type and reeler mice exhibit differential morphology. Further, we found an increase in alpha‐smooth muscle actin and keratin 14 and a trending decrease in Ki67 expression, suggesting a decrease in epithelial‐mesenchymal transition in the metastatic progression. We also looked at several markers of immune cells to assess the immune infiltrates in primary tumors. Although our current data is still incomplete, observations in CD11b and F4/80 staining patterns suggest a difference in immune response to mammary tumors raised in reeler mutants. 368 Identification of Novel Inner Membrane Complex Proteins Using a BioID Approach JUSTIN TOH, Josh A. Beck, Allan L. Chen, Elliot W. Kim, and Peter J. Bradley Toxoplasma gondii is an apicomplexan parasite that causes serious disease in immunocompromised patients and neonates. An important organelle in apicomplexans is the Inner Membrane Complex (IMC), which is vital for host cell invasion and replication. The IMC is known to have two distinct components: the IMC membrane, a series of membranous stacks that underly the plasma membrane, and the IMC network, which composes of a cytoskeletal layer that supports the IMC membrane. Determining protein‐protein interactions for both membrane and cytoskeletal proteins are challenging because the detergents used for these experiments are likely to disrupt weak interactions. To overcome these difficulties, we1 have adapted a newly‐developed protein interaction identification method known as BioID for T. gondii. This technique consists of creating a protein fusion of a promiscuous biotin protein ligase to an IMC membrane protein (ISP3). BioID features proximity‐dependent biotinylation of proteins near the fusion protein. Biotinylated proteins will then be isolated by streptavidin affinity capture chromatography and identified by mass spectrometry. Using this approach, we have identified many novel IMC network and membrane proteins that did not co‐precipitate by ISP3 immunoprecipitation. This suggests that this process of identifying protein‐protein interactions will enable researchers in this field to identify important proteins that can lead to the characterization of a whole new bank of proteins that were not previously accessible. 109
SPD 2014 SESSION THREE 369 Does OEC Transplantation Enhance Serotonergic Axon Regeneration After Complete Spinal Cord Transection? JAMES HAGGERTY‐SKEANS, Rana Khankan and Patricia E. Phelps After spinal cord injury (SCI) the formation of an astroglial scar and expression of growth‐inhibitory factors reduces the regenerative capacity of neurons in the adult nervous system. Additionally, mature neurons have a limited potential to regenerate compared to young neurons. Therapies that aid in axon regeneration must overcome this inhibition at the injury site. A unique CNS glial cell type capable of enhancing axon outgrowth is olfactory ensheathing cells (OECs). Injection of a serotonergic receptor agonist (quipazine) into OEC‐treated rats facilitates motor recovery in treadmill stepping, indicating the significance of serotonergic 5‐HT neurons in therapeutic treatment. We suspect that this motor function recovery is due to descending 5‐HT‐labeled axon regeneration, a pathway that originates from the Raphe. To test this hypothesis, we analyzed the extent of regeneration of 5‐HT‐labeled axons in short‐term, fully transected OEC and fibroblast (FB)‐treated rat spinal cords. We found that OEC‐treated animals had a significantly larger number of axon bundles at the astroglial border than FB‐treated animals. Additionally, the average length of the furthest‐traveling 5‐HT‐labeled axon in FB‐treated controls was significantly less than in OEC transplanted spinal rats at 2‐weeks post injection. The average difference in axon density between FB and OEC‐treated spinal rats showed a positive trend for OECs at 2 weeks. No differences were found in the average area of 5‐HT‐labeled axons between OEC and FB‐treated spinal rats at any time point. These results at 2 weeks suggest that OEC transplantation allows bundles of 5‐HT‐positive axons to extend further into the lesion than in FB controls.
370 Intraperitoneal Injections of Gp100 Vault Nanoparticles for Glioblastoma Multiforme Demonstrate an Improved Overall Survival in Treated Mice BRITTANY L. VOTH, Lawrance Chung, Kim Thill, Nolan Ung, and Isaac Yang Glioblastoma Multiforme (GBM) is the most common and malignant primary adult brain tumor. Glycoprotein 100 (gp100) has been found to be expressed by glioma cells and display immunogenicity. Priming dendritic cells in vivo using engineered vault nanoparticles containing gp100 to direct an immune response against GBM presents a novel line of inquiry for both immunotherapy and nanotechnology. In our study, 36 C57BL/6 mice received intracranial implantation of GL261 glioma cells. The mice were then separated into four treatment groups: saline (PBS) only, empty PVI‐vaults, soluble gp100, and gp100 within PVI‐vaults. The mice in each treatment group received 6 intraperitoneal injections throughout the course of 20 days. The median survival time of mice treated with saline only was 24 days while mice treated with empty PVI‐vaults survived for 29.5 days. Mice treated with soluble gp100 had a median survival time of 29 days while mice treated with gp100 within PVI‐vaults survived for 33 days. Our data suggest that mice treated with gp100 vault nanoparticles had a longer survival time than the control treatments, indicating the potential of a directed immune response against GBM with this novel treatment. Gp100 vault nanoparticles may stimulate an anti‐tumor immune response which contributes to prolonged overall survival in treatment mice. PVI modified vaults may enhance this effect. Further studies may help elicit a stronger trend between the different treatments. 371 Bisphenol A and Methylparaben Have Proliferative Effects on Breast Cancer Stem Cells CYDNEY M. NICHOLS, Stephanie Runke, Gustavo A. Miranda‐Carboni, and Susan A. Krum Two endocrine disruptors; Bisphenol A (BPA), present in polycarbonate plastics, and methylparaben (mePB), a preservative used in cosmetics, are detectable in breast tissue of over 95% of women. The magnitude and mechanism by which BPA and mePB affect breast cells is unknown. We hypothesize that BPA and mePB induce growth of breast cancer stem cells, leading to, or accelerating, tumor formation in humans. Methods: MCF‐7 breast cancer cells and normal mouse epithelial cells isolated from mouse mammary glands were grown in three‐dimensional spherical colonies called mammospheres, as a method to propagate stem cells. Mammospheres were treated on days 4 and 7 after being seeded, with vehicle control or either 1 or 10 nM of 17beta‐Estradiol (positive control), BPA, or mePB. The spheres were embedded in histogel for sectioning. Cross sections of spheres were immunostained to assess the presence of pluripotency markers SOX2, ALDH, Nanog, Oct4, and pCREB. Results: We have observed an increase in the number and size of spheres treated with BPA and mePB, compared to EtOH. Our analysis using immunostaining has shown differences in staining for pluripotent markers between treatment groups, especially the marker Oct4. Conclusion: BPA and mePB appear to have proliferative effects on breast cancer mammospheres, potentially increasing the incidence of breast cancer in humans. Further experiments will elucidate the mechanism of action of these environmental estrogens on stem cells. 110
SPD 2014 SESSION THREE 372 Mes‐4 Regulates Growth in Drosophila melanogaster CHRISTINE SUTANTO and Julian Martinez‐Agosto Post‐translational modifications of histones play a role in the remodeling of chromatin structures and the regulation of gene expression. In Drosophila melanogaster, the Mes‐4 gene encodes for a methyl transferase required for the dimethylation of lysine 36 of histone H3 (H3K36). Mes‐4 is the Drosophila homologue of NSD1, mutations in which cause Sotos syndrome, an overgrowth condition characterized by mental retardation and cancer predisposition. Genomic duplications of the NSD1 gene causes a reciprocal phenotype associated with undergrowth, microcephaly and intellectual disability. As the molecular pathway by which Mes‐4 regulates development in Drosophila still remains unknown, our research utilizes the Drosophila Gal4/UAS system to up regulate and down regulate Mes‐4 expression in the developing imaginal discs of Drosophila third instar larvae to determine its effects on tissue growth. Our research has shown that overexpressing Mes‐4 in Drosophila resulted in undergrowth. Overexpression of Mes‐4 in the developing wing imaginal disc causes a decrease in adult wing size. This is associated with increased H3K36 dimethylation. Interestingly, overexpressing mTOR increases cell apoptosis while down regulating mTOR decreases cell apoptosis. These show that Mes‐4 could be interacting with the mTOR pathway to regulate growth. Our increased understanding of the role of post translational modifications in the context of gene expression and how it affects growth will be key to the development of new therapies to treat diseases. 373 Temporospatial Expression of Nell‐1 in Developing Murine Cartilaginous Tissue MONISH H. PATEL, Alireza Hourfar, and Kang Ting The repair of cartilage due to joint trauma remains difficult due to inefficient healing capacity of cartilage and adverse effects related to current growth factor‐based strategies. Nell‐1 is a growth factor that is proven to promote chondrogenesis, or cartilage formation, and stabilize hyaline cartilage specifically in articular joints, thus leading to a more selective treatment in cartilage repair. However, the mechanism by which it does so is unclear, as is the specific role of Nell‐1 in normal fetal development. To determine the temporospatial pattern of Nell‐1 expression during development, we are evaluating Nell‐1 gene expression in wild‐type and RunX2 deficient murine fetuses at various fetal stages. Furthermore, to determine whether Nell‐1 plays a regulatory role in chondrogenesis, we will look at gene and protein expression levels of genes related to early chondrogenesis and chondrocyte hypertrophy, or growth. Thus far, wild type and RunX2 deficient fetuses have been produced, harvested, and sectioned at consecutive daily time intervals from 11.5 days post coitum to birth. In‐situ hybridization and immunohistochemistry will soon pinpoint expression levels of Nell‐1, early chondrogenesis genes, and cartilage hypertrophy genes in developing appendicular skeleton. We expect to see Nell‐1 foster maturation of chondrocytes in endochondral ossification sites while maintaining the chondrocytes in the articular regions and inhibiting their growth towards terminal hypertrophy, vascularization and ossification. 374 The Role of the miR‐17~92 Cluster in HIV‐induced CD4 T Cell Depletion JULIO C. SILVA, Juan Cubillos‐Ruiz and Bruce D. Walker The hallmark feature of HIV‐1 infection is the substantial loss of CD4 T cells in patients; however, the primary cause and the mechanisms behind this depletion remain poorly understood. Although not well characterized in the context of HIV, recent evidence suggests that microRNAs (miRNAs) may be involved in the progression of retroviral infections. Interestingly, expression of the polycistronic miR‐17~92 cluster has been shown to be suppressed in HIV‐1 infected cells in vitro. Here we show that miR‐17~92 is dramatically downregulated in CD4 T cells isolated from HIV‐1 infected individuals and in human splenic CD4 T cells undergoing in vitro depletion caused by the virus. Consistently, the pro‐
apoptotic miR‐17~92 targets, BIM and E2F1, were upregulated in CD4 T cells of HIV‐1 infected individuals. Our results suggest that HIV‐1 co‐opts the miRNA machinery of the host to promote T cell elimination and progression to AIDS. 111
SPD 2014 SESSION THREE 375 Defining MLLT3‐Regulated Molecular Pathways in Hematopoietic Stem Cell Self‐Renewal ANDREW T. NGUYEN, Vincenzo Calvanese and Hanna K.A. Mikkola Hematopoietic stem cells (HSCs) are multipotent blood cells that can self‐renew and reconstitute the entire hematopoietic system when transplanted into an irradiated recipient. Limited availability of HLA‐matched HSCs, however, restrict the number of patients who can benefit from HSC transplantation therapies. To this end, pluripotent stem cells such as human embryonic stem cells (hESC) are potential unlimited sources of HSCs that can bypass the problem of HLA‐matching and graft‐versus‐host disease. Currently, hESCs‐derived HSCs lack self‐renewal potential and are largely restricted to myeloid and erythroid lineages. We analyzed intrinsic genetic differences between fetal liver HSCs (FL‐HSCs) and hESC‐derived HSCs, identifying the gene MLLT3 to be highly expressed in FL‐HSCs while downregulated in hESCs. Furthermore, MLLT3 is highly expressed in HSCs in many stages of development, such as cord blood and the bone marrow. Thus, we hypothesize that MLLT3 is a candidate transcriptional regulator that promotes HSC self‐renewal. Preliminary experiments have shown that overexpressing MLLT3 in FL‐HSCs prolongs the maintenance of the self‐renewing HSC population over time, while shRNA knockdown of MLLT3 in FL‐HSCs diminishes the self‐renewing population. Together, the preliminary data indicates that MLLT3 functions to promote HSC self‐
renewal. As such, it is of prime interest to determine specific binding targets of MLLT3 to characterize how it regulates the expression of downstream targets to coordinate HSC self‐renewal.
376 IL‐32 as a correlate of protection against tuberculosis through induction of the vitamin D antimicrobial pathway MARCOS MUNOZ, Poorva Vaidya, Harris Choe, Susan Realegeno, Megan Inkeles, Dennis Montoya and Robert Modlin. Biomarkers for tuberculosis (TB) have been defined for the identification of correlates of risk for active disease or pathogenesis, however it remains instrumental to pinpoint correlates of protection. Ninety percent of TB patients have latent TB which display no symptoms and contain the disease. Inferring that latent TB imparts protection against active TB, gene expression profiles of whole blood from latent TB patients were overlapped to find a signal common gene from four separate patient cohorts. That single gene, IL‐32, was determined that it plays a significant role in the vitamin D antimicrobial pathway. IL‐32 was sufficient to induce the vitamin D response pathway similarly to levels expressed by IFN‐gamma and IL‐15. In addition, IFN‐gamma induction of CYP27b1, the enzyme required for converting inactive vitamin D into the active form, was IL‐32 dependent. IL‐32 was also sufficient to induce vitamin D‐dependent antimicrobial peptides such as CATH and DEFB4. Importantly, IL‐32 induces antimicrobial activity against virulent M. tuberculosis in human macrophages. Furthermore the induction of antimicrobial activity by IL‐32 in human macrophages was dependent on sufficient serum levels of inactive vitamin D highlighting the importance of vitamin D levels to mount an appropriate antimicrobial response. The significance of IL‐32 in the vitamin D immune response pathway highlights IL‐32 as a key biomarker of protection preventing latent tuberculosis to progress to its more active and aggressive form. 377 Isobutanol Production at Elevated Temperature in Thermophilic Geobacillus Thermoglucosidasius KOUKI M. YOSHINO, Paul P. Lin and James C. Liao With increased demand to address global environmental and energy problems, biofuel productions from cellulosic material have attracted attention. Biofuel production at elevated temperatures provides several advantages including lower chance of contamination, reduced enzyme quantity for cellulose degradation, and cheaper separation cost. However, enzyme stability and lack of suitable expression systems limit the high temperature production of biofuel in non‐native thermophilic hosts. Here we engineer a thermophile, Geobacillus thermoglucosidasius, to produce isobutanol at 50 degrees C. We first prospected the enzymes involved in the isobutanol biosynthesis pathway and characterized their thermostabilities. Following that, we also constructed an expression system using the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and expression system, 3.3 g/L of isobutanol from glucose and 0.6 g/L of isobutanol from cellobiose has been produced in G. thermoglucosidasius within 48 hours at 50 degrees C. 112
SPD 2014 SESSION THREE 378 Enhanced motor neuron survival following spinal transplantation of neural progenitors secreting GDNF but not IGF‐1 or VEGF in an animal model of ALS CHRISTINE D. CHANG, ANDREW K. LIN, Gen Gowing, Jessica Latter, Pablo Avalos, Brandon C. Shelley, Kevin Staggenborg, Jacklyn McHugh, M. Suzuki, and Clive N. Svendsen Amyotrophic lateral sclerosis (ALS) is a rapidly progressing and fatal motor neuron disease. Therapeutic approaches such as the delivery of growth factors and stem cell transplantation have been shown to be neuroprotective in animal models of ALS. Here, we have combined these powerful methods by genetically modifying cortical‐derived human neural progenitor cells (hNPCs) to produce glial cell‐line derived neurotrophic factor (GDNF). Thus, these cells can both serve as a source of replacement glia and be used for the delivery of trophic molecules to the CNS. In this study, we unilaterally transplanted hNPCs secreting GDNF or control cells directly into the lumbar spinal cord of the SOD1G93A rat model of ALS. Animals were monitored bi‐weekly for body weight loss and motor function using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale and sacrificed at disease end point. Statistical analysis did not reveal any significant effect of transplantation therapy on motor neuron function or lifespan of grafted SOD1G93A rats compared to controls. However, analysis of motor neuron degeneration in grafted and control animals at 5 weeks post‐
transplantation revealed a significant effect of GDNF‐secreting hNPCs on ChAT+ a‐motor neurons when compared to the contralateral non‐transplanted side and control animal groups. 379 How Genetic Variation Contributes to Altered Chromatin Structure: Insights from Analysis of Cardiac Histones RACHEL LOPEZ, Emma Monte and Thomas Vondriska Heart failure is a condition in which blood output is insufficient to meet the oxygen needs of a healthy person and affects approximately 5 million people in America. During disease, the fetal gene program is reactivated, such that normally silent genes become active and genes usually expressed in the normal healthy heart are down regulated. These gene expression changes may be regulated by chromatin. The Vondriska lab hypothesizes that heterochromatin is regulated as a protective mechanism to prevent reactivation of the fetal gene program, and that differences in chromatin structure regulate susceptibility to heart failure. Cardiac chromatin will be measured in a strain susceptible to isoproterenol‐induced heart failure (BALB/cJ) and a resistant strain (BUB/BnJ) as determined by preliminary data collected by a collaborating lab. Acid‐extraction (AE) was performed on whole heart isolated from healthy BALB/cJ mice to test for efficient histone enrichment. Enrichment of Actin in the cytosol, Nucleolin in the nucleoplasm, and Histone H3 and Histone H3 lysine‐27 trimethylation in the AE was expected, however, histones were not enriched in the AE. To optimize histone enrichment, AE was performed on mouse fibroblast 3T3 cells. The three fractions were compared by Western blot and after various trials consistent enrichment of histones was seen in the AE fraction when comparing multiple samples. Future studies will apply this method to test cardiac chromatin in BALB/cJ and BUB/BnJ mouse strains. 380 Identification of Cell Adhesion Markers in Melanoma Cancer Stem Cells CHRISTOPHER G. PHILLIPS, Elyse J. Berlinberg, Leah Goodstein and Ramin K. Nazarian Multiple known genomic alterations can lead to the development of melanoma, including a BRAF V600E mutation, which is found in over half of all melanoma cases. This point mutation leads to uncontrolled cell signaling governed by the MAP kinase pathway. PLX4032/vemurafenib, a novel small molecule inhibitor of BRAF kinase activity, is used as a targeted therapeutic agent to treat melanomas that harbor a BRAF V600E mutation. Although PLX4032 is shown to dramatically improve survival rates, some cancer cells are able to circumvent the treatment and acquire resistance to this drug by utilizing other pathways. Discovering the pathway by which these cells are able to gain resistance could potentially benefit melanoma patients. In order to identify cell adhesion molecules involved in development of melanoma cancer stem cells, we examined the expression levels and subcellular localization of cell adhesion proteins. Melanoma cell lines were plated on multi‐chamber slides followed by immunofluorescence analysis to detect specific cell adhesion markers such as E‐cadherin, N‐cadherin, laminin, vimentin, integrin and collagen. Comparing several different PLX4032 drug‐resistant sub‐lines to their isogenic parental cell lines identified adhesion molecules that play a key role in development of melanoma derived cancer stem cells. Identifying cell adhesion markers involved in melanoma cancer stem cells derived from drug‐resistant sublines will aid in the development of novel therapeutic modalities, including targeted therapy. 113
SPD 2014 SESSION THREE 381 The Role of Quorum Sensing in Regulating Iron and Phosphate Acquisition in Free‐living Sinorhizobium meliloti HOANG Q. VUONG and Ann M. Hirsch The process in which bacteria nodulate plants to fix nitrogen for the plant in exchange for other nutrients is well studied. However, factors in the soil that may influence whether the free‐living bacteria nodulate are not well studied. For example, bacterial signal molecules in the soil may induce modulation of nutrient acquisition, which in nutrient‐
deprived soils may determine whether the bacteria will survive until nodulation. In this study, we asked whether quorum sensing (QS), i.e. cell concentration‐dependent gene regulation may modulate S. meliloti's (Sm) ability to acquire nutrients. To assess the involvement of QS to nutrient acquision, procurement of iron and capacity to solubilize phosphate were compared in sinI and sinR mutants of both wild‐type Sm8530 (wt) and Sm1021 (expR) S. meliloti backgrounds. SinI proteins produce QS signal molecules while SinR and ExpR are proteins that bind the signal molecules to regulate target genes. Iron complex Chrome Azurol S agar which dissociates when overlaid upon bacteria plates, assayed iron procurement. On minimal medium, wt and expR procured iron within 2 days. The other strains' procurement was delayed by 1 day but equaled expR's activity, while wt showed a 50% reduction. Clear zones around from Sm grown upon insoluble phosphate plates assayed phosphate solubilization. ExpR exhibited the greatest degree of phosphate solubilization followed by wt, expR/sinI, sinI, sinR, and expR/sinR. Our data supports our hypothesis that QS does regulate nutrient acquisition. 382 Equatorial Electron Acceleration and Transport Towards the Inner Magnetosphere Modeled with Superposed Transient Electric Fields CAMILLA HARRIS, Christine Gabrielse, and Vassilis Angelopoulos Particle injections in the magnetotail are signatures of particle energization and transport towards the inner magnetosphere and radiation belts. Recent observations by the Time History of Events and Macroscale Interactions during Substorms (THEMIS) mission suggest that the transport and acceleration mechanism behind injections is localized and impulsive in nature, unlike the azimuthally wide acceleration site previously suggested. We therefore model particle transport analytically using the guiding‐center equations of motion under the effects of localized, impulsive electric fields superposed on background fields. During an injection, THEMIS spacecraft observe increases in particle energy flux. We simulate injections by altering parameters of the electric field model to agree with observations, constraining the location, width, and magnitude of the electric fields. We explain features of the simulated energy flux spectrograms, and therefore features of observed spectrograms, by examining particle trajectories prior to and during the injection. We apply this technique to model an event and investigate the source populations and trajectories of particles observed by spacecraft during the event. Recent statistical observations indicate that during enhanced geomagnetic activity, substorm injections populate the radiation belts with energetic particles. Modeling events provides insight on the physical processes of injections and therefore on the nature of fluctuations in the particle populations of the radiation belts. 383 The Role of Carbonic Anhydrase in Trypanosoma brucei Social Motility WALTER HARDESTY, Edwin Saada, Hunter Bennett, and Kent L. Hill Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness, a disease of high mortality rates in sub‐Saharan Africa. T. brucei has a complex lifecycle that alternates between a tsetse fly vector and a mammalian host. In the insect vector, migration through several tissues is required for maturation into mammalian‐infectious forms. Despite the prevalence of surface interaction throughout the T. brucei lifecycle, little is known about how surface contact impacts parasite behavior because African trypanosomes have been traditionally studied in suspension culture. The insect‐stage parasite engages in a form of social motility (SoMo) when cultivated on agarose surfaces. SoMo is affected by changes in CO2 levels and regulated by flagellar adenylate cyclases (ACs), which catalyze the synthesis of cAMP. In many organisms, adenylate cyclases function indirectly as CO2 sensors via carbonic anhydrases (CA), enzymes that interconvert CO2 and bicarbonate. T. brucei has a single carbonic anhydrase, which is putatively located in the flagellum. Here, the role of CA as a regulator of SoMo is investigated. To test this hypothesis, a knockdown of CA was generated using tetracycline‐inducible RNA interference. Social motility assays indicate that knockdown of CA results in a significant impact on SoMo, but does not impact cell viability or growth in vitro. Further protein characterization, which includes identifying subcellular localization, are ongoing studies. 114
SPD 2014 SESSION THREE 384 Regulation of Histone H4 Lysine 20 Monomethylation by Amino Acid Availability ZACHARY PETTERSON and Siavash Kurdistani Histone modifications play a major role in regulating DNA‐based processes such as DNA replication, transcription, and DNA repair. Recently, our lab has uncovered a link between the levels of amino acid and global monomethylation of histone H4 lysine 20 (H4K20me1). We have shown that a reduction in the levels of amino acids results in a global decrease in H4K20me1. This finding has raised a number of questions, one of which is to understand the underlying mechanism regulating the global level of H4K20 monomethylation. To investigate the possible mechanism(s), we measured the levels of the methyltransferase PR‐Set7, known to monomethylate lysine 20, under varying conditions of amino acid starvation. Western Blotting analysis was used to assess the kinetics of the change in H4K20me1 and the levels of PR‐Set7 over a time course of amino acid starvation and subsequent re‐addition in two breast cancer cell lines, MCF‐7 and MDA‐MB‐453. Here, we show amino acid depletion results in a down‐regulation of PR‐Set7. Our data reveal a correlation between amino acid availability and the levels of PR‐Set7, thus, linking the enzyme levels to changes in H4K20me1. We believe the lower levels of PR‐Set7 could explain the decreased monomethylation observed in H4K20 upon amino acid depletion. These findings provide insight into the novel role of amino acid sensing and availability in regulating the cellular levels of H4K20me1. Our findings have the potential to uncover the link between cellular metabolism and epigenetic modifications. 385 Investigating Interactions between the Parkinson's Disease Linked Pesticide, Paraquat, and the Dopamine Transporter (DAT) YOSEPHINE P. LUMINTANG, Ciara A. Martin, and David E. Krantz, MD, Ph.D. Parkinson's disease (PD) is a neuromuscular degenerative disease which can be caused by a combination of genetic and environmental factors. Epidemiological data demonstrates a link between variants in the dopamine transporter (DAT) gene and an increase in PD in pesticide (paraquat) exposed individuals. Paraquat causes oxidative stress and is a substrate of DAT. Thus, decreased DAT function should protect dopaminergic cells against the toxic accumulation of paraquat. Drosophila melanogaster was used as a model to explore this gene‐environment interaction. Specifically, we assess survival and dopamine cell loss, the pathological hallmark of PD. The DAT mutant fly line fumin has already been characterized and is known to lack DAT function. We tested eight independent treatment groups: two different genotypes (DAT mutant and wild type) exposed to four different paraquat concentrations each: 0 mM, 4 mM, 2 mM and 400 µM. We assessed survival over 60 days and found a significant difference between the control and DAT mutant groups at 2 mM paraquat. However, we saw no significant difference between the control and DAT mutant groups at 400 µM paraquat with our dopamine cell counts. Thus, mutation in DAT combined with paraquat exposure causes an increase in mortality rate, but does not affect the specific phenotype of dopamine cell loss in the brain. Overall, loss of DAT function is not protective against paraquat exposure at the doses tested. Ongoing work will investigate other potential pesticide exposures that may interact with DAT 386 Western Diet Induces High Plasma 5‐HETE Levels while HDL Mimetic Peptides Reduce This Lipid Mediator of Inflammation VICTOR FUNG, Sepideh Shakeri, Mohaddese Sharifzadeh, Loris Orbelians, Arash Meshkat, Fatemeh Barkhordarian, Ladan Vakili, Ania Gapeleh, Greg Hough, and Mohamad Navab Introduction. Hyperlipidemia has been known to cause atherosclerotic cardiovascular disease. More recently, it has been shown that inflammation links the two diseases. Lipid oxidation products can induce potent inflammatory molecules. Arachidonic acid and linoleic acid oxidation products give rise to powerful reactive oxidation species including HPETEs and HPODES. Our organism has evolved to protect its systems by converting these hydroperoxides into less toxic hydroxides HETEs and HODEs, including 5‐HETE. 5‐HETE is known to be involved in a variety of metabolic pathways. Objective. In the current study, we sought to determine the effect of a high fat‐high cholesterol diet on circulating levels of 5‐HETE in a mouse model of atherogenesis. LDL receptor deficient mice, at 5 months of age, were maintained on a high fat‐high cholesterol diet for 2 weeks. At this time, the mice were anesthetized, with blood removed, and their plasma prepared. Using LC‐ESI‐MS, oxidized lipids were isolated and identified. Results. The mice that received the high fat‐high cholesterol diet had significantly higher circulating levels of 5‐HETE as compared with the group on the laboratory rodent chow diet (p<0.01). When the hyperlipemic group was treated with the peptide 5F, there was a significant decrease (p<0.02) in the plasma levels of 5‐HETE. Conclusion. Feeding a high fat‐
high cholesterol diet increases the plasma levels of the lipid mediator 5‐HETE, while the HDL mimetic 5F reduces 5‐
HETE plasma levels in a mouse model of atherosclerosis.
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SPD 2014 SESSION THREE 387 Acceleration Behavioral Experiments Using C.Elegans DAVID WANG, Steve Mendoza, Shijia Lu, and Katsushi Arisaka C. elegans are a nematode worm that has been extensively studied for its simplicity and ease of working with. In particular, it is widely used in behavioral studies such as touch and other mechanical stimulations. In this experiment, we study the sensitivity of C. elegans to shaking via a motorized stage. While our experiment has not been directly studied, this behavior is similar to plate tapping and in the plate tapping experiments, C. elegans show an increased frequency of turning. The application of our research is relevant to experiments that use motorized stages to track C. elegans during certain experiments. We do not see significant behavioral differences between our C. elegans during shaking than before shaking when using an acceleration equivalent to 0.05g. 388 Male Behavior and Social Network Attributes on Female Reproductive Success in Yellow MATTHEW DEJONG, MARIA K. SHAFER and Daniel T. Blumstein Many mammals are socially polygynous, but the importance of male social variation on female fitness in these societies is not fully understood. We use contemporary social network statistics to determine how specific male social attributes affect female fitness in a free‐ranging population of yellow‐bellied marmots (Marmota flaviventris), a facultatively social, polygynous rodent. Using mixed effects models, we estimated the effects of centrality, expansiveness, attractiveness, and strength on annual female reproductive success. These results shed new insights on the adaptive value of variation in male sociality in mammals. 389 Social Network Analysis ‐ A New Approach to Explain Reproductive Skew in the Yellow Bellied Marmot (Marmota flaviventris) XINPING ZHANG, Adriana Maldonado, Daniel T. Blumstein Reproductive skew is widely observed across social mammals and is characterized by a dominant member's ability to secure more resources for themselves at the expense of subordinates, through mechanisms such as displaying aggression, decreasing mating rates, and suppressing hormones to prevent pregnancies. Several skew models have been proposed to explain the mechanism behind reproductive skew. The incomplete control model predicts that as group size increases, the dominant individual loses complete control of reproduction, which results in subordinate reproduction. We aimed to explain the variation observed in the reproductive output of yellow‐bellied marmot (Marmota flaviventris) social groups and to better understand the proximate correlations of skew. We used global reaching centrality (GRC), a social network statistic, as a measure of power within groups because a central individual has more influence within the network. We found, that as predicted by the incomplete control model, reproductive skew decreases as a group size increases and increases as the social groups become more central. These results show the utility of using social network statistics to understand the causes and consequences of mammalian sociality. 116
SPD 2014 SESSION THREE 390 The Role of Hydrogen Sulfide as a Reducing Agent to Protect Breast Cancer Cells from Oxidative Stress and Prevent Cell Death DIVYA GUPTA, Brian Kawahara, Suvajit Sen and Gautam Chaudhuri Hydrogen sulfide (H2S) is synthesized naturally from cysteine by several enzymes including cystathionine beta lyase (CSE), cystathionine beta synthetase (CBS) and 3‐mercaptosulfurtransferase (3‐MST) in a wide range of mammalian and non‐mammalian cells. Hydrogen sulfide has been shown to have many potential roles in cells and biochemical pathways because of its strong reducing and nucleophilic properties that can be involved in cancerous biochemical pathways. Though effects of exogenous hydrogen sulfide have been tested on breast cancer proliferation, the role of hydrogen sulfide's endogenous production is still unexplored. Using cell viability assays, we determine how well the cells cope with the oxidative stress inflicted upon them. Western blots reveal that the breast cancer cells over‐
expressed CBS as compared to normal endothelial cells (HUVEC). These breast cancer cells showed elevated levels of hydrogen sulfide as compared to HUVEC, and elevated hydrogen sulfide's presence has shown to be exclusive to breast cancer cells and tumors. We proceeded with experiments with silenced CBS MCF7 cell line and control scrambled CBS MCF7 cell line by co‐culturing macrophages with breast cancer cells where the cells showing elevated levels of hydrogen sulfide have shown preliminary resistance to macrophage's induced oxidative stress. Learning hydrogen sulfide's role in the mechanism of protecting breast cancer cells from macrophages is important for developing effective targeted therapy against breast cancer tumors. 391 Comparison of Dendritic Arbor Morphologies in Vestibular Epithelia of Rana catesbeiana JAKE SMITH, LESLIE WILLIAMS and Larry Hoffman Previous studies have shown that afferent neuron dendrites innervating the vestibular semicircular canal cristae and utricle exhibit heterogeneous morphologies that are associated with innervation region within respective epithelia. However, a comparative study examining dendritic morphologies in analogous regions across these epithelia has yet to be performed. In this study, we tested the hypothesis that dendritic morphologies of large caliber afferents (>2 micrometers) projecting to the isthmus region of the vertical cristae (anterior and posterior) and horizontal cristae, and the striola of the utricle are similar. Afferent neurons were labeled with extracellular injections of tract tracer (Neurobiotin Plus) into the root of the vestibuloauditory nerve in anesthetized bullfrogs (Rana catesbeiana). Dendritic arbors were imaged via confocal microscopy and reconstructed with neural reconstruction software. Afferents innervating the vertical cristae exhibited more branch points (nodes) compared to afferents in the horizontal cristae and utricular striola, which is associated with more terminal branches in the afferent arbors of the vertical cristae. Variation between these parameters in comparable areas of the four epithelia implies that the factors that guide the growth of the arbors vary amongst the epithelia. Further delineation of these factors could lead to strategies to rehabilitate afferents that have been damaged by ototoxins. 392 Exploring the Structure and Role of Methyltransferases Involved in Eukaryotic Elongation KYLE J. TRAVAGLINI, Maria C. Dzialo and Steven G. Clarke The elongation phase of eukaryotic translation is heavily assisted by various elongation factors that guide incoming aminoacyl‐tRNAs through the ribosome. Elongation Factors (EFs) 1A, 2, and 3 are notable because there are a combined total of ten lysine residues that are mono, di, and tri‐methylated. The role of these modifications is unknown, but many of the methylation sites are found on critical motifs, indicating that these modifications may have functional relevance. In order to narrow the search for these unknowns we analyzed the sequence of putative methyltransferases (MTs). This led us to one enzyme, YJR129C, which had a conserved DXXY motif in common known lysine MTs. We then created a computational model of YJR129C and compared it with the crystal structure of its human homolog, METTL21D. We found that the DXXY motif is near the substrate‐binding pocket in both structures, providing cause for further analysis. Using immunoblotting with methyl‐lysine antibodies, we identified YJR129C as a probable trimethylating enzyme that acts on a 100‐kDa substrate. We also assessed the potential functional relevance of methylation on EF1A, EF2, and EF3 using antibiotic assays that inhibit elongation. There was increased sensitivity in the knockouts of known EF MTs, and YJR129C in several antibiotics, indicating YJR129C may also methylate the elongation factors. Further research is needed to confirm the substrate for YJR129C, possibly either EF2 or EF3, and to clarify further how methylation influences elongation. 117
SPD 2014 SESSION THREE 393 ABSTRACT RETRACTED 394 Characterizing the Overexpression of Linc340 in B‐cell Acute Lymphoblastic Leukemia Cell Lines JASMINE J. GAJETON, Thilini R. Fernando and Dinesh S. Rao Long intergenic non‐coding RNAs, or lincRNAs, are long non‐coding pieces of RNA which have conservation in mammals and vertebrates, with roles in regulating transcription factors. Since B‐cell development is regulated by transcription factors, it is possible that lincRNAs may also affect B‐cell development. Our lab has described the first data showing dysregulation of lincRNAs in B‐cell acute lymphoblastic leukemia (B‐ALL). Preliminary data from patient samples shows that Linc340 in humans has an increased expression in B‐ALL as compared to normal pre‐B‐cells. We hypothesized that Linc340 is oncogenic and will be testing if overexpression of Linc340 in pre‐B cell lines causes an increase in cell growth in B‐ALL. In parallel, we will use murine stem cell virus (MSCV) vectors into which Linc340 is cloned along with green fluorescent protein (GFP) as a visual indicator. Following overexpression of Linc340 in murine pre‐B cell lines, we will measure proliferation (MTS Assay) and apoptosis (Caspase Assay) to define the contribution of this lincRNA to cellular homeostasis. In future studies, we will use these vectors to overexpress Linc340 in mouse bone marrow to determine if overexpression causes the development of leukemia in vivo. These findings will provide better insights into B‐cell development, B‐cell leukemia, and begin to define new therapeutic avenues for treating B‐ALL in patients. 395 Targeted Analysis of the Mexican Hypertriglyceridemia Locus on Chromosome 18 LUIS GONZALEZ, Alejandra Rodriguez and Paivi Pajukanta Mexican populations are more susceptible to developing dyslipidemias and CHD than European populations. The first Mexican GWA study for lipids revealed a new locus associated with high serum TGs on human chromosome 18. Within this locus, the trait‐associated SNPs lie within the TMEM241 gene region. Therefore, the purpose of this study was to identify all regional variants in tight LD with the key associated SNPs for future functional studies. We used the PLINK software to determine which SNP variants were in LD with the lead variant in the newly identified TG locus. The UCSC genome browser and RegRNA2.0 databases were utilized to determine whether any of the variants contributed to or altered regulatory elements. Rs1759126 was amplified by PCR and genotyped in Mexican low TG controls to characterize the regional LD patterns between rs17259126 and other genotyped variants around the TMEM241 gene. We observed that most variants in LD with the lead SNP result in RNA motif modification due to changes in transcriptional regulatory motifs along with alteration in functional RNA sequences and ncRNA hybridization regions. After identifying the variants with potential regulatory roles, we will conduct functional studies, such as building a construct with the most promising variants to test if they affect TMEM241 expression levels. Taken together these bioinformatics analyses and functional studies should help elucidate the molecular mechanisms underlying the genetic association signal of high TG levels in theTMEM241 gene region. 118
SPD 2014 SESSION THREE 396 Open Field Spatial Pattern Learning in Rats ELIZABETH D. CABANA, Julia Schroeder and Aaron P. Blaisdell Classic studies with rats navigating mazes suggest that rats develop and maintain spatial memory of paths to a goal. We were interested in whether rats could also learn spatial patterns. Rats were given a task in which five circles were projected onto the floor of an open field. The array of circles was presented such that two lines‐‐horizontal and vertical‐‐each containing three circles, intersected randomly across trials. This resulted in two perpendicular lines forming a "+", "T", or "L" shape. Subjects were rewarded with food for visiting all three targets in either the horizontal or vertical array, with assignment to one array randomized across subjects. The remaining two circles were distractors, which were not reinforced. After learning this task, one of the target locations will be displaced. One previously rewarded location will no longer be rewarded, whereas a former distractor will become a rewarded target location. This displacement creates a new rewarded array with no pattern. We predict that rats will continue to move in a horizontal or vertical line and be unable to learn the change in reward contingency, demonstrating that rats are using the previously learned spatial pattern to guide their actions. The results of this study will give us insight into the cognitive abilities and processes of rats, which can be used to model basic human cognition. 397 Changes in Hippocampal Place Cell Activity Across Days in Virtual Reality Environments SAM KAZEMIAN, Ashley Kees and Mayank R. Mehta The hippocampus contains large populations of place cells, neurons which fire selectively to a particular space in an animal's environment. To study place cells in vivo, six Long‐Evans rats were implanted with a customized, hand‐built 24‐tetrode drive. We used a multisensory virtual reality (VR) system to study navigation and place cell firing in selected environments. Rats were placed on a rotating ball with movement sensors, and a 360 degree projector was used to display the surrounding environment on the walls of the trial room. Surround sound speakers were also used to deliver auditory cues allowing us to load predesigned environments for the rat to navigate as well as selectively remove cues and stimuli from the environment to record changes in brain activity. Recorded, raw electrical data was then filtered in MATLAB using signal analysis techniques to find place cell activity. The rats were exposed to the same environment in the VR each day so that place cell activity to a particular space in a particular environment could be accurately analyzed over extended periods. Similar experiments in real world environments show that the same cell will fire in the same location across days. We anticipate that our results will show that place selectivity remains intact in the same cell across multiple days in the same environment demonstrating that the VR accurately replicates the sensory modalities necessary for the creation of the cognitive map; however, the place field of these cells will shift slightly after experience. 398 Efficacy of a CD133+ Glioma Lysate Pulsed Dendritic Cell Vaccine NICOLE CREMER, Lawrance Chung, Daniel Nagasawa, Kim Thill, and Isaac Yang Introduction‐ Despite advances in surgery and radiation, the median survival in glioblastoma multiforme (GBM) patients remains poor. GBM cancer stem‐like cells (CSCs) with their proliferative potential may represent an ideal target for GBM immunotherapy. Here, we elucidate the efficacy of a CD133+ glioma lysate‐pulsed dendritic cell (DC) vaccine in a murine model. Methods‐ CD133+ cells were sorted from the heterogeneous GL261 tumor bulk via magnetic activated cell sorting (MACS), lysed and pulsed with C57BL/6 bone marrow‐derived DCs. Six mice received 1 x 1,000,000 CD133+ glioma lysate‐pulsed DCs and five mice received 1 x 1,000,000 non‐pulsed DCs. 13 days after vaccination, all mice were challenged with an intracranial implantation of 1x1,000 CD133+ GL261 cells. Results‐ Kaplan‐Meier survival curves demonstrated an increased median survival time for the mice that received the CD133+ pulsed DCs. The mice that received the CD133+ glioma lysate‐pulsed DCs, followed by intracranial implantation, had a median survival of 62 days whereas the mice that received the non‐pulsed DCs followed by intracranial implantation had a median survival of 44 days. Conclusion‐ This study suggests that CD133+ glioma lysate‐pulsed DC vaccines have potential anti‐tumor effects that may increase overall survival. CD133 enriched GBM cancer stem‐like cells may represent an ideal tumor target for glioma immunotherapy. 119
SPD 2014 SESSION THREE 399 The Effect of Ziram on the Dopaminergic Metabolic Pathway CHASE YAMASHIRO, Aaron Lulla and Jeff Bronstein Previous studies suggest that ziram, a pesticide still widely used in rural areas, is linked to the development of Parkinson's disease (PD). While the pathogenesis of the disease remains unclear, there is a potential correlation between loss of dopaminergic neurons and development of PD. Here we investigate the effect of ziram on the dopaminergic metabolic pathway and it's metabolites in zebrafish. Tyrosine hydroxylase (TH) is a key enzyme that converts tyrosine to dopamine. We will use immunoblot to measure TH levels in a wildtype (WT) zebrafish model. We expect to observed a significant increase in TH levels in ziram‐treated zebrafish vs. untreated control zebrafish. In addition, we will use high performance liquid chromatography (HPLC) to measure dopamine and dopamine metabolite
concentrations in our WT zebrafish model. We expect to see an increase in dopamine levels in ziram‐treated zebrafish as well as an increase in concentration of the toxic dopamine metabolite DOPAL. This would suggest a mechanism by which ziram induces dopaminergic cell death and increases the likelihood of developing PD. 400 Structural Studies of the 'Core' Subunits Mmm1 and Mdm12 in the ER‐Mitochondrial Tethering Complex XUAN‐KHOI T. DANG, Young A. Andrew, and Egea F. Pascal The Endoplasmic Reticulum Mitochondria Encounter Structure (ERMES) is an organelle‐tethering complex that was first identified in the yeast Saccharomyces cerevisiae. It is composed of the two outer‐mitochondrial membrane proteins Mdm10 and Mdm34, the ER‐resident protein Mmm1, and the peripheral membrane protein Mdm12. Membrane contact sites between ER and mitochondria are regions where small molecules such as phospholipids and calcium are exchanged. Furthermore, Mmm1, Mdm12 and Mdm34 contain an SMP domain, a conserved membrane‐
binding domain found exclusively in proteins present at membrane contact sites. Besides subunit composition, nothing is known about the structure of the ERMES. The Egea laboratory uses the ERMES as a model system for the structural and functional characterization of inter‐organelle tethers. We hypothesize that the SMP domains present in ERMES are involved in lipid transfer, complex assembly and membrane‐tethering. Using various biochemical and biophysical methods, we report here the reconstitution and characterization of a stable yeast Mdm12/Mmm1 sub‐complex co‐
expressed in E. coli. Single particle electron microscopy imaging reveals an elongated crescent‐shaped hetero‐
tetrameric assembly of equimolecular stoichiometry where two Mdm12 subunits occupy opposite and distal positions on a central Mmm1 homodimer. 401 Investigating HIV Entry Across an In Vitro Model of the Blood‐Brain Barrier FAYE MENDOZA, Woytek Bartkowski, Nicholas E. Webb, and Benhur Lee NeuroAIDS, a complication in 30 to 75 percent of HIV‐infected individuals, is characterized by a series of neurodegenerative symptoms such as motor impairment, sensory loss, dementia, and an increased susceptibility to opportunistic infections in the central nervous system (CNS). These symptoms are linked to disruptions in the blood‐
brain barrier (BBB), a highly selective permeable division between the body's circulatory system and the CNS. Despite being one of the most selective barriers of the human body, HIV is able to infiltrate the BBB through an unknown pathway, causing infection in the brain. The goal of this study is to examine the potential mechanisms of HIV movement through the BBB. I simulated an in vitro model of the BBB by co‐culturing a monolayer of brain microvascular endothelial cells (BMECs) on a permeable Transwell membrane suspended above a layer of Ghost R5 cells, which served as indicators of HIV particles that transited the BMEC layer. Expression of the characteristic BMEC markers PECAM‐1 and GLUT‐1 was verified via immunohistochemistry and BMEC monolayer integrity was measured using transendothelial electrical resistance (TEER) followed by a Dextran Permeability Assay. Membrane permeability and infection assays were used to study transcytosis as a mechanism of HIV migration across the BBB. The successful establishment of this in vitro model of the blood‐brain barrier is crucial to elucidating the entry mechanism used by HIV to infect the brain. 120
SPD 2014 SESSION THREE 402 Quantification of Electric Field Sensory Behavior in Caenorhabditis elegans YU AN LIN, Michelle Kao, and Katsushi Arisaka Electric field sensitivity is present in some organisms such as elasmobranchii and monotremes due to their evolutionary advantages. Papers published previously by Gabel et al. claim that Caenorhabditis elegans are also sensitive to electric field and are inclined to crawl towards the negative pole using a set up similar to an electrophoresis chamber. Other studies indicate that C. elegans are sensitive to ion concentration gradients as well as electrical current stimulations. Due to its environment, C. elegans do not appear to gain advantages from sensitivity to electric field, we therefore hypothesize that C. elegans are in fact not sensitive to a pure electric field and that previous electrotaxis claims are caused by ions sensitivity from the electrophoresis solutions. We test this hypothesis by replicating Gabel's experiment and creating our own stimulation with pure electric field using an aluminum parallel plate capacitor. If C. elegans are sensitive to electric field, both stimulations should incite responses in the worms. Using a worm tracking software, we analyzed and quantified the trajectory of individual worms navigating on agar plates of the electrophoresis chamber and compared it to the ones of the parallel plate capacitor. We observed that the worms crawled towards the negative pole in the electrophoresis chamber but showed no response in the capacitor, suggesting that the worms are not sensitive to pure electric field and may be sensitive to the drifting ions in the chambers. 403 C. Elegans' Inability to Detect Uniform Magnetic Fields ERICK GARCIA, PETER RACIOPPO and Katsushi Arisaka Some organisms have the ability to detect magnetic fields and move as a response to these fields. The nematode Caenorhabditis elegans (C. elegans) was believed to use sensory neurons to detect the earth's magnetic filed in order to orient itself and burrow down within the soil, as opposed to the expected direction of gravity. To test this theory washed, young adult C. elegans were placed in a cage that creates a uniform magnetic field in all three directions of space. They were video recorded under different magnetic field strengths. The cage cancelled the earth's magnetic field for 60 seconds and then a strong magnetic field of five to ten gauss was generated in the same direction as that of the earth for another five to thirty minutes. The strength of the magnetic field generated by the cage was verified by using a gaussmeter, and the direction of the magnetic field was controlled by LabVIEW computer software. C. elegans showed no response to the fields and did not move in the predicted direction or any other constant direction. These results suggest that C. elegans does not possess the sensory abilities to detect uniform magnetic fields, and that another mechanism allows them to orient within the soil. 404 Ubiquitin Protein Modification as a Regulator of Neurotransmission PHILIP N. SHAMASH, Katherine M. Myers and Felix E. Schweizer Calcium‐evoked exocytosis, the nervous system's primary means of intercellular communication, uses a tightly regulated protein network to achieve appropriate timing and magnitude of presynaptic neurotransmitter release. Our lab has found ubiquitination, a post‐translational modification, to be a novel regulator of synaptic transmission and identified two ubiquitination sites on VAMP2 that could mediate some of its effects. I have used site‐directed mutagenesis on VAMP2‐pHluorin to mutate these two lysines into arginines that cannot be ubiquitinated. I have established cell membrane capacitance measurements from neuroendocrine PC12 cells to be a sensitive and rapid method for quantifying exocytosis in single cells. My data show that interfering with protein ubiquitination using either the E1 ubiquitin‐activating enzyme inhibitor Ziram or the de‐ubiquitination inhibitor G5 diminishes evoked exocytosis without decreasing calcium currents. I am now in an excellent position to determine how VAMP2 ubiquitination modulates synaptic function. Using capacitance measurements and pHluorin imaging assays, I will determine whether the mutant protein affects the readily releasable vesicle pool size, the probability of calcium‐
dependent vesicle release, and the rate of calcium‐independent exocytosis. Next I will determine whether the mutation interferes with the effects of Ziram and G5. These experiments will provide insight into the function and molecular mechanisms of ubiquitination's regulatory role in neurotransmission 121
SPD 2014 SESSION THREE 405 Effects of Nfatc2 Knockdown on Chondrogenesis within Murine Mesenchymal Cells STEVEN R. SHEN, Jie Jiang, Monica Li, Xinli Zhang, and Kang Ting In the process of chondrogenesis, mesenchymal stem cells differentiate into chondrocytes, condensing to form the extracellular matrix essential to the establishment of bone and cartilage. Previously, our lab has identified Nell1 as an osteochondrogenic factor in regulating chondrogenic differentiation. However, one of Nell1's primary response genes, Nfatc2, has been shown to suppress early stages of chondrogenesis. Our current study aims to further investigate Nell1's chondrogenic effects in murine mesenchymal cells with Nfatc2 knocked down. C3H10T1/2 cells, an embryonic murine mesenchymal cell line, were transfected with shRNA of Nfatc2 to knock down (KD) gene expression. Nfatc2 knockdown clones were selected for using clonal isolation and the antibiotic Puromycin. Real time PCR analysis was used to confirm the Nfatc2 gene knockdown and relative expression levels. Subsequently, proliferation and chondrogenic differentiation of the clonal Nfatc2‐KD and parental cell lines were examined using MTT and micromass culturing model followed by real time PCR analysis. As shown in the real time PCR results, cell colonies A5, A6, and A9 have been confirmed to express Nfatc2 at reduced levels compared to their parental controls. Based on the previous research, we expect these KD cells to show enhanced chondrogenic differentiation compared to their parental cell lines when treated with Nell1 without significantly affecting cell proliferation. We would conclude that Nfatc2 is an inhibitory molecule in Nell1 mediated chondrogenesis. 406 Dissecting the Presence of AlphaB‐crystallin in Membrane Microdoamins ISHANEE S. DIGHE, Rajendra K. Gangalum and Suraj P. Bhat Mutations in the alphaB‐crystallin gene, a small heat shock protein gene known to be associated with many neurodegenerative diseases (van Noort et al., 1995), have been linked to Desmin‐related cardiomyopathy and cataracts. However, the physiological function of this protein remains unknown. Previous work in our laboratory has shown that alphaB‐crystallin associates with Golgi membranes in developing rat lens, human U373MG glioblastoma cells and retinal pigment epithelial cells (Gangalum et al., 2010). We wanted to investigate if alphaB‐crystallin had any preferential association with or within specific membrane compartments, or domains, in developing heart cells and tissues. Employing various biochemical, molecular and cell‐biological techniques we have characterized lipid raft's (specialized membrane microdomains) in the developing rat heart, C2C12 cells (mouse myoblast cell lines) in culture and alphaB‐crystallin null mice. Using established DRM markers (Alix, Tsg 101, Caveolin‐1, Flotillin‐1 and Hsp70) we find that alphaB‐crystallin not only associates with detergent resistant microdomains (DRMs) but it's presence is critical for the organization of these specialized membrane domains in the heart tissue as well as in the cultured cells. The DRM associated membrane markers were significantly decreased in alphaB silenced epithelial cells as well as in the rat heart, suggesting that alphaB‐crystallin is involved in orchestrating and maintaining membrane heterogeneity. 407 Difunctionalized Carboranes on Gold Surfaces OLIVIA R. IRVING, John C. Thomas, Harsharn Auluck, Jonny Dadras, Anastassia Alexandrova, and Paul S. Weiss Self‐assembly, defined as the spontaneous organization of a disordered molecular system, has proven to be a viable route for bottom‐up design approaches in nanotechnology. Recently, carboranethiols and carboraneselenols have been shown to assemble on Au{111} substrates into two‐dimensional plastic lattices, forming monolayers with minimal defects and made rigid through intermolecular dipole‐dipole interactions after chemisorption. These assemblies are significantly different and simpler than the prototypical and well‐studied n‐alkanethiol system on Au{111}, which has a multitude of distinguishable domains and defects. Within this new family of molecules, carboranedithiols and caboranediselenols are molecules that are terminated by ortho thiol and selenol groups, respectively, both strongly interact with the underlying gold substrate. These surfaces have been studied and explored with scanning tunneling microscopy (STM) and polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Scanning tunneling micrographs reveal a hexagonally close‐packed arrangement with two distinct binding modalities, and PMIRRAS experiments reaffirm monolayer stability. While these experimental techniques have provided structural and local orientation, we also utilize plane wave density functional theory (PWDFT) to elucidate the energetics of different binding preferences of both molecular structures on gold. 122
SPD 2014 SESSION THREE 408 Vasoactive Intestinal Peptide Regulates Body Composition and Orexigenic and Anorexigenic Metabolic Hormones of Mice Placed on a High Fat Diet LEON LUONG, John P. Vu, Patrizia M. Germano, and Joseph R. Pisegna Vasoactive Intestinal Peptide (VIP) is expressed in the gut and arcuate nucleus (ARC) of the hypothalamus to mediate food behavior, body composition and energy metabolism. Our aim was to determine the effects of a high fat diet on the VIP(‐/‐) physiology. VIP knockout (VIP‐/‐) and wildtype (WT) mice (8‐12 weeks of age) were placed on either a control diet consisting of 20% protein, 70% carbohydrates, and 10% fat (VIP‐/‐ n=8, WT n=8) or a high fat diet (HFD) consisting of 20% protein, 35% carbohydrate and 45% fat (VIP(‐/‐) n=8, WT n=8) for 12 weeks. Body weight, food and water weight, and EchoMRI , which was used to calculate body fat and lean mass of the mice, were calculated and conducted biweekly. After the 12‐week duration of the study, plasma levels of a‐Ghrelin, glucagon, insulin and PYY were collected under either postprandial or fasting conditions. We found that VIP (‐/‐) mice exhibited significantly lower percent weight gain, lower body fat gain, lower lean mass loss, lower fat mass composition, and lower epididymal fat when compared to its WT counterpart on the HFD despite both groups eating similar amounts of food. VIP(‐/‐) also had lower ghrelin, glucagon, and insulin levels. We conclude that VIP inhibition ameliorates weight and fat mass gain and affects satiety by regulating metabolic hormone secretion. Our research signifies that VIP is a potential therapeutic target for obesity. 409 In Vivo Provision of Human Cytokines to Enhance Lymphoid Cell Development in a Humanized Mouse Model NAVDEEP SAINI, Eric H. Gschweng, Michael L. Kaufman, and Donald B. Kohn Modifications to the humanized mouse model are continuously being made to further improve the system for study. One of the problems with the model is the inability of human lymphocyte populations to successfully proliferate and achieve homeostasis in the mouse due to a lack of cross reactivity between murine cytokines and human lymphocytes. Provision of human cytokines by virus or injection of recombinant cytokines is technically difficult and expensive, but this problem can potentially be solved by the injection of cells secreting cytokines essential to support cells of the immune system. Two such cytokines, interleukin‐7 (IL‐7) and interleukin‐15 (IL‐15), help to maintain T‐cell pools. Here we show that K562 cells transduced with IL‐15 and IL‐7 vectors are able to grow normally and secrete both cytokines. Since K562 cells are a leukemic cell line, it is necessary to inactivate them before injecting them into an immunocompromised host. We tested several irradiation doses delivered to parental and IL‐7/IL‐15 producer K562 cell lines to examine the feasibility of this approach. Inactivating irradiation did not affect the ability of these cells to secrete cytokines and studies are currently underway to test the efficacy of this cell line in vivo. Our data demonstrate the concept of a cytokine producer cell line that can potentially be used to support human T‐cells in vivo in a humanized mouse model. 410 Fibroblast Growth Factor Receptor 1 and Fibroblast Growth Factor Receptor 2 are involved in Outflow Tract Development in Mouse Embryo SNE S. KANJI and Atsushi Nakano Specified roles of the Fgfr1 and Fgfr2 were examined using the Nkx2.5/Cre marker at various stages of embryonic development. Effects were examined at stages E. 8.5 twice, E.9.0, and E. 16.0. Immunohistology staining of CD31 at E.8.5 for Nkx2.5Cre/+; FGFR1fl/+; FGFR2fl/+ and Nkx2.5Cre/+; FGFR1fl/fl; FGFR2fl/fl showed significantly reduced presence of cardiac jelly, which according to previous studies, produces signal along with myocardium, to transform endothelial cells in the endocardium into mesenchyme cells. Immunohistological staining of endothelial CD31 in the conditional knockout showed a reduction in CD31 expression. The conditional double knockout of FGFR 1 and FGFR 2 at this stage showed more pronounced reduction in endothelial CD31 expression than in the conditional single knockout of FGFR1 and FGFR2 (KHI), implying the same involvement of both receptors in the formation of endothelial lining. Understanding of the relationship between FGFR1 and FGFR2 will aid in the development of therapeutic strategies of diseases such as severe persistant truncus arteriosus. 123
SPD 2014 SESSION THREE 411 ABSTRACT RETRACTED 412 A Theozyme for a Novel Synthesis of Tamiflu ALEX YEH, Jeffrey Vinokur, Colin Lam, Gonzalo Jiménez‐Osés, and K. N. Houk Oseltamivir, or Tamiflu, is a potent antiviral medication used to prevent pandemic flu outbreaks. Despite its simple structure, an efficient and environmentally friendly synthesis of oseltamivir remains challenging. We propose a convergent synthesis where all stereocenters of oseltamivir are installed by a late‐stage Diels‐Alder reaction aided by a designed asymmetric catalyst. Quantum mechanical calculations predicted the desired regioisomer of the Diels‐Alder reaction to be the preferred product, but with poor stereoselectivity. Further calculations have found the optimal positions for activating hydrogen bond donors surrounding the transition state, thus forming a theoretical active site model or theozyme. Future work will consist of placing this theozyme in the appropriate scaffold to stereoselectively form the desired oseltamivir precursor. 413 Analysis of Multiwavelength Photometry of Violently Variable Gamma‐Ray Sources JENNIFER Y. KADOWAKI and Matthew A. Malkan Flat Spectrum Radio Quasar (FSRQ) is a subclass of active galactic nuclei with aligned relativistic jets. Not fully understanding the mechanism relating the FSRQ's accretion disk activity to its rapidly varying, non‐thermal jet radiation, a monitoring campaign was conducted on 15 gamma‐ray loud FSRQs with big blue bumps at zËœ1. Selected quasars were observed in the optical, infrared (IR), and and gamma‐ray energy bands using Lick Observatory's 40‐inch Nickel Telescope, Kitt Peak National Observatory's 2.1 meter Telescope, and NASA's Fermi Gamma‐ray Space Telescope for roughly 20 nights over a 12 month period. Differential photometry on a half dozen bright, non‐variable stars in each field yielded measurements with 1‐2% level precision. Jets generally dominate the redder emission spectrum due to non‐thermal synchrotron radiation and Compton scattering of gamma‐rays off high energy electrons, while accretion disks dominate the bluer emission spectrum with rest frame ~2000 Angstroms. Most of the targeted FSRQs varied significantly over the 12 month monitoring period, with varying levels of fluctuation for each observed wavelength. Some correlations between gamma‐ray and optical wavelengths were observed. 124
SPD 2014 SESSION THREE 414 Genetic Diversity and Plant‐Microbe Symbiosis within the Rhizosphere of Cycas revoluta CYNTHIA LIEM, Jason Varasteh, Greg Wong, Sridharshi Hewawitharana, Kris Reddi, Jared Liu, and Giorgia Pirino Recent studies have indicated that Cycas revoluta is capable of producing compounds with antimicrobial properties. A community sampling of the soil microorganisms within the plants rhizosphere was obtained to analyze phylogenetic relationships and phenotypes of bacteria present. Because C. revoluta is known to have symbiotic relationships with nitrogen‐fixing bacteria and cyanobacteria, the identity of associated bacteria and their potential to resist plant‐based defense mechanisms were examined. Bacterial strains showed a variety of colony and cellular morphologies, pigmentations, and motilities. Metagenomic analysis of bacterial 16S rDNA and genomic DNA, from sixteen pure cultures, was isolated for phylogenetic analysis. A subset of bacterial isolates was subjected to antibiotic resistance assays to determine their susceptibilities to both standard and plant‐based compounds. Comparative analysis of resistance profiles was also used to further characterize the antimicrobial agents produced by the plant. The isolates were grown on media that supported growth of nitrogen‐fixing bacteria or cyanobacteria. 415 Oscillatory Flame Response in Acoustically Driven Fuel Droplet Combustion BRETT LOPEZ, Cristhian Sevilla, Phouc Hai Tran, Ari Ekmekji, Owen Smith and Ann Karagozian This experimental study focuses on droplet combustion characteristics for various liquid fuels during exposure to external acoustical perturbations generated within an acoustic waveguide. The study examines combustion during excitation conditions in which the droplet is situated in the vicinity of a pressure node (PN). In response to such acoustic excitation, the flame surrounding the droplet is deflected, on average, with an orientation depending on the droplet's relative position with respect to the PN. Flame orientation is always found to be consistent with the sign of a theoretical bulk acoustic acceleration, analogous to a gravitational acceleration (Tanabe, et al., PCI, 2000). Yet experimentally measured acoustic accelerations based on mean flame deflection differ quantitatively from that predicted by the theory. Phase‐locked OH* chemiluminescence imaging reveals temporal oscillations in flame standoff distance from the droplet as well as chemiluminescent intensity which are especially pronounced when the droplet is situated close to the PN. Quantification of combustion‐acoustic coupling via the Rayleigh index reveals a more detailed understanding of dynamical phenomena. 416 Effects of the Rbfox Proteins on hnRNP C and hnRNP M Motif‐Mediated Alternative Splicing ADRIAN L. HERNANDEZ, Andrey Damianov and Douglas L. Black Alternative pre‐mRNA splicing is a highly regulated process which effects expression of genes with multiple exons. Currently, there are many known groups of alternative splicing factors, including the heterogeneous nuclear ribonucleoproteins (hnRNPs), the serine‐arginine (SR) proteins, and the RNA binding Fox (Rbfox) proteins. However, the mechanisms which these proteins use to regulate alternative exons are poorly understood. Previous data found the Rbfox proteins in multisubunit complexes with some of the hnRNPs. The Rbfox proteins are known to regulate alternative pre‐mRNA splicing in a position‐dependent manner when they recognize a UGCAUG RNA element. However, association of the Rbfox proteins with hnRNP proteins may alter or lead to different alternative splicing mechanisms, possibly using the hnRNP proteins to recruit Rbfox proteins to non‐UGCAUG sites. Here we examine how transient Rbfox 1 and 3 expressions affect hnRNP C and M motif‐mediated alternative splicing of DUP51 EK minigene transcripts, in a human embryonal kidney (HEK) cell line. These transcripts contain known hnRNP C and M binding elements within an exon, and were compared to mutated hnRNP binding elements within the same DUP51 EK mini gene. Studying the effects of Rbfox proteins on hnRNP mediated complexes may lead to insights on other mechanisms by which the Rbfox proteins regulate alternative splicing; preventing association of RNA binding proteins to their substrates, or preventing the recruitment of other proteins to inhibit complex formation. 125
SPD 2014 SESSION THREE 417 The Role of Metabolic Enzymes in Genetically Unstable Cells SHAWNA CHAN, Nicholas Graham and Thomas Graeber In cancer cells, genomic instability leads to copy number alterations (CNA) that contribute to the aberrant proliferation of cancer cells by deletion of tumor suppressor genes and amplification of oncogenes. Bioinformatic analysis from the Graeber lab has suggested that glycolytic genes may be important targets of genomic amplification or deletion, where high glycolytic tumors show recurrent patterns of CNAs leading to the enrichment of glycolytic pathways. Previous studies in mouse embryonic fibroblasts (MEFs) have shown that overexpression of exogenous HK2 or ENO2 leads to the deletion of the endogenous region along with other neighboring genes (incuding KRAS), yet it is unknown what property of HK2 or ENO2 is responsible. Through the creation of expression vectors for other hexokinase and enolase isoforms, as well as HK2 D209A D657A, which lacks activity, and HK2 T473A, which cannot be phosphorylated by AKT, we are testing which enzymatic properties of hexokinase and enolase can alter CNAs in MEF cells. A better understanding of the genetic events underlying tumorigenesis will aid the development of effective targeted therapeutics for human cancers. 418 Characterization of Newly Identified Niche‐like Cells in Drosophila Hematopoietic Lymph Glands ELITZANDER ALEGRIA‐LEAL, Jesse M. Zaretsky, Cory J. Evans, and Utpal Banerjee The lymph gland is Drosophila's main hematopoietic organ. It consists of bilaterally symmetric primary, secondary, and tertiary lobes. The primary lobes consist of quiescent progenitor cells in the medullary zone (MZ), differentiating cells in the peripheral cortical zone (CZ), and signaling cells called the posterior signaling center (PSC). The PSC functions as a supportive niche that keeps blood cell progenitors quiescent in the MZ. Serrate (Ser) and Pvf1, components of the Notch and Pvr/Pvf1 signaling pathways, respectively, are vital to the PSC's function. Here we show that Ser and Pvf1, previously considered exclusive to the PSC, also express in the anterior domain of the tertiary lobes. Preliminary loss of function analysis, using RNA interference (RNAi) techniques, indicates that Ser in these cells positively regulates the expression of crystal cells (lz‐positive) in the distant primary lobes while Pvf1 appears to positively regulate the expression of non‐crystal differentiated cells in those lobes. This is interesting because Pvf1 from the PSC is thought to be a negative regulator of progenitor differentiation. Thus, this study suggests that a niche‐like population within the tertiary lobes regulates progenitor maintenance, not within the vicinity, but rather in the distant MZ in a manner that differs from that of the nearby PSC‐derived signals. These preliminary results have the potential to expand upon our understanding of the molecular basis for the interaction between blood cell progenitors and their niche. 419 Cancer Stem Cells Derived from Drug‐Resistant Melanoma Sub‐Lines Exhibit Epithelial to Mesenchymal Transition Biomarkers MEGHANA ATTALURI, Elyse J. Berlinberg, Leah Goodstein, and Ramin Nazarian Melanoma is a deadly form of skin cancer characterized by deregulated melanocyte growth. Melanomas often harbor a BRAF Kinase mutation V600E, which results in the activation of MAP Kinase pathway and uncontrolled cell proliferation. Patients with this mutation are treated with targeted therapy drug vemurafenib, which has shown unprecedented tumor inhibition, however, some tumors acquire resistance. Resistant cell lines, which were derived by chronic treatment with vemurafenib, have altered cell morphology and increased receptor tyrosine kinase (PDFGRB) expression, suggesting that the resistant cells are undergoing the epithelial to mesenchymal transition (EMT). This phenomenon results in a dedifferentiated state, potentially leading to development of cancer stem cells. We used immunocytochemistry and protein expression analysis to examine key EMT biomarkers in parental and resistant cell lines to determine whether EMT mechanism is involved in acquired drug resistance. When compared to their parental cells, resistant cell lines showed a differential expression of transcription factors such as Oct 3/4, Nanog, Slug, Snail, and cell adhesion molecules N‐cadherin and E‐cadherin. These altered expressions of biomarkers are often indicative of cells undergoing EMT, including cancer stem cells. Beyond determining the mechanism of resistance, these results can be used to identify novel targets to overcome acquired resistance, which can be used as a potential valuable diagnostic tool to treat melanoma patients. 126
SPD 2014 SESSION THREE 420 Characterization of 1,1‐Dichloroethylene's Effect on 1,4‐Dioxane Biodegradation RAJANI BANSAL, Shu Zhang, and Shaily Mahendra 1,4‐Dioxane, a probable human carcinogen, is an emerging groundwater contaminant at industrial and military sites across the US. It is used as a stabilizer for chlorinated solvents, such as 1,1,1‐trichloroethane (TCA). TCA and its breakdown product 1,1‐dichloroethylene (1,1‐DCE) are co‐contaminants of 1,4‐dioxane, but potential effect of 1,1‐DCE on 1,4‐dioxane degradation has not been thoroughly studied. Pseudonocardia dioxanivorans CB1190 (CB1190) was previously reported to degrade 1,4‐dioxane as its carbon and energy source. In this study, 1,4‐dioxane biodegradation by CB1190 with the presence of 0.05mg/L to 100mg/L 1,1‐DCE was investigated. Concentrations of 1,1‐DCE in the headspace and 1,4‐dioxane in the aqueous phase were measured periodically using a gas chromatograph (GC) equipped with a flame ionization detector (FID) to characterize 1,4‐dioxane degradation. An ATP‐based assay was used to estimate the growth of CB1190. Our results indicated that 10mg/L or higher concentrations of 1,1‐DCE inhibited the biodegradation of 100 mg/L 1,4‐dioxane. No bacterial growth was observed when 1,1‐DCE was supplied in the absence of 1,4‐dioxane. While further experiments are needed to determine the critical concentration of 1,1‐DCE that affects 1,4‐dioxane degradation, this study provides useful information to decide if bioremediation is possible as an in‐situ, economical, and sustainable technology for simultaneous remediation of these groundwater contaminants. 421 Monocarboxylate Transporter 1 Inhibition Promotes the Survival of Dissociated Human Embryonic Stem Cells by Decreasing Reactive Oxygen Species ALANNA B. CHAN, Wen Gu, and Heather R. Christofk Human embryonic stem cells (hESCs) undergo cell death when dissociated. Single hESC culture is important for regenerative medicine because single cell genetic manipulations can be performed and then grown for clonal expansion. Monocarboxylate transporter 1 (MCT1) transports monocarboxylates, such as pyruvate and lactate, across the plasma membrane. We found that MCT1 loss‐of‐function decreases lactate export and glucose consumption rates in hESCs, indicating an important role for MCT1 in hESC glycolytic metabolism. Interestingly, MCT1 inhibition also promotes survival of dissociated hESCs. Since early time points of MCT1 inhibition can decrease levels of intracellular reactive oxygen species (ROS), we hypothesized that MCT1 loss‐of‐function promotes dissociated hESC survival through mitigating oxidative stress. In support of this hypothesis, we found that antioxidants such as N‐acetyl cysteine, which decrease intracellular ROS levels, also promote survival of dissociated hESCs. These findings suggest that reducing oxidative stress is another survival mechanism through which dissociated hESCs can survive, and suggest use of antioxidants as a novel tool to study clonal expansion of dissociated hESCs. 422 Lysosomal Biogenesis Induction by MITF BRIAN PEREZ, Diego Ploper and Edward M. De Robertis Microphthalmia‐associated transcription factor (MITF) and Transcription Factor E‐Box (TFEB) belong to the MITF/TFE family of basic helix‐loop‐helix‐Zipper (bHLH‐Zip) transcription factors. The MITF/TFE family of transcription factors are important for regulating nutrient sensing and energy metabolism, proliferation and differentiation, cellular clearance, and organelle biogenesis. Aberrant signaling of these transcription factors have been linked to many types of cancers including melanoma and TFEB‐associated kidney cancer. MITF and TFEB contain a DNA binding domain that recognizes and binds to E‐box sequence (CANNTG) located in the promoter region of target genes. Active TFEB transcription factors bind to a 10 base pair sequence (GTCACGTGAC) known as the Coordinated Lysosomal Expression and Regulation (CLEAR) element which contain the E‐box sequence. This allows for the simultaneous expression of lysosomal genes upon TFEB activation. Therefore it is possible for MITF to also bind to CLEAR elements and induce the expression of lysosomal genes. We hypothesize that overexpression of MITF can induce the expression of TFEB regulated lysosomal genes. To test this hypothesis, human MITF was overexpressed in C32 using the Tetracyclin Inducible Expression System. Analysis of gene expression levels by Real Time‐quantitative Polymerase Chain Reaction (RT‐qPCR) show that MITF overexpression increases expression of lysosomal genes. 127
SPD 2014 SESSION THREE 423 The Efficacy of Mindfulness‐Based Relapse Prevention for Stimulant Users JESSICA NGOON and Suzette Glasner‐Edwards Substance abuse is a complex condition that pervades every part of a user's life, and consequently is one of the most prevalent sources of morbidity. Mindfulness‐based relapse prevention (MBRP), a meditation‐based treatment, has been shown to be effective in decreasing potential relapse risk. By increasing awareness of cognitive patterns and teaching alternative reactions to discomfort, these techniques can lessen the conditioned linkage between craving and negative affective mood states. In a randomized control trial designed to establish the feasibility of employing MBRP as augmentation to traditional relapse prevention, we examined the effects of this therapy (N=31) in a population of stimulant dependent adults. Potential neural and cognitive mechanisms of action for Mindfulness seem to effectively reduce physiological changes, and thus decrease substance relapse risk and stress reactivity. By examining the effects of MBRP techniques on individuals withdrawing from addiction, we hypothesized that the indices of blood pressure, heart rate, salivary cortisol, and perceived anxiety/craving during an experimental stressor (The Trier Social Stress Test; Kirschbaum et al., 1993) would be lower following an eight‐week mindfulness intervention. T‐test analyses indicated positive results for cortisol as well as subjective stress ratings, but not for heart rate or blood pressure measures. If this therapy can be shown as efficacious for stimulant users, it can be implemented to alleviate relapse risk in a difficult‐to‐treat population. 424 Co‐localization and enrichment of Limbal Stem Cell population by Fz7 expression FELIX V. CHEN, Hua Mei and Sophie X. Deng Limbal stem cells, which locate at the basal limbal epithelium and in the limbal crypts of the Palisades of Vogt, are the main source of corneal epithelial cells, especially during the would‐healing process. The Wnt signaling pathway plays an important role in the regulation of these human limbal stem cells (LSCs). The Frizzled 7 (Fz7) receptor has been shown to be preferentially expressed at the basal limbal epithelium and has been suggested to help maintain the undifferentiated stem cell status of limbal stem cells. This study involves the investigation of the co‐expression of Fz7 with the putative LSC markers including p63a, N‐cadherin and keratin (K) 14 and the corneal maturation marker K12. This study further characterizes the Fz7‐expressing cells. Moreover, it is investigated in this study whether and how limbal stem cells can be enriched through their membrane expression of Fz7. By adjusting the extracellular Ca2+/Mg2+ level, the exposure of Fz7 epitopes is maximized to the antibody so that the enrichment of Fz7‐positive limbal epithelial cells can be achieved. 128
Undergraduate Research Awards 2014
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Tochukwu Andrew
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Andrew Lin
Laura Liu
Jeffrey Shen
Dorit Stein
Alison Thach
Gabriel Toral
Jose Torres
Albert Yen
Victor Yu
Ellyn Gray
David Shia
Zijun Zhao
Dr. James W Gober
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Shankar Iyer
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Yeon Sun Kim
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Maral Bakir
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Alex Yang
Sonia Zaheer
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Chase Yamashiro
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Dr. David Shackelford
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Neda Bionghi
Le Chang
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Trenton James Otto
Amit Kartar Singh Sumal
Tatsuya Araki
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Jason Lin Chang
Erik B Jue
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Wai Man Yu
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Laurel Clare
Jane Wang
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Dr. Martha Blum
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Elisabeth Hodara
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Rajani Bansal
Dewal Gupta
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Joan Chou
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Jennifer Kadowaki
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Dr. Louis-Serge Bouchard
Dr. Benhur Lee
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Dr. Shaily Mahendra
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Joseph Conovaloff
Ishanee Dighe
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Khadij Assani
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Saundra Albers
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Zoe MacDowell Kaswan
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Matthew Abrams
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Naomi So
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Joshua Weinreb
Tianna Wilson
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Hoda Ahmed
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Taylor Brown
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Alanna Chan
Leslie Chang
Jordana Churchill
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Elyse Hartnett
Jason Kerr
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Dr. Bennett Novitch
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Ashley Kim
Michael Reitman
David Shia
Albert Yen
Joon Ha Lee
Mericien Venzon
Kevin Wang
Daniel Wong
Shannon Wongvibulsin
Dr. David Teplow
Dr. Hanna Mikkola
Dr. S. Thomas Carmichael
Dr. Michael E. Jung
Dr. Dean Ho
Dr. Elissa A. Hallem
Dr. Michael E. Alfaro
Dr. Peter J. Bradley
Dr. Julian Antonio Martinez
Dr. Benjamin M. Wu
Rebecca McGillivary
Justin Ondry
Claire Ostertag-Hill
Kenneth Pessino
Georgiana Salant
Ashley So
Michelle Tibbs
C. Y. Kimberly Tsui
Stephanie Wang
Tiffany Wang
Nadezhda Zolotova
Dr. Margot Quinlan
Dr. Sarah Tolbert
Dr. David Dawson
Dr. Karen Lyons
Dr. Steven Clarke
Dr. Michael Levine
Dr. Carrie Miceli
Dr. Lawrence Zipursky
Dr. Daniel Kamei
Dr. Aldons Jake Lusis
Dr. Mark Frye
133
UNDERGRADUATE POSTER AWARD WINNERS 2013-2014
Science Poster Day 2013
Dean’s Prize Winners
2013 SACNAS (Society for the
Advancement of Native Americans
and Chicanos in Science) Conference
Student
Faculty Mentor
Student
Faculty Mentor
Arsineh Amirkhanian
Sahar Askarinam
Shiela Afrahimi
Neda Bionghi
Alexandra Cappiello
Kevin Young Chao
Katherine Chen
Vivian K. Chen
Samantha Clark
Joseph Conovaloff
Dr. Blaire Van Valkenburgh
Dr. Rhonda Reneevoskuhl
Dr. David Teplow
Dr. Marcus Horowitz
Dr. Rhonda Reneevoskuhl
Dr. Xianjie Yang
Dr. Istvan Mody
Dr. Utpal Banerjee
Dr. Richard Kaner
Dr. David Teplow
Daniel Foster
Joseph Fowler
Samantha Mohammad
Dr. James Bisley
Dr. Joseph Loo
Dr. Istvan Mody
Liana Dallalzadeh
Stephanie Kay Fong
Shobhit Garg
Derek Eno Go
Jacqueline Graniel
Ricardo Gray
Saadia Hasan
Milad Javaherian
Jennifer Y. Kadowaki
Hyun Ju Kim
Aaron James Lao
Joon Ha Lee
Michelle Lissner
Connie Yen Liu
Harding H. Luan
Kelly Marie Lucchesi
Peter Nauka
Ronald Ga Mun Ng
Tandre Jean-Paul Oey
Regan Patterson
Jennifer Peña
Jonathan W. Rick
Christopher J. Rodman
Lisa Hieu Ta
Thuy Thi Tran
Poorva Vaidya
Mericien M. Venzon
Kevin Wang
Nicholus Warstadt
Ian M. Williams
Daniel M. Wong
Cameron D. Yamanishi
Dr. Kelsey Martin
Dr. Ann M. Hirsch
Dr. Maha Ashour-Abdalla
Dr. Dino Di Carlo
Dr. David Walker
Dr. Thomas M. Vondriska
Dr. Kelsey C. Martin
Dr. Peyman Golshani
Dr. Matthew A. Malkan
Dr. Xinli Zhang
Dr. Sophie Sokolow
Dr. Elissa A. Hallem
Dr. Stephen Smale
Dr. Lily Wu
Dr. Ting-Ting Wu
Dr. Scott H. Chandler
Dr. Heather D. Maynard
Dr. Min Lee
Dr. Guarav Sant
Dr. Yifang Zhu
Dr. Elissa A. Hallem
Dr. Scott Kitchen
Dr. Tatiana Segura
Dr. Caius Gabriel Radu
Dr. Randolph Hampton
Dr. Robert L. Modlin
Dr. Michael Alfaro
Dr. Peter J. Bradley
Dr. Paul M. Thompson
Dr. Marie-Francoise Chesselet
Dr. Julian Antonio Martinez
Dr. Daniel T. Kamei
2014 Emerging Researchers
National Conference
134
Student
Faculty Mentor
Maria Pedraza
Dr. Steve Clarke
2013 ABRCMS Conference
Student
Nancy Lopez
Jennifer Peña
Brian Perez
Michael Rale
C.Y. Kimberly Tsui
Benni Vargas
Faculty Mentor
Dr. Karen Reue
Dr. Elissa A. Hallem
Dr. Edward de Robertis
Dr. Luisa Iruela-Arispe
Dr. Lawrence Zipursky
2014 California Alliance for
Minority Participation
Student
Brandon Matthews
Faculty Mentor
Dr. Paul Weiss
SCIENCE POSTER DAY 2014 WAS SPONSORED BY UCLA Undergraduate Science Journal 2013‐2014 Editorial Staff Editors in Chief: Leslie Chang Peter Nauka Managing Editor of Research Managing Editor of Reviews Harding Luan Noor Al‐Alusi Asst. Managing Editors of Research Tiasha Shafiq Vish Ramesh Editorial Board Joseph Conovaloff Nicola Overstreet Grace Parker Ruiting Qin Katherine Sheu Sara Shu Taylor Whitaker Managing Editor of Layout Andrew Chen Review Board Suzan Arasheben Rasika Deshpande Karen Hoi Vincent Huang Lawrence Lo Laura Lui Marco Mravic Andrew Nguyen Kanav Saraf Melissa Truong Jaime Vicente Faculty Advisor Dr. Tama Hasson 135 Asst. Managing Editors of Research Paulina Young David Shia Layout Board Andrew Chen Lilian Chou Jessica Leung Tang Jiurui Vishaka Sriniwasan Society for the Advancement of Chicanos and Native Americans in the Sciences (SACNAS) UCLA Chapter Board Co‐Vice Presidents Ellen Harju Samantha Mohammad Social Co‐Chairs Ivan Flores Ervin Herrera Undergraduate Representative Angel Fulgencio President Jacqueline Graniel Media Manager Brian Perez Treasurer Jaime de Anda Fundraising Manager Hoda Ahmed Faculty Advisor Dr. Diana Azurdia Secretary Nancy Quintanilla Outreach Co‐Coordinators Chrystal Murrieta Dami Oshinuga Graduate Representative Angelica Riestra Citylab at UCLA Chapter Board Co‐Directors of Curriculum Development Christine Joseph Alina Naqvi Director of Finances Sunjong “Sunny” Ji Webmaster Steven Shen Co‐Executive Directors Ankit Patel Vincent Truong Co‐Laboratory Directors Miriam Beyder Kelly Lucchesi Director of Publicity Danh Le Faculty Advisor Dr. Dwayne Simmons 136 Co‐Directors of Education Tim Ibrahim Adam Quaal Director of Programming Scarlett Chen Index of Presenters in the Biomedical Research Minor
SESSION ONE
Student Last
ARAKI
BESADA
BHATT
CHAIKOVSKY
DERAKHSHESH
GHAFFARI RAFI
HERRERA
HODARA
HOI
HUANG
KESSLER
KRIBAKARAN
MONTOYA
PELLIONISZ
RAMESH
SCHOENBERG
SHU
TSUI
UPTON
VOROS
WENG
WILSON
YU
12 ‐ 1:10 PM
Student First
Poster #
TATSUYA
132
RANA HANY
134
KUSH VIJAY
95
ANDREA 21
MATTHEW 77
ARASH
39
ERVIN
129
EMMANUELLE
37
KIMBERLY KAM
31
VINCENT HUASHI
113
JASON LLOYD
93
SAHANA
118
TONATIUH
103
PETER ANDRAS
43
VISHWAJITH
54
BENJAMEN ELLIOT
33
SARA MARIANNA
60
KIMBERLY
23
KATHLEEN
47
TIMOTHY CARTER
94
EMILY
84
TIANNA TERESE
78
AMY KATHERINE
107
SESSION TWO
Student Last
BARBER
BEYDER
BROWN
CHENG
FAMENINI
FOSTER
HE
IYER
JI
KEYES
LE
LIN
LIU
MANCIA
PANNELL
PHAN
QUACH
RALE
RAMESH
1:15 ‐ 2:25PM
Student First
Poster #
REBECCA 271
MIRIAM
195
TAYLOR 267
MARSHA 199
SINA
202
DANIEL JACOB
268
CHONGBIN
191
SHANKAR
260
SUN JONG
257
JONATHAN 172
DANH THANH
152
YING
261
LAURA BIN
190
WALTER RENE
209
KATIE CARIN
173
TIEN MINH‐THUY
248
BILL
171
MICHAEL JOSEPH
264
NAVNEET KRISHNA
258
137
SESSION TWO CONT.
Student Last
SHAFIQ
SINDHAR
SINGH
SYED
WANG
YAMAGUCHI
YEN
YUFA
1:15 ‐ 2:25PM
Student First
TIASHA AYUMI
SAMPAT
RASHI
PHILIP SALEEM
JANE
KYOKO
YU‐HSIN
ANN RACHEL
SESSION THREE
Student Last
ALEGRIA‐LEAL
CHAN
GONZALEZ
GUPTA
HAGGERTY‐SKEANS
HARDESTY
LUAN
MCGILLIVARY
NGUYEN
NICHOLS
PATEL
PENA
ROSENBERG
SUTANTO
TOH
TRAVAGLINI
TRUONG
WANG
WANG
WEINREB
WONG
WONGVIBULSIN
WOODRUFF
ZAI
2:30 ‐ 3:40 PM
Student First
Poster #
ELITZANDER
418
ALANNA
421
LUIS ANGEL
395
DIVYA
390
JAMES 369
WALTER 383
HARDING HENG
366
REBECCA 338
ANDREW THAI
375
CYDNEY 371
MONISH 373
JENNIFER 323
MARCI FAYE
316
CHRISTINE
372
JUSTIN
368
KYLE JOSEPH
392
MELISSA 300
ERIC YAO‐DEH
335
KEVIN
358
JOSHUA
302
DANIEL 322
SHANNON
365
ERIK MARTIN
363
ALEXANDER
361
Poster #
217
219
208
247
198
178
256
157
INDEX of COURSE BASED RESEARCH
MCDB 140 AL / BL (first author listed only)
Student Last
Student First
Poster #
BUNN
JASON GRANGER
65
CHEN
FELIX VICTOR
259
CHEN
KELLY YISHU
128
COHEN
ZACK
87
FARIDMOAYER
ERFAN
364
GHESH
TAMER
53
HO
JAMIE
70
KIM
JOONHEE
160
NGO
THONG ANH
241
NGUYEN
BRIAN KHOA DUY
73
TAN
JONATHAN FRANS
122
ENVIRON 180C (first author listed only)
Student Last
Student First
Poster #
JAEGER
JESSIE MARIE
226
EEB FIELD RESEARCH QUARTER (FMBQ)
Student Last
Student First
Poster #
KEELEY
KATHRYN NICOLE
303
HUBBARD
LILAH M
310
PATEL
PRIYA DILIP
170
TRAN
DIEM CHI
313
MIMG 109AL/BL (first author listed only)
Student Last
Student First
Poster #
HUYNH
KRISTINE ANH
194
KONG
ANGEL KA HEI
165
138
Index of Student Presenters
Student Last
Student First
ABRAMS
ALVAREZ
AMIN
ARAKI
ASHBY
ASSANI
BAKIR
BESADA
BHATT
BIONGHI
BOODAIE
BRIONES
BUNN
CHAIKOVSKY
CHANG
CHANG
CHEN
CHEN
CHOU
CHU
COHEN
CONOVALOFF
CONTRERAS
DARMAWAN
DE ANDA
DERAKHSHESH
DESHPANDE
ETCHISON
FALZONE
FLORES
FOWLER
GAMMARIELLO
GAO
GHAFFARI
GHAFFARI RAFI
GHASSEMI
GHESH
GIFFORD
GRANIEL
GREEN
GUTIERREZ
HERRERA
MATTHEW CLINTON
MARCUS FERNANDO
SIMA D
TATSUYA
DAVID SCOTT
KHADIJ
MARAL
RANA HANY
KUSH VIJAY
NEDA
BENJAMIN DAVID
BRANDY ANGELINE
JASON GRANGER
ANDREA CHRISTINE
LE
ELEANOR YIAYUAN
CLARK JIA‐LONG
KELLY YISHU
JOSHUA ARNG
DAVID BRIAN
ZACK
JOSEPH LUKE
ELY GUADALUPE
KELLY FRANCISCA
JAIME
MATTHEW ISAAC
RASIKA RAJENDRA
ANA ISA
GABRIEL DAVID
STEVE
JOSEPH ROBERT THOMAS
JUN
LEYLA
ARASH
NEDA
TAMER
ANDREW PHILLIP
JACQUELINE REJOYCE DENISE
YOSELIN
ERVIN
Poster #
12 ‐ 1:10 PM SESSION ONE Student Last Student First
131
96
51
132
59
1
133
134
95
45
12
19
65
21
8
49
10
128
109
66
87
88
110
3
79
77
38
7
82
40
44
105
29
100
39
81
53
32
124
89
108
129
HESSELL
HO
HODARA
HODARA
HODUL
HOFFMAN
HOI
HUANG
HWANG
JANG
JAYSON
JIN
JUNG
KESSLER
KEUNG
KIM
KIM
KIM
KIM
KRIBAKARAN
LAM
LANGMAIER
LE
LEE
LEE
LEUNG
LIN
LIN
LIU
LORENZO
LU
MATTHEWS
MONTOYA
NAZARIAN
NG
NGUYEN
NGUYEN
NGUYEN
NGUYEN
NGUYEN
NUNEZ
ORTIZ
139
BRITTANY ALEXIS
JAMIE
ELISABETH
EMMANUELLE
MOLLY STACY
ALEXANDRIA KIMBERLY KAM
VINCENT HUASHI
JODI CHRISTINE
MICHELLE SUK
CHRISTINA KONAMI
BENITA MIKYUNG
ERIC EUGENE
JASON LLOYD
LAP‐WOON
YEON SUN
CHAE YOON
JUNG
YEON JOO
SAHANA
VINSON CURTIS
PAMELA JERRY
PHUONGANH JONATHAN BRYAN
JESSICA
SHANNON JEANIE
ANDREW H
DANIEL
NATALIE MICHELLE
MALTISH MISSAEL
SHIJIA WINSON
BRANDON TONATIUH
MEENELY
MELINDA HUYTRAM NHAT
BRIAN KHOA DUY
EILEEN KHANH BRANDON KHOA
BINH KIEN
VANESSA
JOSE ALEJANDRO
Poster #
35
70
36
37
11
91
31
113
119
130
63
114
41
93
90
9
20
68
126
118
58
125
99
26
102
48
42
120
56
61
69
15
103
25
117
18
73
75
86
98
4
121
Index of Student Presenters
Student Last
Student First
PATEL
PEDRAZA
PELLIONISZ
PENG
PHAM
RAMESH
RANDHAWA
RANDHAWA
RAVICHANDRAN
RUDBERG
SANDOUK
SASANINIA
SCHOENBERG
SHABSOVICH
SHAH
SHEN
SHIA
SHMORGON
SHU
SILVA
SIMKO
SO
TALKIN
TAN
TELLEZ
TIBBS
TRAN
TRAN
TRINH
TSUI
UPTON
VOROS
WANG
WANG
WENG
WILSON
XU
YEN
YOUNG
YU
YU
YU
KRISHAN JIGNESH
MARIA ELIZABETH
PETER ANDRAS
MAVIS SONG
MINHHAN NGUYEN
VISHWAJITH
DARSHAN SINGH
ANANTBIR SINGH
SANDHIYA
KRISTA NICOLE
ERIC JOE
KAYVAN CONFESOR
BENJAMEN ELLIOT
DAVID SIMON
SAJAN HARISH
JEFFREY ZHAN
DAVID WEIMING
GARY M
SARA MARIANNA
WENDY
SARAH ANNE
NAOMI AMANDA
REBECCA MAGGIE
JONATHAN FRANS
GERARDO ANTONIO
MICHELLE DEVEREUX
DANIEL QUANG
TAM THANH
HAMILTON N
CHUNG YIN KIMBERLY
MONTANA KATHLEEN
TIMOTHY CARTER
ALICE SHIH‐YU
CYNTHIA
EMILY
TIANNA TERESE
YIQIONG
ALBERT TIEN‐YU
PAULINA MICHI
JINGQI
VICTOR SAN HOU
AMY KATHERINE
Poster #
80
104
43
34
55
54
5
97
101
52
64
24
33
74
106
116
85
27
60
62
76
2
14
122
92
50
6
30
22
23
47
94
16
57
84
78
83
46
28
17
67
107
140
12 ‐ 1:10 PM SESSION ONE Student Last
Student First Poster #
YU
ZAHEER
ZHOU
ZHU
WAI MAN
SONIA
SELENA
JEANETTE
112
115
72
111
Index of Student Presenters Student Last
Student First
ALBERS
AMIN
ARAM
BARBER
BECKETT
BERG
BERG
BEYDER
BHAT
BRARD
BROWN
CARPENTER
CHAN
CHANG
CHEN
CHENG
CHIN
CHITTOOR
CHOI
CLARE
COPP
CRUZ
DISMUKES
ESHAGHZADEH
FAMENINI
FLORES
FOSTER
FRESHMAN
GARCHA
GARCIA
GRAY
GUPTA
GUZMAN
GYALAY
HALL
HE
HEAL
HENRIKSON
HO
HUYNH
IM
IYER
JAEGER
JANIO
JEONG
JI
SAUNDRA MORGAN
SIMA D
NEGAR
REBECCA MAE
CALEB TAYLOR
BRANDON ANDREW RAYMOND
MIRIAM
PRADHAN UDAY
ANDREA TAYLOR JASON MATTHEW
DAVID HOKTIM
YI
FELIX VICTOR
MARSHA RAVICHA
PAOKUAN
JAY R
DAVID LAUREL EILEEN
AUSTIN MICHAEL
RENAE LOPEZ
AVALON HOPE
EDWIN
SINA
IVAN
DANIEL JACOB
RYAN DAVID
JASPREET KAUR
STEPHANIE
ELLYN
DEWAL
STEVE DEREK
SZILARD
CHARLES DOUGLAS
CHONGBIN
KATHRYN WILLIAM MICHAEL
PATRIC JING‐CHI
KRISTINE ANH
CHOONG SUNG
SHANKAR
JESSIE MARIE
EMILY ANN
IL SEOK
SUN JONG
Poster #
SESSION TWO 1:15 ‐ 2:25 PM
Student Last Student First
240
182
222
271
262
187
272
195
211
154
267
207
179
273
259
199
250
215
193
236
196
245
183
175
202
251
268
181
263
230
238
252
253
231
223
191
169
254
229
194
184
260
226
228
168
257
KANG
KEYES
KIM
KIM
KIM
KOMMERS
KONG
KUMTONG
LAI
LE
LEE
LEE
LIEN
LIN
LIN
LINKER
LIU
LIU
MA
MAKKAR
MANCIA
MARVIZI
MATHUR
MEI
MORADIAN
MOYER
MRAVIC
NANIA
NAQVI
NGO
NGO
NGUYEN
NGUYEN
ONDRY
OTTO
PANNELL
PARK
PATEL
PATEL
PERCIVAL
PHAN
QUACH
RALE
RAMESH
RONQUILLO
ROTH
141
JANE JINA
JONATHAN JOONHEE
TANYA SUE
DAE WOONG
CODY EDWARD
ANGEL KA HEI
CRYSTAL CHIEAN
LILLIAN YEN YAN
DANH THANH
JI SUN
JOON HA
IRVIN CHI‐YANG
MICHELLE
YING
KAY ELIZABETH
JIACHEN
LAURA BIN
EVA
NUPUR NIMMI
WALTER RENE
GABRIELLA MONICA BAHADUR
YU TANG
ZANIAR
DANIEL CHENG
MARCO
KANE R
ALINA ZEHRA
JENNIFER
THONG ANH
THALIA MINH‐THU
REGINALD TAN
JUSTIN CARL
TRENTON
KATIE CARIN
SUN YOUNG
AKASH BIPIN
PRIYA DILIP
THEA ELIZABETH
TIEN MINH‐THUY
BILL
MICHAEL JOSEPH
NAVNEET KRISHNA
EMILIO MENDOZA
MAXWELL TAYLOR
Poster #
201
172
160
200
212
177
165
156
224
152
153
269
220
186
261
270
188
190
189
192
209
218
234
225
249
221
174
206
233
214
241
158
244
155
151
173
216
235
170
204
248
171
264
258
227
180
Index of Student Presenters Student Last
Student First
SHAFIQ
SINDHAR
SINGH
STEIN
SU
SUMAL
SUN
SYED
TABIBIAN
TOLUIE
TRAN
TRANG
VENZON
VILLALOBOS
VOLLMER
WAILES
WANG
WANG
WANG
WONG
YAMAGUCHI
YANG
YEE
YEN
YINDEEYOUNGYEON
YIP
YOUN
YUFA
ZAVER
ZHANG
ZHAO
TIASHA AYUMI
SAMPAT
RASHI
DORIT TALIA
BOWEI
AMIT KARTAR FRANK
PHILIP SALEEM
MOJDEH
SHERWIN
CONNY HONG
AMY
MERICIEN AZUCENA
TYLER DENSON
SHANNON HARRIS
SHUYI
STEPHANIE JOY
JANE
CHRISTOPHER KYOKO
SAM
HALINA MAY
YU‐HSIN
VIRATA
ALLISON TIN‐YAN
HAYOUNG
ANN RACHEL
SHIVAM ANOOP
KERMIT SHENG
ZIJUN
Poster #
217
219
208
166
203
205
185
247
239
246
242
213
210
237
266
161
159
197
198
265
178
255
243
256
164
176
163
157
167
162
232
142
SESSION TWO 1:15 ‐ 2:25 PM
Index of Student Presenters Student Last
Student First
ALEGRIA‐LEAL
ATTALURI
AULAKH
BANSAL
CABANA
CASSAR
CHAN
CHAN
CHANG
CHANG
CHANG
CHEN
CHENG
CHOE
CHOU
CREMER
DANG
DERN
DIGHE
FARIDMOAYER
FLORES
FUNG
GAJETON
GARCIA
GONZALEZ
GREEN
GUPTA
HAGGERTY‐SKEANS
HARDESTY
HARRIS
HARVEY
HENRY
HENTSCHEL
HERNANDEZ
HOANG
HUBBARD
IAIA
IRVING
JUE
KADOWAKI
KANJI
KAZEMIAN
KEELEY
KIM
LAM
LE
ELITZANDER
MEGHANA
ROZI
RAJANI RAMA
ELIZABETH DIZON
ISAAC RUSSELL
SHAWNA TAK LONG
ALANNA
SUSAN NICOLE
JASON LIN
CHRISTINE
FELIX VICTOR
SHIRLEY HIU TUNG
HARRIS
JOAN WANG
NICOLE MARISSA
XUAN KHOI TRAN
KATHRYN RENEE
ISHANEE SANDEEP
ERFAN
RICHARD ESTIVEN
VICTOR
JASMINE
ERICK
LUIS ANGEL
MELINDA REGAN
DIVYA
JAMES ROBERT
WALTER CAMILLA DODGE
BRIGIT DANAE
DREW JOSEPH
PATRICK
ADRIAN LINO
JONATHAN
LILAH M
VITO MARIANO
OLIVIA RAE
ERIK B
JENNIFER YUKARI
SNE SHANTIDAS
SAM
KATHRYN NICOLE
RINA
HEI TONG
DEREK JIAJUN
Poster #
SESSION THREE 2:30 ‐ 3:40 PM
Student Last Student First
418
419
321
420
396
340
417
421
308
337
378
424
347
344
367
398
400
325
406
364
327
386
394
403
395
393
390
369
383
382
360
351
362
416
332
310
328
407
353
413
410
397
303
326
333
317
143
LEE
LEE
LEE
LEE
LIEM
LIN
LIN
LOPEZ
LOPEZ
LOW
LUAN
LUMINTANG
LUONG
MACDOWELL MAHAJAN
MCGILLIVARY
MEHTA
MENDOZA
MINASYAN
MISHRA
MUNOZ
NAUKA
NAZEMI
NGO
NGOON
NGUYEN
NGUYEN
NGUYEN
NICHOLS
PATEL
PATEL
PENA
PEREZ
PETTERSON
PHILLIPS
REITMAN
ROSENBERG
SAINI
SHAFER
SHAH
SHAMASH
SHEN
SHERRY
SILVA
SMITH
SUCHILT
BENJAMIN
JU‐YEON
GRACE JIN
KATRINA HANA
CYNTHIA TIFFANY YUWEN
YU‐AN
RACHEL
BRETT THOMAS
JASON
HARDING HENG
YOSEPHINE LEON
ZOE ALEXANDRA
AKSHAT
REBECCA PUJA
FAYE GUZMAN
ANI
PRATIK ANANT
MARCOS PETER AZADEH NASIM
ANDREW JESSICA
AN VIET
PHUONG
ANDREW THAI
CYDNEY MORGAN
PRIYA KIRANKANT
MONISH HITESH
JENNIFER MARIE
BRIAN
ZACHARY MARK
CHRISTOPHER MICHAEL ERIC
MARCI FAYE
NAVDEEP
MARIA SAUMYA MIHIR
PHILIP NURI
STEVEN ROBERT
TIMOTHY RYAN
JULIO CESAR
JAKE MARTIN
LUISANA
Poster #
307
341
352
356
414
331
402
379
415
312
366
385
408
359
318
338
357
401
349
354
376
304
346
311
423
306
336
375
371
324
373
323
422
384
380
301
316
409
388
329
404
405
319
374
391
345
Index of Student Presenters Student Last
Student First
SUN
SUTANTO
TAO
THACH
TOH
TORRES
TRAN
TRAVAGLINI
TRUONG
TRUONG
TSAN
UEMURA
VAN
VOTH
VUONG
WANG
WANG
WANG
WEINREB
WINTERS
WONG
WONGVIBULSIN
WOODRUFF
YAMASHIRO
YANG
YANG
YEH
YEH
YOSHINO
ZAI
ZHANG
ZHOU
BEATRICE JIAYING
CHRISTINE
ALBERT
ALISON VIVIAN
JUSTIN
JOSE J
DIEM CHI
KYLE JOSEPH
MELISSA ELIZABETH
BRIAN
LINDA
SPENCER HIROAKI
KELLY PHAN
BRITTANY LYNN
HOANG QUANG
ERIC YAO‐DEH
KEVIN
JIAN
JOSHUA
VALERIE ABIGAIL
DANIEL MICHAEL
SHANNON
ERIK MARTIN
CHASE YUKIO
ESTHER WANHSIN
ALEX
TIFFANY
ALEXANDER JAMES
KOUKI MATTHEW
ALEXANDER
XINPING
ALICE LILY
Poster #
355
372
343
314
368
334
313
392
300
348
309
305
339
370
381
335
358
387
302
330
322
365
363
399
320
350
315
412
377
361
389
342
144
SESSION THREE 2:30 ‐ 3:40 PM
Index of Faculty Mentors, by Session
SESSION ONE
Faculty Last
Aldave
Arisaka
Bearden
Bensinger
Bertoni
Bjork
Blum
Blumstein
Bradley
Bronstein
Butler
Byrne
Carmichael
Castel
Chesselet
Clarke
Clarke
Colwell
De Robertis
Deng
Di Carlo
Diaconescu
Edgerton
Eisenberg
Fanselow
Fears
Freimer
French
Gimzewski
Gomperts
Gorin
Harris
Hartenstein
Hewison
Hill
Ho
Horwitz
Huang
Johnson
Jung
Kasahara
Kodambaka
Koehler
Kornblum
Krantz
12 ‐ 1:10 PM
Faculty First
Poster #
57, 130
Anthony 43, 69
Katsushi 41
Carrie E 107
Steven J.
66
Carmen 17
Elizabeth
121
Martha Lewis
106
Daniel T 42
Peter John 98
Jeff 38
Peter Cawood 33
James A 97
Stanley Thomas 25
Alan D.
12
Marie‐Francoise 104
Steven G 55, 84
Catherine F 77, 115
Christopher S 132
Edward M 49, 133
Mario C 67
Dino 29
Paula Loredana 114, 117
Victor R 22, 109
David S 11, 30
Michael S 81
Scott Clark 90
Nelson B 56
Samuel Wheeler 64
James K 3, 9
Brigitte N. 75, 93
Michael B. 35
Neil G 6
Volker 51
Martin 72
Kent L 46
Dean 45
Marcus 10
Jiaoti 108
Scott P.
85
Michael E 14
Noriyuki 59
Suneel 27, 63
Carla Marie 54
Harley I 1, 4
David E SESSION ONE
Faculty Last
Lai
Laski
Lee
Lee
12 ‐ 1:10 PM
Faculty First
Albert Frank A Edward Wolfgang Pei‐Yun Lee
Levine
Ley
Lin
Londhe
Loo
Lotfipour
Maynard
Miceli
Micevych
Mikkola
Miller
Minor
Miranda‐Carboni
Modlin
Nakano
Narins
Navab
Nelson
Ozcan
Pajukanta
Ping
Portera‐Cailliau
Prause
Ray
Repetti
Rissman
Sagasti
Sant
Shackelford
Shams
Shivkumar
Singh
Srivatsan
Tang
Teitell
Teplow
Ting
Tirado
Tiwari‐Woodruff
Benhur Michael S Eric J.
Shuo Vedang A. Joseph Shahrdad
Heather D M Carrie Paul E Hanna K. A. Jeffery Floyd Thomas R.
Gustavo Robert L Atsushi Peter M Mohamad Linda Aydogan Paivi Elisabeth Peipei
Carlos Nicole
Lara Allison Rena L.
Jesse
Alvaro Gaurav David B Ladan
Kalyanam Ram Raj Eri S Yi Michael Alan David B Kang Carlos A Seema K. 145
Poster #
18
39
2
53, 65, 70, 73,
87, 122, 128
94
52
26, 86
78
95
44
47
61
50
131
103, 126
24
19
62
99
113, 134
28
100
119, 125
32
96
102
118
48
89
7
120
92
82
76
111
80
5
101
112
21
31, 36, 88
8
74
129
Index of Faculty Mentors, by Session
SESSION ONE
Faculty Last
Torres
Tripati
Voskuhl
Walker
Wassum
Weiss
White
Wong
Wu
Zack
Zhang
Zhu
Zipursky
12 ‐ 1:10 PM
Faculty First
Jorge Aradhna K Rhonda Renee David William Kate M.
Paul S.
Stephanie Ann Gerard Chee Lai Ting‐Ting Jerome A Xinli Quansheng Stephen Lawrence Poster #
37, 110
40, 105
34, 91
124
16, 83
15
20
79
60
116
68
58
23
SESSION TWO
Faculty Last
Aghaloo
Alfaro
Amarasekare
Arisaka
Arumugaswami
Banerjee
Bertoni
Bertozzi
Bisley
Blaisdell
Blum
Blumstein
Bouchard
Bradley
Bradley
Brian Odegaard Carmichael
Carpenter
Carter
Castel
Charles
Cheng
Christofk
Clark
Cohen
Cohn
Colwell
Coppola
Crooks
1:15 ‐ 2:25 PM
Faculty First
Tara Lyn Michael Edward Priyanga A. Katsushi Vaithi
Utpal Carmen Andrea James Warwick Aaron P.
Martha Lewis Daniel T Louis‐Serge Peter John Kenneth Alan Ladan Shams
Stanley Thomas Ellen M Troy A. Alan D.
Andrew C Genhong Heather R Amander Therese Mark S Daniel
Christopher S Giovanni Gay M. Poster #
175
210
262
193, 239, 250
201
257, 271
225
169, 221
268
153
236
170
151
214
245
207
272
212
188
223
220
167
267
217
228, 235
229
152
200
191
146
SESSION TWO
Faculty Last
Deng
Di Carlo
Dunkel Eagle
Edgerton
Ghez
Glanzman
Gorin
Graeber
Griesbach
Hallem
Harran
Henning
Hill
Horwich
Hubbell
Iruela‐Arispe
Johnson
Juvonen
Kahn
Kamei
Kaner
Kohn
Koon
Laski
Lazazzera
Lee
Lee
Lee
Lee
Levine
Liao
Lowry
Lusis
Lyons
Mackenzie
Martinez
Navab
Nguyen
Paige
Peault
Pirino
Plath
Prins
Qin
1:15 ‐ 2:25 PM
Faculty First
Poster #
Mario C 215
Dino 252
Christine 237
Robert 254
Victor R 172, 253
Andrea M 240
David L 208
Michael B. 243
Thomas G 232
Grace S 263
Elissa A 269
Patrick G 163
Susanne 213
Kent L 199
Tamara
182
Wayne L 186
Luisa M 181, 264
Scott P.
154
Jaana
230
Daniel 158
Daniel T 176, 197
Richard B 183
Donald 178
Hon Wai 203
Frank A 219
Beth Ann 179
160, 241, 259
Pei Yun
Kuk‐Wha 168
Min 184, 196
Benhur 205
Michael S 233, 242
James C. 206, 265
William 261
Aldons J 166
Karen Marie 248
Allan 161
Julian 202, 258
Mohamad 218, 222, 246, 249
Thao 260
David A 231
Bruno 174, 256
Giorgia
165, 194
Kathrin 209
Robert M 211
Jing 159
Index of Faculty Mentors, by Session
SESSION TWO
Faculty Last
Reue
Rissman
Rowat
Sandhofer
Shapiro
Shipe
Shivkumar
Slamon
Sork
Stutz
Sun
Tang
Tawil
Tidball
Tolbert
Tripati
Walwyn
Wang
Wassum
Watson
Williams
Wu
Wu
Yang
Yang
Zack
Zhen
Zheng
Zipursky
1:15 ‐ 2:25 PM
Faculty First
Karen Jesse
Amy Catherine
David Rebecca Fe Kalyanam Dennis J Victoria Jochen Peter Ren Yi Nabil J James G Sarah H Aradhna K Wendy
Yibin Kate M.
Rachelle Gary A Hong Ting‐Ting Xianjie Isaac Jerome A Anjie
Zhong Stephen SESSION THREE
Faculty Last
Aldave
Angelopoulos
Arisaka
2:30‐3:40 PM
Faculty First
Poster #
Anthony 317
Vassilis
382
Katsushi 319, 347, 387,
Baker
Banerjee
Bhat
Black
Blaisdell
Blumstein
Bruce L.
362
Utpal
418
Suraj P 406
Douglas L. 416
Aaron
320, 396
Daniel T 303, 310, 313,
Poster #
227
177
255
156
216
226
192
157
204
238
198
185
164
251
155
266
180
273
270
189, 244
187
171
190
224
234
173
247
162
195
393, 402, 403
388, 389
Bradley
Peter John 358, 368
SESSION THREE
Faculty Last
Bronstein
Byrne
Carmichael
Carpenter
Chaudhuri
Christofk
Clarke
Clarke
Cohn
Colicelli
Colwell
Deng
Derobertis
Dorshkind
Dunn
Egea
Eisenberger
Evseenko
Feusner
Glasner‐Edwards
Graeber
Grundfest
Guo
Hallem
Hartenstein
Hill
Hirsch
Hoffman
Houk
Huang
Jones
Juvonen
Kamei
Karagozian
Kasko
Kaufman
Kohn
Krantz
Kurdistani
Lazazzera
Lee
Lee
Lee
Ley
Liao
147
2:30‐3:40 PM
Faculty First
Jeff James A Stanley Ellen M Gautam Heather R Catherine F Steven G Daniel
John J Christopher S Sophie
Edward M. Kenneth A James C Y Pascal Francois Naomi Ilana Denis A Jamie Donald Suzette
Thomas G Warren Zhefeng Elissa A Volker Kent L Ann M Larry F Kendall N Jing Dana Leanne Jaana Daniel T Ann R Andrea M Daniel L Donald Barry David E Siavash K Beth Ann Benhur Min Pei Yun
Eric
James C. Poster #
399
348
301
367
390
421
333
392
356
302
327, 359
424
422
363
315
400
305
329
361
423
417
312, 354
331
323
357
326, 383
381
391
412
334
300
345
314, 325, 336, 353
415
330
306
409
385
384
339
401
341
364
355
377
Index of Faculty Mentors, by Session
SESSION THREE
Faculty Last
Lin
Liu
Lu
Mahajan
Mahendra
Malkan
Martinez
Maynard
Mehta
Mikkola
Miller
Miranda
Modlin
Murphy
Nakano
Navab
Nazarian
Novitch
Nurmi
Ozcan
Pajukanta
Phelps
Pirino
Pisegna
Quinlan
Rao
Sagasti
Schlinger
Schweizer
Shackelford
Svendsen
Ting
Tripati
Vondriska
Walker
Walwyn
Weiss
Weiss
White
Williams
Wu
Wu
Yang
Yang
Zheng
2:30‐3:40 PM
Faculty First
Chentao Wentai Hongjing Aman Shaily Matthew A Julian Antonio Heather D Mayank R Hanna K. A. Jeffrey H Susan Amy Robert L Niall P Atsushi Mohamad Ramin K Bennett G Erika Lynn Aydogan Paivi Elisabeth Patricia E. Giorgia
Joseph R Margot Elizabeth Dinesh Subba Alvaro Barnett Felix Erich David B Clive N Kang Aradhna K Thomas M. Bruce D.
Wendy Shimon Paul S.
Stephanie Ann Gary A Benjamin M. Ting‐Ting Otto Orlean Isaac Zhong Poster #
307
340
352
332
420
413
322, 372
304
397
375
342
371
344, 376
309
410
386
380, 419
335
308
343
395
369
414
408
338
394
316
360
404
350
378
373, 405
318, 351
379
374
349
337
407
321
328
346, 365
366
324
370, 398
311
148
UCLA Undergraduate Research Center‐Sciences Staff Program Representatives Desiree Alaniz Amalia Castaneda Cindy Mosqueda Undergraduate Workers Maria Campos Ada Madigan Alexandria Ray Sara Villalon Bridget Webb Director: Dr. Tama Hasson Assistant Director Diana Azurdia Administrative Assistant Miriam Gamboa Graduate Student Mentors Jenny Chen Maria Dzialo Xavier Gaeta David Lopez Alexander Patananan Eriko Shimada UCLA Undergraduate Research Center‐Sciences Faculty Advisory Board Dr. Whitney Arnold Dr. Diana Azurdia Dr. Paul Barber Dr. Ira Clark Dr. Catherine Clark Dr. Tama Hasson Dr. Richard Kaner Dr. Pirouz Kavehpour Dr. Adrienne Lavine Dr. Patty Phelps (Chair) Dr. Francoise Queval Dr. Steve Smale Dr. Felix Schweizer Dr. Dwayne D. Simmons Dr. Richard L. Weiss Dr. Benjamin Wu 149 UCLA ANNUAL UNDERGRADUATE RESEARCH POSTER SESSIONS Department of Psychology 22nd Annual Psychology Undergraduate Research Conference (PURC) Friday, May 9, 2014 8:30 am‐ 5:30 pm Franz Hall/Kerckhoff Grand Salon Open to the public http://www.psych.ucla.edu/undergraduate/purc Department of Molecular, Cell & Developmental Biology th
10 Annual Molecular, Cell & Developmental Biology Undergraduate Poster Session Friday, May 23, 2014 2:00‐4:00 pm 158 Hershey Hall For more information, please contact Connie Firestone ([email protected]) Department of Ecology and Evolutionary Biology 17th Annual Biology Research Symposium Wednesday, May 14, 2014 4:00 pm Poster Session 158 Hershey Hall/Cortile (Inner Courtyard) Area of Hershey Hall For more information, please contact Jessica Angus ([email protected] or 310‐825‐1680) Department of Integrative Biology and Physiology Integrative Biology and Physiology Undergraduate Research Poster Day Wednesday, May 21, 2014 158 Hershey Hall 2:00‐4:00 pm For more information, please contact Inna Gergel ([email protected])