summer undergraduate research … the gunderson 1988 fractionator method for stereological analysis,...

$: SUMMER UNDERGRADUATE RESEARCH … the Gunderson 1988 fractionator method for stereological analysis, as implemented in the Stereoinvestigator software system (Microbrightfield), neurons,$
SUMMER UNDERGRADUATE RESEARCH PROGRAM

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Schedule of Events July 28, 2017

8:30am Registration Table Opens HPEB LL Lobby 9:00am-12:00pm Poster Viewing

HPEB LL43 A&B 12:00pm-1:00pm Lunch

HPEB LL11 A&B 1:00pm-2:00pm SRP Keynote Speaker

Dr. Gerard Coté Director, Center for Remote Health Technologies and Systems Charles H. and Bettye Barclay Professorship in Engineering Texas A&M University HPEB LL46

2:00pm-3:00pm Presentation of Certificates Dr. Brett Mitchell, Summer Research Program Director HPEB LL46

Presentation of Dean’s Recognition Awards HPEB LL46

3:00pm Group Picture & Adjourn

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Speaker’s Biography

Gerard L. Coté, PhD Director, Center for Remote Health Technologies and Systems

Holder of the Charles H. and Bettye Barclay Professorship

Dr. Gerard L. Cote holds the Charles H. and Bettye Barclay Professorship in Engineering at Texas A&M University and is director of the Center for Remote Health Technologies and Systems. His primary research interests include the use of Optics for medical diagnostics and biomedical sensing.

In particular, the research within his lab focuses on the development of macro-scale to nano-scale biomedical systems and point-of-care devices using lasers, optics and electronics. Some research applications include development of innovative, noninvasive and minimally invasive ways to test blood sugar levels in diabetes; to detect other body chemicals such as cardiac biomarkers for cardiovascular disease or blood toxins such as PCBs or BPA; use of cell phone technology to detect malaria and the point of care; and to monitor perfusion and oxygenation for tissue implants and wearable devices.

The research is multidisciplinary and involves several investigators from across the world including medical doctors, life science faculty, faculty from other engineering disciplines, national laboratory staff and industry personnel.

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Acknowledgements The Texas A&M Health Science Center College of Medicine’s Summer Research Program continues to attract the top students from the best colleges and universities all across the country. This year we had 42 participants who completed the 10-week program. These students were selected from a large pool of applicants based on their research experience, desire to attend graduate and/or medical school, grades, exam scores, and letters of recommendation. I would like to thank the selection committee who dedicated their time to read through the applications.

Kayla Bayless, Ph.D. Sharon DeMorrow, Ph.D. David Huston, M.D. Xu Peng, Ph.D. Samba Reddy, Ph.D.

Mendell Rimer, Ph.D. Andreea Trache, Ph.D. Emily Wilson, Ph.D. Warren Zimmer, Ph.D.

I would also like to thank the faculty that gave their time as mentors. You have provided each of these students with a valuable experience that will undoubtedly help them achieve their career goals. The program would not be able to sustain its quality without you.

The program was made possible by the following people who provided funding and sponsorships:

Dean Paul Ogden, M.D. – Texas A&M Health Science Center Dean Carrie Byington, M.D. – Texas A&M Health Science Center Van Wilson, Ph.D. – Texas A&M Health Science Center Jack Hart, Ph.D. – Temple Health and Bioscience District Robert Dearth, Ph.D. – University of Texas Rio Grande Valley Dennis Daniels, MPH, Dr.PH – Prairie View A&M University Adebayo Oyekan, DVM, Ph.D. – Texas Southern University Filomeno Maldonado, M.A. – Texas A&M Health Science Center Warren Zimmer, Ph.D. – Texas A&M Health Science Center American Heart Association Summer Undergraduate Research Fellowship

Each week we had Roundtable Discussions in which students got to individually engage with faculty mentors at their respective locations regarding numerous “behind the scenes” aspects of science. This was made possible by the time and dedication of the following Site Coordinators:

Temple – Drs. Xu Peng and David Dostal Houston – Drs. David Huston and Margie Moczygemba

Finally, I would like to thank the SRP Coordinator Mary Ann Wolff who did a ton of work arranging the arrival, housing, registration, processing, and weekly feeding of the students. Thank you as well to Monica Flores and Angelica Guerra in Bryan/College Station, Tammy Kocurek in Houston, and Manuela Smith and Tami Annable in Temple who kept each of the sites running smoothly. Thank you to our poster judges who had an extremely difficult task of picking the best out of the best. Thank you students for your hard work and everyone for a memorable summer – Gig ‘em!

Brett Mitchell, Ph.D., F.A.H.A. Director, Summer Research Program

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Abstracts

Apgar, Christopher page 7 Sterological Study of Neuronal and Glial Cells in BA11/BA4 in PTSD

Bass, Brittany page 8 Presenilin-1: A Link Between Alzheimer’s and Heart Failure

Beckman, Katharine page 9

Morphine-Induced Cell Death in a Rodent Model of Spinal Cord Injury Bommareddy, Vaibhav page 10

Correlation of Cortical Electrographic and Behavioral Motor Seizures in the Mouse Model of Temporal Lobe Epilepsy Cheruvu, Sai Sammitha page 11

A Pilot Study on the Efficacy of Synthetic Neurosteroid Analogs on the Development of Chronic Epilepsy in Rats Chroust, Zachary page 12

Identification of Novel Cell Wall Anchored Proteins in S. Epidermidis Clinical Isolates from Patients Clark, Mary-Catherine page 13

Pioneering Anti-Fibrotic Pathways: Activation of Hepatic Stellate Cell Signaling by Mechanically Stressed Cholangiocytes Difranco, Erin page 14

Efficacy of Imipridones, ONC201 and ONC206, on Cholangiocarcinoma Elmer, Sarah page 15

Effect of Neurosteroid Therapy on Neurodegeneration in the Mouse Pilocarpine Model of Chronic Epilepsy Farley Nieves, Christian page 16

Characterization of Lymphatic Expansion Upon Doxycycline Delivery to an Inducible Mouse Model of Renal Lymphangiogenesis in Hypertension

Farooq, Aaminah page 17

MIR-20A-3P Promotes Blood-Brain Barrier Endothelial Cell Function in an OGD Model Glass, Melissa page 18

CF3DODA-ME Downregulates Specificity Protein Transcription Factors in Glioblastoma Via a ROS-Dependent Pathway Ighofose, Pauline page 19

Changes in Renal Lymphatic Vessels During Hypertension Kakkilaya, Akash page 20

The Effect of ERBB Family on Colorectal Cancer Kancharla, Srujan page 21

Ganaxolone Reduces Electrophysiological High-Frequency Oscillations (HFOs) in a Rat Model of Temporal Lobe Epilepsy Kokkapuni, Nikhil page 22

Antimicrobial Activity of a Novel Class of Compounds Against Multi-Drug Resistant Pathogens Kumar, Amy page 23

Substance P Regulates Inflammatory Pathways in Lymphatic Muscle

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Lee, Chi-Hung page 24

Characterization of Mouse NLRC5 Promoter

Lee, Michael page 25 Significance of Type XII Collagen in Promoting Bone Regeneration

Li, Catherine page 26

The Role of LRRK2 in Type 1 Interferon Production and Signaling Lindner, Nicholas page 27

Preliminary Pursuit of Intervention and Outcome Retrieval from Medical Record Systems Mathur, Srishti page 28

ISO-1 as a Novel Therapy for Reducing Chronic Effects of the Neuroinflammatory Response Following Traumatic Brain Injury Medler, Dawson page 29

A High Throughput Drug Screen Identified Both Pro- and Anti- Angiogenic Compounds with Therapeutic Potential Morris, Shea page 30

Role of Guinea Pig Cytomegalovirus Glycoprotein Complexes in Placental Trophoblast Infection and Congenital Model Nagamalla, Vineela page 31

Post-Traumatic Epileptogenesis in Extrasynaptic GABA-A Receptor Knockout Mice Oliver, Lauren page 32

Evaluating the Effects of Lipopolysaccharide on Mice with a Microdeletion Homologous to the Human 15Q13.3DS Patel, Krishna page 33

Generation of P. Maniculatis IPSCS for the Genetic Engineering of Disease Resistance of the North American Deer Mouse Peng, Jasmine page 34

Mechanisms that Regulate NLRP3 Inflammasome Activation During Blood-Brain Barrier Endothelial Cell Hyperpermeability

Perumal Ramasamy, Dheepthi page 35

Effect of Neurosteroid Therapy on Neuroinflammation in the Mouse Pilocarpine Model of Chronic Epilepsy Polamraju, Vinathi page 36

RNA Secondary Structure Visualization of Betacoronaviruses Reddy, Sandesh page 37

Magnetic Resonance Imaging (MRI) Investigation of Volumetric Changes and Neuronal Lesions from Op-Induced Epilepsy in Rats

Saathoff, Micaela page 38

Renal Lymphatic Alterations in Angiotensin II-Induced Hypertension

Shi, Eric page 39 Observing Drug Resistance and Homologous Recombination in Liver Cancer Cells

Sorrell, S. Stephen page 40

A Ubiquitination Inhibitor, PYR-41, Reduces IL-5 and IL-3 Stimulated signal Transduction in Human Eosinophils

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Stewart, Kyndhal page 41

Optimizing PLGA Nanoparticles Through Nanoprecipitation

Thomas, Alicia page 42 Cellular Localization of Intranasally Administrated MSC-Derived A1-Exosomes in the Rat Hippocampus Tucker, Ashley page 43

Exosomes: A Potential Therapeutic Delivery Device for Multiple Sclerosis (MS)

Wang, Andrew page 44 Purification and Structure Identification of Mycobacterium Autoinducer Molecules

Wilson, Hannah page 45 Borrelia Burgdorferi Markerless Deletions Utilizing an Inducible CRE-LOX Recombinase System

Zaragoza, Javier page 46 Treatment of Cathepsin Inhibitor in the Pilocarpine-Induced Model of Temporal Lobe Epilepsy

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TAMHSC 2017 SUMMER RESEARCH PROGRAM

STEROLOGICAL STUDY OF NEURONAL AND GLIAL CELLS IN BA11/BA4 IN PTSD

CHRISTOPHER J. APGAR, KEITH A. YOUNG

Department of Psychiatry and Behavioral Science Texas A&M Health Science Center College of Medicine

Temple, TX PTSD may impair brain function of the executive system, fear conditioning, and emotional regulation. The medial-orbital prefrontal cortex (Brodmann Area 11; BA11), links emotional processing of the limbic system and “fight or flight” responses. The present investigation will provide an initial quantitative morphological survey of cellular populations in this region in comparison to a control area of the motor cortex (BA4), which has not been identified as an area where functional or structural deficits exist in PTSD. 11 post-mortem human brains collected by the National PTSD Brain Bank were used in the study. The right hemisphere was serially cut and stained with Nissl. Using the Gunderson 1988 fractionator method for stereological analysis, as implemented in the Stereoinvestigator software system (Microbrightfield), neurons, astrocytes and oligodendrocytes in BA11 and BA4 across all lamina were counted using a Zeiss AXIO microscope. The experimenter was blinded to the classification of the brains until the data had been collected. The findings showed that there was no difference in the ratios of neurons, astrocytes or oligodendrocytes in PTSD in either area. Examination of available covariates (age, gender, PMI, smoking history) using analysis of covariance (ANCOVA) did not alter the findings. This is of interest because BA11 has been identified as a unique site of anatomical deviation in PTSD in structural MRI studies harboring synaptic deficits in PTSD (Young et al., 2015). Further research into the morphology of these cells may provide a deeper understanding of how cellular pathways are dysfunctional without changes in cell populations.

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PRESENILIN-1: A LINK BETWEEN ALZHEIMER’S AND HEART FAILURE

Brittany Bass, Michelle Kaspick, MinJia Liu, John Jacob, Michaela Ploof, Joe Dostal,

David E. Dostal Department of Medical Physiology

Temple Health and Bioscience District Texas A&M Health Science Center College of Medicine

Central Texas Veterans Health Care System Temple, TX

Heart Failure (HF) is one of the most common symptoms associated with Alzheimer’s and is a leading cause of morbidity and mortality in the Veteran population. Current therapies associated with heart disease focus on treating the symptoms rather than detecting the passages and markers held responsible. Previous studies have led us to believe that abnormal regulation of the protein, presenilin-1 (PSEN-1), could advocate the deadly link between Alzheimer’s and Heart Failure from dysregulation of cardiac Ca2+ metabolism in the sarcoplasmic/endoplasmic reticulum (SR/ER) in neurons. We postulated the mechanism involving Presenilin-1 (JNK-p38-PSEN-1) serves as a pathway to adjust intracellular calcium and contractile performance in cardiac myocytes, depending on the level of myocardial stress. To test this hypothesis, development of a cardiac-targeted PSEN-1 knockout mouse was created and the in vivo temporal changes in cardiac function were observed before and after dosages of Tamoxifen. After performing baseline echocardiography and Tamoxifen dosing, serial echocardiography at one and two weeks, it was determined that additional time is needed in order to detect the loss of cardiac function and associated dilated cardiomyopathy as reported in Alzheimer’s patients. Echocardiography will continue for twelve weeks, during which Langendorff, cell signaling, mass spectroscopy and histological experiments will be performed on mice at various times to assess longitudinal effects on cardiac function and alterations in intracellular calcium and JNK signaling pathways in the absence of cardiac cardiomyocyte PSEN-1 signaling. The end result will provide a better understanding of how a loss in cardiac PSEN-1 function leads to heart disease. This work is supported by the U.S. Department of Veteran’s Affairs

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MORPHINE-INDUCED CELL DEATH IN A RODENT MODEL OF SPINAL CORD

INJURY

Katharine Beckman, Mabel Terminel, Miriam Aceves, Michelle Hook Department of Neuroscience and Experimental Therapeutics

Texas A&M Health Science Center College of Medicine Bryan, TX

Opioids are the most effective analgesics for the treatment of pain during the acute phase of spinal cord injury (SCI). However, we have shown that, irrespective of the route of administration, morphine given at this early time point of injury can undermine locomotor recovery, significantly increase lesion size, increase mortality, and suppress weight gain. We hypothesize that attenuation of function may be due to morphine-induced inflammation and neurotoxicity, increasing cell loss at the injury site. To test this, the current study used the apoptotic marker, Caspase-3 together with specific neuronal and astrocytic markers to analyze temporal sequence of cell loss with morphine administration. Previously our studies have shown after 7 days of morphine administration, markers for neurons, astrocytes and microglia are significantly decreased. The current study suggests that morphine-induced neuronal cell death can be observed with as few as 3 days of administration. Morphine-treated subjects also had increased co-localization between the apoptotic marker Caspase-3 and neurons at this early time point. Conversely, there has no decrease in expression of the astrocyte marker GFAP following 3 days of morphine administration. Instead, morphine appears to attenuate the natural proliferation of astrocytes associated with SCI. Additional studies are needed to understand why there is decreased expression of astrocytes after 7 days of morphine administration and the mechanisms mediating morphine-induced cell death. There is an urgent need to identify the mechanisms mediating adverse effects of opioids to develop effective therapeutic strategies for the use of clinical opioid analgesics in pain management after SCI.

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CORRELATION OF CORTICAL ELECTROGRAPHIC AND BEHAVIORAL MOTOR

SEIZURES IN THE MOUSE MODEL OF TEMPORAL LOBE EPILEPSY

Vaibhav Bommareddy, Bryan Clossen, D. Samba Reddy Department of Neuroscience and Experimental Therapeutics

Texas A&M University Health Science Center Bryan, TX

Epilepsy is a neurological disorder characterized by spontaneous seizures. Epilepsy develops through a dynamic and poorly understood process known as epileptogenesis. EEG detection of spontaneous seizures is a front-line method for evaluating epileptogenesis in experimental models, and video verification of EEG seizures and epileptic activity is a critical for accurate experimental analysis. Therefore, we investigated the extent of correlation between EEG seizures and motor seizures in the pilocarpine model of temporal lobe epilepsy. To validate EEG seizures, we reviewed video and characterized seizures based on their severity according to modified Racine scale: generalized seizures (Lvl. 3-5), partial seizures (Lvl. 1-2), and false positives. Mice were implanted with wireless telemetry for 24-hour video-EEG monitoring. We also investigated the effect of finasteride on the progression of epilepsy. Animals underwent monitoring for 90 days. EEG-seizures were analyzed using DSI’s Neuroscore Software and this data was used for video screening. Overall, our analysis shows a correlation of 0.83 between all motor seizures and EEGs, with a correlation of 0.57 for generalized motor seizures only. However, there was no significant differences in the onset of motor vs. EEG seizures in control- and finasteride-cohorts. There was a strong correlation for incidence of epilepsy and cumulative seizure frequency counts between motor and EEGs. Within motor seizures, generalized seizures were significantly longer in duration than partial seizures in both control and finasteride groups. These results reinforce the importance of video verification of spontaneous EEG seizures for accurate characterization of epileptogenesis following SE.

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A PILOT STUDY ON THE EFFICACY OF SYNTHETIC NEUROSTEROID ANALOGS ON

THE DEVELOPMENT OF CHRONIC EPILEPSY IN RATS

Sai Sammitha Cheruvu, Ramkumar Kuruba, Xin Wu, D. Samba Reddy Department of Neuroscience and Experimental Therapeutics


Epilepsy is a chronic neurological condition characterized by recurrent seizures. Neurosteroids are powerful anticonvulsants with therapeutic potential for epilepsy, which is often intractable to current medications. However, little is known regarding the efficacy of neurosteroid analogs on the development of epilepsy. The objective of this study was to investigate the effect of synthetic neurosteroid analogs (NS21, NS21A and NS21B) on the development of epilepsy in rats. Chronic epilepsy with spontaneous recurrent seizures (SRS) was induced in rats by exposure to the organophosphate GD. They received a test drug at 10 mg/kg dose. SRS were monitored by continuous video-EEG recordings from the hippocampus and frontal cortex for 120 days. The SRS frequency was assessed by a validated MatLab algorithm followed by visual verification of seizure traces. Our results indicate that test neurosteroid analogs showed positive outcomes with delay in latencies for epilepsy development. NS21-treated group exhibited SRS with a latency for first seizure of 38 days compared to 26 days in control epileptic group. Both NS21A and NS21B were comparable in their effect on SRS outcomes. However, NS21B in combination with MDZ had the best outcome among the groups with fewer seizure occurrences. The total cumulative frequency and duration of seizures were also reduced with NS21A and B therapy. These pilot results indicate the promising potential of synthetic neurosteroids for preventative treatment of chronic epilepsy and the overall outcomes are consistent with our recent findings of antiepileptogenic effect of ganaxolone in limbic epilepsy.

Supported by NIH Grant U01-NS083460 to DSR

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IDENTIFICATION OF NOVEL CELL WALL ANCHORED PROTEINS IN S. EPIDERMIDIS

CLINICAL ISOLATES FROM PATIENTS

Zachary Chroust, Srishtee Arora, Dr. Magnus Höök Center for Infectious and Inflammatory Diseases

Institute of Bioscience and Technology Texas A&M Health Science Center College of Medicine

Houston, TX

The gram-positive, coagulase-negative bacteria, S. epidermidis, is an opportunistic pathogen that colonizes human skin, especially in moist areas. Cell wall anchored (CWA) bacterial proteins are critical for establishing infection in the host. The goal of this project is to identify novel CWA proteins in clinical isolates of S. epidermidis collected from blood of cancer patients at MD Anderson. These isolates were investigated for the presence of LPXTG motif proteins. Six clinical isolates were studied and each isolate contained 9-13 LPXTG motif protein. Six of these proteins have not been described before. Studies were focused on a putative collagen binding protein. Through protein structure prediction software, it was determined putative collagen binding protein might have structural similarities to collagen binding protein in Streptococcus as well as Enterococcus. Additionally, S. epidermidis is known for carrying methicillin-resistance gene in a mobile genetic element called SCCmec. Next, we determined the presence of LPXTG motif proteins in known SCCmec elements. By using genomic browser software, identification of CWA LPXTG motif containing protein in 35 different SCCmec types was conducted. These studies revealed 1 LPXTG motif proteins named Plasmin senstitive protein. In conclusion, hypothetical CWA protein with unknown functions were identified in clinical isolates. Further investigation of the clinical isolates is required to generate a comprehensive list of novel protein in S. epidermidis. Also, further studies are warranted to determine if putative collagen binding protein interacts with collagen.

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PIONEERING ANTI-FIBROTIC PATHWAYS: ACTIVATION OF HEPATIC STELLATE

CELL SIGNALING BY MECHANICALLY STRESSED CHOLANGIOCYTES

Mary-Catherine Clark, April O’Brien, Matthew Lopez, Shannon Glaser Department of Internal Medicine

Texas A&M Health Science Center College of Medicine Temple, TX

Liver disease is one of the leading causes of death in the United States affecting one in every ten Americans. Twelve hundred people die annually waiting for a liver transplant. Hepatic fibrosis is a common complication in most liver injuries, and discovering ways to prevent fibrosis as opposed to treating fibrotic symptoms remains a pressing issue in Gastroenterology. On the cellular level, researchers continue to investigate the role of hepatic stellate cells during onset and pathogenesis of liver failure. Hepatic stellate cells (HSCs) are liver cells, that in a healthy microenvironment store Vitamin A and retinal in their lipid droplet, but in the event of hepatic injury become activated. This HSC activation involves a phenotypic change into a myofibroblastic-like morphology and upregulation of collagen for fibrotic tissue construction. Many activation pathways have been previously explored; however, previous literature yields very few studies on the correlation of stressed cholangiocytes and HSC activation. Cholangiocytes comprise a small fraction of the liver, and regulate bile in addition to other various functions. In order to mimic cholangiocyte injury, mechanical stress was applied and the supernatant collected and used to treat HSCs in vitro. A trans-well model was used in the mechanical stress system along with individual cholangiocyte stretching. Using various assays, in addition to immunofluorescence and qPCR, allowed for cell-cell signaling elucidation in order to discover anti-fibrotic pathways that may translate to innovative liver treatments, and mitigate hepatic fibrosis as opposed to simple symptom management.

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EFFICACY OF IMIPRIDONES, ONC201 AND ONC206, ON CHOLANGIOCARCINOMA

Erin DiFranco, Gabriel Frampton, Stephanie Grant, Anca Petrescu, Sharon DeMorrow

Dept. of Internal Medicine Texas A&M Health Science Center College of Medicine

Temple, TX

Cholangiocarcinoma is an uncommon malignancy arising from the epithelial cells of the biliary tract. Symptoms are usually evident only after blockage of the bile duct by the tumor, and at this advanced stage, survival and response to treatment are low. We have previously shown that cholangiocarcinomas produce increased amounts of dopamine, which have growth-promoting effects. The G protein-coupled receptor, dopamine D2, has been indicated as an originator for this over-expression, facilitating tumor metastasis. In an effort to selectively antagonize D2DR and stimulate anti-proliferative effects, a founding member of the impridone class, ONC201, was used. ONC201 is the first D2DR antagonist under clinical development for oncology, which binds GPCRS as a bitopic antagonist. After Oncoceutics developed ONC201, they also produced a second generation model, ONC206, proclaiming improved efficacy. The primary objective of this study was to determine the comparative competencies of ONC201 and ONC206 in mitigating cholangiocarcinoma growth, both in-vitro and in-vivo. The next objective was to investigate the combinatorial synergism between the D5 and D2 receptors in tumor pathology. The findings revealed ONC201 as a more potent, anti-cancer treatment method. ONC201 decreased proliferation in each of the six human cholangiocarcinoma cell lines used. Furthermore, our studies suggested an invaluable role that D5DR expression has in regulating disease development, an area largely understudied. This work was supported by the Central Texas Veterans Health Care System, Temple, Texas.

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EFFECT OF NEUROSTEROID THERAPY ON NEURODEGENERATION IN THE MOUSE

PILOCARPINE MODEL OF CHRONIC EPILEPSY

Sarah Elmer, Bryan Clossen, Victoria Golub, Xin Wu, D. Samba Reddy Department of Neuroscience and Experimental Therapeutics


Epilepsy is characterized by the occurrence of repeated unprovoked seizures and a spectrum of neurodegenerative pathology. It affects about 3 million Americans and approximately 65 million people worldwide. Despite such wide incidence of the disease and the number of drugs available to treat seizures, there is no cure for epilepsy. The objective of this study was to investigate the extent of neurodegeneration in mice with chronic epilepsy, induced by pilocarpine-status epilepticus (SE). Additionally, we sought to determine the effect of augmenting or diminishing available neuroprotective neurosteroids in the brain following SE by administering either a synthetic neurosteroid, ganaxolone (GX), or a 5α reductase inhibitor, finasteride (Fn). Neurosteroids preferentially bind extrasynaptic GABAA receptors that contribute to tonic inhibition in the hippocampus, a key structure in limbic epileptogenesis. At 90 days after experimental epilepsy induction by pilocarpine-SE, coronal serial sections (30-µm) were cut and processed for Nissl, NeuN+, and PV+ immunoreactivity. An unbiased, quantitative neurostereology protocol was utilized to determine the extent of principal neuron loss (NeuN+) and GABAergic interneuron loss (PV+) in hippocampal subfields CA1, CA2, CA3, DG, & DH. Pilocarpine-epilepsy was associated with massive neurodegeneration (65% NeuN+ & 55% PV+) in the hippocampal subfields. GX-treated animals had significantly less neuronal loss (15% NeuN+ & 10% PV+) compared to the non-treated cohort. However, there was no exacerbation of neurodegeneration following finasteride therapy. Our results suggest that neurosteroid therapy provides beneficial neuroprotection against neurodegeneration, preventing the loss of principal neurons and interneurons, often evident in chronic human epilepsy.

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CHARACTERIZATION OF LYMPHATIC EXPANSION UPON DOXYCYCLINE

DELIVERY TO AN INDUCIBLE MOUSE MODEL OF RENAL LYMPHANGIOGENESIS IN HYPERTENSION

Christian A. Farley Nieves, Joseph M. Rutkowski

Department of Medical Physiology Texas A&M Health Science Center College of Medicine

College Station, TX

Renal inflammation has been identified during chronic high blood pressure, or hypertension. Reducing inflammation or immune cell numbers in the kidney has been shown to reduce blood pressure in pre-clinical models of hypertension. Inflammation-associated lymphangiogenesis helps maintain fluid balance and aids in the clearance of immune cells and cytokines from the inflamed tissue. We hypothesized that increasing renal lymphatic vessel density would ameliorate inflammation and thereby reduce blood pressure. To test this, we employed a kidney-specific, doxycycline (dox)-inducible, mouse model that overexpresses the lymphangiogenic protein VEGF-D in the kidney (KidVD mouse). We tested routes of delivery and dox dosing on high salt (to mimic the hypertensive model) diet on renal VEGF-D induction by qPCR and protein analysis. The impact of VEGF-D overexpression was also assessed by quantifying lymphatic vessel density in each delivery condition after 3 weeks. We found that VEGF-D expression and lymphatic vessel expansion was most increased in mice eating high salt diet with 200 mg/L dox in their drinking water versus the dox supplied in the food. This is because the mice drank more water under a high salt diet which, in turn, led to a greater consumption of doxycycline whenever it was in the water. Despite what an increased effective dose, 200mg/L of doxycycline along with a 4% NaCl diet was the most effective delivery method tested to drive renal lymphangiogenesis in a mouse model of salt-induced hypertension. Christian was supported by an American Heart Association Summer Undergraduate Research Fellowship

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MIR-20A-3P PROMOTES BLOOD-BRAIN BARRIER ENDOTHELIAL CELL FUNCTION

IN AN OGD MODEL

Aaminah Farooq, Shameena Bake, Farida Sohrabji Department of Neuroscience and Experimental Therapeutics


Stroke is a leading cause of death and long-term disability, with older women at increased risk for stroke and greater post-stroke morbidity and mortality. Previous research by our laboratory showed reduced miR-20a-3p levels in astrocytes from middle-aged female rats. Compared to younger females, middle-aged female rats have a worse stroke outcome, with more infarct and blood-brain barrier permeability. In the present study, we hypothesized that miR-20a-3p is critical for maintaining barrier integrity, and loss of this miRNA in middle-aged rats is related to poor barrier function. To test this hypothesis, human brain microvascular endothelial cells were grown under normoxia (5% CO2), then subjected to oxygen-glucose deprivation (OGD). Concurrent to OGD, cultures were treated with miR-20a-3p mimic or scrambled-control oligos. A media-LDH assay was used to measure necrotic cell death. TEER (trans-epithelial electrical resistance) testing was conducted, to measure the cell monolayer integrity. Cultures were fixed and stained with fluorescent-labeled lectin, then photographed to visualize cell-cell contact. Media from these cultures was analyzed for zymogen activity. Our results showed that while OGD increased LDH, miR-20a-3p did not reduce cell death. However, OGD decreased TEER, with a statistically non-significant trend towards improvement due to miR-20a-3p treatment. MiR-20a-3p treatment maintained cell-cell contact under OGD as the continuous pattern of lectin stained cells indicated. MMP activity examination via zymography is in progress. These results suggest miR-20a-3p may provide protection during ischemia-like conditions by maintaining cellular integrity of the blood-brain barrier, thus improving stroke outcome. Future studies will focus on validating molecular targets of miR-20a-3p. Supported by NIH grant AG042189 to FS

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CF3DODA-ME DOWNREGULATES SPECIFICITY PROTEIN TRANSCRIPTION

FACTORS IN GLIOBLASTOMA VIA A ROS-DEPENDENT PATHWAY

Melissa Glass, Keshav Karki, Stephen Safe Dept. of Veterinary Physiology & Pharmacology

Texas A&M Health Science Center College of Medicine College Station, TX

Glioblastomas are brain tumors that often contain a mixed grade of cells. The treatment options are limited to surgery, radiation therapy, or chemotherapy, with the 5-year survival rate at only 10%. CF3DODA-Me is a new synthetic triterpenoid anticancer drug that induces reactive oxygen species (ROS) and downregulates specificity protein (Sp) transcription factors (TFs). Sp1, Sp3, Sp4 and pro-oncogenic Sp-regulated genes in cancer cells and tumors. Sp TFs are highly expressed in glioblastomas and we investigated the effects of CF3DODA-Me in glioblastoma cell lines. Initial studies show that 0.5 to 2 µM concentrations of CF3DODA-Me inhibited the growth of A172, U87-MG, T98G cells after treatment for 24 or 48 hours. The same treatment of CF3-DODA-Me also induced apoptosis and ROS. These responses and inhibition of cell proliferation were attenuated after co-treatment with antioxidant glutathione (GSH). CF3DODA-Me also downregulated expression of Sp1, Sp3 and Sp4 and several other pro-oncogenic Sp-regulated gene products. These responses are also attenuated after treatment with antioxidant glutathione. Our experiments work to support the mechanism used by this drug treatment to induce apoptosis in glioblastoma cells. The proposed mechanism could lead to further drug research that takes advantage of this established pathway. The results demonstrate that CF3DODA-Me and its subservient induction of ROS effectively inhibits glioblastoma cell proliferation and survival, suggesting that this compound may have clinical applications for treatment of this deadly neurological cancer. This work was supported by the Syd Kyle Endowment and Texas AgriLife Research.

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CHANGES IN RENAL LYMPHATIC VESSELS DURING HYPERTENSION

Onoriode P. Ighofose, Dakshnapriya Balasubbramanian, Micaela E. Saathoff, Catalina

Lopez Gelston, Brett M. Mitchell Dept. of Medical Physiology


Hypertension is associated with infiltration of immune cells and inflammation in the kidney. The lymphatic system functions to drain these immune cells out of the renal interstitial space and reduce inflammation. The body’s natural response to chronic inflammation is lymphangiogenesis; however, it is unknown how renal lymphatic vessels (RLV) are altered during hypertension. We hypothesized that RLV density will increase in hypertensive mice. We used two models of hypertension: L-NAME and salt-sensitive hypertension (SSHTN). Mice (n= 3 females; 3 males) were administered L-NAME, a nitric oxide synthase inhibitor and vasoconstrictor, in their drinking water for 2 weeks. These mice became hypertensive (SBP: 143 ± 2 mmHg) and developed significantly more RLVs compared to control mice (n=3 females; 3 males) given tap water for 2 weeks. In the SSHTN model, after a 2 week administration of L-NAME, mice (n=3 females; 3 males) were given regular tap water (washout) and then fed a 4% high-salt diet for 3 weeks. Following the high-salt diet, mice were hypertensive (SBP: 152.1 ± 3 mmHg) and exhibited a significant increase in RLV density compared to the normotensive control group (SBP: 103 ± 3 mmHg). Compared to their respective control groups, mice with L-NAME HTN and mice with SSHTN had increased renal gene expression of Cyr61 and Tgfbi. Both are known to be associated with lymphangiogenesis. Our findings indicate that hypertension causes an increase in RLV density. It is possible that further augmenting renal lymphatic vessel density could prevent the development of hypertension. Pauline was supported by an American Heart Association Summer Undergraduate Research Fellowship

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THE EFFECT OF ERBB FAMILY ON COLORECTAL CANCER

Akash Kakkilaya, Carolina Mantilla-Rojas, Evann Bourgeois, Jorge Jaimes, David

Threadgill Dept. of Molecular and Cellular Medicine


Colorectal cancer (CRC) kills over 50,000 people in the United States annually. CRC is most often initiated with mutations in the APC tumor suppressor gene and progresses with an overexpression of Erbb family of receptors and activating mutations in oncogenes like KRAS. Molecular inhibition of epidermal growth factor receptor (EGFR), a member of the Erbb family, in clinical trials has shown primary and secondary resistance. This has led to the hypothesis that there are EGFR-independent mechanisms in CRC progression. We have evidence that other Erbb receptors, like ERBB3 and ERBB4, can activate compensatory pathways involved in CRC. We used the Cre/loxP genetic engineering system for intestinal-specific knockout of Egfr, Erbb3, or Erbb4 within the ApcMin/+ mouse model of CRC. Mice were euthanized when approximately 100 days old, the colons were extracted, splayed open, and fixed in formalin for tumor analysis. Methylene blue staining was used to visualize tumors, and the number, size, and location of tumors from each mouse was recorded. Our results show that intestinal knockout of Egfr and Erbb4 displayed a decrease in intestinal polyp and colon tumor count, indicating their role in tumor development. In contrast, ApcMin/+ mice with intestinal knockout of Erbb3 displayed an increase in intestinal polyp count, indicating its potential role in tumor suppression. This study will advance our understanding of Erbb family biology during colonic tumorigenesis, ultimately contributing to better therapies for CRC.

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GANAXOLONE REDUCES ELECTROPHYSIOLOGICAL HIGH-FREQUENCY OSCILLATIONS (HFOs) IN A RAT MODEL OF TEMPORAL LOBE EPILEPSY

Srujan Kancharla, Ramkumar Kuruba, D. Samba Reddy

Department of Neuroscience and Experimental Therapeutics Texas A&M Health Science Center College of Medicine

Bryan, TX

Temporal lobe epilepsy (TLE) is characterized by the progressive increase in spontaneous recurrent seizures (SRS), which often do not respond to currently available antiepileptic drugs. New and effective drugs are needed to control TLE. Limited biomarkers are available at present for epilepsy detection and therapy monitoring; however, there is evidence suggesting that high frequency oscillations (HFOs) can be EEG biomarkers of epilepsy. The HFOs including ripples (80-200 Hz) and fast ripples (250-500 Hz) are considered as biomarkers in human epilepsy because they represent pathological network activity underlying epileptogenesis in TLE. In this study, we investigated the effect of the synthetic neurosteroid ganaxolone (GX) on interictal spikes (IS) and HFOs in the dentate gyrus. TLE was induced in rats by injecting the organophosphate DFP and animals were monitored by video-EEG system. Rats with SRS were treated with ganaxolone (5 and 10 mg/kg) for one week. From EEG, epochs of 10-min were taken during light and dark periods (non-REM sleep), and analyzed for HFOs by a custom-built MATLAB algorithm. Our data shows increased HFOs (ripples & fast ripples) and IS in epileptic rats. GX treatment significantly reduced fast ripples without affecting ripples and IS. The overall HFOs incidence was lower in GX-groups. Withdrawal from GX treatment triggered a rebound increase in fast ripples, indicating greater seizure susceptibility. Diurnal analysis shows GX reduced HFOs in dark periods only. These results demonstrate that GX can reduce fast ripples without affecting IS, indicating distinct role of neurosteroid-mediated tonic inhibition in HFOs. Supported by NIH Grant U01-NS083460

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ANTIMICROBIAL ACTIVITY OF A NOVEL CLASS OF COMPOUNDS AGAINST MULTI-

DRUG RESISTANT PATHOGENS

Nikhil Kokkapuni, Oluwadara Ogun, Kush Shah, Carolyn Cannon Microbial Pathogenesis and Immunology


Multi-drug resistant pathogens have become a huge problem in the medical field. They are usually the result of misuse or overuse of antibiotics and are very difficult to treat. Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that is found in about 60% S. aureus strains in hospitals in the United States (Sakoulas 2008). It is also one of the main causes of infective endocarditis (IE), an inflammation of the inner heart tissue, which is found in about 3 to 7 people per 100,000 (Baddour 2015). Current standard-of-care (SoC) drugs, such as Vancomycin and Clindamycin, have shown to be largely ineffective when treating MRSA strains, but are more effective against methicillin sensitive Staphylococcus Aureus (MSSA). Our lab has collaborated with the Kurti Lab at Rice University to develop a novel class of compounds based on a natural material, C58, which shows promise in treating MRSA infections. Using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), we were able to determine the drug concentration required to inhibit and eradicate MRSA and MSSA bacterial strains. Our experiments show that Clindamycin was ineffective against most MRSA strains, but was more effective against MSSA. Our results demonstrate that C58 is superior to SoC antimicrobial, clindamycin, and comparable to the gold standard, vancomycin. C58 shows great promise to become a SoC drug in the future.

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SUBSTANCE P REGULATES INFLAMMATORY PATHWAYS IN LYMPHATIC MUSCLE

Amy Kumar, Yang Lee, Mariappan Muthuchamy, Sanjukta Chakraborty

Dept. of Medical Physiology Texas A&M Health Science Center College of Medicine

College Station, TX

Substance P (SP), a neuropeptide that acts through the neurokinin receptors is associated with the sensory innervation of lymphoid tissue and modulates lymphatic contractile function and lymph flow. SP signaling is associated with several inflammatory pathologies. The role of lymphatics in body fluid homeostasis, lipid absorption and inflammation and immunity rely on the contractility of the lymphatic muscle cells (LMCs). We have shown that lymphatic contractile dysfunction is associated with perturbations in inflammatory mechanisms and that SP activates both inflammatory and contractile pathways in lymphatics. However, the specific inflammatory pathways and the resultant cytokines activated by SP in LMCs is unknown. We hypothesized that SP stimulates the pro-survival AKT and MAPK pathways, and increases pro-inflammatory cytokines. Cultured rat mesenteric LMCs were treated with

SP (1M) and total RNA and protein were isolated at different time points (6hr and 24hr). Western analysis was carried out to detect the total and phosphorylated forms of AKT, JNK, GSK3, -catenin, FOXO1 and PDCD4. -actin was used as control. The relative levels of inflammatory cytokines and chemokines, and specific inflammatory microRNAs (miRNAs) were analyzed by real time PCR. Our results indicate that SP activates the AKT and JNK pathways to significantly increase inflammatory cytokine and chemokine expression, and induces expression of miR 17-5p, miR19b and miR186 in the LMCs. Additionally, SP stimulates expression of genes, involved in lymphatic remodeling. Taken together our data demonstrate that SP activates multiple inflammatory signaling pathways in the LMCs that contribute to an inflammatory response in lymphatics during pathological conditions. Amy was supported by an American Heart Association Summer Undergraduate Research Fellowship

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CHARACTERIZATION OF MOUSE NLRC5 PROMOTER

Chi-Hung Lee, Suhail Yousuf, Jason Yeung, Koichi Kobayashi

Dept. of Microbial Pathogenesis and Immunology Texas A&M Health Science Center College of Medicine

College Station, TX

NLRC5, also known as CITA (MHC class I transactivator), is an NLR family protein that in complex with other DNA binding proteins and transcription factors activates the promoters of MHC class I genes. Proper presentation of antigens by MHC class I genes to cytotoxic cells is necessary for the elimination of any foreign or cancer antigen. One of the major reasons for the impaired MHC class I expression is decreased or insufficient Nlrc5 expression possibly either due to somatic mutations or other epigenetic factors as observed in different types of cancers. One such mechanism operates through de novo hyper methylation of CpG sites around the Nlrc5 promoter region. Hence, it is important to understand the architecture and regulation of Nlrc5 promoter. We are characterizing mouse and human Nlrc5 promoter elements to understand the regulation of this immunologically important gene. We have already cloned mouse Nlrc5 probable promoter elements around exon 1 in pGL3 basic vector. Further studies will decipher the exact location of promoter elements and various regulatory mechanisms operational on this promoter.

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SIGNIFICANCE OF TYPE XII COLLAGEN IN PROMOTING BONE REGENERATION

Michael Lee, Suzanne Zeitouni, Eoin McNeill, Simin Pan, Roxanne Reger, Carl Gregory

Molecular and Cellular Medicine Department Texas A&M Health Science Center College of Medicine

College Station, TX

Over 6 million people in the United States experience some form of bone fracture annually. A remarkable portion do not fully recover due to surgical technique, substance abuse, or other factors. While human mesenchymal stem cells (hMSCs) look promising as a treatment for these non-union defects, they appear to lose efficacy shortly after the inflammatory phase. However, hMSCs coadministered with extracellular matrix (ECM) drastically improved the potency of the treatment, upwards of 80-100% healing of the lesion. Previously, type XII collagen had been determined to have a significant impact through immunoblot assays. We hypothesized that type XII collagen is a crucial part of the ECM that promotes bone regeneration, and that its absence reduces the ability for a bone defect to repair. Using hMSCs with Type XII collagen knocked down, we ascertain the importance of Type XII for bone regeneration. By performing cDNA synthesis and qRT-PCR, we confirm the knockdown of the collagen gene on a transcriptional level first. Next, the absence of protein was affirmed with our western blots. Quantification of calcium was performed with an Alizarin Red Solution stain and examined with a spectrophotometer. These data suggest that the absence of Type XII Collagen produces a significant decrease in calcium deposition in vitro. Future experimentation would involve testing the collagen knockdowns in IPS-MSCs for similarity to the hMSCs, which could potentially allow for commercialization. This work was supported by NIH R01AR066033-01 awarded by the National Institute of Arthritis, Musculoskeletal and Skin Diseases.

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THE ROLE OF LRRK2 IN TYPE 1 INTERFERON PRODUCTION AND SIGNALING

Catherine Li, Catherine G. Weindel, Samantha Bell, Kristin Patrick, Robert O. Watson


Bryan, TX Tuberculosis is a major infectious disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis. DNA released by M. tuberculosis into the cytosol activates the DNA sensing pathway, inducing Type 1 interferon production and signaling, a pro-bacterial response upregulated during human active tuberculosis disease. The multifunctional protein LRRK2 is phosphorylated during M. tuberculosis infection and is a known substrate of the kinase TBK1, an actor in the DNA sensing pathway. Our goal was to determine whether

LRRK2 is required for IFN production when the DNA sensing pathway is activated by different stimuli. Wild-type, LRRK2 T1348N mutant (GTPase inactive), and LRRK2 knockout RAW 264.7 mouse macrophages were transfected with dsDNA or treated with DMXAA, two molecules that stimulate the DNA sensing pathway at different points. RNA from these cells was isolated and used to generate cDNA in order to measure expression of IFN and Interferon Stimulated Genes (ISGs) including MX1, ISG15, and GBP2 by qPCR. Compared to wild-type cells, induction of IFN and ISGs was significantly decreased after both dsDNA transfection and DMXAA stimulation in LRRK2 knockout cells but not T1348N mutant cells. These results suggest that the presence of LRRK2 but not its catalytic function is required for IFN production and signaling at or downstream of the level of STING (the receptor of DMXAA in the DNA sensing pathway). Further characterization of the function of LRRK2 in the Type 1 interferon response and other pathways activated by M. tuberculosis could improve understanding of tuberculosis pathogenesis and help identify potential therapeutic targets. This work was supported by the Michael J. Fox Foundation

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PRELIMINARY PURSUIT OF INTERVENTION AND OUTCOME RETRIEVAL FROM

MEDICAL RECORD SYSTEMS

Nicholas Lindner, Abhishek Dave, Muppala Narasim Raju, Duane Steward Dept. of Bioinformatics


Medical records are replete with data that is valuable in reviewing and assessing encounter intervention outcomes in order to individualize health care. This study focuses on developing an ontology for better categorization and retrieval of the ontological elements that can be used to achieve this goal. This study seeks to define ontological labels to then be assigned to medical record data in order to support closed-loop feedback in future intervention encounters. Since most medical records are subject to redaction of personal data to ensure patient confidentiality, data must be cohesively assigned some anonymous identification factors. Data from two domains was analyzed: Hospice Brazos Valley (BHV) and Texas A&M Veterinary Clinic (VMIS). Ontological terms were defined and assigned to changes in medical intervention in order to gauge the number and forms of therapy changes and ultimately if the therapy was successful. Structured data was reformatted to enhance the records and more easily provide access to the ontology elements for analysis. The analysis of the data from the two domains revealed the weaknesses in existing medical records for identifying these ontology elements and thus prevent more optimized and individualized models of therapy. However, deeper data mining revealed that deriving an ontology from existing record data is possible, albeit difficult to judge the accuracy of given incomplete or confusing records. Further study is proposed to elucidate the domains examined in terms of the developing ontology.

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ISO-1 AS A NOVEL THERAPY FOR REDUCING CHRONIC EFFECTS OF THE

NEUROINFLAMMATORY RESPONSE FOLLOWING TRAUMATIC BRAIN INJURY

Srishti Mathur, Chenghao Deng, Jaclyn Jenkins, Sonia Lopez, Javier Zaragoza, Lee Shapiro


Temple, TX

Traumatic brain injury (TBI) can cause an increased prevalence of seizures and epilepsy. Previous studies suggest epileptogenesis may be facilitated by the neuroinflammatory response following TBI in which astrocytes play a major role. Of particular interest is a specific class of astrocytes located at the border between the hilus and granule cell layer (GCL) in the hippocampal dentate gyrus. These astrocytes are among the few neuronal progenitor cells that persist in the adult mammalian brain and have a unique morphology in which their radial glial processes extend through the GCL into the molecular layer (ML). Alterations to these processes may provide a scaffold for basal dendrites of granule cells to grow into the hilus, resulting in a pro-epileptogenic circuitry. The present research served to investigate whether ISO-1, an antagonist of macrophage inhibitory factor (MIF), administered 30 minutes after fluid percussion TBI (FPI) would prevent chronic changes to the radial glial astrocytes and newborn neurons in the dentate gyrus. Mice were given either FPI and ISO-1 (n=3), Sham surgery and ISO-1 (n=4), or FPI and Vehicle (n=3). The results indicate that treatment with ISO-1 reverses the chronic decrease in astrocyte radial glial processes 30 days after FPI. However, there was no significant decrease in the number of immature neurons in the dentate gyrus 30 days after FPI. Additional studies should investigate the morphology of these newborn neurons, their synaptic integration, and whether the number of these neurons further decreases with time. This work was supported by Citizens United for Research in Epilepsy (CURE).

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A HIGH THROUGHPUT DRUG SCREEN IDENTIFIED BOTH PRO- AND ANTI-

ANGIOGENIC COMPOUNDS WITH THERAPEUTIC POTENTIAL

Dawson Medler, David Howell, Colette Abbey, Kayla Bayless Department of Molecular and Cellular Medicine


Angiogenesis is the formation of new blood vessels from existing blood vessels and is associated with a multitude of diseases including rheumatoid arthritis, diabetic retinopathy, cardiovascular disease, skin disease and cancer. Ideally, compounds that inhibit and stimulate angiogenesis are needed to treat a variety of diseases where angiogenesis is either excessive or deficient, and the development of additional candidate drugs would be useful to this endeavor. Our focus is on the observation of angiogenesis in 3D environments in response to physiological pro-angiogenic factors comprised of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and sphingosine 1-phosphate (S1P), which combine to induce angiogenesis in vitro and in vivo. We utilized a high throughput drug screen of 800 FDA-approved drugs to observe which compounds had pro- or anti-angiogenic effects on human umbilical vein endothelial cells that were seeded on a collagen gel in the presence of the pro-angiogenic factors VEGF, bFGF, and S1P. Following qualitative and quantitative analysis of invasion responses in duplicate experiments, we categorized drugs based on whether they stimulated blood vessel formation or blocked it without harming the endothelial cells. As expected, using this methodology, we found compounds with known pro- and anti-angiogenic activity, thus validating our screen. Furthermore, the screen yielded unexpected results, revealing potential candidates for new therapeutics. This work was supported by American Heart Association Southwest Affiliate Grant-In-Aid (16GRNT27760154) to KJB.

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ROLE OF GUINEA PIG CYTOMEGALOVIRUS GLYCOPROTEIN COMPLEXES IN

PLACENTAL TROPHOBLAST INFECTION AND CONGENITAL MODEL

Shea Morris, K. Yeon Choi, Alistair McGregor Department of Microbial Pathogenesis and Immunology

Texas A&M University Health Science Center College of Medicine College Station, TX

Congenital human cytomegalovirus (HCMV) affects 1% of live births in the US causing mental retardation and hearing loss in newborns. Development of a vaccine against congenital HCMV is a high priority. The guinea pig is the only small animal model for congenital cytomegalovirus because of the similarity in placental structure and congenitally infected pups can have sensorineural hearing loss. This study evaluated guinea pig cytomegalovirus (GPCMV) glycoproteins and their roles in virus cell spread and infectivity. The homolog viral glycoprotein complexes gB, gM/gN, gH/gL/gO, and the pentameric complex gH/gL/GP129/GP131/GP133 (PC) are important for cell entry and also neutralizing antibody targets similar to HCMV. GPCMV BAC mutants of specific knockout glycoprotein genes were generated and evaluated on guinea pig fibroblast and placental trophoblast cells for growth. Similar to HMCV, the gO glycoprotein was essential for infection on fibroblast cells, but not placental trophoblast cells. Glycoprotein gB was necessary for viral infection/spread on all cell types. PC specific component, GP131 mutant, blocked infection on trophoblasts. Similarly, GP129 and GP133 PC specific mutants prevented trophoblast infection. Therefore, all five PC glycoproteins were necessary for trophoblast tropism. PC+/ gO- virus was impaired for infection of trophoblast and implied an additional role for the gH/gL/gO triplex for enhancing virus infection of trophoblasts. Interestingly, WT GPCMV stock generated on fibroblast cells had low infectivity on epithelial cells compared to stocks prepared on epithelial cells, despite both stocks being PC+. This potentially implies that additional factors control maturation of PC+ virus particles in a cell dependent manner.

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POST-TRAUMATIC EPILEPTOGENESIS IN EXTRASYNAPTIC GABA-A RECEPTOR

KNOCKOUT MICE

Vineela Nagamalla, Victoria Golub, Bryan Clossen, D. Samba Reddy Department of Neuroscience and Experimental Therapeutics


Traumatic brain injury is a highly heterogeneous condition that often includes varying degrees of contusion, brain injury, inflammation, as well as cognitive and motor impairment. Traumatic brain injury is a leading cause of acquired epilepsy—referred to as post-traumatic epilepsy (PTE)—and accounts for up to 10% of all symptomatic epilepsies. Synaptic GABA-A receptors are primary mediators of rapid phasic inhibition in the brain and have a critical role in the pathophysiology of epilepsy. The -subunit of the GABA-A receptor is typically found extra-synaptically in the dentate gyrus of the hippocampus and has been found to contribute to tonic inhibition and the reduction of seizure susceptibility in electrophysiology studies. In this study, we investigated the role of tonic inhibition on TBI-induced epilepsy development using -subunit knockout (DKO) mice. TBI was induced by a controlled cortico-hippocampal impact (CCI) model and seizure progression between wild-type and DKO mice was monitored by 24/7 video-EEG for 120 days. Our results show that DKO mice were found to have a shorter latency to seizures (WT=39 days, DKO=30 days), higher incidence (100% vs. 83% in WT), higher frequency of seizures, and a significantly higher seizure duration compared to the WT cohort, suggesting greater susceptibility to epilepsy in DKO mice. The overall survival rates and impact outcome was lower in DKOs. A diurnal analysis of seizure occurrence shows a greater seizure frequency during dark periods than light periods in both genotypes. Collectively, these results provide first evidence of a functional role for extrasynaptic GABA-A receptor-mediated tonic inhibition in TBI-induced epileptogenesis.

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EVALUATING THE EFFECTS OF LIPOPOLYSACCHARIDE ON MICE WITH A MICRODELETION HOMOLOGOUS TO THE HUMAN 15Q13.3DS

Lauren Oliver, Amal Halawa, Katherine Rees, Ursula Winzer-Serhan


Bryan, TX

The 15q13.3 microdeletion syndrome (15q13.3DS) includes six genes within a 1.2-megabase region on human chromosome 15. It causes neurological disorders such as autism, schizophrenia, and seizures, but there is incomplete penetrance of the syndrome, and an environmental factor is suspected for the development of the behavioral phenotype. One of the affected genes is the CHRNA7, coding for an α7 nicotinic receptor, which is implicated in the neurological problems. The receptor is also thought to mediate anti-inflammatory functions, which might be compromised in a heterozygous mouse model (Df(h15q13)/+). We hypothesize that these mice have an increased immune response to an LPS challenge and more activation of brain areas involved in sickness behavior. We used expression of immediate early genes to evaluate neuronal activation. We treated both wild type mice and heterozygous mice with 0.1 mg/kg LPS and brains were harvested two hours later, sectioned and processed for in situ hybridization with specific probes for c-Fos, NPAS4, and Arc. Autoradiograms were analyzed in the area postrema (AP), solidary tract nucleus (NST), central nucleus of the amygdala (CeA), and paraventricular hypothalamic nucleus (PVN). Results showed that LPS caused activation of these areas, with a trend of decreased expression in the AP and NST in heterozygous mice, whereas in other areas had no genotype effect. Heterozygous animals did not exhibit increased neuronal activation in areas associated with sickness, but further cytokine analyses and behavioral studies are needed to further evaluate the immune response in Df(h15q13)/+) to see if inflammation could contribute to the phenotype.

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GENERATION OF P. MANICULATIS IPSCS FOR THE GENETIC ENGINEERING OF

DISEASE RESISTANCE OF THE NORTH AMERICAN DEER MOUSE

Krishna N. Patel, Julia L. Popp, Lauren C. Farris, David W. Threadgill Department of Molecular and Cellular Medicine


Peromyscus maniculatus, the native mouse species in North America, serves as a major reservoir for human diseases including Hantavirus and Lyme disease. Since P. maniculatus is not amenable to assisted reproductive techniques and has not been successfully genetically engineered, we are exploring alternative options to manipulate their genome to manage and reduce the spread of human diseases. By combining the non-integrating and transient Sendai Virus (SeV) reprogramming vectors with specific culture conditions, we found that P. maniculatus cells, which are known to be non-permissive to reprogramming, can be driven into a pluripotent-like state similar to that found in embryonic stem cells (ESCs). Induced pluripotent stem cells (iPSCs) are generated from somatic cells that are reprogrammed with key transcription factors expressed in ESCs. Since Mus musculus, the common lab mouse, has the most extensive genetic resources for any animal model, it is an ideal candidate to generate chimeras with P. maniculatus. Creation of a chimera by injecting P. maniculatus iPSCs into M. musculus blastocysts could provide an opportunity to introduce disease resistance into the P. maniculatus genome.

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MECHANISMS THAT REGULATE NLRP3 INFLAMMASOME ACTIVATION DURING

BLOOD-BRAIN BARRIER ENDOTHELIAL CELL HYPERPERMEABILITY

Jasmine Peng, Amira Lambertis, Bobby Robinson, Binu Tharakan Department of Surgery

Texas A&M Health Science Center College of Medicine/Baylor Scott and White Health Temple, TX

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Upon injury, the blood-brain barrier (BBB), which separates brain from intravascular components, is damaged, leading to hyperpermeability and cerebral edema. However, mechanisms that regulate TBI-induced BBB breakdown remain unknown. The NLRP3 inflammasome (NOD-like receptor (NLR) family, pyrin domain–containing protein 3) is a macromolecular complex that releases inflammatory cytokines such as IL-1β, which mediate BBB hyperpermeability. Recent studies suggest that oxidative stress from reactive oxygen species (ROS) formation and cathepsin activation is a key regulator of NLRP3 inflammasomes. Our objective was to investigate the molecular pathways leading to inflammasome activation after TBI. Rat brain microvascular endothelial cells were treated with NLRP3 inflamasome inducers– hydrogen peroxide (H2O2) and Cathepsin-B– or isoliquirtigenin (ILG), an inflammasome inhibitor, in Transwell inserts, chamber slides, and 96-well plates. Results show that Cathepsin-B induced monolayer hyperpermeability (P<0.05) and tight junction disorganization, evidenced by ZO-1 dislocation. H2O2 (100uM) induced a significant increase in ROS formation (P<0.05) that was attenuated with ILG (30uM) treatment (P<0.05). Finally, in silico docking results suggested that ILG favorably interacts with several enzymes upregulated in TBI– Cathepsin-B, MMP-9, Calpain, and Caspase-1– with the highest affinity for MMP-9. In conclusion, NLRP3 inflamamsome activators such as Cathepsin-B and H2O2 induced BBB endothelial cell hyperpermeability by disrupting tight junction proteins. NLRP3 inflammasome inhibition by ILG protected against hyperpermeability and was independent of cell death. Since ILG showed high binding affinity for MMP-9, future research could focus on a NLRP-3-MMP-9 relationship in regulating BBB hyperpermeability. This work was financially supported by Baylor Scott & White Research Division and Jasmine was supported by an American Heart Association Summer Undergraduate Research Fellowship

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EFFECT OF NEUROSTEROID THERAPY ON NEUROINFLAMMATION IN THE MOUSE

PILOCARPINE MODEL OF CHRONIC EPILEPSY

Dheepthi Perumal Ramasamy, Victoria Golub, Bryan Clossen, D. Samba Reddy Department of Neuroscience and Experimental Therapeutics


Epilepsy is a serious neurological disorder marked by spontaneous recurrent seizures (SRS), extensive neurodegeneration, and severe inflammation. Epilepsy can develop from a host of underlying conditions, through a heterogeneous process known as epileptogenesis. Treatments aimed at preventing epilepsy must address the seizure dynamic and the neuronal injury from neurodegeneration. There is no cure for epilepsy, and currently anti-epileptic therapeutics only provide symptomatic relief. Ganaxolone is a synthetic analog of the progesterone-derived endogenous neurosteroid allopregnanolone. Administration of GX has been shown to exert anticonvulsant activity and provide evidence for the modulation of epileptogenesis in animal models such as kindling and pilocarpine-SE. In this study, we chemically induced epilepsy in mice by injecting pilocarpine. Mice were perfused 90 days-post SE, and coronal slices of the brain containing the hippocampus and amygdala were stained for GFAP(+) and IBA1(+) immunohistochemistry. The extent of neuroinflammation was analyzed by area fractionation densitometry from images taken of the hippocampus subfields dentate gyrus, CA1, CA3, and amygdala with an Olympus microscope system. Overall, GX treatment showed a significant reduction in neuroinflammation and damage in epileptic mice in the hippocampus and amygdala; however, there was a region-specific variation in its protection. Mice with chronic epilepsy displayed increased astrocyte and microglia expression in most regions of interest. GX post-treatment altered overall astrocyte and microglia activation, with a significant reduction in GFAP(+) astrocytosis in the hippocampus CA3 and amygdala. These results suggest that post- treatment with GX has the potential to reduce secondary astrocyte activation during epileptogenesis, but does not affect levels of microglia infiltration measured 90 days after SE.

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RNA SECONDARY STRUCTURE VISUALIZATION OF BETACORONAVIRUSES

Vinathi Polamraju, Drew Nunn, Julian Leibowitz

Department of Microbial Pathogenesis and Immunology Texas A&M Health Science Center College of Medicine

College Station, TX

Betacoronaviruses are a subgroup of viruses in the family Coronaviradae known to cause an array of diseases in humans and animals. In this study, we aim to determine the RNA secondary structures of Mouse Hepatitis Virus, strain A59 (MHV-A59), the best studied betacoronavirus, and closely related betacoronaviruses, BCoV and MERS-CoV to identify novel, conserved secondary structures within their genomes. To accomplish this, we infected HRT cell cultures with BCoV. Upon viral clarification and titration, we obtained virus titers between 1.0 and 1.42x107 pfu/mL and purified BCoV via differential and sucrose density gradient centrifugation. Subsequently, we extracted the viral RNA and reacted it with SHAPE-MaP reagent 1-methyl-7-nitroisatoic anhydride (IM7) which probes for and forms adducts with conformationally flexible ribose 2’-hydroxyl groups in the RNA. The derivatized RNA is reverse transcribed in the presence of Mn++ causing misincorporation at adduct sites. This induces mutations in the cDNA transcripts which are incorporated into a cDNA library. Thus, deep sequencing of this cDNA library provided us with an avenue to create relatively accurate RNA secondary structure models using Shannon entropy and pairing probability models. High-confidence regions, characterized by low Shannon entropy and low SHAPE reactivity, were selectivity visualized. The folding model generated by SHAPE-MaP is generally consistent with a previously published model of the BCoV 5’ and 3’ cis-acting regions. Moreover, a potentially noteworthy pseudoknot was revealed ~ 900 – 1550 nucleotides downstream from the 5’ UTR. Further studies are being conducted to complete SHAPE-MaP analysis of MHV-A59 and MERS-CoV and initiate comparative analysis. This work was supported by NIAID grant #R21AI121807�

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MAGNETIC RESONANCE IMAGING (MRI) INVESTIGATION OF VOLUMETRIC

CHANGES AND NEURONAL LESIONS FROM OP-INDUCED EPILEPSY IN RATS

Sandesh Reddy, R. Kuruba, V. Sridhar, A. Glowczwksi, D. Samba Reddy Department of Neuroscience and Experimental Therapeutics


Magnetic resonance imaging (MRI) is an increasingly popular in-vivo imaging technique to investigate anatomic, functional, and neuronal lesions in the brain. However, there has been limited MRI study of epileptogenic progression in rodents, especially in organophosphate (OP) models. The objective of this study is to investigate long-term structural and neuronal lesion abnormalities in rats with OP-induced chronic epilepsy. We hypothesize striking changes in brain regions of interest for epilepsy, particularly in the hippocampus. Epilepsy was induced in adult rats by exposure to the organophosphate GD 90 days before scanning. T2-weighted MR images of ten control and seventeen epileptic rats were acquired using a Siemens 3T scanner (TIPS facility). Each animal produced fifteen coronal and ten sagittal slices of 1.5 mm thickness. Quantification of brain tissue volumes and T2 signal intensity was conducted on Inveon Research Workplace software. Epileptic rats showed drastic hippocampal atrophy, indicating severe damage and neuronal loss, but had similar T2 relaxation times to the control, suggesting comparable tissue composition despite the volume decrease. Conversely, epileptic rats displayed significant increases in lateral ventricle volumes and T2 times, signifying strong cerebrospinal fluid expansion in compensation for tissue atrophy. Total brain volumes, thalamic volumes, and thalamic T2 times were similar in both groups, however, indicating that substantial brain regions remained unaffected long-term during epileptogenesis. Overall, these results provide powerful insight on region-specific neurodegenerative patterns of OP-induced epileptogenesis and demonstrate the feasibility of chronic in-vivo imaging to monitor neuropathology and guide therapeutic response following OP intoxication. Supported by NIH Grant U01-NS083460

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RENAL LYMPHATIC ALTERATIONS IN ANGIOTENSIN II-INDUCED HYPERTENSION

Micaela E. Saathoff, Dakshnapriya Balasubbramanian, Catalina Lopez Gelston, Onoriode P. Ighofose, Brett M. Mitchell Dept. of Medical Physiology


Hypertension (HTN) is a pervasive health concern affecting one in three American adults, and is associated with renal immune cell infiltration and inflammation. De novo renal lymphatic vessel (RLV) formation, exhibited in cases of human renal allografts and kidney disease, is triggered by inflammation. Therefore, RLV formation is considered a compensatory mechanism to drain immune cells and excess interstitial fluid from sites of renal injury. We hypothesized that RLVs would increase in number during angiotensin II-induced HTN, which would be corroborated by gene expression changes of markers for immune cells, kidney injury, and RLV formation. Osmotic pumps containing either saline or angiotensin II were surgically implanted in mice and euthanization occurred after three weeks. Blood pressure readings were obtained weekly via the tail-cuff method and confirmed the development of HTN (SBP: 156 ± 3 vs. 101 ± 2 mmHg; p<0.05 vs. controls). RT-qPCR and immunofluorescence were employed for analysis of gene expression and RLV density, respectively. Angiotensin II treatment for three weeks significantly increased gene expression of the T cell marker Cd4, the Th1 cell marker Tbx21, interstitial fibrosis marker Fn1 (fibronectin), the acute phase protein Cp (ceruloplasmin), the Vegfr3 receptor for the lymphangiogenic ligands VEGF-C and VEGF-D, and the lymphatic endothelial cell marker Pdpn (podoplanin). However, RLV density did not increase, the reason for which is unknown. In summary, angiotensin II caused HTN, renal immune cell infiltration, and kidney injury. The effects of angiotensin II on RLVs will be determined. Micaela was supported by an American Heart Association Summer Undergraduate Research Fellowship

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OBSERVING DRUG RESISTANCE AND HOMOLOGOUS RECOMBINATION IN LIVER

CANCER CELLS

Eric Shi, Robert Tsai Center for Translational Cancer Research

Texas A&M Health Science Center College of Medicine Houston, TX

Cancer is a prevalent disease in society, and researchers have long tried to discover ways to understand and treat it. It is pertinent first to understand mechanisms that lead to the uncontrolled proliferation and drug resistance of cancer cells. Hepatocarcinoma impairs DNA damage response and genome maintenance, so we aimed to see that the use of transfection could show correct HR repair to better understand hepatocellular carcinoma’s chemoresistance and active metastasis. Another objective of this project was to view drug resistance of certain strains of Huh7 cells. The transfection process utilized three different plasmids (CBA, I-Sce1, and DS Green) on both U2OS and Huh7 DR-GFP cells, and each sample was analyzed with FACS flow cytometry. The U2OS served as a standard to show how the I-Sce1 transfected cells showed GFP positive cells. With the Huh7 DR-GFP construct, however, there was nothing positive above the established baseline. To measure drug resistance, different Huh7 cell lines were used in a clonogenic assay with various concentrations of different drugs. Survival rates changed among the different cells and decreased with a greater concentration of the drug. With the data collected, further experimentation testing different transfection conditions can be done to establish a better transfection protocol in order to further expand knowledge about the mechanisms behind the cancer.

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A UBIQUITINATION INHIBITOR, PYR-41, REDUCES IL-5 AND IL-3 STIMULATED

SIGNAL TRANSDUCTION IN HUMAN EOSINOPHILS

Sidney Stephen Sorrell, Sevinj Isgandarova, Jonathan T. Lei, Margarita Martinez-Moczygemba

Center for Infectious and Inflammatory Diseases Institute of Biosciences and Technology

Texas A&M Health Science Center College of Medicine Houston, TX

Eosinophils are multifunctional leukocytes whose effector functions have been implicated in a number of inflammatory, pathogenic disorders such as asthma, allergies, and various hypereosinophilic syndromes. Eosinophil functions are dependent on activation by the c-engaging cytokines, interleukin-5 (IL-5),interleukin-3 (IL-3), and granulocyte-macrophage colony stimulating factor (GM-CSF), as well as their cognate receptors (IL-5R, IL-3R, and GMCSFR), which are comprised of a cytokine specific chain and a c chain. Previous studies in eosinophils have shown that receptor ubiquitination and activation of the JAK/STAT and LYN kinase signal transduction pathways are critical for inducing their inflammatory effector functions. However, to date, very little is known about the potential of using pharmacological inhibitors to block these signaling and regulatory pathways. In this study, we tested the hypothesis that PYR-41, a ubiquitination inhibitor used in cancer research, could block IL-5 and IL-3 stimulated signaling pathways in human eosinophils, and thereby reduce their inflammatory response. To this end, human eosinophils from whole blood leukopaks were sorted using a FACSFusion sorter, treated with or without a non-toxic dose of PYR-41, and analyzed for phospho-STAT5 and phospho-LYN activity by flow cytometry. Our study reveals that pretreatment of human eosinophils with PYR-41 decreased phospho-STAT5 activity more than 5 fold, whereas phospho-Lyn activity was reduced 1.8 fold, indicating that PYR-41 has a higher potency for the JAK/STAT pathway than the Lyn kinase in human eosinophils. Together, our findings demonstrate a novel mechanism for potently blocking key inflammatory signaling pathways in human eosinophils, by repurposing a drug commonly used to kill cancer cells.

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OPTIMIZING PLGA NANOPARTICLES THROUGH NANOPRECIPITATION

Kyndhal Stewart, Bhagath Chirra, Kush Shah, Carolyn Cannon


College Station, TX In the United States, multi-drug resistant bacteria have caused 2 million serious infections and cost our country tens of billions of dollars. Created by the overuse of antibiotics in clinics combined with the shrinking antibiotic pipeline, these pathogens, including Staphlyococcus auerus and Pseudomonas aeruginiosa, are resistant to even the strongest antibiotics. Standard of Care (SoC) antimicrobials are losing their effectiveness, as well as posing other threats to patients, such as disrupting gut microbiota. To combat these issues, our lab has pioneered the development of novel nanodevices which will limit system toxicity, have greater biocompatibility, and will be able to be administered in multiple ways. In our lab we create PLGA-PEG coated PLGA nanoparticles loaded with rhodamine using the nanoprecipitation technique. Nanoprecipitation was chosen for its ability to form particles with uniform characteristics with a size that is easily manipulated. Our goal is to optimize a nanoparticle formulation to yield 100 nm particles, as this size allows them to pass more easily through cell membranes and other biological barriers. The analysis of multiple nanoparticle formulations yielded particles ranging from 700nm to 15 microns in size. We will continue to manipulate the concentrations of PLGA, PLGA-PEG, PVA, and rhodamine until we get the desired results. Once we fine-tune our formula we hope to engineer nanoparticle formulations with enhanced biofilm penetration properties as well as with a maximum attachment to E. coli.

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CELLULAR LOCALIZATION OF INTRANASALLY ADMINISTRATED MSC-DERIVED

A1-EXOSOMES IN THE RAT HIPPOCAMPUS

Alicia Thomas, Olagide Wagner de Castro, Maheedhar Kodali, Raghavendra Upadhya, Bing Shuai, Dong-Ki Kim, Darwin J. Prockop, Ashok K. Shetty

Institute for Regenerative Medicine Department of Molecular and Cellular Medicine

Texas A&M Health Science Center College of Medicine College Station, Texas

Intranasal (IN) administration of mesenchymal stem cell (MSC)-derived, anti-inflammatory A1-exosomes has promise for modulating neuroinflammation and improving cognitive, memory function in conditions such as aging, traumatic brain injury, epilepsy, and Alzheimer’s disease. Since the hippocampus is one of the major brain regions involved in learning, memory, and mood, it is of interest to examine the extent of targeting of IN administered exosomes into the hippocampus. We evaluated the biodistribution of exosomes in hippocampal neurons, microglia and astrocytes of naïve F344 rats, 6 hours after an IN administration of 10 billion, PKH-26 labeled A1-exosomes. Thirty-micrometer thick serial sections through the hippocampus were processed for neuron-specific nuclear antigen (a neuronal marker), IBA-1 (a microglial marker) and glial fibrillary acidic protein (an astrocyte marker) immunofluorescence. Randomly chosen sections were examined and imaged using a confocal microscope. Z-stacks were collected from different hippocampal subfields, and percentages of neurons and microglia containing PKH26+ exosomes were quantified. A large number of exosomes reached the hippocampus and were internalized by neurons, microglia and astrocytes in all hippocampal subfields. Internalization of exosomes was, however greater in CA1 pyramidal neurons of dorsal and ventral hippocampi, and microglia of ventral CA3 region. Exosomes were also seen in a fraction of astrocytes throughout the hippocampus. Thus, IN administered exosomes quickly get incorporated into neurons, microglia and astrocytes in the hippocampus with an increased affinity for CA1 pyramidal neurons and CA3 region microglia. Such distribution in the hippocampus augurs well for improving function following brain injury or disease through IN exosome therapy. This work was supported by a grant from the State of Texas (Emerging Technology Funds to A.K.S.) and the Department of Defense (CDMRP grant to A.K.S.).

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EXOSOMES: A POTENTIAL THERAPEUTIC DELIVERY DEVICE FOR MULTIPLE

SCLEROSIS (MS)

Ashley Tucker, Megha Bijalwan, Colin R. Young, C. Jane Welsh Department of Veterinary Integrative Biosciences

College of Veterinary Medicine and Biomedical Sciences Texas A&M University

College Station, TX Muscle paralysis, impaired coordination, and vision loss are some of the many symptoms of Multiple Sclerosis (MS). With over 2.3 million individuals diagnosed with MS in the United States and with the drawbacks of current medications and other drug delivery devices, there is a critical need for a new treatment for MS. Exosomes are nanovesicles ranging from 40-120nm in size with the ability of passing the blood brain barrier (BBB) while being undetected by the immune system. These nanovesicles can be harvested from available cell lines and be loaded with common MS medications for small but direct treatment of MS. In this pilot study, we investigated whether cerebrovascular endothelial cells (CVE) produce exosomes which could then be used for a drug delivery application. Through the usage of electron microscopy (EM) and antibody to CD81 in immunofluorescence studies, we were successfully able to image exosomes within our CVEs. These exosomes were then harvested through the process of ultracentrifugation, producing a small, but pure yield of exosomes. Through the successful imaging and purification of exosomes from CVEs, we look forward to increasing our yield through future experimentation of current purification techniques and exploration into different designs of tissue flasks, so that we can characterize our exosomes for drug delivery. This work was supported by the WHIN grant.

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PURIFICATION AND STRUCTURE IDENTIFICATION OF MYCOBACTERIUM

AUTOINDUCER MOLECULES

Andrew Wang, Panatda Saenkham, Jeffrey D. Cirillo Department of Microbial Pathogenesis and Immunology Texas A&M Health Science Center College of Medicine

Bryan, TX

Currently, there is little known regarding signaling in Mycobacterium tuberculosis. This process, known as quorum sensing, allows for communication between cells, and is achieved by the diffusion of chemical signaling molecules known as auto inducers. There are approximately 10,000 new cases of tuberculosis (TB) each year in the United States alone, and because quorum sensing is likely to play a role in virulence, understanding the processes behind this communication mechanism can provide insight into how this pathogen causes disease and provide novel strategies for clinical intervention. This study focuses on determining the structure of mycobacterial autoinducers (MAI) using high performance liquid chromatography (HPLC) to purify potential autoinducer molecules and testing their activity using Streptomyces coelicolor-based sensor bioassays. The results from our bioassays suggest that there are two different MAI molecules. Further purification must be done to obtain uncontaminated, purified samples for structure determination. With the correct structures of these MAIs, further investigation can be done to better understand the signaling system involved in Mycobacterium tuberculosis pathogenesis, which can lead to the development of novel therapeutics for tuberculosis.

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BORRELIA BURGDORFERI MARKERLESS DELETIONS UTILIZING AN INDUCIBLE

CRE-LOX RECOMBINASE SYSTEM

Hannah Wilson, Vanessa Ante, Elizabeth Supatra, Jenny A. Hyde Dept. of Microbial Pathogenesis and Immunology


Lyme disease, caused by spirochetal bacterium Borrelia burgdorferi, is the leading tick-borne disease in the United States, resulting in a multi-systemic infection with cardiac, neurologic, and arthritic complications. In 2013, the Centers for Disease Control (CDC) estimated 300,000 cases of Lyme disease per year, indicating this is a significant emerging disease in the United States. B. burgdorferi is a unique pathogen due to its segmented genome comprised of a linear chromosome plus 21 circular and linear plasmids. Genes encoded on the borrelial plasmids are involved in a variety of functions, including metabolism, virulence, and immune invasion, that are important for pathogenesis. The borrelial genome encodes numerous paralogous gene families and open reading frames (ORFs) of redundant function that can impede the characterization of pathogenic mechanisms. Furthermore, elucidating the specific roles of genes in the pathogenic life cycle has been challenging due to the loss of borrelial plasmids during in vitro cultivation, resistance to genetic manipulation, and limited number of genetic tools available for B. burgdorferi. The low transformation efficiency of B. burgdorferi makes antibiotic selection essential for isolating clonal mutants. Currently, only three antibiotic resistant genes are available for laboratory use to alter the genome, thus restricting the number of possible genetic manipulations. We are developing an inducible markerless deletion system in B. burgdorferi utilizing two previously proven techniques, IPTG induction and the Cre-lox recombination system. A gene of interest is deleted by allelic exchange and replace with an antibiotic cassette flanked by loxP sites. The expression of cre is driven by IPTG inducible promoter removes a floxed antibiotic cassette by recombination when treated. This tool will expand the ability to evaluate the pathogenic contribution of multiple borrelial ORFs by allowing the reuse of antibiotic resistant cassettes at independent sites, thus improving the genetic characterization of B. burgdorferi.

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TREATMENT OF CATHEPSIN INHIBITOR IN THE PILOCARPINE-INDUCED MODEL

OF TEMPORAL LOBE EPILEPSY

Javier Zaragoza, Chengaho Deng, Jaclyn Jenkins, Sonia Lopez, Srishti Mathur, Damir Nizamutdinov, Lee Shapiro

Dept. of Neuroscience and Experimental Therapeutics & Dept. of Surgery

Texas A&M Health Science Center College of Medicine Temple, TX

Temporal lobe epilepsy (TLE) is the most common form of epilepsy. Numerous animal models are used to model TLE, including the pilocarpine model. In this model, pilocarpine is administered i.p. after which status epilepticus (SE) develops within about 30-60 mins. After the initial SE, a latent period occurs for ~2-3 weeks, during which epileptogenic processes occur. These include a variety of cellular changes in the hippocampus. In particular, neuron death and astrocytic activation may play a role in the facilitation of spontaneous seizures and the development of epilepsy. Considering that over 30% of male and female TLE patients are pharmacoresistant, new therapeutic strategies are needed. Immunomodulatory therapies are becoming more prevalent for the treatment of a variety of neurological disorders, although such treatments are not yet approved for TLE. To assess the potential role of immune mechanisms in TLE, we examined neurodegeneration and astrocytosis following pilocarpine-induced SE in female CD74 deficient mice, and wild type (WT) male C57/Bl6 mice that received an i.p. injection of cathepsin inhibitor prior to SE. Cathepsin proteases can cleave CD74, and CD74 is a protein involved in several components of the innate and adaptive immune responses. Astrocytosis and neurodegeneration were examined in the hippocampus. Our results show that female CD74def mice and male WT mice treated with Cathepsin inhibitor had altered morphology of the radial glial processes. Furthermore, neurodegeneration was greatest in female CD74def mice.

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2017 Texas A&M Health Science Center

Summer Research Program Participants

Bryan/College Station Katharine Beckman Ave Maria University Dr. Michelle Hook Vaibhav Bommareddy University of Texas at Dallas Dr. Samba Reddy Sammitha Cheruvu Baylor University Dr. Samba Reddy Sarah Elmer Texas A&M University Dr. Samba Reddy Christian Farley Prairie View A&M University Dr. Joe Rutkowski Aaminah Farooq University of Texas at Dallas Dr. Farida Sohrabji Melissa Glass Texas A&M University Dr. Stephen Safe Pauline Ighofoase Prairie View A&M University Dr. Brett Mitchell Akash Kakkilaya University of Texas at Austin Dr. David Threadgill Srujan Kancharla Texas A&M University Dr. Xin Wu Nikhil Kokkapuni University of Texas at Dallas Dr. Carolyn Cannon Amy Kumar University of Texas at Austin Dr. Sanjukta Chakraborty Michael Lee Rice University Dr. Carl Gregory Chi-Hung Lee Baylor University Dr. Koichi Kobayashi Catherine Li Cornell University Dr. Robert Watson Nicholas Lindner Vanderbilt University Dr. Duane Steward Dawson Medler Dallas Baptist University Dr. Kayla Bayless Shea Morris Georgia College and State University Dr. Alistair McGregor Vineela Nagamalla Nova Southeastern University Dr. Samba Reddy Lauren Oliver Oklahoma State University Dr. Ursula Winzer-Serhan Krishna Patel Austin College Dr. David Threadgill Dheepthi Perumal-Ramasamy Texas A&M University Dr. Samba Reddy Vinathi Polamraju Texas A&M University Dr. Julian Leibowitz Sandy Reddy Texas A&M University Dr. Samba Reddy Morgan Riba Texas A&M University Dr. Robert Watson Micaela Saathoff Truman State University Dr. Brett Mitchell Max Sheffield Whitman College Dr. Helene Andrews-Polymenis Kyndal Stewart Colby College Dr. Carolyn Cannon Alicia Thomas Talladega College Dr. Ashok Shetty Ashley Tucker Texas A&M University Dr. Jane Welsh Andrew Wang University of Texas at Austin Dr. Jeff Cirillo Hannah Wilson Texas A&M University Dr. Jenny Hyde Houston Zachary Chroust University of Texas at Dallas Dr. Magnus Hook Eric Shi Rice University Dr. Robert Tsai Stephen Sorrell Davidson University Dr. Margie Moczygemba Temple Christopher Apgar University of Texas at Austin Dr. Keith Young Brittany Bass Texas A&M University Dr. David Dostal Mary-Catherine Clark Texas A&M University Dr. Shannon Glaser Erin Difranco Villanova University Dr. Sharon DeMorrow Srishti Mathur University of Texas at Dallas Dr. Lee Shapiro Jasmine Peng Cornell University Dr. Binu Tharakan Javier Zaragoza University of Mary Hardin-Baylor Dr. Lee Shapiro

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2017 Texas A&M Health Science Center

Summer Research Program Seminar Series

Date Time Topic Presenter

5/26 10:00 AM Roundtable - Record Keeping

5/30 12:00 PM TAMHSC Graduate School Overview Dr. Warren Zimmer

6/2 10:00 AM Roundtable – Fitting in a Laboratory

6/6 12:00 PM CST*R Grand Rounds Dr. Peter Davies

6/9 10:00 AM Roundtable – Literature Review

6/13 12:00 PM Biotechnology & Ethics Dr. Jim Samuel

6/16 10:00 AM Roundtable – Writing an Abstract

6/20 12:00 PM Scientific Method Dr. David McMurray

6/22 12:00 PM TAMHSC MD/PhD Track Dr. Carolyn Cannon

6/23 10:00 AM Scientific Misconduct Dr. Vernon Tesh

6/30 10:00 AM TAMHSC Molecular Pathogenesis & Immunology Track

TAMHSC Cardiovascular Track Dr. Robert Watson

Dr. Cindy Meininger

7/7 10:00 AM Roundtable – Organizing your Poster

7/11 12:00 PM TAMHSC Neuroscience Track TAMHSC Biochemistry Track

Dr. Ursula Winzer-Serhan Dr. Sarah Bondos

7/14 10:00 AM Roundtable – Giving a 10 Minute Talk

7/18 12:00 PM TAMHSC Clinical & Translational Science Track Applying to Medical School

Dr. David Huston Mr. Filomeno Maldonado

7/21 10:00 AM TAMHSC Cell & Molecular Biology Track Roundtable – Applying to Graduate School

Dr. Kayla Bayless

7/24 10:00 AM Student Presentations




7/28 9:00 AM- 3:00 PM Research Day (Poster Presentations, Reception, Keynote Speaker Dr. Gerard Cote, and Awards Ceremony)

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Program Director

Dr. Brett Mitchell Department of Medical Physiology Texas A&M Health Science Center

College of Medicine Rm. 361A Reynolds Medical Building

College Station, TX, 77843-1114 Email: [email protected]

Phone: 979.436.0751

PLEASE KEEP US UPDATED WITH YOUR CONTACT INFORMATION AND CAREER/SCHOOL DECISIONS AFTER GRADUATION

THANK YOU AND THANKS FOR YOUR HARD WORK THIS SUMMER!

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