2016 Abstracts - Basic Sciences - Faculty

BMS 1: Anesthetic Preconditioning in Larval Zebrafish 

Norbert Seidler, Julie Mustard, Matthew Strope, Christopher Havey, and Lucy Wang 

Kansas City University 

Anesthetic preconditioning is a process by which neuronal cells previously exposed to volatile anesthetics exhibit a selective advantage, particularly against future cellular insult. The exact molecular mechanism by which this occurs still remains elusive. The hypothesis is that preconditioning evokes molecular events that result in downstream changes that offer a selective advantage in terms of neuronal function. This current project exposed zebrafish larvae to TFE. Larval zebrafish (3 DPF) were exposed to TFE (0.1mM for 3hr) then tested after 24hrs for select markers of neuronal function (i.e. cognitive, behavioral, and social). The study protocol involved observational endpoints as an indicator of neuronal changes that would evoke a selective advantage. A light-dark test was performed. At 4min, a startle noise was generated at the polar end of the lit area, and the average number of larvae in each section was again determined. Additionally, at 5 DPF, social behavior was tested under standard lighting; we quantitated four types of schooling and shoaling behaviors. We observed that TFE-exposed larvae spent more time in the dark area than control larvae. The larvae also exhibited several differences in social behaviors, including single-direction schooling and shoaling (two larvae: cross shoaling; three larvae: group shoaling). While the long-term effects have yet to be determined, these results shed light on the mechanism of anesthetic preconditioning. These complex zebrafish behaviors normally develop with age and therefore represent, in the TFE-exposed group, a pattern of accelerated maturation of neuronal function, which is attributed to the preconditioning effect of TFE.  

BMS 2: Relationship Between Muscle Attachments and Reduction in Carpal Elements in Selected Anurans 

Marshall Andersen 

Kansas City University 

Early tetrapods and larval forms of some extant anurans typically have twelve carpal elements. Extant adult anurans have three to seven carpal elements. This study examines patterns in the attachment of deep muscles relative to the reduction in the number of adult carpal elements. Examinations and dissections of 331 specimens of 37 species suggest that the third distal carpal element (associated with the fifth metacarpal) present in the archaeobatrachids carries no muscle attachments and has not been preserved as a separate element in the more recently evolved anurans. The remaining two distal carpalia serve both for the attachment of muscles and ligaments. The distal carpal element associated with the third and fourth metacarpals is lost in most of the more recent anurans, and its muscle attachments are transferred to the preaxial centrale, the postaxial centrale or both. The last distal carpal element, associated with the second metacarpus and pollex, is preserved in most anurans and serves for the attachment of muscles associated with the pollex and second digit. These observations tend to support the suggestions of many workers that the reduction of carpal elements in extant anurans is the result of fusion rather than the loss of embryonic anlagen. 

BMS 3: Metamorphosis-A Process of Transformation: Librarians’ Develop Wings and Fly to New Heights 

Lori Fitterling and Marilyn De Geus  

Kansas City University 

Purpose: to demonstrate how the implementation of three initiatives established new pathways for library services. Librarians collaborated to create course syllabi for clerkship students; decreased holdings of printed materials for a transition to a library with more study space; and developed a Medical Informatics course taught by a librarian. Methods: The library partnered with the Clinical Education Department to create nine new course syllabi for MSIII/MSIV clinical programs. Statistics and surveys were collected. In 2014, librarians decreased holdings by critically evaluating titles for retention/withdrawal in a transition to a more virtual collection. Additional study space was created to accommodate increased student enrollment and students were observed as to the use of this newly created space. A new one credit course was added to MSI curriculum with a librarian as course instructor and a focus on medical informatics, information literacy, and student grand rounds case presentations. Students were evaluated and surveys were used to measure course effectiveness. Results: Through observation, surveys, and usage statistics we have been able to measure the effectiveness of these new pathways of service. We recorded sharp increases in E-book usage and virtual services. There was positive feedback with the newly created space in the library, and we are now into the second year of Medical Informatics in the MSI curriculum and are monitoring outcomes. Conclusions: The three programs, when implemented, have allowed librarians to grow new skills, develop working relationships with other departments, and work beyond the walls of the library using virtual services. 

BMS 4: Inhibition of TGF-B/Smad3 Pathway Protects the Function of Doxorubicin Treated Endothelial Cells 

Jill Schriewer, Nicole Cabalo, and Eugene Konorev  

Kansas City University 

Cardiovascular disease is the leading cause of morbidity and mortality in long-term cancer survivors. Doxorubicin (Dox) and other chemotherapeutic drugs cause permanent cardiac damage. We previously detected decreased capillary density and gene expression changes that were indicative of the increased activity of the TGF-β pathway in hearts from Dox treated mice. To further elucidate the effects of Dox on cardiac tissues, we turned to in vitro models. Treatment with either Dox or TGF-β2 suppressed proliferation, migration and vascular network formation by human endothelial cells in vitro as measured using primary human cardiac cells in mono-culture and co-culture. Cells were treated, stained and visualized by automated microscopy. We also measured markers of proliferation in cultured cells by isolating RNA, generating cDNA then measuring the relative abundance of mRNA for a group of cyclin dependent kinase inhibitors known to inhibit proliferation. Treatment with Dox or TGF-β2 increased the expression of these inhibitors, reducing proliferation. SB431542, a small molecule inhibitor of the TGF-β pathway, reversed the suppression. SB431542 blocks the phosphorylation and nuclear translocation of Smad3, an intermediate transcription factor in the TGF-β pathway. We used shRNA introduced with a viral construct to knock down the expression of Smad3 in human endothelial cells. The results paralleled the effects of treatment with SB431542, restored function of endothelial cells. Collectively, this data suggests a role for TGF-β2/Smad3 in endothelial cell function. If the TGF-β2/Smad3 pathway can be targeted to restore endothelial cell function, we may be able to more effectively treat patients with cardiac damage. 

BMS 5: Information Literacy and Grand Rounds: Adding Information Skills and Case Presentation Experience to the First Year Curriculum 

Lori Fitterling and Cheryl McCormick 

Kansas City University 

With an increasing emphasis on health information literacy and guided by a new administration, a Medical Informatics and Information Literacy course for MSI was created with an active learning focus on medical informatics skills and student-lead grand rounds case presentations. A one credit, pass/fail course was adopted into MSI curriculum in the fall of 2014, with a librarian as course instructor and a two-part focus: (1) to instruct students how to search, access, evaluate, and utilize digital medical information, and (2) to facilitate student-led grand rounds case presentations. Four lectures were given in the first 4 weeks and case presentations began 6 weeks into the semester with 2 weeks for case preparation. Students were tested on their knowledge of identifying point-of-care databases, recognizing quality of evidence, valid research studies, and evaluating accuracy of medical literature. Presentations were evaluated by faculty. A post-survey was given to assess student attitudes about information retrieval and literacy. After working through scheduling problems and fine tuning procedures for grand rounds, the acceptance of the Medical Informatics and Information Literacy course by students and faculty has been positive. Collaboration between the library, the Dean/Provost, Academic Affairs and faculty enabled an innovative course model that transformed the library’s role from a custodian of information to an agent of knowledge production and a facilitator of change. The challenge going forward is to continue to design pathways within course curriculum that encourage students to embrace the importance of information literacy in medical education. 

BMS 6: Attenuation of Dilated Cardiomyopathy in DMD Mice Expressing Retinal Dystrophin in Muscle 

Robert White, Amber Wiggins-McDaniel, and Eugenie Hong 

Kansas City University 

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by lack of muscle protein dystrophin. Almost all patients with DMD develop dilated cardiomyopathy. Our lab generated a transgene to allow expression of retinal dystrophin isoform (Dp260) in skeletal muscle where it is not normally expressed. DMD mice with the Dp260 transgene (Tg+) have attenuated skeletal muscle disease. We hypothesize that expression of Dp260 in cardiac muscle will attenuate the dilated cardiomyopathy in DMD mice. Dp260 expression in cardiac muscle was detected by western protein blotting which showed that Dp260 is expressed in hearts DMD, Tg+ mice while retinal dystrophin expression is absent in the hearts of DMD mice. Histopathology of trichrome stain of whole heart cross-sections was completed. Histopathology of DMD mice shows thinning of the left ventricular wall with extensive fibrosis typical of dilated cardiomyopathy, while DMD, Tg+ hearts showed normal phenotype. Functional EKG data was collected showed significant decrease in heart rate, left ventricular mass, and fractional shortening in DMD mice versus DMD, Tg+ mice, which show a similar presentation as control normal mice. Impact on lifespan of DMD, Tg+ versus DMD mice was calculated. The impact on lifespan is that DMD, Tg+ mice survive a normal lifespan as compared to control mice, whereas DMD mice are deceased by 5 months of age. Dilated cardiomyopathy can be vastly improved by expression of Dp260 in DMD cardiac muscle. A novel therapy involving the expression of Dp260 in skeletal and cardiac muscle would be beneficial to DMD patients.  

2016 Abstracts - Basic Sciences - Students

BMS 7: Duchenne Muscular Dystrophy Gene Therapy: Impact on Cardiomyopathy 

Jourdan Harkless, Amber Wiggins-McDaniel, Beau Wakefield, Stephanie Myer, and Robert White  

Kansas City University 

Duchenne Muscular Dystrophy (DMD) is a common neuromuscular disease characterized by progressive muscle degeneration and caused by mutations resulting in the absence of dystrophin. A 260 kDa human retinal dystrophin isoform (Dp260) was discovered which plays a role similar to the mechanical support mechanism for muscle dystrophin. A human Dp260 transgene (Tg+) has been produced and tested in DMD mice. Muscle-specific expression of Dp260 in transgenic DMD-Tg+ animals has striking palliative effects on spinal curvature, pathology, electrophysiology, and lifespan. Cardiomyopathy is present in over 90% of DMD patients and is responsible for 40% of all deaths. DMD mice also exhibit cardiomyopathy. Structural and functional analyses were carried out of the effect of expressing the Dp260 transgene in the hearts of rescued mice. Expression of human Dp260 from the Dp260 transgene in cardiac muscle was determined using western blotting. Histopathology analyses were used to determine its effects on pathology. The status of necrosis and pathology in DMD, Tg+ mice versus DMD mice was examined by light microscopy. Analysis of Dp260 expression on heart function was conducted on DMD-Tg+, DMD, and control mice. There were measurements of the myocardium size, cardiac output, ejection fraction, and heart function during systole and diastole. The effect of Dp260 expression in DMD mice has been examined and positive effects on spinal curvature, pathology, electrophysiology, and lifespan are observed. 

This project was funded by the KCU Summer Fellowship Research Grant Program.  

BMS 8: Biomarker Development for Neurodegenerative Diseases: Expression, Isolation, and Characterization of TDP-43 

Garrett Gilbert, Jessica Sage, Rishi Sharma, Viren Rana, LaSharice Hall, and Abdulbaki Agbas  

Kansas City University 

The major obstacle to progress in the field of human neurodegenerative diseases (NDD) is molecular assessment of the longitudinal progress of proteinopathy in living AD patients long before appearance of the clinical manifestations. A development of blood-based biomarker is urgently needed. The status of platelet surrogate markers is a new way of looking at the mechanisms of proteinopathy in the AD brain, and may help in a better understanding of the etiology of NDDs such as Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, etc. Recently re-discovered TDP-43 protein modification is common in many neurodegenerative diseases. Current quantitative assay methods are limited to measuring total TDP-43 but not hyperphosphorylated TDP-43 due to the limitation of the assay components such as site-specific anti-phosphorylated TDP-43 Ab and phosphorylated TDP-43 protein for establishing the calibration curve for the quantitation. We are well positioned to address this issue since we have been working on obtaining recombinant TDP-43 from bacterial expression system and we have identified the sources for anti-phosphorylated TDP-43 antibodies. The overall strategies of this study are (i) to optimize the expression conditions for obtaining a stable recombinant TDP-43 protein (ii) to isolate and characterize the expressed TDP-43. This approach can be used to quantitate both total TDP-43 and site-specific phosphorylated TDP-43 protein in AD patients’ platelets, and longitudinally monitoring the disease progress.  

This project was funded by the KCU Summer Fellowship Research Grant Program. 

BMS 9: Ketogenic Diet Influences Hepatic Bioenergetic Pathways in Association with Altered Expression of Sirtuins, Protein Acetylation and Succinylation 

Lewis Hutfles1, Heather Wilkins2, Scott Koppel2, Ian Weidling2, Eva Selfridge2, and Russell Swerdlow2  

1Kansas City University, 2University of Kansas Medical Center 

Caloric restriction is a dietary strategy that has been shown to extend life span. However, the mechanisms underlying this benefit are not well understood. Ketogenic diets mimic certain aspects of caloric restriction by reducing carbohydrate intake, insulin levels and inducing ketogenesis. In this study, we apply a ketogenic and standard diet to C57BI/6J male mice for one month to evaluate the effects of ketosis at the molecular level of hepatocytes. Notably, we investigated the effects of a ketogenic diet on sirtuins (Sirt), a class of NAD+ dependent deacetylases that have been implicated as major role players in the regulation of bioenergetics and metabolism via post-translational modifications. Mice fed a ketogenic diet exhibited a state of hyperacetylation and differential succinylation at the whole cell and mitochondrial levels when compared to a standard diet. Reductions in Sirt3, the major mitochondrial deacetylase, were observed at the protein level in ketogenic diets. Sirt5, recognized to possess desuccinylase activity, remained unchanged. We also identified distinct changes in key proteins involved in gluconeogenesis, the electron transport chain, and mitochondrial biogenesis pathways. Overall, these data suggest that ketogenic diets influence bioenergetic pathways in association with altered expression of sirtuins and their respective post-translational modifications. This study informs upon molecular pathways associated with ketone biology that may provide new avenues for interventions that seek to enhance healthy aging and lifespan.  

BMS 10: Loss of Neuronal Calcium Pump Function in Models of Parkinson’s Disease 

Shaneisha Williams1, Paul Ramlow1, John Stanford2, Asma Zaidi1  

1Kansas City University, 2University of Kansas Medical Center 

The precise mechanism underlying the selective death of dopaminergic (DA) neurons in Parkinson’s disease (PD) remains unclear. Strong evidence suggests that mitochondrial dysfunction and consequent increase in reactive oxygen species (ROS) play a key role. ROS can damage key proteins causing cellular dysfunction. The plasma membrane calcium-ATPase (PMCA) plays an important role in cell survival by pumping out calcium against a 106-fold gradient across the plasma membrane to maintain calcium homeostasis. Our previous studies have shown that PMCA is a target of ROS. The current study was conducted to determine the effects of the PD mimetic 6-hydroxydopamine (6-OHDA) on PMCA. We hypothesize that 6- OHDA will have an inhibitory effect on PMCA activity. SH-SY5Y cells were exposed to increasing doses of 6-OHDA. Sprague Dawley rats were anesthetized and injected with 9 µg of 6-OHDA delivered into the right median forebrain bundle. PMCA activity was performed in cell lysate and neuronal-rich P2 brain fractions using Malachite Green Method and protein levels determined by immunoblotting. Exposure to 6-OHDA caused a dose-dependent inhibition of PMCA activity and proteolytic breakdown in cells (n = 6). In vivo injections caused a significant decline in Vmax of PMCA activity in ipsilateral side compared to untreated contralateral side (n = 6) with no change in Kact. Immunoblots showed no change in protein levels suggesting that under in vivo conditions the OHDA influences PMCA function without altering its protein levels. Reduction in PMCA activity is likely to increase calcium overload and cause toxicity and cell death.  

BMS 11: Identification of Human Retinal Dystrophin Promoter for Treatment of DMD Patients 

Megan Woods, Amber Wiggins-McDaniel, and Robert White  

Kansas City University 

Duchenne Muscular Dystrophy (DMD) is a common neuromuscular disease for which there is no cure. DMD is caused by mutations in the gene for dystrophin protein (427 kDa). Expression of a dystrophin isoform, retinal dystrophin (260 kDa; Dp260) in muscle may represent a therapy for treatment of DMD patients by activation of the Dp260 promoter in diseased muscle. The purpose of this study is to identify the minimal DNA sequence that constitutes the human Dp260 promoter. Nested deletion clones of the putative human Dp260 promoter inserted into a reporter gene vector (luciferase plasmid), with the ability to luminesce, will be used to determine the presence of promoter activity in each of the nested deletion plasmids. Generation of these reporter plasmids requires genomic PCR, restriction enzyme digestion, directional cloning into pLuc4.17, transformation of bacteria, plasmid DNA miniprep and DNA sequencing of the insert. The series of clones will be transfected into Y79 human retinoblastoma cells for testing promoter activity. The goal of this portion of the study is to generate 8 nested clones to be used for identification of the Dp260 promoter. Two clones have been successfully prepared. After identification of the minimal DNA of the Dp260 promoter, a final clone with this region will be stably transfected into a human kidney cell line which will be used for high-throughput drug screening followed by testing drugs in our severe DMD mouse model. Our long range goal is to bring this treatment from the lab bench to the bedside of DMD patients.  

BMS 12: Identification of the Mouse Retinal Dystrophin Promoter 

Beau Wakefield, Amber Wiggins-McDaniel, and Robert White  

Kansas City University 

Duchenne Muscular Dystrophy (DMD) is a common neuromuscular disorder. It is caused by mutations in the dystrophin gene that encodes dystrophin protein (427 kD). Our lab has discovered a retinal dystrophin isoform (Dp260; 260 kD) which is expressed in various tissues. Expression of human Dp260 in DMD model mice changes their lethal, severe muscle disease into a mild, viable myopathy. Activation of the Dp260 promoter can possibly treat DMD. We have created 9 nested deletions containing the putative DNA sequence of the mouse Dp260 promoter. These were created by genomic PCR and insertion into a luciferase gene reporter plasmid. Analysis of clones included restriction enzyme digests, DNA plasmid minipreps, and sequencing to assure perfect identity of insert sequence with the putative mouse promoter. Luciferase plasmids will be transfected into human retinoblastoma Y79 cells followed by luciferase assays to determine the activity of each insert. This will determine the minimal DNA region of the mouse Dp260 promoter. All 9 of the nested deletion clones have been generated. A final clone will be generated and its activity tested in the luciferase assay. The long range goal is to transfect the plasmid into a cell line not expressing Dp260 for high-throughput screening of drugs and compounds which may induce the activity of the Dp260 promoter and expression of retinal dystrophin in muscle tissue as a potential treatment for patients with DMD. The next experiment will be to treat our DMD model mice with drugs to determine the efficacy of this strategy. 

BMS 13: PGE2 Receptor /EP3 Receptor Inhibits Water Reabsorption and Contributes to Polyuria and Kidney Injury in a Streptozotocin-induced Mouse Model of Diabetes 

Ramzi Hassouneh1, Rania Nasrallah2, Joe Zimpelmann2, Alex Gutsol2, David Eckert2, Jamie Ghossein2, Kevin Burns2, and Richard Hebert2 

1Kansas City University, 2University of Ottawa 

Hypothesis: The first clinical manifestation of diabetes is polyuria. The PGE2 receptor EP3 antagonizes arginine vasopressin (AVP)-mediated water reabsorption and its expression is increased in the diabetic kidney. The purpose of this work was to study the contribution of EP3 to diabetic polyuria and renal injury. Methods: Male EP3-/- mice were treated with streptozotocin (STZ) to generate a mouse model of diabetes. Renal function was evaluated after 12 weeks. Isolated collecting ducts (CDs) were microperfused to study the contribution of EP3 to AVP-mediated fluid reabsorption. Results: EP3-/-STZ mice exhibited attenuated polyuria and increased urine osmolality compared with wild-type STZ (WT-STZ) mice, suggesting enhanced water reabsorption. Compared with WT-STZ mice, EP3-/-STZ mice had increased protein expression of aquaporin-1, aquaporin-2, and urea transporter-A1, and reduced urinary AVP excretion, but increased medullary V2 receptors. In vitro microperfusion studies indicated that EP3-/- and WT-STZ CDs responded to AVP stimulation similarly to those of wild-type mice, with a 60% increase in fluid reabsorption. In wild-type and WT-STZ mice, EP3 activation with sulprostone (PGE2 analogue) abrogated AVP-mediated water reabsorption; this effect was absent in mice lacking EP3. A major finding of this work is that EP3-/-STZ mice showed blunted renal cyclooxygenase-2 protein expression, reduced renal hypertrophy, reduced hyperfiltration, reduced albuminuria, and diminished tubular dilatation and nuclear cysts. Conclusions: The data suggests that EP3 contributes to diabetic polyuria by inhibiting expression of aquaporins, and that it promotes renal injury during diabetes. EP3 may prove to be a promising target for more selective management of diabetic kidney disease.  

BMS 14: Effect of Smad3 Deficiency on Human Endothelial Cells 

Nicole Cabalo, Jill Schriewer, and Eugene Konorev  

Kansas City University 

Introduction: Increased cardiac levels of transforming growth factor-beta (TGF-beta) superfamily ligands have been detected both in patients and animal models of heart failure, and their levels are positively correlated with the severity of the condition. Our in vitro studies using human cardiac endothelial cells have shown that a TGF-beta pathway inhibitor increases endothelial cell proliferation and vascular network formation. Hypothesis: We hypothesize that TGF-beta suppresses the formation of vascular networks by increasing Smad3 activity in endothelial cells. Therefore, Smad3 deficiency in endothelial cells will then enhance their angiogenic potential. Methods: We created a stable human umbilical vein endothelial cell (HUVEC) line expressing Smad3 short hairpin ribonucleic acid (shRNA). Results: We showed that TGF-beta2 reduced proliferation in control but not in Smad3 deficient HUVEC. Similarly, we observed increased proliferation of the Smad3 knockdown cell line after one day in co- culture with cardiac fibroblasts. Gene expression analysis revealed that TGF-beta2 increased the abundance of messenger RNAs (mRNAs) for cyclin-dependent kinase inhibitors CDKN1A and CDKN1B (p21 and p27 proteins, respectively) in control but not in Smad3 deficient endothelial cells. Additionally, migration on fibronectin was substantially enhanced in the HUVEC line with Smad3 knockdown. Conclusions: Thus, Smad3 deficient endothelial cells were resistant to the TGF-beta2-induced suppression of proliferation. These cells exhibited enhanced migration and decreased expression of cyclin-dependent kinase inhibitors. We conclude that Smad3 deficiency will likely increase the angiogenic potential of endothelial cells. This hypothesis is further tested in vascular sprouting experiments on aortic explants from Smad3 knockout mice. 

This project was funded by the KCU Summer Fellowship Research Grant Program.  

BMS 15: Death of Dopaminergic Neurons in the Substantia Nigra in Parkinson’s Disease - Is Calcium the Silent Killer? 

Sidrah Sheikh, Paul Ramlow, and Asma Zaidi  

Kansas City University 

Parkinson’s disease (PD) is associated with the loss of dopaminergic (DA) neurons in the subtstantia nigra (SN). The mechanism underlying selective death of DA neurons remains unclear. Spontaneous firing of DA neurons brings in extracellular calcium via calcium channels. Calcium transporters located in the plasma membrane [the plasma membrane Ca2+-ATPase (PMCA) and the sodium-calcium exchanger (NCX)] counteract this by extruding Ca2+ against the 106 - fold gradient, critical for neuronal survival. We hypothesize that PMCA and NCX are endogenously low in SN compared to other brain regions and that they are further reduced in PD. To test our hypothesis, we used post-mortem human brain samples from PD patients and controls. PMCA activity was assessed by measuring Pi released upon calcium-dependent ATP hydrolysis. PMCA protein levels were measured by immunoblotting. Our results show region- specific differences in both proteins with significantly lower PMCA activity (50%, p<0.002) and protein levels (20%, p <0.02) in SN compared to frontal cortex (FC) and cerebellum (CBM) in controls (n = 8). A significant reduction was observed in PD (15% less PMCA, p<0.02 and 36% less NCX, p<0.05) compared to age-matched controls (n = 8). Low levels of endogenous Ca2+ transporters coupled with hyperactive Ca2+ influx is likely to predispose DA neurons to high risk 2+ for Ca2+- overload. Reduction in these key proteins in PD may tilt the threshold to Ca - induced toxicity and cell death. Our studies are the first to show that defects in Ca2+ transporters may be causal in DA cell death in PD.  

BMS 16: scaRNAs Role in Congenital Heart Disease 

Mindy Ward1 and Douglas Bittel2 

1Kansas City University, 2Childrens Mercy Hospital Kansas City 

Purpose: We analyzed alternations in the processing of messenger RNA in heart tissue from babies with congenital heart defects, CHDs. We are characterizing the role played by scaRNAs that participate in maturation of components of the spliceosome. We believe scaRNAs are playing a role in regulating splicing. We hypothesize that scaRNAs directly regulate spliceosomes by controlling splicesomal RNA maturation. Methods/Material: We’re using in vitro and in vivo model systems, this vector will be used to transfect developing quail embryos. My role was to test the first vector that incorporated an antisense shRNA targeting the scaRNA ACA35, it’s activated by a heart muscle specific promoter. The promoter function contained GFP that can be tested by light microscopy. The first phase was to test the function of the vector. We transfected the vector into the quail cells and used light microscopy tip determine if green cells were present. Results: We examined transfection experiments using multiple different concentrations of transfection reagent and vector quantity. We clearly were able to see GFP labeled cells, our best efficiency was approximately 10%. This demonstrates that the vector is intact and the promoter functions as expected. Conclusions: This is the first step in clarifying the role that scaRNAs play. They are important for assessing how scaRNAs may contribute to developmental integrity and congenital heart defects. This is a novel concept that may be important for understanding of epigenetic processes that contribute to developmental regulation which may help in understanding the complexity of these processes. 

This project was funded by the KCU Summer Fellowship Research Grant Program.  

BMS 17: Downstream Effects of Microglial Activation on the Plasma Membrane Calcium Pump 

Shruti Sharma, Shivani Patel, Paul Ramlow, and Asma Zaidi  

Kansas City University 

Microglial cells are the major immune cells of the CNS. Stimulation of microglia produces reactive oxygen species (ROS) which can damage critical proteins and influence cellular function. The plasma membrane calcium-ATPase (PMCA) is an ATP-dependent calcium pump that is critical for cell survival. The neuronal PMCA is a sensitive target of ROS in brain aging and in Parkinson’s Disease (Zaidi, 2015). The present study was designed to determine the effects of lipopolysaccharide (LPS) on microglial PMCA. We hypothesize that the microglial activating agent LPS will inhibit PMCA activity. Cultured BV-2 cells were exposed to LPS (1 pg/ml – 1 ug/ml). PMCA activity was assessed by measuring Pi released upon calcium- dependent ATP hydrolysis. PMCA protein levels were measured by immunoblotting. LPS has a biphasic effect on PMCA activity with a ~40% increase observed at 1 - 10 pg/ml and a dose- dependent decrease at higher concentrations (n=3). Immunoblots show ~40% increase in PMCA protein levels (n = 5). It is likely that LPS-mediated ROS induce structural changes in PMCA, which at low doses cause dissociation of the autoinhibitory domain accounting for increased activity. At higher concentrations, ROS cause oxidative damage and inactivation of PMCA which may trigger an upregulation of protein via increased transcription/translation. Current studies are addressing the mechanisms underlying altered activity and protein levels of PMCA. Loss of PMCA function is likely to cause Ca2+ overload and cell death thus exacerbating the neurotoxic milieu in the CNS. Our findings are pertinent to the pathogenesis of diseases such as Alzheimer’s and Parkinson’s.  

BMS 18: Beneficial Effects of Maternal Exercise on Offspring Cardiac Tissue Mitochondrial Efficiency 

Tara Notarianni1, Megan Liberty1, Daniel Barrera1, Ki Suk Eum1, Robert Liu1, Sean Newcomer2, Linda May3, Jessica Sage1, and Abdulkbaki Agbas1 

1Kansas City University , 2California State University San Marcos, 3East Carolina University 

Regular physical training improves cardiac autonomic tone. Besides changes in autonomic control, there are compensatory adaptations that occur within the tissue at the biochemical level. It is known that exercise influences cardiac myocyte biochemistry that enables mitochondria to work more efficiently. Exercising during pregnancy is thought to be beneficial to the cardiovascular health of both mother and fetus. Here we test the hypothesis that regular maternal exercise during the pregnancy increases the mitochondrial efficiency of offspring heart tissue as evidenced by mitochondrial electron transport chain (ETC) enzyme concentration and kinetics. We have used the pregnant Yorkshire pig as an animal model. ETC enzyme protein levels as well as the select ETC enzyme kinetics were analyzed in 2 days, 3, 6 and 9 month old offspring cardiac tissues. Standard immunoblotting analysis employed with the battery of antibodies specific to ETC enzyme proteins. Our results showed less amount of Complex-I and Complex-IV proteins in exercised group whereas other members of ETC did not change. Further, the enzyme kinetics of Complex-I and Complex-IV were analyzed and Vmax values were calculated. In the exercised group, Vmax values of Complex-IV were low as compared to those of the sedentary group, and complex-I Vmax values were slightly higher than those of the sedentary group. Our data may suggest that exercise-induced oxygen utilization alters Complex-IV, and less active Complex-IV will generate less free superoxide radicals which may have a positive impact on the long-term cardiac health.  

BMS 19: Novel Gene Expression Changes Dependent on 2 Mitochondrial Enzymes in a Rescued Model of Parkinson’s Disease 

Kelley Perry, William Kogel, and Christopher Theisen  

Kansas City University 

The pathogenesis of Parkinson’s disease (PD) involves insufficient formation of dopamine as a result of dopaminergic cell death in the midbrain. The cause of demise of these cells is poorly understood, but mitochondrial dysfunction has been implicated as a central pathway in the neurodegenerative process. We have observed cellular protection in a model of PD by increasing the levels of 2 important mitochondrial enzymes, NAMPT and SIRT3. Our current model of Parkinson’s disease employs the use of immortalized dopamine progenitor neurons and zebrafish larvae. In this study, we examined the effects of overexpressing Nampt in dopaminergic neurons as well as neurons stably expressing lentiviral mediated shRNA against Nampt and the essential mitochondrial sirtuin gene, Sirt3. After establishing stable populations of dopaminergic neurons with either increased or decreased levels of NAMPT or SIRT3 we have identified various genes that may be involved in the pathways of neuroprotection in dopaminergic neurons susceptible to cell death in the progression of PD. We compared the expression levels of major genes involved in neurotoxicity. A sub cohort of genes has been identified as over expressers when NAMPT and SIRT3 have reduced expression. There are 15 genes that have significant changes in gene expression consistent with our hypothesis. 7 genes have dramatic changes reciprocal to NAMPT over vs under expression. Our results indicate that NAMPT and SIRT3 alter similar pathways. These mitochondrial enzymes that have shown to prevent neuronal dysfunction are also being examined in a zebrafish model of PD. 

This project was funded by the KCU Summer Fellowship Research Grant Program. 

BMS 20: Characterization of Risk Factors Associated with Increased Prevalence of Nasal Colonization of Methicillin Resistant Staphylococcus Aureus in Homeless Populations in Kansas City, MO 

Megan Ottomeyer1, Charles Graham2, Chad Law1, Mariam Molani1, Karine Matevossian1, Jerry Marlin1, Charlott Williams3, Ramon Newman1, Jason Wasserman4, and Tracey Taylor4 

1Kansas City University, 2University of Nevada School of Medicine, 3Children’s Mercy Hospital Kansas City, 4Oakland University William Beaumont School of Medicine 

Nasal colonization of methicillin-resistant Staphylococcus aureus (MRSA) plays an important role in the epidemiology and pathogenesis of disease. Situations of close-quarter contact in groups are generally regarded as a risk factor for community acquired MRSA strains due to transmission via fomites and person to person contact. With these criteria for risk, a group of homeless individuals using shelter facilities should be considered high risk for colonization and infection. The aim of this study was to determine the prevalence of nasal colonization of MRSA in a homeless population compared to established rates of colonization within the public and a control group, and to analyze phylogenetic diversity among the MRSA strains. Nasal samples were taken from the study population of 332 adult participants, and analyzed. In addition, participants were surveyed about various lifestyle factors in order to elucidate potential patterns of behavior associated with MRSA colonization. These data increase understanding of key differences in MRSA characteristics within homeless populations as well as risks for MRSA associated with being homeless, which may then be a useful tool in guiding more effective prevention, treatment, and healthcare for homeless patients.  

BMS 21: Novel Treatment of Iron Overloading in Hereditary Hemochromatosis 

Whitney Kelly, Amber Wiggins-McDaniel, and Robert White  

Kansas City University 

Hereditary hemochromatosis (HH) is a recessive disease caused by a defect (C282Y) in the HFE (Hereditary iron) gene resulting in uncontrolled intestinal iron absorption. As iron levels increase, storage iron accumulates in liver and heart becoming toxic. Without intervention, this leads to organ failure and death. The treatment for HH patients is frequent phlebotomy or using iron chelating drugs that cause serious side effects including nephrotic syndrome and death. A mutation in mouse gene Ttc7 causes iron depletion in flaky skin (fsn) mutants. The cardinal feature of these mice is that they excrete 100X more urinary iron. The fsn mutation represents a pharmaceutical target for treating iron overload. In this proof of concept study, homozygous fsn mice were mated with Hfe mouse models (Hfe C282Y/C282Y and Hfe -/- knockout mice) to determine if the Ttc7 mutation could result in loss of iron overload in double mutant HH mice. The Hfe C282Y/C282Y mice result from insertion of the C282Y mutation found in humans, whereas Hfe -/- mice are knockout mutants with no HFE protein. Initially, mice of various types were examined for tissue iron overload (normal controls, homozygous fsn mice, Hfe C282Y/C282Y, Hfe -/-). Livers were collected to measure the amount of dry weight tissue iron. It was concluded that Hfe C282Y/C282Y mice were not the optimum model of HH to test in this proof-of-concept study as data demonstrate the Hfe -/- mice exhibited higher tissue iron overload. Therefore, Hfe -/- mice represents a more appropriate clinical model to test. 

This project was funded by the KCU Summer Fellowship Research Grant Program.  

BMS 22: Toward the Development of Novel DNA Methyltransferase 1 Inhibitors 

James Cox1, Nathan Duncan2, Sydney Stoops2, and Jonathan White2  

1Kansas City University, 2MRIGlobal 

Childhood cancer is a devastating disease that affects more than 175,000 people each year. Of pediatric brain tumors, medulloblastoma is the most common malignant primary brain tumor. Current treatment strategies for medulloblastoma include surgery, radiation and chemotherapy. While treatments, particularly combination treatments, have shown success, the toxic effects often lead to downstream developmental problems including delays in physical and cognitive development and higher rates of cardiac disease. In order to better understand this disease and attempt to development more advanced treatment options, our lab is currently investigating the role of DNA methyltransferase (DNMT), specifically DNMT1, in medulloblastoma. DNMT is a family of enzymes responsible for catalyzing the methylation of DNA, which is an important epigenetic regulatory function in cells. It has been reported that hypermethylation via DNMT is an important control factor in medulloblastoma and cancer progression; and therefore, inhibition of DNMT may serve as a potential therapeutic pathway. To study this process, our lab is using molecular modeling (SYBYL-X) to generate and screen non-nucleoside based small molecule libraries for theoretical affinity in the binding pocket of DNMT1 active site. Promising compounds were synthesized and analyzed in an in vitro study which utilized mass spectrometry to quantify DNA methylation by DNMT1. Compounds that inhibit DNMT1, and the subsequent methylation of DNA, will be used as probes to further study the role of epigenetic modulation within the DAOY medulloblastoma cell line as it relates to tumorigenesis, the results of which will be reported.  

BMS 23: Study of microRNAs Role in Medulloblastoma 

Alexander Vavra1, Sydney Stoops2, and Jonathan White2  

1Kansas City University, 2MRIGlobal 

Cancer is often thought as a disease associated with aging, as people age 50 or older account account for 86% of all cancers diagnosed in the U.S. However, every year roughly 14,000 children are diagnosed with cancer in the U.S. alone. Of these 14,000 children roughly 20% will be diagnosed with a brain tumor, of which medulloblastoma is the most common malignant primary brain tumor. The current treatment plan consists of surgery, radiation, and chemotherapy. Despite, the effectiveness of this combination treatment, issues still exist in the form of reoccurrence and severe side effects that hinder physical and cognitive development. In order to better understand this disease and attempt to develop more advanced treatment options, our lab is currently investigating the role of microRNA (miRNA) in medulloblastoma. miRNAs are small noncoding RNA molecules which function as a regulator of post-transcriptional gene expression and RNA silencing. Aberrant miRNA expression has been documented in several cancers, including medulloblastoma. As such, further investigations into treatment strategies are warranted. We are investigating the role of up-regulated miRNAs in modulating DAOY medulloblastoma cells by means of RT-PCR. We have chosen 18 miRNA, known to be associated with medulloblastoma, and determined their expression level as compared to a miRNA Brain Reference Standard. Based on our findings, we will focus our studies toward identifying the putative protein target(s) of selected upregulated miRNAs, which will be investigated with knockdown studies. The collected data will assist in potentially identifying novel targets for improved therapeutic intervention.