Abstracts - Basic Medical Sciences

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    BMS1 | Developing a Biomarker for Early Amyotrophic Lateral Sclerosis DetectioN
    BMS2 | Amyotrophic Lateral Sclerosis Transgenic Mice with Altered Calcineurin and SOD1 Activities Showed Increased Zinc Activity Along Their Spinal Cord Segments
    BMS3 | Interaction of TDP-43 and SOD1 in Spinal Cord of G93A Transgenic Mouse
    BMS4 | Nampt Overexpression as a Protector of Mitochondria in a Parkinson's Disease Model
    BMS5 | Exploratory Multivariate Analysis of Shape in Commingled Fossil Assemblages
    BMS6 | Unexpected Anatomical Variations of the Anterior Talofibular Ligament
    BMS7 | Screening of New Inducers of Fetal Hemoglobin
    BMS8 | Movement-Paired Vagus Nerve Stimulation Improves Motor Recovery Following Endothelin-1 Ischemic Brain Damage
    BMS9 | A Role for Raptor Phosphorylation in the Mechanical Activation of mTOR Signaling
    BMS10 | Effects of Chloride Ions on GAPDH Conformation
    BMS11 | Inflammatory Response of Astrocytes Within a Co-Culture Model Involving Monocytes Exposed to CMV
    BMS12 | Development of a Rapid Flow Cytometry Assay to Examine Cell Adherence of the Gastrointestinal Pathogen Plesiomonas shigelloides
    BMS13 | Development of a Rapid Flow Cytometric Assay to Quantitate Human Astrovirus Particles
    BMS14 | Identification of a Novel Factor Impacting Erythropoiesis
    BMS15 | Characterization of Biofilm Properties in Candida albicans Species
    BMS16 | Astrovirus Blocks Interferon-alpha Induced STAT2 Nuclear Localization
    BMS17 | Antibacterial Properties of the Gastrointestinal Commensal Bifidobacterium bifidum Inhibit Growth of the Human Pathogen Plesiomonas shigelloides
    BMS18 | Investigation of the Role of Carcinoma-Associated Fibroblasts in Tumor Angiogenesis
    BMS19 | The Effects of Lipopolysaccharide on the Calcium Pump in BV-2 Microglial Cells
    BMS20 | Neuroprotective Strategies for Parkinson’s Disease: A Novel Approach
    BMS21 | Selective Targeting the EP2 and EP4 Signaling Pathways May Provide a Strategy for Chemotherapy of COX-2-Positive Colon Cancers
    BMS22 | Comparing the Mechanical Properties of Differently Embalmed Human Soft Tissues
    BMS23 | Alternative Splicing and Autism Spectrum Disorder: JARID1 Genes
    BMS24 | Functional Morphology of the Lesser Trochanter and Structural Correlates of the Proximal Femur in Primates
    BMS25 | Colorectal Cancer Stem Cells as Potential Targets for Immunotherapeutic Cancer Therapies 

    BMS1

    Developing a Biomarker for Early Amyotrophic Lateral Sclerosis Detection 

 
    A. Agbas
 
    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.  

    The accumulation of aberrant proteins is a key feature of neurodegenerative diseases (NDD). Factors, such as biochemical complexity of the NDD, definite post-mortem diagnosis, and the absence of a reliable biomarker/surrogate marker has impeded the early diagnosis of NDD before the clinical manifestations. Amyotrophic lateral sclerosis (ALS) holds a critical status, since there is no cure and credible early biomarker for ALS and all diagnostic tests are post-mortem. Therefore, to identify an early biomarker for ALS remains an elusive goal. The sampling from human brain and spinal cord tissues is not an option from the living ALS patients; hence, we turned to platelets that may be a feasible in which a biomarker protein that mutually present in the brain and in platelets, Transactive Response DNA Binding Protein 43 (TDP-43). Monitoring the characteristics of TDP-43 and its interaction with cellular machinery in ALS patient’s platelets may mirror the same/similar post-translational modifications in the brain and in spinal cord as well. Our hypothesis is that “the occurrences of hyper-phosphorylated TDP-43 in platelets mirror the aberrant TDP-43 accumulations which will interfere with the cellular functions (i.e. proteasome activity and mitochondrial malfunctioning) in the central nervous system of individuals with ALS”. This approach will help the researchers to advance the understanding of the biology of ALS , the clinicians longitudinally to monitor the progress of ALS, and to test the new pharmaceutical interventions designed to provide protein stability in the course of treatment of ALS in particular and NDD’s in general.

    BMS2

    Amyotrophic Lateral Sclerosis Transgenic Mice with Altered Calcineurin and SOD1 Activities Showed Increased Zinc Activity Along Their Spinal Cord Segments 


    E. Arneman, M.Taylor, I. Monast, J. Thomas, and A. Agbas
    
Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of motor neurons. Although the molecular mechanisms of the onset of ALS are not well known, the presence of mutant (Cu/Zn) superoxide dismutase (SOD1) plaques, a decrease in calcineurin (CaN) enzyme activity, and the accumulation of cellular labile zinc (Zn) to toxic levels have all been observed along the spinal cord. These observations lead us to hypothesize that defective SOD1 and CaN interactions will lead to a labile zinc accumulation in the spinal cord of ALS subjects/patients in a region-specific fashion. We used G93A SOD1 transgenic mice as a model animal for ALS and utilized western blot analysis, calcineurin cellular activity assay, SOD1 activity assay, and zinc analysis assay to test the hypothesis. We have found that the transgenic mice had an increase in SOD1 and a decrease in CaN enzyme activity as compared to the control mice, and that there were slight differences in the protein expression of these two proteins along the spinal cord section in both the control and transgenic mice. We also found that zinc levels were elevated in all regions of the spinal cord sections of transgenic mice as compared to the control. The most pronounced Zn elevation was observed in the lumbar region (<50% of control) and it was statistically significant (t-test; P= 0.009). Decreased CaN activity was more pronounced in thoracic and lumbar regions; however, we observed statistical significance in the thoracic region (t-test; P=0.008). We conclude that mutant SOD1 and CaN don’t interact efficiently and this event leads to zinc elevation in G93A mutant mice.

    BMS3

    Interaction of TDP-43 and SOD1 in Spinal Cord of G93A Transgenic Mouse 


    A. Bouzid, M. Nicastle, C. Nguyen, J. Sage, and A. Agbas

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.
     

    Recent studies suggest that the ubiquitous proteins [Cu/Zn] Superoxide dismutase (SOD1) and Transactive Response DNA/RNA Binding Protein (TDP-43; MW: 43 kDA), which have been independently identified as players in the pathogenesis of amyotrophic lateral sclerosis (ALS), physically interact and may be cross-talking in the progression of ALS. The nature and pattern of this interaction involves mutant (monomer) SOD1, but the interaction is not yet fully understood. Previous experimentation was either by cell culture or with brain tissue for evaluation of fronto-temporal lobar degeneration (FTLD-U) and ALS. The goal of this study is to assess the spinal cord of G93A transgenic mouse (a mouse model for ALS) for physical interaction of TDP-43 and mutant SOD1 by employing co-immunoprecipitation and immunoblotting methods, and to test whether this interaction involves aberrant TDP-43 (e.g. phosphorylated TDP-43). We employed immunoaffinity chromatography using Protein A/G agarose resin and antibody cross-linking to isolate TDP-43 and SOD1. Additionally, we searched for aberrant TDP-43 in detergent-insoluble, guanidine-soluble fractions (without immunoaffinity purification) as described in the published protocol for human brain tissue, but we were unsuccessful. The immunoprecipitation results showed complications of low-yield crosslinking, but with proper controls and the assumption that TDP-43 and mutant SOD1 interact in the detergent-soluble fraction, we have concluded that TDP-43 and mutant (monomer) SOD1 physically interact in G93A transgenic mouse thoracic spinal cord. This is in concordance with published data from G93A transgenic mouse brain tissue, as well as human brain tissue.

    BMS4

    Nampt Overexpression as a Protector of Mitochondria in a Parkinson's Disease Model 


    M. Boyd, R. Kahle, and C. Theisen
    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Parkinson’s disease (PD) is a progressive disorder of the central nervous system for which there is no cure. The pathogenesis of 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. Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme catalyzing the rate-limiting step of the salvage pathway for NAD+, an important mitochondrial coenzyme. By promoting the activity of cell survival pathways, through the Sirtuin family of enzymes, and enhancing mitochondrial biogenesis, Nampt has immense potential to protect against mitochondrial dysfunction and the subsequent neurodegeneration found in Parkinson’s disease. In this study, we examined the effects of overexpressing Nampt in a neuronal cell model to determine if mitochondrial protection occurred through a Nampt-mediated pathway. By measuring cellular metabolic activity and gene markers of mitochondrial biogenesis, we were able to examine how mitochondrial function is affected in cells overexpressing Nampt. Our results indicate that Nampt alters mitochondrial function and increases mitochondrial biogenesis. Nampt’s ability to promote cellular energy production and maintain mitochondrial function makes it a potentially powerful target for neuroprotective therapy.

    BMS5

    Exploratory Multivariate Analysis of Shape in Commingled Fossil Assemblages


    T. Cole III, M. Cole, and D. Cunningham

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106 and University of Missouri - Kansas City.

    Fossil assemblages are often small, fragmentary, and commingled, posing considerable challenges to the multivariate study of size and shape. In this context, we must find or develop new methods appropriate to the nature of the data. We focus here on exploratory methods, searching for interesting patterns in shape variation. We use a novel method where complete observations (“composites”) are simulated by randomly combining isolated fragments. We scale the data by defining logshapes, which are in transformed ratios of a composite’s measurements to their geometric mean. Principal components analysis summarizes logshape variation in relatively fewer dimensions. We use interactive graphics methods, including scatterplot matrices and parallel coordinates plots, to search the PCA scores for patterns. These are linked together and patterns are sought by interactively highlighting specific composites with distinctive colors and symbols. To illustrate, we study dental shape variation in Homo erectus sensu lato from China, Indonesia, Africa, and Europe. Mesiodistal and buccolingual diameters of permanent teeth were analyzed in 1000 random composite dentitions. The first ten principal components of logshape were explored using the GGobi software package. Most components describe contrasts between the relative sizes of incisors and molars and between different molars. In comparison, there appears to be relatively less variation in the shapes of individual teeth. When the distribution is explored graphically, the distinctiveness of European specimens from Dmanisi is effectively highlighted. We conclude that these methods show promise for detecting patterns in fossil assemblages that could reflect possible variations in sex, time, geography or taxonomy.

    BMS6

    Unexpected Anatomical Variations of the Anterior Talofibular Ligament 


    E.Czer and B.Wright 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    The anterior talofibular ligament (ATFL) is among the most frequently injured ligaments of the body. The bulk of the research on this ligament to date has focused on anatomical variation, noting the possibility of three distinct forms, ranging from single band to a trifurcate form. Our goal was to augment this literature by measuring the toughness and stiffness of the ATFL and its component bands, and ultimately identify a predisposition to injury by correlating anatomy and mechanics with body habitus (e.g. height, weight). Our results revealed no significant correlations among our measured variables and anthropometric measures. In turn males and females had comparable ligament mechanics (t-test; p > 0.05) and ligament bands were also comparably stiff and tough (one-way ANOVA, p > 0.05). Our most compelling finding was the unforeseen variation in the anatomy of the ATFL. Along with variation in band number, intrinsic, extrinsic and intracapsular forms were identified, something which does not appear to have been detailed in prior literature. Coupling our findings with histological analysis of ATFL tissues may better identify injury predisposition.

    BMS7

    Screening of New Inducers of Fetal Hemoglobin

    J. Edge, R. Brown, J. Raval, D. Anderson, R. White, and F. Costa

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Sickling of red blood cells in sickle cell disease (SCD) is a consequence of a single nucleotide substitution at the adult -globin gene, producing abnormal hemoglobin S (HbS). Affected individuals present moderate to severe anemia, splenic infarction, and pain due to vaso-occlusion. Current SCD treatments include blood transfusion and hydroxyurea administration. Hydroxyurea (HU) increases fetal hemoglobin (HbF – gamma-globin gene) levels and improves clinical symptoms in SCD patients. High levels of HbF have been shown to ameliorate the clinical symptoms of SCD due to its ability to inhibit the polymerization of HbS. However, there is a need to identify new or more effective therapies to treat SCD since HU therapy has limited effect on HbF induction, has suboptimal efficacy and has an uncertain long-term effect on organ damage and stroke. Thus, the goal of this research is to identify new transcription regulators that will target HbF induction in adulthood. Utilizing an approach that combines drug treatment and developmental regulation of transcription, 10 new potential HbF transcription regulators were identified. To further investigate the role of these transcription regulators in the induction of the -globin gene (HbF induction), K562 cells carrying enforced expression plasmids with three transcription regulators (EDF-1, NAMPT, ERH) were generated. Quantitative real-time RT-PCR (qPCR) analysis showed that -globin transcription was increased two- to three-fold. Our data demonstrates that transcription regulators are altered when HbF levels are changed, suggesting that these pathways may represent potential new targets for HbF induction in SCD patients.

    BMS8

    Movement-Paired Vagus Nerve Stimulation Improves Motor Recovery Following Endothelin-1 Ischemic Brain Damage 


    T. Fayyaz, N. Khodaparast, and M. Kilgard 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Stroke is the second-most common cause of disability and death worldwide. A variety of physical rehabilitation methods have been developed to improve recovery of motor function following stroke. These methods do not generate sufficient neural plasticity to provide complete recovery. New approaches are being developed to enhance physical rehabilitation by activating brain mechanisms to direct more effective neural plasticity. Left vagus nerve stimulation (VNS) triggers a precisely timed burst of neurotransmitters and enhances plasticity. We have demonstrated that repeatedly pairing VNS with two distinct forelimb movements results in movement-specific map plasticity within primary motor cortex. In the current study, we predicted that repeatedly pairing VNS with a specific movement would result in motor recovery following ischemic brain damage. To test this hypothesis, we paired VNS with movements of the distal forelimb in two groups of rats, including an isometric pull and a dyskinesia assessment task. After five days of VNS movement pairing, an ischemic stroke was induced by injecting endothelin-1 directly into the cortex. In order to evaluate the effectiveness of VNS on motor rehabilitation and recovery from cortical ischemic damage, one group of rats from each task was given VNS for five weeks. Rats receiving identical motor training without VNS pairing exhibited some recovery. On the other hand, the group with the VNS paired movement showed a far greater recovery and performance level reverted to pre-surgery standards. These results suggest that pairing VNS with a specific event may significantly improve recovery from an ischemic episode.

    BMS9

    A Role for Raptor Phosphorylation in the Mechanical Activation of mTOR Signaling
    

J. Frey and T. Hornberger

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    It is well recognized that the maintenance of skeletal muscle mass contributes significantly to disease prevention and issues associated with the quality of life. Skeletal muscle mass is regulated by a variety of stimuli, one of which is mechanical loading. Recent studies have provided strong evidence that the activation of mTOR signaling is necessary for mechanically-induced changes in skeletal muscle mass. However, the mechanisms that regulate the mechanical activation of mTOR signaling remain poorly defined. In this study, we set out to determine if changes in the phosphorylation of Raptor, an mTOR-associated protein, contribute to the mechanical activation of mTOR. To accomplish this goal, mouse skeletal muscles were subjected to mechanical stimulation via a bout of eccentric contractions (EC). Using mass spectrometry and Western blot analysis, we found that ECs induced an increase in Raptor S696, T706, and S863 phosphorylation, and this effect was not inhibited by rapamycin. This observation suggested that changes in Raptor phosphorylation might be an upstream event in the pathway through which mechanical stimuli activate mTOR. To test this, we employed a phospho-defective mutant of Raptor (S696A/T706A/S863A) and found that the EC-induced activation of mTOR signaling was significantly blunted in muscles expressing this mutant. Furthermore, mutation of the three phosphorylation sites altered the interactions of Raptor with PRAS40 and p70S6k, and it also prevented the EC-induced dissociation of Raptor from p70S6k. Combined, these results suggest that changes in the phosphorylation of Raptor play an important role in the pathway through which mechanical stimuli activate mTOR signaling.

    BMS10

    Effects of Chloride Ions on GAPDH Conformation
    

O. Gorshkalova and N. Seidler

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    The multifunctional protein GAPDH, is known for its involvement in many functions in cells. Several mechanisms may control the multifunctionality of GAPDH, including chemical modification and small molecule binding. The binding of chloride ions may be a trigger for GAPDH in cells such as neurons. We hypothesize that chloride ions alter the conformation of GAPDH and change its functionality. Computationally, we examined several crystal structures' internal movements of interfacial amino acids that may contribute to the conformational changes allowing alteration in function. Experimentally, we cross-linked GAPDH in the presence and absence of chloride ions, and then examined fluorescence intensity and cross-linked protein bands using SDS-PAGE. In the computational approach, three GAPDH crystal structures were compared. All of the structures were obtained from Staphylococcus aureus: NAD+ only, NAD+ and phosphate, and NAD+ and chloride. The computed space for each was obtained. The structure with only NAD+ had the greatest volume, phosphate had no effect, but chloride appeared to decrease this space. We also compared chloride and phosphate structures, measuring the distance from the C5 atom of Leucine-44 to each of the five atoms of the pyrrolidine ring of Proline-190. These distances were significantly greater in the chloride structure. For the experimental approach, the relative distribution of the dimeric forms of GAPDH differed and tryptophan emission fluorescence showed similar results. In summary, our results suggest that chloride ions disrupt the subunit-subunit interactions of GAPDH. The disruption of this interfacial region of tetrameric GAPDH may promote dimerization that could contribute to functional heterogeneity.

    BMS11

    Inflammatory Response of Astrocytes Within a Co-Culture Model Involving Monocytes Exposed to CMV 


    Q. Haque, R. Duncan, J. Taylor, and C. Harrison 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106 and Children’s Mercy Hospital
    .

    Cytomegalovirus (CMV) can invade the central nervous system (CNS) by breaching the blood-brain barrier resulting in CNS injury. This injury is believed to be secondary to direct viral effects plus excess inflammatory response from the innate immune system and glial cells such as activated astrocytes . To better understand this process, a co-culture model was developed to study the inflammatory effects on a human astrocyte cell line (STTG-1) from soluble factors released by CMV-exposed monocyte cell line (THP-1). Immunocytochemistry, PCR and confocal microscopy were used to quantitatively measure inflammatory markers (IL-6, IL-8 and TNF-α) along with neuro-injury markers (e.g. s100β). We detected a significant increase in inflammatory cytokines (from astrocytes) when the astrocyte cells were exposed to soluble factors secreted by CMV exposed THP-1 cells, thus demonstrating activated astrocytes. Dexamethasone (a compound that has broad immune suppressing effects) treatment of monocyte cells was then utilized in the same model. Dexamethasone treatment of monocytes significantly diminished IL-6 and IL-8 expression from activated astrocytes which had exposure to soluble factors from CMV exposed monocytes. The mechanism appears to be induction of IκBα, which inhibits the activation of NFKβ. Activation of the NFKβ pathway is essential for production of multiple inflammatory markers. Selective reduction in CMV-induced inflammation could be neuroprotective.

    BMS12

    Development of a Rapid Flow Cytometry Assay to Examine Cell Adherence of the Gastrointestinal Pathogen Plesiomonas shigelloides 


    L. Harriett, J. Taylor, M. Jeranko, and T. Taylor 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Plesiomonas shigelloides are Gram-negative bacteria that cause gastroenteritis as well as various extraintestinal infections, and are of particular concern in immune-compromised individuals. Although some virulence factors of P. shigelloides have been recognized, mechanisms of pathogenesis by this organism are largely uncharacterized to date. The overall goal of the laboratory is to identify and characterize virulence factors of P. shigelloides. These bacteria are known to adhere and invade human cells, and the goal of this project is to develop a rapid assay to test adherence of P. shigelloides to human cells. We hypothesize that the adherence mechanisms of P. shigelloides can be studied using a flow cytometry-based adherence assay. The human gastrointestinal cell line Caco-2 was infected for various times with green fluorescent labeled-P. shigelloides, and cultures were analyzed using flow cytometry to determine the degree of bacterial adherence. This flow cytometry adherence assay was also performed on various pathogenic bacteria including Staphylococcus epidermidis, Listeria monocytogenes, Serratia marcescens, Escherichia coli, and Aeromonas hydrophila. We further successfully used this flow cytometry assay with other gastrointestinal cell lines (HT-29 and T84) in addition to HeLa cells in order to compare the adherence characteristics of other pathogens with those of P. shigelloides. The use of flow cytometry demonstrated rapid, sensitive and consistent P. shigelloides adherence to various cell lines, and adherence was similar to other known gastrointestinal pathogens. This successfully optimized flow cytometry-based adherence assay can be implemented for the investigation of cell adherence and other virulence factors of P. shigelloides.

    BMS13

    Development of a Rapid Flow Cytometric Assay to Quantitate Human Astrovirus Particles 


    R. Herdes, Z. Rahim, T. Taylor, and J. Taylor 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Viral pathogens cause 80 percent of infectious gastroenteritis cases worldwide, yet these agents are often difficult to study. One such virus, human astrovirus, is a common cause of childhood diarrhea and vomiting, but little is known about astrovirus pathogenesis or replication, and the tools to study astroviruses are not well-developed. For instance, the traditional method used to quantitate viruses is the plaque-forming assay; astroviruses, however, do not readily form plaques, and an efficient method is lacking for simple astrovirus quantitation. To develop an improved assay to quantify infectious astrovirus particles, we hypothesized that flow cytometry could be effectively used to rapidly quantify astrovirus from in vitro samples. Human gastrointestinal CaCo-2 cells were infected with trypsin-activated human astrovirus-1, fixed and stained with anti-astrovirus-capsid antibody to detect viral capsid protein, and cells were analyzed by flow cytometry. To validate flow cytometry data, cells were co-stained with DAPI to stain nuclei and visualized by fluorescent microscopy. Flow cytometry was extremely accurate and sensitive to detect infected cells, and peak expression of astrovirus capsid protein was seen at 24 hours post-infection. Overall, viral titers from flow cytometry data were nearly identical to immunofluorescence data, validating the method. Our data demonstrate that flow cytometry is readily applicable for quantifying infectious astrovirus, and is superior to immunofluorescence in speed and accuracy, since there is no user bias during cell counting. These improvements in methodologies will facilitate our ability to study the pathogenic mechanisms employed by these viruses.

    BMS14

    Identification of a Novel Factor Impacting Erythropoiesis
    

H. Morrison, F. Costa, and R. White 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Identification of novel erythropoietic factors which induce the production of red blood cells is an innovative approach to discovering pharmaceutical targets to treat many types of anemia. The Xpna (X-linked pre and neonatal anemia) mutant mouse exhibits a transient, severe anemia which is resolved by adulthood through an unknown mechanism. The anemia in Xpna mutant mice is caused by a dominantly inherited splicing mutation in the Gata1 gene, an important master control protein absolutely required for the development of mature red blood cells. Despite the lack of GATA1 protein in half of the erythroid progenitor cells due to X-chromosome inactivation, these mutant GATA1-deficient cells produce mature red blood cells as indicated by a polymorphic red blood cell marker Pgk1, phosphoglycerate kinase 1. qRT-PCR was used to indicate that there is one-half the level of GATA1 protein in reticulocytes derived from Xpna mice compared to normal female litter mates. Currently, we are performing FLOW cytometery to confirm there are GATA1 negative erythroid red blood cells present. This finding is highly significant as red blood cells should not be produced from GATA1-deficient erythroid precursors. Therefore, we hypothesize there is a developmentally expressed compensatory gene replacing GATA1 in Xpna erythroid precursors. Analysis is underway to identify the novel gene inducing erythropoiesis in these mutant mice. Discovery of this compensatory gene, whose expression replaces GATA1, is likely to result in the isolation of a potent pharmaceutical target for treating many different types of anemia.

    BMS15

    Characterization of Biofilm Properties in Candida albicans Species 


    S. Parsel, D. Wade, C. McArthur, and S. Gustafson 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    The pathogenesis of many microbial infections is in part due to the production of a biofilm. Candida albicans, a major opportunistic pathogen, has been shown to produce extensive biofilms, which may lead to prolonged infection, enhanced pathogenicity, and contribute to antimicrobial resistance. Our research centers on developing a better understanding of the unique role biofilms play in disease processes. In order to characterize the physical and kinetic properties of biofilms in five strains of C. albicans, we utilized a magnetic biofilm ring (BR) assay. Cultures of each strain were diluted in YPD media to equal UV spectrophotometric absorbance values and then diluted 1000-fold. 100 µL of each diluted strain (an average of 21 CFUs) and 1.2 µL of charged nanoparticles (+ or -) were added to 96-well strips. The strip wells were incubated at 37 ˚C, and the assay was performed at consecutive time points (t = 0, 2, 4, 8, 12, 18, and 24 hours) to determine when biofilm formation occurred. Additionally, a crystal violet (CV) assay was used to verify the presence of biofilm within the strip wells and 100 µL of L- and D-temporin A (2.0 mg/mL), known fungicidal peptides, were added to separate wells to observe their antimicrobial activity against biofilm maintenance and formation. The BR assay showed that biofilm formation was sufficient to inhibit the diffusion of magnetic nanoparticles between 12 and 18 hours using both positive and negative charged particles. Additionally, the CV assay confirmed the presence of a negatively charged substance remaining in the strip wells following complete aspiration of the cultures. Both L- and D-temporin A failed to inhibit yeast proliferation over 48 hours and there was no evidence of biofilm destruction. In conclusion, the C. albicans strains produced significant biofilm between 12 and 18 hours, though the accuracy of these findings is limited to the time point intervals. Second, the crystal violet assay provided visualization of an extensive negatively charged biofilm; however there was no apparent effect on charge of the nanoparticles, suggesting that the biofilm is composed of heterogeneously charged macromolecules. Finally, the antimicrobial properties of L- and D-temporin A were ineffective against all five strains at a concentration previously shown to be fungicidal, suggesting that the observed biofilm production conferred intrinsic resistance to the peptides.

    BMS16

    Astrovirus Blocks Interferon-alpha Induced STAT2 Nuclear Localization
    

P. Reddy, Z. Rahim, and J. Taylor
    
Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    BACKGROUND. The innate interferon (IFN) system is critical for eliciting antiviral effects including enzyme induction and up-regulation of MHCI molecules in infected cells. Most pathogenic viruses, however, can block IFN induction and IFN-stimulated gene expression, essentially evading this host defense system. The pathogenesis of astroviruses, a common causative agent of gastroenteritis in children, is relatively unknown. To understand how astroviruses can replicate within the host, we investigated whether astroviruses could modulate the IFN pathway by assessing the activation of a critical signaling protein in the cascade, STAT2. Hypothesis: We hypothesized that astroviruses may delay the human interferon response by impeding IFN-alpha stimulated STAT2 nuclear localization. METHODS. CaCo-2 cells were infected with human astrovirus for 24 hours, treated with IFN-alpha for 1 hour, fixed, and permeabilized. Cells were stained with anti-STAT2, anti-astrovirus capsid antibody (8E7), and DAPI. Cellular infection and STAT2 nuclear localization were analyzed using fluorescent microscopy. RESULTS. Microscopy and flow cytometry revealed that astrovirus can replicate normally with IFN-alpha treatment. While treatment of uninfected CaCo-2 cells with IFN-alpha (8 ng/ml or 25 ng/ml) yielded STAT2 nuclear localization, astrovirus-infected cells treated with 8 ng/ml displayed decreased nuclear STAT2 accumulation. CONCLUSION. Our results suggest that astrovirus infection can inhibit STAT2 nuclear accumulation as a possible mechanism to evade the host immune response. We are currently investigating how astroviruses can inhibit the activation of STAT2 and other IFN-activated factors. Understanding astrovirus pathogenesis will enhance our ability to develop preventative and therapeutic measures against infection.

    BMS17

    Antibacterial Properties of the Gastrointestinal Commensal Bifidobacterium bifidum Inhibit Growth of the Human Pathogen Plesiomonas shigelloides 


    N. Ridolfi, T. Taylor, and J. Marlin 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Bifidobacteria bifidum (B. bifidum) are Gram-positive anaerobic commensal bacteria that are found within the intestinal tract and have been shown to have probiotic health benefits. These bacteria release numerous substances, such as acid products, bacteriocins, and other metabolites, that prevent bacterial infections within the gastrointestinal system, while also maintaining the integrity of gut mucosa. Plesiomonas shigelloides (P. shigelloides) are Gram-negative bacteria that can cause gastrointestinal infections, and more rarely extra-gastrointestinal infections, especially among the immunocompromised population. The goal of these experiments was to determine whether B.bifidum supernatants were able to inhibit the growth of P. shigelloides. Previous research has demonstrated that B. bifidum supernatant leads to growth inhibition of other gastrointestinal pathogens, such as Listeria monocytogenes, Helicobacter pylori, Salmonella spp., and Escherichia coli, however it is not known if this inhibition also occurs with P. shigelloides. Preliminary data from our lab demonstrated that concentrated B. bifidum supernatant does indeed inhibit the growth of P. shigelloides. Because P. shigelloides infections can potentially be life-threatening, the finding that these supernatants have the ability to inhibit growth is significant. Although we are unsure of the molecule produced by the B. bifidum that is responsible for this growth inhibition, this study supports the notion that probiotics are important in the prevention of gastrointestinal infections, such as those caused by P. shigelloides.

    BMS18

    Investigation of the Role of Carcinoma-Associated Fibroblasts in Tumor Angiogenesis 


    K. Schnarr and E. Konorev 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    INTRODUCTION. It is known that tumor growth is dependent on angiogenesis; however, tumor cells themselves cannot provide the support necessary for growth. It has become more evident that other cell types contribute to this process, namely carcinoma-associated fibroblasts and endothelial cells. We hypothesize that breast cancer enhances the formation of vascular networks via carcinoma-associated fibroblasts. It is our goal to elucidate the role of carcinoma-associated fibroblasts contribution to tumor angiogenesis. METHODS. Human dermal fibroblasts (HDFs), human umbilical vein endothelial cells (HUVECs), and MDA-MB-231 triple negative breast cancer cells were plated separately in cell culture flasks and expanded in their appropriate media conditions prior to culturing cell types together. Upon co-culturing of cells, we visualized formation of vascular networks by staining for endothelial cell marker CD31 and imaged using fluorescence microscopy. We also stained for fibronectin, an extracellular matrix protein produced by fibroblasts. Quantitative PCR was used to assess expression of pro-angiogenic factors after co-culture treatments. RESULTS/CONCLUSION. After co-culturing HUVECs with breast cancer cells, no vascular networks were formed. However, in the presence of fibroblasts, significant vascular networks were present. Breast cancer cells, therefore, do not directly support angiogenesis but rather indirectly by activation of carcinoma-associated fibroblasts. Next, we looked at production of pro-angiogenic factors by tumor-associated fibroblasts. Fibroblasts significantly increased expression of fibronectin in the presence of breast cancer cells conditioned media. Furthermore, we quantitatively assessed these data and found a three-fold increase in VEGF expression and nearly two-fold increase in fibronectin. Thus, we concluded that breast cancer cells enhance production of pro-angiogenic factors by carcinoma-associated fibroblasts. Lastly, we assessed the effect of breast cancer cells on the formation of vascular networks. We co-cultured fibroblasts and endothelial in control starving vs. breast cancer cells conditioned media. Compared to the control, we found that conditioned by breast cancer cells, media significantly enhanced the formation of vascular networks. We conclude that breast cancer does enhance angiogenesis in vitro via carcinoma-associated fibroblasts. Future studies will include using 3D models to more accurately recapitulate the tumor environment and to identify factors secreted by breast cancer cells that activate fibroblasts.

    BMS19

    The Effects of Lipopolysaccharide on the Calcium Pump in BV-2 Microglial Cells
    

S. Sharma, S. Patel, P. Ramlow, and A. Zaidi
    
Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Microglial cells are the major immune cells of the CNS. Stimulation of microglia activates inflammatory pathways, producing cytokines and reactive oxygen species. The resultant oxidative stress can damage critical proteins. The plasma membrane calcium-ATPase (PMCA) is a Ca2+ pump that maintains Ca2+ homeostasis, critical for cell survival. We have previously shown that neuronal PMCA is very sensitive to oxidative stress. The goal of this study was to determine the effects of the inflammatory cascade on microglial PMCA. Cultured BV-2 cells were stimulated with lipopolysaccharide (LPS) (1 pg/ml – 1 µg/ml). Total protein in cell lysate was measured by bicinchoninic acid assay. PMCA activity was assessed by measuring inorganic phosphate released upon Ca2+ -dependent ATP hydrolysis. PMCA protein levels were measured by immunoblotting. Free intracellular calcium [Ca2+]i was measured by flow cytometry using Fluo3. Our results show that LPS caused morphological changes in BV-2 cells indicative of toxicity. PMCA activity showed biphasic response with ~40% increase at 1 pg/ml and dose-dependent decline at higher concentrations (n=3). There was a steady increase in [Ca2+]i till 5 pg/ml after which it plateaued off (n=2). PMCA protein levels were increased by LPS (n=5). In conclusion, LPS has significant effects on microglial PMCA. Low doses stimulate PMCA, whereas higher concentrations cause inhibition and consequent elevation in [Ca2+]i. In contrast, PMCA protein levels increase, presumably to compensate for inactivation. Loss of PMCA function and Ca2+ overload would exacerbate the neurotoxic milieu in the CNS. Our studies are pertinent to pathogenesis of diseases such as Alzheimer’s and Parkinson’s.

    BMS20

    Neuroprotective Strategies for Parkinson’s Disease: A Novel Approach 


    J. Sheth, P. Ramlow, and A. Zaidi 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Parkinson’s disease (PD) is characterized by loss of dopaminergic (DA) neurons in the substantia nigra (SN). DA neurons are unique in having autonomous pace-making activity which continuously brings in extracellular calcium. To prevent calcium-mediated toxicity and maintain homeostasis, DA neurons need an efficient calcium extrusion system. The plasma membrane calcium ATPase (PMCA) is the major transporter that pumps calcium out of cells against its 10,000-fold gradient. We have observed lower PMCA protein levels in SN compared to other brain regions. Additionally, we have observed a significant decline in PMCA in aging and in PD. Reduced PMCA leads to accelerated cell death. Therefore, strategies that increase PMCA protein levels would be neuroprotective. Polyphenol compounds have been shown to upregulate pro-survival genes. The goal of the current study was to determine if dietary flavonoids altered PMCA. SH-SY5Y cells were cultured in DMEM and FBS and treated with resveratrol, curcumin and fisetin at different doses for 24 hours. Flow cytometry was used to determine cell viability. Total protein was measured by bicinchoninic acid assay. PMCA activity was assessed by measuring inorganic phosphate released upon calcium-dependent ATP hydrolysis. PMCA protein levels were measured by immunoblotting and mRNA levels by qRT-PCR. Our results show a dose dependent increase in PMCA activity and protein levels upon exposure to flavonoids. There is no change in mRNA levels suggesting that the upregulation may be due to increase in PMCA mRNA and/or protein stability. Studies are in progress to determine the pathway underlying increased PMCA expression.

    BMS21

    Selective Targeting the EP2 and EP4 Signaling Pathways May Provide a Strategy for Chemotherapy of COX-2-Positive Colon Cancers
    A. Sindel, S. Reifeiss, P. Braunlich, A. Walters, and A. Shnyra 

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    INTRODUCTION. Poor prognosis in various human carcinomas correlates with the extent of Prostaglandin E2 (PGE2) production. Experimental and clinical evidence shows that cyclooxygenase-2 (COX-2) activity and increased levels of PGE2 can contribute colorectal tumorigenesis. These findings provide the rationale for the use of COX-2 inhibitors for chemotherapy. However, therapeutic utility of COX-2 inhibitors is limited due to the side effects. However, selective inhibition of PGE2 effects via pharmacologic targeting of PGE2 receptor(s) may augment anti-cancer chemotherapies. PGE2 signaling through EP2 and EP4 receptors stimulate tumor growth by suppressing apoptosis. Thus, antagonists of the EP receptors may represent an alternative therapeutic approach to cancer treatment. HYPOTHESIS: We hypothesized that targeting E2 and EP4 receptors with their selective antagonists may potentiate induction of the intrinsic and extrinsic apoptosis in tumor cells. METHODS. To test our experimental hypothesis we used COX-2-positive HCA-7 human carcinoma cells which have been established from primary human colorectal adenocarcinoma. When cells were analyzed in our laboratory, HCA-7 cells strongly expressed COX-2 protein and secreted significant amounts of PGE2. The extrinsic apoptosis was induced by treating HCA-7 cells with Tumor Necrosis Factor (TNF) whereas the intrinsic apoptotic pathway was activated by an exposure to ultraviolent light (UV). Induction of apoptosis in carcinoma cells was assessed by using a CellTiter 96® AQ Assay and Proteome ProfilerTM Human Apoptosis Array. RESULTS. Our data show a strong suppression of apoptosis-inducing potential of TNF (extrinsic pathway) in HCA-7 cells treated with Butaprost, a selective agonist for the EP2 receptors. Specifically, decreased protein levels of pro-apoptotic Bad, Bax, pro-caspase 3, cleaved caspases 3, and SMAC/Diablo were detected in TNF/Butaprost- treated HCA-7 cells as compare to that in cells treated with TNF only. However, neither EP2 nor EP4 antagonists significantly affected TNF-induced apoptosis in tumor cells. In contrast, EP2 and EP4 antagonists were equally effective in rendering the resistance to UV-induced apoptosis which were further elaborated by Proteome ProfilerTM Human Apoptosis Array. Of importance, EP2 and EP4 antagonists showed the capacity to reduce levels of pro-apoptotic pro-caspase 3, cleaved caspases 3, and cytochrome c and to increase amounts of anti-apoptotic protein survivin and enzyme catalase. CONCLUSIONS. Taken together, our findings suggest that selective targeting the EP2 or EP4 signaling pathways may provide a strategy for the chemoprevention/chemotherapy of COX-2 colon cancers.

    BMS22

    Comparing the Mechanical Properties of Differently Embalmed Human Soft Tissues 


    R. Skold, B.Wright, and A. Olinger

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    
Most medical professional training involves anatomical dissection and the use of embalmed cadavers. There have been advances in perfecting embalming solutions and techniques; however, the body of literature comparing the mechanical properties of human soft tissue between treatment groups seems insufficient. The purpose of this project is to determine whether there is a significant difference in the toughness of human soft tissue when exposed to various embalming solutions. Seven different soft tissues were collected from fresh cadavers: skin, muscle, artery, vein, nerve, tendon, and ligament. Tissues from the right side were harvested less than 72 hours postmortem, while the left side tissues were harvested at greater than 72 hours. Specimens were divided into the following treatment groups: unembalmed and embalmed with formaldehyde, alcohol-glycerin, and Thiel solutions. Tissue was submerged in solution for 1 week. Samples were tested using a mechanical properties tester to evaluate toughness. The data shows that there is an increase in the toughness of brachial artery (BA), great saphenous vein (GSV), median nerve (MN), and rectus sheath (RST) after being exposed to various embalming solutions. There is also a tendency for soft tissues to increase in toughness when embalmed after 72 hours postmortem. Being aware of this difference is important for students, residents, and health professionals to keep in mind when practicing procedures or performing studies with embalmed cadavers. One next step to further explore this difference would be to perfuse whole cadavers with the different embalming solutions before harvesting tissue and performing mechanical tests.

    BMS23

    Alternative Splicing and Autism Spectrum Disorder: JARID1 Genes 


    J. Steib, A. Gadashova, and Z. Talebizadeh
    
Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106 and Children’s Mercy Hospital.

    Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with a prevalence of 1 in 88 children. ASD is considered a complex heritable disorder with the etiology still unknown in most cases. Some of the undetected disease-causing mutations may alter gene regulation processes such as alternative splicing, which may result in functionally distinct transcripts. Abnormalities in splicing may contribute to the underlying mechanism of diseases. The potential role of alternative splicing in the etiology of ASD has already been suggested for two genes, NLGN3 and NLGN4. Current evidence supports the potential role of the circadian system and clock genes in autism. The objective of the current project was to investigate the alternative splicing profile of JARID1, a circadian gene family, in autism using exon array and RT-PCR experiments. First, data for three autosomal JARID1 genes was obtained from exon array [lymphoblastoid cell line (LCL) n=6 autism, n=3 control]. Differential exon expression was identified for multiple exons in autism compared with controls. Primers were designed for the differentially expressed exons using IDT PrimerQuest. RT-PCR was performed using LCL-derived RNAs followed by gel electrophoresis and direct DNA sequencing. One primer set showed an unexpected product size, which by sequencing and alignment using BLAST was identified to be a product of JARID1a. Compared to the wild-type sequence, the product was missing exons 2-4. While needing replication, this preliminary data is in line with our hypothesis that changes at the alternative splicing level of this circadian gene family may play a role in ASD.

    BMS24

    Functional Morphology of the Lesser Trochanter and Structural Correlates of the Proximal Femur in Primates 


    T. Tobin and K. Wright
    
Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    This study examines lesser trochanter variation and shape of the proximal femur in a sample of behaviorally diverse primates, with the goal of relating morphological variation to locomotor and postural behavior. Previous studies have demonstrated that proximal femoral shape is an accurate predictor of locomotor behaviors in hominoid primates (Harmon, 2007). Additionally, other studies have suggested that specific features of the proximal femur, such as femoral neck length, are also good predictors of locomotor behavior (Janson & Boinski, 1992, Anemone, 1993). We hypothesized that, for neotropical primates (n=98, 12 genera/26 species), lesser trochanter size would also co-vary with other morphological features of the proximal femur in ways that would group species into general locomotor categories. We used both linear and three-dimensional morphometric methods to measure the proximal femoral morphology (8 linear measurements and 1 index; 17 landmark coordinates). Our comparisons of linear morphometric measurements of the proximal femur within a diverse sample of neotropical primate species suggest that the position of the lesser trochanter is similar within phylogenetically related groups, and within these groups there appears to be a strong allometric effect. However, smaller scale within-group comparisons suggest variation that may relate to locomotor behavior. Our geometric morphometric comparisons, which consist of a larger sample that also includes old world monkey species (n=50, 7 genera/16 species), also appear to reflect body size related variation for some groups, but also suggest a possible convergence between leapers and suspensory/climbing forms for overall shape of the proximal femur, irrespective of body size.

    BMS25

    Colorectal Cancer Stem Cells as Potential Targets for Immunotherapeutic Cancer Therapies 


    A. Walters, A. Sindel, P. Braunlich, S. Reifeiss, and A. Shnyra

    Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106.

    Cancer is the second leading cause of death in the United States. The failure of conventional therapies to definitively eradicate cancer suggests a need for alternative approaches for treating the disease. A growing body of evidence points to cancer stem cells (CSCs) as the cells responsible for the initiation, proliferation, and perpetuation of cancer. The failure of conventional chemotherapeutics to eliminate CSCs may explain the relapses and resistances that often arise after clinical remissions. Immunotherapeutic approaches may be the key to targeting CSCs and decisively eradicating cancer. The purpose of this study was to isolate and analyze HCA-7 colorectal cancer stem cells as a means of evaluating their potential as targets for novel, immunomodulatory anti-cancer therapies. Utilizing FlowComp™ Dynabeads®, CD44+ CSCs were isolated from a general population of HCA-7 tumor cells. CSCs were cultured in ultra-low adherent plates to yield CSC spherules, as well as in an alginate 3D culturing system with comparisons of cell viability being made. RNA extraction and RT-PCR of cDNA was performed, comparing the expression of tumorigenic genes by CSCs and general population tumor cells via tumor metastasis array. Apoptotic responses to ultraviolet radiation were also compared between CSCs and general population tumor cells via protein array. Our findings suggest that HCA-7 CSCs represent a distinct subpopulation of cells with gene expression profiles and apoptotic responses that differ from general population HCA-7 tumor cells and may contribute to tumorigenesis, making them potential targets for immunomodulatory therapeutic approaches for treating cancer.