«Strengthening the Nation through Diversity, Innovation & Leadership in STEM San Antonio,Texas · October 3-6, 2013 Get Connected! Connect with the ...»
Observed decreases in BiFC between LMP1 and prospective proteins suggest a critical role for these proteins in LMP1 signaling of infected cells. Such insights can be used to better understand the LMP1-signaling complex and design novel therapeutic approaches to inhibit EBV-associated diseases.
PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO THE EBOLA VIRUSHumberto Hernandez, Barry Rockx.
University of Texas Medical Branch, Galveston, TX.
Ebolavirus hemorrhagic fever is a severe, often fatal disease in humans and nonhuman primates, with a case fatality rate of up to 90%. There is no cure, established drug therapy, or vaccine licensed for human use. There are 5 identified species of Ebola virus that include Bundibugyo, Ivory Coast, Reston, Sudan, and Zaire. There is limited cross-protection among the 5 Ebola virus species. One of the key steps in any virus infection occurs when a virus binds to and enters a cell. The Ebola virus glycoprotein mediates viral attachment and entry into host cells. Based on sequence homology between virus strains, we hypothesize that conserved epitopes are present on the Ebola virus glycoprotein that can be targeted by monoclonal antibodies binding to all known Ebola virus species. We tested this hypothesis by generating monoclonal antibodies from splenocytes of Balb/c mice vaccinated with vesicular stomatitis virus expressing Zaire Ebola virus glycoproteins (rVSV Zebov-GP) and boosting with a Sudan Ebola virus GP expressing VSV. ELISA was used to test reactivity of monoclonal antibodies against the different Ebola virus GP.
We performed western blots to determine whether these antibodies recognized conformational or linear epitopes.
Competitive ELISA assays were also performed to characterize the antibody binding sites. Interestingly, we found 4 antibodies that cross-react with all known species of Ebola virus and recognize Zebov-GP as a linear epitope.
Studies involving phage display libraries are underway. Using computational analysis and X-ray crystallography data, epitope mapping of these antibodies will give us important insight into the conserved epitopes of these cross-reactive monoclonal antibodies.
THE ROLE OF THE SOLO LUXR GENE IN DICKEYA SPECIESChrista Landowski, MS1, Martyna Błaszczyk2,3, Sylwia Jafra2,3.
University of Houston-Downtown, Houston, TX, 2University of Gdansk, Gdansk, PL, 3Medical University of Gdansk, 1 Gdansk, PL.
Dickeya is a genus of enterobacteria that is responsible for many phytopathogenic outbreaks affecting crop and food production. The pathogenicity of Dickeya spp. is partly attributed to the production of signal molecules (acylhomoserine lactones-AHLs) by LuxI synthase and delivered to the bacteria’s environment. If AHL reaches the threshold level in the environment, they are sensed by LuxR proteins, a process referred to as quorum sensing.
Recent studies have shown that plant-associated Pseudomonas spp. possess the LuxR proteins but lack the LuxI homolog necessary for the AHL signal production; this is a solo luxR gene. This genetic makeup suggests that such bacteria can only receive signals, indicating that solo luxR may play a role in inter-species and inter-kingdom signaling. The aim of this study is to investigate the role of solo luxR in Dickeya pathogenicity. For this, solo luxR from Dickeya was amplified using PCR with specific primers and ligated into a vector conferring resistance to ampicillin. Then E. coli competent cells were transformed with the obtained construct. The next steps of the study are placing a gene conferring resistance to chloramphenicol within solo luxR, thus inactivating solo luxR to obtain pMBsolo construct. After introduction of pMBsolo to Dickeya sp. cells, homologous crossing over is expected to occur.
Obtained Dickeya sp. mutant affected in solo luxR will be compared with the wild-type Dickeya sp. for the siderophore production, motility, and virulence, allowing the determination of the role solo luxR plays in the pathogenicity of Dickeya.
124 UNDERGRADUATE POSTER ABSTRACTS
Biological Sciences FRI-573
CHARACTERIZING POSTMORTEM MICROBIAL ECOLOGY OF THE MAGGOT MASS THROUGH MEASURES
OF PH, OXIDATION-REDUCTION POTENTIAL, AND TEMPERATURELauren Chun, David Carter.
Chaminade University of Honolulu, Honolulu, HI.
As a corpse decomposes and the postmortem interval increases, the evidence present at crime scenes diminishes. To compensate for this loss, many investigative techniques use decomposition processes as investigative tools. Recent research has shown that microorganisms, particularly bacteria, have the potential to assist investigators, but a deeper understanding of postmortem microbiology is necessary. We aimed to characterize culturable postmortem microbial communities and present trends that will allow investigators to use microbes as physical evidence. We began with the conspicuous habitat of the maggot mass. We hypothesized that the chemistry and microbiology of the maggot mass will not change as fly larvae develop. To test this, we conducted a field experiment to decompose swine (Sus scrofa domesticus) carcasses and characterize postmortem microbial communities throughout decomposition. We placed the swine in a tropical savanna ecosystem in Palolo Valley near Honolulu, Hawaii, which we replicated 3 times. We scored gross decomposition and characterized maggot masses though measures of pH, oxidation-reduction potential, and temperature. To characterize the microbial community, we swabbed the maggot masses and streaked samples onto standard nutrient agar, which we incubated at 22 °C. Isolated bacteria were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Through 153 hours postmortem, we observed a discrete habitat characterized by high temperature (35 to 39 °C), moderate and decreasing pH (7.4 to 6.6), and highly reducing conditions (approximately -250 mV to -190 mV). The taxonomy of the bacteria will be presented. This information will be important to entomologists and investigators because it provides a better understanding of corpse breakdown.
DESIGN OF MOLECULAR CHEMISTRIES TO EVALUATE SYNECHOCOCCUS IRON LIMITATION IN MONTEREYBAY Andrea Reyes-Ortiz, Julie Robidart., Jonathan Zehr.
University of California, Santa Cruz, Santa Cruz, CA.
The cyanobacteria Synechococcus are significant contributors to the synthesis of organic compounds from carbon dioxide in the ocean, but the factors limiting their distribution in Monterey Bay are unknown. Iron is often a limiting nutrient for phytoplankton, and an examination of idiA, an iron-deficiency induced protein, will indicate whether iron stress occurs in Monterey Bay. Degenerate primers were used on archived samples collected from 4 different seasons within Monterey Bay, and a phylogenetic tree was constructed to examine the local diversity of the Synechococcus clades present. Quantitative PCR assays were designed for the idiA clades, and each was quantified throughout the year using archived samples. The idiA gene expression will be used to evaluate differential iron limitation over time for each clade. These assays will be optimized for deployment on the environmental sample processor (ESP), a robotic eco-genomic sensor for autonomous in situ gene quantification. With these data, we are able to determine the factors contributing to the biogeography and ecology of this important organism over space and time.
APOLIPOPROTEIN E 4/4 SERUM INHIBITS GROWTH OF PLASMODIUM FALCIPARUMElizabeth Perry1, Clyde Phelix1, George Perry1, Xiongwei Zhu2, Hisashi Fujioka2.
University of Texas at San Antonio, San Antonio, TX, 2Case Western Reserve University, Cleveland, OH.
1 Our evolutionary history has been characterized by a constant war between pathogenic microorganisms and various defense mechanisms to counter their pathogenicities. For instance, heterozygotic individuals with sickle cell trait are protected against severe falciparum malaria infections, and the high frequency of hemoglobin S in Africa is due to the selective advantage of the balanced polymorphism. The apolipoprotein E4 (ApoE4) allele has been linked to the pathogenesis of Alzheimer’s disease, cardiovascular disease, and atherosclerosis, but these occur late in life when the force of natural selection has become attenuated. However, the frequency of ApoE4 is highest in the African subcontinent (especially in sub-Saharan Africa) and in certain other isolated populations such as in Papua New Guinea, all areas which exhibit endemic malaria. One hypothesis is that ApoE4 may give a selective advantage against falciparum malaria. In these studies, we determine the growth of malaria in human serum with different ApoE isoforms. We find malaria growth is greatly inhibited by ApoE4/4 serum, suggesting its maintenance in modern humans is a balance between early-life protection from malaria and increased risk of late-life diseases. (Support
provided by a grant from the National Institute on Minority Health and Health Disparities G12MD007591 from the National Institutes of Health. Support provided by the Semmes Foundation.) SAT-583
COMPARISON OF THREE DNA EXTRACTION KITS FOR DETECTION OF MYCOPLASMA SUIS DNA FROM
BLOOD AS ASSESSED BY QUANTITATIVE PCRNahimarys Colón1, Naila Nascimento2, Andrea Santos2, Kathy Johnson2, Joanne Messick2.
University of Puerto Rico, Aguadilla, Aguadilla, PR, 2Purdue University, West Lafayette, IN.
1 Mycoplasma suis is a blood-born pathogen of pigs. Both chronic and acute forms of infection have been reported.
Chronically infected animals show few clinical signs of disease; acute infection is characterized by fever, listlessness, anorexia, and in severe cases, by a life-threatening anemia. The detection of M. suis in blood samples using quantitative PCR (qPCR) depends on the efficiency of the extraction method used. This study evaluated 3 commercially available DNA extraction kits (Zymo Quick-gDNA™ Blood MiniPrep, Qiagen DNeasy Blood & Tissue Kit;
and Promega ReliaPrep™ Blood gDNA Miniprep System) to determine the relative recovery of M. suis from pig blood.
Serial dilutions of whole EDTA blood from an M. suis infected pig were subjected to DNA extraction using the various kits as per the manufacuturer’s instructions. Yield and purity of total genomic DNA extracted from the blood were compared spectrophotometrically while detection of the 16S rRNA gene of M. suis was performed using a Taqman qPCR assay specific for M. suis. Expected results are for the 3 DNA extraction kits to have a different yield and qPCR results.
THE ROLE OF CARBONIC ANHYDRASE IN CAMP SIGNALING AND SOCIAL MOTILITY IN T. BRUCEIWalter Hardesty, Kent Hill, Edwin Saada.
University of California, Los Angeles, Los Angeles, CA.
Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness, a disease with high mortality rates in sub-Saharan Africa. T. brucei has a complex lifecycle that alternates between a tsetse fly vector and a mammalian host. In the insect vector, migration through several tissues is required for maturation into mammalianinfectious forms. However, little is known about how surface contact impacts parasite behavior because African trypanosomes have been traditionally studied in suspension culture.The insect form of T. brucei engages in social motility (SoMo) when cultivated on semisolid agarose surfaces.It is found that SoMo is affected by changes in CO2 levels and regulated by flagellar adenylate cyclases (ACs), which catalyze the synthesis of cAMP. In many organisms, ACs function as CO2 sensors and rely on the activity of carbonic anhydrases (CA), which are enzymes that interconvert CO2 and bicarbonate. Here the role of the T. brucei CA as a mediator of cAMP signaling and social motility is investigated. To test this hypothesis, a knockdown of CA was first generated using tetracycline-inducible RNA interference. Cumulative growth data was collected on several clonal KD lines during uninduced and induced conditions. This data indicated that CA was not required for viability. Additionally, qPCR data further verified CA depletion of clones upon RNAi induction. SoMo assays are in progress to assess KD level and its impact on social motility. These studies could explain the role of carbonic anhydrases as an early step in cAMP signaling, thereby providing a novel target for therapeutic agents.
SCREENING FOR SMALL MOLECULE INHIBITORS OF THE DENGUE VIRUS PROTEASE AND TESTING FOR
DIS3L2 EXORIBONUCLEOLYTIC ACTIVITY IN S. CEREVISIAE IN VITRONorma Marie Elizaga, Daniel Engel.
University of Virginia Medical College, Charlottesville, VA.
Currently, there are no effective treatments for infections caused by dengue virus. The incidence of dengue infections is about 50 to 100 million people per year, making it a clear public health concern. These infections result in dengue fever and potentially lethal dengue shock syndrome. Our aim is directed toward drug development to cure dengue infection. The virus’s compact genome requires viral and host factor collaboration for virus propagation within the cell.
This project involves activity analyses of two proteins: the virus NS3 protease and host exoribonuclease Dis3L2. The NS3 protease, with cofactor NS2B, cleaves the viral polyprotein made early during infection, leading to production of mature viral proteins. We hypothesize that a small molecular weight, soluble, drug-like compound will inhibit protease activity. Assay plates were prepared containing NS3-NS2B purified from E. coli, artificial substrate Ac-nKRR-pNA, and 50 uM solutions of potential inhibitors. Absorbance readings of p-nitroaniline, a product of cleaved substrate,