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«Strengthening the Nation through Diversity, Innovation & Leadership in STEM San Antonio,Texas · October 3-6, 2013 Get Connected! Connect with the ...»

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The plant pathogenic fungus Verticillium dahliae is a causal agent of verticillium-wilt disease. Verticillium wilt is difficult to control because the pathogen is capable of cross infecting multiple plant hosts, and persists for long periods in the field. The fungus produces melanized microsclerotia, which can survive for years in the soil. The microsclerotia also serve as the primary inoculum in the field. We previously identified over 200 genes that were differentially expressed in microsclerotial cultures of V. dahliae, suggesting the importance of these genes in the development and maintenance of microsclerotia. Nearly 50% of the genes identified encode hypothetical proteins of unknown function.

The objective of this work was to prepare knockout constructs for several of the genes highly differentially expressed in the V. dahliae microsclerotial cultures and to assess the role of these genes by functional analyses. The method of one-step construction of Agrobacterium tumefaciens recombination ready plasmids was applied for generation of the gene knockout constructs. Following fungal transformation, additional analyses will be conducted to determine whether the genes are important for morphogenesis and pathogenicity as well as microsclerotial development.

Assessing the roles of genes involved in microsclerotia development or germination may provide insight into alternative verticillium-wilt control strategies.




Ana Diaz, Richard LeBaron, Robert Moritz.

University of Texas at San Antonio, San Antonio, TX.

Since diabetes is a prevalent issue in my community, the purpose of our project is to gain insight into BIGH3 biology in type II diabetes. BIGH3 is a proapoptotic protein that is made in large amounts by aortic and renal cells under diabetic conditions. Based on previous studies, our lab has shown that the C-terminal end of BIGH3 protein is cleaved, inducing apoptosis. Therefore, the logical assumption is that there is a lot of cell death by apoptosis in type II diabetes and that BIGH3 cleavage promotes disease progression. The goal of our research is to study biological mechanisms that can stop this C-terminus cleavage. Our hypothesis is that if the C-terminus of BIGH3 could not be cleaved, there would be less cell death by apoptosis. To test this hypothesis, our research includes two approaches for blocking BIGH3 C-terminal cleavage and then testing for the extent of cleavage. One approach is mixing BIGH3, produced by aortic cells and renal cells, with anti-BIGH3 antibody (Bleed 8). The second approach tests whether extracellular matrix molecules that are known to bind BIGH3, e.g., fibronectin, collagen, will block BIGH3 cleavage. Techniques used include cell culture, protein electrophoresis, western blots, immunoblots, one-way ANOVA to quantify results, and densitometry analysis to measure the extent of cleavage. Preliminary results suggest that fibronectin and antiBIGH3 antibody prevent BIGH3 C-terminal cleavage, although more tests need to be done to quantify results. If our hypothesis is true, it is expected to lead to novel targets to develop agents to combat diabetes.



Nyka Tucker, Ana Gervassi.

Horton Lab, Seattle Biomedical Research Institute, Seattle, WA.

With an estimated 33.3 million individuals infected with HIV-1 at the end of 2010 and 2.5 million new infections per year, the need for a prophylactic HIV-1 vaccine is the biggest global health challenge this century. The variability and high mutation rate of HIV makes it difficult to design effective vaccines. Recent studies in individuals at early stages of infection have shown that the presence of HIV-specific CD8+ T cells recognizing conserved regions of the virus correlates with longer survival. However, the majority of HIV regions targeted during vaccination in healthy seronegatives are variable rather than conserved. These data led us to hypothesize that immunogens should be redesigned to contain only conserved regions of HIV-1 in order to increase the likelihood that T cells induced by vaccination will be able to recognize diverse incoming viral species. Therefore, the goal of this study is to assess the effectiveness of T cells targeting conserved versus variable HIV-1 epitopes to suppress viral replication. We have designed immunogens that contain only conserved regions of the virus and will compare these to current immunogens that also contain variable viral regions. Recombinant Lentivirus encoding all of the immunogens will be prepared and used to induce HIV-specific T cells by in vitro priming. HIV-specific T cells induced by conserved immunogens will then be compared to those induced by current immunogens to determine which are better at suppressing viral replication.

To date, experiments have focused on optimization of lentiviral expression of immunogens in monocyte derived dendritic cells.



Elena Caceres, Mark Huising.

Salk Institute for Biological Studies, San Diego, CA.

Type 1 diabetes (T1D) is an autoimmune disorder that targets insulin-producing β-cells in the pancreatic islets of Langerhans resulting in an inability to regulate blood glucose levels. As there is no cure for T1D, people living with the disease are treated with injections of insulin to prevent dangerously high blood glucose levels. However, insulin injections will not cure T1D; therefore, it is important for researchers to find ways to recover functional β-cell mass which will restore the ability to regulate blood glucose. In order to test the efficacy of any recovered β-cell mass or function, it is necessary to define how glucose profiles and pancreatic morphometry change with respect to diabetic models. Wild-type mice were administered a β–cell selective apoptotic agent, streptozotocin (STZ), or citrate and were monitored for 12 weeks with once weekly measurements by handheld glucometer in order to correlate loss of β-cell mass with the inability to maintain normal glucose profile. Mice treated with STZ demonstrated an increase in hyperglycemia (blood glucose measured 250 mg/dL at 2 consecutive readings). Analysis of β–cell mass showed a decrease in total insulin-positive surface area in STZ-treated mice. In addition to weekly measurements of blood

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Jesse Collins, Kristie D. Ibarra, Sara A. Watson, Eva Harris.

School of Public Health, University of California, Berkeley, Berkeley, CA.

Dengue is the most prevalent mosquito-borne viral illness of humans worldwide, with 100 million cases annually. Four related serotypes of dengue virus (DENV) cause unapparent infections and a range of disease manifestations, from classic dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome. Previous infection with one serotype results in life-long immunity to that same serotype, but increases the risk of severe disease on infection with a different (heterologous) serotype, possibly due to serotype cross-reactive antibodies or T cells. In antibody-dependent enhancement (ADE), cross-reactive, nonneutralizing antibodies raised during the first infection bind the second infecting serotype at subneutralizing levels and facilitate increased viral entry into target cells through Fcγ receptor-mediated endocytosis and phagocytosis. FcγRIIB is the only inhibitory Fcγ receptor. We hypothesized that the absence of FcγRIIB would lead to increased susceptibility to ADE, and increased disease severity in vivo. To test this, we obtained FcγRIIB-/- mice on a C57BL/6 and 129/Sv hybrid background. We are currently backcrossing these mice to C57BL/6 mice deficient in the interferon alpha/beta receptor (IFNAR-/-) to obtain IFNAR-/FcγRIIB-/- double knockout mice on a full C57BL/6 background. The interferon deficiency is necessary for mice to be infected with DENV, which has proteins that incapacitate the human interferon antiviral pathway but not the mouse counterparts. Characterization of serum from infected mice will reveal the breadth and specificity of the IFNAR-/FcγRIIB-/- antibody response, and infections performed under enhancing conditions should reveal the role of FcγRIIB in modulating dengue disease severity.




Yasmin Fahim1, Kathleen Borgmann2, Lin Tang2, Anuja Ghorpade2.

St. Mary’s University, San Antonio, TX, 2University of North Texas Health Science Center, Fort Worth, TX.

1 In the postantiretroviral therapy (ART) era, human immunodeficiency virus (HIV)-1-infected patients are living longer. There is growing concern pertaining to altered aging in these individuals in the setting of HIV-associated neurocognitive disorders (HAND) and combined ART (cART). Astrocytes play a critical role in neuronal health and disease. They are not productively infected with virus, but are highly active participants in neuropathogenesis. The effects of antiretrovirals on noninfected/activated neural cells in the aging, HIV-infected central nervous system (CNS) are understudied. We hypothesized that, during chronic HIV infection and inflammation, neural cells have altered inflammatory responses on long-term treatment with ART. Three classes of ART drugs were used in therapeutically prescribed combinations. Altered responses in primary human macrophages and astrocytes were determined by measuring metabolic activity and secretion of proinflammatory mediators. Primary human monocyte derived macrophage (MDM) culture methods were standardized for purity, differentiation, and infectability. The cultures expressed macrophage-specific markers as measured by both immunocytochemical staining and real-time PCR. On treatment with cART, metabolic activity and tumor necrosis factor-α expression were altered during inflammation.

Cultured human astrocytes were also treated with cART. Metabolic activity and the secretion of proinflammatory cytokines, CCL2, interleukin-6, and CXCL8, were significantly altered during inflammation. These data suggest that cART may affect the responses of healthy, noninfected MDM and astrocytes in the context of chronic inflammation, as observed in the brain during HIV CNS infection. These altered responses may contribute to increased astrocyte dysregulation and lead to neuronal toxicity and dysfunction, ultimately resulting in HAND.



Meagan Aguirre1, Peter Thomas2, Yosi Aizen2.

University of Texas at Austin, Austin, TX, 2University of Texas Marine Science Institute, Port Aransas, TX.

1 Growth factors such as insulin-like growth factors (IGFs) play a large, critical role in oocyte development and maturation in vertebrates. IGFs have been shown to be potent inducers of oocyte meiotic maturation (OM), the final

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stage of oocyte development. However, their sites and mechanisms of action are unknown. In this study, we tested the hypothesis that IGFs act directly on fully grown oocytes to induce OM using an in vitro zebrafish model of OM.

Treatment with IGF-1 or IGF-2 (100 µM) for 3 to 6 hours increased in vitro OM of follicle-enclosed oocytes severalfold compared to vehicle-treated controls. Both IGFs retained their ability to induce OM after removal of the follicle cells. We conclude from these experiments that IGFs act directly on oocytes to induce OM. Interestingly, IGFs also induced OM of smaller oocytes that were unresponsive to other hormones that regulate OM. The mechanisms of IGF stimulation of OM of both large and smaller zebrafish oocytes are currently being examined. An understanding of the control of OM in fish by IGFs and other growth factors and hormones could lead to improved procedures to control the onset of OM, ovulation, and spawning of captive broodstock and, therefore, could potentially be very valuable to the aquaculture industry.




Paul Bump1, Colin Du2, Andrea Bodnar2.

University of Hawaii at Manoa, Honolulu, HI, 2Bermuda Institute of Ocean Sciences, St.George’s, Bermuda.

1 Our global aging population poses one of the most significant challenges of the 21st century. Understanding the molecular and cellular mechanisms that underlie aging is an area of intense investigation. The sea urchin is a unique model organism to study aging as these animals grow indeterminately, a condition in which age is not correlated to functional decline or increasing mortality. Some species are extremely long-lived, living up to 200 years without showing signs of aging, cancer, or other age-related diseases. Sea urchins may achieve their extraordinary life history through continued tissue regeneration with age as a result of maintenance of stem cell populations. To test this hypothesis, we investigated the expression of a panel of stem cell genes in embryos and adult tissues of the sea urchin Lytechinus variegatus. Primers were designed for classic markers of stemness such as the transcription factors Sox2, Oct4, Ronin, myc, and a component of telomerase, TERT. We detected expression of these stem cell markers in RNA from four developmental stages and in adult coelomocytes, ampullae, gonad, esophagus, radial nerve, and Aristotle’s-lantern muscle tissue using qRT-PCR. These initial findings suggest that stem cells may be present in adult sea urchins, and future work will be conducted to understand their role in tissue regeneration. Understanding the mechanisms by which these animals maintain good health throughout their lives may ultimately lead to new avenues for prevention or treatment of age-related diseases in humans.




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