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These results offer new insights into the ability of embryonic cells to regulate pluripotency during development.
VALIDATION OF PICHIAPRO, A PROGRAM FOR AUTOMATED PROTEIN EXPRESSION AND FERMENTATION
OF PICHIA PASTORIS
University of Utah, Salt Lake City, UT, 2University of Utah, Howard Hughes Medical Institute, Salt Lake City, UT.
1 Pichia pastoris is a methanotrophic strain of yeast widely used for expression of recombinant proteins. Induction of methanol utilization is controlled by the AoxI promoter, which is activated by the presence of methanol and suppressed by the presence of other carbon sources such as glycerol. Because the concentration of methanol and glycerol control the level of AoxI transcription and growth rate, optimization of carbon source ratios and feed rate can be used to tune protein expression levels and growth rate by placing the desired protein coding sequence under the control of the AoxI promoter. While feed rates can be crudely controlled without automation, it becomes a laborious and inaccurate process. The Capecchi lab has used mathematical models of Pichia metabolism to create a program, PichiaPro, which interacts with a benchtop fermentor. These models allow PichiaPro to predict the current amount of methanol and glycerol necessary to achieve a set growth rate. Monitoring the continuously changing culture density and volume, while simultaneously calculating methanol and glycerol demands, enable complete growth rate control and automation. Similar systems are currently available only for industrial use; we hope to make this technology accessible to others for a wide variety of applications. To first validate the program’s ability to control culture growth and AoxI induction (protein yield), we compared expression of tdTomato under a variety of programmed culture conditions. The data presented here demonstrate that by enabling growth-rate limited feeds, PichiaPro allows for precise control of growth conditions for optimization of protein expression.
SCREENING NOVEL SYNTHETIC COMPOUNDS FOR THEIR CYTOTOXICITY AND MECHANISM OF CELLDEATH Reyna Valdez, DeAnna Ayupova, Kafayat Busari.
University of Houston-Downtown, Houston, TX.
Cancer is a collection of diseases hallmarked by uncontrolled cell division. Cancer treatment involves varying combinations of surgery, radiation, chemotherapy, and hormone therapy. Chemotherapy employs the use of drugs that kill rapidly dividing cells, a characteristic of cancer cells. Various chemotherapeutic drugs such as paclitaxel and vinblastine interrupt cell division by binding to tubulin, a protein responsible for spindle formation which is a critical step in cell division. For more than 50 years, tubulin-binding drugs have been used to treat cancer. Scientists are still in search of novel anti-cancer compounds targeting tubulin for two reasons: 1) patients develop resistance to existing drugs, and 2) tubulin is one of the most validated targets for cancer treatment. Eighteen analogues of coscinamides were screened for their cytotoxic effects on 5 different cancer cell lines. Our initial screen identified three compounds (TMCOS-3, 6, and 11) that caused cell death with IC50 less than 1µM. TMCOS-3, 6, and 11 caused cell death via apoptosis when investigated for their mechanism of cell death using DNA fragmentation as an apoptotic marker.
66 UNDERGRADUATE POSTER ABSTRACTS
Biological Sciences SAT-511
SURVEY AND DETECTION OF THE WESTERN BLACKLEGGED TICK (IXODES PACIFICUS) AND THE LYMECAUSING SPIROCHETE (BORRELIA BURGDORFERI) IN UTAHSeth Drury, Ryan Davis, Ricardo Ramirez, Scott Bernhardt.
Utah State University, Logan, UT.
According to the Centers for Disease Control and Prevention, Lyme disease is the most reported vector-borne disease in the United States. In Utah, an average of 7 Lyme disease cases are reported annually; however, no cases are believed to have originated in Utah. In 2010, 36 individuals from Lehi, Utah claimed to be infected with Lyme disease, Borrelia burgdorferi. This prompted the Utah Health Department to create a Lyme Disease Taskforce to investigate these cases. As a taskforce member, Utah State University surveyed for and tested western blacklegged ticks (WBLT), Ixodes pacificus, for the presence of B. burgdorferi using PCR techniques. Ticks were collected by dragging a 1 m x 1.25 m white felt cloth (flag) through tick-conducive areas. Collected ticks were placed into a 70% ethanol solution, identified, and imaged. A total of 119 I. pacificus and 188 Dermacentor andersoni were collected.
Zero I. pacificus tested positive for B.burgdorferi. Since D. andersoni ticks are not competent vectors of Lyme disease, they were not tested. Weather, temperature, elevation, GPS coordinates, and habitat/vegetation data were also collected and summarized.
DEREGULATED MICRORNA EXPRESSION IN THE PROGRESSION OF SALIVARY GLAND CANCERHao Zheng1, Alan Kiang1, Weg Ongkeko2.
Veterans Administration Medical Center, San Diego, San Diego, CA, 2University of California, San Diego, La Jolla, 1 CA.
Salivary gland cancer constitutes 1 of the 5 main cancers in the head and neck region. Nonresectable malignant tumors demonstrate a low 20-year survival rate due to radioresistance and a high propensity for metastasis. The molecular mechanisms underlying pathogenesis of salivary cancer remain poorly understood. MicroRNAs (miRNA) are small noncoding RNAs that are predicted to regulate up to 30% of protein-encoding genes. Signature miRNA expression profiles have been identified in various malignancies, implicating miRNAs in the progression of human cancer. In order to better understand the role of miRNAs in salivary cancer, we profiled the miRNA expression levels of normal salivary tissue, benign salivary tumor, and salivary cancer. A total of 17 formalin-fixed, paraffinembedded salivary tissues, including 4 normal, 4 pleomorphic adenoma (PA), 3 squamous cell carcinoma (SCC), 3 mucoepidermoid carcinoma (ME) and 3 adenocarcinoma (AC), were collected and the endogenous expression of 95 miRNAs was analyzed by microarray. A large number of miRNAs were observed to be aberrantly expressed. An unsupervised clustering and a Student t-test were performed with a threshold p-value less than 0.05, resulting in the identification of 22 miRNAs differentially expressed at a statistically significant level. Eight candidate miRNAs were selected and further validated by RT-qPCR. The results suggest 3 miRNAs in SCC (miR-200a, let-7-family, and miRand 3 miRNAs in AC (miR-107, miR-15b and miR-200a) to be highly involved in the carcinogenetic process of salivary cancers. The miRNAs identified in this study may serve as potential biomarkers and targets for future miRNA based therapy.
THE EFFECTS OF GLYCOPROTEINS IN HANTAAN PSEUDOVIRIONS ON INFECTION OF VERO E6 CELLSAsra Khan, Meda Higa.
York College of Pennsylvania, York, PA.
Hantaviruses (family Bunyaviridae) are carried by rodents and can infect humans through aerosolized feces.
Hantavirus infection can lead to death by producing hemorrhagic fever with renal syndrome and pulmonary syndrome.
Glycoproteins Gn and Gc are two surface proteins encoded by the hantavirus genome. Gn and Gc are involved in the virus-host interactions, however the function of the glycosylation sites within these proteins remains unclear. To study this role, pseudovirions will be used. Pseudovirions provide a safer alternate by expressing the glycoproteins on the surface of a vesicular stomatitis virus (VSV) core modified with the reporter gene, luciferase but lacking the ability to replicate in its entirety. We have seen that hantavirus pseudovirions are capable of infecting Vero E6 cells and we are now poised to evaluate the function of the glycosylation sites on the Gn protein. Glycoprotein constructs will be expressed and deglycosylated with different enzymes. After incorporating these modified glycoproteins onto the VSV viral core, infection rate will be determined by fluorescence. We anticipate viruses containing deglycosylated
Gn proteins will display lower levels of infection. Our expected findings will help understand the function of the glycosylation sites on hantavirus infection and hopefully aid researchers in finding an effective vaccine in the future.
TWO-HYBRID ANALYSIS OF PAC1P INTERACTIONS WITH BINDING PROTEINSKayla Davis, Annabel Alonso, Rita Miller.
Oklahoma State University, Stillwater, OK.
The mitotic spindle is the cytoskeletal machinery that separates genetic information stored in chromosomes from the mother cell to the daughter cells during cell division. The positioning of the mitotic spindle is dependent on microtubule lengths and orientation. In the model organism Saccharomyces cerevisiae, the protein Pac1 helps in the localization of dynein to the plus end of the microtubule. The recruitment of dynein to the plus end of the microtubule is necessary for the sliding of microtubules along the bud cortex, and it has been shown that there is no microtubule sliding in cells lacking Pac1p. Pac1p interacts with the small ubiquitin-like modifier (SUMO), and ubiquitin itself. SUMO is an important post-translational modification of proteins in the cell that regulates many critical cellular processes, including nuclear transport, transcription, chromosome segregation, and DNA repair. It is not known how the attachment of SUMO to Pac1p alters its function or the function of dynein. Our lab has identified 2 sites of Pac1p modification, 2KàR mutants. Using 2-hybrid analysis, we have identified changes in protein-protein interaction in the 2KàR mutants compared to wild type.
FRI-495 MID1/MID2-DEPENDENT REGULATION OF P100 PROTEIN LEVELS Edmundo Vides, Jorge Torres.
University of California, Los Angeles, Los Angeles, CA.
Cell division is an essential step for cancer cell proliferation and cancer progression. Through understanding cell division, targeted therapeutics can be developed to help in the treatment of cancer. The MID1 and MID2 ubiquitin E3 ligases have been implicated as regulators of cell division. However, the mitotic proteins that they ubiquitinate remain unknown. Recently, we determined that MID1 and MID2 bind to p100, a well-known microtubule bundling protein, during mitosis. This led us to hypothesize that MID1/MID2 act to target p100 for degradation during mitosis.
Here, we show that MID1/MID2 immunoprecipitate with p100. Using immunofluorescence microscopy we determined that MID1/MID2 colocalize with p100 to the cytokinetic bridge during cytokinesis. Depletion of MID1/MID2 by siRNA treatment led to the stabilization of p100 protein levels, indicating that MID1/MID2 are involved in regulating the levels of p100. Additionally, MID1/MID2 were found to ubiquitinate p100 in in vitro ubiquitination assays. These data are consistent with a model where MID1 and MID2 regulate p100 protein levels by ubiquitinating and targeting it for degradation by the proteasome. P100 degradation then allows for cell abscission to be completed and the release of microtubule bundling that is not required during interphase of the cell cycle. Our studies have increased our understanding of the molecular mechanisms by which MID1/MID2 are critical for cancer cell division and highlight the possibility of developing small molecule inhibitors to MID1/MID2 to inhibit cancer cell division.
COLD-INDUCIBLE RNA BINDING PROTEIN IN THE DEVELOPMENT OF BREAST CANCERSelina Garcia, Rebecca S. Hartley.
University of New Mexico, Albuquerque, NM.
Cold-inducible RNA binding protein (CIRP) moves from the nucleus to the cytoplasm in response to stress, where it regulates cellular proliferation. CIRP is overexpressed in 30% of 33 breast tumors examined and is overexpressed in breast cancer cells in vitro. The goals of this study are to determine if CIRP expression is elevated in breast tumors compared to matched normal tissue, differs among breast cancer subtypes, and correlates with proliferation. Indirect immunofluorescence analysis was performed for CIRP and Ki67, a proliferation marker, in 6 breast tissue microarrays.
Microarrays included 25 tumors and their matched normal tissue for three cancer subtypes. The subtypes were based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). ER+PR+HER2- tumors are least aggressive, ER-HER2+ tumors are more aggressive, and ERPR-HER2- tumors are most aggressive. CIRP and Ki67 were assessed and results show that ER+PR+HER2- and ER-PR-HER2- tumors have significantly higher nuclear CIRP compared to matched normal tissue, while ER-HER2+ tumors have significantly lower cytoplasmic CIRP than matched normal tissue. The ratio of nuclear versus cytoplasmic CIRP in ER+PR+HER2- and ER-HER2+ tumors was significantly higher than their matched normal pairs. Both
THE ROLE OF SMAD6 AND SMAD7 IN CARDIOGENESISRana Besada, Andrew Harmon, Atsushi Nakano.
University of California, Los Angeles, Los Angeles, CA.