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ALL-TRANS RETINOL DELIVERY BY IRBPAntonio Pena, Brandi Betts, Andrew Tsin.
University of Texas at San Antonio, San Antonio, TX.
The cone cycle involves the constant recycling of all-trans retinol (ATOL) and 11-cis-retinol (11COL). This occurs between Müller cells and cone photoreceptors cells within the retina. One abundant protein found in the interphotoreceptor matrix is interphotoreceptor-retinoid-binding protein (IRBP). IRBP has the ability to bind to these
HUR AND MIR-16 REGULATION OF CYCLIN E1 IN BREAST CANCERMelanie Connick1, Rebecca S. Hartley2.
University of New Mexico, Albuquerque, NM, 2University of New Mexico School of Medicine Cancer Center, 1 Albuquerque, NM.
Breast cancer is the second leading cause of cancer deaths in women and is the most common female cancer in the United States. RNA-binding proteins (RBPs) and microRNAs (miRNAs) have been shown to post-transcriptionally regulate gene expression in the development of cancer. Cyclin E1 is a cell-cycle regulator that is overexpressed in 30% of breast cancers. Overexpression correlates with cancer aggressiveness and poor prognosis. We have shown that the RBP HuR, also overexpressed in 30% of breast cancers, stabilizes Cyclin E1 mRNA leading to protein overexpression. MiR-16, a small, noncoding miRNA that is decreased in breast cancer, destabilizes and therefore decreases expression of Cyclin E1 mRNA. We set out to test the hypothesis that HuR impedes miR-16 destabilization of Cyclin E1 mRNA. To determine if Cyclin E1 mRNA bound by HuR can also bind miR-16, HuR was immunoprecipitated from MCF-7 cells, a human breast adenocarcinoma cell line in which Cyclin E1 and HuR are overexpressed. Argonaut 2 (Ago2), a protein that associates with miRNAs and is necessary for their function, was also immunoprecipitated. Immunoprecipitates were analyzed by western blot to determine if HuR and Ago2 are associated. Real-time PCR on RNA obtained from the cell lysates will further determine if Cyclin E1 mRNA is bound by Ago2 and HuR, and if miR-16 is bound by Ago2 and/or HuR. Determining the association of RBPs and miRNAs is important for understanding how their interaction plays a role in the development of breast cancer.
EFFECTS OF BIGH3 PROTEIN ON RHESUS MONKEY RETINAL ENDOTHELIAL CELLSJennifer Espinoza1, Andrew Mendiola2, Brandi Betts2, Andrew Tsin2.
Texas A&M International University, Laredo, TX, 2University of Texas at San Antonio, San Antonio, TX.
1 Diabetic retinopathy (DR) results from microvascular retinal changes that lead to vision loss. The diabetic milieu promotes inflammation that results in retinal tissue damage. The link between DR and inflammation remains unsolved;
however, we have recently shown that macrophages cultured in hyperglycemia and hyperlipidemia secrete TGF-β, which induces TGFβ-induced gene human clone 3 (BIGH3), a proapoptotic protein. The aim of this study is to assess cell viability of a continuous cell line of rhesus monkey retinal endothelial cells (RhREC) in response to the addition BIGH3. We will examine the hypothesis that BIGH3 will induce a time- and dose-dependent effect on cell viability.
Our general strategy is to culture RhREC and introduce recombinant BIGH3 protein at different concentrations in the condition media and measure cell viability over 96 h via trypan dye-exclusion assay. The results obtained from this experiment will provide information on the development of diabetic retinopathy.
INVESTIGATING THE ROLE OF ASCORBIC ACID IN MAINTAINING GENOMIC INSTABILITY IN HUMANINDUCED PLURIPOTENT STEM CELLSBrian Truong, Patrick Lee, Jason Awe, Agustin Vega Crespo, James Byrne.
University of California, Los Angeles, Los Angeles, CA.
Human-induced pluripotent stem (iPS) cells hold significant promise for future personalized treatments, but mitochondrial and nuclear genetic aberrations have been widely observed following the in vitro manipulation and culture of human iPS cells, making these cells unacceptable for personalized cellular therapeutics. Ascorbic acid is an
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antioxidant that has been used to improve the efficiency of both mouse and human iPS cell generation and facilitated the first generation of all-iPS-cell mice from terminally differentiated B cells. This property indicates its potential role in maintaining a normal karyotype capable of sustaining normal murine development. In this study, we will investigate the hypothesis that augmenting iPS cell-culture media with ascorbic acid can significantly enhance genomic stability following the stressful conversion of human iPS cells to clinical-grade (xeno-free substrates). This will be determined via gamma-histone 2AX immunocytochemistry, single nucleotide polymorphisms (SNP)-based loss of heterozygosity analysis, and karyotypic analysis after clinical-grade conversion in the presence of different concentrations of ascorbic acid.
SURVIVIN GENE EXPRESSION IN MULTIPLE MYELOMA CELLS UNDER TREATMENT WITH RESVERATROLAnthony Robateau, Lizbeth Romero.
Inter-American University of Puerto Rico, Arecibo Campus, Arecibo, PR.
Multiple myeloma is a type of cancer characterized by malignant plasma cells in the bone marrow. Patients with multiple myeloma are prone to lytic bone lesions and deficient immunity. Survivin, a survival gene, codes for a protein involved in the inhibition of apoptosis. It has been shown that cancer cells can become resistant to chemotherapy by multiple routes, including inhibition of apoptosis. Our objectives were to investigate the expression levels of the survivin gene in myeloma cells treated with resveratrol, a natural antioxidant found in grapes that has been shown to be a potential anticancer treatment. In this study, we analyze gene expression using real time quantitative PCR.
Results showed that the level of expression of survivin declined after 24 and 48 hrs of treatment with resveratrol.
Further studies are required to determine if the decrease in survivin expression is inhibiting apoptosis.
DEVELOPING A NEW SYSTEM AND IMPROVED TOOLS FOR THE STUDY OF NOVEL BACTERIOPHAGESMonica Zepeda1, Grant Hartzog2.
Gavilan College, Gilroy, CA, 2University of California, Santa Cruz, Santa Cruz, CA.
1 Bacteriophages are viruses that infect bacteria. They first bind to the surface of their specific bacterial host, then inject their DNA into the bacterium where their genome is replicated. Bacteriophage proteins are expressed and the replicated bacteriophage DNA is assembled into new viral particles. The infectious cycle concludes as the cell lyses and the new phages are released from the host. Bacteriophages are of particular interest for their potential as therapeutic and diagnostic agents of bacterial infections and for their potential uses to manipulate the genomes of their bacterial hosts. The objective of this study is to establish procedures for isolation of bacteriophages that infect the bacterial species Actimycetales arthrobacter for which only a few phages have been isolated. Soil from the University of California, Santa Cruz (UCSC) campus will be sampled for arthrobacter-specific bacteriophages.
These viruses will be purified and characterized by electron microscopy and isolation and restriction analysis of their DNA genomes. In the second project, proteomic analysis of bacteriophage Dori will be performed. This novel bacteriophage was isolated at UCSC using Mycobacterium smegmatis as the host. By performing mass-spectrometry on purified viral particles, Dori’s structural proteins will be identified and compared to previous genomic annotations.
These experiments will allow a better understanding of the genetics and structure of bacteriophage.
DETERMINING MRNA TARGETS AND FUNCTION OF CPEB4 IN ADIPOCYTES USING BIOINFORMATICSAPPROACHES Kimberly Insigne1, Mete Civelek2, Aldons Lusis2.
University of California, San Diego, La Jolla, CA, 2University of California, Los Angeles, Los Angeles, CA.
1 Metabolic syndrome (MetSyn) is a group of metabolic conditions that occur together and promote the development of cardiovascular disease (CVD) and type 2 diabetes. The incidence of MetSyn is predicted to increase as obesity has become a worldwide epidemic. Genome-wide association studies have identified over 400 genomic loci that are associated with obesity, CVD, and other MetSyn conditions. However, most of the underlying genes and the roles they play in the disease pathology remain unknown. Preliminary results have identified a causal gene called cytoplasmic polyadenylation element binding protein 4 (CPEB4), which encodes an RNA binding protein. It binds to target mRNAs to control translation. CPEB4 is significantly associated with waist-to-hip ratio in humans, one indicator of obesity. The function of CPEB4 has not been extensively explored, and its exact role in MetSyn is unknown. The main objective is to identify the mRNAs that are potentially targeted by CPEB4 using bioinformatics approaches. Using known
CHARACTERIZATION OF PLURIPOTENCY GENE EXPRESSION IN BABOON INDUCED PLURIPOTENT STEMCELLS Stephanie Ikediobi, John R. McCarrey.
University of Texas at San Antonio, San Antonio, TX.
Induced pluripotent stem cells (iPSCs) are typically derived from fibroblasts that are induced with pluripotency factors to be reprogrammed to a state similar to embryonic stem cells (ESCs). ESC lines have been established from humans; however, these lines are of concern due to ethical issues. Induced pluripotent stem cells (iPSCs) are derived from adult somatic cells, so they do not carry the same ethical concern but must still be optimized for therapeutic use.
Nonhuman primate-derived iPSCs provide a clinically relevant model for studies of the efficacy and safety of stem cell-based therapy because baboons share 92% genome sequence homology with humans. Many factors involving the therapeutic application of iPSCs need to be optimized prior to initiating clinical trials in humans. Baboons provide an ideal model system for this purpose. To be characterized as iPSCs, reprogrammed cells must express the OCT4, SOX2, and NANOG pluripotency genes. Primers were designed specifically for the baboon sequence of these genes plus β-actin (as a control), using the Rhesus macaque genome sequence as a reference. Multiple baboon iPSC (biPSC) lines were derived by transduction with pluripotency factors (OCT3/4, SOX2, NANOG, and KLF4). We plan to use RT-PCR to confirm the presence of mRNA transcripts for these pluripotency factors in biPSC lines. To date, we have optimized RT-PCR primers and conditions for detection of the β-actin and Oct4 transcripts in biPSCs. Our goal is to validate each of our baboon iPSC lines for future use in preclinical studies of patient-specific stem cell-based therapies.
THE ROLE OF ZFP148 IN VASCULAR FORMATION IN MOUSE SMOOTH MUSCLE CELLSValeria De La Rosa Reyes1, Morgan Angulo2, Gilbert R. Upchurch2, Gorav Ailawadi2.
Universidad de Puerto Rico en Humacao, Humacao, PR, 2University of Virginia School of Medicine, Charlottesville, 1 VA.
Aortic aneurysms (AAs) are an abnormal widening of a portion of an artery caused by smooth muscle cell death and extracelluar matrix degradation. In the US, AAs are the 15th leading cause of death, and around 12,000 patients die each year. Since AAs are largely asymptomatic and surgery is the treatment option, the development of novel diagnostic tools and treatments is critical to improve patient mortality. Zinc finger protein 148 (ZFP148) is a krüppel type zinc-finger transcription factor that plays a key role in hematopoiesis, skeletal muscle development, and gastric development; however, the role of ZFP148 in vascular development and disease remains unknown. The role of the current study is to determine the role in vitro of ZFP148 following elastase treatment in cells important for AA formation. We will knock down the expression of ZFP148 using siRNAs and then examine, using qPCR and zymography, the expression of: 1) smooth muscle marker genes (SM-actin, SM22, and smooth muscle myosin heavy chain); 2) inflammatory markers (MCP1, IL1β and IL6, TNFα); and 3) matrix metalloproteinases (MMP2 and 9) in smooth muscle cells and macrophages as appropriate. We predict that knockdown of ZFP148 will attenuate inflammatory gene expression and MMP activation during AA formation. From these studies, we hope to conclude that ZFP148 has a role in AA formation and could represent a viable treatment target for human disease. Future studies will include comparing ZFP148 and krüppel-like factor 4 (KLF4), a stem cell pluripotency factor whose deletion in smooth muscle cells results in attenuated aneurysm formation during AA formation.
CHARACTERIZING THE PLASTICITY OF NEURAL ECTODERM IN XENOPUS LAEVISKrissie Tellez, Brigette Jong, Carmen Domingo.
San Francisco State University, San Francisco, CA.
During embryogenesis, the 3 embryonic germ layers give rise to distinct cell types that form specific tissues and structures within the adult organism. In the young embryo, cells are plastic and are thus able to differentiate into many different cell types. However, as development progresses, cells lose their plasticity and eventually give rise
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