Mapping Functional Epitopes of Dengue Virus E2 DIII for Binding to Broadly-Neutralizing Antibody 4E11

Characterization of the EGFR signaling in acquired endocrine resistance using an MCF7 in vitro model Julia L. Alba, Esther A. Peterson, Paraic A. Kenny Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461 The ERBB family of receptors are transmembrane tyrosine kinases involved in the activation of complex signaling pathways that controls a variety of functions, including proliferation, adhesion, migration and differentiation. When there is an abnormality somewhere in this pathway it can promote tumorigenesis. Several ligands are responsible for the activation of the receptors including EGF, TGF-alpha, Betacellulin, Epiregulin, Neuregulin and Amphiregulin (AREG). There is evidence that after initially responding to endocrine therapy, many ER+ tumors progress to resistance to endocrine therapies and are frequently characterized by higher levels of EGFR , although the identity of the ligand(s) responsible remains unclear. For this study I am using sublines of an estrogen receptor (ER) positive breast cancer cell line, MCF7, which are resistant to either Fulvestrant (FulvR) or Tamoxifen (TamR). My goals are: (1) To test the expression of the EGFR ligands in each endocrine resistant line in comparison to parental MCF7 cells. (2) To assess the activation of the EGFR signaling pathway by testing the phosphorylation levels of EGFR and downstream effectors of the pathway (e.g. ERK1/2, AKT). The first approach used to test expression of the ligands was qRT-PCR. The data shows that FulvR line has higher Betacellulin expression compared to the parental control, while TamR has lower expression of all ligands tested when compared to the parental control. We used western blotting to determine the expression and/or activation of effectors of the EGFR signaling pathway in the endocrine resistant lines. The expression and/or phosphorylation status of ERα, EGFR, ERK1/2 and AKT in MCF7 parental, FulvR and TamR was determined in starved, steady-state conditions (complete growth media) and upon treatment with endocrine inhibitors, Fulvestrant (ICI) and Tamoxifen (OHT). In all cases, GAPDH was used as a loading control. Analysis of blots shows higher activation of EGFR signaling in FulvR cell lines under starved and steady-state conditions. Future experiments will include the use of siRNA techniques to knockdown the expression of the ligands expressed in the resistant lines. This will allow us to test if the resistant lines are dependent on specific ligands for survival and/or proliferation. In addition, targeting the EGFR signaling pathway using inhibitors will be performed to see if the endocrine resistant cells could potentially be resensitized to endocrine therapy. In summary, understanding the role of EGFR signaling in endocrine resistant lines could potentially help to develop alternative therapies for ER-alpha positive breast cancers that become resistant to endocrine therapy. Snf1-Regulated Transcription Factors are Required for Proteasome Mediated Lifespan Extension in Saccharomyces cerevisiae Sonia Barakat, Yanhua Yao, Marion Schmidt Department of Biochemistry at Albert Einstein College of Medicine Bronx, New York The AMPK pathway is highly conserved and plays a pivotal role in longevity and cellular response to caloric restriction. Our data demonstrates that proteasome mediated lifespan extension results in the deregulation of the AMPK/Snf1 signaling pathway causing an increase in respiratory capacity. We further propose an interconnected network containing the proteasome, Sir2, and AMPK/Hxk2 signaling that regulates lifespan in Saccharomyces cerevisiae.The switch from fermentation to respiration is controlled by AMPK/Snf1 signaling, Transcriptional regulators downstream of AMPK repress/activate genes in response to carbon source availability. A deregulation of the pathway leads to premature induction of respiration, increasing levels of ROS and affects aging. Increased respiration and oxidative stress induces the activity of proteolytic systems responsible for preventing the accumulation of aggregates that can lead to cell death. In aging cells, however, we find that the activity of the proteasome declines due to increased oxidative damage. We have shown previously that upregulation of proteasome activity in aging yeast cells has a beneficial effect on lifespan. This beneficial factor can be due to increased protein homestasis or caused by increased degradation of a negative regulator of lifespan downstream of AMP-kinase, namely Mig1 and Hxk2. In this project we test the hypothesis that increased degradation of Mig1 might contribute to proteasome mediated effects on lifespan. Mutant strains were constructed and allowed to grow on various media inducing different proteotoxic stresses and incubated at 30°C.Expression changes in response to proteotoxic stress prove that there is an interaction between these genes. Increased levels of proteotoxic stress, however, do not necessarily pose strictly harm to the cells. As you can see, cells were still able to grow even after being treated with different stresses. This helps to indicate that some doses of stress can be of valuable use to the cell as they activate different stress responses and pathways. Rpn4 is a transcription factor that stimulates the expression of proteasome genes and is transcriptionally regulated by various stress responses. Ubr2 is a cytoplasmic ubiquitin-protein ligase required for the ubiquitylation of Rpn4p. In ubr2∆ cells, Mig1 levels were lower when compared to rpn4∆ cells that have reduced proteasome activity. Based on these observations, it would suggest that Mig1 is targeted and degraded by the proteasome, necessary for proteasome-mediated lifespan extension, and is incorrectly processed under repressive conditions in cells with increased proteasome abundance. Increased degradation of Mig1 leads to the inactivation of Mig1 and premature activation of the AMPK/Snf1 pathway. Mig1 was further deleted in two other strains (Sir2 and Hxk2) that allow for lifespan extension. Sir2 is a conserved NAD dependent histone deacetylase involved in negatively regulating the initiation of DNA replication. Overexpression of Sir2 allows for increased longevity. Hxk2 directly regulates Mig1 and deletion of Hxk2, which acts as an activator for Mig1, leads to increased respiration and extends replicative and chronological lifespan in yeast. Because Hxk2 is upstream from Mig1, loss of MIG1 would not have any consequences on lifespan extension in Hxk2 cells. Having established that increased proteasome activity leads to a deregulation of the AMPK/Snf1 pathway, we investigated that the proteasome activity also affects Sir2 and Hxk2 mediated lifespan extension. In both strains, it was found that increased proteasome activity concurred an additive effect, while on the other hand, decreasing levels of proteasome activity completely blocked lifespan extension for both strains. Increased proteasome capacity improves viability under proteotoxic stress and elevated proteasome activity reduces the aggregation of misfolding proteins in aging cells. Elevated proteasome capacity also results in reduced abundance and nuclear localization of AMPK/Snf1-regulated Mig1, increasing respiratory activity, and increased oxidative stress response. Reduced or impaired proteasome activity limits lifespan extension in cells overexpressing SIR2 or in cells lacking Hxk2. We can therefore propose that a network between the proteasome, Sir2, and AMPK/Hxk2 signaling exists and impacts the longevity in Saccharomyces cerevisiae. Allelic Diversity of IFNAR-1 in African Patients with Malaria Jacqueline Benayoun, Lucas R. Cusumano,Seungjin Ryu, Catherine Manix Feintuch, Daouda Ndiaye, Esther Gondwe, Karl Seydel, Terrie Taylor, Yousin Suh, Johanna P. Daily Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, N.Y. Malaria is a serious and sometimes fatal disease that caused 207 million clinical episodes and 627,000 deaths in 2012 alone (World Health Organization). Genetic predispositions can modulate the risk for severe malaria. Prior studies have found the association of mutations within the Type I Interferon Receptor (IFNAR1) and disease outcomes in Plasmodium falciparum malaria. To further explore the role of mutations in IFNAR1 we carried out an in-depth Single Nuclear Polymorphism (n=21 SNPs) analysis at the IFNAR1 locus. We examined genomic DNA from children with cerebral malaria from Malawi and tested associations with disease outcomes. In addition we examined the IFNAR1 allelic diversity between West Africa (Senegal) and East Africa (Malawi). Type I IFNs are involved in host response to malaria. Type I IFNs prime macrophage proinflammatory responses, enhance intracellular killing, dendritic cell (DC) maturation and T helper 1 cell responses; and promote lymphocyte activity. Type 1 IFN has been associated with modulating malaria infection outcomes in the animal model of cerebral malaria and in human malarial disease. Type I IFN receptor (IFNAR1) polymorphisms are associated with disease outcomes in malaria. This will be tested through an association study of IFNAR1 alleles in patients with mild versus severe malaria. Genomic DNA was extracted from dry blood spots of African Patients using a DNeasy extraction kit. The samples were quantified by nanodrop and amplified using PCR. Gel electrophoresis was run to confirm the presence of the DNA. Then, Iplex was performed to determine the alleles present in the African cohorts in 21 SNPs. Finally, a Typer program was used to generate the patient spectra and allele call rate. After testing 21 SNPs, a 94% call rate from 20 SNPs was achieved. Out of the 20 primers that worked, 7 primers were monomorphic. From the remaining 13 primers, 4 primers showed no significant differences between the acute and mild cohorts, and. 3 were statistically significant. Significant differences are