ALBERT EINSTEIN COLLEGE OF MEDICINE OF YESHIVA UNIVERSITY GRADUATE DIVISION OF BIOMEDICAL SCIENCES SUMMER UNDERGRADUATE RESEARCH PROGRAM 2009

Cardiovascular disease (CVD) dominates as the leading cause of death in western-style societies. Inflammatory CVDs such as atherosclerosis and restenosis may be affected by allograft inflammatory factor-1 (AIF-1), which is reported to function as a pro-migratory, proproliferative, and pro-survival protein in vascular smooth muscle cells (VSMCs) and macrophages. Several preceding studies suggest that, through these effects on VSMCs and macrophages, AIF-1 acts to induce greater accumulation of neointima. We used a surgical carotid arterial ligation model to mimic aspects of the pathogenesis of atherosclerosis using AIF-1 knockout (KO) and WT mice on a C57BL/6J genetic background (n = 6 KO, n = 4 WT). We hypothesized that AIF-1 would induce greater neointima accumulation, thus the KO model would have smaller neointimas. The mice were anesthetized, and the ventral aspect of the neck was shaved, and cleaned with ethanol. A longitudinal incision was made in the skin overlying the left carotid artery. The artery was completely occluded by ligation near its bifurcation. The skin incision was closed using super glue, and the mice recovered amongst their cagemates. After 28 days, the mice were killed and the ligated arteries were harvested. The arteries were perfused and fixed with 4 % paraformaldehyde for 16 hours and embedded in parrafin. The paraffinembedded arteries were sectioned 5 μm thick using a microtome. The sections were mounted and stained with hemotoxylin-eosin and Verhoeff’s Elastic Stain. Morphometric analysis was performed with Photoshop® by calculating the area of the lumen and the areas inside the internal elastic and external elastic laminae. Contrary to our hypothesis, the KO mice showed a trend toward more neointima formation than WT mice, although this difference was not statistically significant (neointima/media area 0.5155 ± 0.1892 vs. 0.1446 ± 0.04673, p = 0.1599). These data suggest that AIF-1 may act to limit neointimal formation. AIF-1 has been implicated, based on its overexpression in VSMCs, as a promoter of neointima accumulation in previous publications. Our findings using mice completely lacking AIF-1 suggests that AIF-1 has a protective function that limits neointimal formation since total loss of AIF-1 was hypothesized, based on previous literature, to increase neointima. Future investigations would benefit by examining overexpressed AIF-1, AIF-1 KO, and WT mice. These studies could compare a broad range of AIF-1 expression levels, because it may be that AIF-1 functions best under optimal expression conditions. I would like to thank the Sibinga Laboratory, Nicholas Sibinga, MD, Yueting Shang, MD, PhD, Isabel Casimiro, MS, Prameladevi Chinnasamy, MS, and Dario Riascos-Bernal, MD. Jessica Brown was supported by NIH Grant No. T34 GM008718 and Isabel Casimiro was supported by the Cell Molecular Biology & Genetics Training Grant T32 GM007491. Swine Influenza or Seasonal Influenza? An Evaluation of Pediatric Patients at the Beginning of the 2009 Pandemic Bryk, D. , Zitter, S. , Narlieva, M. MS, Akid, I. , Pan, Q. PhD, Fox, A. MD, MS Montefiore Medical Center Bronx, NY 10467 There have been four major influenza pandemics in the twentieth century. The most recent one was the swine influenza pandemic in 1977 until novel swine influenza was identified in the United States in April 2009. Over the next two months, the situation escalated and the World Health Organization declared a swine influenza pandemic on June 11. The first 40 Pediatric Emergency Room (PER) patients who were positive on the rapid influenza A test (Quidel Corp., San Diego, CA) at the beginning of the pandemic were located in the hospital’s database. Thirty-one specimens (viral transport media) were available for testing on an automated platform. The dates of these samples ranged from April 22 to May 18. Viral RNA from the 31 specimens was extracted using the Abbott m2000sp (Abbott Molecular, Chicago, IL) and then was analyzed by RT-PCR using the Abbott m2000rt with CDC approved primers and probes. The primers and probes were for influenza A, swine influenza A, swine H1N1, and an internal positive control, RnaseP. Cycle number threshold (Ct) < 37 indicated a positive result. Of the 31 patients, 13 had swine influenza while 18 had seasonal influenza. From April 22 to May 13, 0/13 cases were swine flu. The first swine influenza case was detected on May 14. Following this, there were 12 more swine influenza cases and only 5 seasonal influenza cases. The prevalence of swine influenza increased as the month progressed ultimately becoming the dominant strain. Acknowledgements: SURP 2009 for support. Dr Gavin Cloherty and Dr. Danijela Lucic of Abbott Molecular Laboratories for advice and technical assistance. Dr. Tylis Chang for advice and technical support. ∗ For research use only 1 http://www.who. int/csr/resources/publications/swineflu/realtimeptpcr/en/index.html Novel biosensor for Cdc42 – N-WASP interaction, based on solvatochromic dyes Fay Burekhovich, Louis Hodgson Stern College for Women, Yeshiva University, New York, NY 10016 Gruss-Lipper Biophontics Center, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461 Cdc43, a member of Rho-family GTPase, regulates critical cellular functions including cell polarity maintenance and actin cytoskeleton rearrangement. Interaction of Cdc42 with one of its key downstream effectors, neuronal isoform of Wiskott Aldrich Syndrome Protein (N-WASP), has important implications with respect to cancer, where Cdc42-N-WASP binding plays a principal role in the establishment and function of invadopodia, invasive membrane extensions with matrix degrading activity vital for metastatic invasion of carcinoma cells. Studies utilizing fluorescent biosensors in live cancer cells have shown that N-WASP is active only at invadopodia, and does not impact normal cellular operations. Thus, inhibiting Cdc42-N-WASP interaction could be useful as a possible anti-metastatic therapy. Based on previous research that detected endogenous Cdc42 activation in living cells by its binging to the Cdc42 binding domain (CBD) of hematopoietic WASP. The derivatized GBD is attached to a solvent-sensitive dye that undergoes a great change in fluorescence emission intensity upon binding to activated, endogenous Cdc42. The new probe will be valuable in development of potential approaches for high-throughput screening of inhibitor libraries targeting Cdc42-N-WASP interaction, while minimizing spurious inhibition of hematopoietic WASP. Using recombinant DNA techniques, a novel biosensor for Cdc42-N-WASP interaction was produced and characterized in vitro. FOCUSED ULTRASOUND-INDUCED UNFOLDED PROTEIN RESPONSE Gaurab Chakrabarti, Subhrajit Saha and Chandan Guha Department of Radiation Oncology, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY Unfolded protein response (UPR) is a stress response induced in cells because of defects in protein folding during protein synthesis, in the endoplasmic reticulum. UPR induces the synthesis of chaperone molecules, such as , heat-shock proteins that attempt to correct misfolded proteins. On prolonged stress, unfolded proteins are targeted for proteosomal degradation. We hypothesized that therapeutic ultrasound would generate mechanical vibrational and thermal stress in cells, thereby, inducing UPR. The murine prostate cancer cell line RM1 and rat oval cells (ROC, hepatic stem cells) were treated with low intensity (LOFU: focal intensity ~500 W/cm2, frequency, 1 Mhz) and high intensity focused ultrasound (HIFU: focal intensity ~1300-2000 W/cm2 at 4 Mhz). Using qRT-PCR, we found that LOFU increased the expression of UPR target genes (GRP78 and EDEM) by 10-20 folds in RM1 cells, which was futher augmented by combination therapy of LOFU-HIFU to 200-800 folds. ELISA demonstrated an increase in HSP70 protein levels with subsequent release of HSPs from LOFU-HIFU treated cells (390±62.9ng/mil and 8-fold increase). LFU alone increased membrane bound HSP70 levels in ROC by 7.5 folds, as noted in FACS. The results indicate that focused ultrasound induces a UPR stress response in tumor and stem cells. Our long-term goals is to exploit the ultrasound-induced UPR stress response for generating tumor-derived peptide antigens for an autologous in situ tumor vaccine. Further investigation of UPR on differentiation of hepatic ROC progenitor cells is underway.