2012 Keynote Video	2012 Presentations	2012 Poster Session

Oral Presentation & Poster Abstracts

Oral Presentation Abstracts
Oral presentation abstracts will be reviewed by the student committee and posted here in July.

Poster Presentation Abstracts
Poster presentation abstracts will be posted as they are submitted.

1) Katherine Amato, "Targeting EphA2 in Tyrosine Kinase Inhibitor Resistant Lung Cancer," Nathanael Gray, Jin Chen

Lung cancer is the leading cause of cancer-related deaths in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers with an overall five-year survival rate of approximately 15%. However, NSCLC is not a single disease, but rather represents a composition of unique molecular aberrations. Identifying the relevant molecular subtypes of this heterogeneous disease and matching patients with appropriate targeted agents is of critical importance in the treatment of NSCLC. Although significant progress has been made over the past decade to develop new, molecularly-targeted therapeutics, challenges still remain in targeting the most commonly occurring mutations in lung cancer, KRAS and EGFR. Overcoming the large composite of challenges related to targeting KRAS and EGFR mutant lung tumors will require astute, robust strategies to identify and inhibit novel kinase targets required for cell viability in these subtypes of lung cancer. To this end, we have shown that the EphA2 receptor tyrosine kinase plays a unique role in the maintenance of cell viability in lung cancer, especially in lung cancers with KRAS and EGFR activating mutations. EphA2 is generally overexpressed in NSCLC, and it is often further overexpressed in KRAS and EGFR T790M mutant NSCLC. Preliminary data from our lab has shown that knockdown of EphA2 expression in a large number of human NSCLC cell lines inhibits tumor cell viability, most dramatically in cell lines bearing KRAS and EGFR mutations. To further assess the clinical relevance and utility of pharmacological inhibition of EphA2 to affect cell viability and tumor growth, we identified and characterized a novel, EphA2-specific kinase inhibitor, ALW-II-41-27. This EphA2 inhibitor has shown striking reduction of cell viability in vitro on an array of NSCLC cell lines including but not limited to KRAS and EGFR T790M mutant cell lines. ALW-II-41-27 has also induced tumor regression in KRAS and EGFR mutant NSCLC xenografts. Our preliminary data strongly suggests that targeting EphA2 holds unique promise for a difficult-to-treat subset of NSCLC. Based on our preliminary data, we hypothesize that EphA2 provides a novel target for modulating cell viability in lung cancers with KRAS or EGFR mutations associated with limited therapeutic options.

2) Stephanie H. DeLuca, "RosettaEPR: Combining Computational Rigid Body Sampling with Sparse EPR Data to Improve Membrane Protein Structure Determination," Nathan S. Alexander, Hassane S. Mchaourab, Jens Meiler

Membrane proteins remain a particular challenge in structural biology. Only about 1.5% of reported tertiary structures and sixty unique membrane protein topologies consisting of more than one transmembrane span are represented in the PDB. However, these proteins make up over 30% of the entire proteome, and over half of all therapeutics target this group. Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is often used for the structural characterization of proteins that elude X-ray crystallography and NMR, but high-resolution structures are difficult to obtain via EPR due to uncertainty in the spin label location and sparseness of experimental data. RosettaEPR has been designed to combine the strengths of both computation and EPR spectroscopy. We have demonstrated the feasibility of using RosettaEPR with soluble proteins by testing the method on T4-lysozyme; after full-atom refinement, a 1.7Å model of the protein was produced (Hirst, et al., 2011). RosettaEPR was also benchmarked on eleven membrane proteins of known structure in combination with simulated EPR distance data. While it was generally observed that de novo folding in the presence of EPR restraints enriched the recovery of the proteins’ correct topology compared to when folding with no restraints, it was clear that an innovative conformational sampling strategy was still needed. The preliminary work presented here demonstrates how a new membrane protein sampling protocol, in which transmembrane helices are moved as rigid bodies, can further enhance RosettaEPR’s performance. By moving entire helices in relation to one another, Rosetta is able to form more long-range, high contact order interactions, which are extremely under-sampled when using the traditional fragment assembly folding protocol. Folding with rigid body sampling increased the percentage of correctly folded models for 10 of 12 helical membrane proteins, including rhodopsin. Rosetta was able to fold rhodopsin, a GPCR, to 4.5Å RMSD₁₀₀ in the transmembrane regions. This newly implemented membrane protein folding algorithm can be combined with EPR data to enhance larger and more complex topologies that are consistent with experimental information.

3) Myriam Díaz Martínez, "Imaging Characterization of Adipose Tissue Before and After Roux-en-Y Gastric Bypass Surgery," E. Brian Welch

Background and Specific Aims: The aim of this study is to characterize adipose tissue (AT) in an obese rat model before and after gastric bypass surgery, utilizing MRI, PET imaging and molecular/cellular analysis. Morbid obesity is an increasing epidemic in the US and is the leading factor in the development of cardiovascular diseases such as coronary atherosclerosis, hypertension, dyslipidemia, and Type 2 diabetes (T2DM). Currently, gastrointestinal bypass surgery, specifically Roux-en-Y bypass (RGBP) surgery, is the most established and effective treatment for substantial and sustained weight loss in morbidly obese subjects. However, the mechanism in which the tissue of the adipose organ changes is not well understood. Strategies have been developed to understand the role and changes of this endocrine organ before, during and after the RGBP surgery, particularly its spatial distribution, triglyceride composition, and mitochondrial density along with molecular and cellular approaches.
Approach: We will investigate AT using MRI and PET imaging on obese rats before and after RGBP surgery. A number of control obese rats will undergo a sham RGBP surgery. MRI-based characterization of AT will include analysis of: (a) spatial distribution (visceral and subcutaneous) using fat-water MRI, (b) triglyceride chain length, unsaturation degree and polyunsaturation degree composition, and (c) R2* relaxation time within AT to detect iron concentration increases associated with inflammation. Also we will perform PET/CT scans using 18F-FDG and TSPO contrast agents to respectively determine glucose uptake and mitochondrial density within brown adipose tissue (BAT) that is distinct from white adipose tissue (WAT). We will compare our results with metabolic blood panels and molecular biology techniques to validate and understand AT changes before, during and after RGBP surgery.
Results & Significance: Many advances have been made to understand the underlying mechanism of prolonged weight loss after Roux-en-Y, but characterization of AT, specifically spatial distribution, triglyceride composition, inflammation status and mitochondrial density before and after surgery, has not been previously measured in a single imaging-based study. Investigating the role played by AT in combination with hormone and cellular signaling will provide a better understanding of weight loss mechanisms after gastrointestinal (GI) bypass intervention.

4) Laura A. Mike, "A small molecule that stimulates endogenous heme biosynthesis is toxic to fermenting Staphylococcus aureus," Brendan F. Dutter, Devin L. Stauff, Jessica L. Moore, Nicholas P. Vitko, Olusegun Aranmolate, Thomas E. Kehl-Fie, Sarah Sullivan, Paul R. Reid, Jennifer L. DuBois, Anthony R. Richardson, Richard M. Caprioli, Gary A. Sulikowski, and Eric P. Skaar

Staphylococcus aureus is a Gram-positive pathogen that causes a wide range of pathologies ranging from superficial skin infections to deep seeded osteomyelitis and endocarditis. In order to be a successful pathogen in diverse niches S. aureus senses and adapts to environmental changes using two-component signaling systems (TCSs). Upon entry into the host S. aureus encounters an environment devoid of free iron. S. aureus satisfies its iron and heme requirements by scavenging host heme from hemoglobin. While heme is a valuable iron source and co-factor, the reactivity of the molecule is a liability to the cell. The S. aureus TCS heme sensor system (HssRS) is activated in the presence of heme and up-regulates the heme regulated transporter (HrtAB), which protects the bacteria from heme toxicity. In order to understand the mechanism by which S. aureus senses and responds to heme toxicity during infection, a high-throughput screen for small molecule activators of HssRS was performed. The most potent HssRS activator, VU0038882 (‘882), was found to stimulate endogenous heme biosynthesis by perturbing central metabolism. Furthermore, the ‘882-mediated alterations to central metabolism are toxic to fermenting S. aureus, including clinically relevant small colony variants (SCVs). Targeting fermentation in a facultative anaerobe has therapeutic utility as ‘882 prevents the emergence of antibiotic resistance, enhances phagocyte killing and reduces S. aureus pathogenesis. This small molecule has revealed fundamental insights into staphylococcal heme homeostasis as well as provided a novel therapeutic strategy for combating this aggressive pathogen.

5) Md. Imam Uddin, "SLC7A11 and SLC1A5 Targeted Amino Acids− ¹⁸F-FP-GLU and 18F-GLN as Imaging-Biomarkers for Colon-Cancer," Yiu-Yin Cheung, Matthew R. Hight, Jason R. Buck, Sam A. Saleh, Mike Nickels, Eliot T. McKinley, George H. Wilson, Noor Tantawy, H. Charles Manning

Background and Specific Aim: [¹⁸F]-PET is one of the sensitive noninvasive modalities that can quantitatively analyze the tracer bio-distribution and tissue-uptake and could measure the activity of oncogenic events and have broad impact on clinical needs. Recent studies have shown that amino-acid transporter proteins SLC7A11 and SLC1A5 play an important role in transporting ¹⁸F-4FP-GLU and ¹⁸F-4F-GLN respectively, across the cell membrane. Though, ¹⁸F-FDG PET is used clinically for visualization of tumors in colon and other lesions, measures are more often confounded by glucose uptake in normal tissues that results in modest to poor tumor-to-background ratios. We envisioned to use¹⁸F-4FP-GLU and ¹⁸F-4F-GLN as new radiotracers for the detection of colon-cancer in xenograft models, with near term clinical applications.
Approach: Precursor was chemically synthesized that is structurally suitable for ¹⁸F-incorporation and PET-imaging studies. Human CRC cell line overexpressing SLC7A11 and SLC1A5 were used to generate tumor bearing athymic nude mice by implanting the cells. In vivo [¹⁸F]-PET imaging was performed by radiochemical synthesis and intravenous injection of the ¹⁸F-4FP-GLU and ¹⁸F-4F-GLN in tumor bearing xenograft models and PET-image analysis.
Results and Significance: Successful incorporation of ¹⁸F by radiochemical synthesis using a tosylate or a nosylate-precursor, and retro orbital injection followed by in vivo PET-imaging showed significant uptake of the radiotracer in tumor compared to normal tissues. These PET-radiotracers thus visualized and translated the proliferative tissues in xenograft model. In vivo stability of the probes and bio-distribution results reveled, these new tracers as promising PET ligand for evaluating proliferation in colon-cancers and potentially other cancer types.

6) Andrew Cognata, “Real-Time Electrochemical Monitoring of Metabolic Processes In Hollow Fiber Bioreactor Cellular Cultures,” David Cliffel

Two dimensional cell culturing are the primary means for investigation of animal tissue cells. The utility of this approach is limited by a number of constraints, most notably the limited lifespan of individual cultures and a failure to reproduce the in vivo conditions that cells and tissues would typically experience. Hollow Fiber Bioreactors (HFBx), a three dimensional culturing methodology that that simulates the physical stresses and selective behavior of biological membranes allowing for more realistic in vitro conditions when observing cell behavior, are an emerging solution to these limitations. Preliminary research using HFBx indicates that these conditions can dramatically affect the metabolic activity of cells, in many cases significantly increasing the yield of many biomolecules on a per cell basis. Awareness of the metabolic behavior of cells is essential both for the in-lab maintenance of cell cultures and observation of cellular behavior during experimental investigations. Current quantitative sampling techniques for HFBx center are predominately off-line, periodic methods, or else utilize expensive instrumentation. Such methods fail to provide a low-cost, real-time monitoring solution. To address this deficiency, a screen-printed electrode (SPE) array encased in a microfluidic flow chamber has been developed for use in electrochemical monitoring of cellular metabolites including glucose, lactate, oxygen, and extracellular acidification in the effluent released from the HFBx chamber. This approach, which utilizes a variety of modifiable electrodes, allows a selection of metabolic processes to be monitored in real time with excellent selectivity. The SPE is comprised of five platinum electrodes, which can be modified to electrochemically detect a wide number of analytes, including those mentioned above, through the application of a variety of selective enzyme and metal films. Individual sensors are inexpensive, reusable during their lifetime of several weeks, and easily configured using little or no modification to preexisting procedures. The capabilities of the monitoring method are demonstrated by real-time monitoring of both basal metabolic rates and responses to well-studied metabolic stimuli in immortalized cell lines and validated by comparison to similar experiments previously conducted using an established real-time detector for 2D cultures (such as the Multianalyte Microphysiometer) which is not compatible with the HFBx. This demonstrates the utility and validity of electrochemical detectors for the monitoring of HFBx, an important step in taking full advantage of the opportunities provided by bioreactors and over 2D culturing environments.

7) Amanda Duran, "Computational Design for a Symmetric Glycerol Facilitator Protein," Steven Combs, Jens Meiler

Proteins that exhibit structural symmetry (despite divergent amino acid sequences) are proposed to be the result of gene duplication, fusion, and diversification events originating from a monomeric gene. Several membrane protein structures exhibit an inverted symmetric topology. The Glycerol Facilitator protein (GlpF) from Escherichia coli is a membrane protein that exhibits a two-fold inverted pseudo-symmetry. The crystal structure for GlpF (PDBID:1fx8) is of high resolution with a C α root mean square deviation (RMSD) of 1.8 Å between the inverted halves which initially made it ideal for this study. Using the Combinatorial Extension alignment server and the Rosetta Protein Modeling Suite, GlpF was computationally engineered to be perfectly symmetric in sequence and presumably in structure. The symmetric gene was assembled, cloned, and expressed; however, many challenges have prevented structural characterization. In an attempt to better understand this poorly behaving engineered protein, Rosetta Membrane and Design are currently being used to further re-design this protein.

8)  David P. Nannemann, "Human neutralizing monoclonal antibodies that recognize respiratory droplet transmissible H5N1 influenza viruses," Natalie J. Thornburg, David L. Blum, Jessica A. Belser, Terrence M. Tumpey, Shyam Deshpande, Gloria A. Fritz, Jens C. Krause, Jeong Hyun Lee, Andrew B. Ward, David E. Lee, Sheng Li, Katie L. Winarski, Benjamin W. Spiller, Jens Meiler, and James E. Crowe Jr.

Recent research described the experimental adaptation of influenza H5 hemagglutinins (HAs) that confers respiratory droplet transmission to influenza virus in ferrets, which may mimic the future development of a highly pathogenic pandemic H5 “bird flu” virus in nature. This research set off a national debate on publication of research with potential dual use in bioterrorism. Human hybridomas from peripheral blood mononuclear cells from patients who had participated previously in clinical trials for an H5N1 vaccine were generated and characterized for antigen binding and neutralization of avian influenza viruses. Antibodies that bind the HA head domain and recognize both circulating and respiratory droplet transmissible H5 HAs have been identified. Using a hybrid methods approach to synthesize a number of structural techniques, including X-ray crystallography, electron microscopy, hydrogen/deuterium exchange mass spectrometry, and binding affinity assay, we defined a mechanism of antibody binding to the head of HA in a manner that retains molecular recognition of HAs with mutations that render H5N1 viruses transmissible by respiratory droplet in mammalian models. The model was validated through characterization of HA mutations predicted to confer antibody escape and explains naturally-occurring escape mutations. Rational design of novel H5 HA immunogens to protect against mammalian adapted mutants should consider incorporation or retention of this critical antigenic site. Furthermore, engineering of these antibodies may allow for therapeutic application in the event of avian influenza pandemic.

9)  William R. Birmingham," Bioretrosynthetic Evolution of a Didanosine Biosynthetic Pathway," Timothy D. Panosian, David P. Nannemann, Chrystal A. Starbird, Tina M. Iverson, Brian O. Bachmann

The next logical step in the engineering of biocatalysts is their concatenation into multi-step non-natural biosynthetic pathways. We have combined the conceptual design paradigm of retrosynthesis and the theory of retrograde evolution in ‘bioretrosynthesis’, an applied method for constructing an in vitro biosynthetic pathway, and evaluate the strategy in vitro using the nucleoside analog didanosine as a model target. As a retrograde pathway extension from an engineered purine nucleoside phosphorylase, we have evolved phosphopentomutase activity toward the non-natural substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate specificity and 3-fold increased activity in cell lysate. An additional retrograde pathway extension toward engineering a dideoxyribokinase provided a 50-fold total increase in didanosine production and ~9,500-fold change in nucleoside production specificity in a five step biosynthetic pathway beginning from a simple sugar analog 2,3-dideoxyribose and including an ATP regeneration cycle. Unexpectedly, the biosynthetic productivity of this engineered ribokinase is the result of a new phosphorylation regioselectivity, creating a pathway shortening bypass by directly forming a substrate for purine nucleoside phosphorylase. This previously uncharacterized activity was detected through testing for final product formation in the bioretrosynthesis evolution strategy rather than possibly having gone overlooked by screening for the anticipated dideoxyribose 5-phosphate intermediate.