MABEC 2003 UMCP MABEC 2003 Home Registration Abstract Submission Program Keynote Abstracts Participants Travel Sponsors UMCP Links UMCP College of Engineering Chemical Engineering Timothy Barbari William Bentley Tracey Holoman Maria Klapa Nam Sun Wang

Mid-Atlantic Biochemical Engineering Consortium MABEC 2003 Abstracts Keynote Presentation Development and Scale-Up of Synagisâ David A. Lindsay MedImmune, Inc. Gaithersburg, MD 20878 Time-to-market has always been critical to the success of a biopharmaceutical product journeying down an R&D pipeline. Process development plays a central role in setting the pace of a product development program. The speed of development needs to be carefully balanced with the performance of the resulting production process as measured by its robustness, efficiency, and ease of scale-up. Prior to market approval, a manufacturing strategy should be in place to anticipate demand growth and secure product supply. An emphasis on continuous yield enhancement can steadily improve the process economics. In this presentation, we will use Synagisr, a humanized monoclonal antibody against Respiratory Syncytial Virus, as a case study to highlight the key issues involved in advancing from development stage to commercial manufacturing of a biotech product. Oral Presentations The Creation of Allostery by Domain Fusion Gurkan Guntas and Marc Ostermeier Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore, MD, 21218 Molecular evolution has proven to be a powerful approach for engineering proteins with improved and novel functions. A key step in applying molecular evolution is the creation of DNA diversity by such methods as error prone PCR or DNA shuffling. One method of creating diversity that has not been extensively explored, particularly in a combinatorial fashion, is that of domain insertion: the insertion of one protein domain into another. We have created domain insertion libraries between two proteins: TEM-1 beta-lactamase and the E.coli maltose binding protein (MalE). Using a selection scheme involving MalE- auxotroph, we have selected and characterized bifunctional fusion proteins of beta-lactamase inserted into MalE that confer resistance to beta-lactam antibiotics and are capable of transporting maltose in E.coli. In a random sampling of 408 bifunctional fusions two exhibited a stimulation of beta-lactamase activity in the presence of maltose indicating the establishment of allostery between maltose binding and beta-lactam hydrolysis. Kinetic and biochemical characterization of these two "protein molecular switches" suggest a role for conformational changes in the mechanism of switching behavior. Increasing Recombinant Protein Production in Fungal Fermentation by a Modified Feeding Strategy of Limiting Carbon Source Swapnil Bhargava1, Mark Marten1, and Kevin Wenger2 1Department of Chemical and Biochemical Engineering University of Maryland Baltimore County Baltimore, MD 21205 2Novozymes, North America, Inc. NC A wide spectrum of biological products including: bulk chemicals, antibiotics, industrial enzymes and recombinant proteins, are produced in filamentous fungal fermentations. The world market for industrial enzymes alone is estimated at nearly a billion dollars a year. However, many fungal fermentations suffer from the problem of high broth viscosity, attributed to their filamentous morphology. High broth viscosity results in poor mixing, which leads to insufficient oxygen mass transfer and ultimately can decrease productivity. Numerous attempts to resolve this problem have been made, yet none of these solutions appear to be consistently effective or reliable. In the current study, we present an alternative means to reduce fungal broth viscosity without a considerable change in process setup. In this approach, fed-batch fermentations are carried out with limiting carbon fed in controlled pulses (instead of traditional continuous feeding). Pulse feeding of limiting carbon source results in smaller fungal mycelia, which in turn leads to reduced broth viscosity. Reduced viscosity is then used to improve recombinant enzyme productivity by strategic changes in the feeding control strategy. An increase as high as 70% was achieved using the adopted process strategy. If these results are found to be broadly applicable, this strategy might provide a simple means of improving productivity significantly in fungal fermentations. Factors Controlling Filtrate Flux and Sieving Characteristics of Virus Filtration Membranes during Protein Filtration David M. Bohonak and Andrew L. Zydney Department of Chemical Engineering The Pennsylvania State University University Park, PA 16801 Although virus filtration is increasingly used in the biopharmaceutical industry, capacity and protein fouling remain problematic. Experimental studies were conducted in dead-end, stirred ultrafiltration cells with hydrophilized Viresolve 180 polyvinylidene fluoride membranes using several proteins. Flux and sieving data were obtained for membranes in two different flow orientations, with the selective "skin" layer oriented on either the upstream surface or downstream relative to the fluid flow. Compaction of the substructure occurs when the skin layer is downstream, leading to an increase in membrane resistance. Concentration polarization in the bulk solution or within the membrane substructure caused a substantial increase in the protein sieving coefficient, with this effect being greatest when the flow entered through the substructure. Fouling is primarily due to the deposition of large protein aggregates. The effect of this fouling on the flux was reduced when the skin layer was oriented downstream since the substructure acted as a prefilter. These results demonstrate the critical role that membrane morphology and orientation play in determining the overall performance of virus filtration membranes. Using Computational Fluid Dynamics (CFD) to Predict Particle Retention and Shear in Industrial Continuous-Flow Centrifuges Madhava Paranandi and William J. Kelly Department of Chemical Engineering Villanova University Villanova, PA 19085 Local Shear rates in industrial continuous flow centrifuges can effect performance in bioprocessing. Animal cells and large biological macromolecules can be damaged upon exposure to regions of high shear. Most biological solutions are non-Newtonian, so the viscosity varies spatially with shear rate. Viscosity is a critical parameter dictating the potential for the particles in a centrifuge to settle, per stokes law. In this research, Computation Fluid Dynamics (CFD) is being employed to model 3D flow in specific industrial continuous flow centrifuges (i.e. Sharples #16 Supercentrifuge). The resulting CFD models can be used to identify the location and size of high shear regions, where cell damage can occur. CFD modeling can be also be used to predict particle retention for Newtonian and non-Newtonian fluids, and may be especially useful for scale-up and with complex bowl geometries (i.e. scroll decanter centrifuges) where the plug flow assumption implicit in Sigma theory is invalid. Interaction of Apolipoprotein A-I with Lecithin-Cholesterol Vesicles in Model Bile Manasa V. Gudheti and Steven P. Wrenn Department of Chemical Engineering Drexel University Philadelphia, PA 19104 Gallstones are formed due the nucleation of cholesterol from lecithin-cholesterol vesicles of cholesterol-supersaturated bile. Lecithin-cholesterol vesicles are the primary transporters of cholesterol in bile and they can accommodate a cholesterol:lecithin ratio of 2:1. The vesicles are thermodynamically metastable and eventually revert to an equilibrium lamellar phase which accommodates a maximum cholesterol:lecithin ratio of unity. The excess cholesterol present nucleates out to form microscopic cholesterol monohydrate crystals that are precursors to gallstones. Cholesterol supersaturation exists in both healthy and diseased individuals. Therefore, cholesterol supersaturation is a necessary but not sufficient condition for gallstone formation. Hence, thermodynamics alone cannot explain the lithogenicity of bile. The rate of cholesterol nucleation (the kinetics) also influences the process. The kinetics of cholesterol nucleation are influenced by various biliary species, two of them being Apolipoprotein A-I (Apo A-I), an anti-nucleating agent and Phospholipase C (PLC), a pro-nucleating agent. In this study, the influence of Apolipoprotein A-I on lecithin-cholesterol vesicles of supersaturated bile is examined in the presence of Phospholipase C. Supersaturated vesicles of a specific cholesterol loading were subjected to different PLC and Apo A-I loadings. The effect of varying the cholesterol loading is also studied. Absorbance, dynamic light scattering and fluorescence measurements were taken. The fundamental understanding gained can be potentially applied to the study of heart disease as both the diseases share a similar mechanistic pathway. Generation of Functional Hepatocytes from Stem Cell Populations Eric Novik, Tim Maguire, Rene Schloss, Martin Yarmush Department of Chemical Engineering, Biomedical Engineering Rutgers University Piscataway, NJ 08854 Our group is investigating the potential of embryonic stem cells to differentiate into functional hepatocytes in vitro. Differentiated hepatocytes are characterized by the degree of albumin and urea synthesis. We are currently looking at the effects of culture conditions on the degree of diffentiation, specifically the temporal addition of growth factors, as well as the presence of an extracellular matrix components. Imaging and Spatial Analysis of Phosphoinositide 3-Kinase Signaling in Living Fibroblasts Ian Schneider and Jason Haugh Department of Chemical Engineering North Carolina State University Raleigh, NC 27695-7905 During wound healing, fibroblasts from connective tissue are directed to migrate to sites of clotting by gradients of platelet-derived growth factor (PDGF). Mammalian cells detect chemical gradients by spatial sensing, in which the cell can discriminate between the levels of signaling through cell surface receptors at its front and rear. The phosphoinositide (PI) 3-kinase enzyme generates specific phospholipid second messenger products in the cell membrane, a signal transduction event required for directed migration, in a manner that is polarized by gradients of PDGF and other chemoattractants. The pattern of 3' PIs in the plasma membrane is governed by generation, lateral diffusion, and turnover. Using a real-time fluorescence imaging technique in conjunction with a generalized reaction-diffusion model, we have devised a methodology to estimate constant parameters that describe the rates of these concurrent molecular processes in fibroblasts under uniform stimulation conditions. In concert with future experiments analyzing cells exposed to PDGF gradients, we expect to develop an integrated model of directed fibroblast migration at the level of intracellular processes. Quantification of Bacterial Transport Properties at Elevated Temperatures Kevin Kusy and Roseanne M. Ford Department of Chemical Engineering University of Virginia at Charlottesville Charlottesville, VA 22904-4741 We are interested in designing an experimental assay to quantify the motility and chemotactic responses of hyperthermophilic archaea with the intention of explaining the implications of these adaptations on cell survival. Preliminary experiments were initiated to test the capabilities of the high-temperature assays by evaluating the transport properties of the well-characterized mesophilic organism, Escherichia coli. A series of capillary assays were performed at multiple temperatures and were assessed via plate count enumerations and dark-field light scattering. During the investigation, problems were observed with elevating the temperature of the experiments. These problems were associated with the expansion of air inside the sealed capillaries, responses to oxygen gradients within the chamber, and heat and density related convection. The effects were exaggerated with increasing temperature and steps were taken to minimize the contributing factors. The capillaries were completely filled with liquid, and all air pockets were removed from the inside of the chamber. The adjustments improved the reproducibility of the assay system; however, caution should still be used when interpreting observations from the capillary assays at elevated temperatures. Electro-Deposited Chitosan as a Scaffold for Biomolecule Assembly Hyunmin Yi, Li-Qun Wu, Sheng Li, Reza Ghodssi, Gary W. Rubloff, Gregory F. Payne, and William E. Bentley Department of Chemical Engineering University of Maryland at College Park College Park, MD 20742 Obtaining and utilizing a biologically reactive surface at a defined location is crucial for the development of biosensors. We are investigating the use of biopolymer chitosan as a scaffold for generating such surfaces. A primary amine group at each monomer unit of chitosan gives this carbohydrate biopolymer several unique properties. First, chitosan is soluble at low pH and insoluble at higher pH due to the amine group�_s low pKa (= 6.5) value. This property enabled a simple, effective and reproducible way of depositing chitosan on an electrode surface by applying voltage. A patterned surface of chitosan with a fine spatial resolution was generated using this technique. Second, this amine group is an excellent starting point for various covalent modifications of the biopolymer, making chitosan a covalent coupling substrate for biomolecules such as nucleic acids, proteins and microbial cells. A variety of amine group reactive chemical crosslinking strategies can be employed for this purpose. For example, we have grafted fluorescent molecules and DNA oligonucleotides to chitosan surfaces for nucleic acid hybridization assays in a 96-well microtiter plate format. Further, we are investigating the use of such techniques to generate biosensor surfaces by chemically manipulating electrically deposited chitosan surfaces. Poster Presentations The Development of a Targeted Ultrasound Contrast Agent Justin D. Lathia1, Brian E. Oeffeinger1, Kenneth A. Barbee1, Flemming Forsberg2, Ji-Bin Liu2, Dan Merton2, Barry Goldberg2, and Margaret A. Wheatley1,2 1School of Biomedical Engineering, Science and Health Systems, and Department of Chemical Engineering Drexel University, Philadelphia, PA 19104 2Department of Radiology, Thomas Jefferson University Philadlephia, PA 19107 In the development of a targeted ultrasound contrast agent, several parameters must be considered including the effect of contrast agent composition on both image enhancement and the efficiency of ligand binding to targeted cells. Once a composition has been determined, it can be used as a platform for conjugating various targeting ligands. Polymer composition, the ratio of lactic acid (LA) to glycolic acid (GA) monomer, was evaluated for its contribution to the acoustic enhancement of a poly (co-lactic-glycolic) acid (PLGA) ultrasound contrast agent. Contrast agents were prepared from four different polymer compositions (LA:GA): 100:0, 85:15, 75:25, 50:50 and tested in vivo. The results indicate that all contrast agents enhance Doppler images in vivo but the 50:50 had the shortest clinical useful life-time (~ 1 min). The duration of enhancement by the contrast agents was dependent on the ratio of LA to GA monomer and was considered in the design of the targeted contrast agent. Fibronectin peptide fragments were then conjugated to pure PLA contrast agents to develop a targeted contrast agent. The microcapsules were conjugated with generic GRGDS and REDV peptide sequences that target integrins over-expressed in angiogenesis, avb3 and avb5. The contrast agents bound to human breast cancer cells in vitro within 3 minutes under flow conditions. The GRGDS and REDV peptide modified polymeric ultrasound contrast agents show promise as candidates for targeted therapeutic imaging and have potential to be used as a drug delivery vehicle. Peptide Surface Modification of PLA Ultrasound Contrast Agent for Targeting Lauren M. Leodore1, Justin D. Lathia2, and Margaret A. Wheatley2,3 Department of Bioscience and Biotechnology1, School of Biomedical Engineering, Science and Health Systems2, and Department of Chemical Engineering3 Drexel University Philadelphia, PA 19104 Angiogenesis is the formation of new vasculature and is enhanced in tumor growth. Cellular adhesion, particularly in angiogenesis, has been shown to be dictated through integrin receptors. The fibronectin fragments GRGDS and REDV are peptide sequences that target integrins over-expressed in angiogenesis, avb3 and avb5. Pure PLA microcapsules were conjugated with both peptide sequences to develop a targeted contrast agent and were tested in vitro on breast cancer cells within 5 minutes. The results of this study show that the modified PLA microcapsules bound to the cells better than the unconjugated PLA microspheres. Since the microspheres have buoyancy because they are filled with gas, their attachment suggests that the peptide is facilitating the attachment process. The results of this study demonstrate that peptide modified microspheres can be used in targeted imaging. Alginate Strings with Genetically Engineered Fibroblasts and their Application in Spinal Cord Regeneration Saravanan Kanakasabai, Margaret A. Wheatley, M. Murray, and I. Fisher Biomedical Engineering, Science and Health Systems Drexel University Philadelphia, PA 19104 We have optimized a method for producing strings of alginate bioconjugated with a laminin pentapeptide that aids in cell adhesion. These strings are 400-500mm in thickness and could be bundled to act as a graft at the site of injury. Invitro studies have shown that rat (NB2a) and human (SHSY5Y) neuroblastoma cell lines adhere to, and differntiate on these modified alginate strings. The strings also have the capacity to hold genetically modified fibroblasts that release neurotrophins that would aid in neuronal regeneration. These strings are strong enough to be surgically transplanted to the spinal cord, and may ultimately help in bridging the gap at the injury site. Alginate Strings Acting as a Directional Gradient in Spinal Cord Regeneration Argjenta Orana, Saravanan Kanakasabai, Ryan Murphy, and Margaret A. Wheatley School of Biomedical Engineering, Science and Health Systems Drexel University Philadelphia, PA 19104 Functional insufficiencies following spinal cord injury are due to interruption of ascending and descending axons and lack of successive regeneration. We are designing a graft that would aid in directionally specific neuronal regeneration. In our experiments we plan to use genetically engineered cells that release neurotrophic factors encapsulated in modified alginate strings. Thus far, we have a method for making strings of alginate bioconjugated with a laminin pentapeptide (YIGSR), which support cell adhesion, and the strings are also coated with a high molecular weight Poly-L-Ornithine that protects the encapsulated fibroblasts from host immune reaction while allowing them to release neurotrophic factors. These strings are 400-500�m in thickness. In vitro studies have shown that rat (NB2a) and human (SHSY5Y) neuroblastoma cell lines adhere, and differentiate on these modified alginate strings. Currently we are aiming to establish a growth factor gradient to promote neuronal differentiation in a desired direction. When these modified strings are surgically transplanted, we look towards having this graft design set up a physical gradient providing directional regeneration of the adherent neuronal cells. Evaluation of the Effect of Monomer Ratio on the Ultrasound Contrast Agent Performance Ehren Carine, Justin D. Lathia, Dalia El-Sherif, and Margaret A. Wheatley School of Biomedical Engineering, Science and Health Systems Drexel University Philadelphia, PA 19104 Our lab specializes in microencapsulation and ultrasound (US) contrast agents (CA). Recent efforts have focused on developing a versatile polymeric CA that can be used for targeted imaging and drug delivery. The end goal of this project is to develop a therapeutic ultrasound CA which can bind to specific cells, and be used as a drug delivery vehicle. The objective of this study was to evaluate the ratio of lactic acid (LA) to glycolic acid (GA) components on the performance of a poly (co-lactic-glycolic) acid (PLGA) contrast agent. LA is more hydrophobic than GA and the greater the proportion of LA, the more hydrophobic the CA. Contrast agents were prepared from two different polymer compositions with varying ratios of lactic (LA) to glycolic acid (GA) (LA:GA): 50:50 and 100:0 and tested in vitro at room temperature and 37°C. The CA were tested for echogenicity (amount of US reflected to the receiver) at 5MHz. When comparing the dose response curves of PLGA 50:50 and PLA, both polymer blends show diagnostically usable acoustic enhancement at room temperature (e.g. ~ 19 dB @ a dose of 0.015mg/ml), but the PLGA 50:50 shows less acoustic enhancement at 37°C (e.g. ~ 11 dB vs. 17.5 dB for 0.015 mg/ml). The time response curves of PLGA 50:50 and PLA show acoustic stability over a 15 minutes interval at room temperature. However at 37°C, the PLGA 50:50 shows a dramatic loss of enhancement with time (~ 90% loss in 6 min). The dose and time response curves for both blends of polymer contrast agents suggest that the in vitro performance of the contrast agent is dependent on the composition of the polymer, namely the amount of LA to GA. Furthermore, the contrast agent with the greater amount of LA shows more acoustic stability at 37°C. For the creation of a targeted CA, further agent processing is required. An integrin-specific peptide was chemically linked to the CA surface. The peptide modified PLA sample was evaluated using a dose response curve at both room temperature and 37°C. The results indicate that the processing has not compromised the acoustic stability; however, a higher dose of the contrast agent is required. 0These results demonstrate the importance of polymer composition on contrast agent performance and should be considered in the design of polymeric ultrasound contrast agents. The results also indicate that the polymer can be modified with a peptide and still remain effective in vitro. Measurement of Cholesterol Crystal Nucleation from Low Density Lipoproteins Utilizing Fluorescence Energy Transfer from Dehydroergosterol to Dansylated Lecithin Andrew J. Guarino and Steven P. Wrenn Department of Chemical Engineering Drexel University Philadelphia, PA 19104 Cardiovascular disease is the primary cause of mortality in the U.S. and is on the rise. Low density lipoproteins (LDL) are known to be the carriers of cholesterol in the blood and accumulate in atherosclerotic lesions along with cholesterol crystals. However, the mechanism of cholesterol deposition onto the artery walls is still unknown. Native LDL or enzymatically modified LDL may generate crystals extracellularly, which are then deposited onto the arteries or the crystals could be generated intracellularly through uptake by macrophages. Fluorescence energy transfer between dehydroergosterol (DHE) and dansylated lecithin (DL), which takes advantage of the separation between fluorophores, can be used to track the onset of nucleation much sooner than traditional microscopy. This technique was used to determine the onset of nucleation from vesicles modeling LDL particles and from LDL labeled with the fluorophores and pretreated with cholesterol esterase (CEase) upon exposure to sphingomyelinase (Smase). Dynamic light scattering showed the LDL aggregated increasing in size from 25nm to over 100nm for a loading of 0.5 units of Smase, which is a precursor to crystal formation. The absorbance at 450nm for LDL that was pretreated with CEase and then exposed to Smase was 5 times greater than LDL that was only exposed to Smase, reflecting an increase in aggregation. Also, about a 20% alleviation of energy transfer was seen after several hours for the LDL pretreated with CEase as the DHE peak increased at the expense of the DL peak signaling nucleation, while cholesterol crystals were not visible under a fluorescent microscope until after 24hr. Engineering Nanoparticulate Prodrugs Meredith Hans and Anthony M. Lowman Department of Chemical Engineering Drexel University Philadelphia, PA, 19104 Nanoparticles prepared from biodegradable polymers have the potential to serve as vehicles for improved methods for delivery of bioactive agents. One major advantage of polymeric nanoparticles is their small size (in many cases < 100 nm), which renders them nearly invisible to the reticuloendothelial system and allows for long-term circulation in the bloodstream. Another advantage of these systems is their synthetic versatility which allows for addition of functionalities such as polyethylene glycol that will also provide for increased in vivo half-life ("stealth" nature), target specific tissues or organs or provide for a unique release profile to match a clinical need. Additionally, these particles have the ability to deliver a wide range of drugs to varying areas of the body for sustained periods of time. In our initial work we physically incorporated haloperidol, a model hydrophobic antipsychotic used to treat schizophrenia, into PLGA nanoparticles using the traditional emulsification solvent diffusion technique. However, limitations such as poor drug loading and a large initial burst release prevent these nanoparticles from reaching their full potential as drug delivery vehicles. One way to avoid these issues is to create polymeric prodrugs by chemically conjugating the drug to the polymer. These prodrugs can be formulated into biodegradable nanoparticles, creating a "prodrug nanoparticle." We have developed several strategies to chemically conjugate haloperidol to polymers to create prodrug delivery devices. Detection of Pathogen E. Coli 0157:H7 Using the Piezoelectric Microcantilevers Gossett Campbell and Rajakannu Mutharasan Department of Chemical Engineering Drexel University Philadelphia, PA, 19104 The Lead Zirconate Titanate/Stainless Steel (PZT/Stainless Steel) unimorph microcantilevers ( 2-4 mm length) were fabricated and characterized for their mass change detection sensitivity with the goal of measuring ultra low concentration of bacteria and/or proteins in solutions. The cantilever tip was prepared to immoblize antibody using Protein G for detection of pathogen, E. coli 0157:H7. Binding of the pathogen to the cantilever tip alters its resonance frequency which quantitatively relates to the pathogen concentration. Selectivity of the detector to the specific pathogen is demonstrated by experiments conducted under various contaminating conditions. Because of the relative large size of the antigen, binding is enhanced by linking the antibody to cantilever via a ligand. Sensitivity of the microcantilever is established using binding experiments at various pathogen concentration in presence of a non-pathogen variant. To enhance visualization of pathogen detection, plasmid coding for green fluorescent protein was introduced into JM101 strain of E. coli and was used as a contaminating species in test samples. Presentation will also include physics of detection and measurement methodology. Protein Adsorption into Polymersomes: Effect of Chain Length on Circulation Time in vivo Veena Pata and Nily Dan Department of Chemical Engineering Drexel University Philadelphia, PA 19104 The adsorption of immunoproteins onto drug-carrying nano-particles such as liposomes enables their recognition by reticuloendothelial cells which mediate the clearance process in vivo. The attachment of polyethylene glycol (PEG) chains to the liposomes has been shown to reduce protein adsorption and enhance circulation time in vivo. Circulation time was found to increase either with PEG molecular weight or with the percentage of PEG-carrying lipids in the bilayer. Previous analysis of the effect of PEG on protein adsorption focused on supported monolayers or bilayers, thereby ignoring one of the essential features of lipid bilayers, namely, self-assembly. We show here that bilayer reorganization significantly affects the equilibrium concentration of proteins in bilayers in general, and PEGylated bilayers in particular, elucidating the effect of the chain length and concentration. In this study we present a simple model of proteins embedded or adsorbed onto polymeric bilayers, as a function of the polymer chain length (N). We find that the probability of protein adsorption into the bilayer peaks at a specific bilayer thickness, which, most likely, corresponds to natural bilayers' dimensions. As a result, we predict that the concentration of proteins decreases and in vivo circulation time will increase as a function of polymer molecular weight. Fitting our results to a power law yield a relationship where circulation time roughly scales as N0.4. Engineering of Sialylation Pathway in Insect Cells Karthik Viswanathan1, Shawn Lawrence1, Stephan Hinderlich2, Yuan C. Lee3, Michael Betenbaugh1 1Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore, Maryland 21218 2Institut f�r Molekularbiologie und Biochemie Freie Universit�t; 3Department of Biology; Johns Hopkins University Baltimore, Maryland 21218 Sialylation has been shown in cells to be directly related to the levels of sialylation substrate, namely N-acetylneuraminic acid (Neu5Ac). Previous studies have indicated negligible intracellular levels of both sialic acids including Neu5Ac and CMP-sialic acids in a number of insect cell lines grown in serum-free medium. In this study we identified the bottlenecks in the sialic acid synthesis pathway and were able to overcome this by overexpression of the genes of the pathway enzymes combined with appropriate substrate feeding. An alternative sialic acid 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid (KDN) can also be generated in insect cells. By suggesting the potential for controlling not only the production of sialic acids but also the type of sialic acid that is generated. The results of these studies can be used to optimize the sialylation process in insect as well as mammalian cell culture systems. An Optimized One-Hybrid System for Evolving Zinc Finger Specificity A. Bosley1, S. Durai1,2, A. Bridgeman3, S. Chandrasegaran2, M. Ostermeier1 1Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore, Maryland 21218 2Department of Environmental Health Sciences Johns Hopkins University Bloomberg School of Public Health Baltimore, MD 21205 3Thomas C. Jenkins Department of Biophysics Johns Hopkins University Baltimore, Maryland 21218 Zinc fingers are proteins that recognize a specific three base pair sequence of DNA, and are of great interest to researchers working in the field of gene therapy. Their potential uses range from conferring DNA binding to chimeric nucleases, to creating artificial transcription factors. To utilize these proteins, there must be a method that can be used to quickly and accurately evaluate and evolve their affinities. Hochschild and coworkers1 have previously described a one-hybrid system in which DNA binding domains are fused to the a-subunit of RNA polymerase. We have adapted and optimized this system for interrogating zinc finger-DNA interactions. In this system, the nine base pair binding site for the zinc finger is located upstream of the reporter gene which is either green fluorescent protein (GFP) or the chloramphenicol resistance gene (Cm). The binding of the zinc finger domain, to its target site, localizes the a-subunit, which recruits the rest of the polymerase and increases gene transcription. To optimize this system, we have used incremental truncation to create a library of reporter plasmids with varying distances between the binding site and the beginning of the reporter gene, as well as a library of zinc fingers-a-subunit fusion proteins with varying linker lengths between the two domains. From these libraries, we have identified constructs that result in improved transcription in our model system and we have shown that this level of transcription is sufficient to differentiate between binding sites differing in only one base. 1) S. Dove, J. Joung and A. Hochschild Nature 386 (1997), pp. 627-630. Predictions of the Docking of an Engineered Antibody to Anthrax Toxin Carlos A. Castaneda and Jeffrey Gray Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore, MD 21211 The proliferation of the anthrax bacterium can be prevented by the use of antibiotics such as ciprofloxacin; however, no suitable therapeutic has been designed to combat the toxins released by the bacteria. Thus, there is a need to develop treatments for unexpected, acute exposure to the bacteria. One route currently being explored is the development of an antidote derived from antibodies which block the activity of the toxin. The anthrax toxin contains three components: EF, LF, and PA. PA (Protective Antigen) is vital to the anthrax toxin's ability to infect a cell via endocytosis. The murine monoclonal antibody 14B7 shows limited binding ability to PA capable of prohibiting PA from binding to a surface cellular receptor. Maynard et. al. used directed evolution techniques on 14B7 to develop the 1H antibody, which binds to PA with 50 times higher affinity. However, no experimental structure for the antibodies or the antibody/toxin complexes have been determined, hindering further efforts to optimize the antibody�s efficacy and interpret mutational studies. Therefore, we have predicted the structure of the antibody/toxin complex using a recently developed protein-protein docking algorithm. The docking algorithm combines a low-resolution search and a high-resolution refinement using Monte Carlo cycles of side-chain packing and energy minimization methodologies. A generous number of docking "decoys" were generated, and these predicted structures were subsequently grouped into 14 clusters of similar, low-free energy solutions. Using in silico models for each of the antibodies, we were successful in predicting structures of PA docked to the 14B7 and 1H antibodies. One particular structure was preferred over all docking runs. The most common residue interactions (common to at least 5 clusters) between the antibodies (Ab) and toxin (Ag) were: AbH:104Y-Ag:654E, AbL: 53R-Ag:683D, AbL: 92N-Ag:690S, AbL:92N-Ag:688Y, AbL:32Y-Ag:688Y, AbL:30R-Ag:688Y, and AbL:30R-Ag:690S. Subsequently determined mutagenes AbL:92N near the docking interface. We are investigating various approaches to improving the docking algorithm�s performance. An aspect that played a key role in the 1H prediction set came with better packing of the H/L chains around the a-barrel of the antibody configuration. Generating Small-Molecule-Controlled Inteins for Applications in Biotechnology Georgios Skretas and David W. Wood Department of Chemical Engineering Princeton University Princeton, NJ We describe a methodology for generating small-molecule-controlled inteins. A rationally engineered estrogen receptor-intein chimera was genetically fused to a metabolic enzyme required for cell growth. The appropriate E. coli auxotrophs were grown in the presence and absence of estrogen agonists and antagonists. Enhanced cell growth that correlates well with ligand binding affinity was observed. Subsequent replacement of the estrogen receptor with the ligand binding domain of the thyroid receptor resulted in thyroid hormone-dependent cell growth that was insensitive to the existence of estrogen. We are hopeful that our approach will prove to be a generic methodology for constructing small-molecule activated inteins. Such inteins are very likely to find numerous applications in biotechnology varying from the costruction of biosensors, drug discovery and delivery systems to the generation of cellular computation tools. Micropatterning of Stretched and Aligned Molecules of DNA and Applications in Template-Directed Bottoms-Up Assembly Cecilia A. Petit and Jeffrey D. Carbeck Department of Chemical Engineering Princeton University Princeton, NJ, 08544 Molecular combing is a process by which molecules of DNA are stretched and aligned on a surface. This process relies on the tethering of one end of a DNA molecule and the extension of the rest of the molecule by the movement of an air-liquid interface. DNA immobilized in this manner retains some of its biological activity, allowing for example applications in optical restriction mapping and genetic diagnostics, such as quantifying the lengths of microdeletions and the copy number in the replication of oncogenes. If molecular combing is to be widely used as an efficient scientific and diagnostic tool for genetic analyses, these methods must be automated to allow parallel processing. We have developed a new method for creating addressable arrays of stretched and aligned DNA on the microscale by performing molecular combing in micro-fabricated channels. Because the shape of the receding air-water interface determines the direction of the stretched molecules, controlling thi! s meniscus allowed us to control the orientation of the chains. We have shown that the wall chemistry and geometry of the microchannel combine with the speed of withdrawal of the fluid in determining the shape of the meniscus. Careful balancing of these factors allowed us to align DNA on the surface parallel to the walls of the channel. We also demonstrate control over the placement of stretched DNA by micropatterning hydrophobic silanes on the substrate via photolithography. Over-Expression of Tyrosine Decarboxylase in California Poppy Cell Suspension Culture Jintae Lee and Henrik Pedersen Department of Chemical and Biochemical Engineering Rutgers University Piscataway, NJ 08854 The production of benzophenanthridine alkaloids from suspension cell cultures of Eschscholzia californica (California poppy) has been extensively studied with an emphasis on strategies to overcome low productivity. Recently, genetic engineering methods have allowed for the cloning genes and stable genetic transformation of plants. We have developed a genetic transformation protocol suited to E. californica that generates transgenic cells expressing the modified green fluorescent protein (GFP), a popular reporter protein. The transformed cells have been selected for high level expression, maintained, and analyzed in terms of cell growth and alkaloid production in suspension cell culture. Since the biosynthetic pathway of benzophenanthridine alkaloids has been completely elucidated at the enzyme level, we are investigating the effect of over-expression of tyrosine decarboxylase (TYDC), an elicitor-inducible enzyme in the alkaloid pathway, on the production of end product compounds. This work will demonstrate manipulation of gene expression in order to improve the production yields of benzophenanthridine alkaloids, and further provide insights on flux control in the biosynthetic pathway. Generally, it will be a good example of genetic plant metabolic engineering to produce alkaloids from plant cell suspension cultures. Optimizing the Endoplasmic Reticulum for Increased Secretion of Pyrococcus Furiosus Beta-Glucosidase in Yeast Jason D. Smith and Anne S. Robinson Department of Chemical Engineering University of Delaware Newark, DE 19716 Efficient protein folding is essential for high level protein production. For eukaryotes the endoplasmic reticulum (ER) is the site for secretory protein folding. An array of ER-resident proteins called chaperones and foldases aid in protein folding as well as act as a quality control system allowing only properly folded proteins to exit the ER. Overexpression of secretion-competent foreign proteins can often lead to bottlenecks in the ER due to sub-optimal folding conditions and/or overloading of ER machinery. Improved secretion yields have been achieved by overexpression of ER chaperones and foldases, however optimization of these effects has not been studied in great detail. We found that overexpressing a secretion-competent tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus in Saccharomyces cerevisiae led to an intracellular ER bottleneck, likely due to misfolding of the monomer. Co-overexpression of ER-resident proteins BiP and PDI increased secretion levels ~ 60%. In an effort to optimize this interaction, a library created with variable BiP and PDI levels was then screened for improved beta-glucosidase secretion. From a screen of 140 transformants we isolated engineered strains with up to 3-fold improved secretion levels. By quantitating BiP and PDI levels in the 15 best strains, we observed that increasing BiP levels tended to decrease secretion whereas increasing PDI levels increased secretion. Future work will seek to more fully understand the mechanism for the improvement. The Use of a Green Fluorescent Protein Fusion Tag to Engineer Yeast Cells for Optimal Expression of Membrane Proteins Ronald T. Niebauer, James Butz, and Anne Skaja Robinson Department of Chemical Engineering University of Delaware Newark, DE 19716 The G-protein coupled receptors (GPCRs) are an important class of transmembrane proteins that mediate cellular response to diverse stimuli. Many diseases including cancer and heart disease have been linked to GPCR function. GPCRs represent the target for the majority of present pharmaceuticals, but little is known about expression, folding, and interactions of these proteins. Biophysical studies to elucidate structural and functional properties are limited by an inability to produce high levels of functional protein. The goal of this research is to develop a Saccharomyces cerevisiae expression system for efficient expression of functional protein. The particular GPCRs used in this study are the human A2a receptor, which is believed to play a cardioprotective role in the body, and the mouse substance P receptor (SPR), which is from the receptor class that has been linked to asthma and Alzheimer's disease. The green fluorescent protein (GFP) has been used as a reporter of expression and to help monitor protein trafficking. Initial studies show that the A2a-GFP protein is correctly localized to the plasma membrane, as determined by confocal microscopy, and is functional, as determined by a radioligand binding assay. In contrast, the SPR-GFP does not reach the plasma membrane and is not functional. Current studies are focused on optimizing A2a protein expression levels and gaining an understanding of what limits overexpression of this class of proteins. Using Proteomics to Understand How Rapidly Changing Nutrient Environments Can Affect E. coli Babu Raman and Mark R. Marten Department of Chemical and Biochemical Engineering University of Maryland Baltimore County Baltimore, MD 21250 It has been known for many years that non-ideal mixing in large-scale bioreactors can lead to oxygen limitations and substrate gradients. When this occurs, cells will experience changing concentrations of oxygen and substrate as they travel through different regions of the tank. While this condition exists for many different types of cells, in many different reactor configurations, surprisingly little is known about how cells respond to rapidly changing nutrient environments. This is because most lab-scale research is done on "ideally" mixed (i.e., homogeneous) systems that are not representative of production scale conditions. We have simulated the non-ideal mixing found in large-scale E. coli fermentations, by operating a 20 L fermentor in fed-batch mode with an intermittent or "pulsed" carbon feed. As a control, an identical fermentation was operated with a constant carbon feed (same total amount of carbon fed to both cultures). Two dimensional gel electrophoresis and MALDI-TOF mass spectrometry was used to perform a proteome analysis of these cultures, which shows the expression level of a large number of proteins is affected by intermittent carbon feeding. Morphological Changes and Extent of Autolysis in Filamentous Fungi as a Response to Pulse-Feeding of Nutrients Judith Kadarusman and Mark R. Marten Department of Chemical and Biochemical Engineering University of Maryland Baltimore County Baltimore, MD 21250 Filamentous fungal fermentations are used to produce nearly $1 billion in industrial enzymes annually, yet many suffer from high broth viscosity, resulting in reduced productivity. Recently, we found that pulsed feeding during fed-batch fermentation led to smaller mycelia, reduced viscosity, and increased productivity. The goal in this study was to study morphological changes during pulse-feeding. A model fungi, Aspergillus oryzae, was grown in a narrow (50 micron) gap, parallel plate flow chamber mounted on a microscope stage. Video microscopy was used to observe the mycelial growth over time, and image analysis techniques were used to quantify the changes in morphology, vacuolation, and other cellular degradation phenomena within mycelial elements. Results of these observations will be discussed. Adhesion of Staphylococcus Aureus to Red Blood Cells under Hydrodynamic Shear Conditions Pyong Kyun Shin1, Parag Pawar2, Konstantinos Konstantopoulos2, and Julia M. Ross1 1Department of Chemical and Biochemical Engineering University of Maryland Baltimore County Baltimore, MD 21055 2Department of Chemical Engineering Johns Hopkins University Baltimore, MD 21205 S. aureus is a well known pathogenic organism which causes a variety of blood-borne infectious diseases such as infective endocarditis and sepsis. The dynamic adhesion of S. aureus to various substrates such as collagen, platelets, and endothelial cells has been intensively studied to elucidate the underlying mechanisms and to prevent the infection by this pathogen. During our study on the adhesion of S. aureus to platelets in whole blood, we found that S. aureus also adheres to red blood cells (RBC). The adhesion of S. aureus to RBCs is specific. And plasma protein(s) is required. The adhesion showed maximum value at the shear rate range of 100s-1 to 400 s-1 and was partly maintained even at such a high shear rate of 2000 s-1. The results suggest that under physiological shear, S. aureus can bind to RBC, which affects the binding of this bacterium to other cells in blood. Quantification Studies Involving Fibrinogen as a Bridging Protein in Mediating Staphylococcus Aureus - Platelet Interractions in Dynamic Shear Environments Niraj P.E. George and Julia M. Ross Department of Chemical and Biochemical Engineering University of Maryland Baltimore County ECS Bldg, 1000 Hilltop Circle, Baltimore, MD 21229 Staphylococcus aureus is an important pathogen that causes a variety of infections ranging from superficial skin infections to more serious and potentially fatal illnesses such as acute infectious endocarditis, osteomyelitis, septic arthritis, pneumonia and septicemia. The molecular pathogenesis of these infections involves bacterial adherence via surface structures called adhesins that bind to ligands in the host. Often, the bacterial adhesion takes place in the blood stream where fluid shear stress may influence binding events. We hypothesize that shear stress will affect the process of bacterial adhesion to platelets. My research involves characterization of the adhesive interactions between S. aureus adhesins and platelet receptors to determine the relative importance of each under varying shear stress. Although there are numerous surface proteins on platelets, the initial focus of this research is on those that are most abundant on the cell surface, namely GPIIb-IIIa (�IIB/�3). The interactions between adhesins and platelet receptors are mediated via fibrinogen-binding proteins in S. aureus such as clumping factor A (ClfA), fibronectin binding protein A (FnbpA) and secreted proteins coagulase A (CoaA) and Eap. The study of these proteins will also be undertaken. Initially fibrinogen will be examined as the sole bridging protein for S. aureus - platelet interactions. Understanding and identifying the relative importance of adhesins, bridging molecules and platelet receptors under specific shear conditions is an important step in developing new therapeutics to combat S. aureus cardiovascular infections. An in vitro Study of the Effects of ClfA on Staphyloccal-Collagen Binding Interactions in Whole Blood Under Physiological Fluid Shear Conditions Michael A. Johnson and Julia M. Ross Department of Chemical and Biochemical Engineering University of Maryland Baltimore County Baltimore, MD 21250 In staphyloccal bloodborne infections, bacteria and platelets often aggregate on the sub-endothelium where the extracellular matrix is exposed. In past studies the S. aureus adhesin, clumping factor A (ClfA), has been shown to be a critical virulence factor in several experimental models of infections. However, the extent of the role of ClfA in infections is uncertain due to many unknown protein-protein interactions. In this study the role of interaction between ClfA and a possible bridging molecule in S. aureus-collagen binding interactions is studied. The S. aureus-collagen binding interactions were evaluated using a ClfA positive and mutant strain of S. aureus and antibody-blocking techniques. The bacterial cell adhesion location (at collagen surface or above in platelet aggregate) was measured using confocal laser microscopy. Results demonstrated S. aureus ClfA-binding interactions through a possible bridging molecule under physiological shear conditions. In conclusion, the role of ClfA, bridging molecules, and antibody-blocking effects should be considered when developing novel therapeutics for bloodborne infections. Efficacy of Different Targeting Agents in the Photolysis of Interleukin-2 Receptor Bearing Cells Jennifer Ruiz Pacheco, Roberto Linares, and Theresa Good Department of Chemical and Biochemcial Engineering University of Maryland Baltimore County (UMBC) Baltimore, MD 21250 The multichain interleukin-2 receptor (IL-2R) has been proposed as a target for immunotherapy in cancer treatment and autoimmune diseases. Normal, non-activated T cells do no express this receptor, but some leukemias and lymphomas including adult T-cell leukemia, cutaneous T-cell lymphoma, and Hodgkin�s disease abnormally express the IL-2R. Immunoconjugates use monoclonal antibodies or natural cell ligands coupled to therapeutic agents to selectively target and kill over-reactive cells. In order to enhance selectivity and efficacy of immunoconjugates, the use of photosensitizers has been suggested. This report describes the selective photolysis of activated and non-activated IL-2R expressing cells using two photosensitizers, hematoporphyrin (HP) and chlorin e6 (ce6), covalently linked to IL-2 or anti-IL-2R antibodies. Selective destruction of IL-2R bearing cells was achieved after irradiation with both photosensitizers. Chlorin containing conjugates were more effective, by a factor of 4 or more, than hematoporphyrin containing conjugates. Conjugates made with IL-2 were under some conditions, more than 30 times more effective than conjugates that used a monoclonal antibody against the IL-2R for targeting. Activation of the cells to increase IL-2R expression decreases the internalization time required for optimal therapeutic efficacy; however, stimulation of the cell to increase IL-2 secretion greatly reduces effectiveness. Hsp20, a Novel a-Crystallin, Prevents Ab fibril Formation and Toxicity Sungmun Lee2, Kenneth Carson1,3, Allison Rice-Ficht1,3, and Theresa Good1,3 1Department of Chemical and Biochemcial Engineering University of Maryland Baltimore County (UMBC) Baltimore, MD 21250 2Department of Chemical Engineering Texas A&M University College Station, TX 3Department of Medical Biochemistry and Genetics Texas A&M University System Health Science Center College Station, TX b-Amyloid (Ab) is a major protein of senile plaques in Alzheimer's disease, and it has neurotoxicity when it is aggregated. It is still controversial what size of Ab aggregation form has the most toxicity to the neuron cell, and no consensus mechanism of Ab aggregation has yet been proposed. From pharmaceutical point of view, however, it is very good to reduce or retard the neurotoxicity of Ab by preventing the aggregation. Even delaying Ab aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. a-crystallins are molecular chaperones, able to prevent aggregation of other proteins. A novel protein, Hsp20, isolated from the bovine erythrocyte parasite Babesia bovis has a-crystallin like properties. In this research, turbidity assay showed that this novel protein prevented the aggregation of denatured alcohol dehydrogenase! (ADH). Ab aggregation was also prevented by addition of Hsp20 at the beginning of Ab incubation or in the middle of Ab incubation prior to form fibril. Hsp20 had the best activity to reduce the Ab aggregation at specific mole concentration ratio (Ri) between Hsp20 and Ab in the range of 0~0.5 (Ri = mole concentration of Hsp20 / mole concentration of Ab). In the case of 100mM of Ab, 0.1mM of Hsp20 prevented the Ab fibril formation the most by 92.16% compared to the Ab fibril formation without Hsp20. The effect of Hsp20 on the reduction of Ab aggregation was related to the cell viability. The toxicity of Ab to SY5Y cells was decreased by the addition of Hsp20 by 20~30%. The results of electron micrographs showed that the addition of Hsp20 to Ab formed globular species compared to the long fibrils of Ab only. Two hypothesis- Hsp20 aggregation model and irreversible best fit model- were suggested to elucidate the phenomena that Hsp20 has the best activity to prevent Ab aggregation at some specific mole concentration ratio. Study of Ocular Transport in the Vitreous Using Magnetic Resonance Imaging Hyuncheol Kim1,2, Martin Lizak2, Ginger Tansey2, Michael Robinson2, Robert Lutz2, Nam Sung Wang1 1Department of Chemical Engineering University of Maryland College Park, MD 20742 2National Institute of Health Bethesda, MD 90892 Posterior segment eye diseases such as retinitis, lymphoma and age-related macular degeneration (AMD) are main causes of blindness. About 10 million Americans suffer from AMD and 25,000 new cases of blindness are reported in the US each year. Traditional methods of drug delivery, such as topical administration, systemic injection, or even intravitreal injections are often ineffective or have serious side effects. With advances in controlled-release drug delivery technology, interest has increased in developing long-term, drug-releasing ocular implants for treating chronic eye diseases. To develop the most effective treatment regimens, it is important to understand the mechanisms of ocular drug transport from sustained release implants. In this study, we use magnetic resonance imaging (MRI) to track the movement of a drug surrogate, Gd-DTPA (an MRI image enhancer), from prototype intravitreal implants that we have developed. Image data from implants in the vitreous of New Zealand white rabbits is converted to Gd-DTPA concentration as a function of time in regions of the eye. We have also developed a finite element mathematical model of the rabbit eye to correlate with the experimental data, which assists our fundamental understanding of the transport mechanisms and allows for the development of optimum treatment strategies. To date, the results of our studies indicate that diffusion is the primary mode of transport of hydrophilic, small molecular weight drug through the vitreous, with minimal convective contributions from vitreal fluid flow toward the posterior segment of the eye. Aqueous fluid flow toward the anterior compartment of the eye can be a significant source of drug elimination. Continued studies are underway. Nanopore Enzyme Enhanced Devices (NEEDs) Allan E. David, Patricia Gonzales, Arthur Yang, Nam Sun Wang Department of Chemical Engineering University of Maryland College Park, MD 20742 The biotechnology field has been growing by leaps and bounds over the past few years. Rapid developments in the biochemical field have created both the availability and demand of novel bio-molecules. Many of these new bio-molecules, acting as enzymes, can improve current processes if it were cost effective. The immobilization of enzymes provides tremendous cost reductions by 1) allowing for repeated uses of the enzymes and 2) reducing downstream purification needs through separation of enzymes from the products. The goal of this research is to develop an effective method for the immobilization of bio-molecules onto silica surfaces. Such materials can find use in heterogeneous catalysis, affinity chromatography, membrane reactors, bio-sensors, and drug delivery. While the immobilization of enzymes has been studied for a number of years, it has been done mainly through the modification of a pre-fabricated gel. This method can be limited to lower enzyme loading on the gel as it depends on diffusion of the bio-molecule into the pores. Greater loadings can be achieved if the enzyme is introduced before gel formation. Preliminary results have been obtained for the immobilization of invertase in silica gels. The immobilization is done using silicic acid a precursor. Silica gels formed by simply raising the pH of silicic acid contain a significant number of hydroxyl groups on the surface. These reactive groups can be used for the addition of various surface functionalities with silane coupling agents (e.g. aminopropyltriethoxysiliane-APTES). Initial results on the immobilization of invertase on an APTES modified, glutaraldehyde linked gel have shown promise. Invertase hydrolyzes sucrose to from glucose and fructose. Enzyme loadings of approximately 32,000 U/g, where the unit is defined as the production of one mol glucose per minute, using this method of immobilization. Results obtained from immobilized thermolysin, a protease, have also been significant. Nanoparticle Based Metal Affinity Separation of Histidine Tagged Protein Chi-Wei Hung and Tracey R. Pulliam-Holoman Department of Chemical Engineering University of Maryland College Park, MD 20742 A nanoparticle-based protein separation method derived from the principles of immobilized metal-ion affinity chromatography (IMAC) was developed in this study. Silica nanoparticles and silica/iron dioxide magnetic nanoparticles (MNPs) were utilized to separate recombinant histidine-tagged proteins from crude cell lysates. Silylation reactions were performed to immobilize different species of metal ions on the silica particle surfaces. Nanoparticles, due to their high surface area, provide more binding capacity than traditional IMAC particles. The separation method presented here could be applied to all histidine-tagged proteins, and should be scalable. Isolation and Characterization of Phenanthrene (PHE)-Degrading Microbes under Methanogenic Conditions Youngsoon Um, Wook Chang, and Tracey R Pulliam Holoman Department of Chemical Engineering University of Maryland College Park, MD 20742 This research focuses on the isolation and characterization of anaerobic phenanthrene (PHE)-degrading microbes under methanogenic conditions. In a previous study from our research group, degradation of and PHE under anaerobic methanogenic conditions was detected utilizing Baltimore harbor sediments to initiate anaerobic enrichment cultures. To characterize anaerobic PHE-degrading enrichment cultures, the microbial consortia of PHE-degrading cultures were monitored via comparative sequence analysis of genes coding for 16S rDNA with "universal primers" and "archaeal primers". This information was then utilized to design targeted isolation strategies for methanogens and a sulfate-reducing consortium. To screen PHE-degrading microbes, two modified plating methods were utilized. Method 1 involved inoculating enrichment cultures with 0.5% molten agar onto a visible white coating of PHE which was made by spreading PHE:acetone solutions on a solidified agar plate. Method 2 involved spreading an agar overlayer containing enrichment cultures and ethanol solutions of PHE. Agar overlayer becomes cloudy due to the evenly dispersed fine PAH particles. After the plates were incubated in glass jars under N2: CO2: H2 (15:4:1), colonies exhibiting PHE degradation were detected by the appearance of clear zones surrounding the colony due to PHE uptake and utilization. Based on 16S rDNA analysis, the isolated colony was a mixed colony related to uncultured eubacterium R1 found in the xanthan-degrading anaerobic cultures, Acholeplasma genus, and methanogens. The isolated colonies will be tested for the ability to degrade PHE under the methanogenic conditions in the liquid cultures by monitoring CH4 production. Development of Species-Specific Oligonucleotide Probes for the Detection of 16S rDNA in Environmental Samples by Using DNA Microarrays Wook Chang, Youngsoon Um, and Tracey R. Pulliam-Holoman Department of Chemical Engineering University of Maryland College Park, MD 20742 Previously the predominant microbial species in anaerobic enrichment cultures initiated with Baltimore Harbor (BH) sediments and polycyclic aromatic hydrocarbons such as naphthalene, phenanthrene, and pyrene were identified utilizing comparative sequence analysis. Based on this information, we developed species-specific oligonucleotide probes for the creation of DNA microarrays to detect 16S rDNA in soil extracts. Although new molecular screenings were performed on other cultures, the predominant species were successfully found in the pool of the previously identified ones, thus indicating that the pool is large enough to represent the most dominant anaerobic populations in BH sediments. The specificity of the oligonucleotide probes was tested by the utilization of mixed and pure 16S rDNA populations and by comparison with the previous comparative sequence analysis. As a result, we could employ DNA microarrays as more efficient tools to directly identify the relative abundance of each predominant species in microbial community structure. Interactions between Magnetic Nanoparticles and Escherichia coli: An X-Ray Scattering Study Isaac Koh, Bani H. Cipriano, Sheryl H. Ehrman, Tracey R. Pulliam-Holoman, and Lus J. Martinez-Miranda Department of Chemical Engineering University of Maryland Collge Park, MD, 20742 Magnetic nanoparticles (MNPs) have increasing applicability in drug delivery, cancer treatment, and immunoassays. There is a need for an improved understanding of how MNPs interact with cell membranes in applied magnetic fields in order to use them effectively. The interactions between Escherichia coli (E. coli) and SiO2/Fe2O3 composite particles in magnetic fields were studied using X-ray diffraction in this work. Three magnetic field strengths of 227, 298 and 423 mT were applied to the samples and the XRD results were compared with those in the absence of a magnetic field. We observed the del(intensity)3 showed a change for 423 mT and comparatively none for 298 mT, which suggests that 298 mT is a changing point. A broad peak of Del(intensity)3 in 423 mT between 0.5 and 0.85 in two theta suggests that d, lattice spacing, in Bragg's law was increased likely due to the rotation of the molecules in the cell membrane bilayers under the influence of an applied magnetic field of 423 mT. There was no peak in 227 and 298 mT in this region of two theta. A peak shift from 1.0 to 1.1 in two theta was observed in 298 mT. This peak shift in 298 mT seems to be associated with a large change of Del(intensity)3 in 7.5 of two theta. The results showed preliminary but promising clues in understanding the interactions between E. coli and the MNPs in the magnetic fields. Understanding and Manipulating Cell-to-cell Communication: Role of Glucose on Autoinducer Synthesis Liang Wang1,3 and William E. Bentley2,3 1Department of Cell Biology and Molecular Genetics 2Department of Chemical Engineering 3Center for Biosystems Research3 University of Maryland College Park, MD 20742 Bacteria have evolved complex genetic circuits to modulate their physiological states and behaviors in response to a variety of extracellular signals or stimulations. In a process termed quorum sensing, or density-dependent gene regulation, bacteria produce, release and respond to hormone-like signaling molecules (autoinducers), which accumulate as a function of cell density. Quorum sensing confers on bacteria the ability to communicate with each other and coordinate their activities as a muticellular organism. The discovery of an interspecies cell-to-cell communication signal in E. coli (autoinducer-2, AI-2) raises the question of how AI-2 biosynthesis and degradation is controlled. Here we use Northern blot and lacZ fusion to show that glucose increases the transcription of luxS, a gene encoding AI-2 synthase in the pathway of AI-2 production. Furthermore, the effect of glucose on luxS transcription was attenuated by the addition of 10mM cAMP into the cell culture, confirming a glucose or energy level related mechanism. Additionally, the mutation of crp, a gene encoding cAMP receptor protein, resulted in a significantly higher expression of luxS gene relative to the controls although the cells were grown in medium without glucose. In accordance with this, we found that mutants in crp or cya gene (encoding adenylate cyclase), both produce much higher levels of AI-2 than the wildtype. These data suggest that cAMP and cAMP receptor protein (CRP) are involved in the downregulation of the luxS transcription and AI-2 synthesis. Simple Protein Purification Using Polysaccharide Conjugation David A. Small, Tianhong Chen, Gregory F. Payne, and William E. Bentley Department of Chemical Engineering University of Maryland College Park, MD 20742 The purification step of protein production and isolation is the most time consuming and complex step in processing. Optimization of the purification process requires rapid, economical, and cost-effective methods to purify proteins at both the small-scale and large-scale. A tyrosine tag with an enterokinase enzymatic cleavage site was added to the C-terminus of green fluorescent protein (GFP). The tyrosine tagged protein was grown in E. coli, harvested, and lysed. The lysate containing the tyrosine tagged GFP was coupled to a polysaccharide (chitosan) using tyrosinase enzyme. The GFP in the protein-polysaccharide conjugate was then liberated using enterokinase enzyme. This method was effective in conjugating and purifying the protein using a centrifuge with a limited number of processing steps. Integrated Genomic and Metabolic Analyses of Arabidopsis Thaliana Physiology Maria I. Klapa1, Tara Vantoai2,3, Linda Moly2, Lara Linford2, Harin Kanani1, Bhaskar Dutta1, Kinjal Suchak1, Jeremy Hasseman2, John Quackenbush2 1Department of Chemical Engineering University of Maryland College Park, MD 20742 2The Institute for Genomic Research (TIGR) Rockville, MD 20850 3Department of Food, Agricultural, and Biological Engineering Ohio State University Columbus, OH 43210 It is presently clear that the analysis of a biological system requires the integration of all fingerprints of cellular function: gene expression, total protein production and in vivo enzymatic activity. The value of knowing one of the three is undermined without equally detailed information about the others. Integration of all profiles of a systematically perturbed cellular system can provide insight about the function of unknown genes, the relationship between gene and metabolic regulation and even the reconstruction of the gene regulation network. In this context, we will demonstrate an integrated analysis of the Arabidopsis thaliana physiology, in which the response of the plant to increased CO2 concentration in its environment was measured simultaneously at the genomic and metabolic level. Specifically, two sets of plants, grown for 12 days under constant light in liquid cultures, were fed continuously for a day with air of ambient composition and 1% CO2 concentration, respectively. In both cases CO2 was 10% labeled. Plants were harvested at various time points and their average gene expression profile was measured using full-genome DNA microarrays, while their metabolic profile using Gas Chromatography - Mass Spectrometry (GC-MS). The metabolic profile comprises the intracellular concentrations of sugars, organic acids and amino acids, while, when possible, measurements of the distribution of label in the various metabolite pools provided additional information about the in vivo enzymatic activity. Comparison of the two cellular profiles is expected to similarities and differences between the genomic and metabolic response of the plants to the external stress, while the derived conclusions will prove beneficial for the design of new more specific experiments. Microbial Synthesis of Vanillin Nyan Win and Rachel Chen Department of Chemical Engineering Virginia Commonwealth University 601 W. Main Street Richmond, VA23284 Vanillin is one of the most widely used flavor compounds. Natural vanillin from vanilla plant can only supply 0.2% of the demand. Synthetic, petroleum-based vanillin is being used as the substitute. The synthetic manufacturing process involves toxic chemicals, particularly a carcinogen, dimethyl sulfate. The presentation will highlight our recent efforts in developing a renewable resource based, environmentally benign vanillin synthesis process. Bioconversion with cells immobilized in calcium alginate matrix showed that the activity could be maintained for nearly 200 hr, but the bioconversion rate was significantly lower than the suspension culture. Product inhibition was observed at vanillin concentration as low as 0.5 g/l. Above 1 g/l vanillin concentration, rapid product degradation was observed. In-situ product recovery with XAD2 resin successfully curtailed the product inhibition and product degradation, resulting in an 80% increase in bioconversion rate and an increase of product yield from 65% to 87%. Surface Chemistries and Blocking Strategies for DNA Microarrays Scott G. Taylor3, Stephanie Smith3, Brad Windle2,3 and Anthony Guiseppi-Elie1,3 Department of Chemical Engineering Virginia Commonwealth University Richmond, VA, 23284-3028 The surfaces and immobilization chemistries of DNA microarrays are the foundation for high quality gene expression data. Four surface modification chemistries were evaluated using cDNA and oligonucleotide microarrays for signal character and immobilization properties. cDNA and oligonucleotides were spotted on glass microscope slides modified with either poly-L-Lysine (PLL), 3-glycidoxypropyltrimethoxysilane (GPS), DAB-AM-poly(propyleminime hexadecaamine) dendimer (DAB) (linked to the glass surface via GPS), or a-aminopropyltrimethoxysilane (APS). Two unmodified glass surfaces, i) RCA-cleaned, and ii) RCA-cleaned and immersed in tris-EDTA buffer were also studied. The microarrays were composed of three subarrays of amino terminated oligonucleotides (30mers) specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and three subarrays of amine terminated GAPDH PCR product (~600 bp). A total of six 10 x 6 sub-arrays were printed on each slide. The sub-arrays consisted of replicates (Rows) in each of six columns. Each column represented one of six spotting concentrations (1~1,000 ng/ml). DNA on amine surfaces (PLL, APS, DAB) was immobilized by UV crosslinking (90mJ/cm2), while DNA on the GPS surface immobilized by incubation at 50% Rh and 42oC for eight hours. Arrays were blocked prior with either succinic anhydride (SA), bovine serium albumin (BSA), or were left unblocked prior to hybridization with labeled GAPDH PCR product. Microarray quality factors evaluated were surface affinity for cDNA and oligonucleotides, spot and background intensity, spotting concentration and blocking chemistry. Contact angle measurements (water and hexadecane) and Atomic Force Microscopy were preformed to characterize wettability and gross morphology of the surfaces. Overall the GPS surface exhibited the lowest background intensity regardless of whether the slides were blocked with SA, BSA, or not blocked. The raw signal intensity of oligos and cDNA on the GPS surface was comparable to the raw intensity values obtained on the amine surfaces. cDNA spots yielded greater intensity compared to oligonucleotides (same concentration). Blocking did not have a significant effect on raw spot intensity, however it did have a negative impact on background intensity. Oligonucleotide and cDNA sectors from the unblocked group of GPS slides gave the highest overall spot to background intensity ratio compared to oligos printed on amine surfaces. BSA blocking produced the lowest background, while succinic anhydride yielded high background and produced a smearing effect. It was concluded the unblocked GPS surface was the most appropriate surface for contact printing of microarrays. Preclinical Investigation of the Mechanism of Action of Novel Platinum Compounds in Malignant Glioma Using Microarray Gene Expression Monitoring Derk Bemeleit1,2, Dietmar Blohm2, Oliver Bogler3, and Anthony Guiseppi-Elie1 1Department of Chemical Engineering and Center for Bioelectronics, Biosensors and Biochips (C3B) Virginia Commonwealth University 1601 W. Main Street Richmond, VA 23284 2Center for Environmental Research and Technology (UFT) University of Bremen Leobener Strasse 28359 Bremen, Germany 3Neurosurgery and Hermelin Brain Tumor Center Henry Ford Hospital 2799 W. Grand Blvd. Detroit, MI 48202 Primary brain tumors cause an estimated 22,000 deaths annually in the US, with astrocytic gliomas representing the largest group. An important observation is that the earliest and most common known genetic event in astrocytic tumors is the mutation of the tumor suppressor gene p53, which leads to the production of aberrant p53 proteins that have lost tumor suppressor activity. This kind of mutation is represented in the cell line LNZ308, which is under survey in these studies. While cisplatin as a chemotherapeutic agent showed some clinical promise in the treatment of glioma, the problems encountered with delivery and toxicity among others have meant that it is no longer a drug of choice for this disease. The novel platinum compound BBR3464, which is based on a trinuclear structure, causes different types of DNA crosslinks than cisplatin, and also has vastly superior efficacy and pharmacological characteristics. In order to determine the molecular nature of the different cellular responses observed with different platinum compounds, we intend to analyze gene expression profiles of treated cells using the C3B10k microarray, representing 10,000 genes of the human genome. It is anticipated, in view of the clinical data showing lack of cross-resistance between cisplatin and BBR3464, that the mechanisms of action of each compound, and hence the repertoire of gene expression changes that each induces will be distinct. These experiments are designed to test this hypothesis and also, by identifying the genes in question, to provide important first concrete molecular evidence of mechanistic differences between cisplatin and the new platinum compound BBR3464. Quartz Crystal Microbalance-Based Biosensor for the Quantitative Analysis of Solid Phase DNA Immobilization and Hybridization Tin Christopher Hang and Anthony Guiseppi-Elie Chemical Engineering and Center for Bioelectronics, Biosensors, and Biochips (C3B) Virginia Commonwealth University Richmond, VA 23284-3038 We are concerned with the study of immobilizing 30-mer single stranded DNA (ssDNA) probes onto quartz crystal oscillators and monitoring hybridization with the complementary target sequences for the development of a piezoelectric DNA diagnostic sensor. In the present work, we report on the surface processes and chemistries that are necessary to prepare electrodes as substrates for DNA hybridization. The first step in this process involves the oxidation of platinum electrodes of AT-cut quartz crystal microbalance (QCM) devices to generate a surface amenable to organosilane chemistry. The electrodes were solvent cleaned and chemically oxidized in a 2.5% solution of K2Cr2O7 in 15% HNO3. Preliminary QCM data reveals that the chemical oxidation procedure yields an average mass change of ca. 23.12 ng, corresponding to the generation of oxide functionalities on the cleaned electrode surface. 3-glycidoxypropyltrimethoxysilane (GPS) was then covalently coupled to the oxidized platinum using common organosilane chemistry techniques. Epoxy-derivatized platinum electrodes are produced that are available for the subsequent binding of amine-terminated ssDNA to the sensor surface. Approximately 271 ng of GPS was deposited onto the platinum electrodes. Furthermore, atomic force microscopy (AFM) was used to examine surface morphologies and Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR) was used to verify surface functionality of the oxide and silane layers. Comparative Evaluation of `Bio-smart' Electroconductive Hydrogel Composites for Biosensors Gymama Slaughter, Sean Brahim, and Anthony Guiseppi-Elie Department of Chemical Engineering Virginia Commonwealth University Richmond, Virginia 23284-3038 Two groups of polymers that have been the focus of widespread research are hydrogels and conducting electroactive polymers (CEPs). "Intelligent" hydrogels are materials whose properties change in response to specific environmental stimuli such as pH, temperature, chemical species, magnetic fields, etc. Conducting electroactive polymers such as polypyrrole, polyaniline and polythiophene have accelerated advances in polymer sensing technologies. We had previously formed composites of inherently conductive polypyrrole within highly hydrophilic poly(2-hydroxyethyl methacrylate)-based hydrogels. These materials retain the hydration characteristics of hydrogels as well as the electroactivity and electronic conductivity of CEPs and are thus called `electroconductive hydrogels'. The enhanced biosensing capabilities of these composite films have been demonstrated in the fabrication of glucose, cholesterol and galactose biosensors. In the present work, we modified the composition of the hydrogel composites in two ways to tailor these materials for in vivo biosensing. First, a phospholipid containing monomer, 2-methacrylooyloxyethyl phosphorylcholine (MPC), was covalently linked to the cross-linked hydrogel network. One of the most challenging problems afflicting the performance of biosensors in vivo is the lack of biocompatibility. Our strategy to improve biocompatibility involves incorporating MPC into the formulation, thereby mimicking the primary chemical entities of the outer leaflet of a cell biomembrane, so that the composite gel will present to the extracellular matrix the same chemical functionality and structure that emulates the extracellular matrix. The second modification involved the use of the prepolymer, poly(ethylene glycol) methacrylate (PEGMA). This hydrophilic prepolymer was incorporated into the formulation for the purposes of stabilization of protein activity and to reduce protein denaturation. We evaluated the capability of these modified "bio-smart" materials to function as amperometric biosensors for glucose. A comparative approach was taken in which polypyrrole was not incorporated in the modified hydrogel matrix. The biosensors still displayed extended linear response ranges (0.10-13.0 mM ) to glucose in phosphate buffer with rapid response times (< 60 s), but however, exhibited poor screening of the common electrooxidizable interferents ascorbic acid, uric acid, and L-cysteine. These findings confirm that the incorporation of polypyrrole results in significant screening against indigenous interferents. These composite biosensors are being further developed for in-vivo sensing applications. Further miniaturization to a scale suitable for subcutaneous implantation and modification of the existing biosensor design to accommodate two working electrodes on one biochip will be performed, allowing continuous sensing of glucose and lactate.

©2003 Nam Sun Wang nsw@eng.umd.edu