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1. High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells

Author

Cesar A. Patino, Prithvijit Mukherjee, Eric J. Berns, Elamar Hakim Moully, Liliana Stan, Milan Mrksich, and Horacio D. Espinosa

Subjects
Electroporation, Microfluidics, General Engineering, General Physics and Astronomy, General Materials Science, Gold, RNA, Small Interfering, Plasmids
Abstract

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, we report a

Published
2022
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2. Surface potential modulation as a tool for mitigating challenges in SERS-based microneedle sensors

Author

Vitor Brasiliense, Ji Eun Park, Eric J. Berns, Richard P. Van Duyne, and Milan Mrksich

Subjects
Multidisciplinary, Adsorption, Spectrum Analysis, Raman, Nanostructures
Abstract

Raman spectroscopic-based biosensing strategies are often complicated by low signal and the presence of multiple chemical species. While surface-enhanced Raman spectroscopy (SERS) nanostructured platforms are able to deliver high quality signals by focusing the electromagnetic field into a tight plasmonic hot-spot, it is not a generally applicable strategy as it often depends on the specific adsorption of the analyte of interest onto the SERS platform. This paper describes a strategy to address this challenge by using surface potential as a physical binding agent in the context of microneedle sensors. We show that the potential-dependent adsorption of different chemical species allows scrutinization of the contributions of different chemical species to the final spectrum, and that the ability to cyclically adsorb and desorb molecules from the surface enables efficient application of multivariate analysis methods. We demonstrate how the strategy can be used to mitigate potentially confounding phenomena, such as surface reactions, competitive adsorption and the presence of molecules with similar structures. In addition, this decomposition helps evaluate criteria to maximize the signal of one molecule with respect to others, offering new opportunities to enhance the measurement of analytes in the presence of interferants.

Published
2022
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3. Exploration of the Nanomedicine-Design Space with High-throughput Screening and Machine Learning*

Author

Albert Y. Xue, Eric J. Berns, Chad A. Mirkin, Andrew Lee, Milan Mrksich, Neda Bagheri, and Gokay Yamankurt

Subjects
0301 basic medicine, Computer science, High-throughput screening, Oligonucleotides, Biomedical Engineering, Medicine (miscellaneous), Bioengineering, Machine learning, computer.software_genre, Article, Cell Line, Machine Learning, Structure-Activity Relationship, 03 medical and health sciences, 0302 clinical medicine, Nucleic Acids, Animals, Humans, Screening tool, Analysis method, business.industry, Extramural, Scale (chemistry), Alkaline Phosphatase, High-Throughput Screening Assays, Computer Science Applications, Nanomedicine, 030104 developmental biology, Liposomes, Nanoparticles, Artificial intelligence, business, Design space, computer, 030217 neurology & neurosurgery, Biotechnology, Immune activation
Abstract

Only a tiny fraction of the nanomedicine-design space has been explored, owing to the structural complexity of nanomedicines and the lack of relevant high-throughput synthesis and analysis methods. Here, we report a methodology for determining structure–activity relationships and design rules for spherical nucleic acids (SNAs) functioning as cancer-vaccine candidates. First, we identified ~1,000 candidate SNAs on the basis of reasonable ranges for 11 design parameters that can be systematically and independently varied to optimize SNA performance. Second, we developed a high-throughput method for making SNAs at the picomolar scale in a 384-well format, and used a mass spectrometry assay to rapidly measure SNA immune activation. Third, we used machine learning to quantitatively model SNA immune activation and identify the minimum number of SNAs needed to capture optimum structure–activity relationships for a given SNA library. Our methodology is general, can reduce the number of nanoparticles that need to be tested by an order of magnitude, and could serve as a screening tool for the development of nanoparticle therapeutics.

Published
2020
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4. Traceless Immobilization of Analytes for High-Throughput Experiments with SAMDI Mass Spectrometry

Author

Milan Mrksich, Eric J. Berns, Kazi Y. Helal, and Azmain Alamgir

Subjects
Molecular Structure, Azirines, Surface Properties, 010405 organic chemistry, Chemistry, General Chemistry, Enzymes, Immobilized, 010402 general chemistry, Mass spectrometry, 01 natural sciences, Biochemistry, Chemical reaction, Combinatorial chemistry, Catalysis, High-Throughput Screening Assays, 0104 chemical sciences, Adduct, Matrix-assisted laser desorption/ionization, Colloid and Surface Chemistry, Cytochrome P-450 Enzyme System, Covalent bond, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Desorption, Monolayer, Molecule
Abstract

[Image: see text] Label-free assays, and particularly those based on the combination of mass spectroscopy with surface chemistries, enable high-throughput experiments of a broad range of reactions. However, these methods can still require the incorporation of functional groups that allow immobilization of reactants and products to surfaces prior to analysis. In this paper, we report a traceless method for attaching molecules to a self-assembled monolayer for matrix-assisted laser desorption and ionization (SAMDI) mass spectrometry. This method uses monolayers that are functionalized with a 3-trifluoromethyl-3-phenyl-diazirine group that liberates nitrogen when irradiated and gives a carbene that inserts into a wide range of bonds to covalently immobilize molecules. Analysis of the monolayer with SAMDI then reveals peaks for each of the adducts formed from molecules in the sample. This method is applied to characterize a P450 drug metabolizing enzyme and to monitor a Suzuki–Miyaura coupling chemical reaction and is important because modification of the substrates with a functional group would alter their activities. This method will be important for high-throughput experiments in many areas, including reaction discovery and optimization.

Published
2018
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5. In vivomigration of endogenous brain progenitor cells guided by an injectable peptide amphiphile biomaterial

Author

H. Georg Kuhn, Samuel I. Stupp, Piyush M. Patel, Reza Motalleb, Eric J. Berns, and Julie Gold

Subjects
Male, 0301 basic medicine, Doublecortin Protein, animal structures, Rostral migratory stream, Nanofibers, Biomedical Engineering, Medicine (miscellaneous), Biocompatible Materials, Neocortex, Article, Rats, Sprague-Dawley, Biomaterials, 03 medical and health sciences, Neural Stem Cells, Neuroblast, Cell Movement, medicine, Peptide amphiphile, Animals, Progenitor cell, Microglia, Chemistry, Tenascin, Cell migration, Neural stem cell, Rats, Cell biology, 030104 developmental biology, medicine.anatomical_structure, nervous system, Brain Injuries, Peptides
Abstract

Biomaterials hold great promise in helping the adult brain regenerate and rebuild after trauma. Peptide amphiphiles (PA) are highly versatile biomaterials, gelling and forming macromolecular structures when exposed to physiological levels of electrolytes. We are here reporting on the first ever in vivo use of self-assembling peptide amphiphile carrying a Tenascin-C signal (E(2)Ten-C PA) for the re-direction of endogenous neuroblasts in the rodent brain. The PA forms highly aligned nanofibers, displaying the migratory sequence of Tenascin-C glycoprotein as epitope. In this in vivo work, we have formed in situ a gel of aligned peptide amphiphile nanofibers presenting a migratory Tenascin-C signal sequence in the ventral horn of the rostral migratory stream, creating a track reaching the neocortex. Seven days post-transplant, doublecortin positive cells were observed migrating inside and alongside the injected biomaterial, reaching the cortex. We observed a 24-fold increase in number of redirected neuroblasts for the E(2)Ten-C PA injected animals compared to control. We also found injecting the E(2)Ten-C PA to cause minimal neuroinflammatory response. Analyzing GFAP(+) astrocytes and Iba1(+) microglia activation, the PA does not elicit a stronger neuroinflammatory response than would be expected from a small needle stab wound. Re-directing endogenous neuroblasts and increasing the number of cells reaching a site of injury using peptide amphiphiles may open up new avenues for utilizing the pool of neuroblasts and neural stem cells within the adult brain for regenerating damaged brain tissue and replacing neurons lost to injury.

Published
2018
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6. Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells

Author

Milan Mrksich, Elamar Hakim Moully, and Eric J. Berns

Subjects
Phosphopeptides, Lysis, Protein tyrosine phosphatase, 010402 general chemistry, 01 natural sciences, Article, Analytical Chemistry, Flow cytometry, Cell Line, Tumor, Lysis buffer, medicine, Humans, Sulfhydryl Compounds, Enzyme Assays, medicine.diagnostic_test, Phosphopeptide, Chemistry, 010401 analytical chemistry, HEK 293 cells, Membranes, Artificial, Flow Cytometry, 0104 chemical sciences, Matrix-assisted laser desorption/ionization, HEK293 Cells, Cell culture, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Biophysics, Ethylene Glycols, Protein Tyrosine Phosphatases, Single-Cell Analysis
Abstract

Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS). Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation—during which the PTP enzymes act on the peptide substrate—the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.

Published
2019

7. Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry

Author

Maria D. Cebezas, Eric J. Berns, and Milan Mrksich

Subjects
0301 basic medicine, chemistry.chemical_classification, Chromatography, Chemistry, Nanotechnology, General Chemistry, Mass spectrometry, 01 natural sciences, Article, Mass Spectrometry, Enzymes, High-Throughput Screening Assays, Substrate Specificity, 0104 chemical sciences, Biomaterials, 010404 medicinal & biomolecular chemistry, 03 medical and health sciences, 030104 developmental biology, Enzyme, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, High-content screening, Substrate specificity, General Materials Science, Biotechnology
Abstract

Cell-based, high-throughput screening (HTS) assays are increasingly important tools used in drug discovery, but frequently rely on readouts of gene expression or phenotypic changes and require development of specialized, labeled reporters. Here a cell-based, label-free assay compatible with HTS is introduced that can report quantitatively on enzyme activities by measuring mass changes of substrates with matrix-assisted laser desorption/ionization mass spectrometry. The assay uses self-assembled monolayers to culture cells on arrays presenting substrates, which serve as reporters for a desired enzyme activity. Each spot of cells is treated with a compound, cultured and lysed, enabling endogenous enzymes to act on the immobilized peptide substrate. It is demonstrated that the assay can measure protein tyrosine phosphatase (PTP) activity from as few as five cells and a screen is described that identifies a compound that reduces PTP activity in cell lysates. This approach offers a valuable addition to the methods available for cell-based screening.

Published
2016
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8. Enzyme Sampling: Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry (Small 26/2020)

Author

Prithvijit Mukherjee, Cesar A. Patino, John A. Kessler, Horacio D. Espinosa, Eric J. Berns, Lingqian Chang, S. Shiva P. Nathamgari, Elamar Hakim Moully, and Milan Mrksich

Subjects
Biomaterials, chemistry.chemical_classification, Enzyme, Chromatography, chemistry, Electroporation, Microfluidics, Sampling (statistics), General Materials Science, General Chemistry, Mass spectrometry, Biotechnology
Published
2020
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9. Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels

Author

Samuel I. Stupp, Shuming Zhang, John A. Kessler, Joshua E. Goldberger, Shantanu Sur, Sunitha C. Suresh, Eric J. Berns, and Liuliu Pan

Subjects
Scaffold, Patch-Clamp Techniques, Materials science, Neurite, Nanofibers, Biophysics, Bioengineering, Synaptic Transmission, Article, Biomaterials, Mice, Microscopy, Electron, Transmission, Dorsal root ganglion, Cell Movement, Neurites, Peptide amphiphile, medicine, Animals, Neural cell, Cells, Cultured, Tissue Scaffolds, Regeneration (biology), Neural stem cell, medicine.anatomical_structure, Mechanics of Materials, Neural tissue regeneration, Microscopy, Electron, Scanning, Ceramics and Composites, Gels, Oligopeptides, Biomedical engineering
Abstract

Regeneration of neural tissues will require regrowth of axons lost due to trauma or degeneration to reestablish neuronal connectivity. One approach toward this goal is to provide directional cues to neurons that can promote and guide neurite growth. Our laboratory previously reported the formation of aligned monodomain gels of peptide amphiphile (PA) nanofibers over macroscopic length scales. In this work, we modified these aligned scaffolds specifically to support neural cell growth and function. This was achieved by displaying extracellular matrix (ECM) derived bioactive peptide epitopes on the surface of aligned nanofibers of the monodomain gel. Presentation of IKVAV or RGDS epitopes enhanced the growth of neurites from neurons encapsulated in the scaffold, while the alignment guided these neurites along the direction of the nanofibers. After two weeks of culture in the scaffold, neurons displayed spontaneous electrical activity and established synaptic connections. Scaffolds encapsulating neural progenitor cells were formed in situ within the spinal cord and resulted in the growth of oriented processes in vivo. Moreover, dorsal root ganglion (DRG) cells demonstrated extensive migration inside the scaffold, with the direction of their movement guided by fiber orientation. The bioactive and macroscopically aligned scaffold investigated here and similar variants can potentially be tailored for use in neural tissue regeneration.

Published
2014
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10. Nanofiber-mediated inhibition of focal adhesion kinase sensitizes glioma stemlike cells to epidermal growth factor receptor inhibition

Author

John A. Kessler, Eric J. Berns, Maya Srikanth, Samuel I. Stupp, Sunit Das, and Juno Kim

Subjects
Cancer Research, Blotting, Western, Nanofibers, Fluorescent Antibody Technique, Apoptosis, Mice, SCID, Biology, Real-Time Polymerase Chain Reaction, Immunoenzyme Techniques, Focal adhesion, Mice, Cell Movement, Mice, Inbred NOD, Glioma, Cell Adhesion, Tumor Cells, Cultured, medicine, Animals, Humans, Immunoprecipitation, RNA, Messenger, Epidermal growth factor receptor, Cell adhesion, In Situ Hybridization, Fluorescence, Cell Proliferation, Brain Neoplasms, Reverse Transcriptase Polymerase Chain Reaction, Kinase, Cell growth, Integrin beta1, Flow Cytometry, medicine.disease, Peptide Fragments, ErbB Receptors, Oncology, Focal Adhesion Protein-Tyrosine Kinases, Basic and Translational Investigations, Neoplastic Stem Cells, Cancer research, biology.protein, Laminin, Neurology (clinical), Stem cell
Abstract

Background. Glioblastoma multiforme is the most common glioma in adults and carries a poor prognosis, due to tumor recurrence despite aggressive treatment. Such relapse has been attributed to the persistence of glioma stemlike cells (GSCs), a subpopulation of glioma cells with stem cell properties. Thus, targeting these cells will be critical to achieving meaningful improvement in glioblastoma multiforme survival. We investigated the role of b1-integrin signaling as one such potential target. Methods. We used GSCs isolated from primary human gliomas and maintained in stem cell conditions. We manipulated b1-integrin signaling using a self-assembling peptide amphiphile (PA) displaying the IKVAV (isoleucine-lysine-valine-alanine-valine) epitope as well as lentiviral overexpression, and we assayed the effects on downstream effectors and apoptosis using immunofluorescence. Results. We show that b1-integrin expression correlates with decreased survival in glioma patients and that b1integrin is highly expressed by GSCs. The IKVAV PA potently increases immobilized b1-integrin at the GSC membrane, activating integrin-linked kinase while inhibiting focal adhesion kinase (FAK). The IKVAV PA induces striking apoptosis in GSCs via this FAK inhibition, which is enhanced in combination with inhibition of epidermal growth factor receptor (EGFR). Conversely, lentiviral overexpression of b1-integrin renders GSCs resistant to EGFR inhibition, which was overcome by FAK inhibition. Conclusions. These observations reveal a role for b1-integrin signaling through FAK in GSC treatment resistance and introduce self-assembling PAs as a novel new therapeutic approach for overcoming this resistance.

Published
2013
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11. Measuring Drug Metabolism Kinetics and Drug-Drug Interactions Using Self-Assembled Monolayers for Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

Author

Pradeep Bugga, Alfred L. George, Milan Mrksich, Eric J. Berns, and Lyndsey L. Anderson

Subjects
Drug, media_common.quotation_subject, Tolbutamide, Kinetics, 02 engineering and technology, 010402 general chemistry, Mass spectrometry, 01 natural sciences, Analytical Chemistry, In vivo, New chemical entity, medicine, Humans, Hypoglycemic Agents, Drug Interactions, media_common, Cytochrome P-450 CYP2C9, Molecular Structure, Chemistry, 021001 nanoscience & nanotechnology, 0104 chemical sciences, High-Throughput Screening Assays, Matrix-assisted laser desorption/ionization, Biochemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Biophysics, 0210 nano-technology, Drug metabolism, medicine.drug
Abstract

The competition of two drugs for the same metabolizing enzyme is a common mechanism for drug-drug interactions that can lead to altered kinetics in drug metabolism and altered elimination rates in vivo. With the prevalence of multidrug therapy, there is great potential for serious drug-drug interactions and adverse drug reactions. In an effort to prevent adverse drug reactions, the FDA mandates the evaluation of the potential for metabolic inhibition by every new chemical entity. Conventional methods for assaying drug metabolism (e.g., those based on HPLC) have been established for measuring drug-drug interactions; however, they are low-throughput. Here we describe an approach to measure the catalytic activity of CYP2C9 using the high-throughput technique self-assembled monolayers for matrix-assisted laser desorption-ionization (SAMDI) mass spectrometry. We measured the kinetics of CYP450 metabolism of the substrate, screened a set of drugs for inhibition of CYP2C9 and determined the Ki values for inhibitors. The throughput of this platform may enable drug metabolism and drug-drug interactions to be interrogated at a scale that cannot be achieved with current methods.

Published
2016

12. Cell-Based Screening: Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry (Small 28/2016)

Author

Milan Mrksich, Maria D. Cabezas, and Eric J. Berns

Subjects
chemistry.chemical_classification, Chemistry, Cell Survival, Cell, Analytical chemistry, General Chemistry, Mass spectrometry, Mass Spectrometry, High-Throughput Screening Assays, Biomaterials, Membrane, medicine.anatomical_structure, Enzyme, Cell Line, Tumor, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Monolayer, Lysis buffer, medicine, Biophysics, Humans, General Materials Science, Cell adhesion, Biotechnology, Cell based
Abstract

On page 3811, M. Mrksich and co-workers culture cells using self-assembled monolayers presenting cell adhesion ligands and enzyme substrates. A lysis buffer disrupts the cell membranes, releasing enzymes that modify the immobilized substrates. These modifications can be measured with SAMDI mass spectrometry, giving a high-throughput, cell-based assay.

Published
2016

13. The National Lung Screening Trial: Overview and Study Design

Author

Natalie Cunningham, Michael Khalili, John Waltz, Ralph Weiben, Deb Gurtner, Linda DeAlmeida, Sanjay Gupta, Sharon Maxfield, Crissy Kibic, Kathleen DeWitt, David DeMets, Walter Allen Bowman, Robert Epstein, Mia Burkhard, Stephen J. Swensen, Hattie Cromwell, Kianoush Rezai, Steadman Sankey, Lisa Scott Wasson, Rita Musanti, Tamim Malbari, Joy Ferola, Qimei He, Patty Trapnell, Melba Francis, Sam Quattlebaum, Joanice Thompson, Ana Birofka, Robin Griggs, Elizabeth Johnson, Margaret R. Spitz, Nicole Richardson, Yuting Liang, Lawrence G. Hutchins, Mirjana Tecmire, Lila Camara, James J. Navin, Eileen Frost, Diane Romano, Carrie Petkus, Eric J. Berns, Pei Jan P Lin, Steve D. Uttecht, Marian Acerra, Lawrence R. Ragard, Leo P. Lawler, Christopher M. Rogers, Alan Lee Goodwin, L. Ellen Martinusen, Melissa Ford, Michael T. Fisher, Beverly Powell, Cindy Lin, Jamie Downs, Brent Fodera, Bonita Wohlers, Michael Brangan, Peggy Bradley, Todd B. Burt, Susan Allen, Shiva Borgheian, Mingying Zeng, Thomas Riley, Danielle Gherardini, Steven Shiff, Olivia Campa, Wahied Gendi, Fang F. Xu, Ivana K. Kazda, Anne Chung, Briar Doi, Helen Price, Maria Vlachou, Alan Morgan, Simone Vuong, Pierre P. Massion, Darcy Watson, Debbie William, Esther Nakano, Karen Broski, David Creed, Melanie Bvorak, Lakisha Hawkins, Gladys Hino, Raymond Dauphinais, Michele Sallas, Helene Shiratori, Venus Brown, Denise Brooks, Heather Porter, Ilana F. Gareen, Tracy Lee, Melissa Cates, Kyle Turner, Tiffanie Hammond, Margaret Paquette, Lorraine Kerchum, Barbara Lewis, Douglas J. Reding, Thomas E. Hartman, Cathy Longden, Melissa Laron, Reza Abaya, Beborah Robertson, J W Semenkovich, Christine Holland, Hugh McGinley, Chani Montalbo, Karen Zubena, Vanessa Ralda, Adam C. Stein, Jennifer Ott, Lawrence M. Kotner, Jing Lee, Arnold Ssali, Michael Young, Quinn A. DeMordaunt, Linda V. White, Steve Dubinett, Pearl Chan, Roxana Phillips, Mallory Kolich, Brent B. Nelson, Phi Do, Jill Spivak, Angele LaFleur, Kesha Smith, Elayne Weslowsky, Patricia Nieters, Maurice LeBlanc, Satinder Singh, Lonna Matthews, Quentin McMullen, Karen Lappe, Sharon Longacre, Cindy Cobb, Jane A. Zehner, Michael Teepe, Pamela M. Marcus, Kathleen Bow, Wendy Francis, Mary Gemmel, Robert S. Fontana, Linda Jurjans, Barbara Ginther, Jonathan B. Clapp, Monica Richel, Scott F. Pickering, Brenda Edwards, Kendrick Looney, Randy Marshall, Roni Atkins, Danielle Wicks, Julie Peterson, Dcanna Cape, Albert J. Cook, Jerry Brekke, Louisa Turner, Larry Stoller, Mark B. Salerno, Bavid E. Midthun, Mark Delano, Minnetta Belyea, Deborah Greene, Jonathan Goldin, Terry Lewis, Virginia Fischer, Andrea Chapman, Shari Jordan, Deb Warren, Demetria Johnson, Rekha Khatri, Lisa Sirianni, Guillermo Geisse, Michael A. Fuchs, Kanya Kumbalasiri, Jeremy J. Erasmus, Vicki Shambaugh, Denise Boyles, Sarah Hallsky, Anna Nanovski, Jill Heinz, Mollie King, Kay Vydareny, Olga Soukhanova, Patricia Rueweler, Perry G. Pernicano, Regina Rendas-Baum, Phyllis Pirotte, Russell Harris, Neil Argyle, Miyoung Kim, June Krebsbach, Audrey Gallego, Sheila Wein, Mukesh F. Karwat, Karla Myra-Bloom, Pamela Byrnes, Mitchell D. Schnall, Hector Ahumada, Eric Sanchez, Donna DesMarais, Julie Maderitz, Cindy Lavergne, Lori Kirchoff, Patricia C. Sanders, Elizabeth Thielke, Michael Sullivan, Jennifer Gaegler, Janet Manual, Jennifer R. Heinz, Ray Zisumbo, Diane C. Strollo, Candace Mueller, Irene Mahon, Brenda Delfosse, Carolyn M. Johnson, William E. Grizzle, Merideth Stanley, Sylvan Green, Pamela Harvey, Lindsay Richardson, Brenda K. Brewer, Philip Costello, Deanna Zapolski, John Worrell, Jeffrey G. Schragin, David S. Alberts, Edward L. Korn, Tamara Owens, Hank Brastater, Kay Mathiesen-Viergutz, Mark Broschinsky, Paul W. Spirn, Grace Isaacs, John S. Waltz, Mitch Goodsitt, Christi Newton-Foster, Sharlene Snowden, Barbara Voight, Gail Bizer, Kathy McDonough, William Huynh, Eduard Van Stam, Robert A. Carlson, Mike Florzyk, Paula M. Jacobs, Joan Fuller, Mauren Grunenwald, Ann Bangerter, Jacksonville, Adriane Andersen, Tess Thompson, Kenneth Nowers, Stephanie Helwi, Martin J. Edelman, Emmanuel Omoba, Rubenia Flores, Kevin T. White, Patrick W. Wolfe, Michael Milacek, Sharon Gard, Brandon B. Bigby, Cynthia H. McCollough, Andrew Burnside, Sheryl L. Ogden, Maisha Pollard, Thomas K. Pilgram, Sydney Laster, Claudia J. Kasales, Bruce W. Turnbull, Cheri Haselhuhn, Laura N. Myers, Jean Jacobsen, Melissa Love, Gavin D. Watt, Cheryl Love, Gerald F. Abbott, Susanne Kozakowski, Jerry L. Montague, Cynthia Hill, Neil F. O'Donnell, Anna Sear, Thomas M. Beck, Jean Wegner, Chrispina Wray, Edward M. Brown, Louise Ledbetter, Karen Bellware, Julie Moody, Noel Bahr, Matthew T. Freedman, Thomas Hensley, John E. Madewell, Leanne Hadfield, David R. Maffitt, Lisa Cottrell, John J. Warner, Deborah Graham, Krystal Arnold, Alejandra Reyes, Kristin Lieberman, Derek Omori, Donna Garland, Mike Burek, Mel Johnson, Judith Harkins, Martha Fronheiser, M. Y. M. Chen, Dawn Simmons, Kathleen Voight, Aaron O. Bungum, Marianne Rice, Lakeshia Murray, Tami Krpata, Donna Sammons, Leslie Kmetty, Catherine Duda, Carissa Krzeczkowski, Anne Nguyen, Richard H. Lane, Cynthia Mack, Loren C. Macey, Eddy Wicklander, Kelly McDaniel, Sue Zahradka, Hassan Bourija, Cristina Farkas, Jincy George, Renae Kiffmeyer, Wendell Christie, Catherine Engartner, John Crump, Mimi Kim, Carol Steinberg, Reginald F. Munden, Deb Kirby, Jo Ann Stetz, Barbara O'Brien, Sally Tenorio, Laura Multerer, Carlotta McCalister-Cross, Jessica Silva-Gietzen, Tamara Saunders, Harvey Glazer, Cam Vashel, Maria Oh, Rodkise Estell, Steven M. Moore, Tara Riley, Grant Izmirlian, D. Claire Anderson, James Burner, Steven Peace, Phil Hoffman, Angela Del Pino, Brian Irons, Carlos Jamis-Dow, John K. Lawlor, Edward F. Patz, Jay Afiat, Amber Barrow, Bawn M. Beno, Melissa S. Fritz, Lynn Coppage, Scott J. Sheltra, Tim Swan, Jerry Bergen, Charlie Fenton, Eric Deaton, Marilyn J. Siegel, Korinna Vigeant, Kerry Engber, Sarah Merrill, Buddy Williams, Kimberly Stryker, Bradley S. Snyder, Christina Romo, Andrea Hugill, Michael J. O'Shea, Linda White, Gail Fellows, Yasmeen Hafeez, Joe Woodside, Shauna Dave Scholl, Philip C. Prorok, Sharon Carmen, Kelly Hatton, Steven V. Marx, Sooah Kim, Robert Kobistek, Dawn Thomas, Lea Momongan, Chris Steward, Kari Bohman, Holly Bradford, Bradley S. Sabloff, Phillip Peterson, William C. Black, Lisa Pineda, James G. Ravenel, Karen Taylor, Beverly Trombley, Mona N. Fouad, Amber McDonald, Lauren J. Ramsay, Lisa Harmon, Jeffrey Geiger, David L. Spizarny, Jeffrey S. Klein, Xizeng Wu, Heather Tumberlinson, Joy Espiritu, Gina Varner, Dawn Fuehrer, Eric A. Hoffman, Sheila Moesinger, Nina Wadhwa, Steve King, Patricia Lavernick, Paola Spicker, Timothy R. Church, Cheryl Whistle, Sheila Greenup, Patricia Fantuz, Stephanie Levi, Peter Balkin, Mary E. Johnson, Johanna Ziegler, Susan Hoffman, Kathy L. Clingan, Craig Kuhlka, Maria Marchese, Lawrence F Cohen, Cylen Javidan-Nejad, Wilbur A. Franklin, Kevin J. Leonard, Tim A. Parritt, Jade Quijano, Kathleen Poler, Jennifer Rosenbaum, Xiuli Zhang, Christine Brown, Terri David-Schlegel, Susan M. Peterson, James R. Jett, Kenneth W. Clark, Edward P. Gelmann, Arthur Migo, Patricia Fox, Lori Hamm, Janie McMahon, Darlene Guillette, Robert C. Young, Patty Beckmann, Jerome Jones, Nikki Jablonsky, Roberta Yoffie, Heather L. Bradley, Darlene Higgins, Francine L. Jacobson, Christine B. Berg, Mark Bramwitt, Constantine N. Petrochko, Karen Stokes, Jennifer Rowe, Kathy McKeeta-Frobeck, Brenda Sleasman, Courtney Bell, Dave Tripp, Saundra S. Buys, Susan Walsh, Jo Rean D. Sicks, Richard G. Barr, Kirk Midkiff, Tom Caldwell, Elisabeth A. Grady, Subbarao Inampudi, Marilyn Calulot, Paul A. Kvale, Alice DuChateau, Kathy Berreth, Ruth Holdener, Katie Kuenhold, Thomas E. Warfel, David P. Naidich, Mandie Leming, Fraser Wilton, Leanne Franceshelli, Kathleen McMurtrie, Elaine Bowman, Donald F. Bittner, Helen Kaemmerer, Merri Mullennix, Adelheid Lowery, Andrew Karellas, Jenny Hirschy, Kate Naughton, Ashley B. Long, Kristin M. Gerndt, Kathleen Young, Richard M. Schwartzstein, Wendy Smith, Joseph Aisner, Shane Ball, Kathleen Krach, Cathy Mueller, Virginia May, Christopher Blue, Marsha Lawrence, Ronald S. Kuzo, Colleen McGuire, Alisha Moore, Sara Cantrell, Christie Leary, Pamela Allen, Maryann Trotta, Clifford Caughman, Peggy J. Gocala, Brian Mullen, Janan Alkilidar, Maryann Duggan, Lin Mueller, Alesis Nieves, Fenghai Duan, Frederick Olson, Edwin G. Williams, Jo Ann Hall Sky, Grant Izmirilian, Peggy Joyce, Judy Preston, Cristine Juul, Julianne Falcone, Bruce Neilson, Fla Lisa Beagle, Beth Evans, Jamie Mood, Janet Bishop, Jean Tsukamoto, Vivien Gardner, Gillian Devereux, Minesh Patel, Sally Fraki, Celia Stolin, Ami Lyn Taplin, Stephenie Johnson, Saeed Matinkhah, Jenna Bradford, Sanjeev Bhalla, Charles Jackson, Julie Haglage, Darlene R. Fleming, Allie M. Bell, Paul A. Bunn, Gail Orvis, Andrew J. Bierhals, Julie Ngo, Belores K. Prudoehl, Elaine N. Daniel, Peggy Olson, Paul F. Pinsky, Glenna M. Fehrmann, Aras Acemgil, Andrea Hamilton-Foss, Leeta Grayson, Smita Patel, Scott Emerson, Carl J. Zylak, James R. Maxwell, Jennifer Fleischer, Suzanne Smith, Jacqueline R. Sheeran, Alan Williams, Scott Gaerte, John Fletcher, Sonya Clark, Nancy Gankiewicz, Stuart S. Sagel, Jason Spaulding, Nancy E. Hanson, Nicole Fields, Richard D. Nawfel, Dinakar Gopalakrishnan, Margaret Oechsli, Susan Wenmoth, Isabelle Forter, Elizabeth Morrell, Jessica Rider, Letitia Clark, Michael Woo, Cynthia A. Brown, Camille Mueller, Mark T. Dransfield, Lois M. Roberts, Anne Randall, Eduard J. Gamito, Carrie O'Brien, Carolyn Palazzolo, Julie Schach, Robert Falk, Melissa Hudson, Jennifer Garcia Livingston, Cynthia L. Andrist, Tammy Fox, Elliott Drake, Tanya Zeiger, Renee Metz, Kevin Thomas, Neha Kumar, Elizabeth Couch, Beborah Bay, Mei Hsiu Chen, Jason Bronfman, Philip Dennis, Deb Engelhard, Pamela McBride, Daniel Kimball, Amy Haas, Pamela M. Mazuerk, Marlea Osterhout, Venetia Cooke, Tina Taylor, Amy St.Claire, Joe Hughes, Becky McElsain, Beverly Brittain, Michele Adkinson, Paige Beck, Martha Maineiro, Paula R. Beerman, Jackie Seivert, Mary M. Pollock, Donald Corle, Tina Herron, Marcella Petruzzi, Natalie F. Scully, Kenneth A. Coleman, Jennifer Yang, Debra Loria, Wendy Moss, Alan Brisendine, Cheryl M. Lewis, Dalphany Blalock, Lonni Schultz, Douglas Bashford, Nora Szabo, David Shea, Amanda Devore, Karen Schleip, Judy Netzer, Barry Clot, Gerald M. Mulligan, Nancy E. Krieger Black, David Schultz, Jim Pool, Craig E. Leymaster, Kathryn Rabanal, Kay Bohn, Tara Berg, Marisol Furlong, Stacey Mitchell, Donna Biracree, Laura Jones, Cassie Olson, Robin Stewart, Jeremy Pierce, Marilyn Bruger, Valene Kennedy, Stephanie Davis, Colin O'Donnell, Glenn A. Tung, Shannon Wright, William Lake, Sharon Jones, Vincent Girardi, Brad Benjamin, Veenu Harjani, Drew A. Torigian, Kevin Edelman, Sue Frederickson, Paul E. Smart, Michelle Wann Haynes, D S Gierada, Glenn Fletcher, Rosalie Ronan, Patricia Ann Street, Eleace Eldridge-Smith, Lynly Wilcox, Cindy Lewis-Burke, La Tonja Davis, Rachel Black Thomas, Dawn Shone, Evangeline Griesemer, Tim Budd, Lindsey Dymond, Marlene Semansky, Amy Rueth, Constantine Gatsonis, Kay H. Vydareny, Usha Singh, Amy Lita Evangelista, Angelica C. Barrett, Bethany Pitino, Shirley Wachholz, Angela M. Williams, Sandra Fiarman, Karen Luttrop, David Chellini, Michael Bradley, Helen Fink, Aaron Zirbes, Roger Inatomi, Joon K. Lee, Heather Bishop Blake, Lisa Woodard, Craig Hritz, Sarah Neff, Aine Marie Kelly, Deborah Harbison, Baigalmaa Yondonsambuu, Amy Lloyd, Christine Gjertson, Erin Cunningham, Angelee Mean, June Morfit, Ping Hu, William Thomas, Jazman Brooke, Paul Marcus, Jeremy Gorelick, Erin Lange, William Stanford, Denise R. Aberle, Lena Glick, Annabelle Lee, Ian Malcomb, Deanna L. Miller, Mary Mesnard, Jacqueline Jackson, Jhenny Hernandez, Desiree E. Morgan, Howard I. Jolies, Jacquie Marietta, Teresa Lanning, Debra Rempinski, Amanda C. Davis, Karen Mathews Batton, Mahadevappa Mahesh, Erik Wilson, Deana Nelson, Sharan L. Campleman, William Manor, Julie Sears, Howard Mann, E. David Crawford, Carl Krinopol, Greg Gambill, Margo Cousins, Rex C. Yung, Sangeeta Tekchandani, Thomas Vahey, Ann D. McGinnis, Kimberly Nolan, Kaylene Crawford, Kelli P. Rockwell, Dana Roeshe, Fred W. Prior, Kari Ranae Kramer, Heidi Nordstrom, Frank Stahan, Shawn Sams, Cherie Baiton, Joy Tani, Thomas J. Watson, Angela Cosas, Diane Kowalik, Pritha Dalal, Ann Jolly, Jeanine Wade, Laura Bailey, Julie Varner, Glen K. Nyborg, Christopher Toyn, David Gemmel, Susanna N. Dyer, Laurie Amendolare, Mary Ellen Frebes, Judy Ho, Adele Perryman, John Keller, D. Sullivan, George Mahoney, Scott Cupp, Linda L. Welch, Peter Greenwald, Robert Sole, Marcello Grigolo, Caroline Chiles, Patricia Sheridan, Deborah M. Chewar, Vijayasri Narayanaswami, Susan Blackwell, Suzanne B. Lenz, Alphonso Dial, Melvin Tockman, Carolyn Hill, John Stubblefield, Catherine E. Smith, Judith Lobaugh, Rosa M. Medina, Jackie Meier, Nandita Bhattacharjee, Robert Tokarz, Lisa Clement, Nancy Caird, Cindy Masiejczyk, Patricia Shwarts, Laura Springhetti, Sandra Schornak-Curtis, Edwin F. Donnelly, Patricia Tesch, Laurie Rathmell, Pamela K. Woodard, Edward A. Sausville, David R. Pickens, Kylee Hansen, Paulette Williams, Barbara Ferris, Rachel L. McCall, Nicole M. Carmichael, Dawn Whistler, Ramachandra Chanapatna, Glynis Marsh, Mary Wiseman, Tony DeAngelis, L. Heather, Vicki Prayer, Robin Laura, Priscilla Bland, Gregory W. Gladish, Amy Garrett, Kelly McNulty, Daniel J. Pluta, Mylene T. Truong, Serelda Young, Crista Cimis, Gordon Jacob Sen, Rhonda Rosario, Anthony B. Miller, Edward Hunt, Juanita Helms, Jill K. Bronson, Jeff Yates, Ginette D. Turgeon, Bo Lu, Nancy Fredericks, Pam Senn, Ryan Pena, Hakan Sahin, Mary Lynn Steele, Jill E. Cordes, Noel Maddy, R. Adam DeBaugh, Hope Hooks, Zipporah Lewis, Robert L. Berger, Shani Harris, Natalie Gray, Jennifer Kasecamp, Elizabeth King, Jacinta Mattingly, Hrudaya Nath, Kathy Torrence, Christine Cole Johnson, Sara Mc Clellan, Kalin Albertsen, Kim Sprenger, Ryan Norton, Jody Wietharn Kristopher, Linda Warren, Byung Choi, Casey O'Quinn, Mark K. Haron, Chris J. Jennings, Karen Robinson, Joan Molton, Dorothy Hastings, Robert I. Garver, Christopher J. Cangelosi, Jeannette Lynch, Peter Ohan, Angela Campbell, Dawn Mead, Miriam Galbraith, Divine Hartwell, Natalya Portnov, Gene L. Colice, Andetta R. Hunsaker, Analisa Somoza, Todd Risa, Daniel C. Sullivan, Karthikeyan Meganathan, Tammy DeCoste, Peter Zamora, Richard M. Fagerstrom, Iiana Gareen, Phyllis J. Walters, Barbara L. Carter, Alem Mulugeta, Rob Bowman, Kavita Garg, Andrea Franco, Mary Adams Zafar Awan, Edward Reed Smith, Rachel Phillips, Michelle Aganon-Acheta, Fred R. Hirsch, Peter Jenkins, Pamela Taybus, Joy Knowles, Karen M. Horton, Cheryl Spoutz-Ryan, Sarah Landes, William G. Hocking, Laura B. Schroeder, Erini Makariou, Jered Sieren, Kaylene Evans, Erin Nekervis, Brenda Polding, Tonda Robinson, Joel L. Weissfeld, Terry J. Sackett, Michael F. McNitt-Gray, Leslie Dobson, Raymond Weatherby, Randell Kruger, Revathy B. Iyer, Mary Krisk, Anthony Levering, Susan Collins, Alison Schmidt, William M. Hanson, Patricia Schuler, Karen Glanz, Morgan Ford, Beatrice Trotman-Bickenson, Richard Guzman, Paul Koppel, Judith K. Amorosa, Meredith Slear, Dayna Love, Carol Vaughn, Kellyn Adams, Celeste Monje, Garry Morrison, Sherri Mesquita, Paul Cronin, Tony Blake, Constance Elbon-Copp, Robert A. Clark, Felix Mestas, Erich Allman, Armen Markarian, Cheryl Souza, Karen O’Toole, Elliot K. Fishman, Karen Augustine, Jane Hill, Bonnie Kwit, Ralph Drosten, Susan Foley, Stacy E. Smith, Angie Bailey, Jennifer Bishop Kaufmann, Shelly Meese, Phillip M. Boiselle, Howard Morrow, Thomas D. Hinke, Barry Edelstein, Erin Schuler, William C. Bailey, Donna Letizia, David S. Gierada, Frederick J. Larke, Robin Haverman, Sarah Baum, Sally Hurst, Richard L. Morin, Ben Dickstein, William Russell, J. Anthony Seibert, Sophia Sabina, Mary Alyce Riley, Michael A. Taylor, Katherine BeAngelis, Robert A. Hawkins, Fernando R. Gutierrez, Amie Welch, Heather Lancor, George Armah, James Blaine, Eric Henricks, Joel Dunnington, Carole Walker, Laura Motley, Melody Kolich, Bruce J. Hillman, David W. Sturges, Mindy Lofthouse, Amy Warren, Michael Black, Mark Kolich, Lisa A. Holloway, Shannon M. Pretzel, Susan Shannon, Yassminda Harts, Dallas Sorrel, Lance A. Yokochi, Diana Wisler, Arthur Sandy, Roberta Clune, Shirley Terrian, Shalonda Manning, Bradley Willcox, Thomas J. Payne, James L. Tatum, Dale Brawner, Sandy Morales, Rodolfo C. Morice, Amy Vieth, Emily Jewitt, Chelsea O'Carroll, Theresa C. McLoud, John E. Langenfeld, Chris H. Cagnon, Lisa B. Hinshaw, Gena Kucera, Helena R. Richter, Drew Torigian, June McSwain, Courtney Eysmans, Vinis Salazar, David Spizarny, Mary Kelly-Truran, Mark Whitty, Henry Albano, Connie L. Sathre, William R. Geiser, Barnett S. Kramer, Marianna Gustitis, Gordon C. Jones, Neil E. Caporaso, Timothy Welsh, Roger Tischner, Ana Maria Mendez, Dominick A. Antico, Cathy L. Bornhorst, Carla Chadwell, Stephanie Pawlak, Kelli M. West, Joe V. Selby, Randall Kruger, Jodi Hildestad, Elaine Freesmeier, Nicole Rivas, Andrew Goodman, Naima Vera-Gonzalez, Stuart Lutzker, Eric M. Hart, Melanie Yeh, Shane Sorrell, Deb Multerer, Sharon Jacoby, Debbie Gembala, Elizabeth Fleming, Myrle Johnson, Michael J. Flynn, Frank Tabrah, Martin L. Schwartz, Deanna Mandley, Brad Siga, Guillermo Marquez, Jeffrey Koford, Victoria Jenkins, Janice Pitts, Constantine A. Gatsonis, Natalie Baptiste, Edith M. Marom, Gina Sammons, Anne Burrough, Martha Ramirez, Jack Cahill, Carl Jaffe, Linda Heinrichs, Aura Cole, Paul Rust, Alon Coppens, Gregg Hamm, Lisa Conklin, Kathleen A. Robbins, Carleaner Williams, Gwen Chalom, Winston Sterling, Colleen Hudak, Lea Matous, Ella A. Kazerooni, Denise Kriescher, David A. Lynch, Liz Bolan, Jacob Wolf, Jonathan G. Goldin, Roberta Quinn, L. A. Schneider, Kathleen A. Murray, Erica Sturgeon, Jennifer Avrin, Michelle T. Biringer, Mark Hinson, Cynthia Reiners, Brian Chin, Amy Brunst, Ann M. Lambrecht, Katherine Lohmann, Jennifer Bacon, Ulander Giles, Diane Shepherd, William T. Corey, Timothy Cosgrove, Lana C. Walters, Nancy Kadish, Hilary C. Nosker, Christine D. Berg, Thomas Payne, Jackie Becker, Kanistha Sookpisal, Lyn Seguin, Todd R. Hazelton, Roy Adaniya, James Fisher, Annmarie Walsh, Shirleen Hyun, Laura Stark, Kenneth Hansen, Carolyn Nelson, Martin Tammemagi, Mary A. Wolfsberger, Barry H. Gross, Valentina Ortico, Marge Watry, Jeff Childs, Gabe Herron, Loretta Thorpe, Lisa Damon, Evanthia Papadopoulos, Denise Moline, Voula E. Christopoulos, John D. Minna, Tony Jones, Mitchell Machtay, Michael Plunkett, Melissa Laughren, Luis Zagarra, Adam Leming, Eda Ordonez, Chris Howell, Marissa Peters, Wendy Mosiman, Joanne Gerber, Alfonso Lorenzo, Barbara L. McComb, Laura Hill, Gale Christensen, Hanna Comer, Carmen Guzman, Kathy Taylor, Misty Oviatt, Malcolm King, Lily Stone, Rex Welsh, Bernadette Pennetta, Cristina Raver, Jan E. Hyder, Stephanie Clabo, Peggy Lau, Jacqueline Fearon, Patricia Pangburn, Pamela Dow, William K. Evans, Victor De Caravalho, Mike Wirth, Brooke Johnson, Meridith Blevins, Lisa H. Gren, Sharon L. Kurjan, James P. Evans, Kirk E. Smith, Donna King, John A. Worrell, Mindy S. Geisser, Philip F. Judy, Richard Barr, Sue Misko, Stanley R. Phillips, Jillian Nickel, Christine M. McKey, Joe Austin, Donna Hartfeil, Laura Young, Shovonna White, Alexis K. Potemkin, Anthony Boulos, Tawny Martin, Karen Kofka, Heather McLaughlin, Matthew K. Siemionko, Melissa Houston, Angela Lee Rowley, Adys Fernandez, Murray Backer, Jagdish Singh, Mary Weston, Nancy Payte, Charles Apgar, John K. Gohagan, Jeff Fairbanks, Wylie Burke, David Chi, Michael Nahill, Kevin DeMarco, Karen Patella, Beverly Rozanok, Carol M. Moser, Nicole Matetic Mac, Karen Boyle, Dinah Lorenzo, Elanor Adkins, Phyllis Olsson, Amanda M. Adams, Sujaya Rao, K.E. Jones, Polly Kay, D. Lynn Werner, John B. Weaver, Sally Anne Kopesec, Jennifer Frye, Victoria Chun, Cathy Francow, Cheri Whiton, Jo Ann Nevilles, Andrew Bodd, Barbara Galen, Sabrina Chen, Cindy Cyphert, Stephen M. Moore, Petra J. Lewis, Shanna Nichols, Mareie Walters, Thea Palmer Zimmerman, Warren B. Gefter, Peter Dubbs, Ann Reinert, Holly Washburn, Renee MacDonald, Boleyn R. Andrist, Dianalyn M. Evans, Marvin Flores, Tricia Adrales-Bentz, Claudine Isaacs, Regina C. MacDougall, Greg M. Silverman, Nichoie Cadez, Lynne Bradford, Rochelle Williams, Angela M. McLaughlin, Ellen Sandberg, Cheryl Crozier, Robert Mayer, Richard P. Remitz, Sheron Bube, Leroy Riley, Vish Iyer, Sophie Breer, Stephen Baylin, Anna Boyle, Shannon Williams, Kristen Keating, Martin M. Oken, Gerald L. Andriole, Bruce E. Hubler, Eric T. Goodman, David Engelhart, Bonna Au, Brianne Whittaker, Tricia Hoffa, Eng Brown, Tammy Wolfsohn, Denise L. Foster, Barry H. Cohen, Linda Galocy, Matthew T. Bee, Jacqueline Matuza, Leslie Henry, Katherine Meagher, Mona Fouad, Beth McLellan, Troy Cook, John Sheflin, Lilian Villaruz, Marcella Moore, Brandy Mack-Pipkin, Vanessa Graves, Ryan Weyls, William T. Herbick, Geoffrey McLennan, Lynn Hoese, Janise Webb, Terrie Kitchner, Michele Lee, Robert T. Greenlee, Charles C. Matthews, Nicole Spiese, Jeffrey Heffernon, Dianna D. Cody, Patricia Blair, Kathy Garrett, Michael A. Sullivan, and Loretta Granger

Subjects
Oncology, medicine.medical_specialty, business.industry, Mortality rate, medicine.disease, law.invention, Quality-adjusted life year, Randomized controlled trial, law, Internal medicine, medicine, Radiology, Nuclear Medicine and imaging, National Lung Screening Trial, Radiology, Overdiagnosis, business, Lung cancer, Lung cancer screening, Mass screening
Abstract

The National Lung Screening Trial (NLST) is a randomized multicenter study comparing low-dose helical computed tomography (CT) with chest radiography in the screening of older current and former heavy smokers for early detection of lung cancer, which is the leading cause of cancer-related death in the United States. Five-year survival rates approach 70% with surgical resection of stage IA disease; however, more than 75% of individuals have incurable locally advanced or metastatic disease, the latter having a 5-year survival of less than 5%. It is plausible that treatment should be more effective and the likelihood of death decreased if asymptomatic lung cancer is detected through screening early enough in its preclinical phase. For these reasons, there is intense interest and intuitive appeal in lung cancer screening with low-dose CT. The use of survival as the determinant of screening effectiveness is, however, confounded by the well-described biases of lead time, length, and overdiagnosis. Despite previous attempts, no test has been shown to reduce lung cancer mortality, an endpoint that circumvents screening biases and provides a definitive measure of benefit when assessed in a randomized controlled trial that enables comparison of mortality rates between screened individuals and a control group that does not undergo the screening intervention of interest. The NLST is such a trial. The rationale for and design of the NLST are presented.

Published
2011
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14. Creating a stem cell niche in the inner ear using self-assembling peptide amphiphiles

Author

Charles A. Miller, Zachery D. Morrissey, Zafar A. Sayed, Tomoji Mashimo, Anil R. Wadhwani, Alexandra N. Edelbrock, Shun Kobayashi, Eric J. Berns, Tammy L. McGuire, Akihiro J. Matsuoka, Duncan M. Chadly, John A. Kessler, Samuel I. Stupp, and Nicholas Stephanopoulos

Subjects
0301 basic medicine, Cell Transplantation, Cellular differentiation, lcsh:Medicine, Biochemistry, Animal Cells, Medicine and Health Sciences, Blood and Lymphatic System Procedures, Stem Cell Niche, lcsh:Science, Cells, Cultured, Neurons, Multidisciplinary, Chemistry, Stem Cells, Cell Differentiation, Cochlea, Cell biology, medicine.anatomical_structure, Inner Ear, Cytochemistry, Anatomy, Cellular Types, Stem cell, Neuronal Differentiation, Immunocytochemistry, Research Article, Neurite, Surgical and Invasive Medical Procedures, 03 medical and health sciences, Neurites, medicine, Peptide amphiphile, Animals, Humans, Spiral ganglion, Transplantation, Regeneration (biology), lcsh:R, Biology and Life Sciences, Cell Biology, Neuronal Dendrites, Embryonic stem cell, Rats, 030104 developmental biology, Ears, Ear, Inner, Cellular Neuroscience, lcsh:Q, Peptides, Head, Developmental Biology, Stem Cell Transplantation, Neuroscience
Abstract

The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.

Published
2017
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15. A bioengineered peripheral nerve construct using aligned peptide amphiphile nanofibers

Author

Andrew Li, Mark T. McClendon, Samuel I. Stupp, Akishige Hokugo, Eric J. Berns, Igor Spigelman, Luis A. Segovia, Anisa Yalom, Reza Jarrahy, and Nicholas Stephanopoulos

Subjects
Peripheral nerve repair, Nerve guidance conduit, Nanofibers, Biocompatible Materials, Neurodegenerative, Regenerative Medicine, Rats, Sprague-Dawley, chemistry.chemical_compound, Tissue engineering, Polylactic Acid-Polyglycolic Acid Copolymer, Nerve conduit, Nanotechnology, Tissue Scaffolds, Self assembly, Sciatic Nerve, PLGA, medicine.anatomical_structure, Mechanics of Materials, Neurological, Sciatic nerve, Development of treatments and therapeutic interventions, Epineurial repair, Oligopeptides, Biotechnology, Materials science, Biophysics, Biomedical Engineering, Schwann cell, Bioengineering, Article, Cell Line, Biomaterials, medicine, Peptide amphiphile, Animals, Lactic Acid, Peripheral Neuropathy, Alignment, Transplantation, 5.2 Cellular and gene therapies, Tissue Engineering, Guided Tissue Regeneration, Regeneration (biology), Neurosciences, Nanofiber, Peptide Fragments, Nerve Regeneration, Rats, Quality Education, chemistry, Ceramics and Composites, Sprague-Dawley, Laminin, Schwann Cells, Polyglycolic Acid, Biomedical engineering
Abstract

Peripheral nerve injuries can result in lifelong disability. Primary coaptation is the treatment of choice when the gap between transected nerve ends is short. Long nerve gaps seen in more complex injuries often require autologous nerve grafts or nerve conduits implemented into the repair. Nerve grafts, however, cause morbidity and functional loss at donor sites, which are limited in number. Nerve conduits, in turn, lack an internal scaffold to support and guide axonal regeneration, resulting in decreased efficacy over longer nerve gap lengths. By comparison, peptide amphiphiles (PAs) are molecules that can self-assemble into nanofibers, which can be aligned to mimic the native architecture of peripheral nerve. As such, they represent a potential substrate for use in a bioengineered nerve graft substitute. To examine this, we cultured Schwann cells with bioactive PAs (RGDS-PA, IKVAV-PA) to determine their ability to attach to and proliferate within the biomaterial. Next, we devised a PA construct for use in a peripheral nerve critical sized defect model. Rat sciatic nerve defects were created and reconstructed with autologous nerve, PLGA conduits filled with various forms of aligned PAs, or left unrepaired. Motor and sensory recovery were determined and compared among groups. Our results demonstrate that Schwann cells are able to adhere to and proliferate in aligned PA gels, with greater efficacy in bioactive PAs compared to the backbone-PA alone. In vivo testing revealed recovery of motor and sensory function in animals treated with conduit/PA constructs comparable to animals treated with autologous nerve grafts. Functional recovery in conduit/PA and autologous graft groups was significantly faster than in animals treated with empty PLGA conduits. Histological examinations also demonstrated increased axonal and Schwann cell regeneration within the reconstructed nerve gap in animals treated with conduit/PA constructs. These results indicate that PA nanofibers may represent a promising biomaterial for use in bioengineered peripheral nerve repair. © 2014 Elsevier Ltd.

Published
2014
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