47 results on '"Chad Ray"'
Search Results
2. Supplementary Methods from Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy
- Author
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Manfred Kraus, Joseph Tumang, Xianxian Zheng, Li-Chin Yao, Martin Wythes, Benoit J. Van den Eynde, Konstantinos Tsaparikos, Vince R. Torti, Nicole Streiner, Chad Ray, Virginie Rabolli, Romain Pirson, Nichol Miller, Reece Marillier, Karen Maegley, Wenlin Li, Marie-Claire Letellier, Jie Guo, Valeria R. Fantin, Christopher P. Dillon, Sofie Denies, Deepak Dalvie, Stefano Crosignani, Sandra Cauwenberghs, Danying Cai, Derek Bartlett, Gregory Driessens, and Bruno Gomes
- Abstract
Supplementary Methods
- Published
- 2023
3. Table S1, Table S3, Figures S1-S8 from Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy
- Author
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Manfred Kraus, Joseph Tumang, Xianxian Zheng, Li-Chin Yao, Martin Wythes, Benoit J. Van den Eynde, Konstantinos Tsaparikos, Vince R. Torti, Nicole Streiner, Chad Ray, Virginie Rabolli, Romain Pirson, Nichol Miller, Reece Marillier, Karen Maegley, Wenlin Li, Marie-Claire Letellier, Jie Guo, Valeria R. Fantin, Christopher P. Dillon, Sofie Denies, Deepak Dalvie, Stefano Crosignani, Sandra Cauwenberghs, Danying Cai, Derek Bartlett, Gregory Driessens, and Bruno Gomes
- Abstract
Table S1 shows the plasma protein binding and blood to plasma ratio in mouse, rat, dog and human. Table S3 shows the plasma concentrations following a single oral dose of PF-06840003. Figure S1 shows that PF-06840003 does not impact cell viability. Figure S2 shows that PF-06840003 rescues T-cell proliferation in a co-culture assay. Figure S3 shows tumor growth inhibition of CT26 tumors at different dose levels. Figure S4 shows that IDO1 inhibitor mediated tumor growth inhibition depends on CD8+ T cells. Figure S5 shows a combination benefit with anti-PD-L1 treatment in CT26 tumors. Figure S6 shows IDO1i mediated tumor growth inhibition of MDA-MB-231 breast tumors in humanized mice. Figure S7 shows that PF-06840003 improves the TGI of avelumab in a humanized mouse breast tumor model. Figure S8 shows the fractions of CT26 tumor immune infiltrating cells.
- Published
- 2023
4. Data from Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy
- Author
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Manfred Kraus, Joseph Tumang, Xianxian Zheng, Li-Chin Yao, Martin Wythes, Benoit J. Van den Eynde, Konstantinos Tsaparikos, Vince R. Torti, Nicole Streiner, Chad Ray, Virginie Rabolli, Romain Pirson, Nichol Miller, Reece Marillier, Karen Maegley, Wenlin Li, Marie-Claire Letellier, Jie Guo, Valeria R. Fantin, Christopher P. Dillon, Sofie Denies, Deepak Dalvie, Stefano Crosignani, Sandra Cauwenberghs, Danying Cai, Derek Bartlett, Gregory Driessens, and Bruno Gomes
- Abstract
Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro. In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti–PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.
- Published
- 2023
5. A quantitative LC–MS/MS approach for monitoring 2′-fluoro-2′-deoxy-D-glucose uptake in tumor tissue
- Author
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Scott Fountain, Chad Ray, Nanni Huser, Cathy Zhang, Wenlin Li, and Erick Kindt
- Subjects
Analyte ,Clinical Biochemistry ,Breast Neoplasms ,Mass spectrometry ,Rhamnose ,01 natural sciences ,Analytical Chemistry ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Tandem Mass Spectrometry ,Lc ms ms ,medicine ,Animals ,Protein precipitation ,Mouse tumor ,General Pharmacology, Toxicology and Pharmaceutics ,Fluorodeoxyglucose ,Chromatography ,Chemistry ,010401 analytical chemistry ,General Medicine ,Tumor tissue ,0104 chemical sciences ,carbohydrates (lipids) ,Medical Laboratory Technology ,Positron-Emission Tomography ,030220 oncology & carcinogenesis ,Female ,2-Deoxy-D-glucose ,Chromatography, Liquid ,medicine.drug - Abstract
Purpose: Develop a quantitative LC–MS/MS method for FDG, FDG-monophosphate, glucose and glucose-monophosphate in mouse tumor models to assist in validating the use of [18F]FDG-positron emission tomography (PET) imaging for anticancer therapies in a clinical setting. Methodology/results: Analytes were isolated from tumors by protein precipitation and detected on a Sciex API-5500 mass spectrometer. Improved assay robustness and selectivity were achieved through chromatographic separation of FDG-monophosphate from glucose-monophosphate, selection of a unique ion transition and incorporation of stable isotope labeled internal standards. In a mouse JIMT-1 tumor model, FDG-monophosphate levels measured by LC–MS/MS correlated with [18F]FDG-PET imaging results. Conclusion: LC–MS/MS analysis of FDG-monophosphate accumulation in tumors is a cost-effective tool to gauge the translational potential of [18F]FDG-PET imaging as a noninvasive biomarker in clinical studies.
- Published
- 2021
6. A Flexible Multiplatform Bioanalytical Strategy for Measurement of Total Circulating Shed Target Receptors: Application to Soluble B Cell Maturation Antigen Levels in the Presence of a Bispecific Antibody Drug
- Author
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Hall Michael Christopher, Chad Ray, and Angela Stauffer
- Subjects
Drug ,Bioanalysis ,CD3 ,media_common.quotation_subject ,Pharmacology ,030226 pharmacology & pharmacy ,03 medical and health sciences ,0302 clinical medicine ,Pharmacokinetics ,Tandem Mass Spectrometry ,Antibodies, Bispecific ,Drug Discovery ,Animals ,Humans ,B-Cell Maturation Antigen ,Receptor ,030304 developmental biology ,media_common ,0303 health sciences ,biology ,Chemistry ,Ligand binding assay ,Macaca fascicularis ,Drug development ,biology.protein ,Molecular Medicine ,Chromatography, Liquid - Abstract
B cell maturation antigen (BCMA) is a membrane-bound receptor that is overexpressed on multiple myeloma cells and can be targeted with biotherapeutics. Soluble shed forms of membrane-associated receptors in circulation can act as a drug sink, especially when it is present in high molar ratio compared to drug concentration, potentially derailing the intended pharmacological mechanism and impacting pharmacokinetic (PK) measurements and efficacious dose predictions. In this study, we present a bioanalytical strategy for assessing dynamic levels of total soluble BCMA before and during treatment with a bispecific antibody targeting BCMA and CD3. Implementation of a ligand binding assay was not successful due to extensive bispecific antibody interference. Instead, we explored two types of immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays, one at the protein level and one at the surrogate peptide level. Ultimately, the protein-level IA-LC-MS/MS method was optimized for use in a cynomolgus monkey PK/pharmacodynamic study. In addition, we demonstrated that the method was easily adapted for use with human samples in preparation for translation to the clinic. This work demonstrates the benefit of flexibility and agility in bioanalytical method development in early drug development. Multiplatform suitability assessments enable rapid, resource-sparing identification and qualification of clinically translatable assays. We recommend early adoption of this strategy to provide enough time for critical reagent development and assay validation for analysis of shed targets.
- Published
- 2021
7. Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy
- Author
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Jie Guo, Li-Chin Yao, Christopher P. Dillon, Wenlin Li, Stefano Crosignani, Karen A. Maegley, Danying Cai, Reece Marillier, Sandra Cauwenberghs, Manfred Kraus, Virginie Rabolli, Valeria Fantin, Benoît Van den Eynde, Deepak Dalvie, Joseph Tumang, Derek W. Bartlett, Martin James Wythes, Vince Torti, Xianxian Zheng, Sofie Denies, Gregory Driessens, Bruno Gomes, Nichol Miller, Konstantinos Tsaparikos, Nicole Streiner, Marie-Claire Letellier, Chad Ray, and Romain Pirson
- Subjects
0301 basic medicine ,Cancer Research ,Indoles ,T-Lymphocytes ,medicine.medical_treatment ,Succinimides ,Antineoplastic Agents ,Antibodies, Monoclonal, Humanized ,B7-H1 Antigen ,Substrate Specificity ,Immune tolerance ,Interferon-gamma ,03 medical and health sciences ,chemistry.chemical_compound ,Lymphocytes, Tumor-Infiltrating ,0302 clinical medicine ,Downregulation and upregulation ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Indoleamine-Pyrrole 2,3,-Dioxygenase ,CTLA-4 Antigen ,Enzyme Inhibitors ,Indoleamine 2,3-dioxygenase ,Kynurenine ,Cell Proliferation ,Mice, Inbred BALB C ,Tumor microenvironment ,Chemistry ,Antibodies, Monoclonal ,FOXP3 ,Stereoisomerism ,Immunotherapy ,Immune checkpoint ,Mice, Inbred C57BL ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Biocatalysis ,Cancer research ,Female - Abstract
Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro. In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti–PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.
- Published
- 2018
8. Considerations for Soluble Protein Biomarker Blood Sample Matrix Selection
- Author
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Jon E. Peterson, Daoyu Duan, Yan G. Ni, Connie Wang, Chad Ray, John Allinson, Martha Hokom, Sara Hamon, Joel A Mathews, Xuemei Zhao, and Greta Wegner
- Subjects
Blood Platelets ,Analyte ,Protein biomarkers ,Chemistry ,Sample (material) ,Anticoagulants ,Reproducibility of Results ,Pharmaceutical Science ,Blood Proteins ,Computational biology ,030226 pharmacology & pharmacy ,Biomarkers, Pharmacological ,Protein markers ,03 medical and health sciences ,0302 clinical medicine ,Drug development ,Predictive Value of Tests ,030220 oncology & carcinogenesis ,Immune Cell Activation ,Leukocytes ,Animals ,Humans ,Biomarker (medicine) ,Blood Chemical Analysis ,Selection (genetic algorithm) - Abstract
Blood-based soluble protein biomarkers provide invaluable clinical information about patients and are used as diagnostic, prognostic, and pharmacodynamic markers. The most commonly used blood sample matrices are serum and different types of plasma. In drug development research, the impact of sample matrix selection on successful protein biomarker quantification is sometimes overlooked. The sample matrix for a specific analyte is often chosen based on prior experience or literature searches, without good understanding of the possible effects on analyte quantification. Using a data set of 32 different soluble protein markers measured in matched serum and plasma samples, we examined the differences between serum and plasma and discussed how platelet or immune cell activation can change the quantified concentration of the analyte. We have also reviewed the effect of anticoagulant on analyte quantification. Finally, we provide specific recommendations for biomarker sample matrix selection and propose a systematic and data-driven approach for sample matrix selection. This review is intended to raise awareness of the impact and considerations of sample matrix selection on biomarker quantification.
- Published
- 2020
9. Preventing Cardiovascular Disease Among Urban African Americans With a Mobile Health App (the MOYO App): Protocol for a Usability Study
- Author
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Taylor Jr, Herman A, primary, Francis, Sherilyn, additional, Evans, Chad Ray, additional, Harvey, Marques, additional, Newton, Brittney A, additional, Jones, Camara P, additional, Akintobi, Tabia Henry, additional, and Clifford, Gari, additional
- Published
- 2020
- Full Text
- View/download PDF
10. Preventing Cardiovascular Disease Among Urban African Americans With a Mobile Health App (the MOYO App): Protocol for a Usability Study (Preprint)
- Author
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Herman A Taylor Jr, Sherilyn Francis, Chad Ray Evans, Marques Harvey, Brittney A Newton, Camara P Jones, Tabia Henry Akintobi, and Gari Clifford
- Abstract
BACKGROUND Cardiovascular disease (CVD) disparities are a particularly devastating manifestation of health inequity. Despite advancements in prevention and treatment, CVD is still the leading cause of death in the United States. Additionally, research indicates that African American (AA) and other ethnic-minority populations are affected by CVD at earlier ages than white Americans. Given that AAs are the fastest-growing population of smartphone owners and users, mobile health (mHealth) technologies offer the unparalleled potential to prevent or improve self-management of chronic disease among this population. OBJECTIVE To address the unmet need for culturally tailored primordial prevention CVD–focused mHealth interventions, the MOYO app was cocreated with the involvement of young people from this priority community. The overall project aims to develop and evaluate the effectiveness of a novel smartphone app designed to reduce CVD risk factors among urban-AAs, 18-29 years of age. METHODS The theoretical underpinning will combine the principles of community-based participatory research and the agile software development framework. The primary outcome goals of the study will be to determine the usability, acceptability, and functionality of the MOYO app, and to build a cloud-based data collection infrastructure suitable for digital epidemiology in a disparity population. Changes in health-related parameters over a 24-week period as determined by both passive (eg, physical activity levels, sleep duration, social networking) and active (eg, use of mood measures, surveys, uploading pictures of meals and blood pressure readings) measures will be the secondary outcome. Participants will be recruited from a majority AA “large city” school district, 2 historically black colleges or universities, and 1 urban undergraduate college. Following baseline screening for inclusion (administered in person), participants will receive the beta version of the MOYO app. Participants will be monitored during a 24-week pilot period. Analyses of varying data including social network dynamics, standard metrics of activity, percentage of time away from a given radius of home, circadian rhythm metrics, and proxies for sleep will be performed. Together with external variables (eg, weather, pollution, and socioeconomic indicators such as food access), these metrics will be used to train machine-learning frameworks to regress them on the self-reported quality of life indicators. RESULTS This 5-year study (2015-2020) is currently in the implementation phase. We believe that MOYO can build upon findings of classical epidemiology and longitudinal studies like the Jackson Heart Study by adding greater granularity to our knowledge of the exposures and behaviors that affect health and disease, and creating a channel for outreach capable of launching interventions, clinical trials, and enhancements of health literacy. CONCLUSIONS The results of this pilot will provide valuable information about community cocreation of mHealth programs, efficacious design features, and essential infrastructure for digital epidemiology among young AA adults. INTERNATIONAL REGISTERED REPORT DERR1-10.2196/16699
- Published
- 2019
11. Workshop Report: Crystal City VI—Bioanalytical Method Validation for Biomarkers
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Chad Ray, Brian Booth, Mark E. Arnold, and Lindsay King
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business.industry ,010401 analytical chemistry ,Pharmacology toxicology ,Pharmaceutical Science ,Translational research ,Pharmacology ,030226 pharmacology & pharmacy ,01 natural sciences ,Data science ,0104 chemical sciences ,Food and drug administration ,03 medical and health sciences ,0302 clinical medicine ,Drug development ,Medicine ,Biomarker (medicine) ,State of the science ,business ,Strengths and weaknesses ,Pharmaceutical industry - Abstract
With the growing focus on translational research and the use of biomarkers to drive drug development and approvals, biomarkers have become a significant area of research within the pharmaceutical industry. However, until the US Food and Drug Administration’s (FDA) 2013 draft guidance on bioanalytical method validation included consideration of biomarker assays using LC-MS and LBA, those assays were created, validated, and used without standards of performance. This lack of expectations resulted in the FDA receiving data from assays of varying quality in support of efficacy and safety claims. The AAPS Crystal City VI (CC VI) Workshop in 2015 was held as the first forum for industry-FDA discussion around the general issues of biomarker measurements (e.g., endogenous levels) and specific technology strengths and weaknesses. The 2-day workshop served to develop a common understanding among the industrial scientific community of the issues around biomarkers, informed the FDA of the current state of the science, and will serve as a basis for further dialogue as experience with biomarkers expands with both groups.
- Published
- 2016
12. Preventing Cardiovascular Disease Among Urban African Americans With a Mobile Health App (the MOYO App): Protocol for a Usability Study (Preprint)
- Author
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Taylor Jr, Herman A, primary, Francis, Sherilyn, additional, Evans, Chad Ray, additional, Harvey, Marques, additional, Newton, Brittney A, additional, Jones, Camara P, additional, Akintobi, Tabia Henry, additional, and Clifford, Gari, additional
- Published
- 2019
- Full Text
- View/download PDF
13. 31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016): part two
- Author
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Casey Ager, Matthew Reilley, Courtney Nicholas, Todd Bartkowiak, Ashvin Jaiswal, Michael Curran, Tina C. Albershardt, Anshika Bajaj, Jacob F. Archer, Rebecca S. Reeves, Lisa Y. Ngo, Peter Berglund, Jan ter Meulen, Caroline Denis, Hormas Ghadially, Thomas Arnoux, Fabien Chanuc, Nicolas Fuseri, Robert W. Wilkinson, Nicolai Wagtmann, Yannis Morel, Pascale Andre, Michael B. Atkins, Matteo S. Carlino, Antoni Ribas, John A. Thompson, Toni K. Choueiri, F. Stephen Hodi, Wen-Jen Hwu, David F. McDermott, Victoria Atkinson, Jonathan S. Cebon, Bernie Fitzharris, Michael B. Jameson, Catriona McNeil, Andrew G. Hill, Eric Mangin, Malidi Ahamadi, Marianne van Vugt, Mariëlle van Zutphen, Nageatte Ibrahim, Georgina V. Long, Robyn Gartrell, Zoe Blake, Ines Simoes, Yichun Fu, Takuro Saito, Yingzhi Qian, Yan Lu, Yvonne M. Saenger, Sadna Budhu, Olivier De Henau, Roberta Zappasodi, Kyle Schlunegger, Bruce Freimark, Jeff Hutchins, Christopher A. Barker, Jedd D. Wolchok, Taha Merghoub, Elena Burova, Omaira Allbritton, Peter Hong, Jie Dai, Jerry Pei, Matt Liu, Joel Kantrowitz, Venus Lai, William Poueymirou, Douglas MacDonald, Ella Ioffe, Markus Mohrs, William Olson, Gavin Thurston, Cristian Capasso, Federica Frascaro, Sara Carpi, Siri Tähtinen, Sara Feola, Manlio Fusciello, Karita Peltonen, Beatriz Martins, Madeleine Sjöberg, Sari Pesonen, Tuuli Ranki, Lukasz Kyruk, Erkko Ylösmäki, Vincenzo Cerullo, Fabio Cerignoli, Biao Xi, Garret Guenther, Naichen Yu, Lincoln Muir, Leyna Zhao, Yama Abassi, Víctor Cervera-Carrascón, Mikko Siurala, João Santos, Riikka Havunen, Suvi Parviainen, Akseli Hemminki, Angus Dalgleish, Satvinder Mudan, Mark DeBenedette, Ana Plachco, Alicia Gamble, Elizabeth W. Grogan, John Krisko, Irina Tcherepanova, Charles Nicolette, Pooja Dhupkar, Ling Yu, Eugenie S. Kleinerman, Nancy Gordon, Italia Grenga, Lauren Lepone, Sofia Gameiro, Karin M. Knudson, Massimo Fantini, Kwong Tsang, James Hodge, Renee Donahue, Jeffrey Schlom, Elizabeth Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Cathie Foster, Alan Howell, Leslie Balch, Alyssa Knapp, John E. Leonard, Mark Paris, Terry Fisher, Siwen Hu-Lieskovan, Ernest Smith, Maurice Zauderer, William Fogler, Marilyn Franklin, Matt Thayer, Dan Saims, John L. Magnani, Jian Gong, Michael Gray, George Fromm, Suresh de Silva, Louise Giffin, Xin Xu, Jason Rose, Taylor H. Schreiber, Sofia R. Gameiro, Paul E. Clavijo, Clint T. Allen, James W. Hodge, Kwong Y. Tsang, Jane Grogan, Nicholas Manieri, Eugene Chiang, Patrick Caplazi, Mahesh Yadav, Patrick Hagner, Hsiling Chiu, Michelle Waldman, Anke Klippel, Anjan Thakurta, Michael Pourdehnad, Anita Gandhi, Ian Henrich, Laura Quick, Rob Young, Margaret Chou, Andrew Hotson, Stephen Willingham, Po Ho, Carmen Choy, Ginna Laport, Ian McCaffery, Richard Miller, Kimberly A. Tipton, Kenneth R. Wong, Victoria Singson, Chihunt Wong, Chanty Chan, Yuanhiu Huang, Shouchun Liu, Jennifer H. Richardson, W. Michael Kavanaugh, James West, Bryan A. Irving, Ritika Jaini, Matthew Loya, Charis Eng, Melissa L. Johnson, Alex A. Adjei, Mateusz Opyrchal, Suresh Ramalingam, Pasi A. Janne, George Dominguez, Dmitry Gabrilovich, Laura de Leon, Jeannette Hasapidis, Scott J. Diede, Peter Ordentlich, Scott Cruickshank, Michael L. Meyers, Matthew D. Hellmann, Pawel Kalinski, Amer Zureikat, Robert Edwards, Ravi Muthuswamy, Nataša Obermajer, Julie Urban, Lisa H. Butterfield, William Gooding, Herbert Zeh, David Bartlett, Olga Zubkova, Larissa Agapova, Marina Kapralova, Liudmila Krasovskaia, Armen Ovsepyan, Maxim Lykov, Artem Eremeev, Vladimir Bokovanov, Olga Grigoryeva, Andrey Karpov, Sergey Ruchko, Alexandr Shuster, Danny N. Khalil, Luis Felipe Campesato, Yanyun Li, Adam S. Lazorchak, Troy D. Patterson, Yueyun Ding, Pottayil Sasikumar, Naremaddepalli Sudarshan, Nagaraj Gowda, Raghuveer Ramachandra, Dodheri Samiulla, Sanjeev Giri, Rajesh Eswarappa, Murali Ramachandra, David Tuck, Timothy Wyant, Jasmin Leshem, Xiu-fen Liu, Tapan Bera, Masaki Terabe, Birgit Bossenmaier, Gerhard Niederfellner, Yoram Reiter, Ira Pastan, Leiming Xia, Yang Xia, Yangyang Hu, Yi Wang, Yangyi Bao, Fu Dai, Shiang Huang, Elaine Hurt, Robert E. Hollingsworth, Lawrence G. Lum, Alfred E. Chang, Max S. Wicha, Qiao Li, Thomas Mace, Neil Makhijani, Erin Talbert, Gregory Young, Denis Guttridge, Darwin Conwell, Gregory B. Lesinski, Rodney JM Macedo Gonzales, Austin P. Huffman, Ximi K. Wang, Ran Reshef, Andy MacKinnon, Jason Chen, Matt Gross, Gisele Marguier, Peter Shwonek, Natalija Sotirovska, Susanne Steggerda, Francesco Parlati, Amani Makkouk, Mark K. Bennett, Ethan Emberley, Tony Huang, Weiqun Li, Silinda Neou, Alison Pan, Jing Zhang, Winter Zhang, Netonia Marshall, Thomas U. Marron, Judith Agudo, Brian Brown, Joshua Brody, Christopher McQuinn, Matthew Farren, Hannah Komar, Reena Shakya, Thomas Ludwug, Y. Maurice Morillon, Scott A. Hammond, John W. Greiner, Pulak R. Nath, Anthony L. Schwartz, Dragan Maric, David D. Roberts, Aung Naing, Kyriakos P. Papadopoulos, Karen A. Autio, Deborah J. Wong, Manish Patel, Gerald Falchook, Shubham Pant, Patrick A. Ott, Melinda Whiteside, Amita Patnaik, John Mumm, Filip Janku, Ivan Chan, Todd Bauer, Rivka Colen, Peter VanVlasselaer, Gail L. Brown, Nizar M. Tannir, Martin Oft, Jeffrey Infante, Evan Lipson, Ajay Gopal, Sattva S. Neelapu, Philippe Armand, Stephen Spurgeon, John P. Leonard, Rachel E. Sanborn, Ignacio Melero, Thomas F. Gajewski, Matthew Maurer, Serena Perna, Andres A. Gutierrez, Raphael Clynes, Priyam Mitra, Satyendra Suryawanshi, Douglas Gladstone, Margaret K. Callahan, James Crooks, Sheila Brown, Audrey Gauthier, Marc Hillairet de Boisferon, Andrew MacDonald, Laura Rosa Brunet, William T. Rothwell, Peter Bell, James M. Wilson, Fumi Sato-Kaneko, Shiyin Yao, Shannon S. Zhang, Dennis A. Carson, Cristina Guiducci, Robert L. Coffman, Kazutaka Kitaura, Takaji Matsutani, Ryuji Suzuki, Tomoko Hayashi, Ezra E. W. Cohen, David Schaer, Yanxia Li, Julie Dobkin, Michael Amatulli, Gerald Hall, Thompson Doman, Jason Manro, Frank Charles Dorsey, Lillian Sams, Rikke Holmgaard, Krishnadatt Persaud, Dale Ludwig, David Surguladze, John S. Kauh, Ruslan Novosiadly, Michael Kalos, Kyla Driscoll, Hardev Pandha, Christy Ralph, Kevin Harrington, Brendan Curti, Wallace Akerley, Sumati Gupta, Alan Melcher, David Mansfield, David R. Kaufman, Emmett Schmidt, Mark Grose, Bronwyn Davies, Roberta Karpathy, Darren Shafren, Katerina Shamalov, Cyrille Cohen, Naveen Sharma, James Allison, Tala Shekarian, Sandrine Valsesia-Wittmann, Christophe Caux, Aurelien Marabelle, Brian M. Slomovitz, Kathleen M. Moore, Hagop Youssoufian, Marshall Posner, Poonam Tewary, Alan D. Brooks, Ya-Ming Xu, Kithsiri Wijeratne, Leslie A. A. Gunatilaka, Thomas J. Sayers, John P. Vasilakos, Tesha Alston, Simon Dovedi, James Elvecrog, Iwen Grigsby, Ronald Herbst, Karen Johnson, Craig Moeckly, Stefanie Mullins, Kristen Siebenaler, Julius SternJohn, Ashenafi Tilahun, Mark A. Tomai, Katharina Vogel, Eveline E. Vietsch, Anton Wellstein, Martin Wythes, Stefano Crosignani, Joseph Tumang, Shilpa Alekar, Patrick Bingham, Sandra Cauwenberghs, Jenny Chaplin, Deepak Dalvie, Sofie Denies, Coraline De Maeseneire, JunLi Feng, Kim Frederix, Samantha Greasley, Jie Guo, James Hardwick, Stephen Kaiser, Katti Jessen, Erick Kindt, Marie-Claire Letellier, Wenlin Li, Karen Maegley, Reece Marillier, Nichol Miller, Brion Murray, Romain Pirson, Julie Preillon, Virginie Rabolli, Chad Ray, Kevin Ryan, Stephanie Scales, Jay Srirangam, Jim Solowiej, Al Stewart, Nicole Streiner, Vince Torti, Konstantinos Tsaparikos, Xianxian Zheng, Gregory Driessens, Bruno Gomes, Manfred Kraus, Chunxiao Xu, Yanping Zhang, Giorgio Kradjian, Guozhong Qin, Jin Qi, Xiaomei Xu, Bo Marelli, Huakui Yu, Wilson Guzman, Rober Tighe, Rachel Salazar, Kin-Ming Lo, Jessie English, Laszlo Radvanyi, Yan Lan, Michael Postow, Yasin Senbabaoglu, Billel Gasmi, Hong Zhong, Cailian Liu, Daniel Hirschhorhn-Cymerman, Yuanyuan Zha, Gregory Malnassy, Noreen Fulton, Jae-Hyun Park, Wendy Stock, Yusuke Nakamura, Hongtao Liu, Xiaoming Ju, Rachelle Kosoff, Kimberly Ramos, Brandon Coder, Robert Petit, Michael Princiotta, Kyle Perry, Jun Zou, Ainhoa Arina, Christian Fernandez, Wenxin Zheng, Michael A. Beckett, Helena J. Mauceri, Yang-Xin Fu, Ralph R. Weichselbaum, Whitney Lewis, Yanyan Han, Yeting Wu, Chou Yang, Jing Huang, Dongyun Wu, Jin Li, Xiaoling Liang, Xiangjun Zhou, Jinlin Hou, Raffit Hassan, Thierry Jahan, Scott J. Antonia, Hedy L. Kindler, Evan W. Alley, Somayeh Honarmand, Weiqun Liu, Meredith L. Leong, Chan C. Whiting, Nitya Nair, Amanda Enstrom, Edward E. Lemmens, Takahiro Tsujikawa, Sushil Kumar, Lisa M. Coussens, Aimee L. Murphy, Dirk G. Brockstedt, Sven D. Koch, Martin Sebastian, Christian Weiss, Martin Früh, Miklos Pless, Richard Cathomas, Wolfgang Hilbe, Georg Pall, Thomas Wehler, Jürgen Alt, Helge Bischoff, Michael Geissler, Frank Griesinger, Jens Kollmeier, Alexandros Papachristofilou, Fatma Doener, Mariola Fotin-Mleczek, Madeleine Hipp, Henoch S. Hong, Karl-Josef Kallen, Ute Klinkhardt, Claudia Stosnach, Birgit Scheel, Andreas Schroeder, Tobias Seibel, Ulrike Gnad-Vogt, Alfred Zippelius, Ha-Ram Park, Yong-Oon Ahn, Tae Min Kim, Soyeon Kim, Seulki Kim, Yu Soo Lee, Bhumsuk Keam, Dong-Wan Kim, Dae Seog Heo, Shari Pilon-Thomas, Amy Weber, Jennifer Morse, Krithika Kodumudi, Hao Liu, John Mullinax, Amod A. Sarnaik, Luke Pike, Andrew Bang, Tracy Balboni, Allison Taylor, Alexander Spektor, Tyler Wilhite, Monica Krishnan, Daniel Cagney, Brian Alexander, Ayal Aizer, Elizabeth Buchbinder, Mark Awad, Leena Ghandi, Jonathan Schoenfeld, Elizabeth Lessey-Morillon, Lisa Ridnour, Neil H. Segal, Manish Sharma, Dung T. Le, Robert L. Ferris, Andrew D. Zelenetz, Ronald Levy, Izidore S. Lossos, Caron Jacobson, Radhakrishnan Ramchandren, John Godwin, A. Dimitrios Colevas, Roland Meier, Suba Krishnan, Xuemin Gu, Jaclyn Neely, John Timmerman, Claire I. Vanpouille-Box, Silvia C. Formenti, Sandra Demaria, Erik Wennerberg, Aranzazu Mediero, Bruce N. Cronstein, Michael P. Gustafson, AriCeli DiCostanzo, Courtney Wheatley, Chul-Ho Kim, Svetlana Bornschlegl, Dennis A. Gastineau, Bruce D. Johnson, Allan B. Dietz, Cameron MacDonald, Mark Bucsek, Guanxi Qiao, Bonnie Hylander, Elizabeth Repasky, William J. Turbitt, Yitong Xu, Andrea Mastro, Connie J. Rogers, Sita Withers, Ziming Wang, Lam T. Khuat, Cordelia Dunai, Bruce R. Blazar, Dan Longo, Robert Rebhun, Steven K. Grossenbacher, Arta Monjazeb, William J. Murphy, Scott Rowlinson, Giulia Agnello, Susan Alters, David Lowe, Nicole Scharping, Ashley V. Menk, Ryan Whetstone, Xue Zeng, Greg M. Delgoffe, Patricia M. Santos, Jian Shi, Greg Delgoffe, Misako Nagasaka, Ammar Sukari, Miranda Byrne-Steele, Wenjing Pan, Xiaohong Hou, Brittany Brown, Mary Eisenhower, Jian Han, Natalie Collins, Robert Manguso, Hans Pope, Yashaswi Shrestha, Jesse Boehm, W. Nicholas Haining, Kyle R. Cron, Ayelet Sivan, Keston Aquino-Michaels, Marco Orecchioni, Davide Bedognetti, Wouter Hendrickx, Claudia Fuoco, Filomena Spada, Francesco Sgarrella, Gianni Cesareni, Francesco Marincola, Kostas Kostarelos, Alberto Bianco, Lucia Delogu, Jessica Roelands, Sabri Boughorbel, Julie Decock, Scott Presnell, Ena Wang, Franco M. Marincola, Peter Kuppen, Michele Ceccarelli, Darawan Rinchai, Damien Chaussabel, Lance Miller, Andrew Nguyen, J. Zachary Sanborn, Charles Vaske, Shahrooz Rabizadeh, Kayvan Niazi, Steven Benz, Shashank Patel, Nicholas Restifo, James White, Sam Angiuoli, Mark Sausen, Sian Jones, Maria Sevdali, John Simmons, Victor Velculescu, Luis Diaz, Theresa Zhang, Jennifer S. Sims, Sunjay M. Barton, Angela Kadenhe-Chiweshe, Filemon Dela Cruz, Andrew T. Turk, Christopher F. Mazzeo, Andrew L. Kung, Jeffrey N. Bruce, Darrell J. Yamashiro, Eileen P. Connolly, Jason Baird, Marka Crittenden, David Friedman, Hong Xiao, Rom Leidner, Bryan Bell, Kristina Young, Michael Gough, Zhen Bian, Koby Kidder, Yuan Liu, Emily Curran, Xiufen Chen, Leticia P. Corrales, Justin Kline, Ethan G. Aguilar, Jennifer Guerriero, Alaba Sotayo, Holly Ponichtera, Alexandra Pourzia, Sara Schad, Ruben Carrasco, Suzan Lazo, Roderick Bronson, Anthony Letai, Richard S. Kornbluth, Sachin Gupta, James Termini, Elizabeth Guirado, Geoffrey W. Stone, Christina Meyer, Laura Helming, Nicholas Wilson, Robert Hofmeister, Natalie J. Neubert, Laure Tillé, David Barras, Charlotte Soneson, Petra Baumgaertner, Donata Rimoldi, David Gfeller, Mauro Delorenzi, Silvia A. Fuertes Marraco, Daniel E. Speiser, Tara S. Abraham, Bo Xiang, Michael S. Magee, Scott A. Waldman, Adam E. Snook, Wojciech Blogowski, Ewa Zuba-Surma, Marta Budkowska, Daria Salata, Barbara Dolegowska, Teresa Starzynska, Leo Chan, Srinivas Somanchi, Kelsey McCulley, Dean Lee, Nico Buettner, Feng Shi, Paisley T. Myers, Stuart Curbishley, Sarah A. Penny, Lora Steadman, David Millar, Ellen Speers, Nicola Ruth, Gabriel Wong, Robert Thimme, David Adams, Mark Cobbold, Remy Thomas, Mariam Al-Muftah, Michael KK Wong, Michael Morse, Joseph I. Clark, Howard L. Kaufman, Gregory A. Daniels, Hong Hua, Tharak Rao, Janice P. Dutcher, Kai Kang, Yogen Saunthararajah, Vamsidhar Velcheti, Vikas Kumar, Firoz Anwar, Amita Verma, Zinal Chheda, Gary Kohanbash, John Sidney, Kaori Okada, Shruti Shrivastav, Diego A. Carrera, Shuming Liu, Naznin Jahan, Sabine Mueller, Ian F. Pollack, Angel M. Carcaboso, Alessandro Sette, Yafei Hou, Hideho Okada, Jessica J. Field, Weiping Zeng, Vincent FS Shih, Che-Leung Law, Peter D. Senter, Shyra J. Gardai, Nicole M. Okeley, Jennifer G. Abelin, Abu Z. Saeed, Stacy A. Malaker, Jeffrey Shabanowitz, Stephen T. Ward, Donald F. Hunt, Pam Profusek, Laura Wood, Dale Shepard, Petros Grivas, Kerstin Kapp, Barbara Volz, Detlef Oswald, Burghardt Wittig, Manuel Schmidt, Julian P. Sefrin, Lars Hillringhaus, Valeria Lifke, Alexander Lifke, Anna Skaletskaya, Jose Ponte, Thomas Chittenden, Yulius Setiady, Eva Sivado, Vincent Thomas, Meddy El Alaoui, Sébastien Papot, Charles Dumontet, Mike Dyson, John McCafferty, Said El Alaoui, Praveen K. Bommareddy, Andrew Zloza, Frederick Kohlhapp, Ann W. Silk, Sachin Jhawar, Tomas Paneque, Jenna Newman, Pedro Beltran, Felicia Cao, Bang-Xing Hong, Tania Rodriguez-Cruz, Xiao-Tong Song, Stephen Gottschalk, Hugo Calderon, Sam Illingworth, Alice Brown, Kerry Fisher, Len Seymour, Brian Champion, Emma Eriksson, Jessica Wenthe, Ann-Charlotte Hellström, Gabriella Paul-Wetterberg, Angelica Loskog, Ioanna Milenova, Magnus Ståhle, Justyna Jarblad-Leja, Gustav Ullenhag, Anna Dimberg, Rafael Moreno, Ramon Alemany, Sharad Goyal, Ann Silk, Janice Mehnert, Nashat Gabrail, Jennifer Bryan, Daniel Medina, Leah Mitchell, Kader Yagiz, Fernando Lopez, Daniel Mendoza, Anthony Munday, Harry Gruber, Douglas Jolly, Steven Fuhrmann, Sasa Radoja, Wei Tan, Aldo Pourchet, Alan Frey, Ian Mohr, Matthew Mulvey, Robert H. I. Andtbacka, Merrick Ross, Sanjiv Agarwala, Kenneth Grossmann, Matthew Taylor, John Vetto, Rogerio Neves, Adil Daud, Hung Khong, Stephanie M. Meek, Richard Ungerleider, Scott Welden, Maki Tanaka, Matthew Williams, Sigrun Hallmeyer, Bernard Fox, Zipei Feng, Christopher Paustian, Carlo Bifulco, Sadia Zafar, Otto Hemminki, Simona Bramante, Lotta Vassilev, Hongjie Wang, Andre Lieber, Silvio Hemmi, Tanja de Gruijl, Anna Kanerva, Tameem Ansari, Srividya Sundararaman, Diana Roen, Paul Lehmann, Anja C. Bloom, Lewis H. Bender, Ian B. Walters, Jay A. Berzofsky, Fanny Chapelin, Eric T. Ahrens, Jeff DeFalco, Michael Harbell, Amy Manning-Bog, Alexander Scholz, Danhui Zhang, Gilson Baia, Yann Chong Tan, Jeremy Sokolove, Dongkyoon Kim, Kevin Williamson, Xiaomu Chen, Jillian Colrain, Gregg Espiritu Santo, Ngan Nguyen, Wayne Volkmuth, Norman Greenberg, William Robinson, Daniel Emerling, Charles G. Drake, Daniel P. Petrylak, Emmanuel S. Antonarakis, Adam S. Kibel, Nancy N. Chang, Tuyen Vu, Dwayne Campogan, Heather Haynes, James B. Trager, Nadeem A. Sheikh, David I. Quinn, Peter Kirk, Murali Addepalli, Thomas Chang, Ping Zhang, Marina Konakova, Katsunobu Hagihara, Steven Pai, Laurie VanderVeen, Palakshi Obalapur, Peiwen Kuo, Phi Quach, Lawrence Fong, Deborah H. Charych, Jonathan Zalevsky, John L. Langowski, Yolanda Kirksey, Ravi Nutakki, Shalini Kolarkar, Rhoneil Pena, Ute Hoch, Stephen K. Doberstein, John Cha, Zach Mallon, Myra Perez, Amanda McDaniel, Snjezana Anand, Darrin Uecker, Richard Nuccitelli, Eva Wieckowski, Ravikumar Muthuswamy, Roshni Ravindranathan, Ariana N. Renrick, Menaka Thounaojam, Portia Thomas, Samuel Pellom, Anil Shanker, Duafalia Dudimah, Alan Brooks, Yu-Lin Su, Tomasz Adamus, Qifang Zhang, Sergey Nechaev, Marcin Kortylewski, Spencer Wei, Clark Anderson, Chad Tang, Jonathan Schoenhals, Efrosini Tsouko, John Heymach, Patricia de Groot, Joe Chang, Kenneth R. Hess, Adi Diab, Padmanee Sharma, David Hong, James Welsh, Andrea J. Parsons, Jardin Leleux, Stephane Ascarateil, Marie Eve Koziol, Dina Bai, Peihong Dai, Weiyi Wang, Ning Yang, Stewart Shuman, Liang Deng, Patrick Dillon, Gina Petroni, David Brenin, Kim Bullock, Walter Olson, Mark E. Smolkin, Kelly Smith, Carmel Nail, Craig L. Slingluff, Meenu Sharma, Faisal Fa’ak, Louise Janssen, Hiep Khong, Zhilan Xiao, Yared Hailemichael, Manisha Singh, Christina Vianden, Willem W. Overwijk, Andrea Facciabene, Pierini Stefano, Fang Chongyung, Stavros Rafail, Michael Nielsen, Peter Vanderslice, Darren G. Woodside, Robert V. Market, Ronald J. Biediger, Upendra K. Marathi, Kevin Hollevoet, Nick Geukens, Paul Declerck, Nathalie Joly, Laura McIntosh, Eustache Paramithiotis, Magnus Rizell, Malin Sternby, Bengt Andersson, Alex Karlsson-Parra, Rui Kuai, Lukasz Ochyl, Anna Schwendeman, James Moon, Weiwen Deng, Thomas E. Hudson, Bill Hanson, Chris S. Rae, Joel Burrill, Justin Skoble, George Katibah, Michele deVries, Peter Lauer, Thomas W. Dubensky, Xin Chen, Li Zhou, Xiubao Ren, Charu Aggarwal, Drishty Mangrolia, Roger Cohen, Gregory Weinstein, Matthew Morrow, Joshua Bauml, Kim Kraynyak, Jean Boyer, Jian Yan, Jessica Lee, Laurent Humeau, Sandra Oyola, Susan Duff, David Weiner, Zane Yang, Mark Bagarazzi, Douglas G. McNeel, Jens Eickhoff, Robert Jeraj, Mary Jane Staab, Jane Straus, Brian Rekoske, Glenn Liu, Marit Melssen, William Grosh, Nikole Varhegyi, Nadejda Galeassi, Donna H. Deacon, Elizabeth Gaughan, Maurizio Ghisoli, Minal Barve, Robert Mennel, Gladice Wallraven, Luisa Manning, Neil Senzer, John Nemunaitis, Masahiro Ogasawara, Shuichi Ota, Kaitlin M. Peace, Diane F. Hale, Timothy J. Vreeland, Doreen O. Jackson, John S. Berry, Alfred F. Trappey, Garth S. Herbert, Guy T. Clifton, Mark O. Hardin, Anne Toms, Na Qiao, Jennifer Litton, George E. Peoples, Elizabeth A. Mittendorf, Lila Ghamsari, Emilio Flano, Judy Jacques, Biao Liu, Jonathan Havel, Vladimir Makarov, Timothy A. Chan, Jessica B. Flechtner, John Facciponte, Stefano Ugel, Francesco De Sanctis, George Coukos, Sébastien Paris, Agnes Pottier, Laurent Levy, Bo Lu, Federica Cappuccini, Emily Pollock, Richard Bryant, Freddie Hamdy, Adrian Hill, Irina Redchenko, Hussein Sultan, Takumi Kumai, Valentyna Fesenkova, Esteban Celis, Ingrid Fernando, Claudia Palena, Justin M. David, Elizabeth Gabitzsch, Frank Jones, James L. Gulley, Mireia Uribe Herranz, Hiroshi Wada, Atsushi Shimizu, Toshihiro Osada, Satoshi Fukaya, Eiji Sasaki, Milad Abolhalaj, David Askmyr, Kristina Lundberg, Ann-Sofie Albrekt, Lennart Greiff, Malin Lindstedt, Dallas B. Flies, Tomoe Higuchi, Wojciech Ornatowski, Jaryse Harris, Sarah F. Adams, Todd Aguilera, Marjan Rafat, Laura Castellini, Hussein Shehade, Mihalis Kariolis, Dadi Jang, Rie vonEbyen, Edward Graves, Lesley Ellies, Erinn Rankin, Albert Koong, Amato Giaccia, Reham Ajina, Shangzi Wang, Jill Smith, Mariaelena Pierobon, Sandra Jablonski, Emanuel Petricoin, Louis M. Weiner, Lorcan Sherry, John Waller, Mark Anderson, Alison Bigley, Chantale Bernatchez, Cara Haymaker, Harriet Kluger, Michael Tetzlaff, Natalie Jackson, Ivan Gergel, Mary Tagliaferri, Patrick Hwu, Mario Snzol, Michael Hurwitz, Theresa Barberi, Allison Martin, Rahul Suresh, David Barakat, Sarah Harris-Bookman, Charles Drake, Alan Friedman, Sara Berkey, Stephanie Downs-Canner, Robert P. Edwards, Tyler Curiel, Kunle Odunsi, Tullia C. Bruno, Brandon Moore, Olivia Squalls, Peggy Ebner, Katherine Waugh, John Mitchell, Wilbur Franklin, Daniel Merrick, Martin McCarter, Brent Palmer, Jeffrey Kern, Dario Vignali, Jill Slansky, Anissa S. H. Chan, Xiaohong Qiu, Kathryn Fraser, Adria Jonas, Nadine Ottoson, Keith Gordon, Takashi O. Kangas, Steven Leonardo, Kathleen Ertelt, Richard Walsh, Mark Uhlik, Jeremy Graff, Nandita Bose, Ravi Gupta, Nitin Mandloi, Kiran Paul, Ashwini Patil, Rekha Sathian, Aparna Mohan, Malini Manoharan, Amitabha Chaudhuri, Yu Chen, Jing Lin, Yun-bin Ye, Chun-wei Xu, Gang Chen, Zeng-qing Guo, Andrey Komarov, Alex Chenchik, Michael Makhanov, Costa Frangou, Yi Zheng, Carla Coltharp, Darryn Unfricht, Ryan Dilworth, Leticia Fridman, Linying Liu, Milind Rajopadhye, Peter Miller, Fernando Concha-Benavente, Julie Bauman, Sumita Trivedi, Raghvendra Srivastava, James Ohr, Dwight Heron, Uma Duvvuri, Seungwon Kim, Heather Torrey, Toshi Mera, Yoshiaki Okubo, Eva Vanamee, Rosemary Foster, Denise Faustman, Edward Stack, Daisuke Izaki, Kristen Beck, Dan Tong Jia, Paul Armenta, Ashley White-Stern, Douglas Marks, Bret Taback, Basil Horst, Laura Hix Glickman, David B. Kanne, Kelsey S. Gauthier, Anthony L. Desbien, Brian Francica, Justin L. Leong, Leonard Sung, Ken Metchette, Shailaja Kasibhatla, Anne Marie Pferdekamper, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John Tallarico, Steven Bender, Chudi Ndubaku, Sarah M. McWhirter, Elena Gonzalez Gugel, Charles J. M. Bell, Adiel Munk, Luciana Muniz, Nina Bhardwaj, Fei Zhao, Kathy Evans, Christine Xiao, Alisha Holtzhausen, Brent A. Hanks, Nathalie Scholler, Catherine Yin, Pien Van der Meijs, Andrew M. Prantner, Cecile M. Krejsa, Leia Smith, Brian Johnson, Daniel Branstetter, Paul L. Stein, Juan C. Jaen, Joanne BL Tan, Ada Chen, Timothy Park, Jay P. Powers, Holly Sexton, Guifen Xu, Steve W. Young, Ulrike Schindler, Wentao Deng, David John Klinke, Hannah M. Komar, Gregory Serpa, Omar Elnaggar, Philip Hart, Carl Schmidt, Mary Dillhoff, Ming Jin, Michael C. Ostrowski, Madhuri Koti, Katrina Au, Nichole Peterson, Peter Truesdell, Gillian Reid-Schachter, Charles Graham, Andrew Craig, Julie-Ann Francis, Beatrix Kotlan, Timea Balatoni, Emil Farkas, Laszlo Toth, Mihaly Ujhelyi, Akos Savolt, Zoltan Doleschall, Szabolcs Horvath, Klara Eles, Judit Olasz, Orsolya Csuka, Miklos Kasler, Gabriella Liszkay, Eytan Barnea, Collin Blakely, Patrick Flynn, Reid Goodman, Raphael Bueno, David Sugarbaker, David Jablons, V. Courtney Broaddus, Brian West, Paul R. Kunk, Joseph M. Obeid, Kevin Winters, Patcharin Pramoonjago, Edward B. Stelow, Todd W. Bauer, Osama E. Rahma, Adam Lamble, Yoko Kosaka, Fei Huang, Kate A. Saser, Homer Adams, Christina E. Tognon, Ted Laderas, Shannon McWeeney, Marc Loriaux, Jeffery W. Tyner, Brian J. Druker, Evan F. Lind, Zhuqing Liu, Shanhong Lu, Lawrence P. Kane, Gulidanna Shayan, Julia Femel, Ryan Lane, Jamie Booth, Amanda W. Lund, Anthony Rodriguez, Victor H. Engelhard, Alessandra Metelli, Bill X. Wu, Caroline W. Fugle, Rachidi Saleh, Shaoli Sun, Jennifer Wu, Bei Liu, Zihai Li, Zachary S. Morris, Emily I. Guy, Clinton Heinze, Jasdeep Kler, Monica M. Gressett, Lauryn R. Werner, Stephen D. Gillies, Alan J. Korman, Hans Loibner, Jacquelyn A. Hank, Alexander L. Rakhmilevich, Paul M. Harari, Paul M. Sondel, Erica Huelsmann, Joseph Broucek, Dorothee Brech, Tobias Straub, Martin Irmler, Johannes Beckers, Florian Buettner, Elke Schaeffeler, Matthias Schwab, Elfriede Noessner, Alison Wolfreys, Andre Da Costa, John Silva, Andrea Crosby, Ludovicus Staelens, Graham Craggs, Annick Cauvin, Sean Mason, Alison M. Paterson, Andrew C. Lake, Caroline M. Armet, Rachel W. O’Connor, Jonathan A. Hill, Emmanuel Normant, Ammar Adam, Detlev M. Biniszkiewicz, Scott C. Chappel, Vito J. Palombella, Pamela M. Holland, Annette Becker, Manmohan R. Leleti, Eric Newcomb, Joanne B. L. Tan, Suthee Rapisuwon, Arash Radfar, Kellie Gardner, Geoffrey Gibney, Michael Atkins, Keith R. Rennier, Robert Crowder, Ping Wang, Russell K. Pachynski, Rosa M. Santana Carrero, Sarai Rivas, Figen Beceren-Braun, Scott Anthony, Kimberly S. Schluns, Deepali Sawant, Maria Chikina, Hiroshi Yano, Creg Workman, Elise Salerno, Ileana Mauldin, Donna Deacon, Sofia Shea, Joel Pinczewski, Thomas Gajewski, Stefani Spranger, Brendan Horton, Akiko Suzuki, Pamela Leland, Bharat H. Joshi, Raj K. Puri, Randy F. Sweis, Riyue Bao, Jason Luke, Marie-Nicole Theodoraki, Frances-Mary Mogundo, Haejung Won, Dayson Moreira, Chan Gao, Xingli Zhao, Priyanka Duttagupta, Jeremy Jones, Massimo D’Apuzzo, and Sumanta Pal
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0301 basic medicine ,Pharmacology ,Cancer Research ,medicine.medical_specialty ,business.industry ,medicine.medical_treatment ,Immunology ,Cancer ,Immunotherapy ,medicine.disease ,3. Good health ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Oncology ,030220 oncology & carcinogenesis ,Family medicine ,Molecular Medicine ,Immunology and Allergy ,Medicine ,business - Abstract
O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1
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- 2016
14. 2013 White Paper on recent issues in bioanalysis: ‘hybrid’ – the best of LBA and LCMS
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Sam Haidar, Stacy Ho, Lauren Stevenson, Surinder Kaur, Jeff Duggan, Alvydas Mikulskis, Jian Wang, Judy Shih, Magnus Knutsson, Lakshmi Amaravadi, Boris Gorovits, Rand Jenkins, Steve Lowes, Hanlan Liu, Mark Ma, Binodh DeSilva, Ronald Shoup, Emma Whale, Chad Ray, Christopher P. Evans, Marian Kelley, Jean W. Lee, An Song, João Tavares Neto, Bob Nicholson, Laixin Wang, Dominique Gouty, Rafiq Islam, Suzanne Martinez, Mike Losauro, Isabelle Dumont, Adrien Musuku, Dawn Dufield, Vincenzo Pucci, Shefali Patel, Eric Ormsby, Laura Wright, Margarete Brudny-Kloeppel, Valerie Theobald, Gary Schultz, Stephanie Fraser, Timothy V Olah, Noriko Katori, Richard Houghton, Mark E. Arnold, Eric Fluhler, Catherine Dicaire, Eric Woolf, Sherri Dudal, Fabio Garofolo, Jan Welink, Mario Rocci, Laurence Mayrand-Provencher, Heather Myler, Raymond Naxing Xu, Jason Wakelin-Smith, Brian Booth, Roger Hayes, Craig Simon, Surendra Bansal, and Theingi M. Thway
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Medical Laboratory Technology ,Bioanalysis ,White paper ,Political science ,Clinical Biochemistry ,Nanotechnology ,Engineering ethics ,General Medicine ,Validation Studies as Topic ,General Pharmacology, Toxicology and Pharmaceutics ,Analytical Chemistry - Abstract
The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These ‘hot’ topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.
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- 2013
15. Discovery fit-for-purpose ligand-binding PK assays: what’s really important?
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Chad Ray, Lindsay King, and Sheldon Leung
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Chemistry ,Ligand binding assay ,Clinical Biochemistry ,General Medicine ,Pharmacology ,Ligands ,Antibodies ,Analytical Chemistry ,Medical Laboratory Technology ,Pharmaceutical Preparations ,Biochemistry ,Pharmacokinetics ,General Pharmacology, Toxicology and Pharmaceutics ,Half-Life - Published
- 2013
16. Feasibility of Singlet Analysis for Ligand Binding Assays: a Retrospective Examination of Data Generated Using the Gyrolab Platform
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Chad Ray, Aidong Wu, Kathleen Pelletier, Lindsay King, Yiqun Zhang, Allison Given Chunyk, Tracey Clark, Alison Joyce, Jo-Ann Wentland, Petar Pop-Damkov, Elizabeth A. Dreher, Laurie Tylaska, and Phillip D. Yates
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Databases, Factual ,Statistics as Topic ,Cmax ,Pharmaceutical Science ,Ligands ,030226 pharmacology & pharmacy ,01 natural sciences ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Statistics ,Animals ,Singlet state ,Retrospective Studies ,Chemistry ,Ligand binding assay ,010401 analytical chemistry ,Replicate ,PK Parameters ,Haplorhini ,0104 chemical sciences ,Rats ,Standard curve ,Concordance correlation coefficient ,Pharmaceutical Preparations ,Feasibility Studies ,Analysis of variance ,Protein Binding - Abstract
There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.
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- 2016
17. Fit-for-Purpose Validation
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Chad Ray
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Accuracy and precision ,Reproducibility ,Process (engineering) ,Computer science ,Stability (learning theory) ,Data mining ,computer.software_genre ,computer ,Normal range - Abstract
This chapter serves as an introduction to the terms and definitions of biomarkers as well as the validation assay process which includes: a standard curve, validation samples, accuracy and precision (reproducibility), limits of quantification, parallelism, dilution linearity, specificity and interference, stability, and normal range. Different biomarkers exist for the various stages of the drug discovery and development process. The main objective of this chapter is to introduce concepts that will facilitate the successful use of biomarkers in drug development—from selection, to validation, and then implementation.
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- 2016
18. Association Kinetics from Single Molecule Force Spectroscopy Measurements
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Boris B. Akhremitchev, Senli Guo, Chad Ray, and Nimit Lad
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Models, Molecular ,Spectroscopy, Imaging, and Other Techniques ,Normal Distribution ,Biophysics ,Analytical chemistry ,Biotin ,02 engineering and technology ,Activation energy ,Microscopy, Atomic Force ,010402 general chemistry ,Kinetic energy ,01 natural sciences ,Dissociation (chemistry) ,Motion ,Steric factor ,Molecule ,Probability ,Reaction step ,Chemistry ,Spectrum Analysis ,Intermolecular force ,Uncertainty ,Force spectroscopy ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Kinetics ,Chemical physics ,Thermodynamics ,Streptavidin ,0210 nano-technology ,Algorithms ,Protein Binding - Abstract
Single molecule force spectroscopy is often used to study the dissociation of single molecules by applying mechanical force to the intermolecular bond. These measurements provide the kinetic parameters of dissociation. We present what to our knowledge is a new atomic force microscopy-based approach to obtain the activation energy of the association reaction and approximate grafting density of reactive receptors using the dependence of the probability to form molecular bonds on probe velocity when one of the interacting molecules is tethered by a flexible polymeric linker to the atomic force microscopy probe. Possible errors in the activation energy measured with this approach are considered and resulting corrections are included in the data analysis. This new approach uses the same experimental setup as traditional force spectroscopy measurements that quantify dissociation kinetics. We apply the developed methodology to measure the activation energy of biotin-streptavidin association (including a contribution from the steric factor) and obtain a value of 8 ± 1 kT. This value is consistent with the association rate measured previously in solution. Comparison with the solution-derived activation energy indicates that kinetics of biotin-streptavidin binding is mainly controlled by the reaction step.
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- 2009
19. Anisotropy of Pairwise Interactions between Hexadecanes in Water Measured by AFM Force Spectroscopy
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Chao Gu, Chad Ray, Jason R. Brown, Andrea Kirkpatrick, and Boris B. Akhremitchev
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Force spectroscopy ,Activation energy ,Hexadecane ,Dissociation (chemistry) ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Accessible surface area ,Hydrophobic effect ,chemistry.chemical_compound ,Crystallography ,General Energy ,chemistry ,Chemical physics ,Molecule ,Physical and Theoretical Chemistry ,Anisotropy - Abstract
The pulling coordinate dependence of hexadecane dimer dissociation in water was studied using AFM-based single molecule force spectroscopy. Hexadecanes were covalently bound to both the AFM cantilever and to the glass substrates through hydrophilic poly-(ethylene glycol) tethers. The polymer tether was attached either to the end of hexadecane or in the middle of the molecule. Experimentally studied configurations of hexadecanes tethered to the AFM probe and to the glass substrate include a symmetric end-attached configuration (EE), an asymmetric end-attached vs middle-attached configuration (ME), and a symmetric middle-attached configuration (MM). Kinetic parameters of the distance to the transition state barrier (barrier width) and activation energy of dissociation were extracted from the statistical analysis of double tether rupture events. The rupture force analysis employs a recently introduced two-bond model that corrects for errors induced by potential multiple simultaneous rupture events and accounts for the tether stiffening effects. Effects of the shape of intermolecular potential were considered by using the Bell−Evans and Hummer−Szabo force spectroscopy models. The activation energies to dissociation were similar for all configurations while the barrier width was significantly shorter for the MM and ME configurations than for EE configurations. Primitive models that include touching or merging spherical or cylindrical shapes were considered. These models were inconsistent with the extracted kinetic parameters. It is suggested that the observed anisotropy may be a result of conformational transition of hexadecane from extended to collapsed state during dimerization. A flexible four-bead model of hexadecane was introduced to account for conformational flexibility. Using the length and solvent accessible surface area of hexadecane, the four-bead model gave molecular dissociation parameters consistent with the experimental data. This suggests that conformational flexibility is an important factor in hydrophobic interactions between alkane chains.
- Published
- 2008
20. Effects of Multiple-Bond Ruptures on Kinetic Parameters Extracted from Force Spectroscopy Measurements: Revisiting Biotin-Streptavidin Interactions
- Author
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Senli Guo, Chad Ray, Andrea Kirkpatrick, Boris B. Akhremitchev, and Nimit Lad
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Streptavidin ,Models, Molecular ,Analytical chemistry ,Biophysics ,Biotin ,02 engineering and technology ,010402 general chemistry ,Kinetic energy ,01 natural sciences ,Molecular physics ,Dissociation (chemistry) ,chemistry.chemical_compound ,Molecular dynamics ,Spectroscopy, Imaging, Other Techniques ,Molecule ,Computer Simulation ,Quantitative Biology::Biomolecules ,Spectrum Analysis ,Force spectroscopy ,Energy landscape ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Biomechanical Phenomena ,Kinetics ,chemistry ,Covalent bond ,0210 nano-technology ,Caltech Library Services - Abstract
Force spectroscopy measurements of the rupture of the molecular bond between biotin and streptavidin often results in a wide distribution of rupture forces. We attribute the long tail of high rupture forces to the nearly simultaneous rupture of more than one molecular bond. To decrease the number of possible bonds, we employed hydrophilic polymeric tethers to attach biotin molecules to the atomic force microscope probe. It is shown that the measured distributions of rupture forces still contain high forces that cannot be described by the forced dissociation from a deep potential well. We employed a recently developed analytical model of simultaneous rupture of two bonds connected by polymer tethers with uneven length to fit the measured distributions. The resulting kinetic parameters agree with the energy landscape predicted by molecular dynamics simulations. It is demonstrated that when more than one molecular bond might rupture during the pulling measurements there is a noise-limited range of probe velocities where the kinetic parameters measured by force spectroscopy correspond to the true energy landscape. Outside this range of velocities, the kinetic parameters extracted by using the standard most probable force approach might be interpreted as artificial energy barriers that are not present in the actual energy landscape. Factors that affect the range of useful velocities are discussed.
- Published
- 2008
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21. Effects of Multiple-Bond Ruptures in Force Spectroscopy Measurements of Interactions between Fullerene C60 Molecules in Water
- Author
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Chad Ray, Chao Gu, Boris B. Akhremitchev, Senli Guo, and Andrea Kirkpatrick
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Quantitative Biology::Biomolecules ,Fullerene ,Force spectroscopy ,Kinetic energy ,Molecular physics ,Dissociation (chemistry) ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,chemistry.chemical_compound ,Crystallography ,General Energy ,chemistry ,Covalent bond ,Molecule ,Physical and Theoretical Chemistry ,Bifunctional ,Ethylene glycol - Abstract
Interactions between fullerene C_(60) molecules in water were measured by force spectroscopy. Fullerene molecules were covalently connected to bifunctional water-soluble poly(ethylene glycol) (PEG) linkers and subsequently tethered to the substrate and to the tip of the atomic force microscope to facilitate single molecule detection and avoid spurious surface effects. The distributions of rupture forces for substrates prepared with different incubation times of C_(60)-PEG-NH_2 exhibit high rupture forces that cannot be explained by the theoretical distribution of single molecule binding. Moreover, the relative amplitude of the high force peak in the histogram increases with incubation time. These observations are explained by attributing the measured high forces to the rupture of multiple bonds between fullerene molecules. Force spectroscopy data analysis based on the most probable forces gives significantly different dissociation rates for samples that exhibit different amplitudes of the high force peak. An approximate analytical model that considers ruptures of two bonds that are simultaneously loaded by tethers with different lengths is proposed. This model successfully fits the distributions of the rupture forces using the same set of kinetic parameters for samples prepared with different grafting densities. It is proposed that this model can be used as a common tool to analyze the probability distributions of rupture forces that contain peaks or shoulders on the high force side of the distribution.
- Published
- 2008
22. TGF-β signalling-related markers in cancer patients with bone metastasis
- Author
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Carlos L. Arteaga, Thomas Daly, Ann Cleverly, Daphne L. Farrington, José Baselga, Michael Lahn, Susanne Kloeker Rhoades, Joan Seoane, Jeffrey Fill, Chad Ray, Brandi Berry, Jonathan M. Yingling, Mace L. Rothenberg, Lisa Anne Wallace, Michael A. Carducci, and Josep Tabernero
- Subjects
Male ,Health, Toxicology and Mutagenesis ,Clinical Biochemistry ,Bone Neoplasms ,Enzyme-Linked Immunosorbent Assay ,Smad Proteins ,Platelet Factor 4 ,Plasma biomarkers ,Biochemistry ,Peripheral blood mononuclear cell ,Transforming Growth Factor beta ,Blood plasma ,Humans ,Medicine ,Tgf β signalling ,Aged ,business.industry ,Parathyroid Hormone-Related Protein ,Bone metastasis ,Cancer ,Middle Aged ,Interleukin-11 ,medicine.disease ,Immunology ,Female ,Signal transduction ,business ,Biomarkers ,Signal Transduction ,Transforming growth factor - Abstract
We measured transforming growth factor (TGF)-beta-dependent biomarkers in plasma and in peripheral blood mononuclear cells (PBMCs) to identify suitable pharmacodynamic markers for future clinical trials with TGF-beta inhibitors. Forty-nine patients with bone metastasis were enrolled in the study, including patients with breast (n=23) and prostate cancer (n=15). Plasma TGF-beta1 levels were elevated in more than half of the cancer patients (geometric mean 2.63 ng ml(-1)) and positively correlated with increased platelet factor 4 (PF4) levels, parathyroid-related protein (PTHrP), von Willebrand Factor (vWF) and interleukin (IL)-10. PBMC were stimulated ex vivo to determine the individual biological variability of an ex vivo assay measuring pSMAD expression. This assay performed sufficiently well to allow its future use in a clinical trial of a TGF-beta inhibitor.
- Published
- 2008
23. Single-Molecule Force Spectroscopy Measurements of Interactions between C60 Fullerene Molecules
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Chao Gu, Boris B. Akhremitchev, Senli Guo, and Chad Ray
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Quantitative Biology::Biomolecules ,Fullerene ,Chemistry ,Anharmonicity ,Force spectroscopy ,Kinetic energy ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Hydrophobic effect ,chemistry.chemical_compound ,General Energy ,Chemical physics ,Computational chemistry ,Molecule ,Physical and Theoretical Chemistry ,Solubility ,Ethylene glycol - Abstract
The hydrophobic effect is important for many biological and technological processes. Despite progress in theory, experimental data clarifying the structure of water and the interactions between hydrophobic solutes at the nanometer scale are scarce because of the very low solubility of hydrophobic species. This article describes single-molecule force spectroscopy measurements of interactions between single fullerene C60 molecules in water. The C60 molecules were tethered by flexible poly(ethylene glycol) linkers to AFM probes and substrates, removing the uncertainty of the aggregation state of solution-based approaches and spurious surface effects. Our analysis of the dependence of the measured most-probable rupture force on the most-probable loading rate considered the deviations from the conventional Bell−Evans model caused by the effects of the anharmonic tether, as well as by the finite depth and shape of the potential well. The kinetic parameters of the activation barrier width, the dissociation rate ...
- Published
- 2007
24. Rupture Force Analysis and the Associated Systematic Errors in Force Spectroscopy by AFM
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Chad Ray, Jason R. Brown, and Boris B. Akhremitchev
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Systematic error ,Analytical expressions ,Atomic force microscopy ,Chemistry ,Theoretical models ,Force spectroscopy ,Nanotechnology ,Surfaces and Interfaces ,Mechanics ,Condensed Matter Physics ,Nonlinear system ,Force analysis ,Electrochemistry ,Loading rate ,General Materials Science ,Spectroscopy - Abstract
Force spectroscopy is a new and valuable tool in physical chemistry and biophysics. However, data analysis has yet to be standardized, hindering the advancement of the technique. In this article, treatment of the rupture forces is described in the framework of the Bell-Evans model, and the systematic errors associated with the tether effect for approaches that utilize the most probable, the median, and the mean rupture forces are compared. It is shown that significant systematic errors in the dissociation rate can result from nonlinear loading with polymeric tethers even if the apparent loading rate is used in the analysis. Analytical expressions for the systematic errors are provided for the most probable and median forces. The use of these expressions to correct the associated systematic errors is illustrated by the analysis of the measured rupture forces between single hexadecane molecules in water. It is noted that the measured distributions of rupture forces often contain high forces that are unaccounted for by theoretical models. Experimental data indicate that the most significant effect of the high forces "tail" is on the dissociation rate obtained from the median force analysis whereas the barrier width appears to be unaffected.
- Published
- 2007
25. Correction of Systematic Errors in Single-Molecule Force Spectroscopy with Polymeric Tethers by Atomic Force Microscopy
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Chad Ray, Jason R. Brown, and Boris B. Akhremitchev
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chemistry.chemical_classification ,Quantitative Biology::Biomolecules ,Chemistry ,Intermolecular force ,Analytical chemistry ,Force spectroscopy ,Stiffness ,Polymer ,Molecular physics ,Dissociation (chemistry) ,Surfaces, Coatings and Films ,Condensed Matter::Soft Condensed Matter ,Hydrophobic effect ,Materials Chemistry ,medicine ,Molecule ,Dewetting ,Physical and Theoretical Chemistry ,medicine.symptom - Abstract
Single-molecule force spectroscopy has become a valuable tool for the investigation of intermolecular energy landscapes for a wide range of molecular associations. Atomic force microscopy (AFM) is often used as an experimental technique in these measurements, and the Bell-Evans model is commonly used in the statistical analysis of rupture forces. Most applications of the Bell-Evans model consider a constant loading rate of force applied to the intermolecular bond. The data analysis is often inconsistent because either the probe velocity or the apparent loading rate is being used as an independent parameter. These approaches provide different results when used in AFM-based experiments. Significant variations in results arise from the relative stiffness of the AFM force sensor in comparison with the stiffness of polymeric tethers that link the molecules under study to the solid surfaces. An analytical model presented here accounts for the systematic errors in force-spectroscopy parameters arising from the nonlinear loading induced by polymer tethers. The presented analytical model is based on the Bell-Evans model of the kinetics of forced dissociation and on the asymptotic models of tether stretching. The two most common data reduction procedures are analyzed, and analytical expressions for the systematic errors are provided. The model shows that the barrier width is underestimated and that the dissociation rate is significantly overestimated when force-spectroscopy data are analyzed without taking into account the elasticity of the polymeric tether. Systematic error estimates for asymptotic freely jointed chain and wormlike chain polymer models are given for comparison. The analytical model based on the asymptotic freely jointed chain stretching is employed to analyze and correct the results of the double-tether force-spectroscopy experiments of disjoining "hydrophobic bonds" between individual hexadecane molecules that are covalently tethered via poly(ethylene glycol) linkers of different lengths to the substrates and to the AFM probes. Application of the correction algorithm decreases the spread of the data from the mean value, which is particularly important for measurements of the dissociation rate, and increases the barrier width to 0.43 nm, which might be indicative of the theoretically predicted hydrophobic dewetting.
- Published
- 2007
26. Optimization of analytical and pre-analytical variables associated with an ex vivo cytokine secretion assay
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Ian Gourley, Viswanath Devanarayan, Peter J. O'Brien, Carmen M. Dumaual, Mark B. Willey, Chad Ray, Jeffrey Fill, and Robert J. Konrad
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Lipopolysaccharides ,Chemistry, Pharmaceutical ,medicine.medical_treatment ,Clinical Biochemistry ,Pharmaceutical Science ,Stimulation ,Pharmacology ,Peripheral blood mononuclear cell ,Chemistry Techniques, Analytical ,Dexamethasone ,Analytical Chemistry ,Inhibitory Concentration 50 ,Drug Discovery ,medicine ,Humans ,Spectroscopy ,Whole blood ,Immunoassay ,medicine.diagnostic_test ,Tumor Necrosis Factor-alpha ,Chemistry ,Temperature ,Anticoagulants ,Cytokine ,Drug Design ,Cytokines ,Cytokine secretion ,Ex vivo ,medicine.drug - Abstract
Purpose Measurements of cytokine release in whole blood after ex vivo stimulation are useful in drug development. The components contributing to variation within such assays have not been clearly defined. Therefore, we characterized the sources of variability within an ex vivo stimulation assay for TNF-α release. Method Fresh whole blood or mononuclear cells from a cell preparation tube were added to silanized, screw-top tubes with a final concentration of 1 μg/mL lipopolysaccharide (LPS). Each tube was purged with 95% air/5%CO 2 and incubated 4 or 6 h at 37 °C in a metabolic water bath. Plasma TNF-α was next measured in supernatants by immunoassay. Total method variability was assessed in 10 normal donors each drawn in the morning and afternoon over 3 days. Four additional samples were pre-treated with dexamethasone to investigate inhibition of TNF-α release. Results Our analysis indicated precise temperature control, the timing and duration of stimulation, and the surface properties of the stimulation vessel most significantly influenced assay performance. A comparison of multiple anticoagulants indicated that careful consideration should be taken in selecting the optimal anticoagulant. The estimated total assay CV for all anticoagulants tested was less than 33.81%. The analytical variability (stimulation and measurement) was less than 25.88% CV. The one exception was mononuclear cells collected in sodium heparin. The total variability estimate incorporated day-to-day, diurnal, inter-donor, tube-to-tube and immunoassay variability. Using our optimized conditions, TNF-α release was inhibited by dexamethasone with a mean IC 50 of 33.3 ± 4.6 nM. Conclusions We have described an optimal set of conditions for collection, storage and processing of an ex vivo cytokine stimulation assay. These conditions were selected for operational feasibility, minimal imprecision and elimination of potential confounding factors. The end result is a more robust method that can be applied to clinical drug development.
- Published
- 2006
27. Teva Pharmaceuticals v. Sandoz: availability of generic glatiramer acetate and the impact to patent litigation claim construction
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Chad Ray and Louis E Fogel
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Pharmacology ,Drug Industry ,Deference ,Commerce ,General Medicine ,Glatiramer Acetate ,Trial court ,Drug Costs ,United States ,Supreme court ,Patents as Topic ,Patent troll ,Law ,Drug Discovery ,medicine ,Drug and Narcotic Control ,Drugs, Generic ,Humans ,Business ,Glatiramer acetate ,Peptides ,Immunosuppressive Agents ,medicine.drug - Abstract
In the upcoming case of Teva Pharmaceuticals v. Sandoz, the U.S. Supreme Court will address how much deference the appellate court should afford to a trial court’s claim construction ruling. The effect of this decision will be far-reaching, as how claims are construed can determine whether a patent is infringed or not infringed, valid or invalid.
- Published
- 2014
28. Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum
- Author
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Chad Ray, Robert A. Dean, Mark B. Willey, Wendell C. Smith, Viswanath Devanarayan, Ronald R. Bowsher, and John T. Brandt
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Immunoassay ,Analyte ,Chromatography ,medicine.diagnostic_test ,Chemistry ,Clinical Biochemistry ,Limits of agreement ,Analytical chemistry ,Pharmaceutical Science ,Analytical Chemistry ,Microsphere ,Elisa kit ,Sample volume ,Concordance correlation coefficient ,Drug Discovery ,medicine ,Cytokines ,Humans ,Multiplex ,Spectroscopy - Abstract
Quantification of biomarkers can provide important information about the safety and efficacy of candidate drugs. Unfortunately, limited sample volume and excess costs often limit analysis of multiple biomarkers. We developed, optimized, validated, and implemented a multiplex immunoassay for simultaneous measurement of multiple circulating cytokines: IL-1beta, TNFalpha, IL-6, IL-8, and IL-10. Multiplex immuoassays were performed using the Luminex LabMAP instrument. Capture antibodies for each cytokine were covalently bound to distinct microsphere subsets distinguished by differing dye ratios. The concentration of each individual cytokine determined by measuring orange fluorescence produced by a complex of a biotinylated cytokine-specific antibody and streptavidin-phycoerythrin. The lower limit of quantification for all assays was 20 pg/mL with the exception of IL-8 which was 100 pg/mL. The inter-assay precision was less than 25%CV for all analytes at all control levels both pre-study and in-study. The percent recovery ranged from 83 to 108% pre-study and 90 to 125% in-study. In a linearity assessment, a 15,000 pg/mL multi-analyte control could be diluted 1:50 and maintain expected accuracy. We measured the cytokine concentrations in more than 2000 serum samples from patients with sepsis. Multiplex results for IL-6 were compared to a conventional commercially available ELISA kit. The degree of agreement between the two methods as measured by the concordance correlation coefficient was 84.5%. Multiplex results were 2.36-fold higher than ELISA values on the average. After adjusting for this mean difference, the 95% empirical limits of agreement for the ratio of individual sample values were 0.33, 2.65. This multiplex immunoassay provided simultaneous measurement of circulating cytokines using 80% less patient specimen compared to traditional approaches and at a significantly decreased cost. Efficient use of this platform requires process improvements to fully maximize the positive impact of multiplex assays in clinical drug development.
- Published
- 2005
29. Biomarker accuracy: exploring the truth
- Author
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Chad Ray
- Subjects
Pre analytical ,business.industry ,Clinical Biochemistry ,Clinical Chemistry Tests ,General Medicine ,Computational biology ,Analytical Chemistry ,Medical Laboratory Technology ,Medicine ,Biomarker (medicine) ,Humans ,General Pharmacology, Toxicology and Pharmaceutics ,business ,Artifacts ,Biomarkers - Published
- 2014
30. Tissue bioanalysis of biotherapeutics and drug targets to support PK/PD
- Author
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Eric Fluhler, Chad Ray, Mireia Fernández Ocaña, Chengjie Ji, Boris Gorovits, Scott Fountain, Kimberly Long, Tracey Clark, Yan Weng, Joe Palandra, Lindsay King, Sheldon Leung, Jianqing Chen, Wenlin Li, Denise O'Hara, Mengmeng Wang, and Hendrik Neubert
- Subjects
Drug ,Bioanalysis ,Drug discovery ,Chemistry ,media_common.quotation_subject ,Clinical Biochemistry ,General Medicine ,Computational biology ,Pharmacology ,Chemistry Techniques, Analytical ,Analytical Chemistry ,Biomarker (cell) ,Biopharmaceutics ,Medical Laboratory Technology ,Target site ,Pharmaceutical Preparations ,Animals ,Humans ,General Pharmacology, Toxicology and Pharmaceutics ,Target binding ,PK/PD models ,Biomarkers ,media_common - Abstract
Contemporary drug discovery leverages quantitative modeling and simulation with increasing emphasis, both to gain deeper knowledge of drug targets and mechanisms as well as improve predictions between preclinical models and clinical applications, such as first-in-human dose projections. Proliferation of novel biotherapeutic modalities increases the need for applied PK/PD modeling as a quantitative tool to advance new therapies. Of particular relevance is the understanding of exposure, target binding and associated pharmacology at the target site of interest. Bioanalytical methods are key to informing PK/PD models and require assessment of both PK and PD end points. Where targets are sequestered in tissues (noncirculating), the ability to quantitatively measure drug or biomarker in tissue compartments becomes particularly important. This perspective provides an overview of contemporary applications of quantitative bioanalysis in tissue compartments as applied to PK and PD assessments associated with novel biotherapeutics. Case studies and key references are provided.
- Published
- 2012
31. Ligand binding assays in the 21st Century laboratory: platforms
- Author
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Franklin Spriggs, Zhandong Don Zhong, Chad Ray, Saloumeh K Fischer, Jenny Ly, Martin Boissonneault, Darshana Jani, Narasaiah Dontha, Nathalie Rouleau, Anita Kant, Lia Brilando, Afshin Safavi, and Karolina Österlund
- Subjects
Flexibility (engineering) ,Drug Industry ,Computer science ,Event (computing) ,business.industry ,Clinical Laboratory Techniques ,media_common.quotation_subject ,SIGNAL (programming language) ,Pharmaceutical Science ,Reproducibility of Results ,Nanotechnology ,Ligands ,Automation ,Variety (cybernetics) ,Product life-cycle management ,Systems engineering ,Commentary ,Table (database) ,Quality (business) ,business ,media_common ,Biotechnology ,Protein Binding - Abstract
The 21st Century Bioanalytical Laboratory Platforms initiative in the BIOTEC section of the American Association of Pharmaceutical Scientists (AAPS) began with a pre-conference workshop held in Seattle, WA in June of 2009. This workshop brought together members of the pharmaceutical and biotechnology industries, with instrument and reagent manufacturers to discuss the current and potential future state of the bioanalytical laboratories supporting biologics development. At the conclusion of the workshop, four sub-teams were formed to further develop the ideas and concepts raised during the 2-day workshop. The sub-teams are reagents, automation, e-solutions, and platforms. This paper discusses the critical attributes of a research and development ligand binding assay (LBA) platform and the desired characteristics new platforms should strive to offer in the future. This paper is not intended to be a review and comparison of the current platforms on the market, as this has been done and published elsewhere (1–9). The platforms team consists of a balanced cross-section of the industry with representatives from pharmaceutical, biotechnology, contract research organizations, and instrument manufacturers. The Platforms team have collaborated to discuss and arrive at a consensus regarding the most useful characteristics of a bioanalytical platform for biologics. We present here the results of these discussions. A platform is the technology employed in an analytical method to transduce a biochemical event into a measureable output or signal. This signal allows the bioanalytical scientist to accurately and reproducibly make measurements to analyze different aspects of a specific biologic target (therapeutic, biomarker, and anti-drug antibody) such as its pharmacokinetics, immunogenicity, potency, or effect of biomarkers. An instrument is the tool utilized minimally to measure a platform’s output and convert the resultant signal into interpretable information the analytical scientist can use but can incorporate other aspects such as liquid handling. Many platforms employ optical signals including the absorbance of light through a medium (10) or the emission of fluorescence (10) or luminescence (11). A variety of light detectors are used to measure these optical signals including photo diodes, charge-coupled device cameras, and photo-multiplier tubes (Table I). Sections in this paper provide details on the desirable analytical characteristics, multiplexing, platform flexibility and throughput, desirable instrument characteristics, and finally, life cycle management of the ideal LBA platform. Table I Commonly Used Platforms The analytical characteristics of today’s ligand binding assays are primarily influenced by three major factors—the quality of the reagents, assay format, and the choice of the analytical platform. This paper describes only those aspects derived from the analytical platform.
- Published
- 2012
32. Ligand binding assays in the 21st century laboratory-a call for change
- Author
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Ago B. Ahene and Chad Ray
- Subjects
Process management ,Binding Sites ,Standardization ,business.industry ,Event (computing) ,Best practice ,media_common.quotation_subject ,Pharmaceutical Science ,Ligands ,Automation ,Task (project management) ,Biopharmaceutical ,Editorial ,Order (business) ,Income ,Quality (business) ,Business ,Cooperative Behavior ,Laboratories ,Simulation ,media_common - Abstract
In June 2009, a group of ligand binding assay experts met in Seattle, WA to discuss the need for greater efficiency in bioanalytical laboratories. The central premise was increased utilization of technological innovation will lead to increased quality, throughput, and efficiency. Currently, there are very few laboratories that have reached the ideal or utopian level desired. The biggest challenge currently facing the ligand binding community is a reliance on manual processes and paper-based systems, ineffective integration, and a lack of standardization in many areas. In order to address these gaps, we identified speakers who had developed solutions to parts of the overall problem. The event spanned 2 days with participation from three distinct groups: biopharmaceutical companies, contract research organizations, and research equipment providers. The goal was to share best practices, identify gaps, and define next steps. The outcome of themeeting was the development of four sub-teams that included: reagents, instrument platforms, automation, and electronic solutions. Each sub-team was given the task of identifying the most achievable and high-impact facets to change or improve.
- Published
- 2012
33. Development and validation of an alpha fetoprotein immunoassay using Gyros technology
- Author
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Chad Ray, Pamela Whalen, Allison Given, and Peter J. O'Brien
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Oncology ,medicine.medical_specialty ,Accuracy and precision ,Clinical Biochemistry ,Analytical chemistry ,Pharmaceutical Science ,Mice, SCID ,Antibodies ,Analytical Chemistry ,Mice ,Internal medicine ,Drug Discovery ,medicine ,Animals ,Prognostic biomarker ,Spectroscopy ,Automation, Laboratory ,Immunoassay ,Mice, Inbred ICR ,medicine.diagnostic_test ,Chemistry ,Reproducibility of Results ,medicine.disease ,digestive system diseases ,Sample stability ,Low volume ,Hepatocellular carcinoma ,Biomarker (medicine) ,alpha-Fetoproteins ,Alpha-fetoprotein ,Blood Chemical Analysis - Abstract
Circulating alpha fetoprotein (AFP) is a diagnostic and prognostic biomarker for hepatocellular carcinoma (HCC) with potential utility as a pharmacodynamic endpoint in rodent tumor models. This application is limited, however, by low sample volumes, highlighting the need for sensitive, sample-sparing biomarker assay methods. In order to improve the utility of AFP as an oncology biomarker, we developed a method for AFP using the Gyrolab™, an automated microimmunoassay platform. Commercially available antibodies were screened to identify optimal combinations that were then used in a multi-factorial design of experiments (DOE) to optimize reaction conditions. Analytical validation included assessments of accuracy and precision (A&P), and dilutional linearity/hook effect, as well as reagent and sample stability. The method is reliable, with total error, a measure of accuracy and precision, less than 30% for all concentrations tested. AFP concentrations were measurable in diseased mice and undetectable in normal mice. Therefore, this novel, low volume AFP immunoassay is suitable for pre-clinical drug development, where its miniaturized format facilitates serial sampling in rodent models of cancer.
- Published
- 2011
34. Mechanical distortion of protein receptor decreases the lifetime of a receptor-ligand bond
- Author
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Nan Li, Boris B. Akhremitchev, Nimit Lad, Senli Guo, and Chad Ray
- Subjects
Chemistry ,Atomic force microscopy ,Bond ,Kinetics ,Proteins ,Receptors, Cell Surface ,General Chemistry ,Mechanical force ,Ligands ,Microscopy, Atomic Force ,Biochemistry ,Catalysis ,Dissociation (chemistry) ,Crystallography ,Colloid and Surface Chemistry ,Molecular recognition ,Chemical physics ,Covalent bond ,Receptor - Abstract
Substantial experimental evidence indicates that the mechanical force applied to pull apart non-covalent molecular bonds (such as receptor-ligand pairs) can significantly decrease the bond lifetime. This evidence is often generated in single-molecule experiments that are designed to specifically test effects of pulling forces. However, the effect of compressive forces on the lifetime of receptor-ligand bonds remains largely unexplored. Here we extend the common usage of the atomic force microscopy technique to study whether compressive forces applied to bound streptavidin-biotin species can significantly accelerate the rate of dissociation. Presented experimental data indicate that compressive forces can substantially decrease the lifetime of the molecular bond. Surprisingly, the efficiency of accelerating dissociation by compressive forces sometimes exceeds the enhancement of the dissociation rate measured in pulling experiments, indicating that compressive forces applied to the bound species might be efficiently used to control the lifetime of adhesion bonds.
- Published
- 2010
35. Kinetic parameters from detection probability in single molecule force spectroscopy
- Author
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Jason L. Brown, Chad Ray, Senli Guo, Boris B. Akhremitchev, and Nan Li
- Subjects
Streptavidin ,Force spectroscopy ,Analytical chemistry ,Surfaces and Interfaces ,Condensed Matter Physics ,Kinetic energy ,Molecular physics ,Dissociation (chemistry) ,Displacement (vector) ,chemistry.chemical_compound ,chemistry ,Covalent bond ,Electrochemistry ,Range (statistics) ,Molecule ,General Materials Science ,Spectroscopy - Abstract
The detection probability of rupture events in AFM force spectroscopy measurements presents a viable alternative to standard methods for extracting kinetic parameters of dissociation. The detection probability has a maximum as a function of the probe velocity where (1) the probability to form a molecular bond is independent of the probe velocity and (2) the detection of rupture events is limited by noise and performed with a constant density of data points per distance of the probe displacement. This newly developed model indicates that the optimal detection velocity is independent of dissociation rate and depends on the distance to the barrier kinetic parameter. Therefore, the kinetic parameters of bond dissociation can be extracted from the dependence of detection probability on probe velocity and the detection threshold. This approach is sensitive to low rupture forces and therefore is complementary to the common most probable force data analysis approach. The developed approach is tested using rupture forces measured with specific bonds between biotin and streptavidin and with nonspecific bonds between linear alkanes in water. Results for the analysis of specific bonds rupture are consistent with the previous measurements, suggesting that rupture forces spanning a wide range of values originate from the same binding potential. Kinetic parameters obtained for linear alkanes are significantly different from previous measurements suggesting possible heterogeneity of the bound state.
- Published
- 2010
36. Development and validation of a phosphorylated SMAD ex vivo stimulation assay
- Author
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Robert J. Konrad, Jonathan M. Yingling, Thomas Daly, Yue-Wei Qian, Xiliang Wang, Lei Yan, Shou J, Jeffrey Fill, Ehsani Me, Daphne L. Farrington, Ann Cleverly, Michael Lahn, and Chad Ray
- Subjects
Angiogenesis ,Health, Toxicology and Mutagenesis ,Clinical Biochemistry ,Blotting, Western ,Drug Evaluation, Preclinical ,Receptor, Transforming Growth Factor-beta Type I ,Mice, Nude ,Stimulation ,Enzyme-Linked Immunosorbent Assay ,Smad Proteins ,SMAD ,Smad2 Protein ,Biology ,Protein Serine-Threonine Kinases ,Biochemistry ,Transforming Growth Factor beta1 ,Mice ,Cell Line, Tumor ,Neoplasms ,Animals ,Humans ,Smad3 Protein ,Phosphorylation ,Receptor ,Protein Kinase Inhibitors ,Dose-Response Relationship, Drug ,Kinase ,Reproducibility of Results ,Xenograft Model Antitumor Assays ,Rats, Inbred F344 ,Biomarker (cell) ,Rats ,Data Interpretation, Statistical ,Cancer research ,Leukocytes, Mononuclear ,Female ,Activin Receptors, Type I ,Receptors, Transforming Growth Factor beta ,Ex vivo ,Biomarkers - Abstract
Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.
- Published
- 2007
37. Abstract 2594: Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR
- Author
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Paolo Vicini, Marlena Walls, Jie Guo, Sajiv Krishnan Nair, Jennifer Lafontaine, Valeria Fantin, Yuli Wang, Tod Smeal, Brion W. Murray, Gary Li, Zelanna Goldberg, Manli Shi, Pramod P. Mehta, Greg Stevens, Scott L. Weinrich, Wei Tan, Anand Sistla, Simon Paul Planken, Allison Given, Mike Zientek, Jun Li Feng, Sangita M. Baxi, Michelle Hemkens, John Charles Kath, Chad Ray, Liu Yang, Min-Jean Yin, and Henry Cheng
- Subjects
Cancer Research ,business.industry ,Mutant ,Cell ,Wild type ,Cancer ,Pharmacology ,medicine.disease ,respiratory tract diseases ,T790M ,medicine.anatomical_structure ,Oncology ,Mechanism of action ,In vivo ,medicine ,Phosphorylation ,medicine.symptom ,business - Abstract
Activating mutations in EGFR confer constitutive activity providing the oncogenic drive in EGFR-mutant NSCLC. First and 2nd generation EGFR tyrosine kinase inhibitors (TKIs) are effective drugs in this setting, but are constrained by dose-limiting toxicities attributed to inhibition of wild type (WT) EGFR and by drug resistance caused, in the majority of cases, via a T790M secondary mutation in EGFR. We report the pharmacology of a novel irreversible 3rd generation EGFR TKI active against EGFR with activating and T790M mutations, but sparing WT EGFR. Our novel 3rd generation EGFR TKI was studied in a variety of in vitro and in vivo models to determine its inhibitory potencies on different EGFR variants, pharmacokinetics (PK), antitumor efficacy, exposure-response relationships, mechanism of action, and predicted human efficacious dose. In enzyme and cell assays, our compound is a highly potent inhibitor of EGFR double mutants (L858R/T790M and Del/T790M) and EGFR activating mutants (L858R and Del), but a weak inhibitor of WT EGFR (26-fold margin over mutant target potencies). Effects on downstream signaling and function indicate the underlying mechanism of the compound is direct inhibition of EGFR, with subsequent inhibition of downstream signaling that results in apoptosis and viable cell decline. In xenograft mouse models, the compound demonstrates tumor growth inhibition and regression at well-tolerated doses in models driven by EGFR double mutants and EGFR activating mutants. The antitumor efficacy is dose-dependent and strongly correlates with inhibition of EGFR phosphorylation and EGFR-mediated downstream signaling, and induction of apoptosis. Plasma concentrations assumed to be sufficient for efficacy (Ceff) were defined using a mathematical model incorporating the plasma levels of the compound, the associated inhibitory effects on EGFR phosphorylation, and the antitumor efficacy in the double and activating mutant xenograft models. Ceff was in agreement across several models and was used with in vitro human PK properties to calculate required human dose. While our compound possesses a similar profile as other recently disclosed 3rd generation EGFR TKIs, this molecule is distinguished by better potency on the activating mutants and by the widest potency margin on WT EGFR. Given that the target potencies and WT margins of 3rd generation EGFR TKIs have been sufficient for tolerated clinical efficacy in preliminary results, it can be inferred that our compound will have similar promise in the clinic. These results support our compound as a novel EGFR TKI with an inhibitory profile and favorable drug-like properties that suggest utility for treating patients with NSCLC with EGFR activating and resistance mutations. Citation Format: Mike Zientek, Sangita Baxi, Henry Cheng, Valeria Fantin, Jun Li Feng, Allison Given, Zelanna Goldberg, Jie Guo, Michelle Hemkens, John Kath, Jennifer Lafontaine, Gary Li, Pramod Mehta, Brion Murray, Sajiv Nair, Simon Planken, Chad Ray, Yuli Wang, Manli Shi, Anand Sistla, Tod Smeal, Greg Stevens, Wei Tan, Paolo Vicini, Marlena Walls, Liu Yang, Min-Jean Yin, Scott L. Weinrich. Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2594. doi:10.1158/1538-7445.AM2015-2594
- Published
- 2015
38. Single-molecule force spectroscopy measurements of 'hydrophobic bond' between tethered hexadecane molecules
- Author
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Jason R. Brown, Boris B. Akhremitchev, and Chad Ray
- Subjects
Polymers ,Molecular Conformation ,Hexadecane ,Microscopy, Atomic Force ,Dissociation (chemistry) ,Hydrophobic effect ,chemistry.chemical_compound ,Surface-Active Agents ,Alkanes ,Spectroscopy, Fourier Transform Infrared ,Materials Chemistry ,Molecule ,Organic chemistry ,Physical and Theoretical Chemistry ,Solubility ,Probability ,chemistry.chemical_classification ,Chemistry ,Chemistry, Physical ,Force spectroscopy ,Temperature ,Water ,Polymer ,Models, Theoretical ,Surfaces, Coatings and Films ,Hydrophobe ,Chemical physics ,Spectrophotometry ,Thermodynamics ,Dimerization ,Hydrophobic and Hydrophilic Interactions - Abstract
The hydrophobic effect is important for many biological and technological processes. Despite progress in theory, experimental data clarifying water structure and the interaction between hydrophobic solutes at the nanometer scale are scarce due to the very low solubility of hydrophobic species. This article describes an AFM single molecule force spectroscopy method to probe the interaction between molecules with low solubility and reports measurements of the strength and the length scale of the "hydrophobic bond" between hexadecane molecules. Hexadecane molecules are tethered by flexible poly(ethylene glycol) linkers to AFM probes and substrates, removing the aggregation state uncertainty of solution-based approaches as well as spurious surface effects. A shorter hydrophilic polymer layer is added to increase the accessibility of hydrophobic molecules for the force spectroscopy measurements. Statistical analysis of the rupture forces yields a barrier width of 0.24 nm, and a dissociation rate of 1.1 s(-1). The results of single molecule measurements are related to the theoretical predictions of the free energy of cavitation in water and to the empirical model of micellization of nonionic surfactants. It is estimated that approximately one-quarter of each molecule's surface is hydrated during forced dissociation, consistent with an extended (nonglobular) conformation of the hexadecane molecules in the dimer.
- Published
- 2006
39. Development and validation of a drug activity biomarker that shows target inhibition in cancer patients receiving enzastaurin, a novel protein kinase C-beta inhibitor
- Author
-
Lisa J. Green, Luna Musib, S. Gail Eckhardt, Carolyn A. Cook, Roy S. Herbst, Philip Marder, Chad Ray, Donald Thornton, Michele Basche, Susan Jaken, Michael A. Carducci, and Carolyn D. Britten
- Subjects
Cancer Research ,Indoles ,Enzyme Activators ,Pharmacology ,Peripheral blood mononuclear cell ,Deoxycytidine ,Sensitivity and Specificity ,Monocytes ,Flow cytometry ,chemistry.chemical_compound ,Structure-Activity Relationship ,Enzastaurin ,Cell Line, Tumor ,Neoplasms ,Antineoplastic Combined Chemotherapy Protocols ,Protein Kinase C beta ,Medicine ,Humans ,Enzyme Inhibitors ,Protein kinase A ,Protein kinase C ,Capecitabine ,Protein Kinase C ,medicine.diagnostic_test ,Clinical Trials, Phase I as Topic ,business.industry ,Kinase ,Reproducibility of Results ,Biological activity ,Flow Cytometry ,Treatment Outcome ,Oncology ,chemistry ,Leukocytes, Mononuclear ,Fluorouracil ,business ,Ex vivo ,Biomarkers ,Follow-Up Studies ,Signal Transduction - Abstract
Purpose: To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent. Experimental Design: The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach. Results: Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin. Conclusion: An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.
- Published
- 2006
40. Conformational heterogeneity of surface-grafted amyloidogenic fragments of alpha-synuclein dimers detected by atomic force microscopy
- Author
-
Boris B. Akhremitchev and Chad Ray
- Subjects
chemistry.chemical_classification ,Alpha-synuclein ,Surface (mathematics) ,Amyloid ,Tethering ,Atomic force microscopy ,Surface Properties ,Molecular Conformation ,Peptide ,General Chemistry ,Atmospheric temperature range ,Microscopy, Atomic Force ,Biochemistry ,Catalysis ,Peptide Fragments ,chemistry.chemical_compound ,Crystallography ,Colloid and Surface Chemistry ,chemistry ,alpha-Synuclein ,Molecule ,Ethylene glycol ,Dimerization - Abstract
A force-spectroscopy-based approach is used to characterize separation between amyloidogenic peptide fragments of alpha-synuclein. Interactions between individual molecules are studied using a scanning-force-microscopy-based technique. Alpha-synuclein fragments are attached to the solid surfaces via flexible long poly-(ethylene glycol) linkers removing aggregation state uncertainty of solution-based approaches and spurious surface effects. Tethering one fragment to the scanning probe tip and another fragment to the second surface ensures that interactions between tethered molecules are studied. Control experiments with only one tethered peptide indicate peptide-peptide interactions as the source of observed interaction forces in the double-tether experiment. The temperature dependence of rupture forces from 17.5 degrees C to 40 degrees C reveals similar molecular parameters indicating that no significant conformational changes occur in the associated molecules over this temperature range. Rate-dependent measurements indicate conformational heterogeneity of joined peptide molecules.
- Published
- 2005
41. Fit-for-purpose method development and validation for successful biomarker measurement
- Author
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Ira Weinryb, Robert Millham, Masood Khan, Jean W. Lee, Marie Green, Yu Chen Barrett, Scott Fountain, Stephen Keller, James A. Rogers, Jeff M. Sailstad, John Allinson, Chad Ray, John A. Wagner, Larry Duan, Peter J. O'Brien, Viswanath Devanarayan, and Russell Weiner
- Subjects
Pharmacology ,Quality Control ,Models, Statistical ,Computer science ,Organic Chemistry ,Pharmaceutical Science ,Data interpretation ,New Drug Approvals ,Reproducibility of Results ,Context (language use) ,Nanotechnology ,Method development ,Drug development ,Risk analysis (engineering) ,Pharmaceutical technology ,Data Interpretation, Statistical ,Drug Design ,Terminology as Topic ,Calibration ,Molecular Medicine ,Biomarker (medicine) ,Pharmacology (medical) ,Drug analysis ,Biomarkers ,Biotechnology - Abstract
Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate. Key differences preclude the direct application of existing validation paradigms for drug analysis to biomarker research. Following the AAPS 2003 Biomarker Workshop (J. W. Lee, R. S. Weiner, J. M. Sailstad, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development. A conference report. Pharm Res 22:499-511, 2005), these and other critical issues were addressed. A practical, iterative, "fit-for-purpose" approach to biomarker method development and validation is proposed, keeping in mind the intended use of the data and the attendant regulatory requirements associated with that use. Sample analysis within this context of fit-for-purpose method development and validation are well suited for successful biomarker implementation, allowing increased use of biomarkers in drug development.
- Published
- 2005
42. [Untitled]
- Author
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Brent Kern, Chad Ray, Eugenia Kraynov, and Li-Fen Lee
- Subjects
medicine.diagnostic_test ,media_common.quotation_subject ,CD3 ,Immunology ,Hematology ,Biology ,Pharmacology ,Biochemistry ,In vitro ,Flow cytometry ,In vivo ,medicine ,biology.protein ,Immunology and Allergy ,Receptor ,Internalization ,Molecular Biology ,Ex vivo ,media_common ,Whole blood - Abstract
In vitro (or ex vivo) quantification of cellular parameters is often employed to inform systems models that define the relationship between drug exposure and response. We developed two flow cytometry based assays to evaluate the interaction between the IL-7 receptor (IL-7R) and an anti-IL-7R monoclonal antibody (Ab1). IL-7R occupancy and rate of internalization in the presence of Ab1 was determined using flow cytometry in monkeys and human whole blood. Two commercially available antibodies were used to detect IL-7R. One detected “free” and the other determined “total” receptor. Receptor internalization was measured ex vivo at various time points at 37 °C. The receptor occupancy assay was used in vivo following administration of Ab1 into cynomolgus monkeys at 0.3, 3, and 30 mg/kg. The amount of Ab1 bound to receptor and the total amount of receptor was quantified. The ex vivo receptor occupancy mean IC50 values in monkey and human whole blood were 5.64 ± 2.38 and 9.39 ± 1.54 ng/ml, respectively. The ex vivo rate of internalization of Ab1-IL-7R complex was characterized by a slope of 384 ± 24 and 2482 ± 319, for monkey and human, respectively. In the in vivo monkey study, no free IL-7R was detectable on the CD3 T cell surface at 0.25 h post-dose in all treatment groups. Free receptor levels returned to 78% on day 8 at 0.3 mg/kg, 55% on day 15 at 3 mg/kg, and 12% on day 15 at 30 mg/kg, compared to pre-dose levels. By Day 22, free IL-7R levels in all dose groups returned to their baseline. Ab1 treatment resulted in a significant reduction in total IL-7R. Effective biomeasures provide a link between species and the ex vivo and in vivo systems. Integration of these values into systems models reduces the number of model assumptions and increases the likelihood of success.
- Published
- 2013
43. Non-localized ligand-to-metal charge transfer excited states in (Cp)2Ti(IV)(NCS)2
- Author
-
Chad Ray, John W. Kenney, Melissa A. Summers, Grant D. Meyer, Jason A. Marshall, Elizabeth L. Patrick, Theodore P. Ortiz, and James A. Brozik
- Subjects
Infrared ,Chemistry ,Infrared spectroscopy ,General Chemistry ,Biochemistry ,Catalysis ,Delocalized electron ,Crystallography ,Colloid and Surface Chemistry ,Nuclear magnetic resonance ,Excited state ,Molecular orbital ,Triplet state ,Phosphorescence ,Ground state - Abstract
The bent d(0) titanium metallocene (Cp)(2)Ti(NCS)(2) exhibits an intense phosphorescence from a ligand-to-metal charge transfer triplet excited state at 77 K in an organic glass substrate and a poly(methyl methacrylate) plastic substrate. Quantum chemical calculations and spectroscopic studies show that the orbital parentage of this triplet state arises from the promotion of an electron from an essentially nonbonding symmetry adapted pi molecular orbital located on the NCS(-) ligands to a d(z)2-(y)2 orbital located on the Ti metal. Standard infrared spectroscopy of (Cp)(2)Ti(NCS)(2) in its ground electronic state at 77 K reveals a pair of closely spaced absorptions at (2072 cm(-1), 2038 cm(-1))(glass) and (2055 cm(-1), 2015 cm(-1))(plastic) that are assigned, respectively, to the symmetric and antisymmetric CN stretching modes of the two coordinated NCS(-) ligands. Low-temperature (77 K) time-resolved infrared spectroscopy that accesses the phosphorescing triplet excited state on the ns time scale shows an IR bleach that is coincident with the two ground state CN stretching bands and an associated grow-in of a pair of new IR bands at slightly lower energies (2059 cm(-1), 2013 cm(-1))(glass) and (2049 cm(-1), 1996 cm(-1))(plastic) that are assigned, respectively, to the symmetric and antisymmetric CN stretches in the emitting triplet state. These transient IR bands decay with virtually identical lifetimes to those observed for the phosphorescence decays when measured under identical experimental conditions. Singular value decomposition analysis of the time-resolved infrared data shows that the observed transient IR features arise from the same electronic manifold as measured through luminescence studies. The close similarity between the ground state and excited-state CN stretching bands in (Cp)(2)Ti(NCS)(2) indicates that symmetry breaking does not occur in forming the charge-transfer triplet excited-state manifold; i.e., electron density is withdrawn from a delocalized pi MO spread across both NCS(-) ligands. Calculations at several levels of theory reveal a delocalized ligand-to-metal charge transfer excited triplet manifold. These calculations closely reproduce the relative intensity ratios and frequencies of the symmetric and antisymmetric transient infrared vibrations in the CN region. This study is the first time-resolved infrared investigation of a ligand-to-metal charge-transfer excited state and the first to be performed at cryogenic temperatures in thin-film organic glass and plastic substrates.
- Published
- 2003
44. Cardiogenic shock temporally associated with COVID-19 vaccination after prior COVID-19 infection: A case report
- Author
-
Elizabeth M. Jean-Marie, Aya Tabbalat, Chad Raymond, Meisam Moghbelli, Keith Armitage, and Ian J. Neeland
- Subjects
Multisystem inflammatory syndrome in children ,Multisystem inflammatory syndrome in adults ,MIS-C ,MIS-A ,Coronavirus 2019 ,Heart failure ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
The introduction of coronavirus 2019 (COVID-19) vaccination has been an integral force in stopping the spread of COVID-19 across the globe. While reported side effects of vaccination have predominantly been mild, in the last year reports have emerged of myocarditis following the BNT162b2 (Pfizer-BioNtech) and mRNA-1273 (Moderna) vaccinations. The adolescent and young adult population have been the population most reported, with over 1000 cases under review by the Centers for Disease Control (CDC) since April 2021. Here we report a case of a previously healthy 21-year-old male who developed Multisystem Inflammatory Syndrome in Adults (MIS-A) and following the second dose of the Pfizer-BioNtech vaccine. The young male initially presented with fever, leukocytosis with high neutrophil-lymphocyte ratio, severe cardiac illness, and positive COVID-19 nucleocapsid serology, consistent with MIS-A diagnosis. His case was complicated by cardiogenic shock, requiring brief venoarterial extracorporeal membrane oxygenation (VA-ECMO) support. While this report does not detract from the overwhelming benefit of vaccination from COVID-19, clinicians should be aware of this possible relationship in the future.
- Published
- 2022
- Full Text
- View/download PDF
45. Single-molecule Force Spectroscopy Measurements of “Hydrophobic Bond” between Tethered Hexadecane Molecules.
- Author
-
Chad Ray, Jason R. Brown, and Boris B. Akhremitchev
- Subjects
- *
ETHYLENE glycol , *SOLUBILITY , *SOLUTION (Chemistry) , *SCISSION (Chemistry) , *SURFACE active agents - Abstract
The hydrophobic effect is important for many biological and technological processes. Despite progress in theory, experimental data clarifying water structure and the interaction between hydrophobic solutes at the nanometer scale are scarce due to the very low solubility of hydrophobic species. This article describes an AFM single molecule force spectroscopy method to probe the interaction between molecules with low solubility and reports measurements of the strength and the length scale of the “hydrophobic bond” between hexadecane molecules. Hexadecane molecules are tethered by flexible poly(ethylene glycol) linkers to AFM probes and substrates, removing the aggregation state uncertainty of solution-based approaches as well as spurious surface effects. A shorter hydrophilic polymer layer is added to increase the accessibility of hydrophobic molecules for the force spectroscopy measurements. Statistical analysis of the rupture forces yields a barrier width of 0.24 nm, and a dissociation rate of 1.1 s-1. The results of single molecule measurements are related to the theoretical predictions of the free energy of cavitation in water and to the empirical model of micellization of nonionic surfactants. It is estimated that approximately one-quarter of each molecule's surface is hydrated during forced dissociation, consistent with an extended (nonglobular) conformation of the hexadecane molecules in the dimer. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
46. Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement.
- Author
-
Jean Lee, Viswanath Devanarayan, Yu Barrett, Russell Weiner, John Allinson, Scott Fountain, Stephen Keller, Ira Weinryb, Marie Green, Larry Duan, James Rogers, Robert Millham, Peter O'Brien, Jeff Sailstad, Masood Khan, Chad Ray, and John Wagner
- Subjects
BIOMARKERS ,DRUG development ,PHARMACOLOGY ,PHARMACY - Abstract
Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate. Key differences preclude the direct application of existing validation paradigms for drug analysis to biomarker research. Following the AAPS 2003 Biomarker Workshop (J. W. Lee, R. S. Weiner, J. M. Sailstad, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development. A conference report. Pharm Res22:499–511, 2005), these and other critical issues were addressed. A practical, iterative, “fit-for-purpose” approach to biomarker method development and validation is proposed, keeping in mind the intended use of the data and the attendant regulatory requirements associated with that use. Sample analysis within this context of fit-for-purpose method development and validation are well suited for successful biomarker implementation, allowing increased use of biomarkers in drug development. [ABSTRACT FROM AUTHOR]
- Published
- 2006
47. Lost in Non-Translation: Politics of Misrepresenting Arabs
- Author
-
Sally Gomaa and Chad Raymond
- Subjects
Oriental languages and literatures ,PJ - Abstract
Undergraduate college students in the USA often encounter the Arab Middle East through novels translated into English. These novels are often presented by instructors and understood by students as stylized but accurate depictions of Arab societies as they currently exist. This article argues that the extremely limited number of translated Arabic novels that have made their way into American classrooms perpetuate stereotypes about Arab societies. These novels present students with themes that are often ahistorical and infused with violence, misogyny, and religious fanaticism. Although students may be highly interested in learning about Arab societies, the literary content they come across encourages affective rather than critical or complex responses.
- Published
- 2014
- Full Text
- View/download PDF
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