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Sample records for nanobody surface substantially

  1. Yeast surface display platform for rapid discovery of conformationally selective nanobodies

    DEFF Research Database (Denmark)

    McMahon, Conor; Baier, Alexander S.; Pascolutti, Roberta

    2018-01-01

    this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover...... conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research....

  2. Spontaneous emission of an atom in the presence of nanobodies

    International Nuclear Information System (INIS)

    Klimov, Vasilii V; Ducloy, M; Letokhov, V S

    2001-01-01

    The effect of nanobodies, i.e., the bodies whose size is small compared to the emission wavelength, on spontaneous emission of an atom located near them is considered. The results of calculations performed within the framework of quantum and classical electrodynamics are presented both in analytic and graphical forms and can be readily used for planning experiments and analysis of experimental data. It is shown that nanobodies can be used to control efficiently the rate of spontaneous transitions. Thus, an excited atom located near a nanocylinder or a nanospheroid pole, whose transition dipole moment is directed normally to the nanobody surface, can decay with the rate that is tens and hundreds times higher than the decay rate in a free space. In the case of some (negative) dielectric constants, the decay rate can increase by a factor of 10 5 -10 6 and more. On the other hand, the decay of an excited atom whose transition dipole moment is directed tangentially to the nanobody surface substantially slows down. The probability of nonradiative decay of the excited state is shown to increase substantially in the presence of na-nobodies possessing losses. (review)

  3. REVIEW: Spontaneous emission of an atom in the presence of nanobodies

    Science.gov (United States)

    Klimov, Vasilii V.; Ducloy, M.; Letokhov, V. S.

    2001-07-01

    The effect of nanobodies, i.e., the bodies whose size is small compared to the emission wavelength, on spontaneous emission of an atom located near them is considered. The results of calculations performed within the framework of quantum and classical electrodynamics are presented both in analytic and graphical forms and can be readily used for planning experiments and analysis of experimental data. It is shown that nanobodies can be used to control efficiently the rate of spontaneous transitions. Thus, an excited atom located near a nanocylinder or a nanospheroid pole, whose transition dipole moment is directed normally to the nanobody surface, can decay with the rate that is tens and hundreds times higher than the decay rate in a free space. In the case of some (negative) dielectric constants, the decay rate can increase by a factor of 105—106 and more. On the other hand, the decay of an excited atom whose transition dipole moment is directed tangentially to the nanobody surface substantially slows down. The probability of nonradiative decay of the excited state is shown to increase substantially in the presence of na-nobodies possessing losses.

  4. Sortase A-mediated functionalization of nanobodies toward surface coupling

    OpenAIRE

    TA, Duy Tien; STEEN REDEKER, Erik; GUEDENS, Wanda; ADRIAENSENS, Peter

    2013-01-01

    The conserved LPETG motif, at which sortase A-catalyzed transpeptidation occurs, is engineered at the C-terminal region of the variable domain of single-domain heavy chain antibody (or nanobody) against Vascular Cell Adhesion Molecule 1 (VCAM1). The recombinant nanobody can then subsequently be ligated, under sortase A catalysis, to a variety of oligoglycine containing targets, including material surfaces, contrast labeling molecules and molecules functionalized for specific chemical coupling...

  5. Nanobody-Displaying Flagellar Nanotubes.

    Science.gov (United States)

    Klein, Ágnes; Kovács, Mátyás; Muskotál, Adél; Jankovics, Hajnalka; Tóth, Balázs; Pósfai, Mihály; Vonderviszt, Ferenc

    2018-02-26

    In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion. Linkers of various lengths and conformational properties were chosen from a linker database and they were randomly attached to both ends of an anti-GFP nanobody to facilitate insertion. Functional fusion constructs capable of forming filaments on the surface of flagellin-deficient host cells were selected by magnetic microparticles covered by target GFP molecules and appropriate linkers were identified. TEM studies revealed that short filaments of 2-900 nm were formed on the cell surface. ITC and fluorescent measurements demonstrated that the fusion protein exhibited high binding affinity towards GFP. Our approach allows the development of functionalized flagellar nanotubes against a variety of important target molecules offering potential applications in biosensorics and bio-nanotechnology.

  6. Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension.

    Science.gov (United States)

    Fan, Kelong; Jiang, Bing; Guan, Zhe; He, Jiuyang; Yang, Dongling; Xie, Ni; Nie, Guohui; Xie, Can; Yan, Xiyun

    2018-04-10

    Nanobodies consist of a single domain variable fragment of a camelid heavy-chain antibody. Nanobodies have potential applications in biomedical fields because of their simple production procedures and low cost. Occasionally, nanobody clones of interest exhibit low affinities for their target antigens, which, together with their short half-life limit bioanalytical or therapeutic applications. Here, we developed a novel platform we named fenobody, in which a nanobody developed against H5N1 virus is displayed on the surface of ferritin in the form of a 24mer. We constructed a fenobody by substituting the fifth helix of ferritin with the nanobody. TEM analysis showed that nanobodies were displayed on the surface of ferritin in the form of 6 × 4 bundles, and that these clustered nanobodies are flexible for antigen binding in spatial structure. Comparing fenobodies with conventional nanobodies currently used revealed that the antigen binding apparent affinity of anti-H5N1 fenobody was dramatically increased (∼360-fold). Crucially, their half-life extension in a murine model was 10-fold longer than anti-H5N1 nanobody. In addition, we found that our fenobodies are highly expressed in Escherichia coli, and are both soluble and thermo-stable nanocages that self-assemble as 24-polymers. In conclusion, our results demonstrate that fenobodies have unique advantages over currently available systems for apparent affinity enhancement and half-life extension of nanobodies. Our fenobody system presents a suitable platform for various large-scale biotechnological processes and should greatly facilitate the application of nanobody technology in these areas.

  7. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

    Directory of Open Access Journals (Sweden)

    Peter Bannas

    2017-11-01

    Full Text Available Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL. The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL. VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv. scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa and nanobody-based human heavy chain antibodies (75 kDa can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb. Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody

  8. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

    Science.gov (United States)

    Bannas, Peter; Hambach, Julia; Koch-Nolte, Friedrich

    2017-01-01

    Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo . Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo . Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy

  9. Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine-Nanobody Complex.

    Science.gov (United States)

    Desmyter, Aline; Spinelli, Silvia; Boutton, Carlo; Saunders, Michael; Blachetot, Christophe; de Haard, Hans; Denecker, Geertrui; Van Roy, Maarten; Cambillau, Christian; Rommelaere, Heidi

    2017-01-01

    The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (V HH ) domains (Nanobodies ® ) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo , as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn's disease, rheumatoid arthritis, and psoriasis.

  10. Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine–Nanobody Complex

    Directory of Open Access Journals (Sweden)

    Aline Desmyter

    2017-08-01

    Full Text Available The heterodimeric cytokine interleukin (IL 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH domains (Nanobodies® with low nanomolar affinity for human interleukin (hIL 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn’s disease, rheumatoid arthritis, and psoriasis.

  11. Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine–Nanobody Complex

    Science.gov (United States)

    Desmyter, Aline; Spinelli, Silvia; Boutton, Carlo; Saunders, Michael; Blachetot, Christophe; de Haard, Hans; Denecker, Geertrui; Van Roy, Maarten; Cambillau, Christian; Rommelaere, Heidi

    2017-01-01

    The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH) domains (Nanobodies®) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn’s disease, rheumatoid arthritis, and psoriasis. PMID:28871249

  12. Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application.

    Science.gov (United States)

    Xu, Chongxin; Yang, Ying; Liu, Liwen; Li, Jianhong; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Liu, Xianjin

    2018-04-30

    Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC 50 ) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC 10 ) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple

  13. A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.

    Science.gov (United States)

    Wendel, Sofie; Fischer, Emil C; Martínez, Virginia; Seppälä, Susanna; Nørholm, Morten H H

    2016-05-03

    Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay for evaluating and developing surface display systems is missing. Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency. We have developed an inexpensive and easy read-out assay for surface display using nanobody:GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies on the mechanism of protein transport to the surface of living cells, as well as the optimisation of applications in industrial biotech.

  14. Modulation of protein properties in living cells using nanobodies.

    Science.gov (United States)

    Kirchhofer, Axel; Helma, Jonas; Schmidthals, Katrin; Frauer, Carina; Cui, Sheng; Karcher, Annette; Pellis, Mireille; Muyldermans, Serge; Casas-Delucchi, Corella S; Cardoso, M Cristina; Leonhardt, Heinrich; Hopfner, Karl-Peter; Rothbauer, Ulrich

    2010-01-01

    Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

  15. Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2

    Science.gov (United States)

    Zhu, Min; Hu, Yonghong; Li, Guirong; Ou, Weijun; Mao, Panyong; Xin, Shaojie; Wan, Yakun

    2014-09-01

    Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.

  16. Nanobody mediated inhibition of attachment of F18 Fimbriae expressing Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Kristof Moonens

    Full Text Available Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site. This D″-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.

  17. Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications.

    Science.gov (United States)

    Wang, Yongzhong; Fan, Zhen; Shao, Lei; Kong, Xiaowei; Hou, Xianjuan; Tian, Dongrui; Sun, Ying; Xiao, Yazhong; Yu, Li

    2016-01-01

    Owing to peculiar properties of nanobody, including nanoscale size, robust structure, stable and soluble behaviors in aqueous solution, reversible refolding, high affinity and specificity for only one cognate target, superior cryptic cleft accessibility, and deep tissue penetration, as well as a sustainable source, it has been an ideal research tool for the development of sophisticated nanobiotechnologies. Currently, the nanobody has been evolved into versatile research and application tool kits for diverse biomedical and biotechnology applications. Various nanobody-derived formats, including the nanobody itself, the radionuclide or fluorescent-labeled nanobodies, nanobody homo- or heteromultimers, nanobody-coated nanoparticles, and nanobody-displayed bacteriophages, have been successfully demonstrated as powerful nanobiotechnological tool kits for basic biomedical research, targeting drug delivery and therapy, disease diagnosis, bioimaging, and agricultural and plant protection. These applications indicate a special advantage of these nanobody-derived technologies, already surpassing the "me-too" products of other equivalent binders, such as the full-length antibodies, single-chain variable fragments, antigen-binding fragments, targeting peptides, and DNA-based aptamers. In this review, we summarize the current state of the art in nanobody research, focusing on the nanobody structural features, nanobody production approach, nanobody-derived nanobiotechnology tool kits, and the potentially diverse applications in biomedicine and biotechnology. The future trends, challenges, and limitations of the nanobody-derived nanobiotechnology tool kits are also discussed.

  18. Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay.

    Science.gov (United States)

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Bhunia, Arun K; Tu, Zhui; Chen, Bo; Liu, Yuan-Yuan

    2015-08-05

    In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL(-1) and 19.97 ± 0.84 ng mL(-1), respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of "nanobody (N-28) - anti-DON MAb" complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 - Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Development of nanobody-based flow injection chemiluminescence immunoassay for sensitive detection of human prealbumin.

    Science.gov (United States)

    Ma, Lei; Sun, Yanyan; Kang, Xuejun; Wan, Yakun

    2014-11-15

    Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000 μg L(-1) with a detection limit of 0.01 μg L(-1). The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

    Directory of Open Access Journals (Sweden)

    Wang Y

    2016-07-01

    Full Text Available Yongzhong Wang,1 Zhen Fan,2 Lei Shao,3 Xiaowei Kong,1 Xianjuan Hou,1 Dongrui Tian,1 Ying Sun,1 Yazhong Xiao,1 Li Yu4 1School of Life Sciences, Collaborative Innovation Center of Modern Bio-manufacture, Anhui University, Hefei, People’s Republic of China; 2Department of Biomedical Engineering, The Ohio State University, Columbus, OH, USA; 3State Key Laboratory of New Drugs and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, Shanghai, 4Department of Microbiology and Parasitology, Anhui Provincial Laboratory of Microbiology and Parasitology, Anhui Key Laboratory of Zoonoses, Anhui Medical University, Hefei, People’s Republic of China Abstract: Owing to peculiar properties of nanobody, including nanoscale size, robust structure, stable and soluble behaviors in aqueous solution, reversible refolding, high affinity and specificity for only one cognate target, superior cryptic cleft accessibility, and deep tissue penetration, as well as a sustainable source, it has been an ideal research tool for the development of sophisticated nanobiotechnologies. Currently, the nanobody has been evolved into versatile research and application tool kits for diverse biomedical and biotechnology applications. Various nanobody-derived formats, including the nanobody itself, the radionuclide or fluorescent-labeled nanobodies, nanobody homo- or heteromultimers, nanobody-coated nanoparticles, and nanobody-displayed bacteriophages, have been successfully demonstrated as powerful nanobiotechnological tool kits for basic biomedical research, targeting drug delivery and therapy, disease diagnosis, bioimaging, and agricultural and plant protection. These applications indicate a special advantage of these nanobody-derived technologies, already surpassing the “me-too” products of other equivalent binders, such as the full-length antibodies, single-chain variable fragments, antigen-binding fragments, targeting peptides, and DNA-based aptamers. In

  1. Nanobody: the "magic bullet" for molecular imaging?

    Science.gov (United States)

    Chakravarty, Rubel; Goel, Shreya; Cai, Weibo

    2014-01-01

    Molecular imaging involves the non-invasive investigation of biological processes in vivo at the cellular and molecular level, which can play diverse roles in better understanding and treatment of various diseases. Recently, single domain antigen-binding fragments known as 'nanobodies' were bioengineered and tested for molecular imaging applications. Small molecular size (~15 kDa) and suitable configuration of the complementarity determining regions (CDRs) of nanobodies offer many desirable features suitable for imaging applications, such as rapid targeting and fast blood clearance, high solubility, high stability, easy cloning, modular nature, and the capability of binding to cavities and difficult-to-access antigens. Using nanobody-based probes, several imaging techniques such as radionuclide-based, optical and ultrasound have been employed for visualization of target expression in various disease models. This review summarizes the recent developments in the use of nanobody-based probes for molecular imaging applications. The preclinical data reported to date are quite promising, and it is expected that nanobody-based molecular imaging agents will play an important role in the diagnosis and management of various diseases.

  2. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F. L.; Leonhardt, Heinrich

    2018-01-01

    Abstract Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. PMID:28913971

  3. A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

    Science.gov (United States)

    Xu, Li; Song, Xiaoyu; Jia, Lingyun

    2017-11-01

    Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  4. L-plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages.

    Directory of Open Access Journals (Sweden)

    Sarah De Clercq

    Full Text Available Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.

  5. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications.

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F L; Leonhardt, Heinrich; Hackenberger, Christian P R

    2018-02-23

    Nanobodies can be seen as next-generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site-specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  6. Nanobody: The “Magic Bullet” for Molecular Imaging?

    Science.gov (United States)

    Chakravarty, Rubel; Goel, Shreya; Cai, Weibo

    2014-01-01

    Molecular imaging involves the non-invasive investigation of biological processes in vivo at the cellular and molecular level, which can play diverse roles in better understanding and treatment of various diseases. Recently, single domain antigen-binding fragments known as 'nanobodies' were bioengineered and tested for molecular imaging applications. Small molecular size (~15 kDa) and suitable configuration of the complementarity determining regions (CDRs) of nanobodies offer many desirable features suitable for imaging applications, such as rapid targeting and fast blood clearance, high solubility, high stability, easy cloning, modular nature, and the capability of binding to cavities and difficult-to-access antigens. Using nanobody-based probes, several imaging techniques such as radionuclide-based, optical and ultrasound have been employed for visualization of target expression in various disease models. This review summarizes the recent developments in the use of nanobody-based probes for molecular imaging applications. The preclinical data reported to date are quite promising, and it is expected that nanobody-based molecular imaging agents will play an important role in the diagnosis and management of various diseases. PMID:24578722

  7. Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies

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    Duy Tien Ta

    2016-07-01

    Full Text Available Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1, an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL. Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I-catalyzed azide-alkyne cycloaddition (CuAAC “click” chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR, respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.

  8. 99mTc labeled anti EGFR Nanobody pentamer for tumor radioimmunoimaging

    International Nuclear Information System (INIS)

    Ding Zhiling; Lan Xiaoli; Li Chongjiao; Pei Zhijun; Zhang Yongxue; Wang Lifei; Gao Bin

    2014-01-01

    Novel Nanobody has small molecular weight and lower affinity. Appropriate polymer would be more suitable for radioimmunoimaging. In this study, we labeled anti EGFR Nanobody pentamer with 99m Tc to prepare tumor targeting imaging agent and to investigate its binding characteristics of tumor cells and tissues in vitro and in vivo, and to explore the feasibility of 99m Tc-EGFR Nanobody pentamer for tumor radioimmunoimaging compared with anti EGFR Nanobody monomer. EGFR Nanobody labeled with 99m Tc through tricarbonyl intermediate. The labeled compounds were purified by an ultra centrifugal filter; The labeling efficiency was determined by thin layer chromatography (TLC), and the radiochemical purity more than 95%. In vitro, 99m Tc-EGFR Nanobody monomer and pentamer have the specific binding capability with EGFR overexpression A431 tumor cell. the binding rate of 99m Tc-EGFR Nanobody monomer higher than that of pentamer (11.32% ± 2.73% vs 5.80% ± 0.92%, P < O.05). In A431 xenografted tumor was clearly displayed after intravenous injection of 99m Tc-EGFR Nanobody pentamer at l.5 h, T/NT maximum was 2.9 (1.5 h), whereas, the tumor tissues was not obviously found using 99m Tc-EGFR Nanobody monomer. The negative EGFR expression OCM-I xenografted tumor was not showed in both monomer and pentamer tracer. The experiment indicated that 99m Tc-EGFR Nanobody pentamer are appropriate for tumor radioimmunoimaging and has the potential value for the further study. (authors)

  9. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

    Science.gov (United States)

    Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

    2017-01-01

    Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

  10. The development of nanobody probes for molecular imaging

    International Nuclear Information System (INIS)

    Ding Zhiling; Lan Xiaoli; Zhang Yongxue

    2014-01-01

    The nanobody is a novel antibody fragment, which has beneficial biophysical and pharmacokinetic properties, such as the small molecular weight, high affinity and specificity for antigen. Nanobody is ideally suitable for molecular imaging as a targeting probe that could label antigen at nmol level in vitro. In animal models of xenografted tumor, atherosclerotic plaques and brain disorders, the target tissues were specifically and clearly detected and the high tumor-to-blood (T/B) ratios were obtained. Structural or chemical modified nanobodies will have higher affinity and retention to target tissues, and be convenient for the application of molecular imaging. With the development of the related research, nanobody-based molecular imaging will be gradually transformed into the clinical applications, and play an important role in early diagnosis and therapeutic assessment. (authors)

  11. A novel nanobody against urease activity of Helicobacter pylori.

    Science.gov (United States)

    Ardekani, Leila Safaee; Gargari, Seyed Latif Mousavi; Rasooli, Iraj; Bazl, Masoumeh Rajabi; Mohammadi, Mohammad; Ebrahimizadeh, Walead; Bakherad, Hamid; Zare, Hamed

    2013-09-01

    Helicobacter pylori infection is associated with gastritis and in some cases with gastric and duodenal ulcers, and even adenocarcinoma. Antibiotic therapy has significant limitations, such as the high cost and the emergence of antibiotic-resistant strains, generating the need for new treatments. The administration of antibody against H. pylori is a new effective therapeutic strategy. In this study, we successfully developed a single-variable domain of heavy chain antibody against recombinant UreC. A VHH phagemid library was constructed from immune camel heavy chain antibodies. The nanobodies were displayed on M13 phage. Library selection was performed against UreC recombinant protein. A specific single-variable domain of heavy chain antibody against UreC was screened in five rounds of panning. The nanobody with the highest score in the phage ELISA was selected for soluble expression. The nanobody was purified with a nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Affinity, specificity, and urease inhibitory properties of the nanobody were assayed. Here we showed the isolation and purification of a specific nanobody with high affinity against UreC recombinant protein that can inhibit urease activity. The isolated UreC nanobody can specifically detect and bind to UreC and inhibit urease activity. This nanobody could be a novel class of treatment measure against H. pylori infection. Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  12. A bi-paratopic anti-EGFR nanobody efficiently inhibits solid tumour growth

    Science.gov (United States)

    Roovers, Rob C.; Vosjan, Maria J.W.D.; Laeremans, Toon; el Khoulati, Rachid; de Bruin, Renée C.G.; Ferguson, Kathryn M.; Verkleij, Arie J.; van Dongen, Guus A.M.S.; van Bergen en Henegouwen, Paul M. P.

    2014-01-01

    The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies (mAbs) have been successfully used, among which cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In the present study, we aimed to improve on these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single bi-paratopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or of cetuximab to EGFR and that did not compete for each others binding. A combination of nanobodies from both epitope groups into the bi-paratopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this bi-paratopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, mono-specific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of bi-paratopic nanobody-based anti-cancer therapeutics may yield potent lead molecules for further development. PMID:21520037

  13. Development of a novel nano-sized anti-VEGFA nanobody with enhanced physicochemical and pharmacokinetic properties.

    Science.gov (United States)

    Khodabakhsh, Farnaz; Norouzian, Dariush; Vaziri, Behrouz; Ahangari Cohan, Reza; Sardari, Soroush; Mahboudi, Fereidoun; Behdani, Mahdi; Mansouri, Kamran; Mehdizadeh, Ardavan

    2017-08-25

    Since physiological and pathological processes occur at nano-environments, nanotechnology has considered as an efficient tool for designing of next generation specific biomolecules with enhanced pharmacodynamic and pharmacodynamic properties. In the current investigation, by control of the size and hydrodynamic volume at the nanoscale, for the first time, physicochemical and pharmacokinetic properties of an anti-VEGFA nanobody was remarkably improved by attachment of a Proline-Alanine-Serine (PAS) rich sequence. The results elucidated unexpected impressive effects of PAS sequence on physicochemical properties especially on size, hydrodynamics radius, and even solubility of nanobody. CD analysis revealed an increment in random coil structure of the PASylated protein in comparison to native one without any change in charge state or binding kinetic parameters of nanobody assessed by isoelectric focusing and surface plasmon resonance measurements, respectively. In vitro biological activities of nanobody were not affected by coupling of the PAS sequence. In contrast, the terminal half-life was significantly increased by a factor of 14 for the nanobody-PAS after single dose IV injection to the mice. Our study demonstrated that the control of size in the design of small therapeutic proteins has a promising effect on the stability and solubility, in addition to their physiochemical and pharmacokinetic properties. The designed new anti-VEGFA nanobody could promise a better therapeutic agent with a long administration intervals and lower dose, which in turn leads to a better patient compliance. Size adjustment of an anti-VEGF nanobody at the nanoscale by the attachment of a natural PAS polymer remarkably improves physicochemical properties, as well as a pharmacokinetic profile without any change in biological activity of the miniaturized antibody.

  14. Nanobodies as novel agents for disease diagnosis and therapy

    Directory of Open Access Journals (Sweden)

    Siontorou CG

    2013-11-01

    Full Text Available Christina G Siontorou Department of Industrial Management and Technology, University of Piraeus, Piraeus, Greece Abstract: The discovery of naturally occurring, heavy-chain only antibodies in Camelidae, and their further development into small recombinant nanobodies, presents attractive alternatives in drug delivery and imaging. Easily expressed in microorganisms and amenable to engineering, nanobody derivatives are soluble, stable, versatile, and have unique refolding capacities, reduced aggregation tendencies, and high-target binding capabilities. This review outlines the current state of the art in nanobodies, focusing on their structural features and properties, production, technology, and the potential for modulating immune functions and for targeting tumors, toxins, and microbes. Keywords: heavy chain antibodies, nanobodies, antibody expression, molecular display, formatting

  15. Generation and characterization of an anti-delta like ligand-4 Nanobody to induce non-productive angiogenesis.

    Science.gov (United States)

    Baharlou, Rasoul; Tajik, Nader; Habibi-Anbouhi, Mahdi; Shokrgozar, Mohammad Ali; Zarnani, Amir-Hassan; Shahhosseini, Fatemeh; Behdani, Mahdi

    2018-03-01

    Antibody-based targeting of angiogenesis is a key approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. It forecasts the prognosis of human malignancies and blocking its signaling can help to inhibit neovascularization and tumor metastasis. Nanobodies are the smallest antigen-binding domains of heavy chain antibodies in camelidae. The aim of this study was to develop a Nanobody against DLL4 and apply binding and functional approaches to target it. In this work, a Nanobody library against human recombinant DLL4 was developed. After panning, the periplasmic-extract (PE) of individual colonies were screened through ELISA. The interactions between Nanobody and DLL4 were assessed using immunohistochemistry and FACS. The functional assessment was carried out via tube formation assay. We selected a Nanobody (3Nb3) with a high binding signal to DLL4, associated with a binding affinity of 3.6 nM. It was demonstrated that 3Nb3 binds to native DLL4 on the surface of MKN cells and gastric carcinoma tissue, and also inhibits the maturation of capillary-like structures in HUVECs. The results were indicative of the potential of Nanobody for DLL4 identification and can broaden the scope for development of cancer diagnosis and treatment techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

    Directory of Open Access Journals (Sweden)

    Keiji Itoh

    Full Text Available Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies, which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

  17. Nanobody mediated crystallization of an archeal mechanosensitive channel.

    Directory of Open Access Journals (Sweden)

    Christian Löw

    Full Text Available Mechanosensitive channels (MS are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

  18. Exploiting sequence and stability information for directing nanobody stability engineering.

    Science.gov (United States)

    Kunz, Patrick; Flock, Tilman; Soler, Nicolas; Zaiss, Moritz; Vincke, Cécile; Sterckx, Yann; Kastelic, Damjana; Muyldermans, Serge; Hoheisel, Jörg D

    2017-09-01

    Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein. We analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally. Some mutations improved the stability of nanobodies by up to 6.1°C, with an average of 2.3°C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some instances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated. The results reveal a mutational strategy to improve the biophysical behavior of nanobody binders and indicate a species-specificity of nanobody architecture. This study illustrates the potential and limitations of engineering nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Cathode deposits in fullerene formation — microstructural evidence for independent pathways of pyrolytic carbon and nanobody formation

    Science.gov (United States)

    Taylor, G. H.; Gerald, J. D. Fitz; Pang, L.; Wilson, M. A.

    1994-01-01

    Microstructures in cathode deposits formed during fullerene production by electrical arcing in helium have been examined in detail. This has provided new information about the mechanisms by which nanobodies (nanotubes and nanoparticles) and pyrolytic carbon are deposited. Nanobodies and pyrolytic carbon form independently; the former probably grow in the plasma then deposit on the electrode but much of the latter deposits directly on the electrode surface.

  20. Anti-c-MET Nanobody - a new potential drug in multiple myeloma treatment.

    Science.gov (United States)

    Slørdahl, Tobias Schmidt; Denayer, Tinneke; Moen, Siv Helen; Standal, Therese; Børset, Magne; Ververken, Cedric; Rø, Torstein Baade

    2013-11-01

    c-MET is the tyrosine kinase receptor of the hepatocyte growth factor (HGF). HGF-c-MET signaling is involved in many human malignancies, including multiple myeloma (MM). Recently, multiple agents have been developed directed to interfere at different levels in HGF-c-MET signaling pathway. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy-chain-only antibodies. In this study, we wanted to determine the anticancer effect of a novel anti-c-MET Nanobody in MM. We examined the effects of an anti-c-MET Nanobody on thymidine incorporation, migration, adhesion of MM cells, and osteoblastogenesis in vitro. Furthermore, we investigated the effects of the Nanobody on HGF-dependent c-MET signaling by Western blotting. We show that the anti-c-MET Nanobody effectively inhibited thymidine incorporation of ANBL-6 MM cells via inhibition of an HGF autocrine growth loop and thymidine incorporation in INA-6 MM cells induced by exogenous HGF. HGF-induced migration and adhesion of INA-6 were completely and specifically blocked by the Nanobody. Furthermore, the Nanobody abolished the inhibiting effect of HGF on bone morphogenetic protein-2-induced alkaline phosphatase activity and the mineralization of human mesenchymal stem cells. Finally, we show that the Nanobody reduced phosphorylation of tyrosine residues in c-MET, MAPK, and Akt. We also compared the Nanobody with anti-c-MET monoclonal antibodies and revealed the similar or better effect. The anti-c-MET Nanobody inhibited MM cell migration, thymidine incorporation, and adhesion, and blocked the HGF-mediated inhibition of osteoblastogenesis. The anti-c-MET Nanobody might represent a novel therapeutic agent in the treatment of MM and other cancers driven by HGF-c-MET signaling. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

    Directory of Open Access Journals (Sweden)

    Sander A. A. Kooijmans

    2016-03-01

    Full Text Available Background: Extracellular vesicles (EVs are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. Methods: EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI anchor signal peptides derived from decay-accelerating factor (DAF. EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. Results: V analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression, but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. Conclusions: We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.

  2. Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

    Science.gov (United States)

    Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

    2016-01-15

    Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Characterization and enzyme-conjugation of a specific anti-L1 nanobody.

    Science.gov (United States)

    Minaeian, Sara; Rahbarizadeh, Fatemeh; Zarkesh Esfahani, Sayyed Hamid; Ahmadvand, Davoud

    2012-01-01

    Persistent infection of the human papillomaviruses (HPV) has been shown to result in cervical cancer and intraepithelial neoplasia. Early detection and screening programs are essential strategies against cervical cancer. A nanobody is the smallest antigen-binding fragment known and is derived from a camelid heavy-chain antibody. This tiny protein shows high solubility and stability. It can be produced cost-effectively with high yield production. In this study, we enriched a nanobody library against the L1 protein of HPV. Several colons were selected from this enriched library using monoclonal phage-enzyme linked immunosorbent assay (phage-ELISA) and analyzed for identification of nanobody genes. The expression of nanobody fragments was performed in Rosetta gami2. The C74 nanobody that showed strong binding to the L1 protein of HPV16 was selected, purified, and characterized by Western blotting and ELISA. The selected nanobody was tested for sensitivity, specificity, and affinity. A nanobody conjugated to horseradish peroxidase (HRP) was selected and used for detection of L1 protein of HPV16. This study demonstrates that the C74-HRP, due to its specificity and good binding affinity for a specific viral antigen, is a potential diagnostic tool that can be used as a promising reagent for the new generation of HPV diagnosis approaches.

  4. Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo.

    Science.gov (United States)

    Maussang, David; Mujić-Delić, Azra; Descamps, Francis J; Stortelers, Catelijne; Vanlandschoot, Peter; Stigter-van Walsum, Marijke; Vischer, Henry F; van Roy, Maarten; Vosjan, Maria; Gonzalez-Pajuelo, Maria; van Dongen, Guus A M S; Merchiers, Pascal; van Rompaey, Philippe; Smit, Martine J

    2013-10-11

    The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

  5. Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

    Science.gov (United States)

    Bakherad, Hamid; Gargari, Seyed Latif Mousavi; Sepehrizadeh, Zargham; Aghamollaei, Hossein; Taheri, Ramezan Ali; Torshabi, Maryam; Yazdi, Mojtaba Tabatabaei; Ebrahimizadeh, Walead; Setayesh, Neda

    2017-09-01

    It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models. Copyright © 2017. Published by Elsevier Masson SAS.

  6. 99m Tc-anti-epidermal growth factor receptor nanobody for tumor imaging.

    Science.gov (United States)

    Piramoon, Majid; Hosseinimehr, Seyed Jalal; Omidfar, Kobra; Noaparast, Zohreh; Abedi, Seyed Mohammad

    2017-04-01

    Nanobodies are important biomolecules for tumor targeting. In this study, we synthesized and labeled anti-epidermal growth factor receptor (EGFR) nanobody OA-cb6 with 99m Tc(CO) 3 + and evaluated its characteristics for targeting the EGFR in the A431 human epidermal carcinoma cell line. Nanobody radiolabeling was achieved with high yield and radiochemical purity, and the radioconjugate was stable. Biodistribution results in nude mice exhibited a favorable tumor-to-muscle ratio at 4-hr postinjection, and tumor location was visualized at 4 hr after injection of radiolabeled nanobody. Our result showed that the OA-cb6- 99m Tc-tricarbonyl radiolabeled nanobody is a promising radiolabeled biomolecule for tumor imaging in cancers with high EGFR overexpression. © 2016 John Wiley & Sons A/S.

  7. Targeted radionuclide therapy with A 177Lu-labeled anti-HER2 nanobody.

    Science.gov (United States)

    D'Huyvetter, Matthias; Vincke, Cécile; Xavier, Catarina; Aerts, An; Impens, Nathalie; Baatout, Sarah; De Raeve, Hendrik; Muyldermans, Serge; Caveliers, Vicky; Devoogdt, Nick; Lahoutte, Tony

    2014-01-01

    RIT has become an attractive strategy in cancer treatment, but still faces important drawbacks due to poor tumor penetration and undesirable pharmacokinetics of the targeting vehicles. Smaller radiolabeled antibody fragments and peptides feature highly specific target accumulation, resulting in low accumulation in healthy tissue, except for the kidneys. Nanobodies are the smallest (MWnanobodies is predominantly dictated by the number of polar residues in the C-terminal amino acid tag. Three nanobodies were produced with different C-terminal amino-acid tag sequences (Myc-His-tagged, His-tagged, and untagged). Dynamic planar imaging of Wistar rats with 111In-DTPA-nanobodies revealed that untagged nanobodies showed a 70% drop in kidney accumulation compared to Myc-His-tagged nanobodies at 50 min p.i.. In addition, coinfusion of untagged nanobodies with the plasma expander Gelofusin led to a final reduction of 90%. Similar findings were obtained with different 177Lu-DTPA-2Rs15d nanobody constructs in HER2pos tumor xenografted mice at 1 h p.i.. Kidney accumulation decreased 88% when comparing Myc-His-tagged to untagged 2Rs15d nanobody, and 95% with a coinfusion of Gelofusin, without affecting the tumor targeting capacity. Consequently, we identified a generic method to reduce kidney retention of radiolabeled nanobodies. Dosimetry calculations of Gelofusin-coinfused, untagged 177Lu-DTPA-2Rs15d revealed a dose of 0.90 Gy/MBq that was delivered to both tumor and kidneys and extremely low doses to healthy tissues. In a comparative study, 177Lu-DTPA-Trastuzumab supplied 6 times more radiation to the tumor than untagged 177Lu-DTPA-2Rs15d, but concomitantly also a 155, 34, 80, 26 and 4180 fold higher radioactivity burden to lung, liver, spleen, bone and blood. Most importantly, nanobody-based targeted radionuclide therapy in mice bearing small estiblashed HER2pos tumors led to an almost complete blockade of tumor growth and a significant difference in event-free survival

  8. Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents.

    Science.gov (United States)

    Yamagata, Masahito; Sanes, Joshua R

    2018-02-27

    Sensitive and specific antibodies are essential for detecting molecules in cells and tissues. However, currently used polyclonal and monoclonal antibodies are often less specific than desired, difficult to produce, and available in limited quantities. A promising recent approach to circumvent these limitations is to employ chemically defined antigen-combining domains called "nanobodies," derived from single-chain camelid antibodies. Here, we used nanobodies to prepare sensitive unimolecular detection reagents by genetically fusing cDNAs encoding nanobodies to enzymatic or antigenic reporters. We call these fusions between a reporter and a nanobody "RANbodies." They can be used to localize epitopes and to amplify signals from fluorescent proteins. They can be generated and purified simply and in unlimited amounts and can be preserved safely and inexpensively in the form of DNA or digital sequence.

  9. In vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody.

    Science.gov (United States)

    Hendrickx, Maarten L V; Zatloukalova, Monika; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge; Gils, Ann; Declerck, Paul J

    2014-05-05

    One of the main disadvantages of current t-PA thrombolytic treatment is the increased bleeding risk. Upon activation, thrombin activatable fibrinolysis inhibitor (TAFI) is a very powerful antifibrinolytic enzyme. Therefore, co-administration of a TAFI inhibitor during thrombolysis could reduce the required t-PA dose without compromising the thrombolytic efficacy. In this study we generated and characterised a nanobody that is inhibitory towards rat TAFI and evaluated its profibrinolytic property in vitro and in vivo. Nanobody VHH-rTAFI-i81 inhibits (at a 16-fold molar ratio nanobody over TAFI) the thrombin/thrombomodulin (T/TM)-mediated activation of rat TAFI (rTAFI) by 83 ± 1.8% with an IC50 of 0.46 (molar ratio nanobody over TAFI). The affinity (KA) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 x 10(10) M(-1) and illustrates a very strong binding. In an in vitro clot lysis assay, administration of VHH-rTAFI-i81 strongly enhances the degree of lysis and reduces time to reach full lysis of t-PA-mediated clot lysis. Epitope mapping discloses that Lys392 is of primary importance for the nanobody/rTAFI interaction besides minor contributions of Tyr175 and Glu183. In vivo application of VHH-rTAFI-i81 in a tissue factor-induced mouse thromboembolism model significantly decreases fibrin deposition in the lungs in the absence of exogenous administered t-PA. Nanobody VHH-rTAFI-i81 is a very potent inhibitor of T/TM-mediated TAFI activation. Co-administration of this nanobody and t-PA enhances the fibrinolytic efficacy. In an in vivo mouse thromboembolism model, VHH-rTAFI-i81 reduces fibrin deposition in the lungs.

  10. A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

    DEFF Research Database (Denmark)

    Wendel, Sofie; Christian Fischer, Emil; Martinez, Virginia

    2016-01-01

    Background: Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay...... to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency.Conclusions: We have developed an inexpensive and easy read-out assay for surface display using...... nanobody: GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies...

  11. Construction and expression of an anti-VEGFR2 Nanobody-Fc fusionbody in NS0 host cell.

    Science.gov (United States)

    Qasemi, Maryam; Behdani, Mahdi; Shokrgozar, Mohammad Ali; Molla-Kazemiha, Vahid; Mohseni-Kuchesfahani, Homa; Habibi-Anbouhi, Mahdi

    2016-07-01

    Angiogenesis is the formation of new blood vessels which is involved in migration, growth and differentiation of endothelial cells. This process regularly occurs during growth and development in children however, in adults is usually part of a disease process such as cancer. The vascular endothelial growth factor (VEGF) is a vital player in the vascular development and angiogenesis in physiological and pathological processes. Camelid's immune system has unique antibodies which are composed of only a heavy chain homodimer and the variable domain (VHH, Nanobody). Nanobodies are small, around 15 kDa and stable. In this study, we engineered and constructed a new Nanobody-Fc fusion protein (fusionbody) composed of an anti-VEGFR2 Nanobody and an Fc fragment of human IgG1 antibody. The recombinant vector was transfected into NS0 host cells. Stable producer clones were developed and the recombinant fusionbody was expressed and purified. Functional assay showed the anti-VEGFR2 fusionbody could bind to VEGFR2 on cell surface via VHH part and could mediate killing the targeted cells through direct cell death and complement-dependent cytotoxicity (CDC). Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization.

    Directory of Open Access Journals (Sweden)

    Anna D Koromyslova

    2017-11-01

    Full Text Available Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42 were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14, allosteric interference (Nano-32, and violation of normal capsid morphology (Nano-26 and Nano-85. Finally, we showed that two Nanobodies (Nano-26 and Nano-85 not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great

  13. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization

    Science.gov (United States)

    2017-01-01

    Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to

  14. Rational Design of Nanobody80 Loop Peptidomimetics

    DEFF Research Database (Denmark)

    Martin, Charlotte; Moors, Samuel L C; Danielsen, Mia

    2017-01-01

    G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized β2 -adrenergic receptor (β2 AR) have been......-hairpin conformation. Syntheses, conformational analysis, binding and functional in vitro assays, as well as internalization experiments, were performed. We demonstrate that peptidomimetics can structurally mimic the CDR3 loop of a nanobody and its function by inhibiting G protein coupling as measured by partial...

  15. Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function.

    Science.gov (United States)

    Kazemi-Lomedasht, Fatemeh; Muyldermans, Serge; Habibi-Anbouhi, Mahdi; Behdani, Mahdi

    2018-03-01

    Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem. Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity. The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis.

  16. Enzymatic functionalization of a nanobody using protein insertion technology.

    Science.gov (United States)

    Crasson, O; Rhazi, N; Jacquin, O; Freichels, A; Jérôme, C; Ruth, N; Galleni, M; Filée, P; Vandevenne, M

    2015-10-01

    Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function

    Directory of Open Access Journals (Sweden)

    Fatemeh Kazemi-Lomedasht

    2018-03-01

    Full Text Available Objective(s: Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.   Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody, evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Results: Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity.  Conclusion: The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis.

  18. A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis.

    Science.gov (United States)

    Van Impe, Katrien; Bethuyne, Jonas; Cool, Steven; Impens, Francis; Ruano-Gallego, David; De Wever, Olivier; Vanloo, Berlinda; Van Troys, Marleen; Lambein, Kathleen; Boucherie, Ciska; Martens, Evelien; Zwaenepoel, Olivier; Hassanzadeh-Ghassabeh, Gholamreza; Vandekerckhove, Joël; Gevaert, Kris; Fernández, Luis Ángel; Sanders, Niek N; Gettemans, Jan

    2013-12-13

    Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit. We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading. With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS). CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers

  19. Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

    Science.gov (United States)

    Braun, Michael B; Traenkle, Bjoern; Koch, Philipp A; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-21

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

  20. (18)F-nanobody for PET imaging of HER2 overexpressing tumors.

    Science.gov (United States)

    Xavier, Catarina; Blykers, Anneleen; Vaneycken, Ilse; D'Huyvetter, Matthias; Heemskerk, Jan; Lahoutte, Tony; Devoogdt, Nick; Caveliers, Vicky

    2016-04-01

    Radiolabeled nanobodies are exciting new probes for molecular imaging due to high affinity, high specificity and fast washout from the blood. Here we present the labeling of an anti-HER2 nanobody with (18)F and its validation for in vivo assessment of HER2 overexpression. The GMP grade anti-HER2 nanobody was labeled with the prosthetic group, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]-SFB), and its biodistribution, tumor targeting and specificity were evaluated in mouse and rat tumor models. [(18)F]FB-anti-HER2 nanobody was prepared with a 5-15% global yield (decay corrected) and a specific activity of 24.7 ± 8.2 MBq/nmol. In vivo studies demonstrated a high specific uptake for HER2 positive xenografts (5.94 ± 1.17 and 3.74 ± 0.52%IA/g, 1 and 3h p.i.) with high tumor-to-blood and tumor-to-muscle ratios generating high contrast PET imaging. The probe presented fast clearance through the kidneys (4%IA/g at 3h p.i.). [(18)F]FB-anti-HER2 nanobody is able to image HER2 expressing tumors when co-administered with the anti-HER2 therapeutic antibody trastuzumab (Herceptin), indicating the possibility of using the tracer in patients undergoing Herceptin therapy. The GMP grade anti-HER2 nanobody was labeled with (18)F. This new PET probe for imaging HER2 overexpression in tumors has ample potential for clinical translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. 18F-nanobody for PET imaging of HER2 overexpressing tumors

    International Nuclear Information System (INIS)

    Xavier, Catarina; Blykers, Anneleen; Vaneycken, Ilse; D'Huyvetter, Matthias; Heemskerk, Jan; Lahoutte, Tony; Devoogdt, Nick; Caveliers, Vicky

    2016-01-01

    Introduction: Radiolabeled nanobodies are exciting new probes for molecular imaging due to high affinity, high specificity and fast washout from the blood. Here we present the labeling of an anti-HER2 nanobody with 18 F and its validation for in vivo assessment of HER2 overexpression. Methods: The GMP grade anti-HER2 nanobody was labeled with the prosthetic group, N-succinimidyl-4-[ 18 F]fluorobenzoate ([ 18 F]-SFB), and its biodistribution, tumor targeting and specificity were evaluated in mouse and rat tumor models. Results: [ 18 F]FB-anti-HER2 nanobody was prepared with a 5–15% global yield (decay corrected) and a specific activity of 24.7 ± 8.2 MBq/nmol. In vivo studies demonstrated a high specific uptake for HER2 positive xenografts (5.94 ± 1.17 and 3.74 ± 0.52%IA/g, 1 and 3 h p.i.) with high tumor-to-blood and tumor-to-muscle ratios generating high contrast PET imaging. The probe presented fast clearance through the kidneys (4%IA/g at 3 h p.i.). [ 18 F]FB-anti-HER2 nanobody is able to image HER2 expressing tumors when co-administered with the anti-HER2 therapeutic antibody trastuzumab (Herceptin), indicating the possibility of using the tracer in patients undergoing Herceptin therapy. Conclusions: The GMP grade anti-HER2 nanobody was labeled with 18 F. This new PET probe for imaging HER2 overexpression in tumors has ample potential for clinical translation.

  2. Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV E2 protein.

    Directory of Open Access Journals (Sweden)

    Tiansen Li

    Full Text Available Bovine viral diarrhea-mucosal disease (BVD-MD is caused by bovine viral diarrhea virus (BVDV, and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3, and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

  3. Pre-Clinical Intravenous Serum Pharmacokinetics of Albumin Binding and Non-Half-Life Extended Nanobodies®

    Directory of Open Access Journals (Sweden)

    Sven Hoefman

    2015-07-01

    Full Text Available Nanobodies are antigen-binding, single variable domain proteins derived from naturally-occurring, heavy chain only antibodies. They are highly soluble, stable, and can be linked to build multi-specific formats. Several Nanobodies are currently in clinical development in different therapeutic areas, for both chronic and acute applications. For the former, prolonged exposure is achieved by half-life extending moieties that target endogenous albumin, while for the latter, non-half-life extended constructs are preferable. To demonstrate the general pharmacokinetic behavior of both formats, serum levels of seven intravenously administered Nanobodies were analyzed in cynomolgus monkeys, mice or rabbits. In monkeys, the total clearance of a monomeric irrelevant Nanobody was rapid (2.0 mL/(min*kg and approximated the species glomerular filtration rate, indirectly suggesting that the Nanobody was mainly eliminated via the kidneys. When linked to an anti-albumin Nanobody, a 376-fold decrease in clearance was observed, resulting in a terminal half-life of 4.9 days, corresponding to the expected species albumin half-life. Similar conclusions were drawn for (non- half-life extended mono-, bi- and trimeric Nanobodies in mice or rabbits, suggesting that these kinetic principles apply across species. Applying this knowledge to species translation and study design is crucial for successful pre-clinical development of novel therapeutic Nanobody candidates.

  4. Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein.

    Science.gov (United States)

    Li, Tiansen; Huang, Meiling; Xiao, Hongran; Zhang, Guoqi; Ding, Jinhua; Wu, Peng; Zhang, Hui; Sheng, Jinliang; Chen, Chuangfu

    2017-01-01

    Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

  5. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  6. Nanobodies as Versatile Tools to Understand, Diagnose, Visualize and Treat Cancer

    Directory of Open Access Journals (Sweden)

    Isabel Van Audenhove

    2016-06-01

    Full Text Available Since their discovery, nanobodies have been used extensively in the fields of research, diagnostics and therapy. These antigen binding fragments, originating from Camelid heavy-chain antibodies, possess unusual hallmarks in terms of (small size, stability, solubility and specificity, hence allowing cost-effective production and sometimes outperforming monoclonal antibodies. In this review, we evaluate the current status of nanobodies to study, diagnose, visualize or inhibit cancer-specific proteins and processes. Nanobodies are highly adaptable tools for cancer research as they enable specific modulation of targets, enzymatic and non-enzymatic proteins alike. Molecular imaging studies benefit from the rapid, homogeneous tumor accumulation of nanobodies and their fast blood clearance, permitting previously unattainable fast tumor visualization. Moreover, they are endowed with considerable therapeutic potential as inhibitors of receptor-ligand pairs and deliverers of drugs or drug-loaded nanoparticles towards tumors. More in vivo and clinical studies are however eagerly awaited to unleash their full potential.

  7. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

    Science.gov (United States)

    Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

  8. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    Science.gov (United States)

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  9. A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment

    International Nuclear Information System (INIS)

    Farajpour, Zahra; Rahbarizadeh, Fatemeh; Kazemi, Bahram; Ahmadvand, Davoud

    2014-01-01

    Highlights: • A novel nanobody directed to antigenic regions on VEGF was identified. • Our nanobody was successfully purified. • Our nanobody significantly inhibited VEGF-induced proliferation of HUVECs in a dose dependent manner. - Abstract: Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae. Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate

  10. A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment

    Energy Technology Data Exchange (ETDEWEB)

    Farajpour, Zahra [Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Kazemi, Bahram [Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2014-03-28

    Highlights: • A novel nanobody directed to antigenic regions on VEGF was identified. • Our nanobody was successfully purified. • Our nanobody significantly inhibited VEGF-induced proliferation of HUVECs in a dose dependent manner. - Abstract: Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae. Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate.

  11. Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis.

    Science.gov (United States)

    Skottrup, Peter Durand; Leonard, Paul; Kaczmarek, Jakub Zbigniew; Veillard, Florian; Enghild, Jan Johannes; O'Kennedy, Richard; Sroka, Aneta; Clausen, Rasmus Prætorius; Potempa, Jan; Riise, Erik

    2011-08-15

    Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors.

    Science.gov (United States)

    Pardon, Els; Betti, Cecilia; Laeremans, Toon; Chevillard, Florent; Guillemyn, Karel; Kolb, Peter; Ballet, Steven; Steyaert, Jan

    2018-05-04

    The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β 2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein–Protein Interaction Analysis, and Protein Function Exploration

    Directory of Open Access Journals (Sweden)

    Els Beghein

    2017-07-01

    Full Text Available Over the last two decades, nanobodies or single-domain antibodies have found their way in research, diagnostics, and therapy. These antigen-binding fragments, derived from Camelid heavy chain only antibodies, possess remarkable characteristics that favor their use over conventional antibodies or fragments thereof, in selected areas of research. In this review, we assess the current status of nanobodies as research tools in diverse aspects of fundamental research. We discuss the use of nanobodies as detection reagents in fluorescence microscopy and focus on recent advances in super-resolution microscopy. Second, application of nanobody technology in investigating protein–protein interactions is reviewed, with emphasis on possible uses in mass spectrometry. Finally, we discuss the potential value of nanobodies in studying protein function, and we focus on their recently reported application in targeted protein degradation. Throughout the review, we highlight state-of-the-art engineering strategies that could expand nanobody versatility and we suggest future applications of the technology in the selected areas of fundamental research.

  14. Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein-Protein Interaction Analysis, and Protein Function Exploration.

    Science.gov (United States)

    Beghein, Els; Gettemans, Jan

    2017-01-01

    Over the last two decades, nanobodies or single-domain antibodies have found their way in research, diagnostics, and therapy. These antigen-binding fragments, derived from Camelid heavy chain only antibodies, possess remarkable characteristics that favor their use over conventional antibodies or fragments thereof, in selected areas of research. In this review, we assess the current status of nanobodies as research tools in diverse aspects of fundamental research. We discuss the use of nanobodies as detection reagents in fluorescence microscopy and focus on recent advances in super-resolution microscopy. Second, application of nanobody technology in investigating protein-protein interactions is reviewed, with emphasis on possible uses in mass spectrometry. Finally, we discuss the potential value of nanobodies in studying protein function, and we focus on their recently reported application in targeted protein degradation. Throughout the review, we highlight state-of-the-art engineering strategies that could expand nanobody versatility and we suggest future applications of the technology in the selected areas of fundamental research.

  15. Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein–Protein Interaction Analysis, and Protein Function Exploration

    Science.gov (United States)

    Beghein, Els; Gettemans, Jan

    2017-01-01

    Over the last two decades, nanobodies or single-domain antibodies have found their way in research, diagnostics, and therapy. These antigen-binding fragments, derived from Camelid heavy chain only antibodies, possess remarkable characteristics that favor their use over conventional antibodies or fragments thereof, in selected areas of research. In this review, we assess the current status of nanobodies as research tools in diverse aspects of fundamental research. We discuss the use of nanobodies as detection reagents in fluorescence microscopy and focus on recent advances in super-resolution microscopy. Second, application of nanobody technology in investigating protein–protein interactions is reviewed, with emphasis on possible uses in mass spectrometry. Finally, we discuss the potential value of nanobodies in studying protein function, and we focus on their recently reported application in targeted protein degradation. Throughout the review, we highlight state-of-the-art engineering strategies that could expand nanobody versatility and we suggest future applications of the technology in the selected areas of fundamental research. PMID:28725224

  16. Nanobody conjugated PLGA nanoparticles for active targeting of African Trypanosomiasis.

    Science.gov (United States)

    Arias, José L; Unciti-Broceta, Juan D; Maceira, José; Del Castillo, Teresa; Hernández-Quero, José; Magez, Stefan; Soriano, Miguel; García-Salcedo, José A

    2015-01-10

    Targeted delivery of therapeutics is an alternative approach for the selective treatment of infectious diseases. The surface of African trypanosomes, the causative agents of African trypanosomiasis, is covered by a surface coat consisting of a single variant surface glycoprotein, termed VSG. This coat is recycled by endocytosis at a very high speed, making the trypanosome surface an excellent target for the delivery of trypanocidal drugs. Here, we report the design of a drug nanocarrier based on poly ethylen glycol (PEG) covalently attached (PEGylated) to poly(D,L-lactide-co-glycolide acid) (PLGA) to generate PEGylated PLGA nanoparticles. This nanocarrier was coupled to a single domain heavy chain antibody fragment (nanobody) that specifically recognizes the surface of the protozoan pathogen Trypanosoma brucei. Nanoparticles were loaded with pentamidine, the first-line drug for T. b. gambiense acute infection. An in vitro effectiveness assay showed a 7-fold decrease in the half-inhibitory concentration (IC50) of the formulation relative to free drug. Furthermore, in vivo therapy using a murine model of African trypanosomiasis demonstrated that the formulation cured all infected mice at a 10-fold lower dose than the minimal full curative dose of free pentamidine and 60% of mice at a 100-fold lower dose. This nanocarrier has been designed with components approved for use in humans and loaded with a drug that is currently in use to treat the disease. Moreover, this flexible nanobody-based system can be adapted to load any compound, opening a range of new potential therapies with application to other diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis

    OpenAIRE

    Skottrup, Peter Durand; Leonard, Paul; Kaczmarek, Jakub Zbigniew; Veillard, Florian; Enghild, Jan Johannes; O'Kennedy, Richard; Sroka, Aneta; Clausen, Rasmus Praetorius; Potempa, Jan; Riise, Erik

    2011-01-01

    Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases, termed gingipains, and in this study we have utilized the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naïve camel nanobody library and used phage display to select one nanobody towards RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for R...

  18. Improved tumor targeting of anti-HER2 nanobody through N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling.

    Science.gov (United States)

    Pruszynski, Marek; Koumarianou, Eftychia; Vaidyanathan, Ganesan; Revets, Hilde; Devoogdt, Nick; Lahoutte, Tony; Lyerly, H Kim; Zalutsky, Michael R

    2014-04-01

    Nanobodies are approximately 15-kDa proteins based on the smallest functional fragments of naturally occurring heavy chain-only antibodies and represent an attractive platform for the development of molecularly targeted agents for cancer diagnosis and therapy. Because the human epidermal growth factor receptor type 2 (HER2) is overexpressed in breast and ovarian carcinoma, as well as in other malignancies, HER2-specific Nanobodies may be valuable radiodiagnostics and therapeutics for these diseases. The aim of the present study was to evaluate the tumor-targeting potential of anti-HER2 5F7GGC Nanobody after radioiodination with the residualizing agent N-succinimidyl 4-guanidinomethyl 3-(125/131)I-iodobenzoate (*I-SGMIB). The 5F7GGC Nanobody was radiolabeled using *I-SGMIB and, for comparison, with N(ε)-(3-*I-iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-GEEEK (*I-IB-Mal-d-GEEEK), another residualizing agent, and by direct radioiodination using IODO-GEN ((125)I-Nanobody). The 3 labeled Nanobodies were evaluated in affinity measurements, and paired-label internalization assays were performed on HER2-expressing BT474M1 breast carcinoma cells and in paired-label tissue distribution measurements in mice bearing subcutaneous BT474M1 xenografts. *I-SGMIB-Nanobody was produced in 50.4% ± 3.6% radiochemical yield and exhibited a dissociation constant of 1.5 ± 0.5 nM. Internalization assays demonstrated that intracellular retention of radioactivity was up to 1.5-fold higher for *I-SGMIB-Nanobody than for coincubated (125)I-Nanobody or *I-IB-Mal-d-GEEEK-Nanobody. Peak tumor uptake for *I-SGMIB-Nanobody was 24.50% ± 9.89% injected dose/g at 2 h, 2- to 4-fold higher than observed with other labeling methods, and was reduced by 90% with trastuzumab blocking, confirming the HER2 specificity of localization. Moreover, normal-organ clearance was fastest for *I-SGMIB-Nanobody, such that tumor-to-normal-organ ratios greater than 50:1 were reached by 24 h in all tissues except lungs

  19. Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3).

    Science.gov (United States)

    Homayouni, Vida; Ganjalikhani-Hakemi, Mazdak; Rezaei, Abbas; Khanahmad, Hossein; Behdani, Mahdi; Lomedasht, Fatemeh Kazemi

    2016-11-01

    As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. We immunized a camel with TIM-3 antigen and then, synthesized a VHH phage: mid library from its B cell's transcriptome using nested PCR. Library selection against TIM-3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA) column, SDS-PAGE and Western blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines. Specific 15kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3 with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-3 expressing HL-60 cell line. Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody with a high affinity and an anti-proliferative activity on an AML cell line in vitro.

  20. Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3

    Directory of Open Access Journals (Sweden)

    Vida Homayouni

    2016-11-01

    Full Text Available Objective(s: As T-cell immunoglobulin and mucin domain 3 (TIM-3 is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. Materials and Methods:We immunized a camel with TIM-3 antigen and then, synthesized a VHH phagemid library from its B cell’s transcriptome using nested PCR. Library selection against TIM-3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA column, SDS-PAGE and Western blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines. Results:Specific 15kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3 with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-3 expressing HL-60 cell line. Conclusion: Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody with a high affinity and an anti-proliferative activity on an AML cell line in vitro.

  1. A general protocol for the generation of Nanobodies for structural biology

    DEFF Research Database (Denmark)

    Pardon, Els; Laeremans, Toon; Triest, Sarah

    2014-01-01

    There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins...... and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage...... display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6-12 months....

  2. Structure of a nanobody-stabilized active state of the β(2) adrenoceptor

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; Choi, Hee-Jung; Fung, Juan Jose

    2011-01-01

    to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β(2) adrenergic receptor (β(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex...

  3. High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement*

    Science.gov (United States)

    De Tavernier, Evelyn; Detalle, Laurent; Morizzo, Erika; Roobrouck, Annelies; De Taeye, Severine; Rieger, Melanie; Verhaeghe, Tom; Correia, Andreia; Van Hegelsom, Rob; Figueirido, Rita; Noens, Jeroen; Steffensen, Søren; Stöhr, Thomas; Van de Velde, Willem; Depla, Erik; Dombrecht, Bruno

    2016-01-01

    Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms. PMID:27226529

  4. Nanobody-Based Biologics for Modulating Purinergic Signaling in Inflammation and Immunity

    Directory of Open Access Journals (Sweden)

    Stephan Menzel

    2018-03-01

    Full Text Available Adenosine triphosphate (ATP and nicotinamide adenine dinucleotide (NAD+ are released as danger signals from cells during infection and sterile inflammation. In the extracellular compartment ATP is converted by CD39, CD73, and other ecto-enzymes into metabolites that modulate the activity of T cells and macrophages. While ATP mediates pro-inflammatory signals via P2X7 and other P2 receptors, adenosine triggers anti-inflammatory signaling via the adenosine 2a receptor (Adora2a and other P1 receptors. The latter also plays a role in maintaining an immunosuppressive tumor microenvironment. NAD+ is converted by CD38, CD203 and other ecto-enzymes to the Ca2+ mobilizing messengers cyclic ADP-ribose and ADP-ribose, and to adenosine. Recent findings on the roles of CD38, CD39, CD73, CD203, P2X7, and Adora2a in inflammation and immunity underscore the potential of these proteins as drug targets. However, available small molecule inhibitors often lack specificity and mediate unwanted off-target toxicity. Nanobodies – single domain antibodies derived from heavy chain antibodies that naturally occur in camelids – display a propensity to bind functional epitopes not accessible to conventional antibodies. Like conventional antibodies, nanobodies and nanobody-based biologics are highly specific and have well-understood, tunable in vivo pharmacodynamics with little if any toxicity. Nanobodies thus represent attractive alternatives to small molecule inhibitors for modulating purinergic signaling in inflammation and immunity. Here we review recent progress made in developing nanobodies against key targets of purinergic signaling.

  5. Nanobody-Based Biologics for Modulating Purinergic Signaling in Inflammation and Immunity.

    Science.gov (United States)

    Menzel, Stephan; Schwarz, Nicole; Haag, Friedrich; Koch-Nolte, Friedrich

    2018-01-01

    Adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD + ) are released as danger signals from cells during infection and sterile inflammation. In the extracellular compartment ATP is converted by CD39, CD73, and other ecto-enzymes into metabolites that modulate the activity of T cells and macrophages. While ATP mediates pro-inflammatory signals via P2X7 and other P2 receptors, adenosine triggers anti-inflammatory signaling via the adenosine 2a receptor (Adora2a) and other P1 receptors. The latter also plays a role in maintaining an immunosuppressive tumor microenvironment. NAD + is converted by CD38, CD203 and other ecto-enzymes to the Ca 2+ mobilizing messengers cyclic ADP-ribose and ADP-ribose, and to adenosine. Recent findings on the roles of CD38, CD39, CD73, CD203, P2X7, and Adora2a in inflammation and immunity underscore the potential of these proteins as drug targets. However, available small molecule inhibitors often lack specificity and mediate unwanted off-target toxicity. Nanobodies - single domain antibodies derived from heavy chain antibodies that naturally occur in camelids - display a propensity to bind functional epitopes not accessible to conventional antibodies. Like conventional antibodies, nanobodies and nanobody-based biologics are highly specific and have well-understood, tunable in vivo pharmacodynamics with little if any toxicity. Nanobodies thus represent attractive alternatives to small molecule inhibitors for modulating purinergic signaling in inflammation and immunity. Here we review recent progress made in developing nanobodies against key targets of purinergic signaling.

  6. Generation and characterization of a human nanobody against VEGFR-2.

    Science.gov (United States)

    Ma, Lin; Gu, Kai; Zhang, Cheng-Hai; Chen, Xue-Tao; Jiang, Yi; Melcher, Karsten; Zhang, Juan; Wang, Min; Xu, H Eric

    2016-06-01

    Nanobody is an antibody fragment consisting of a single monomeric variable antibody domain, which can be used for a variety of biotechnological and therapeutic purposes. The aim of this work was to isolate and characterize a human signal domain antibody against VEGFR-2 domain3 (VEGFR D3) from a phage display library. To produce antigen-specific recombinant nanobodies with high affinity to VEGFR2 D3, a liquid phase panning strategy was used for all rounds of panning. For nanobody expression and purification, four VEGFR2 D3-blocking clones were subcloned into a pETduet-biotin-MBP expression vector. The recombinant proteins carried an MBP tag to facilitate purification by affinity chromatography. Recombinant NTV(1-4) was obtained after an additional gel filtration chromatography step. The interactions between VEGFR2 D3 and NTV(1-4) were assessed with luminescence-based AlphaScreen assay and SPR assay. Anti-angiogenesis effects were examined in human umbilical vein endothelial cells (HUVECs). In the AlphaScreen assay, NTV1 (100 and 200 nmol/L) elicited the highest binding signal with VEGFR2 D3; NTV2 showed moderate interactions with VEGFR2 D3; NTV3 and NTV4 exhibited little or no interaction with VEGFR2 D3. In the SPR assay, NTV1 displayed a high affinity for VEGFR2 D3 with an equilibrium dissociation constant (KD) of 49±1.8 nmol/L. NTV1 (1-1000 nmol/L) dose-dependently inhibited the proliferation of HUVECs and the endothelial tube formation by the HUVECs. The nanobody NTV1 is a potential therapeutic candidate for blocking VEGFR2. This study provides a novel and promising strategy for development of VEGFR2-targeted nanobody-based cancer therapeutics.

  7. Generation of an alpaca-derived nanobody recognizing γ-H2AX

    Science.gov (United States)

    Rajan, Malini; Mortusewicz, Oliver; Rothbauer, Ulrich; Hastert, Florian D.; Schmidthals, Katrin; Rapp, Alexander; Leonhardt, Heinrich; Cardoso, M. Cristina

    2015-01-01

    Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers. PMID:26500838

  8. Hypoxia-Targeting Fluorescent Nanobodies for Optical Molecular Imaging of Pre-Invasive Breast Cancer

    NARCIS (Netherlands)

    van Brussel, Aram S A; Adams, Arthur; Oliveira, Sabrina; Dorresteijn, Bram; El Khattabi, Mohamed; Vermeulen, J. F.; van der Wall, Elsken; Mali, Willem P Th M; Derksen, Patrick W B; van Diest, Paul J; van Bergen En Henegouwen, Paul M P

    PURPOSE: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. PROCEDURES: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated

  9. Hypoxia-Targeting Fluorescent Nanobodies for Optical Molecular Imaging of Pre-Invasive Breast Cancer

    NARCIS (Netherlands)

    van Brussel, Aram S A; Adams, Arthur; Oliveira, Sabrina; Dorresteijn, Bram; El Khattabi, Mohamed; Vermeulen, Jeroen F.; van der Wall, Elsken; Mali, W.P.T.M.; Derksen, Patrick W B; van Diest, Paul J.; van Bergen En Henegouwen, Paul M P

    Purpose: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. Procedures: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated

  10. Structure and binding properties of a cameloid nanobody raised against KDM5B

    DEFF Research Database (Denmark)

    Wiuf, Anders; Kristensen, Line Hyltoft; Kristensen, Ole

    2015-01-01

    The histone demethylase KDM5B is considered to be a promising target for anticancer therapy. Single-chain antibodies from llama (nanobodies) have been raised to aid in crystallization and structure determination of this enzyme. The antigen-binding properties of 15 of these nanobodies have been...

  11. A nanobody targeting carcinoembryonic antigen as a promising molecular probe for non-small cell lung cancer.

    Science.gov (United States)

    Wang, Hao; Meng, Ai-Min; Li, Sheng-Hua; Zhou, Xiao-Liang

    2017-07-01

    Carcinoembryonic antigen (CEA) is a biomarker and therapy target for non‑small cell lung cancer (NSCLC), which is the most common type of lung cancer. Nanobodies with high target specificity are promising candidates to function as anti‑CEA probes. In the present study, the targeting effects of an anti‑CEA nanobody obtained from phage display were investigated using technetium‑99 m (99mTc) and fluorescence labeling. In vitro binding and immunofluorescent staining assays, as well as in vivo blood clearance and biodistribution assays were performed. High specificity and affinity of the nanobody for CEA‑positive H460 cells was observed in vitro. The pharmacokinetics assay of the 99mTc‑nanobody in Wistar rats demonstrated that the nanobody had appropriate T1/2α and T1/2β, which were 20.2 and 143.5 min, respectively. The biodistribution assay using H460 xenograft‑bearing nude mice demonstrated a high ratio of signal in tumor compared with background, which confirmed that the nanobody may be useful as a molecular probe for CEA‑positive cancer, particularly in NSCLC.

  12. Nanobody®-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites

    Science.gov (United States)

    Nguyen-Duc, Trong; Peeters, Eveline; Muyldermans, Serge; Charlier, Daniel; Hassanzadeh-Ghassabeh, Gholamreza

    2013-01-01

    Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid–protein and protein–protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA–protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation. PMID:23275538

  13. Nanobody Based Dual Specific CARs

    Directory of Open Access Journals (Sweden)

    Stijn De Munter

    2018-01-01

    Full Text Available Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR. High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

  14. Nanobody Based Dual Specific CARs.

    Science.gov (United States)

    De Munter, Stijn; Ingels, Joline; Goetgeluk, Glenn; Bonte, Sarah; Pille, Melissa; Weening, Karin; Kerre, Tessa; Abken, Hinrich; Vandekerckhove, Bart

    2018-01-30

    Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

  15. Construction of pRMES and pTMES plasmids to test their expression ability of Nanobodies via the efficient T7 promoter

    International Nuclear Information System (INIS)

    Masoud, H.; Quider, M.; Abbady, A.

    2014-01-01

    Nanobody technology is considered as a promising molecular biology technique performed by means of the genetic engineering of special type of antibodies, existing exclusively in Camelidea. It enables the obtaining of small proteins, referred to as Nanobodies, which are characterized by high stability and solubility, are able to link to their specific antigens. After production, the Nanobody genes are cloned within plasmids of protein expression in bacteria, allowing their stable and continuous production for research and applied purposes. This work aimed to design new plasmids for Nanobody genes cloning in order to ensure a strong expression via the efficient T7 promoter, thus enhancing the quantity of the produced Nanobodies. These plasmids were called pRMES and pTMES and their ability to express Nanobodies, NbBruc02 and Nb16M, was tested. The plasmid pTMES showed an enhanced production condition of this Nanobody. These new plasmids, by their variable characteristics, could represent efficient tools for general production of recombinant proteins, including Nanobodies (author).

  16. Optimization of an anti-HER2 nanobody expression using the Taguchi method.

    Science.gov (United States)

    Farasat, Alireza; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Yazdian, Fatemeh

    2017-09-14

    Despite being widely used in immunotherapy of cancer, whole antibodies are limited by several disadvantages. This has led to the advent of novel biomolecules such as nanobodies. Taguchi method is a statistical experimental design to study the effect of multiple variables in biological processes. In an effort to overexpress a recombinant anti-human epidermal growth factor receptor type 2 (HER2) nanobody, we performed a detailed study to find optimal condition of temperature, induction, culture media, vector, and host strain, using Taguchi methodology. A total of 16 various experiments were designed. Total protein of the formulated cultures were assessed by Bradford test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by size exclusion high performance liquid chromatography to quantify the relative concentration of the nanobody in different expression settings. Western blotting was performed to confirm the expression of the anti-HER2 nanobody. When, individually, optimum parameters determined by Taguchi were applied, including SHuffle strain cultured in LB medium, induced with 0.4 mM isopropyl-β-D-thio-galactoside for 18 h at 24°C, production yield further increased by about 9% (25.4 mg/L), compared to the highest expression setting. Flow cytometry and enzyme-linked immunosorbent assay result indicated improved protein binding in optimized conditions. Overall, our findings provide a basis for further investigations on economical production of recombinant nanobodies to improve production yield and activity.

  17. Peptides in headlock ? a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    OpenAIRE

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a ?-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once boun...

  18. Boosted Electrochemical Immunosensing of Genetically Modified Crop Markers Using Nanobody and Mesoporous Carbon.

    Science.gov (United States)

    Zhang, Mingming; Li, Guanghui; Zhou, Qing; Pan, Deng; Zhu, Min; Xiao, Runyu; Zhang, Yuanjian; Wu, Guoqiu; Wan, Yakun; Shen, Yanfei

    2018-03-23

    The problems of environmental security and the potential risks of human health caused by transgenic crops have attracted much attention. Recent studies reveal 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 protein (CP4-EPSPS), which shows very high resistance to herbicide glyphosate, is a typical biomarker of genetically modified (GM) crops. For this reason, it is highly anticipated to devise a sensitive and convenient strategy to detect CP4-EPSPS protein in crops. Herein, we report a simple electrochemical immunosensor by coupling nanobody, ordered mesoporous carbon (OMC), and thionine (Th). As a capture agent, the nanobody was screened out from an immunized Bactrian camel, and exhibited superior properties with respect to conventional antibody, such as higher stability and stronger heat resistance. Moreover, OMC offered an effective platform with high surface area, electrical conductivity, and biocompatibility, which greatly facilitated the assembly of redox probe Th, and further coupling of large amount of capture nanobodies. As a result, the CP4-EPSPS protein could be determined with high sensitivity and efficiency by differential pulse voltammetry (DPV) in a wide linear range from 0.001 to 100 ng·mL -1 with a low detection limit of 0.72 pg·mL -1 , which was more than 3 orders of magnitude lower than those of previously reported works. As an example, the proposed electrochemical immunosensor was successfully applied to spiked samples, demonstrating its great potential in CP4-EPSPS screening and detection.

  19. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

    Science.gov (United States)

    Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  20. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

    Science.gov (United States)

    Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  1. Combinatorial design of a nanobody that specifically targets structured RNAs.

    Science.gov (United States)

    Cawez, F; Duray, E; Hu, Y; Vandenameele, J; Romão, E; Vincke, C; Dumoulin, M; Galleni, M; Muyldermans, S; Vandevenne, M

    2018-04-11

    Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs (ncRNAs). Most of them adopt a well-defined 3D structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of ncRNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody doesn't alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof-of-concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes. Copyright © 2018. Published by Elsevier Ltd.

  2. Exploiting Nanobodies in the Detection and Quantification of Human Growth Hormone via Phage-Sandwich Enzyme-Linked Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    Hossam Murad

    2017-05-01

    Full Text Available BackgroundMonitoring blood levels of human growth hormone (hGH in most children with short stature deficiencies is crucial for taking a decision of treatment with extended course of daily and expensive doses of recombinant hGH (rhGH or Somatropin®. Besides, misusing of rhGH by sportsmen is banned by the World Anti-Doping Agency and thus sensitive GH-detecting methods are highly welcome in this field. Nanobodies are the tiniest antigen-binding entity derived from camel heavy chain antibodies. They were successfully generated against numerous antigens including hormones.MethodsA fully nanobody-based sandwich ELISA method was developed in this work for direct measurement of GH in biological samples.ResultsTwo major characteristics of nanobody were exploited for this goal: the robust and stable structure of the nanobody (NbGH04 used to capture hGH from tested samples, and the great ability of tailoring, enabling the display of the anti-GH detector nanobody (NbGH07 on the tip of M13-phage. Such huge, stable, and easy-to-prepare phage-Nb was used in ELISA to provide an amplified signal. Previously, NbGH04 was retrieved on immobilized hGH by phage display from a wide “immune” cDNA library prepared from a hGH-immunized camel. Here, and in order to assure epitope heterogeneity, NbGH07 was isolated from the same library using NbGH04-captured hGH as bait. Interaction of both nanobodies with hGH was characterized and compared with different anti-GH nanobodies and antibodies. The sensitivity (~0.5 ng/ml and stability of the nanobody-base sandwich ELISA were assessed using rhGH before testing in the quantification of hGH in blood sera and cell culture supernatants.ConclusionIn regard to all advantages of nanobodies; stability, solubility, production affordability in Escherichia coli, and gene tailoring, nanobody-based phage sandwich ELISA developed here would provide a valuable method for hGH detection and quantification.

  3. Bioscreening and expression of a camel anti-CTGF VHH nanobody and its renaturation by a novel dialysis-dilution method.

    Science.gov (United States)

    Xue, Xiulei; Fan, Xiaobo; Qu, Qingrong; Wu, Guoqiu

    2016-12-01

    The variable regions of the camel heavy chain antibody, also known as nanobody is the smallest antibody with antigen-binding efficiency. CTGF is considered important during extracellular matrix deposition which was involved in the pathogenesis of fibrosis related diseases. There are several anti-CTGF-C nanobody drugs under developing in pharmacy. In this study, we described the screening of a novel anti-CTGF-C nanobody from the peripheral blood of immunized camel by phage display. The screened nanobody was further expressed and purified from E. coli cells. A sophisticated dialysis-dilution method was designed for the in vitro refolding of the nanobody. The results showed that the expressed nanobody was consisted of 135 amino acid and mainly expressed as inclusion body in E. coli cells. The dialysis-dilution method was very effective and the recovery rate of the renaturation was more than 80 %. The ELISA result suggested the nanobody had been well refolded showing a superior CTGF binding activity to the commercial mouse anti-CTGF-C mAb. In conclusion, the anti-CTGF-C nonobody had been successfully screened by phage display. The dialysis-dilution refolding method was very effective and the recovery rate reached over 80 %.

  4. Phage-Mediated Immuno-PCR for Ultrasensitive Detection of Cry1Ac Protein Based on Nanobody.

    Science.gov (United States)

    Liu, Yuanyuan; Jiang, Dongjian; Lu, Xin; Wang, Wei; Xu, Yang; He, Qinghua

    2016-10-11

    The widespread use of Cry proteins in transgenic plants for insect control has raised concerns about the environment and food safety in the public. An effective detection method for introduced Cry proteins is of significance for environmental risk assessment and product quality control. This paper describes a novel phage mediated immuno-PCR (iPCR) for the ultrasensitive determination of Cry proteins based on nanobodies. Three nanobodies against Cry1Ac protein were obtained from a naı̈ve phage displayed nanobody library without animal immunization process and were applied to the iPCR assay for Cry1Ac. The phage-mediated iPCR for Cry1Ac based on nanobodies showed a dynamic range of 0.001-100 ng/mL and a limit detection of 0.1 pg/mL. Specific measurement of this established method was performed by testing cross-reativity of other Cry1Ac analogues, and the result showed negligible cross-reactivity with other test Cry proteins (Cry1Ab, Cry1F, Cry3B). Furthermore, the phage-mediated iPCR based on nanobody should be easily applicable to the detection of many other Cry proteins.

  5. Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

    Directory of Open Access Journals (Sweden)

    Haddad Muhammad

    2016-01-01

    Full Text Available Camelid’ s heavy-chain antibody (HCAb consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody® was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG. Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.

  6. A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment.

    Science.gov (United States)

    Farajpour, Zahra; Rahbarizadeh, Fatemeh; Kazemi, Bahram; Ahmadvand, Davoud

    2014-03-28

    Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae. Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

    OpenAIRE

    Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

    2018-01-01

    Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  8. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants.

    Science.gov (United States)

    Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

    2018-01-01

    Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  9. Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1.

    Science.gov (United States)

    Zhu, Min; Gong, Xue; Hu, Yonghong; Ou, Weijun; Wan, Yakun

    2014-12-20

    Influenza H5N1 is one subtype of the influenza A virus which can infect human bodies and lead to death. Timely diagnosis before its breakout is vital to the human health. The current clinical biochemical diagnosis for influenza virus are still flawed, and the diagnostic kits of H5N1 are mainly based on traditional monoclonal antibodies that hardly meet the requirements for clinical applications. Nanobody is a promising tool for diagnostics and treatment due to its smallest size, high specificity and stability. In this study, a novel Nanobody-based bioassay was developed for rapid, low-cost and sensitive detection of the influenza H5N1 virus. Nanobodies specific to H5N1 virus were selected from a VHH library by phage display technology. In this system, the biotinylated Nanobody was directionally captured by streptavidin coated on ELISA plate, which can specifically capture the H5N1 virus. Another Nanobody conjugated with HRP was used as a detector. A novel directional enzyme-linked immunosorbent assay for H5N1 using specific Nanobodies was established and compared to the conventional undirected ELISA assay. We have successfully constructed a high quality phage display Nanobody library and isolated two Nanobodies against H5N1 with high affinity and specificity. These two Nanobodies were further used to prepare the biosensor detection system. This streptavidin-biotin-based directional double Nanobodies sandwich ELISA for H5N1 detection showed superiority over the commonly undirectional ELISA protocol. The linear range of detection for standards in this immunoassay was approximately 50-1000 ng/mL and the detection limit was 14.1 ng/mL. The average recoveries of H5N1 virus from human serum samples were in the range from 94.58% to 114.51%, with a coefficient of variation less than 6.5%. Collectively, these results demonstrated that the proposed detection system is an alternative diagnostic tool that enables a rapid, inexpensive, sensitive and specific detection of the

  10. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

    Directory of Open Access Journals (Sweden)

    Bianca Baudisch

    2018-02-01

    Full Text Available Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  11. Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Leonard, Paul; Kaczmarek, Jakub Zbigniew

    2011-01-01

    Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display...... epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble Rgp......B. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection...

  12. The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma.

    Directory of Open Access Journals (Sweden)

    Travis McMurphy

    Full Text Available Environmental and genetic activation of a brain-adipocyte axis inhibits cancer progression. Leptin is the primary peripheral mediator of this anticancer effect in a mouse model of melanoma. In this study we assessed the effect of a leptin receptor antagonist on melanoma progression. Local administration of a neutralizing nanobody targeting the leptin receptor at low dose adjacent to tumor decreased tumor mass with no effects on body weight or food intake. In contrast, systemic administration of the nanobody failed to suppress tumor growth. Daily intraperitoneal injection of high-dose nanobody led to weight gain, hyperphagia, increased adiposity, hyperleptinemia, and hyperinsulinemia, and central effects mimicking leptin deficiency. The blockade of central actions of leptin by systemic delivery of nanobody may compromise its anticancer effect, underscoring the need to develop peripherally acting leptin antagonists coupled with efficient cancer-targeting delivery.

  13. The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma.

    Science.gov (United States)

    McMurphy, Travis; Xiao, Run; Magee, Daniel; Slater, Andrew; Zabeau, Lennart; Tavernier, Jan; Cao, Lei

    2014-01-01

    Environmental and genetic activation of a brain-adipocyte axis inhibits cancer progression. Leptin is the primary peripheral mediator of this anticancer effect in a mouse model of melanoma. In this study we assessed the effect of a leptin receptor antagonist on melanoma progression. Local administration of a neutralizing nanobody targeting the leptin receptor at low dose adjacent to tumor decreased tumor mass with no effects on body weight or food intake. In contrast, systemic administration of the nanobody failed to suppress tumor growth. Daily intraperitoneal injection of high-dose nanobody led to weight gain, hyperphagia, increased adiposity, hyperleptinemia, and hyperinsulinemia, and central effects mimicking leptin deficiency. The blockade of central actions of leptin by systemic delivery of nanobody may compromise its anticancer effect, underscoring the need to develop peripherally acting leptin antagonists coupled with efficient cancer-targeting delivery.

  14. Determination of Aspergillus pathogens in agricultural products by a specific nanobody-polyclonal antibody sandwich ELISA.

    Science.gov (United States)

    Wang, Ting; Li, Peiwu; Zhang, Qi; Zhang, Wen; Zhang, Zhaowei; Wang, Tong; He, Ting

    2017-06-28

    Aspergillus and its poisonous mycotoxins are distributed worldwide throughout the environment and are of particular interest in agriculture and food safety. In order to develop a specific method for rapid detection of Aspergillus flavus to forecast diseases and control aflatoxins, a nanobody, PO8-VHH, highly reactive to A. flavus was isolated from an immunized alpaca nanobody library by phage display. The nanobody was verified to bind to the components of extracellular and intracellular antigen from both A. flavus and A. parasiticus. To construct a sandwich format immunoassay, polyclonal antibodies against Aspergillus were raised with rabbits. Finally, a highly selective nanobody-polyclonal antibody sandwich enzyme-linked immunosorbent assay was optimized and developed. The results revealed that the detection limits of the two fungi were as low as 1 μg mL -1 , and that it is able to detect fungal concentrations below to 2 μg mg -1 of peanut and maize grains in both artificially and naturally contaminated samples. Therefore, we here provided a rapid and simple method for monitoring Aspergillus spp. contamination in agricultural products.

  15. Nanobody Technology for Mycotoxin Detection in the Field of Food Safety: Current Status and Prospects.

    Science.gov (United States)

    He, Ting; Zhu, Jiang; Nie, Yao; Hu, Rui; Wang, Ting; Li, Peiwu; Zhang, Qi; Yang, Yunhuang

    2018-04-29

    Mycotoxins, which are toxic, carcinogenic, and/or teratogenic, have posed a threat to food safety and public health. Sensitive and effective determination technologies for mycotoxin surveillance are required. Immunoassays have been regarded as useful supplements to chromatographic techniques. However, conventional antibodies involved in immunoassays are difficult to be expressed recombinantly and are susceptible to harsh environments. Nanobodies (or VHH antibodies) are antigen-binding sites of the heavy-chain antibodies produced from Camelidae. They are found to be expressed easily in prokaryotic or eukaryotic expression systems, more robust in extreme conditions, and facile to be used as surrogates for artificial antigens. These properties make them the promising and environmentally friendly immunoreagents in the next generation of immunoassays. This review briefly describes the latest developments in the area of nanobodies used in mycotoxin detection. Moreover, by integrating the introduction of the principle of nanobodies production and the critical assessment of their performance, this paper also proposes the prospect of nanobodies in the field of food safety in the foreseeable future.

  16. Tumor-targeted Nanobullets: Anti-EGFR nanobody-liposomes loaded with anti-IGF-1R kinase inhibitor for cancer treatment.

    Science.gov (United States)

    van der Meel, Roy; Oliveira, Sabrina; Altintas, Isil; Haselberg, Rob; van der Veeken, Joris; Roovers, Rob C; van Bergen en Henegouwen, Paul M P; Storm, Gert; Hennink, Wim E; Schiffelers, Raymond M; Kok, Robbert J

    2012-04-30

    The epidermal growth factor receptor (EGFR) is a validated target for anti-cancer therapy and several EGFR inhibitors are used in the clinic. Over the years, an increasing number of studies have reported on the crosstalk between EGFR and other receptors that can contribute to accelerated cancer development or even acquisition of resistance to anti-EGFR therapies. Combined targeting of EGFR and insulin-like growth factor 1 receptor (IGF-1R) is a rational strategy to potentiate anti-cancer treatment and possibly retard resistance development. In the present study, we have pursued this by encapsulating the kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The thus developed dual-active nanobody-liposomes associated with EGFR-(over)expressing cells in an EGFR-specific manner and blocked both EGFR and IGF-1R activation, due to the presence of the EGFR-blocking nanobody EGa1 and the anti-IGF-1R kinase inhibitor AG538 respectively. AG538-loaded nanobody-liposomes induced a strong inhibition of tumor cell proliferation even upon short-term exposure followed by a drug-free wash-out period. Therefore, AG538-loaded nanobody-liposomes are a promising anti-cancer formulation due to efficient intracellular delivery of AG538 in combination with antagonistic and downregulating properties of the EGa1 nanobody-liposomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Inhibition of type VI secretion by an anti-TssM llama nanobody.

    Directory of Open Access Journals (Sweden)

    Van Son Nguyen

    Full Text Available The type VI secretion system (T6SS is a secretion pathway widespread in Gram-negative bacteria that targets toxins in both prokaryotic and eukaryotic cells. Although most T6SSs identified so far are involved in inter-bacterial competition, a few are directly required for full virulence of pathogens. The T6SS comprises 13 core proteins that assemble a large complex structurally and functionally similar to a phage contractile tail structure anchored to the cell envelope by a trans-membrane spanning stator. The central part of this stator, TssM, is a 1129-amino-acid protein anchored in the inner membrane that binds to the TssJ outer membrane lipoprotein. In this study, we have raised camelid antibodies against the purified TssM periplasmic domain. We report the crystal structure of two specific nanobodies that bind to TssM in the nanomolar range. Interestingly, the most potent nanobody, nb25, competes with the TssJ lipoprotein for TssM binding in vitro suggesting that TssJ and the nb25 CDR3 loop share the same TssM binding site or causes a steric hindrance preventing TssM-TssJ complex formation. Indeed, periplasmic production of the nanobodies displacing the TssM-TssJ interaction inhibits the T6SS function in vivo. This study illustrates the power of nanobodies to specifically target and inhibit bacterial secretion systems.

  18. Structural insights into a high affinity nanobody:antigen complex by homology modelling

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2017-01-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby...... simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for Rgp...

  19. A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting

    Directory of Open Access Journals (Sweden)

    Wang SM

    2015-04-01

    Full Text Available Shan-Mei Wang,1,* Xian He,2,* Nan Li,1,* Feng Yu,3 Yang Hu,1 Liu-Sheng Wang,1 Peng Zhang,4 Yu-Kui Du,1 Shan-Shan Du,1 Zhao-Fang Yin,1 Ya-Ru Wei,1 Xavier Mulet,5 Greg Coia,6 Dong Weng,1 Jian-Hua He,3 Min Wu,7 Hui-Ping Li1 1Department of Respiratory Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, 2School of Medicine, Suzhou University, SuZhou, 3Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 4Department of Chest Surgery, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 5CSIRO (Commonwealth Scientific and Industrial Research Materials Science and Engineering, Clayton, 6CSIRO Materials Science and Engineering, Parkville, Melbourne, VIC, Australia; 7Department of Basic Sciences, University of North Dakota, Grand Forks, ND, USA *These authors contributed equally to this work Abstract: Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies’ potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high

  20. Capillary electrophoresis-based assessment of nanobody affinity and purity

    NARCIS (Netherlands)

    Haselberg, Rob; Oliveira, Sabrina; van der Meel, Roy; Somsen, Govert W; de Jong, Gerhardus J

    2014-01-01

    Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a

  1. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus.

    Science.gov (United States)

    Doerflinger, Sylvie Y; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna; Hansman, Grant S

    2016-01-01

    Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical setting, the

  2. Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

    Directory of Open Access Journals (Sweden)

    Emmanuel Obishakin

    Full Text Available Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa, costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa, antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392 targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1 of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392 that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.

  3. Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin

    Science.gov (United States)

    Li, Min; Zhu, Min; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun

    2014-01-01

    Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system. PMID:25474492

  4. Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt Cry1Ac Toxin

    Directory of Open Access Journals (Sweden)

    Min Li

    2014-12-01

    Full Text Available Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies sandwich-ELISA (DAS-ELISA assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac were selected as capture antibody (Nb61 and detection antibody (Nb44. The capture antibody (Nb61 was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.

  5. Delivery of an anti-TTR Nanobody to the brain through intranasal administration reveals TTR expression and secretion by Motor Neurons.

    Science.gov (United States)

    Gomes, João R; Cabrito, Inês; Soares, Hugo R; Costelha, Susete; Teixeira, Anabela; Wittelsberger, Angela; Stortelers, Catelijne; Vanlandschoot, Peter; Saraiva, Maria J

    2018-03-12

    Transthyretin (TTR) is a transport protein of retinol and thyroxine in serum and cerebrospinal fluid (CSF), which is mainly secreted in liver and choroid plexus, and in smaller amounts in other cells throughout the body. The exact role of TTR and its specific expression in Central Nervous System (CNS) remains understudied. We investigated TTR expression and metabolism in CNS, through the intranasal and intracerebroventricular delivery of a specific anti-TTR Nanobody to the brain, unveiling Nanobody pharmacokinetics to the CNS. In TTR deficient mice, we observed that anti-TTR Nanobody was successfully distributed throughout all brain areas, and also reaching the spinal cord. In wild type (WT) mice, a similar distribution pattern was observed. However, in areas known to be rich in TTR, reduced levels of Nanobody were found, suggesting potential target-mediated effects. Indeed, in WT mice, the anti-TTR Nanobody was specifically internalized in a receptor-mediated process, by neuronal-like cells, which were identified as motor neurons. Whereas in KO TTR mice Nanobody was internalized by all cells, for late lysosomal degradation. Moreover, we demonstrate that in-vivo motor neurons also actively synthesize TTR. Finally, in-vitro cultured primary motor neurons were also found to synthesize and secrete TTR into culture media. Thus, through a novel intranasal CNS distribution study with an anti-TTR Nanobody, we disclose a new cell type capable of synthesizing TTR, which might be important for the understanding of the physiological role of TTR, as well as in pathological conditions where TTR levels are altered in CSF, such as amyotrophic lateral sclerosis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  6. Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD.

    Science.gov (United States)

    Mireku, S A; Sauer, M M; Glockshuber, R; Locher, K P

    2017-10-30

    Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10 -6 and 10 -9  M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 10 4 and 10 6  M -1 s -1 . For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies.

  7. Neutralization of TNFα in tumor with a novel nanobody potentiates paclitaxel-therapy and inhibits metastasis in breast cancer.

    Science.gov (United States)

    Ji, Xuemei; Peng, Zhengxin; Li, Xiaorui; Yan, Zhonghui; Yang, Yue; Qiao, Zheng; Liu, Yu

    2017-02-01

    Metastatic disease is the major cause of death from cancer, and immunotherapy and chemotherapy have had limited success in reversing its progression. Researchers have suggested that inflammatory factors in the tumor environment can promote cancer invasion and metastasis, stimulating cancer progression. Thus, novel strategies that target cytokines and modulate the tumor microenvironment may emerge as important approaches for treating metastatic breast cancer. Specific neutralization of pathogenic TNF signaling using a TNFα antibody has gained increasing attention. Considering this, a selective human TNFα neutralized antibody was generated based on nanobody technology. A TNFα-specific nanobody was produced in Pichia pastoris with a molecular mass of 15 kDa and affinity constant of 2.05 nM. In the proliferation experiment, the TNFα nanobody could inhibit the proliferation of the breast cancer cell line MCF-7 induced by hTNFα in a dose-dependent manner. In the microinvasion model, the TNFα nanobody could inhibit the migration of the breast cancer cell lines MCF-7, MDA-MB-231 and the invasiveness of MDA-MB-231 induced by hTNFα in a dose-dependent manner. Drug administration of the combination of paclitaxel with the TNFα nanobody in vivo significantly enhanced the efficacy against 4T-1 breast tumor proliferation and lung metastasis; meanwhile, E-cadherin tumor epithelial marker expression was upregulated, supporting the anti-tumor therapeutic relevance of paclitaxel and the TNFα nanobody on EMT. This study highlights the importance of neutralizing low TNFα levels in the tumor microenvironment to sensitize the chemotherapeutic response, which has attractive potential for clinical applications. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Phage-Mediated Competitive Chemiluminescent Immunoassay for Detecting Cry1Ab Toxin by Using an Anti-Idiotypic Camel Nanobody.

    Science.gov (United States)

    Qiu, Yulou; Li, Pan; Dong, Sa; Zhang, Xiaoshuai; Yang, Qianru; Wang, Yulong; Ge, Jing; Hammock, Bruce D; Zhang, Cunzheng; Liu, Xianjin

    2018-01-31

    Cry toxins have been widely used in genetically modified organisms for pest control, raising public concern regarding their effects on the natural environment and food safety. In this work, a phage-mediated competitive chemiluminescent immunoassay (c-CLIA) was developed for determination of Cry1Ab toxin using anti-idiotypic camel nanobodies. By extracting RNA from camels' peripheral blood lymphocytes, a naive phage-displayed nanobody library was established. Using anti-Cry1Ab toxin monoclonal antibodies (mAbs) against the library for anti-idiotypic antibody screening, four anti-idiotypic nanobodies were selected and confirmed to be specific for anti-Cry1Ab mAb binding. Thereafter, a c-CLIA was developed for detection of Cry1Ab toxin based on anti-idiotypic camel nanobodies and employed for sample testing. The results revealed a half-inhibition concentration of developed assay to be 42.68 ± 2.54 ng/mL, in the linear range of 10.49-307.1 ng/mL. The established method is highly specific for Cry1Ab recognition, with negligible cross-reactivity for other Cry toxins. For spiked cereal samples, the recoveries of Cry1Ab toxin ranged from 77.4% to 127%, with coefficient of variation of less than 9%. This study demonstrated that the competitive format based on phage-displayed anti-idiotypic nanobodies can provide an alternative strategy for Cry toxin detection.

  9. Patterning protein complexes on DNA nanostructures using a GFP nanobody.

    Science.gov (United States)

    Sommese, R F; Hariadi, R F; Kim, K; Liu, M; Tyska, M J; Sivaramakrishnan, S

    2016-11-01

    DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies. © 2016 The Protein Society.

  10. Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library.

    Science.gov (United States)

    Shahi, B; Mousavi Gargari, S L; Rasooli, I; Rajabi Bazl, M; Hoseinpoor, R

    2014-08-01

    To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR. The gene coding for specific VHH derived from the camel immunized with binding domain of botulinum neurotoxin E (BoNT/E) was amplified by error-prone PCR. Several biopanning rounds were used to screen the phage-displaying BoNT/E Hc nanobodies. The final nanobody, SHMR4, with increased affinity recognized BoNT/E toxin with no cross-reactivity with other antigens especially with related BoNT toxins. The constructed nanobody could be a suitable candidate for VHH-based biosensor production to detect the Clostridium botulinum type E. Diagnosis and treatment of botulinum neurotoxins are important. Generation of high-affinity antibodies based on the construction of secondary libraries using affinity maturation step leads to the development of reagents for precise diagnosis and therapy. © 2014 The Society for Applied Microbiology.

  11. Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies

    Directory of Open Access Journals (Sweden)

    Marius Terfrüchte

    2017-04-01

    Full Text Available Exploiting secretory pathways for production of heterologous proteins is highly advantageous with respect to efficient downstream processing. In eukaryotic systems the vast majority of heterologous proteins for biotechnological application is exported via the canonical endoplasmic reticulum–Golgi pathway. In the endomembrane system target proteins are often glycosylated and may thus be modified with foreign glycan patterns. This can be destructive for their activity or cause immune reactions against therapeutic proteins. Hence, using unconventional secretion for protein expression is an attractive alternative. In the fungal model Ustilago maydis, chitinase Cts1 is secreted via an unconventional pathway connected to cell separation which can be used to co-export heterologous proteins. Here, we apply this mechanism for the production of nanobodies. First, we achieved expression and unconventional secretion of a functional nanobody directed against green fluorescent protein (Gfp. Second, we found that Cts1 binds to chitin and that this feature can be applied to generate a Gfp-trap. Thus, we demonstrated the dual use of Cts1 serving both as export vehicle and as purification tag. Finally, we established and optimized the production of a nanobody against botulinum toxin A and hence describe the first pharmaceutically relevant target exported by Cts1-mediated unconventional secretion.

  12. TRIM28 and β-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.

    Directory of Open Access Journals (Sweden)

    Ivana Jovčevska

    Full Text Available Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM, is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells. After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass

  13. TRIM28 and β-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.

    Science.gov (United States)

    Jovčevska, Ivana; Zupanec, Neja; Kočevar, Nina; Cesselli, Daniela; Podergajs, Neža; Stokin, Clara Limbaeck; Myers, Michael P; Muyldermans, Serge; Ghassabeh, Gholamreza Hassanzadeh; Motaln, Helena; Ruaro, Maria Elisabetta; Bourkoula, Evgenia; Turnšek, Tamara Lah; Komel, Radovan

    2014-01-01

    Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry

  14. A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

    Science.gov (United States)

    Kijanka, M; van Donselaar, E G; Müller, W H; Dorresteijn, B; Popov-Čeleketić, D; El Khattabi, M; Verrips, C T; van Bergen En Henegouwen, P M P; Post, J A

    2017-07-01

    Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM. Copyright © 2017. Published by Elsevier Inc.

  15. Nanobody-functionalized PEG-b-PCL polymersomes and their targeting study.

    Science.gov (United States)

    Zou, Tao; Dembele, Fatimata; Beugnet, Anne; Sengmanivong, Lucie; Trepout, Sylvain; Marco, Sergio; de Marco, Ario; Li, Min-Hui

    2015-11-20

    We prepared and characterized polymersomes functionalized with nanobodies (VHHs) on the basis of biocompatible, biodegradable and FDA-approved poly(ethylene glycol)-block-poly(ϵ-caprolactone) (PEG-b-PCL). Fluorescein isothiocyanate (FITC) and N-beta-maleimidopropyl-oxysuccinimide ester were allowed reacting with H2N-PEG-b-PCL to produce FITC and maleimide (Mal) functionalized copolymers, Mal-PEG-b-PCL and FITC-PEG-b-PCL. A mixture of MeO-PEG-b-PCL, Mal-PEG-b-PCL and FITC-PEG-b-PCL was used to prepare polymersomes by thin film hydration and nanoprecipitation methods. Morphological studies by cryogenic transmission electron microscopy (Cryo-TEM) showed that the nanoparticles exhibited predominantly vesicular structures (polymersomes). Their mean diameters measured by dynamic light scattering were around 150 nm and the zeta-potentials around -1 mV at pH 7.4. The nanoparticles were functionalized with either anti-HER2 (VHH1) or anti-GFP (VHH2) nanobodies using maleimide-cysteine chemistry. Their particle size and zeta-potential increased slightly after nanobody-functionalization. The specific binding of VHH-functionalized polymersomes and control nanoparticles towards HER2 positive breast cancer cells was analyzed by flow cytometry and confocal microscopy. The collected results represent the first report which experimentally demonstrates that VHH1-functionalized PEO-b-PCL polymersomes can target specifically breast cancer cells expressing HER2 receptors. The detailed morphological and cell-binding studies described herein pave the way for future in vivo studies to evaluate the feasibility to use such nanoparticles for targeted drug delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Pre-clinical studies of toxin-specific Nanobodies: Evidence of in vivo efficacy to prevent fatal disturbances provoked by scorpion envenoming

    International Nuclear Information System (INIS)

    Hmila, Issam; Cosyns, Bernard; Tounsi, Hayfa; Roosens, Bram; Caveliers, Vicky; Abderrazek, Rahma Ben; Boubaker, Samir; Muyldermans, Serge; El Ayeb, Mohamed; Bouhaouala-Zahar, Balkiss; Lahoutte, Tony

    2012-01-01

    Scorpions represent a significant threat to humans and animals in various countries throughout the world. Recently, we introduced Nanobodies (Nbs) to combat more efficiently scorpion envenoming and demonstrated the performance of NbAahIF12 and NbAahII10 to neutralize scorpion toxins of Androctonus australis hector venom. A bispecific Nb construct (NbF12-10) comprising these two Nbs is far more protective than the classic Fab′ 2 based therapy and is the most efficient antivenom therapy against scorpion sting in preclinical studies. Now we investigate the biodistribution and pharmacokinetics of 99m Tc labeled Nbs by in vivo imaging in rodents and compared these data with those of the Fab′ 2 product (PAS). The pharmacodynamics of the Nbs was investigated in rats by in vivo echocardiography and it is shown that NbF12-10 prevents effectively the hemodynamic disturbances induced by a lethal dose of venom. Moreover, even a late injection of NbF12-10 restores the heart rate and brings the blood pressure to baseline values. Histology confirms that NbF12-10 prevents lung and heart lesions of treated mice after envenoming. In conjunction, in this preclinical study, we provide proof of concept that NbF12-10 prevents effectively the fatal disturbances induced by Androctonus venom, and that the Nanobody based therapeutic has a potential to substitute the classic Fab′ 2 based product as immunotherapeutic in scorpion envenoming. Further clinical study using larger cohorts of animals should be considered to confirm the full protecting potential of our NbF12-10. -- Highlights: ► Nanobody therapy prevents the hemodynamic disturbances induced by a lethal dose. ► Late injection of Nanobody restores hemodynamic parameters to baseline values. ► Nanobody therapy prevents lung and heart lesions of treated mice after envenoming. ► Labeled Nanobody and Fab’2 pharmacokinetics curves reach plateau in favour of Nanobody.

  17. Pre-clinical studies of toxin-specific Nanobodies: Evidence of in vivo efficacy to prevent fatal disturbances provoked by scorpion envenoming

    Energy Technology Data Exchange (ETDEWEB)

    Hmila, Issam [Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, 13 Place Pasteur, BP-74, 1002 Tunis (Tunisia); Cosyns, Bernard [Laboratory of In Vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel (Belgium); Tounsi, Hayfa [Service d' Anatomo-Pathologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP-74, 1002 Tunis (Tunisia); Roosens, Bram; Caveliers, Vicky [Laboratory of In Vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel (Belgium); Abderrazek, Rahma Ben [Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, 13 Place Pasteur, BP-74, 1002 Tunis (Tunisia); Boubaker, Samir [Service d' Anatomo-Pathologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP-74, 1002 Tunis (Tunisia); Muyldermans, Serge [Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel (Belgium); Department of Structural Biology, VIB, Brussels (Belgium); El Ayeb, Mohamed [Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, 13 Place Pasteur, BP-74, 1002 Tunis (Tunisia); Bouhaouala-Zahar, Balkiss, E-mail: balkiss.bouhaouala@pasteur.rns.tn [Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, 13 Place Pasteur, BP-74, 1002 Tunis (Tunisia); Faculté de Médecine de Tunis, Université de Tunis-El Manar (Tunisia); Lahoutte, Tony [Laboratory of In Vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel (Belgium)

    2012-10-15

    Scorpions represent a significant threat to humans and animals in various countries throughout the world. Recently, we introduced Nanobodies (Nbs) to combat more efficiently scorpion envenoming and demonstrated the performance of NbAahIF12 and NbAahII10 to neutralize scorpion toxins of Androctonus australis hector venom. A bispecific Nb construct (NbF12-10) comprising these two Nbs is far more protective than the classic Fab′{sub 2} based therapy and is the most efficient antivenom therapy against scorpion sting in preclinical studies. Now we investigate the biodistribution and pharmacokinetics of {sup 99m}Tc labeled Nbs by in vivo imaging in rodents and compared these data with those of the Fab′{sub 2} product (PAS). The pharmacodynamics of the Nbs was investigated in rats by in vivo echocardiography and it is shown that NbF12-10 prevents effectively the hemodynamic disturbances induced by a lethal dose of venom. Moreover, even a late injection of NbF12-10 restores the heart rate and brings the blood pressure to baseline values. Histology confirms that NbF12-10 prevents lung and heart lesions of treated mice after envenoming. In conjunction, in this preclinical study, we provide proof of concept that NbF12-10 prevents effectively the fatal disturbances induced by Androctonus venom, and that the Nanobody based therapeutic has a potential to substitute the classic Fab′{sub 2} based product as immunotherapeutic in scorpion envenoming. Further clinical study using larger cohorts of animals should be considered to confirm the full protecting potential of our NbF12-10. -- Highlights: ► Nanobody therapy prevents the hemodynamic disturbances induced by a lethal dose. ► Late injection of Nanobody restores hemodynamic parameters to baseline values. ► Nanobody therapy prevents lung and heart lesions of treated mice after envenoming. ► Labeled Nanobody and Fab’2 pharmacokinetics curves reach plateau in favour of Nanobody.

  18. Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference.

    Science.gov (United States)

    Liu, Xing; Tang, Zongwen; Duan, Zhenhua; He, Zhenyun; Shu, Mei; Wang, Xianxian; Gee, Shirley J; Hammock, Bruce D; Xu, Yang

    2017-03-01

    A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC 50 of 0.64ng/mL and a linear range (IC 20 -IC 80 ) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Structural insights into a high affinity nanobody:antigen complex by homology modelling.

    Science.gov (United States)

    Skottrup, Peter Durand

    2017-09-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for RgpB binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified. Collectively, the VHH7 homology model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

    Science.gov (United States)

    Che, Tao; Majumdar, Susruta; Zaidi, Saheem A; Ondachi, Pauline; McCorvy, John D; Wang, Sheng; Mosier, Philip D; Uprety, Rajendra; Vardy, Eyal; Krumm, Brian E; Han, Gye Won; Lee, Ming-Yue; Pardon, Els; Steyaert, Jan; Huang, Xi-Ping; Strachan, Ryan T; Tribo, Alexandra R; Pasternak, Gavril W; Carroll, F Ivy; Stevens, Raymond C; Cherezov, Vadim; Katritch, Vsevolod; Wacker, Daniel; Roth, Bryan L

    2018-01-11

    The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Nanobody-Based Apolipoprotein E Immunosensor for Point-of-Care Testing.

    Science.gov (United States)

    Ren, Xiang; Yan, Junrong; Wu, Dan; Wei, Qin; Wan, Yakun

    2017-09-22

    Alzheimer's disease (AD) biomarkers can reflect the neurochemical indicators used to estimate the risk in clinical nephrology. Apolipoprotein E (ApoE) is an early biomarker for AD in clinical diagnosis. In this research, through bactrian camel immunization, lymphocyte isolation, RNA extraction, and library construction, ApoE-specific Nbs with high affinity were successfully separated from an immune phage display nanobody library. Herein, a colorimetric immunosensor was developed for the point-of-care testing of ApoE by layer-by-layer nanoassembly techniques and novel nanobodies (Nbs). Using highly oriented Nbs as the capture and detection antibodies, an on-site immunosensor was developed by detecting the mean gray value of fade color due to the glutaraldehyde@3-aminopropyltrimethoxysilane oxidation by H 2 O 2 . The detection limit of AopE is 0.42 pg/mL, and the clinical analysis achieves a good performance. The novel easily operated immunosensor may have potential application in the clinical diagnosis and real-time monitoring for AD.

  2. Production, crystallization and X-ray diffraction analysis of two nanobodies against the Duffy binding-like (DBL) domain DBL6∊-FCR3 of the Plasmodium falciparum VAR2CSA protein

    International Nuclear Information System (INIS)

    Vuchelen, Anneleen; Pardon, Els; Steyaert, Jan; Gamain, Benoît; Loris, Remy; Nuland, Nico A. J. van; Ramboarina, Stéphanie

    2013-01-01

    Two nanobodies generated against the VAR2CSA DBL6∊-FCR3 domain involved in pregnancy-associated malaria were selected, expressed, purified and crystallized. The VAR2CSA protein has been closely associated with pregnancy-associated malaria and is recognized as the main adhesin exposed on the surface of Plasmodium falciparum-infected erythrocytes. Chondroitin sulfate A was identified as the main host receptor in the placenta. Single-domain heavy-chain camelid antibodies, more commonly called nanobodies, were selected and produced against the DBL6∊-FCR3 domain of VAR2CSA. Crystals of two specific nanobodies, Nb2907 and Nb2919, identified as strong binders to DBL6∊-FCR3 and the full-length VAR2CSA exposed on the surface of FCR3 P. falciparum-infected erythrocytes, were obtained. Crystals of Nb2907 diffract to 2.45 Å resolution and belong to space group C2 with unit-cell parameters a = 136.1, b = 78.5, c = 103.4 Å, β = 118.8°, whereas Nb2919 crystals diffract to 2.15 Å resolution and belong to space group P4 3 2 1 2 with unit-cell parameters a = b = 62.7, c = 167.2 Å

  3. A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy

    NARCIS (Netherlands)

    Kijanka, M; van Donselaar, E G; Müller, W H; Dorresteijn, B; Popov-Celeketic, Dusan; El Khattabi, M; Verrips, C T; van Bergen En Henegouwen, P M P; Post, J A

    Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind

  4. Molecular dynamics simulations and docking enable to explore the biophysical factors controlling the yields of engineered nanobodies

    Science.gov (United States)

    Soler, Miguel A.; De Marco, Ario; Fortuna, Sara

    2016-10-01

    Nanobodies (VHHs) have proved to be valuable substitutes of conventional antibodies for molecular recognition. Their small size represents a precious advantage for rational mutagenesis based on modelling. Here we address the problem of predicting how Camelidae nanobody sequences can tolerate mutations by developing a simulation protocol based on all-atom molecular dynamics and whole-molecule docking. The method was tested on two sets of nanobodies characterized experimentally for their biophysical features. One set contained point mutations introduced to humanize a wild type sequence, in the second the CDRs were swapped between single-domain frameworks with Camelidae and human hallmarks. The method resulted in accurate scoring approaches to predict experimental yields and enabled to identify the structural modifications induced by mutations. This work is a promising tool for the in silico development of single-domain antibodies and opens the opportunity to customize single functional domains of larger macromolecules.

  5. Intraoperative fluorescence delineation of head and neck cancer with a fluorescent anti-epidermal growth factor receptor nanobody.

    Science.gov (United States)

    van Driel, P B A A; van der Vorst, J R; Verbeek, F P R; Oliveira, S; Snoeks, T J A; Keereweer, S; Chan, B; Boonstra, M C; Frangioni, J V; van Bergen en Henegouwen, P M P; Vahrmeijer, A L; Lowik, C W G M

    2014-06-01

    Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections. © 2013 UICC.

  6. Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor.

    Science.gov (United States)

    Solt, Andras S; Bostock, Mark J; Shrestha, Binesh; Kumar, Prashant; Warne, Tony; Tate, Christopher G; Nietlispach, Daniel

    2017-11-27

    A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., G s and β-arrestin. Using 13 C methyl methionine NMR for the β 1 -adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with G s -mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody-receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.

  7. Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

    Science.gov (United States)

    Li, Dongyang; Cui, Yongliang; Morisseau, Christophe; Gee, Shirley J; Bever, Candace S; Liu, Xiangjiang; Wu, Jian; Hammock, Bruce D; Ying, Yibin

    2017-06-06

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.

  8. Peptide Based Targeted Therapeutic Radiopharmaceuticals: A Focus on the Synthesis of Radiolabelled Nanobodies

    International Nuclear Information System (INIS)

    Impens, N.; Campsteyn, A.; Aerts, A.; Baatout, S.; Devoogdt, N.; Caveliers, V.; Xavier, C.; Lahoutte, T.

    2009-01-01

    In 1993, the Vrije Universiteit Brussel (Brussels, Belgium) discovered in the blood of camelidae antibodies consisting of only a heavy chain. Due to the lack of the light chain only the variable part of the heavy chain is important for antigen binding. This variable part of these heavy-chain-only antibodies is a good candidate as a targeted therapeutic radiopharmaceutical and was called a nanobody, having a molecular weight of about 15 kDa. Its dimensions are included in between the small peptides like derived from e.g. somatostatin, and the classical monoclonal antibodies. This makes that some characteristics like the physical behaviour, the chemical stability, the penetration in tumour and in healthy tissues, and the blood clearance lie in between the characteristics of the small peptides and the monoclonal antibodies, therefore taking advantage of both extremes. Nanobodies have been humanised to decrease the immunogenic response. The building blocks of molecules such as the octreotide, nanobodies and monoclonal antibodies are amino acids linked via peptide bonds. The modification reactions are therefore all based on the same 'peptide chemistry'. The functional groups on the present amino acids will determine the possible reactions. In order to link a radionuclide to the nanobodies, we opted to use bifunctional ligands containing DOTA, because this is a suitable chelating agent for the diagnostic radionuclide Ga-68, and for therapeutic radionuclides such as Lu-177 and Y-90, covering short and long range β-particle emitters suitable for attacking a wide range of tumour sizes. The ratio of bifunctional ligand to nanobody can be varied by carefully selecting the functional groups of the peptide involved in the reaction with the bifunctional ligand, avoiding the complementarity determining region (CDR), i.e. the part of the molecule binding to the antigen. This is a first way to predetermine the amount of radionuclides that can be linked to the peptide, or the

  9. Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential.

    Science.gov (United States)

    Yu, Yuan; Li, Jialu; Zhu, Xuejun; Tang, Xiaowen; Bao, Yangyi; Sun, Xiang; Huang, Yuhui; Tian, Fang; Liu, Xiaomei; Yang, Lin

    2017-01-01

    Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdc em26 Il2rg em26 Nju (NCG) mice

  10. A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

    Science.gov (United States)

    Bethuyne, Jonas; De Gieter, Steven; Zwaenepoel, Olivier; Garcia-Pino, Abel; Durinck, Kaat; Verhelle, Adriaan; Hassanzadeh-Ghassabeh, Gholamreza; Speleman, Frank; Loris, Remy; Gettemans, Jan

    2014-01-01

    The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations. PMID:25324313

  11. EGFR targeted nanobody-photosensitizer conjugates for photodynamic therapy in a pre-clinical model of head and neck cancer.

    Science.gov (United States)

    van Driel, Pieter B A A; Boonstra, Martin C; Slooter, Maxime D; Heukers, Raimond; Stammes, Marieke A; Snoeks, Thomas J A; de Bruijn, Henriette S; van Diest, Paul J; Vahrmeijer, Alexander L; van Bergen En Henegouwen, Paul M P; van de Velde, Cornelis J H; Löwik, Clemens W G M; Robinson, Dominic J; Oliveira, Sabrina

    2016-05-10

    Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

    Science.gov (United States)

    Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

    2014-01-01

    The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

  13. Dual color single particle tracking via nanobodies

    International Nuclear Information System (INIS)

    Albrecht, David; Winterflood, Christian M; Ewers, Helge

    2015-01-01

    Single particle tracking is a powerful tool to investigate the function of biological molecules by following their motion in space. However, the simultaneous tracking of two different species of molecules is still difficult to realize without compromising the length or density of trajectories, the localization accuracy or the simplicity of the assay. Here, we demonstrate a simple dual color single particle tracking assay using small, bright, high-affinity labeling via nanobodies of accessible targets with widely available instrumentation. We furthermore apply a ratiometric step-size analysis method to visualize differences in apparent membrane viscosity. (paper)

  14. Production, crystallization and preliminary X-ray diffraction of the Gαs α-helical domain in complex with a nanobody.

    Science.gov (United States)

    Triest, Sarah; Wohlkönig, Alexandre; Pardon, Els; Steyaert, Jan

    2014-11-01

    GPCR-G-protein complexes are one of the most important components of cell-signalling cascades. Extracellular signals are sensed by membrane-associated G-protein-coupled receptors (GPCRs) and transduced via G proteins towards intracellular effector molecules. Structural studies of these transient complexes are crucial to understand the molecular details of these interactions. Although a nucleotide-free GPCR-G-protein complex is stable, it is not an ideal sample for crystallization owing to the intrinsic mobility of the Gαs α-helical domain (AHD). To stabilize GPCR-G-protein complexes in a nucleotide-free form, nanobodies were selected that target the flexible GαsAHD. One of these nanobodies, CA9177, was co-crystallized with the GαsAHD. Initial crystals were obtained using the sitting-drop method in a sparse-matrix screen and further optimized. The crystals diffracted to 1.59 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=44.07, b=52.55, c=52.66 Å, α=90.00, β=107.89, γ=90.00°. The structure of this specific nanobody reveals its binding epitope on GαsAHD and will help to determine whether this nanobody could be used as crystallization chaperone for GPCR-G-protein complexes.

  15. A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

    Science.gov (United States)

    Nüske, Stefan; Scholz, Armin M.; Bogner, Jacqueline; Ruf, Benjamin; Zolghadr, Kourosh; Drexler, Sophie E.; Drexler, Guido A.; Girst, Stefanie; Greubel, Christoph; Reindl, Judith; Siebenwirth, Christian; Romer, Tina; Friedl, Anna A.; Rothbauer, Ulrich

    2016-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair. PMID:26950694

  16. A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.

    Directory of Open Access Journals (Sweden)

    Andrea Buchfellner

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP1 is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa, termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

  17. A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.

    Science.gov (United States)

    Buchfellner, Andrea; Yurlova, Larisa; Nüske, Stefan; Scholz, Armin M; Bogner, Jacqueline; Ruf, Benjamin; Zolghadr, Kourosh; Drexler, Sophie E; Drexler, Guido A; Girst, Stefanie; Greubel, Christoph; Reindl, Judith; Siebenwirth, Christian; Romer, Tina; Friedl, Anna A; Rothbauer, Ulrich

    2016-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

  18. Analysis of Nanobody-Epitope Interactions in Living Cells via Quantitative Protein Transport Assays.

    Science.gov (United States)

    Früholz, Simone; Pimpl, Peter

    2017-01-01

    Over the past few decades, quantitative protein transport analyses have been used to elucidate the sorting and transport of proteins in the endomembrane system of plants. Here, we have applied our knowledge about transport routes and the corresponding sorting signals to establish an in vivo system for testing specific interactions between soluble proteins.Here, we describe the use of quantitative protein transport assays in tobacco mesophyll protoplasts to test for interactions occurring between a GFP-binding nanobody and its GFP epitope. For this, we use a secreted GFP-tagged α-amylase as a reporter together with a vacuolar-targeted RFP-tagged nanobody. The interaction between these proteins is then revealed by a transport alteration of the secretory reporter due to the interaction-triggered attachment of the vacuolar sorting signal.

  19. Development of oligoclonal nanobodies for targeting the tumor-associated glycoprotein 72 antigen

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali

    2013-01-01

    The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specific...

  20. Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential

    Directory of Open Access Journals (Sweden)

    Yu Y

    2017-03-01

    Full Text Available Yuan Yu,1–3 Jialu Li,1–3 Xuejun Zhu,4 Xiaowen Tang,2,5 Yangyi Bao,6 Xiang Sun,6 Yuhui Huang,1,2 Fang Tian,4 Xiaomei Liu,1,2 Lin Yang1–3 1The Cyrus Tang Hematology Center, 2Collaborative Innovation Center of Hematology, Soochow University, 3Suzhou Cancer Immunotherapy and Diagnosis Engineering Center, Suzhou, 4Central Laboratory, Department of Hematology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing, 5Department of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, 6Department of Hematology-Oncology, The First People’s Hospital of Hefei, Hefei, People’s Republic of China Background: Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies], are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6 as well as further truncated the Pseudomonas exotoxin A (PE-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Methods and results: Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that

  1. Facile labelling of an anti-epidermal growth factor receptor nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET

    International Nuclear Information System (INIS)

    Vosjan, Maria J.W.D.; Perk, Lars R.; Stigter van Walsum, Marijke; Roovers, Rob C.; Bergen en Henegouwen, Paul M.P. van; Visser, Gerard W.M.; Dongen, Guus A.M.S. van

    2011-01-01

    The ∝15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies registered ) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with 68 Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for 89 Zr immuno-positron emission tomography (PET). Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified 68 Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml -1 gentisic acid, pH 5.5) at 4 C or in human serum at 37 C, a mixture of 67 Ga and 68 Ga was used. Biodistribution and immuno-PET studies of 68 Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using 89 Zr-Df-Bz-NCS-7D12 as the reference conjugate. The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall 68 Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. 68/67 Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period ( 68 Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate 89 Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study. Via a rapid

  2. Affinity enhancement of nanobody binding to EGFR: in silico site-directed mutagenesis and molecular dynamics simulation approaches.

    Science.gov (United States)

    Farasat, Alireza; Rahbarizadeh, Fatemeh; Hosseinzadeh, Ghader; Sajjadi, Sharareh; Kamali, Mehdi; Keihan, Amir Homayoun

    2017-06-01

    Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, is overexpressed in many cancers such as head-neck, breast, prostate, and skin cancers for this reason it is a good target in cancer therapy and diagnosis. In nanobody-based cancer diagnosis and treatment, nanobodies with high affinity toward receptor (e.g. EGFR) results in effective treatment or diagnosis of cancer. In this regard, the main aim of this study is to develop a method based on molecular dynamic (MD) simulations for designing of 7D12 based nanobody with high affinity compared with wild-type nanobody. By surveying electrostatic and desolvation interactions between different residues of 7D12 and EGFR, the critical residues of 7D12 that play the main role in the binding of 7D12 to EGFR were elucidated and based on these residues, five logical variants were designed. Following the 50 ns MD simulations, pull and umbrella sampling simulation were performed for 7D12 and all its variants in complex with EGFR. Binding free energy of 7D12 (and all its variants) with EGFR was obtained by weighted histogram analysis method. According to binding free energy results, GLY101 to GLU mutation showed the highest binding affinity but this variant is unstable after 50 ns MD simulations. ALA100 to GLU mutation shows suitable binding enhancement with acceptable structural stability. Suitable position and orientation of GLU in residue 100 of 7D12 against related amino acids of EGFR formed some extra hydrogen and electrostatic interactions which resulted in binding enhancement.

  3. Na-no-body : De l’oubli du corps sensible en nanomédecine

    Directory of Open Access Journals (Sweden)

    Noury, Mathieu

    2016-09-01

    Full Text Available This article presents the ethical reflexion of a recent art project, Na-no-body, presented at the Montreal gallery Espace Projet in the context of the exposition Art + Bioéthique. Na-no-body is the result of a collaborative work between the artist Stephanie Coleman and the sociologist Mathieu Noury. The aim of this project was to open an ethical dialogue on nanomedicine and its relationship with the body. To do so, the article examines specifically the notion of “personalized medicine” promoted by nanomedicine. This article proposes that, far from developing an approach reintegrating the person and the personal experience of illness at the heart of the care relationship, nanomedicine brings a simple molecular and technical response to caring. It is argued that two major themes constitute the core of this notion: 1 a molecular conception of personalization, and 2 a technical conception of personalization.

  4. A novel photoelectrochemical immunosensor by integration of nanobody and TiO2 nanotubes for sensitive detection of serum cystatin C

    International Nuclear Information System (INIS)

    Mi, Li; Wang, Pingyan; Yan, Junrong; Qian, Jing; Lu, Jusheng; Yu, Jiachao; Wang, Yuzhen; Liu, Hong; Zhu, Min; Wan, Yakun; Liu, Songqin

    2016-01-01

    Cystatin C (CysC) is a sensitive marker for the estimation of the glomerular filtration rate and the clinical diagnosis of different diseases. In this paper, CysC-specific nanobodies (Nbs) were isolated from a phage display nanobody library. A simple and sensitive photoelectrochemical immunosensor based on TiO 2 nanotube arrays (TNAs) was proposed for the sensitive detection of CysC. The TiO 2 nanotube arrays deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation. After coupling CysC-specific nanobody to TNA (Nb/TNA), the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb. This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM. The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM–3.6 nM. The immunosensor had a correlation coefficient of 0.97 and a detection limit of 0.14 pM at a signal-to-noise ratio of 3. The proposed immunosensor showed satisfactory intra- and inter-assay accuracy, high selectivity and good stability. As a result, this proposed strategy would offer a novel and simple approach for the detection of immunoreactions, provide new insights in popularizing the diagnosis of CysC, and extend the application of TiO 2 nanotubes. - Highlights: • CysC-specific nanobody to CysC is isolated from phage display nanobody library. • A photoelectrochemical immunosensor for CysC develops by Nb modified TNA. • An excellent sensitivity and good selectivity of CysC sensing was obtained.

  5. Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline

    International Nuclear Information System (INIS)

    Zhu, Min; Li, Guanghui; Li, Min; Zhou, Zikai; Liu, Hong; Lei, Hongtao; Shen, Yanfei; Wan, Yakun

    2015-01-01

    We describe an electrochemical immunoassay for the Cry1Ab toxin that is produced by Bacillus thuringiensis. It is making use of a nanobody (a heavy-chain only antibody) that was selected from an immune phage displayed library. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline. PANI can be easily detected by differential pulse voltammetry at a working voltage as low as 40 mV (vs. Ag/AgCl) which makes the assay fairly selective. This immunoassay for Cry1Ab has an analytical range from 0.1 to 1000 ng∙mL -1 and a 0.07 ng∙mL -1 lower limit of detection. The average recoveries of the toxin from spiked samples are in the range from 102 to 114 %, with a relative standard deviation of <7.5 %. The results demonstrated that the assay represented an attractive alternative to existing immunoassays in enabling affordable, sensitive, robust and specific determination of this toxin. (author)

  6. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

    Directory of Open Access Journals (Sweden)

    Daria A Burmistrova

    Full Text Available Developing pathogen-specific recombinant antibody fragments (especially nanobodies is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh, for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection.

  7. Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET.

    Science.gov (United States)

    Vosjan, Maria J W D; Perk, Lars R; Roovers, Rob C; Visser, Gerard W M; Stigter-van Walsum, Marijke; van Bergen En Henegouwen, Paul M P; van Dongen, Guus A M S

    2011-04-01

    The ∼15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies®) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with (68)Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for (89)Zr immuno-positron emission tomography (PET). Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified (68)Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml(-1) gentisic acid, pH 5.5) at 4°C or in human serum at 37°C, a mixture of (67)Ga and (68)Ga was used. Biodistribution and immuno-PET studies of (68)Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using (89)Zr-Df-Bz-NCS-7D12 as the reference conjugate. The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall (68)Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. (68/67)Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate (89)Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study. Via a rapid

  8. Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis.

    Science.gov (United States)

    Duhoo, Yoan; Roche, Jennifer; Trinh, Thi Trang Nhung; Desmyter, Aline; Gaubert, Anaïs; Kellenberger, Christine; Cambillau, Christian; Roussel, Alain; Leone, Philippe

    2017-05-01

    PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) in Porphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

  9. A Novel ¹¹¹In-Labeled Anti-Prostate-Specific Membrane Antigen Nanobody for Targeted SPECT/CT Imaging of Prostate Cancer.

    Science.gov (United States)

    Chatalic, Kristell L S; Veldhoven-Zweistra, Joke; Bolkestein, Michiel; Hoeben, Sander; Koning, Gerben A; Boerman, Otto C; de Jong, Marion; van Weerden, Wytske M

    2015-07-01

    Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single-domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high target specificity and rapid background clearance. We developed a novel anti-PSMA Nanobody (JVZ-007) for targeted imaging and therapy of PCa. Here, we report on the application of the (111)In-radiolabeled Nanobody for SPECT/CT imaging of PCa. A Nanobody library was generated by immunization of a llama with 4 human PCa cell lines. Anti-PSMA Nanobodies were captured by biopanning on PSMA-overexpressing cells. JVZ-007 was selected for evaluation as an imaging probe. JVZ-007 was initially produced with a c-myc-hexahistidine (his) tag allowing purification and detection. The c-myc-his tag was subsequently replaced by a single cysteine at the C terminus, allowing site-specific conjugation of chelates for radiolabeling. JVZ-007-c-myc-his was conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-DTPA) via the lysines, whereas JVZ-007-cys was conjugated to maleimide-DTPA via the C-terminal cysteine. PSMA targeting was analyzed in vitro by cell-binding experiments using flow cytometry, autoradiography, and internalization assays with various PCa cell lines and patient-derived xenografts (PDXs). The targeting properties of radiolabeled Nanobodies were evaluated in vivo in biodistribution and SPECT/CT imaging experiments, using nude mice bearing PSMA-positive PC-310 and PSMA-negative PC-3 tumors. JVZ-007 was successfully conjugated to DTPA for radiolabeling with (111)In at room temperature. (111)In-JVZ007-c-myc-his and (111)In-JVZ007-cys internalized in LNCaP cells and bound to PSMA-expressing PDXs and, importantly, not to PSMA-negative PDXs and human kidneys. Good tumor targeting and fast blood clearance were observed for (111)In-JVZ-007-c-myc-his and (111)In

  10. A potent complement factor C3 specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

    DEFF Research Database (Denmark)

    Jensen, Rasmus K; Pihl, Rasmus; Gadeberg, Trine A F

    2018-01-01

    The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance and inhibition of the complement alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds...... to multiple functional states of C3 with sub-nanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable to C3, and hC3Nb1 is shown to prevent both proconvertase assembly as well as binding of the C3 substrate...... to C3 convertases. Our crystal structure of the C3b-hC3Nb1 complex and functional experiments demonstrate that proconvertase formation is blocked by steric hindrance between the nanobody and an Asn-linked glycan on complement factor B. In addition, hC3Nb1 is shown to prevent factor H binding to C3b...

  11. A novel photoelectrochemical immunosensor by integration of nanobody and TiO{sub 2} nanotubes for sensitive detection of serum cystatin C

    Energy Technology Data Exchange (ETDEWEB)

    Mi, Li [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); Wang, Pingyan; Yan, Junrong [Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 (China); Qian, Jing; Lu, Jusheng; Yu, Jiachao [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); Wang, Yuzhen; Liu, Hong [Plexera LLC, WA 98072 (United States); Zhu, Min [Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 (China); Wan, Yakun, E-mail: ywansystemsbiology@gmail.com [Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 (China); Jiangsu Nanobody Engineering and Research Center, Nantong 226010 (China); Liu, Songqin, E-mail: liusq@seu.edu.cn [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China)

    2016-01-01

    Cystatin C (CysC) is a sensitive marker for the estimation of the glomerular filtration rate and the clinical diagnosis of different diseases. In this paper, CysC-specific nanobodies (Nbs) were isolated from a phage display nanobody library. A simple and sensitive photoelectrochemical immunosensor based on TiO{sub 2} nanotube arrays (TNAs) was proposed for the sensitive detection of CysC. The TiO{sub 2} nanotube arrays deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation. After coupling CysC-specific nanobody to TNA (Nb/TNA), the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb. This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM. The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM–3.6 nM. The immunosensor had a correlation coefficient of 0.97 and a detection limit of 0.14 pM at a signal-to-noise ratio of 3. The proposed immunosensor showed satisfactory intra- and inter-assay accuracy, high selectivity and good stability. As a result, this proposed strategy would offer a novel and simple approach for the detection of immunoreactions, provide new insights in popularizing the diagnosis of CysC, and extend the application of TiO{sub 2} nanotubes. - Highlights: • CysC-specific nanobody to CysC is isolated from phage display nanobody library. • A photoelectrochemical immunosensor for CysC develops by Nb modified TNA. • An excellent sensitivity and good selectivity of CysC sensing was obtained.

  12. Facile labelling of an anti-epidermal growth factor receptor nanobody with {sup 68}Ga via a novel bifunctional desferal chelate for immuno-PET

    Energy Technology Data Exchange (ETDEWEB)

    Vosjan, Maria J.W.D.; Perk, Lars R.; Stigter van Walsum, Marijke [VU University Medical Center, Department of Otolaryngology/Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); Roovers, Rob C.; Bergen en Henegouwen, Paul M.P. van [Utrecht University, Cellular Dynamics, Science Faculty, Utrecht (Netherlands); Visser, Gerard W.M. [VU University Medical Center, Department of Nuclear Medicine and PET Research, Amsterdam (Netherlands); Dongen, Guus A.M.S. van [VU University Medical Center, Department of Otolaryngology/Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); VU University Medical Center, Department of Nuclear Medicine and PET Research, Amsterdam (Netherlands)

    2011-04-15

    The {proportional_to}15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies {sup registered}) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with {sup 68}Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for {sup 89}Zr immuno-positron emission tomography (PET). Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified {sup 68}Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml{sup -1} gentisic acid, pH 5.5) at 4 C or in human serum at 37 C, a mixture of {sup 67}Ga and {sup 68}Ga was used. Biodistribution and immuno-PET studies of {sup 68}Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using {sup 89}Zr-Df-Bz-NCS-7D12 as the reference conjugate. The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall {sup 68}Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. {sup 68/67}Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (<2% radioactivity loss). In biodistribution studies the {sup 68}Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 {+-} 1.3 to 7.2 {+-} 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was

  13. Ultrasonic Nanobubbles Carrying Anti-PSMA Nanobody: Construction and Application in Prostate Cancer-Targeted Imaging.

    Directory of Open Access Journals (Sweden)

    Xiaozhou Fan

    Full Text Available To facilitate prostate cancer imaging using targeted molecules, we constructed ultrasonic nanobubbles coupled with specific anti-PSMA (prostate specific membrane antigen nanobodies, and evaluated their in vitro binding capacity and in vivo imaging efficacy. The "targeted" nanobubbles, which were constructed via a biotin-streptavidin system, had an average diameter of 487.60 ± 33.55 nm and carried the anti-PSMA nanobody as demonstrated by immunofluorescence. Microscopy revealed targeted binding of nanobubbles in vitro to PSMA-positive cells. Additionally, ultrasonography indicators of nanobubble imaging (including arrival time, peak time, peak intensity and enhanced duration were evaluated for the ultrasound imaging in three kinds of animal xenografts (LNCaP, C4-2 and MKN45, and showed that these four indicators of targeted nanobubbles exhibited significant differences from blank nanobubbles. Therefore, this study not only presents a novel approach to target prostate cancer ultrasonography, but also provides the basis and methods for constructing small-sized and high-efficient targeted ultrasound nanobubbles.

  14. Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

    Science.gov (United States)

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

    2016-01-01

    A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

  15. In vivo immunotherapy of lung cancer using cross-species reactive vascular endothelial growth factor nanobodies

    Directory of Open Access Journals (Sweden)

    vFatemeh Kazemi-Lomedasht v

    2017-05-01

    Full Text Available Objective(s: Lung cancer is the main leading cause of cancer death worldwide. Angiogenesis is the main step in proliferation and spreading of tumor cells. Targeting vascular endothelial growth factor (VEGF is an effective approach for inhibition of cancer angiogenesis. Nanobodies (NBs are a novel class of antibodies derived from the camel. Unique characteristics of Nbs like their small size and good penetration to tumor tissues makes them promising tools in drug development.  Development of NBs targeting both human and mouse VEGF is required for understanding their in vivo functions.  Therefore, development of cross-species reactive anti-VEGF Nbs for immunotherapy of lung cancer was the main aim of the current study. Materials and Methods: Here we developed NBs from Camelus dromedarius library with high specificity and binding affinity to both human and mouse VEGF. In vitro and In vivo function of developed NB was evaluated on human endothelial cells and lung epithelial tumor cells (TC-1. Results: A nanobody showed the highest affinity to human and mouse VEGF and potently inhibited VEGF in the ELISA experiment. Anti-VEGF NBs significantly inhibited in vitro human endothelial cell migration through blockade of VEGF (P=0.045. Anti-VEGF NBs also significantly inhibited in vivo TC-1 growth in a dose-dependent manner (P=0.001 and resulted in higher survival rate in the nanobody treated group Conclusion: These findings demonstrate the potential of anti-VEGF NBsin tumor growth inhibition and are promising as novel cancer therapeutic candidate.

  16. A novel 111In-labeled anti-PSMA nanobody for targeted SPECT/CT imaging of prostate cancer

    NARCIS (Netherlands)

    Chatalic, K.L.S.; Veldhoven-Zweistra, J.; Bolkestein, M.; Hoeben, S.; Koning, G.A.; Boerman, O.C.; Jong, M. de; Weerden, W.M. van

    2015-01-01

    Prostate-specific Membrane Antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high target

  17. Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade.

    Science.gov (United States)

    Zhang, Fei; Wei, Hudie; Wang, Xiaoxiao; Bai, Yu; Wang, Pilin; Wu, Jiawei; Jiang, Xiaoyong; Wang, Yugang; Cai, Haiyan; Xu, Ting; Zhou, Aiwu

    2017-01-01

    The use of antibodies to target immune checkpoints, particularly PD-1/PD-L1, has made a profound impact in the field of cancer immunotherapy. Here, we identified KN035, an anti-PD-L1 nanobody that can strongly induce T-cell responses and inhibit tumor growth. The crystal structures of KN035 complexed with PD-L1 and free PD-L1, solved here at 1.7 and 2.7 Å resolution, respectively, show that KN035 competes with PD-1 (programmed death protein 1) for the same flat surface on PD-L1, mainly through a single surface loop of 21 amino acids. This loop forms two short helices and develops key hydrophobic and ionic interactions with PD-L1 residues, such as Ile54, Tyr56 and Arg113, which are also involved in PD-1 binding. The detailed mutagenesis study identified the hotspot residues of the PD-L1 surface and provides an explanation for the stronger (~1 000-fold) binding of KN035 to PD-L1 than PD-1 and its lack of binding to PD-L2. Overall, this study reveals how a single immunoglobulin-variable scaffold of KN035 or PD-1 can bind to a flat protein surface through either a single surface loop or beta-sheet strands; and provides a basis for designing new immune checkpoint blockers and generating bi-specific antibodies for combination therapy.

  18. Sindbis Virus-Pseudotyped Lentiviral Vectors Carrying VEGFR2-Specific Nanobody for Potential Transductional Targeting of Tumor Vasculature.

    Science.gov (United States)

    Ahani, Roshank; Roohvand, Farzin; Cohan, Reza Ahangari; Etemadzadeh, Mohammad Hossein; Mohajel, Nasir; Behdani, Mahdi; Shahosseini, Zahra; Madani, Navid; Azadmanesh, Kayhan

    2016-11-01

    Introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (LVs), is crucial in their systemic administration for cancer gene therapy. The pivotal role of tumor-associated endothelial cells (TAECs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (VEGFR2 or KDR) in TAECs makes them a potent target in cancer treatment. Herein, we report the development of VEGFR2-targeted LVs pseudotyped with chimeric sindbis virus E2 glycoprotein (cSVE2s). For this purpose, either sequence of a VEGFR2-specific nanobody or its natural ligand (VEGF 121 ) was inserted into the binding site of sindbis virus E2 glycoprotein. In silico modeling data suggested that the inserted targeting motifs were exposed in the context of cSVE2s. Western blot analysis of LVs indicated the incorporation of cSVE2s into viral particles. Capture ELISA demonstrated the specificity/functionality of the incorporated cSVE2s. Transduction of 293/KDR (expressing VEGFR2) or 293T cells (negative control) by constructed LVs followed by fluorescent microscopy and flow cytometric analyses indicated selective transduction of 293/KDR cells (30 %) by both targeting motifs compared to 293T control cells (1-2 %). These results implied similar targeting properties of VEGFR2-specific nanobody compared to the VEGF 121 and indicated the potential for transductional targeting of tumor vasculature by the nanobody displaying LVs.

  19. A potent complement factor C3 specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement.

    Science.gov (United States)

    Jensen, Rasmus K; Pihl, Rasmus; Gadeberg, Trine A F; Jensen, Jan K; Andersen, Kasper R; Thiel, Steffen; Laursen, Nick S; Andersen, Gregers Rom

    2018-03-01

    The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance and inhibition of the complement alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds to multiple functional states of C3 with sub-nanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable to C3, and hC3Nb1 is shown to prevent both proconvertase assembly as well as binding of the C3 substrate to C3 convertases. Our crystal structure of the C3b-hC3Nb1 complex and functional experiments demonstrate that proconvertase formation is blocked by steric hindrance between the nanobody and an Asn-linked glycan on complement factor B. In addition, hC3Nb1 is shown to prevent factor H binding to C3b rationalizing its inhibition of factor I activity. Our results identify hC3Nb1 as a versatile, inexpensive, and powerful inhibitor of the alternative pathway in both human and murine in vitro model systems of complement activation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Screening and antitumor effect of an anti-CTLA-4 nanobody

    Science.gov (United States)

    Wan, Ruirong; Liu, Aiqun; Hou, Xiaoqiong; Lai, Zongqiang; Li, Jieping; Yang, Nuo; Tan, Juntao; Mo, Fengzhen; Hu, Zixi; Yang, Xiaomei; Zhao, Yongxiang; Lu, Xiaoling

    2018-01-01

    Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a critical negative regulator of immune responses. CTLA-4 is rapidly upregulated following T-cell activation, and then binds to B7 molecules with a higher affinity than CD28. CTLA-4 may abolish the initiation of the responses of T cells by raising the threshold of signals required for full activation of T cells, and it also may terminate ongoing T-cell responses. This regulatory role has led to the development of monoclonal antibodies (mAbs) designed to block CTLA-4 activity for enhancing immune responses against cancer. mAbs have several disadvantages including high production cost and unstable behavior. Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelid heavy-chain antibodies, which are highly attractive in cancer immunotherapy due to their small size, high specificity, and stability. We selected CTLA-4-specific Nbs from a high quality dromedary camel immune library by phage display technology. Four positive colonies were sequenced and classified based on the amino acids sequences in the CDR3 region. These Nbs recognized unique epitopes on CTLA-4 and displayed high binding rates when used on PHA-stimulated human T cells. Treatment of B16 melanoma-bearing C57BL/6 mice with anti-CTLA-4 nanobody 16 (Nb16) delayed melanoma growth and prolonged the survival time of mice. These data indicate that anti-CTLA-4 Nbs selected from a high quality phage display library may be effective for the treatment of patients with tumors. PMID:29207143

  1. Intraoperative fluorescence delineation of head and neck cancer with a fluorescent Anti-epidermal growth factor receptor nanobody

    NARCIS (Netherlands)

    Van Driel, P.B.A.A.; Van Der Vorst, J.R.; Verbeek, F.P.R.; Oliveira, S.|info:eu-repo/dai/nl/304841455; Snoeks, T.J.A.; Keereweer, S.; Chan, B.; Boonstra, M.C.; Frangioni, J.V.; Van Bergen En Henegouwen, P.M.P.|info:eu-repo/dai/nl/071919481; Vahrmeijer, A.L.; Lowik, C.W.G.M.

    2014-01-01

    Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for

  2. Camelid immunoglobulins and nanobody technology.

    Science.gov (United States)

    Muyldermans, S; Baral, T N; Retamozzo, V Cortez; De Baetselier, P; De Genst, E; Kinne, J; Leonhardt, H; Magez, S; Nguyen, V K; Revets, H; Rothbauer, U; Stijlemans, B; Tillib, S; Wernery, U; Wyns, L; Hassanzadeh-Ghassabeh, Gh; Saerens, D

    2009-03-15

    It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

  3. Quantum-Dot-Based Theranostic Micelles Conjugated with an Anti-EGFR Nanobody for Triple-Negative Breast Cancer Therapy.

    Science.gov (United States)

    Wang, Yuyuan; Wang, Yidan; Chen, Guojun; Li, Yitong; Xu, Wei; Gong, Shaoqin

    2017-09-13

    A quantum-dot (QD)-based micelle conjugated with an anti-epidermal growth factor receptor (EGFR) nanobody (Nb) and loaded with an anticancer drug, aminoflavone (AF), has been engineered for EGFR-overexpressing cancer theranostics. The near-infrared (NIR) fluorescence of the indium phosphate core/zinc sulfide shell QDs (InP/ZnS QDs) allowed for in vivo nanoparticle biodistribution studies. The anti-EGFR nanobody 7D12 conjugation improved the cellular uptake and cytotoxicity of the QD-based micelles in EGFR-overexpressing MDA-MB-468 triple-negative breast cancer (TNBC) cells. In comparison with the AF-encapsulated nontargeted (i.e., without Nb conjugation) micelles, the AF-encapsulated Nb-conjugated (i.e., targeted) micelles accumulated in tumors at higher concentrations, leading to more effective tumor regression in an orthotopic triple-negative breast cancer xenograft mouse model. Furthermore, there was no systemic toxicity observed with the treatments. Thus, this QD-based Nb-conjugated micelle may serve as an effective theranostic nanoplatform for EGFR-overexpressing cancers such as TNBCs.

  4. Energy profile of nanobody-GFP complex under force

    Science.gov (United States)

    Klamecka, Kamila; Severin, Philip M.; Milles, Lukas F.; Gaub, Hermann E.; Leonhardt, Heinrich

    2015-10-01

    Nanobodies (Nbs)—the smallest known fully functional and naturally occuring antigen-binding fragments—have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb-green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that—despite identical epitopes—the Nb binds stronger (41-56 pN) to enhanced GFP than to wild-type GFP (28-45 pN). Measured forces make the Nb-GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo.

  5. Energy profile of nanobody-GFP complex under force.

    Science.gov (United States)

    Klamecka, Kamila; Severin, Philip M; Milles, Lukas F; Gaub, Hermann E; Leonhardt, Heinrich

    2015-09-10

    Nanobodies (Nbs)-the smallest known fully functional and naturally occuring antigen-binding fragments-have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb-green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that-despite identical epitopes-the Nb binds stronger (41-56 pN) to enhanced GFP than to wild-type GFP (28-45 pN). Measured forces make the Nb-GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo.

  6. Evaluation of an Anti-HER2 Nanobody Labeled with 225Ac for Targeted α-Particle Therapy of Cancer.

    Science.gov (United States)

    Pruszynski, Marek; D'Huyvetter, Matthias; Bruchertseifer, Frank; Morgenstern, Alfred; Lahoutte, Tony

    2018-04-02

    Human epidermal growth factor receptor type 2 (HER2) is overexpressed in numerous carcinomas. Nanobodies (Nbs) are the smallest antibody-derived fragments with beneficial characteristics for molecular imaging and radionuclide therapy. Therefore, HER2-targeting nanobodies could offer a valuable platform for radioimmunotherapy, especially when labeled with α-particle emitters, which provide highly lethal and localized radiation to targeted cells with minimal exposure to surrounding healthy tissues. In this study, the anti-HER2 2Rs15d-nanobody was conjugated with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( p-SCN-Bn-DOTA) and radiolabeled with an α-emitter 225 Ac with a high yield (>90%) and a radiochemical purity above 95%. The 225 Ac-DOTA-Nb binding affinity was 4.12 ± 0.47 nM with an immunoreactive fraction above 80%. Binding to low HER2-expressing MDA-MB-231 cells was negligible, whereas HER2-overexpressing SKOV-3 cells could be blocked with an excess of unlabeled nanobody, confirming the specificity of binding. Noncompeting binding to HER2 was observed in the presence of an excess of trastuzumab. The cell-associated fraction of 225 Ac-DOTA-Nb was 34.72 ± 16.66% over 24 h. In vitro, the radioconjugate was toxic in an HER2-mediated and dose-dependent manner, resulting in IC 50 values of 10.2 and 322.1 kBq/mL for 225 Ac-DOTA-Nb and the 225 Ac-DOTA control, respectively, on SKOV-3 cells, and 282.2 kBq/mL for 225 Ac-DOTA-Nb on MDA-MB-231 cells. Ex vivo biodistribution studies, performed in mice bearing subcutaneous HER2-overexpressing and low HER2-expressing tumors, showed a fast uptake in SKOV-3 tumors compared to MDA-MB-231 (4.01 ± 1.58% ID/g vs 0.49 ± 0.20% ID/g after 2 h), resulting also in high tumor-to-normal tissue ratios. In addition, coinjection of 225 Ac-DOTA-Nb with Gelofusine reduced kidney retention by 70%. This study shows that 225 Ac-DOTA-Nb is a promising new radioconjugate for targeted α-particle therapy

  7. Rapid Visualization of Human Tumor Xenografts through Optical Imaging with a Near-Infrared Fluorescent Anti–Epidermal Growth Factor Receptor Nanobody

    Directory of Open Access Journals (Sweden)

    Sabrina Oliveira

    2012-01-01

    Full Text Available Given that overexpression of the epidermal growth factor receptor (EGFR is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.

  8. Homology modeling and docking of AahII-Nanobody complexes reveal the epitope binding site on AahII scorpion toxin.

    Science.gov (United States)

    Ksouri, Ayoub; Ghedira, Kais; Ben Abderrazek, Rahma; Shankar, B A Gowri; Benkahla, Alia; Bishop, Ozlem Tastan; Bouhaouala-Zahar, Balkiss

    2018-02-19

    Scorpion envenoming and its treatment is a public health problem in many parts of the world due to highly toxic venom polypeptides diffusing rapidly within the body of severely envenomed victims. Recently, 38 AahII-specific Nanobody sequences (Nbs) were retrieved from which the performance of NbAahII10 nanobody candidate, to neutralize the most poisonous venom compound namely AahII acting on sodium channels, was established. Herein, structural computational approach is conducted to elucidate the Nb-AahII interactions that support the biological characteristics, using Nb multiple sequence alignment (MSA) followed by modeling and molecular docking investigations (RosettaAntibody, ZDOCK software tools). Sequence and structural analysis showed two dissimilar residues of NbAahII10 CDR1 (Tyr27 and Tyr29) and an inserted polar residue Ser30 that appear to play an important role. Indeed, CDR3 region of NbAahII10 is characterized by a specific Met104 and two negatively charged residues Asp115 and Asp117. Complex dockings reveal that NbAahII17 and NbAahII38 share one common binding site on the surface of the AahII toxin divergent from the NbAahII10 one's. At least, a couple of NbAahII10 - AahII residue interactions (Gln38 - Asn44 and Arg62, His64, respectively) are mainly involved in the toxic AahII binding site. Altogether, this study gives valuable insights in the design and development of next generation of antivenom. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

    Science.gov (United States)

    Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

    2017-09-01

    The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Utilizing nanobody technology to target non-immunodominant domains of VAR2CSA.

    Directory of Open Access Journals (Sweden)

    Sisse B Ditlev

    Full Text Available Placental malaria is a major health problem for both pregnant women and their fetuses in malaria endemic regions. It is triggered by the accumulation of Plasmodium falciparum-infected erythrocytes (IE in the intervillous spaces of the placenta and is associated with foetal growth restriction and maternal anemia. IE accumulation is supported by the binding of the parasite-expressed protein VAR2CSA to placental chondroitin sulfate A (CSA. Defining specific CSA-binding epitopes of VAR2CSA, against which to target the immune response, is essential for the development of a vaccine aimed at blocking IE adhesion. However, the development of a VAR2CSA adhesion-blocking vaccine remains challenging due to (i the large size of VAR2CSA and (ii the extensive immune selection for polymorphisms and thereby non-neutralizing B-cell epitopes. Camelid heavy-chain-only antibodies (HcAbs are known to target epitopes that are less immunogenic to classical IgG and, due to their small size and protruding antigen-binding loop, able to reach and recognize cryptic, conformational epitopes which are inaccessible to conventional antibodies. The variable heavy chain (VHH domain is the antigen-binding site of camelid HcAbs, the so called Nanobody, which represents the smallest known (15 kDa intact, native antigen-binding fragment. In this study, we have used the Nanobody technology, an approach new to malaria research, to generate small and functional antibody fragments recognizing unique epitopes broadly distributed on VAR2CSA.

  11. Structural evaluation of a nanobody targeting complement receptor Vsig4 and its cross reactivity.

    Science.gov (United States)

    Wen, Yurong; Ouyang, Zhenlin; Schoonooghe, Steve; Luo, Siyu; De Baetselier, Patrick; Lu, Wuyuan; Muyldermans, Serge; Raes, Geert; Zheng, Fang

    2017-06-01

    Vsig4 is a recently identified immune regulatory protein related to the B7 family with dual functionality: a negative regulator of T cell activation and a receptor for the complement components C3b and C3c. Here we present a structural evaluation of a nanobody, Nb119, against the extracellular IgV domain protein of both mouse and human recombinant Vsig4, which have a high degree of sequence identity. Although mouse and human Vsig4 bind to Nb119 with a 250 times difference in dissociation constants, the interaction results in a highly identical assembly with a RMSD of 0.4Å. The molecular determinants for Vsig4 recognition and cross reactivity unveiled by the atomic structure of Nb119 in complex with mVsig4 and hVsig4 afford new insights useful for the further optimization of the nanobody for potential use in humans. Additionally, structural analysis of the Vsig4-Nb119 complexes indicates that Nb119 occupies the interface on Vsig4 recognized by the macroglobulin-like domains MG4 and MG5 of C3b. Thus an affinity-improved Nb119 may have the potential to influence the activation of both T cells and complement. Copyright © 2016. Published by Elsevier GmbH.

  12. A novel photoelectrochemical immunosensor by integration of nanobody and TiO₂ nanotubes for sensitive detection of serum cystatin C.

    Science.gov (United States)

    Mi, Li; Wang, Pingyan; Yan, Junrong; Qian, Jing; Lu, Jusheng; Yu, Jiachao; Wang, Yuzhen; Liu, Hong; Zhu, Min; Wan, Yakun; Liu, Songqin

    2016-01-01

    Cystatin C (CysC) is a sensitive marker for the estimation of the glomerular filtration rate and the clinical diagnosis of different diseases. In this paper, CysC-specific nanobodies (Nbs) were isolated from a phage display nanobody library. A simple and sensitive photoelectrochemical immunosensor based on TiO2 nanotube arrays (TNAs) was proposed for the sensitive detection of CysC. The TiO2 nanotube arrays deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation. After coupling CysC-specific nanobody to TNA (Nb/TNA), the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb. This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM. The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM-3.6 nM. The immunosensor had a correlation coefficient of 0.97 and a detection limit of 0.14 pM at a signal-to-noise ratio of 3. The proposed immunosensor showed satisfactory intra- and inter-assay accuracy, high selectivity and good stability. As a result, this proposed strategy would offer a novel and simple approach for the detection of immunoreactions, provide new insights in popularizing the diagnosis of CysC, and extend the application of TiO2 nanotubes. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP

    Science.gov (United States)

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain and multiple cardiovascular related diseases. A variable domain of a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses advantages of small size, high ...

  14. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    Science.gov (United States)

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  15. Improved Debulking of Peritoneal Tumor Implants by Near-Infrared Fluorescent Nanobody Image Guidance in an Experimental Mouse Model.

    Science.gov (United States)

    Debie, Pieterjan; Vanhoeij, Marian; Poortmans, Natalie; Puttemans, Janik; Gillis, Kris; Devoogdt, Nick; Lahoutte, Tony; Hernot, Sophie

    2017-10-31

    Debulking followed by combination chemotherapy is currently regarded as the most effective treatment for advanced ovarian cancer. Prognosis depends drastically on the degree of debulking. Accordingly, near-infrared (NIR) fluorescence imaging has been proposed to revolutionize cancer surgery by acting as a sensitive, specific, and real-time tool enabling visualization of cancer lesions. We have previously developed a NIR-labeled nanobody that allows fast, specific, and high-contrast imaging of HER2-positive tumors. In this study, we applied this tracer during fluorescence-guided surgery in a mouse model and investigated the effect on surgical efficiency. 0.5 × 10 6 SKOV3.IP1-Luc+ cells were inoculated intraperitoneally in athymic mice and were allowed to grow for 30 days. Two nanomoles of IRDye800CW-anti-HER2 nanobody was injected intravenously. After 1h30, mice were killed, randomized in two groups, and subjected to surgery. In the first animal group (n = 7), lesions were removed by a conventional surgical protocol, followed by excision of remaining fluorescent tissue using a NIR camera. The second group of mice (n = 6) underwent directly fluorescence-guided surgery. Bioluminescence imaging was performed before and after surgery. Resected tissue was categorized as visualized during conventional surgery or not, fluorescent or not, and bioluminescent positive or negative. Fluorescence imaging allowed clear visualization of tumor nodules within the abdomen, up to submillimeter-sized lesions. Fluorescence guidance resulted in significantly reduced residual tumor as compared to conventional surgery. Moreover, sensitivity increased from 59.3 to 99.0 %, and the percentage of false positive lesions detected decreased from 19.6 to 7.1 %. This study demonstrates the advantage of intraoperative fluorescence imaging using nanobody-based tracers on the efficiency of debulking surgery.

  16. Discovery of Selective Nanobodies against α-elapitoxin Dpp2c from Black Mamba through Phage Display Screening

    DEFF Research Database (Denmark)

    Milbo, Christina; Laustsen, Andreas Hougaard; Lohse, Brian

    Feared for its highly neurotoxic venom and rapid attack technique, the Black mamba (Dendroaspis polylepis) is Africa’s largest venomous snake. The clinical manifestations of a bitefrom D. polylepis include flaccid paralysis leading to respiratory failure and death due to postsynaptic blockade of ......-neurotoxins. Here, we report the discovery of selective nanobodies targeting α-elapitoxin Dpp2c from D. polylepis through phage display screening....

  17. Expression and extracellular release of a functional anti-trypanosome Nanobody® in Sodalis glossinidius, a bacterial symbiont of the tsetse fly

    Directory of Open Access Journals (Sweden)

    De Vooght Linda

    2012-02-01

    Full Text Available Abstract Background Sodalis glossinidius, a gram-negative bacterial endosymbiont of the tsetse fly, has been proposed as a potential in vivo drug delivery vehicle to control trypanosome parasite development in the fly, an approach known as paratransgenesis. Despite this interest of S. glossinidius as a paratransgenic platform organism in tsetse flies, few potential effector molecules have been identified so far and to date none of these molecules have been successfully expressed in this bacterium. Results In this study, S. glossinidius was transformed to express a single domain antibody, (Nanobody® Nb_An33, that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG of the parasite Trypanosoma brucei. Next, we analyzed the capability of two predicted secretion signals to direct the extracellular delivery of significant levels of active Nb_An33. We show that the pelB leader peptide was successful in directing the export of fully functional Nb_An33 to the periplasm of S. glossinidius resulting in significant levels of extracellular release. Finally, S. glossinidius expressing pelBNb_An33 exhibited no significant reduction in terms of fitness, determined by in vitro growth kinetics, compared to the wild-type strain. Conclusions These data are the first demonstration of the expression and extracellular release of functional trypanosome-interfering Nanobodies® in S. glossinidius. Furthermore, Sodalis strains that efficiently released the effector protein were not affected in their growth, suggesting that they may be competitive with endogenous microbiota in the midgut environment of the tsetse fly. Collectively, these data reinforce the notion for the potential of S. glossinidius to be developed into a paratransgenic platform organism.

  18. Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function

    OpenAIRE

    Fatemeh Kazemi-Lomedasht; serge muyldermans; Mahdi Habibi-Anbouhi; Mahdi Behdani

    2018-01-01

    Objective(s): Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.   Materials and Methods: Here, we designed a humanize...

  19. Nanobodies As Novel Agents for Targeting Angiogenesis in Solid Cancers

    Directory of Open Access Journals (Sweden)

    Roghaye Arezumand

    2017-12-01

    Full Text Available Solid cancers are dependent on angiogenesis for sustenance. The FDA approval of Bevacizumab in 2004 inspired many scientists to develop more inhibitors of angiogenesis. Although several monoclonal antibodies (mAbs are being administered to successfully combat various pathologies, the complexity and large size of mAbs seem to narrow the therapeutic applications. To improve the performance of cancer therapeutics, including those blocking tumor angiogenesis, attractive strategies such as miniaturization of the antibodies have been introduced. Nanobodies (Nbs, small single-domain antigen-binding antibody fragments, are becoming promising therapeutic and diagnostic proteins in oncology due to their favorable unique structural and functional properties. This review focuses on the potential and state of the art of Nbs to inhibit the angiogenic process for therapy and the use of labeled Nbs for non-invasive in vivo imaging of the tumors.

  20. Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning.

    Directory of Open Access Journals (Sweden)

    Tiziano Gaiotto

    Full Text Available Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu, 2009 H1N1 pandemic (swine flu and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our

  1. Safety, tolerability, pharmacokinetics and pharmacodynamics of anti-VWF nanobody® ALX-0681 after single and multiple subcutaneous administrations to healthy volunteers

    NARCIS (Netherlands)

    Abd-Elaziz, Khalid; Kamphuisen, Pieter W; Lyssens, Christophe; Reuvers, Mariska; Den Daas, Izaak; Van Bockstaele, Femke; Vercruysse, Kristof; Ulrichts, Hans; Baumeister, Judith; Crabbe, Patricia; Compernolle, Veerle; Holz, Josefin-Beate

    2009-01-01

    ALX-0681 is a humanized bivalent Nanobody®, that binds to the A1 domain of von Willebrand factor (vWF) and hence blocks its interaction with platelet receptor GPIb-IX-V. Given its mode of action, ALX-0681 could provide an alternative treatment option for thrombotic thrombocytopenic purpura (TTP), a

  2. Novel CD7-specific nanobody-based immunotoxins potently enhanced apoptosis of CD7-positive malignant cells.

    Science.gov (United States)

    Tang, Jinle; Li, Jialu; Zhu, Xuejun; Yu, Yuan; Chen, Dan; Yuan, Lei; Gu, Zhenyang; Zhang, Xingding; Qi, Lin; Gong, Zhishu; Jiang, Pengjun; Yu, Juhua; Meng, Huimin; An, Gangli; Zheng, Huyong; Yang, Lin

    2016-06-07

    Various CD7-targeting immunotoxins have been tested for its potential in treating CD7+ malignant patients but none of those immunotoxins was approved clinically because of lacking enough efficacy and safety. Here we successfully constructed the monovalent and bivalent CD7 nanobody-based immunotoxins PG001 and PG002, both conjugated with a truncated derivative of Pseudomonas exotoxin A respectively. The prokaryotic system expressed immunotoxins not only maintained their binding specificity for CD7-positive cells with a Kd of 16.74 nM and 3.6 nM for PG001 and PG002 respectively, but also efficiently promoted antigen-restricted apoptosis of the CD7-positive leukemia cell lines Jurkat and CEM, and primary T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells with an in vitro cytotoxic activity (EC50) in the range of 23-30 pM for PG002. In NOD/SCID mice transplanted with CEM cells, PG001 and PG002 prevented engraftment of the cells and markedly prolonged mouse survival. Owing to the efficient antigen-restricted anti-leukemic activity of PG002, this CD7 nanobody-based immunotoxin exhibited a superior anti-CD7 positive malignancies activity than previously reported immunotoxins, and may represent a promising therapeutic strategy in treating CD7-positive leukemia and lymphoma, which still remain a significant clinical challenge.

  3. Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma.

    Science.gov (United States)

    Keyaerts, Marleen; Xavier, Catarina; Heemskerk, Johannes; Devoogdt, Nick; Everaert, Hendrik; Ackaert, Chloé; Vanhoeij, Marian; Duhoux, Francois P; Gevaert, Thierry; Simon, Philippe; Schallier, Denis; Fontaine, Christel; Vaneycken, Ilse; Vanhove, Christian; De Greve, Jacques; Lamote, Jan; Caveliers, Vicky; Lahoutte, Tony

    2016-01-01

    Human epidermal growth factor receptor 2 (HER2) status is one of the major tumor characteristics in breast cancer to guide therapy. Anti-HER2 treatment has clear survival advantages in HER2-positive breast carcinoma patients. Heterogeneity in HER2 expression between primary tumor and metastasis has repeatedly been described, resulting in the need to reassess HER2 status during the disease course. To avoid repeated biopsy with potential bias due to tumor heterogeneity, Nanobodies directed against HER2 have been developed as probes for molecular imaging. Nanobodies, which are derived from unique heavy-chain-only antibodies, are the smallest antigen-binding antibody fragments and have ideal characteristics for PET imaging. The primary aims were assessment of safety, biodistribution, and dosimetry. The secondary aim was to investigate tumor-targeting potential. In total, 20 women with primary or metastatic breast carcinoma (score of 2+ or 3+ on HER2 immunohistochemical assessment) were included. Anti-HER2-Nanobody was labeled with (68)Ga via a NOTA derivative. Administered activities were 53-174 MBq (average, 107 MBq). PET/CT scans for dosimetry assessment were obtained at 10, 60, and 90 min after administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor-targeting potential was assessed in primary and metastatic lesions. No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h after injection. Uptake was seen mainly in the kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall, with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above the background level was demonstrated in most identified sites of

  4. In vivo detection of small tumour lesions by multi-pinhole SPECT applying a (99m)Tc-labelled nanobody targeting the Epidermal Growth Factor Receptor.

    Science.gov (United States)

    Krüwel, Thomas; Nevoltris, Damien; Bode, Julia; Dullin, Christian; Baty, Daniel; Chames, Patrick; Alves, Frauke

    2016-02-25

    The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody (99m)Tc-D10 for visualizing small tumour lesions with volumes below 100 mm(3) by targeting EGFR in orthotopic human mammary MDA-MB-468 and MDA-MB-231 and subcutaneous human epidermoid A431 carcinoma mouse models. Use of nanobody (99m)Tc-D10 of a size as small as 15.5 kDa enables detection of tumours by single photon emission computed tomography (SPECT) imaging already 45 min post intravenous administration with high tumour uptake (>3% ID/g) in small MDA-MB-468 and A431 tumours, with tumour volumes of 52.5 mm(3) ± 21.2 and 26.6 mm(3) ± 16.7, respectively. Fast blood clearance with a serum half-life of 4.9 min resulted in high in vivo contrast and ex vivo tumour to blood and tissue ratios. In contrast, no accumulation of (99m)Tc-D10 in MDA-MB-231 tumours characterized by a very low expression of EGFR was observed. Here we present specific and high contrast in vivo visualization of small human tumours overexpressing EGFR by preclinical multi-pinhole SPECT shortly after administration of anti-EGFR nanobody (99m)Tc-D10.

  5. In vivo detection of small tumour lesions by multi-pinhole SPECT applying a 99mTc-labelled nanobody targeting the Epidermal Growth Factor Receptor

    Science.gov (United States)

    Krüwel, Thomas; Nevoltris, Damien; Bode, Julia; Dullin, Christian; Baty, Daniel; Chames, Patrick; Alves, Frauke

    2016-01-01

    The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody 99mTc-D10 for visualizing small tumour lesions with volumes below 100 mm3 by targeting EGFR in orthotopic human mammary MDA-MB-468 and MDA-MB-231 and subcutaneous human epidermoid A431 carcinoma mouse models. Use of nanobody 99mTc-D10 of a size as small as 15.5 kDa enables detection of tumours by single photon emission computed tomography (SPECT) imaging already 45 min post intravenous administration with high tumour uptake (>3% ID/g) in small MDA-MB-468 and A431 tumours, with tumour volumes of 52.5 mm3 ± 21.2 and 26.6 mm3 ± 16.7, respectively. Fast blood clearance with a serum half-life of 4.9 min resulted in high in vivo contrast and ex vivo tumour to blood and tissue ratios. In contrast, no accumulation of 99mTc-D10 in MDA-MB-231 tumours characterized by a very low expression of EGFR was observed. Here we present specific and high contrast in vivo visualization of small human tumours overexpressing EGFR by preclinical multi-pinhole SPECT shortly after administration of anti-EGFR nanobody 99mTc-D10. PMID:26912069

  6. Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET.

    Science.gov (United States)

    Vaidyanathan, Ganesan; McDougald, Darryl; Choi, Jaeyeon; Koumarianou, Eftychia; Weitzel, Douglas; Osada, Takuya; Lyerly, H Kim; Zalutsky, Michael R

    2016-06-01

    The human growth factor receptor type 2 (HER2) is overexpressed in breast as well as other types of cancer. Immuno-PET, a noninvasive imaging procedure that could assess HER2 status in both primary and metastatic lesions simultaneously, could be a valuable tool for optimizing application of HER2-targeted therapies in individual patients. Herein, we have evaluated the tumor-targeting potential of the 5F7 anti-HER2 Nanobody (single-domain antibody fragment; ∼13 kDa) after (18)F labeling by 2 methods. The 5F7 Nanobody was labeled with (18)F using the novel residualizing label N-succinimidyl 3-((4-(4-(18)F-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ((18)F-SFBTMGMB; (18)F-RL-I) and also via the most commonly used (18)F protein-labeling prosthetic agent N-succinimidyl 3-(18)F-fluorobenzoate ((18)F-SFB). For comparison, 5F7 Nanobody was also labeled using the residualizing radioiodination agent N-succinimidyl 4-guanidinomethyl-3-(125)I-iodobenzoate ((125)I-SGMIB). Paired-label ((18)F/(125)I) internalization assays and biodistribution studies were performed on HER2-expressing BT474M1 breast carcinoma cells and in mice with BT474M1 subcutaneous xenografts, respectively. Small-animal PET/CT imaging of 5F7 Nanobody labeled using (18)F-RL-I also was performed. Internalization assays indicated that intracellularly retained radioactivity for (18)F-RL-I-5F7 was similar to that for coincubated (125)I-SGMIB-5F7, whereas that for (18)F-SFB-5F7 was lower than coincubated (125)I-SGMIB-5F7 and decreased with time. BT474M1 tumor uptake of (18)F-RL-I-5F7 was 28.97 ± 3.88 percentage injected dose per gram of tissue (%ID/g) at 1 h and 36.28 ± 14.10 %ID/g at 2 h, reduced by more than 90% on blocking with trastuzumab, indicating HER2 specificity of uptake, and was also 26%-28% higher (P < 0.05) than that of (18)F-SFB-5F7. At 2 h, the tumor-to-blood ratio for (18)F-RL-I-5F7 (47.4 ± 13.1) was significantly higher (P < 0.05) than for (18)F-SFB-5F7 (25.4 ± 10

  7. Introducing site-specific cysteines into nanobodies for mercury labelling allows de novo phasing of their crystal structures

    DEFF Research Database (Denmark)

    Hansen, Simon Boje; Laursen, Nick Stub; Andersen, Gregers Rom

    2017-01-01

    of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend...... phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys-Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its...

  8. A bispecific nanobody approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2-T cells.

    Science.gov (United States)

    de Bruin, Renée C G; Veluchamy, John P; Lougheed, Sinéad M; Schneiders, Famke L; Lopez-Lastra, Silvia; Lameris, Roeland; Stam, Anita G; Sebestyen, Zsolt; Kuball, Jürgen; Molthoff, Carla F M; Hooijberg, Erik; Roovers, Rob C; Santo, James P Di; van Bergen En Henegouwen, Paul M P; Verheul, Henk M W; de Gruijl, Tanja D; van der Vliet, Hans J

    2017-01-01

    Though Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitro and in an in vivo mouse xenograft model. Tumor cell lysis was independent of KRAS and BRAF tumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients.

  9. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

    Science.gov (United States)

    Eden, Thomas; Menzel, Stephan; Wesolowski, Janusz; Bergmann, Philine; Nissen, Marion; Dubberke, Gudrun; Seyfried, Fabienne; Albrecht, Birte; Haag, Friedrich; Koch-Nolte, Friedrich

    2018-01-01

    Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic

  10. A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

    Science.gov (United States)

    2018-01-01

    Polyclonal anti–immunoglobulin G (anti-IgG) secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class. PMID:29263082

  11. Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

    Science.gov (United States)

    Schenck, Stephan; Kunz, Laura; Sahlender, Daniela; Pardon, Els; Geertsma, Eric R; Savtchouk, Iaroslav; Suzuki, Toshiharu; Neldner, Yvonne; Štefanić, Saša; Steyaert, Jan; Volterra, Andrea; Dutzler, Raimund

    2017-08-01

    The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

  12. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies

    DEFF Research Database (Denmark)

    Staus, Dean P; Wingler, Laura M; Strachan, Ryan T

    2014-01-01

    . However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate...... their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed...... on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies...

  13. Limiting the protein corona: A successful strategy for in vivo active targeting of anti-HER2 nanobody-functionalized nanostars.

    Science.gov (United States)

    D'Hollander, Antoine; Jans, Hilde; Velde, Greetje Vande; Verstraete, Charlotte; Massa, Sam; Devoogdt, Nick; Stakenborg, Tim; Muyldermans, Serge; Lagae, Liesbet; Himmelreich, Uwe

    2017-04-01

    Gold nanoparticles hold great promise as anti-cancer theranostic agents against cancer by actively targeting the tumor cells. As this potential has been supported numerously during in vitro experiments, the effective application is hampered by our limited understanding and control of the interactions within complex in vivo biological systems. When these nanoparticles are exposed to a biological environment, their surfaces become covered with proteins and biomolecules, referred to as the protein corona, reducing the active targeting capabilities. We demonstrate a chemical strategy to overcome this issue by reducing the protein corona's thickness by blocking the active groups of the self-assembled monolayer on gold nanostars. An optimal blocking agent, 2-mercapto ethanol, has been selected based on charge and length of the carbon chain. By using a nanobody as a biological ligand of the human epidermal growth factor 2 receptor (HER2), the active targeting is demonstrated in vitro and in vivo in an experimental tumor model by using darkfield microscopy and photoacoustic imaging. In this study, we have established gold nanostars as a conceivable theranostic agent with a specificity for HER2-positive tumors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ.

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    David C Butler

    Full Text Available Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn are closely associated with synucleinopathies, including Parkinson's disease (PD. VH14 is a human single domain intrabody selected against the non-amyloid component (NAC hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.

  15. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

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    Thomas Eden

    2018-01-01

    Full Text Available Nanobodies (Nbs are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully

  16. Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab.

    Science.gov (United States)

    Galli, Valentina; Sebastian, Rafael; Moutel, Sandrine; Ecard, Jason; Perez, Franck; Roux, Aurélien

    2017-10-12

    Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.

  17. A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila.

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    Harmansa, Stefan; Alborelli, Ilaria; Bieli, Dimitri; Caussinus, Emmanuel; Affolter, Markus

    2017-04-11

    The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. We show that the localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, we used the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.

  18. Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting.

    Science.gov (United States)

    Pansieri, Jonathan; Plissonneau, Marie; Stransky-Heilkron, Nathalie; Dumoulin, Mireille; Heinrich-Balard, Laurence; Rivory, Pascaline; Morfin, Jean-François; Toth, Eva; Saraiva, Maria Joao; Allémann, Eric; Tillement, Olivier; Forge, Vincent; Lux, François; Marquette, Christel

    2017-07-01

    Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses. The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy. Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with K D values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues. Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy.

  19. Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library.

    Science.gov (United States)

    Xu, Chongxin; Liu, Xiaoqin; Zhang, Cunzheng; Zhang, Xiao; Zhong, Jianfeng; Liu, Yuan; Hu, Xiaodan; Lin, Manman; Liu, Xianjin

    2017-02-01

    Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL -1 and the medium inhibition of control (IC 50 ) was 0.73 ng mL -1 . It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%-96.6% and with a coefficient of variation (CV) among 2.0%-8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued.

    Science.gov (United States)

    van Lith, Sanne A M; Roodink, Ilse; Verhoeff, Joost J C; Mäkinen, Petri I; Lappalainen, Jari P; Ylä-Herttuala, Seppo; Raats, Jos; van Wijk, Erwin; Roepman, Ronald; Letteboer, Stef J; Verrijp, Kiek; Leenders, William P J

    2016-11-01

    Diffuse gliomas are primary brain cancers that are characterised by infiltrative growth. Whereas high-grade glioma characteristically presents with perinecrotic neovascularisation, large tumor areas thrive on pre-existent vasculature as well. Clinical studies have revealed that pharmacological inhibition of the angiogenic process does not improve survival of glioblastoma patients. Direct targeting of tumor vessels may however still be an interesting therapeutic approach as it allows pinching off the blood supply to tumor cells. Such tumor vessel targeting requires the identification of tumor-specific vascular targeting agents (TVTAs).Here we describe a novel TVTA, C-C7, which we identified via in vivo biopanning of a llama nanobody phage display library in an orthotopic mouse model of diffuse glioma. We show that C-C7 recognizes a subpopulation of tumor blood vessels in glioma xenografts and clinical glioma samples. Additionally, C-C7 recognizes macrophages and activated endothelial cells in atherosclerotic lesions. By using C-C7 as bait in yeast-2-hybrid (Y2H) screens we identified dynactin-1-p150Glued as its binding partner. The interaction was confirmed by co-immunostainings with C-C7 and a commercial anti-dynactin-1-p150Glued antibody, and via co-immunoprecipitation/western blot studies. Normal brain vessels do not express dynactin-1-p150Glued and its expression is reduced under anti-VEGF therapy, suggesting that dynactin-1-p150Glued is a marker for activated endothelial cells.In conclusion, we show that in vivo phage display combined with Y2H screenings provides a powerful approach to identify tumor-targeting nanobodies and their binding partners. Using this combination of methods we identify dynactin-1-p150Glued as a novel targetable protein on activated endothelial cells and macrophages.

  1. A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

    Science.gov (United States)

    Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

    2010-10-01

    Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

  2. Nanobodies As Tools to Understand, Diagnose, and Treat African Trypanosomiasis

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    Benoit Stijlemans

    2017-06-01

    Full Text Available African trypanosomes are strictly extracellular protozoan parasites that cause diseases in humans and livestock and significantly affect the economic development of sub-Saharan Africa. Due to an elaborate and efficient (vector–parasite–host interplay, required to complete their life cycle/transmission, trypanosomes have evolved efficient immune escape mechanisms that manipulate the entire host immune response. So far, not a single field applicable vaccine exists, and chemotherapy is the only strategy available to treat the disease. Current therapies, however, exhibit high drug toxicity and an increased drug resistance is being reported. In addition, diagnosis is often hampered due to the inadequacy of current diagnostic procedures. In the context of tackling the shortcomings of current treatment and diagnostic approaches, nanobodies (Nbs, derived from the heavy chain-only antibodies of camels and llamas might represent unmet advantages compared to conventional tools. Indeed, the combination of their small size, high stability, high affinity, and specificity for their target and tailorability represents a unique advantage, which is reflected by their broad use in basic and clinical research to date. In this article, we will review and discuss (i diagnostic and therapeutic applications of Nbs that are being evaluated in the context of African trypanosomiasis, (ii summarize new strategies that are being developed to optimize their potency for advancing their use, and (iii document on unexpected properties of Nbs, such as inherent trypanolytic activities, that besides opening new therapeutic avenues, might offer new insight in hidden biological activities of conventional antibodies.

  3. Novel recombinant immunotoxin of EGFR specific nanobody fused with cucurmosin, construction and antitumor efficiency in vitro.

    Science.gov (United States)

    Deng, Cuimin; Xiong, Jiani; Gu, Xiaofan; Chen, Xiaoying; Wu, Shuifa; Wang, Zhe; Wang, Duanduan; Tu, Jinjin; Xie, Jieming

    2017-06-13

    Epidermal growth factor receptor (EGFR) overexpression is related to the increased aggressiveness, metastases, and poor prognosis in various cancers. In this study, we successfully constructed a new EGFR nanobody-based immunotoxin rE/CUS containing cucurmosin (CUS), The immunotoxin was expressed by prokaryotic system and we obtained a yield of 5 mg protein per liter expression medium. The percentage of it's binding ability totumor cell lines A549, HepG2, SW116, which highly expressed EGFR was 55.6%, 79.6% and 97.1%, respectively, but SW620 was only 4.45%. rE/CUS has the ability to bind A549, HepG2, SW116 cells specifically, and the antigen binding capability was not affected because of extra part of CUS component. The rE/CUS significantly inhibited the cell viability against EGFR over expression tumor cell lines in a dose-and time-dependent manner. Moreover, rE/CUS also induced apoptosis of HepG2 and A549 mightily. Our results demonstrate that rE/CUS is a potential therapeutic strategy for treating EGFR-positive solid tumors.

  4. Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody

    International Nuclear Information System (INIS)

    Pruszynski, Marek; Koumarianou, Eftychia; Vaidyanathan, Ganesan; Revets, Hilde; Devoogdt, Nick; Lahoutte, Tony; Zalutsky, Michael R.

    2013-01-01

    Introduction: With a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors. Methods: 5F7GGC Nb was radioiodinated with 125 I using Iodogen and with 131 I using the residualizing agent N ε -(3-[ 131 I]iodobenzoyl)-Lys 5 -N α -maleimido-Gly 1 -GEEEK ([ 131 I]IB-Mal-D-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations. Results: The radiochemical yields for Iodogen and [ 131 I]IB-Mal-D-GEEEK labeling were 83.6 ± 5.0% (n = 10) and 59.6 ± 9.4% (n = 15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [ 131 I]IB-Mal-D-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65 ± 0.61% ID/g vs. 2.92 ± 0.24% ID/g at 8 h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios. Conclusions: Taken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [ 131 I]IB-Mal-D-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT ( 129 I) and PET imaging ( 124 I) of patients with HER2-expressing tumors.

  5. Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain.

    Science.gov (United States)

    Nunes-Silva, Sofia; Gangnard, Stéphane; Vidal, Marta; Vuchelen, Anneleen; Dechavanne, Sebastien; Chan, Sherwin; Pardon, Els; Steyaert, Jan; Ramboarina, Stephanie; Chêne, Arnaud; Gamain, Benoît

    2014-12-09

    VAR2CSA stands today as the leading vaccine candidate aiming to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of pregnancy associated malaria (PAM). The rational design of an efficient VAR2CSA-based vaccine relies on a profound understanding of the molecular interactions associated with P. falciparum infected erythrocyte sequestration in the placenta. Following immunization of a llama with the full-length VAR2CSA recombinant protein, we have expressed and characterized a panel of 19 nanobodies able to recognize the recombinant VAR2CSA as well as the surface of erythrocytes infected with parasites originating from different parts of the world. Domain mapping revealed that a large majority of nanobodies targeted DBL1X whereas a few of them were directed towards DBL4ε, DBL5ε and DBL6ε. One nanobody targeting the DBL1X was able to recognize the native VAR2CSA protein of the three parasite lines tested. Furthermore, four nanobodies targeting DBL1X reproducibly inhibited CSA adhesion of erythrocytes infected with the homologous NF54-CSA parasite strain, providing evidences that DBL1X domain is part or close to the CSA binding site. These nanobodies could serve as useful tools to identify conserved epitopes shared between different variants and to characterize the interactions between VAR2CSA and CSA.

  6. Nanobody-Based Delivery Systems for Diagnosis and Targeted Tumor Therapy

    Directory of Open Access Journals (Sweden)

    Yaozhong Hu

    2017-11-01

    Full Text Available The development of innovative targeted therapeutic approaches are expected to surpass the efficacy of current forms of treatments and cause less damage to healthy cells surrounding the tumor site. Since the first development of targeting agents from hybridoma’s, monoclonal antibodies (mAbs have been employed to inhibit tumor growth and proliferation directly or to deliver effector molecules to tumor cells. However, the full potential of such a delivery strategy is hampered by the size of mAbs, which will obstruct the targeted delivery system to access the tumor tissue. By serendipity, a new kind of functional homodimeric antibody format was discovered in camelidae, known as heavy-chain antibodies (HCAbs. The cloning of the variable domain of HCAbs produces an attractive minimal-sized alternative for mAbs, referred to as VHH or nanobodies (Nbs. Apart from their dimensions in the single digit nanometer range, the unique characteristics of Nbs combine a high stability and solubility, low immunogenicity and excellent affinity and specificity against all possible targets including tumor markers. This stimulated the development of tumor-targeted therapeutic strategies. Some autonomous Nbs have been shown to act as antagonistic drugs, but more importantly, the targeting capacity of Nbs has been exploited to create drug delivery systems. Obviously, Nb-based targeted cancer therapy is mainly focused toward extracellular tumor markers, since the membrane barrier prevents antibodies to reach the most promising intracellular tumor markers. Potential strategies, such as lentiviral vectors and bacterial type 3 secretion system, are proposed to deliver target-specific Nbs into tumor cells and to block tumor markers intracellularly. Simultaneously, Nbs have also been employed for in vivo molecular imaging to diagnose diseased tissues and to monitor the treatment effects. Here, we review the state of the art and focus on recent developments with Nbs as

  7. Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

    Science.gov (United States)

    Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

    2018-03-25

    Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

  8. Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection

    Science.gov (United States)

    Stohr, Thomas; Palomo, Concepción; Piedra, Pedro A.; Gilbert, Brian E.; Mas, Vicente; Millar, Andrena; Power, Ultan F.; Stortelers, Catelijne; Allosery, Koen; Melero, José A.; Depla, Erik

    2015-01-01

    Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease. PMID:26438495

  9. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    International Nuclear Information System (INIS)

    Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J.

    2011-01-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  10. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Iri-Sofla, Farnoush Jafari [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [Center of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O (Denmark); Rasaee, Mohammad J. [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  11. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins.

    Directory of Open Access Journals (Sweden)

    Hadrien Peyret

    Full Text Available The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.

  12. Description of a nanobody-based competitive immunoassay to detect tsetse fly exposure.

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    Guy Caljon

    2015-02-01

    Full Text Available Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11 was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao. Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95% and positive predictive values (> 98% but higher negative predictive values and hence increased sensitivity (> 95% and accuracy (> 95%.We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

  13. Generation of Small Single Domain Nanobody Binders for Sensitive Detection of Testosterone by Electrochemical Impedance Spectroscopy.

    Science.gov (United States)

    Li, Guanghui; Zhu, Min; Ma, Lu; Yan, Junrong; Lu, Xiaoling; Shen, Yanfei; Wan, Yakun

    2016-06-08

    A phage display library of variable domain of the heavy chain only antibody or nanobody (Nb) was constructed after immunizing a bactrian camel with testosterone. With the smaller molecular size (15 kDa), improved solubility, good stability, high affinity, specificity, and lower immunogenicity, Nbs are a promising tool in the next generation of diagnosis and medical applications. Testosterone is a reproductive hormone, playing an important role in normal cardiac function and being the highly predictive marker for many diseases. Herein, a simple and sensitive immunosensor based on electrochemical impedance spectroscopy (EIS) and Nbs was successfully developed for the determination of testosterone. We successfully isolated the antitestosterone Nbs from an immune phage display library. Moreover, one of the Nbs was biotinylated according to in vivo BirA system, which showed the highest production yield and the most stable case. Further, the EIS immunosensor was set up for testosterone detection by applying the biotinylated antitestosterone Nb. As a result, the biosensor exhibited a linear working range from 0.05 to 5 ng mL(-1) with a detection limit of 0.045 ng mL(-1). In addition, the proposed immunosensor was successfully applied in determining testosterone in serum samples. In conclusion, the proposed immunosensor revealed high specificity of testosterone detection and showed as a potential approach for sensitive and accurate diagnosis of testosterone.

  14. Establishing Substantial Equivalence: Transcriptomics

    Science.gov (United States)

    Baudo, María Marcela; Powers, Stephen J.; Mitchell, Rowan A. C.; Shewry, Peter R.

    Regulatory authorities in Western Europe require transgenic crops to be substantially equivalent to conventionally bred forms if they are to be approved for commercial production. One way to establish substantial equivalence is to compare the transcript profiles of developing grain and other tissues of transgenic and conventionally bred lines, in order to identify any unintended effects of the transformation process. We present detailed protocols for transcriptomic comparisons of developing wheat grain and leaf material, and illustrate their use by reference to our own studies of lines transformed to express additional gluten protein genes controlled by their own endosperm-specific promoters. The results show that the transgenes present in these lines (which included those encoding marker genes) did not have any significant unpredicted effects on the expression of endogenous genes and that the transgenic plants were therefore substantially equivalent to the corresponding parental lines.

  15. Substantial Union or Substantial Distinction of Mind and Body in Descartes' Metaphysics

    Directory of Open Access Journals (Sweden)

    Fahime Jamei

    2009-01-01

    Full Text Available According to Descartes’ metaphysics there are two different kinds of substances in the world of creatures: “thinking substance” and “extended substance” or soul and matter. In Descartes’ philosophy the soul is equal to the mind and considered as a “thinking substance”. This immaterial substance is the essence of the human being. Body, being considered as a “matter“, is an “extended substance” and entirely distinct from the soul. The soul, therefore, exists and may be known prior to body and, not being corporeal, can exist after human death. Hence, Descartes can prove the immortality of human soul in the framework of the principle of substantial distinction. On the other hand, as a physiologist and psychologist, Descartes indeed believes in mind-body union, so that some causal interactions between mind and body show their substantial union. In this essay, the authors show that Descartes faces a serious problem in combining substantial union of mind and body with their substantial distinction; despite of his efforts in introducing the idea of pineal gland, the problem remains unsolved. Therefore it seems that as he cannot dispense with his only reason for proving the immortality of human soul, he has to hold the mind-body distinction theory in his metaphysics. Indeed, Descartes prefers to support the distinction theory rather than union theory in confronting a thesis and an antithesis stating one of two theories

  16. Substantial :union: or Substantial Distinction of Mind and Body in Descartes\\' Metaphysics

    Directory of Open Access Journals (Sweden)

    f Jamei

    2009-06-01

    Full Text Available According to Descartes’ metaphysics there are two different kinds of substances in the world of creatures: “thinking substance” and “extended substance” or soul and matter. In Descartes’ philosophy the soul is equal to the mind and considered as a “thinking substance”. This immaterial substance is the essence of the human being. Body, being considered as a “matter“, is an “extended substance” and entirely distinct from the soul. The soul, therefore, exists and may be known prior to body and, not being corporeal, can exist after human death. Hence, Descartes can prove the immortality of human soul in the framework of the principle of substantial distinction. On the other hand, as a physiologist and psychologist, Descartes indeed believes in mind-body :union:, so that some causal interactions between mind and body show their substantial :union:. In this essay, the authors show that Descartes faces a serious problem in combining substantial :union: of mind and body with their substantial distinction despite of his efforts in introducing the idea of pineal gland, the problem remains unsolved. Therefore it seems that as he cannot dispense with his only reason for proving the immortality of human soul, he has to hold the mind-body distinction theory in his metaphysics. Indeed, Descartes prefers to support the distinction theory rather than :union: theory in confronting a thesis and an antithesis stating one of two theories.

  17. 24 CFR 902.79 - Substantial default.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Substantial default. 902.79 Section... PUBLIC HOUSING ASSESSMENT SYSTEM PHAS Incentives and Remedies § 902.79 Substantial default. (a) Events or conditions that constitute substantial default. The following events or conditions shall constitute...

  18. Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis.

    Science.gov (United States)

    Arezumand, Roghaye; Mahdian, Reza; Zeinali, Sirous; Hassanzadeh-Ghassabeh, Gholamreza; Mansouri, Kamran; Khanahmad, Hossein; Namvar-Asl, Nabiollah; Rahimi, Hamzeh; Behdani, Mahdi; Cohan, Reza Ahangari; Eavazalipour, Mehdi; Ramazani, Ali; Muyldermans, Serge

    2016-10-01

    Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×10 7 individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p<0.05) with an EC 50 of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC 50 of57nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. [AntiEGFRnano inhibites proliferation and migration of estrogen-dependent Ishikawa cells of human endometrial cancer cell line].

    Science.gov (United States)

    Diao, Zhen-yu; Lu, Wu-guang; Cao, Peng; Hu, Yun-long; Zhou, Xing; Xue, Ping-ping; Shen, Li; Sun, Hai-xiang

    2012-10-01

    Nanobody is a kind of antibody from camel, which misses light chain. Nanobody has the same antigen binding specificity and affinity as mAb. Moreover, because of its small molecular weight, high stability and easy preparation, nanobody has great value of biomedical applications. In this study, we successfully prepared highly pure antiEGFR nanobody in E.coli using genetic engineering techniques. Cell proliferation assay (CCK-8 assay) and migration experiments (cell scratch test and Transwell assay) indicated that the recombinant antiEGFRnano can significantly inhibit the proliferation and migration of endometrial cancer cells. These results provide a new way of thinking and methods for EGFR-targeted therapy of endometrial cancer.

  20. Dynamics of graphite flake on a liquid

    Science.gov (United States)

    Miura, K.; Tsuda, D.; Kaneta, Y.; Harada, R.; Ishikawa, M.; Sasaki, N.

    2006-11-01

    One-directional motion, where graphite flakes are driven by a nanotip on an octamethylcyclotetrasiloxane (OMCTS) liquid surface, is presented. A transition from quasiperiodic to chaotic motions occurs in the dynamics of a graphite flake when its velocity is increased. The dynamics of graphite flakes pulled by the nanotip on an OMCTS liquid surface can be treated as that of a nanobody on a liquid.

  1. Principles of geological substantiation for toxic waste disposal facilities sites selection

    International Nuclear Information System (INIS)

    Khrushchov, D. P.; Matorin, Eu. M.; Shekhunova, S. B.

    2002-01-01

    Industrial, domestic and military activities result in accumulation of toxic and hazardous waste. Disposal of these waste comprises two main approaches: technological processing (utilization and destruction) and landfill. According to concepts and programs of advanced countries technological solutions are preferable, but in fact over 70 % of waste are buried in storages, prevailingly of near surface type. The target of this paper is to present principles of geological substantiation of sites selection for toxic and hazardous waste isolation facilities location. (author)

  2. 20 CFR 404.1675 - Finding of substantial failure.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Finding of substantial failure. 404.1675... DISABILITY INSURANCE (1950- ) Determinations of Disability Substantial Failure § 404.1675 Finding of substantial failure. A finding of substantial failure with respect to a State may not be made unless and until...

  3. 20 CFR 416.1075 - Finding of substantial failure.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Finding of substantial failure. 416.1075... AGED, BLIND, AND DISABLED Determinations of Disability Substantial Failure § 416.1075 Finding of substantial failure. A finding of substantial failure with respect to a State may not be made unless and until...

  4. Substantiation of rate setting of surface contamination with amino acids, labelled with tritium

    International Nuclear Information System (INIS)

    Zhesko, T.V.

    1987-01-01

    For rate setting of surface contamination with the wide-spread biogenic tritium compounds-protein predecessors-experimental study of skin absorption and skin deposit of amino acids labelled with tritium is carried out on rats. While extrapolating data to people and calculating tolerable skin contamination with 3 H- amino acids, it is supposed that people arm skin, 100-500 cm 2 , has no defects and that the skin surface decontamination after radionuclide contact is carried out with a preparation, efficiency of which is not less than 97%. The value of tolerable skin absorption of tritium amino acids, being 110-550 MBq/year or 4.8 kBq/cm 2 per one working day, is calculated

  5. 20 CFR 604.6 - Conformity and substantial compliance.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Conformity and substantial compliance. 604.6... FOR ELIGIBILITY FOR UNEMPLOYMENT COMPENSATION § 604.6 Conformity and substantial compliance. (a) In... for the administration of its UC program. (b) Resolving Issues of Conformity and Substantial...

  6. 19 CFR 10.7 - Substantial containers or holders.

    Science.gov (United States)

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Substantial containers or holders. 10.7 Section 10... Exported and Returned § 10.7 Substantial containers or holders. (a) Substantial containers or holders... domestic products exported and returned. When such containers or holders are imported not containing or...

  7. A novel photoelectrochemical immunosensor by integration of nanobody and ZnO nanorods for sensitive detection of nucleoside diphosphatase kinase-A

    International Nuclear Information System (INIS)

    Liu, Anran; Yin, Kaifei; Mi, Li; Ma, Mengyao; Liu, Yuanjian; Li, Ying; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2017-01-01

    Nucleoside diphosphatase kinase A (NDPK-A) is a metastasis-suppressor protein and a biomarker that act on a wide range cancer cells to inhibit the potential metastasis. Herein, we present a simple photoelectrochemical immunosensor based on ZnO nanorod arrays for the sensitive detection of NDPK-A. The ZnO nanorod arrays cosensitized with CdS nanoparticles and Mn 2+ displayed a high and stable photocurrent response under irradiation. After anti-NPDK-A nanobodies were immobilized to the ZnO nanorod arrays, the proposed immunosensor can be utilized for detecting NPDK-A by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Accordingly, the well-designed immunosensor exhibited a low limit of detection (LOD) of 0.3 pg mL −1 and a wide linear range from 0.5 pg mL −1 to 10 μg mL −1 . The R 2 of the regression curve is 0.99782. Meanwhile, the good stability, reproducibility and specificity of the resulting photoelectrochemical biosensor are demonstrated. In addition, the presented work would offer a novel and simple approach for the detection of immunoreactions and provide new insights in popularizing the diagnosis of NPDK-A. - Highlights: • A photoelectrochemical immunosensor based on ZnO NAs and NBs was fabricated. • Cosensitized structure of ZnO NAs, CdS and Mn improved the photocurrent response. • An excellent sensitivity and selectivity of NDPK-A detection was obtained.

  8. An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase.

    Directory of Open Access Journals (Sweden)

    Steven Odongo

    2016-02-01

    Full Text Available Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system.An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA was shown to detect experimental infections with high Positive Predictive Value (98%, Sensitivity (87% and Specificity (94%. Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i T. congolense soluble proteome, (ii T. congolense secretome preparation and (iii sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase.The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious

  9. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

    2016-01-01

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  11. Minimal surfaces

    CERN Document Server

    Dierkes, Ulrich; Sauvigny, Friedrich; Jakob, Ruben; Kuster, Albrecht

    2010-01-01

    Minimal Surfaces is the first volume of a three volume treatise on minimal surfaces (Grundlehren Nr. 339-341). Each volume can be read and studied independently of the others. The central theme is boundary value problems for minimal surfaces. The treatise is a substantially revised and extended version of the monograph Minimal Surfaces I, II (Grundlehren Nr. 295 & 296). The first volume begins with an exposition of basic ideas of the theory of surfaces in three-dimensional Euclidean space, followed by an introduction of minimal surfaces as stationary points of area, or equivalently

  12. Self-Assembly of Protein Nanostructures to Enhance Biosensor Sensitivity

    Science.gov (United States)

    Olsen, Bradley; Dong, Xuehui; Obermeyer, Allie

    The Langmuir adsorption isotherm predicts that the number of bound species on a surface at a given concentration will be directly proportional to the number of binding sites on the surface. Therefore, the number of binding events in a biosensor may be increased at a given analyte concentration if the surface density of binding domains is increased. Here, we demonstrate the formation of block copolymers where one block is a human IgG antibody or a nanobody and self-assemble these molecules into nanostructured films with a high density of binding sites. The type of nanostructure formed and the rate of transport through the protein-polymer layers are explored as a function of coil fraction of the protein-polymer conjugate block copolymers, showing optima for transport and assembly that depend upon the identity of the protein. For small enough analytes, binding to the antibodies and nanobodies is linear with film thickness, indicating that the entire film is accessible. Consistent with the enhanced number of binding sites and the prediction of the Langmuir isotherm, the films improve sensitivity by several orders of magnitude relative to chemisorbed protein layers used in current sensor designs. Current research is integrating this new material technology into prototype sensors. Work supported by the Air Force Office of Scientific Reesearch (AFOSR).

  13. Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis.

    Directory of Open Access Journals (Sweden)

    Juan D Unciti-Broceta

    2015-06-01

    Full Text Available African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.

  14. A novel photoelectrochemical immunosensor by integration of nanobody and ZnO nanorods for sensitive detection of nucleoside diphosphatase kinase-A

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Anran, E-mail: liuar@seu.edu.cn; Yin, Kaifei; Mi, Li; Ma, Mengyao; Liu, Yuanjian; Li, Ying; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2017-06-22

    Nucleoside diphosphatase kinase A (NDPK-A) is a metastasis-suppressor protein and a biomarker that act on a wide range cancer cells to inhibit the potential metastasis. Herein, we present a simple photoelectrochemical immunosensor based on ZnO nanorod arrays for the sensitive detection of NDPK-A. The ZnO nanorod arrays cosensitized with CdS nanoparticles and Mn{sup 2+} displayed a high and stable photocurrent response under irradiation. After anti-NPDK-A nanobodies were immobilized to the ZnO nanorod arrays, the proposed immunosensor can be utilized for detecting NPDK-A by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Accordingly, the well-designed immunosensor exhibited a low limit of detection (LOD) of 0.3 pg mL{sup −1} and a wide linear range from 0.5 pg mL{sup −1} to 10 μg mL{sup −1}. The R{sup 2} of the regression curve is 0.99782. Meanwhile, the good stability, reproducibility and specificity of the resulting photoelectrochemical biosensor are demonstrated. In addition, the presented work would offer a novel and simple approach for the detection of immunoreactions and provide new insights in popularizing the diagnosis of NPDK-A. - Highlights: • A photoelectrochemical immunosensor based on ZnO NAs and NBs was fabricated. • Cosensitized structure of ZnO NAs, CdS and Mn improved the photocurrent response. • An excellent sensitivity and selectivity of NDPK-A detection was obtained.

  15. Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein

    International Nuclear Information System (INIS)

    Abskharon, Romany N. N.; Soror, Sameh H.; Pardon, Els; El Hassan, Hassan; Legname, Giuseppe; Steyaert, Jan; Wohlkonig, Alexandre

    2010-01-01

    The crystallization of a specific nanobody against mouse PrP C and preliminary diffraction analysis of a crystal that diffracted to 1.23 Å resolution are presented. Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrP C into the pathogenic isoform PrP Sc . Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb-PrP-01) was raised against mouse PrP C . A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 30.04, b = 37.15, c = 83.00 Å, and diffracted to 1.23 Å resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrP C together with the known PrP C structure may help in understanding the PrP C /PrP Sc transition mechanism

  16. Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface.

    Directory of Open Access Journals (Sweden)

    Wouter S P Jong

    Full Text Available Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.

  17. Net-Zero Building Technologies Create Substantial Energy Savings -

    Science.gov (United States)

    only an estimated 1% of commercial buildings are built to net-zero energy criteria. One reason for this Continuum Magazine | NREL Net-Zero Building Technologies Create Substantial Energy Savings Net -Zero Building Technologies Create Substantial Energy Savings Researchers work to package and share step

  18. A Highly Specific Gold Nanoprobe for Live-Cell Single-Molecule Imaging

    Science.gov (United States)

    Leduc, Cécile; Si, Satyabrata; Gautier, Jérémie; Soto-Ribeiro, Martinho; Wehrle-Haller, Bernhard; Gautreau, Alexis; Giannone, Grégory; Cognet, Laurent; Lounis, Brahim

    2013-04-01

    Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

  19. Intrinsically active nanobody-modified polymeric micelles for tumor-targeted combination therapy

    NARCIS (Netherlands)

    Talelli, M.; Oliveira, S.; Rijcken, C.J.; Pieters, E.H.; Etrych, T.; Ulbrich, K.; van Nostrum, C.F.; Storm, Gerrit; Hennink, W.E.; Lammers, Twan Gerardus Gertudis Maria

    2013-01-01

    Various different passively and actively targeted nanomedicines have been designed and evaluated over the years, in particular for the treatment of cancer. Reasoning that the potential of ligand-modified nanomedicines can be substantially improved if intrinsically active targeting moieties are used,

  20. Turbocharger with variable nozzle having vane sealing surfaces

    Science.gov (United States)

    Arnold, Philippe [Hennecourt, FR; Petitjean, Dominique [Julienrupt, FR; Ruquart, Anthony [Thaon les Vosges, FR; Dupont, Guillaume [Thaon les Vosges, FR; Jeckel, Denis [Thaon les Vosges, FR

    2011-11-15

    A variable nozzle for a turbocharger includes a plurality of vanes rotatably mounted on a nozzle ring and disposed in a nozzle flow path defined between the nozzle ring and an opposite nozzle wall. Either or both of the faces of the nozzle ring and nozzle wall include(s) at least one step that defines sealing surfaces positioned to be substantially abutted by airfoil surfaces of the vanes in the closed position of the vanes and to be spaced from the airfoil surfaces in positions other than the closed position. This substantial abutment between the airfoil surfaces and the sealing surfaces serves to substantially prevent exhaust gas from leaking past the ends of the airfoil portions. At the same time, clearances between the nozzle ring face and the end faces of the airfoil portions can be sufficiently large to prevent binding of the vanes under all operating conditions.

  1. 20 CFR 654.13 - Determination of areas of substantial unemployment.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Determination of areas of substantial unemployment. 654.13 Section 654.13 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF... 10582 § 654.13 Determination of areas of substantial unemployment. An area of substantial unemployment...

  2. Monitoring failure rates of commercial implant brands; substantial equivalence in question?

    Science.gov (United States)

    Hujoel, Philippe; Becker, William; Becker, Burton

    2013-07-01

    The aim of this study was to report on the failure rates of two distinct dental implant systems in a clinical practice setting. Date of implant placement and loss were entered prospectively in a data registry system. Failure rates of two commercially pure titanium implants, one with a porous oxydized surface (POS) and the other with a chemically altered surface (CAS), were assessed using a quality control chart and survival analyses. A total of 860 POS and 759 CAS implants were placed. A warning of an increased failure rate of the CAS implant was identified by means of the quality control chart. Survival analyses indicated that the CAS implant failure rate was twice that of the POS implant (Hazard Ratio: 2.08; 95% CI: 1.33-3.28, P-value POS implant was associated with a non-significant 64% drop in the implant failure rate within less than a year (HR: 0.36; 95% CI: 0.12-1.14; P-value POS implant and an 8% failure with the CAS implant appears inconsistent with the assumption of substantial equivalence. © 2012 John Wiley & Sons A/S.

  3. Increased greenhouse effect substantiated through measurements

    International Nuclear Information System (INIS)

    Skartveit, Arvid

    2001-01-01

    The article presents studies on the greenhouse effect which substantiates the results from satellite measurements during the period 1970 - 1997. These show an increased effect due to increase in the concentration of the climatic gases CO 2 , methane, CFC-11 and CFC-12 in the atmosphere

  4. Theory of quasiparticle surface states in semiconductor surfaces

    International Nuclear Information System (INIS)

    Hybertsen, M.S.; Louie, S.G.

    1988-01-01

    A first-principles theory of the quasiparticle surface-state energies on semiconductor surfaces is developed. The surface properties are calculated using a repeated-slab geometry. Many-body effects due to the electron-electron interaction are represented by the electron self-energy operator including the full surface Green's function and local fields and dynamical screening effects in the Coulomb interaction. Calculated surface-state energies for the prototypical Si(111):As and Ge(111):As surfaces are presented. The calculated energies and dispersions for the occupied surface states (resonances) are in excellent agreement with recent angle-resolved photoemission data. Predictions are made for the position of empty surface states on both surfaces which may be experimentally accessible. The resulting surface state gap at Gamma-bar for Si(111):As agrees with recent scanning-tunneling-spectroscopy measurements. Comparison of the present results to eigenvalues from the local-density-functional calculation reveals substantial corrections for the gaps between empty and occupied surface states. This correction is found to depend on the character of the surface states involved

  5. 19 CFR 134.35 - Articles substantially changed by manufacture.

    Science.gov (United States)

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Articles substantially changed by manufacture. 134... substantially changed by manufacture. (a) Articles other than goods of a NAFTA country. An article used in the United States in manufacture which results in an article having a name, character, or use differing from...

  6. 21 CFR 514.4 - Substantial evidence.

    Science.gov (United States)

    2010-04-01

    ... adequate and well-controlled studies, such as a study in a target species, study in laboratory animals... and conditions of use. Substantial evidence of effectiveness of a new animal drug shall demonstrate that the new animal drug is effective for each intended use and associated conditions of use for and...

  7. Methodology for substantiation of the fast reactor fuel element serviceability

    International Nuclear Information System (INIS)

    Tsykanov, V.A.; Maershin, A.A.

    1988-01-01

    Methodological aspects of fast reactor fuel element serviceability substantiation are presented. The choice of the experimental program and strategies of its realization to solve the problem set in short time, taking into account available experimental means, are substantiated. Factors determining fuel element serviceability depending on parameters and operational conditions are considered. The methodological approach recommending separate studing of the factors, which points to the possibility of data acquisition, required for the development of calculational models and substantiation of fuel element serviceability in pilot and experimental reactors, is described. It is shown that the special-purpose data are more useful for the substantiation of fuel element serviceability and analytical method development than unsubstantial and expensive complex tests of fuel elements and fuel assemblies, which should be conducted only at final stages for the improvement of the structure on the whole

  8. Establishing Substantial Equivalence: Proteomics

    Science.gov (United States)

    Lovegrove, Alison; Salt, Louise; Shewry, Peter R.

    Wheat is a major crop in world agriculture and is consumed after processing into a range of food products. It is therefore of great importance to determine the consequences (intended and unintended) of transgenesis in wheat and whether genetically modified lines are substantially equivalent to those produced by conventional plant breeding. Proteomic analysis is one of several approaches which can be used to address these questions. Two-dimensional PAGE (2D PAGE) remains the most widely available method for proteomic analysis, but is notoriously difficult to reproduce between laboratories. We therefore describe methods which have been developed as standard operating procedures in our laboratory to ensure the reproducibility of proteomic analyses of wheat using 2D PAGE analysis of grain proteins.

  9. Light extraction block with curved surface

    Science.gov (United States)

    Levermore, Peter; Krall, Emory; Silvernail, Jeffrey; Rajan, Kamala; Brown, Julia J.

    2016-03-22

    Light extraction blocks, and OLED lighting panels using light extraction blocks, are described, in which the light extraction blocks include various curved shapes that provide improved light extraction properties compared to parallel emissive surface, and a thinner form factor and better light extraction than a hemisphere. Lighting systems described herein may include a light source with an OLED panel. A light extraction block with a three-dimensional light emitting surface may be optically coupled to the light source. The three-dimensional light emitting surface of the block may includes a substantially curved surface, with further characteristics related to the curvature of the surface at given points. A first radius of curvature corresponding to a maximum principal curvature k.sub.1 at a point p on the substantially curved surface may be greater than a maximum height of the light extraction block. A maximum height of the light extraction block may be less than 50% of a maximum width of the light extraction block. Surfaces with cross sections made up of line segments and inflection points may also be fit to approximated curves for calculating the radius of curvature.

  10. Method For Creating Corrosion Resistant Surface On An Aluminum Copper Alloy

    Science.gov (United States)

    Mansfeld, Florian B.; Wang, You; Lin, Simon H.

    1997-06-03

    A method for treating the surface of aluminum alloys hang a relatively high copper content is provided which includes the steps of removing substantially all of the copper from the surface, contacting the surface with a first solution containing cerium, electrically charging the surface while contacting the surface in an aqueous molybdate solution, and contacting the surface with a second solution containing cerium. The copper is substantially removed from the surface in the first step either by (i) contacting the surface with an acidic chromate solution or by (ii) contacting the surface with an acidic nitrate solution while subjecting the surface to an electric potential. The corrosion-resistant surface resulting from the invention is excellent, consistent and uniform throughout the surface. Surfaces treated by the invention may often be certified for use in salt-water services.

  11. Surface Finish after Laser Metal Deposition

    Science.gov (United States)

    Rombouts, M.; Maes, G.; Hendrix, W.; Delarbre, E.; Motmans, F.

    Laser metal deposition (LMD) is an additive manufacturing technology for the fabrication of metal parts through layerwise deposition and laser induced melting of metal powder. The poor surface finish presents a major limitation in LMD. This study focuses on the effects of surface inclination angle and strategies to improve the surface finish of LMD components. A substantial improvement in surface quality of both the side and top surfaces has been obtained by laser remelting after powder deposition.

  12. 26 CFR 1.42-7 - Substantially bond-financed buildings. [Reserved

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 1 2010-04-01 2010-04-01 true Substantially bond-financed buildings. [Reserved] 1.42-7 Section 1.42-7 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY INCOME TAX INCOME TAXES Credits Against Tax § 1.42-7 Substantially bond-financed buildings. [Reserved] ...

  13. Surface phonons

    CERN Document Server

    Wette, Frederik

    1991-01-01

    In recent years substantial progress has been made in the detection of surface phonons owing to considerable improvements in inelastic rare gas scattering tech­ niques and electron energy loss spectroscopy. With these methods it has become possible to measure surface vibrations in a wide energy range for all wave vectors in the two-dimensional Brillouin zone and thus to deduce the complete surface phonon dispersion curves. Inelastic atomic beam scattering and electron energy loss spectroscopy have started to play a role in the study of surface phonons similar to the one played by inelastic neutron scattering in the investigation of bulk phonons in the last thirty years. Detailed comparison between experimen­ tal results and theoretical studies of inelastic surface scattering and of surface phonons has now become feasible. It is therefore possible to test and to improve the details of interaction models which have been worked out theoretically in the last few decades. At this point we felt that a concise, co...

  14. Controlling the surface photovoltage on WSe2 by surface chemical modification

    Science.gov (United States)

    Liu, Ro-Ya; Ozawa, Kenichi; Terashima, Naoya; Natsui, Yuto; Feng, Baojie; Ito, Suguru; Chen, Wei-Chuan; Cheng, Cheng-Maw; Yamamoto, Susumu; Kato, Hiroo; Chiang, Tai-Chang; Matsuda, Iwao

    2018-05-01

    The surface photovoltage (SPV) effect is key to the development of opto-electronic devices such as solar-cells and photo-detectors. For the prototypical transition metal dichalcogenide WSe2, core level and valence band photoemission measurements show that the surface band bending of pristine cleaved surfaces can be readily modified by adsorption with K (an electron donor) or C60 (an electron acceptor). Time-resolved pump-probe photoemission measurements reveal that the SPV for pristine cleaved surfaces is enhanced by K adsorption, but suppressed by C60 adsorption, and yet the SPV relaxation time is substantially shortened in both cases. Evidently, adsorbate-induced electronic states act as electron-hole recombination centers that shorten the carrier lifetime.

  15. 26 CFR 1.528-4 - Substantiality test.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Substantiality test. 1.528-4 Section 1.528-4 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED... residence. Units which are used for purposes auxiliary to residential use (such as laundry areas, swimming...

  16. Toward More Substantial Theories of Language Acquisition

    Science.gov (United States)

    Jenson, Cinnamon Ann

    2015-01-01

    Cognitive linguists argue that certain sets of knowledge of language are innate. However, critics have argued that the theoretical concept of "innateness" should be eliminated since it is ambiguous and insubstantial. In response, I aim to strengthen theories of language acquisition and identify ways to make them more substantial. I…

  17. Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

    Science.gov (United States)

    Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

    2013-01-01

    ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley

  18. Emerging monoclonal antibodies against Clostridium difficile infection.

    Science.gov (United States)

    Péchiné, Séverine; Janoir, Claire; Collignon, Anne

    2017-04-01

    Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

  19. Liquid flow along a solid surface reversibly alters interfacial chemistry.

    Science.gov (United States)

    Lis, Dan; Backus, Ellen H G; Hunger, Johannes; Parekh, Sapun H; Bonn, Mischa

    2014-06-06

    In nature, aqueous solutions often move collectively along solid surfaces (for example, raindrops falling on the ground and rivers flowing through riverbeds). However, the influence of such motion on water-surface interfacial chemistry is unclear. In this work, we combine surface-specific sum frequency generation spectroscopy and microfluidics to show that at immersed calcium fluoride and fused silica surfaces, flow leads to a reversible modification of the surface charge and subsequent realignment of the interfacial water molecules. Obtaining equivalent effects under static conditions requires a substantial change in bulk solution pH (up to 2 pH units), demonstrating the coupling between flow and chemistry. These marked flow-induced variations in interfacial chemistry should substantially affect our understanding and modeling of chemical processes at immersed surfaces. Copyright © 2014, American Association for the Advancement of Science.

  20. Evidence that global evapotranspiration makes a substantial contribution to the global atmospheric temperature slowdown

    Science.gov (United States)

    Leggett, L. Mark W.; Ball, David A.

    2018-02-01

    The difference between the time series trend for temperature expected from the increasing level of atmospheric CO2 and that for the (more slowly rising) observed temperature has been termed the global surface temperature slowdown. In this paper, we characterise the single time series made from the subtraction of these two time series as the `global surface temperature gap'. We also develop an analogous atmospheric CO2 gap series from the difference between the level of CO2 and first-difference CO2 (that is, the change in CO2 from one period to the next). This paper provides three further pieces of evidence concerning the global surface temperature slowdown. First, we find that the present size of both the global surface temperature gap and the CO2 gap is unprecedented over a period starting at least as far back as the 1860s. Second, ARDL and Granger causality analyses involving the global surface temperature gap against the major candidate physical drivers of the ocean heat sink and biosphere evapotranspiration are conducted. In each case where ocean heat data was available, it was significant in the models: however, evapotranspiration, or its argued surrogate precipitation, also remained significant in the models alongside ocean heat. In terms of relative scale, the standardised regression coefficient for evapotranspiration was repeatedly of the same order of magnitude as—typically as much as half that for—ocean heat. The foregoing is evidence that, alongside the ocean heat sink, evapotranspiration is also likely to be making a substantial contribution to the global atmospheric temperature outcome. Third, there is evidence that both the ocean heat sink and the evapotranspiration process might be able to continue into the future to keep the temperature lower than the level-of-CO2 models would suggest. It is shown that this means there can be benefit in using the first-difference CO2 to temperature relationship shown in Leggett and Ball (Atmos Chem Phys 15

  1. Chaotic behavior appearing in dynamic motions of nanoscale particles

    Energy Technology Data Exchange (ETDEWEB)

    Ishikawa, M [Innovation Plaza Tokai, Japan Science and Technology Agency, 23-1 Ahara-cho, Minami-ku, Nagoya 457-0063 (Japan); Harada, R [Department of Physics, Aichi University of Education, Hirosawa 1, Igaya-cho, Kariya 448-8542 (Japan); Kato, M [Innovation Plaza Tokai, Japan Science and Technology Agency, 23-1 Ahara-cho, Minami-ku, Nagoya 457-0063 (Japan); Sasaki, N [Department of Applied Physics, Faculty of Science and Engineering, Seikei University, 3-3-1 Kichijoji Kitamachi, Musashino-shi, Tokyo 180-8633 (Japan); Miura, K [Innovation Plaza Tokai, Japan Science and Technology Agency, 23-1 Ahara-cho, Minami-ku, Nagoya 457-0063 (Japan)

    2007-11-15

    The case of one-directional motion, under which graphite and mica flakes are driven on an octamethylcyclotetrasiloxane (OMCTS) liquid surface, is presented. The dynamical forces needed to move these bodies increase linearly with the logarithm of scanning velocity, which are typical energy dissipation process. A transition from quasi-periodic to chaotic motions occurs in the dynamics of a graphite flake when its velocity is increased. The dynamics of graphite flakes pulled by the nanotip on an OMCTS liquid surface can be treated as that of a nanobody on a liquid. On the other hand, there do not appear chaotic motions in the dynamics of a mica flake because the contact area between a mica flake and an OMCTS liquid surface is larger than that between a graphite flake and an OMCTS liquid surface.

  2. Chaotic behavior appearing in dynamic motions of nanoscale particles

    International Nuclear Information System (INIS)

    Ishikawa, M; Harada, R; Kato, M; Sasaki, N; Miura, K

    2007-01-01

    The case of one-directional motion, under which graphite and mica flakes are driven on an octamethylcyclotetrasiloxane (OMCTS) liquid surface, is presented. The dynamical forces needed to move these bodies increase linearly with the logarithm of scanning velocity, which are typical energy dissipation process. A transition from quasi-periodic to chaotic motions occurs in the dynamics of a graphite flake when its velocity is increased. The dynamics of graphite flakes pulled by the nanotip on an OMCTS liquid surface can be treated as that of a nanobody on a liquid. On the other hand, there do not appear chaotic motions in the dynamics of a mica flake because the contact area between a mica flake and an OMCTS liquid surface is larger than that between a graphite flake and an OMCTS liquid surface

  3. Chaotic behavior appearing in dynamic motions of nanoscale particles

    Science.gov (United States)

    Ishikawa, M.; Harada, R.; Kato, M.; Sasaki, N.; Miura, K.

    2007-11-01

    The case of one-directional motion, under which graphite and mica flakes are driven on an octamethylcyclotetrasiloxane (OMCTS) liquid surface, is presented. The dynamical forces needed to move these bodies increase linearly with the logarithm of scanning velocity, which are typical energy dissipation process. A transition from quasi-periodic to chaotic motions occurs in the dynamics of a graphite flake when its velocity is increased. The dynamics of graphite flakes pulled by the nanotip on an OMCTS liquid surface can be treated as that of a nanobody on a liquid. On the other hand, there do not appear chaotic motions in the dynamics of a mica flake because the contact area between a mica flake and an OMCTS liquid surface is larger than that between a graphite flake and an OMCTS liquid surface.

  4. 76 FR 63846 - Substantially Underserved Trust Areas (SUTA)

    Science.gov (United States)

    2011-10-14

    ... Cost Rural Communities; 10.861, Public Television Station Digital Transition Grant Program; 10.862... rule do not impose substantial unreimbursed direct compliance costs on Indian tribal, Alaska native, or native Hawaiian governments and sovereign institutions or have tribal implications that preempt tribal...

  5. Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook

    Directory of Open Access Journals (Sweden)

    Mehdi Arbabi-Ghahroudi

    2017-11-01

    Full Text Available Tremendous effort has been expended over the past two and a half decades to understand many aspects of camelid heavy chain antibodies, from their biology, evolution, and immunogenetics to their potential applications in various fields of research and medicine. In this article, I present a historical perspective on the development of camelid single-domain antibodies (sdAbs or VHHs, also widely known as nanobodies since their discovery and discuss the advantages and disadvantages of these unique molecules in various areas of research, industry, and medicine. Commercialization of camelid sdAbs exploded in 2001 with a flurry of patents issued to the Vrije Universiteit Brussel (VUB and later taken on by the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB and, after 2002, the VIB-founded spin-off company, Ablynx. While entrepreneurial spirit has certainly catalyzed the exploration of nanobodies as marketable products, IP restrictions may be partially responsible for the relatively long time span between the discovery of these biomolecules and their entry into the pharmaceutical market. It is now anticipated that the first VHH-based antibody drug, Caplacizumab, a bivalent anti-vWF antibody for treating rare blood clotting disorders, may be approved and commercialized in 2018 or shortly thereafter. This elusive first approval, along with the expiry of key patents, may substantially alter the scientific and biomedical landscape surrounding camelid sdAbs and pave the way for their emergence as mainstream biotherapeutics.

  6. SUBSTANTIATION OF SOLUTIONS PERTAINING TO COMPLEX PRODUCTION RECONSTRUCTION

    Directory of Open Access Journals (Sweden)

    V. Y. Gurinovich

    2011-01-01

    Full Text Available While taking an example of reconstruction and modernization of OJSC «Construction and Mounting Trust No.16, Novopolotsk» the paper substantiates solutions for complex production reconstruction. The production reconstruction is divided in three start-up facilities ensuring continuous finished-product output.

  7. Substantiation of Optimum Stoker Number in Potassium Production by Game Method

    Directory of Open Access Journals (Sweden)

    B. A. Bogatov

    2005-01-01

    Full Text Available Dump formation process of hard halite waste in potassium production has been analyzed in the paper. The paper shows high cost of a salt dump formation and an actuality to substantiate number of ОШ-1900-110/150 stokers that are operating simultaneously. A game theory method has been used to substantiate a solution of the problem.

  8. Substantial difference in target surface chemistry between reactive dc and high power impulse magnetron sputtering

    Science.gov (United States)

    Greczynski, G.; Mráz, S.; Schneider, J. M.; Hultman, L.

    2018-02-01

    The nitride layer formed in the target race track during the deposition of stoichiometric TiN thin films is a factor 2.5 thicker for high power impulse magnetron sputtering (HIPIMS), compared to conventional dc processing (DCMS). The phenomenon is explained using x-ray photoelectron spectroscopy analysis of the as-operated Ti target surface chemistry supported by sputter depth profiles, dynamic Monte Carlo simulations employing the TRIDYN code, and plasma chemical investigations by ion mass spectrometry. The target chemistry and the thickness of the nitride layer are found to be determined by the implantation of nitrogen ions, predominantly N+ and N2+ for HIPIMS and DCMS, respectively. Knowledge of this method-inherent difference enables robust processing of high quality functional coatings.

  9. Generation and characterization of the first immortalized alpaca cell line suitable for diagnostic and immunization studies.

    Directory of Open Access Journals (Sweden)

    Valentina Franceschi

    Full Text Available Raising of alpacas as exotic livestock for wool and meat production and as companion animals is growing in importance in the United States, Europe and Australia. Furthermore the alpaca, as well as the rest of the camelids, possesses the peculiarity of producing single-chain antibodies from which nanobodies can be generated. Nanobodies, due to their structural simplicity and reduced size, are very versatile in terms of manipulation and bio-therapeutic exploitation. In fact the biotech companies involved in nanobody production and application continue to grow in number and size. Hence, the development of reagents and tools to assist in the further growth of this new scientific and entrepreneurial reality is becoming a necessity. These are needed mainly to address alpaca disease diagnosis and prophylaxis, and to develop alpaca immunization strategies for nanobody generation. For instance an immortalized alpaca cell line would be extremely valuable. In the present work the first stabilized alpaca cell line from alpaca skin stromal cells (ASSCs was generated and characterized. This cell line was shown to be suitable for replication of viruses bovine herpesvirus-1, bovine viral diarrhea virus and caprine herpesvirus-1 and the endocellular parasite Neospora caninum. Moreover ASSCs were easy to transfect and transduce by several methods. These two latter characteristics are extremely useful when recombinant antigens need to be produced in a host homologous system. This work could be considered as a starting point for the expansion of the biotechnologies linked to alpaca farming and industry.

  10. 32 CFR 48.302 - Substantiating evidence regarding dependency and age of dependents.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 1 2010-07-01 2010-07-01 false Substantiating evidence regarding dependency and age of dependents. 48.302 Section 48.302 National Defense Department of Defense OFFICE OF THE... Designation of Beneficiaries § 48.302 Substantiating evidence regarding dependency and age of dependents. At...

  11. Patients' substantialization of disease, the hybrid symptom and metaphysical care.

    Science.gov (United States)

    Pârvan, Alexandra

    2015-06-01

    In the context of current scholarship concerned with facilitating integration between the biomedical and the patient-centred models of care, the article suggests that disease brings about an ontological disruption in patients, which is not directly addressed in either model, and may interfere with treatment and therapy outcomes if not met with a type of care termed here as 'metaphysical'. The receipt of diagnosis and medical care can give patients the sense that they are ontologically diminished, or less of a human, and along with physicians' approaches to and discourses about disease, may prompt them to seek ontological restoration or security in the same way as psychologically traumatized patients sometimes do: by treating the disease and/or the experience of harm associated with it as a thing that exists per se. I call this 'substantialization' of disease (or harm) and draw on Augustine's theory of non-substantial deficiencies (physiological and moral) and on Plato's and Plotinus's different takes on such defects in order to discuss what substantialization can do for patients. Based on literature that examines patients' ways of talking about and living with their disease, I speculate that substantialization can generate a 'hybrid symptom', consisting in patterns of exercising agency which may predispose to non-adherence. Ways in which physicians could provide metaphysical care are proposed, along with an understanding of chronic patients as hybrid ontological and agentic units, which draws on theories of enactive cognition. I opine that metaphysical care may facilitate integration between the depersonalized and personalized models of care. © 2014 John Wiley & Sons, Ltd.

  12. Child protection decisions to substantiate hospital child protection teams' reports of suspected maltreatment.

    Science.gov (United States)

    Jedwab, Merav; Benbenishty, Rami; Chen, Wendy; Glasser, Saralee; Siegal, Gil; Lerner-Geva, Liat

    2015-02-01

    The present study focuses on the way child protection officers (CPOs) in Israel assess suspected abuse and neglect (SCAN) reports made by hospital child protection teams (CPTs), to determine whether the alleged maltreatment is substantiated. The study was conducted in six medical centers and included 358 reports investigated by CPOs for SCAN. A structured questionnaire was completed by hospital CPTs to capture all relevant information on each child referred to the CPTs. Structured phone interviews were conducted with each of the CPOs who received a CPT report. Bivariate associations and multivariate logistic regressions were conducted to estimate the substantiation rate of cases reported by CPTs and the types of maltreatment substantiated, as well as to identify case characteristics of the child and the family that were associated with the CPOs' substantiation decision. CPO follow-up investigations revealed a substantiation rate of 53.5%. The maltreatment type most commonly substantiated was neglect. The case characteristics associated with substantiation included socio-demographic background, parents' health and functioning, previous contact with social services, characteristics of the hospital referral, medical findings and an assessment of the parents' behaviors. The findings of the study highlighted the importance of cooperation between the health and welfare services and the policy makers. This cooperation is essential for identifying early signs of maltreatment. Enhanced cooperation and effective information transfer between various professionals would help prevent or at least reduce the recurrence of maltreatment and would ensure that the children and their families are treated appropriately. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Cumulative stress and substantiated maltreatment: the importance of caregiver vulnerability and adult partner violence.

    Science.gov (United States)

    Wekerle, Christine; Wall, Anne-Marie; Leung, Eman; Trocmé, Nico

    2007-04-01

    Our goal is to assess the effect of caregiver vulnerabilities, singly and in combination, on the substantiation of child abuse (physical, sexual) and neglect, while controlling for relevant background variables. We test the moderator role of adult partner violence in qualifying the relationship between caregiver vulnerabilities and maltreatment substantiation. Secondary analyses of the 1998 Canadian Incidence Study of Reported Child Maltreatment (CIS) are used to predict child protective service investigation substantiation versus non-substantiation from a range of caregiver vulnerability factors. Involvement in partner violence was examined as a moderator in the relation between caregiver vulnerabilities and maltreatment substantiation. The CIS is an epidemiological survey of first-reported cases to child protective services, using a random sample of child welfare agencies across Canada. Child welfare workers completed a research form on the child, primary caregiver, family, perpetrator, severity and type of maltreatment, as well as services and court outcomes. All maltreatment classifications were assigned according to the Canadian legal definition of child abuse and neglect. Hierarchical logistic regression analyses were used, with stepped entry of: (1) demographic factors, socioeconomic disadvantage, and caregiver's own history of maltreatment; (2) caregiver vulnerability factors; (3) involvement in partner violence; (4) the interaction between caregiver vulnerability and partner violence. Caregiver substance abuse was found to be the single most potent kind of caregiver vulnerability in predicting maltreatment substantiation. When the total number of vulnerabilities was used as the predictor, prediction across all types of maltreatment increased, especially for substantiated neglect. Analyses also showed that the presence of partner violence in the home exacerbated the effect of caregiver vulnerability on substantiation. The total number of caregiver

  14. Design of a surface alloy catalyst for steam reforming

    DEFF Research Database (Denmark)

    Besenbacher, F.; Chorkendorff, Ib; Clausen, B.S.

    1998-01-01

    Detailed studies of elementary chemical processes on well-characterized single crystal surfaces have contributed substantially to the understanding of heterogeneous catalysis. insight into the structure of surface alloys combined with an understanding of the relation between the surface compositi...... and reactivity is shown to lead directly to new ideas for catalyst design, The feasibility of such an approach is illustrated by the synthesis, characterization, and tests of a high-surface area gold-nickel catalyst for steam reforming....

  15. Domestic violence, parental substance misuse and the decision to substantiate child maltreatment.

    Science.gov (United States)

    Victor, Bryan G; Grogan-Kaylor, Andrew; Ryan, Joseph P; Perron, Brian E; Gilbert, Terri Ticknor

    2018-05-01

    Families that experience domestic violence and parental substance misuse are disproportionately involved with the child welfare system. Prior research suggests that child protective services (CPS) caseworkers are more likely to substantiate maltreatment allegations when domestic violence and parental substance misuse are identified during the investigation, pointing to one possible mechanism for this disproportionate involvement. While previous studies have relied on nationally representative data sets, the current study used administrative records from a large Midwestern child welfare agency that accounts for state-level variation in child welfare policy and practice. A total of 501,060 substantiation decisions made between 2009 and 2013 were examined to assess the influence of caseworker-perceived domestic violence and parental substance misuse on the decision to substantiate reported maltreatment. Results from multilevel modeling suggest that the identification of domestic violence and parental substance misuse during an investigation significantly increased the probability that an allegation would be substantiated. The implication of these findings for child welfare practice are considered in light of the fact that many child welfare agencies do not consider exposure to domestic violence and parental substance misuse in and of themselves to constitute child maltreatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Engineering Substantially Prolonged Human Lifespans: Biotechnological Enhancement and Ethics

    NARCIS (Netherlands)

    Derkx, P.H.J.M.

    2006-01-01

    Substantial extension of the human lifespan has recently become a subject of lively debate. One reason for this is the completion in 2001 of the Human Genome Project and the experimental avenues for biogerontological research it has opened. Another is recent theoretical progress in biogerontology.

  17. Bulk and surface loss in superconducting transmon qubits

    International Nuclear Information System (INIS)

    Dial, Oliver; McClure, Douglas T; Poletto, Stefano; Keefe, G A; Rothwell, Mary Beth; Gambetta, Jay M; Abraham, David W; Chow, Jerry M; Steffen, Matthias

    2016-01-01

    Decoherence of superconducting transmon qubits is purported to be consistent with surface loss from two-level systems on the substrate surface. Here, we present a study of surface loss in transmon devices, explicitly designed to have varying sensitivities to different surface loss contributors. Our experiments also encompass two particular different sapphire substrates, which reveal the onset of a yet unknown additional loss mechanism outside of surface loss for one of the substrates. Tests across different wafers and devices demonstrate substantial variation, and we emphasize the importance of testing large numbers of devices for disentangling different sources of decoherence. (paper)

  18. TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production

    DEFF Research Database (Denmark)

    Rennig, Maja; Martinez, Virginia; Mirzadeh, Kiavash

    2018-01-01

    Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome...... and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody. The method is a simple......-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange...

  19. An inflatable surface coil for rectal imaging

    International Nuclear Information System (INIS)

    Martin, J.F.; Hajek, P.C.; Baker, L.L.; Gylys-Morin, V.; Mattrey, R.F.

    1986-01-01

    Surface coils have become ubiquitous in MR imaging of the body because of substantial gains in signal-to-noise ratio. Unfortunately, there are some anatomic regions, such as the prostate, for which surface coils have insufficient depth sensitivity. The authors have developed an inflatable, distributed capacitance, passively decoupled surface coil which is collapsed for insertion and reinflated for imaging. Images of the prostate are dramatically improved due to proximity of the coil. Lesions in cadaver specimens were observed which were not seen with body coil imaging. Clinical trials are expected to begin in September

  20. Self-healing phenomena on corroding steel in simulated pore water and mortar, substantiated via cyclic voltammetry and surface analysis

    Energy Technology Data Exchange (ETDEWEB)

    Koleva, D. A.; Breugel, K. van [Delft University of Technology, The Netherlands Faculty of CiTG, Department Materials and Environment, Delf (Netherlands); Hu, J. [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); Kolev, H. [Bulgarian Academy of Sciences, Institute of Catalysis, Sofia (Bulgaria)

    2013-07-01

    The application of polymeric nano-particles was investigated as an approach to control corrosion and/or self-heal corrosion damage on steel in simulated alkaline medium and reinforced mortar. The “self-healing agent”, present in the closed inner volume of PEO-b-PS vesicles was Ca-based and chosen as such due to the natural predominance of Ca in the investigated system. The vesicles’ concentration was 0.0024 wt.% in the model medium and 0.025 wt.% per cement weight for the case of mortar. Therefore, a “self-repair” or “self-healing” of the steel product layer solely due to the Ca- component is not realistic in view of these minimal concentrations. The most plausible mechanism is the nature of incorporation of the Ca-containing vesicles in the product layer, enhanced chloride binding effects and adsorption on active sites on the steel surface. A more uniform and stable surface layer, initial pitting formation and propagation, but consecutive “healing”, are evidenced by surface analysis and electrochemical response i.e. largely reduced anodic and corrosion currents and no further pit propagation are observed when Ca-containing vesicles are present in the model medium. Corrosion products-free steel/cement paste interface is relevant for the reinforced mortar, containing Ca-rich vesicles in contrast to vesicles-free and empty vesicles-containing matrix. Key words: corrosion; concrete; polymeric nano-particles; CVA; SEM; XRD; XPS.

  1. Tilting the balance between canonical and noncanonical conformations for the H1 hypervariable loop of a llama VHH through point mutations.

    Science.gov (United States)

    Mahajan, Sai Pooja; Velez-Vega, Camilo; Escobedo, Fernando A

    2013-01-10

    Nanobodies are single-domain antibodies found in camelids. These are the smallest naturally occurring binding domains and derive functionality via three hypervariable loops (H1-H3) that form the binding surface. They are excellent candidates for antibody engineering because of their favorable characteristics like small size, high solubility, and stability. To rationally engineer antibodies with affinity for a specific target, the hypervariable loops can be tailored to obtain the desired binding surface. As a first step toward such a goal, we consider the design of loops with a desired conformation. In this study, we focus on the H1 loop of the anti-hCG llama nanobody that exhibits a noncanonical conformation. We aim to "tilt" the stability of the H1 loop structure from a noncanonical conformation to a (humanized) type 1 canonical conformation by studying the effect of selected mutations to the amino acid sequence of the H1, H2, and proximal residues. We use all-atomistic, explicit-solvent, biased molecular dynamic simulations to simulate the wild-type and mutant loops in a prefolded framework. We thus find mutants with increasing propensity to form a stable type 1 canonical conformation of the H1 loop. Free energy landscapes reveal the existence of conformational isomers of the canonical conformation that may play a role in binding different antigenic surfaces. We also elucidate the approximate mechanism and kinetics of transitions between such conformational isomers by using a Markovian model. We find that a particular three-point mutant has the strongest thermodynamic propensity to form the H1 type 1 canonical structure but also to exhibit transitions between conformational isomers, while a different, more rigid three-point mutant has the strongest propensity to be kinetically trapped in such a canonical structure.

  2. Global and regional ocean carbon uptake and climate change: sensitivity to a substantial mitigation scenario

    Energy Technology Data Exchange (ETDEWEB)

    Vichi, Marcello; Masina, Simona; Navarra, Antonio [Centro Euro-Mediterraneo per i Cambiamenti Climatici (CMCC), Bologna (Italy); Istituto Nazionale di Geofisica e Vulcanologia, Bologna (Italy); Manzini, Elisa [Centro Euro-Mediterraneo per i Cambiamenti Climatici (CMCC), Bologna (Italy); Istituto Nazionale di Geofisica e Vulcanologia, Bologna (Italy); Max Planck Institute for Meteorology, Hamburg (Germany); Fogli, Pier Giuseppe [Centro Euro-Mediterraneo per i Cambiamenti Climatici (CMCC), Bologna (Italy); Alessandri, Andrea [Centro Euro-Mediterraneo per i Cambiamenti Climatici (CMCC), Bologna (Italy); ENEA, Rome (Italy); Patara, Lavinia [Centro Euro-Mediterraneo per i Cambiamenti Climatici (CMCC), Bologna (Italy); Leibniz Institute of Marine Sciences (IFM-GEOMAR), Kiel (Germany); Scoccimarro, Enrico [Istituto Nazionale di Geofisica e Vulcanologia, Bologna (Italy)

    2011-11-15

    Under future scenarios of business-as-usual emissions, the ocean storage of anthropogenic carbon is anticipated to decrease because of ocean chemistry constraints and positive feedbacks in the carbon-climate dynamics, whereas it is still unknown how the oceanic carbon cycle will respond to more substantial mitigation scenarios. To evaluate the natural system response to prescribed atmospheric ''target'' concentrations and assess the response of the ocean carbon pool to these values, 2 centennial projection simulations have been performed with an Earth System Model that includes a fully coupled carbon cycle, forced in one case with a mitigation scenario and the other with the SRES A1B scenario. End of century ocean uptake with the mitigation scenario is projected to return to the same magnitude of carbon fluxes as simulated in 1960 in the Pacific Ocean and to lower values in the Atlantic. With A1B, the major ocean basins are instead projected to decrease the capacity for carbon uptake globally as found with simpler carbon cycle models, while at the regional level the response is contrasting. The model indicates that the equatorial Pacific may increase the carbon uptake rates in both scenarios, owing to enhancement of the biological carbon pump evidenced by an increase in Net Community Production (NCP) following changes in the subsurface equatorial circulation and enhanced iron availability from extratropical regions. NCP is a proxy of the bulk organic carbon made available to the higher trophic levels and potentially exportable from the surface layers. The model results indicate that, besides the localized increase in the equatorial Pacific, the NCP of lower trophic levels in the northern Pacific and Atlantic oceans is projected to be halved with respect to the current climate under a substantial mitigation scenario at the end of the twenty-first century. It is thus suggested that changes due to cumulative carbon emissions up to present and the

  3. Apparatus for plasma surface treating and preparation of membrane layers

    NARCIS (Netherlands)

    1990-01-01

    An apparatus suitable for plasma surface treating (e.g., forming a membrane layer on a substrate surface) comprises a plasma generation section which is operable at least at substantially atmospheric pressure and is in communication via at least one plasma inlet (e.g., a nozzle) with an enclosed

  4. Detection of Dew-Point by substantial Raman Band Frequency Jumps (A new Method)

    DEFF Research Database (Denmark)

    Hansen, Susanne Brunsgaard; Berg, Rolf W.; Stenby, Erling Halfdan

    Detection of Dew-Point by substantial Raman Band Frequency Jumps (A new Method). See poster at http://www.kemi.dtu.dk/~ajo/rolf/jumps.pdf......Detection of Dew-Point by substantial Raman Band Frequency Jumps (A new Method). See poster at http://www.kemi.dtu.dk/~ajo/rolf/jumps.pdf...

  5. Effect of strain on surface diffusion and nucleation

    DEFF Research Database (Denmark)

    Brune, Harald; Bromann, Karsten; Röder, Holger

    1995-01-01

    The influence of strain on diffusion and nucleation has been studied by means of scanning tunneling microscopy and effective-medium theory for Ag self-diffusion on strained and unstrained (111) surfaces. Experimentally, the diffusion barrier is observed to be substantially lower on a pseudomorphic...... effect on surface diffusion and nucleation in heteroepitaxy and are thus of significance for the film morphology in the kinetic growth regime....

  6. Nuclear industry in a country with a substantial oil reserve

    International Nuclear Information System (INIS)

    Alvarez, R.; Castillo, H.; Costa, D.; Galan, I.; Martinez, M.

    1981-01-01

    The importance of the development of a nuclear industry in a country like Mexico, with a substantial oil reserve is analyzed, taking into account the technical, economical, political, ecological and social aspects of the problem. (author)

  7. The application of a layer of carbonaceous material to a surface

    International Nuclear Information System (INIS)

    Holland, L.A.

    1981-01-01

    A method of applying a carbonaceous material to a surface is described. It consists of exposing the surface to an ionised gas atmosphere generated in a gas consisting substantially of carbon and hydrogen, and applying to the surface through capacitive means an electrical potential which changes in sign at time intervals of between 5 x 10 -9 seconds and 10 -6 seconds. (author)

  8. Nonthermal Effects of Photon Illumination on Surface Diffusion

    International Nuclear Information System (INIS)

    Ditchfield, R.; Llera-Rodriguez, D.; Seebauer, E.G.

    1998-01-01

    Nonthermal influences of photon illumination on surface diffusion at high temperatures have been measured experimentally for the first time. Activation energies and preexponential factors for diffusion of germanium and indium on silicon change substantially in response to illumination by photons having energies greater than the substrate band gap. Results depend on doping type. Ionization of surface vacancies by photogenerated charge carriers seems to play a key role. The results have significant implications for aspects of microelectronics fabrication governed by surface mobility. copyright 1998 The American Physical Society

  9. Dissolution model for a glass having an adherent insoluble surface layer

    International Nuclear Information System (INIS)

    Harvey, K.B.; Larocque, C.A.B.

    1990-01-01

    Waste form glasses that contain substantial quantities of iron, manganese, and aluminum oxides, such as the Savannah River SRL TDS-131 glass, form a thick, hydrated surface layer when placed in contact with water. The dissolution of such a glass has been modeled with the Savannah River Model. The authors showed previously that the equations of the Savannah River Model could be fitted to published experimental data if a time-dependent diffusion coefficient was assumed for species of diffusing through the surface layer. The Savannah River Model assumes that all of the material dissolved from the glass enters solution, whereas it was observed that substantial quantities of material were retained in the surface layer. An alternative model, presented contains a mass balance equation that allows material either to enter solution or to be retained in the surface layer. It is shown that the equations derived using this model can be fitted to the published experimental data assuming a constant diffusion coefficient for species diffusing through the surface layer

  10. Boiling and quenching heat transfer advancement by nanoscale surface modification.

    Science.gov (United States)

    Hu, Hong; Xu, Cheng; Zhao, Yang; Ziegler, Kirk J; Chung, J N

    2017-07-21

    All power production, refrigeration, and advanced electronic systems depend on efficient heat transfer mechanisms for achieving high power density and best system efficiency. Breakthrough advancement in boiling and quenching phase-change heat transfer processes by nanoscale surface texturing can lead to higher energy transfer efficiencies, substantial energy savings, and global reduction in greenhouse gas emissions. This paper reports breakthrough advancements on both fronts of boiling and quenching. The critical heat flux (CHF) in boiling and the Leidenfrost point temperature (LPT) in quenching are the bottlenecks to the heat transfer advancements. As compared to a conventional aluminum surface, the current research reports a substantial enhancement of the CHF by 112% and an increase of the LPT by 40 K using an aluminum surface with anodized aluminum oxide (AAO) nanoporous texture finish. These heat transfer enhancements imply that the power density would increase by more than 100% and the quenching efficiency would be raised by 33%. A theory that links the nucleation potential of the surface to heat transfer rates has been developed and it successfully explains the current finding by revealing that the heat transfer modification and enhancement are mainly attributed to the superhydrophilic surface property and excessive nanoscale nucleation sites created by the nanoporous surface.

  11. 20 CFR 416.972 - What we mean by substantial gainful activity.

    Science.gov (United States)

    2010-04-01

    ... INCOME FOR THE AGED, BLIND, AND DISABLED Determining Disability and Blindness Substantial Gainful... consider activities like taking care of yourself, household tasks, hobbies, therapy, school attendance...

  12. Surface Potential of Polycrystalline Hematite in Aqueous Medium

    Directory of Open Access Journals (Sweden)

    Tajana Preočanin

    2011-01-01

    Full Text Available The surface potential of polycrystalline hematite in aqueous sodium perchlorate environment as a function of pH was examined. Surface potential of hematite was obtained from measured electrode potential of a nonporous polycrystalline hematite electrode. Acidic solution was titrated with base, and the backward titration with acid was performed. Substantial hysteresis was obtained which enabled location of the point of zero potential and equilibrium values of surface potentials. The theoretical interpretation of the equilibrium data was performed by applying the surface complexation model and the thermodynamic equilibrium constants for the first and the second step of surface protonation was obtained as logK1∘=11.3;logK2∘=2.8.

  13. EXPERIMENTAL SUBSTANTIATION OF ATTRIBUTIONAL STYLE IN TRAFFIC POLICE OFFICERS’ BEHAVIOR

    Directory of Open Access Journals (Sweden)

    Olga Vladimirovna Merkusheva

    2016-02-01

    Full Text Available The article prepared for the ‘Psychological and Methodological Aspects of Professional Personnel Training at Internal Affairs Bodies of the Russian Federation’ scholar tradition looks at the applied aspect of handling security issues by traffic police officers. It presents the results of empirical study of the employees’ psychological characteristics conducted to substantiate their attributional style, which determines the specific character as well as the qua-lity and reliability of traffic police officers’ performance. Traffic police officers’ attributional style of behavior is researched with the help of attributional cognitive techniques for hazard phenomena detection as well as social perception and attribution technique and is viewed as the most stable entity, which reflects the employee’s personal and professional focus.Goal: to provide empirical substantiation of attributional style in traffic police officers’ behavior as an integral personality characteristic that ensures timely detection of hazard phenomena.

  14. SUBSTANTIAL EXCEPTIONS AND (DELIMITATIONS OF THE POWERS OF THE JUDGES ON CIVIL PROCEDURAL LAW

    Directory of Open Access Journals (Sweden)

    Igor Raatz

    2017-08-01

    Full Text Available This essay aims to unveil the role of the substantial exceptions on delimiting the powers of the judges on civil procedural law, especially regarding the ex officio judicial activity. This way, under a phenomenological method and based on a vision of guarantee of rights on procedural law, the article offers a brief explanation of the question concerning the content of the object under litigation and its role of (delimiting the powers of the judges. The work hypothesis is the addition of the substantial exceptions among the content of the object under litigation, along with the claim itself and the cause of action. The results lie on the premise that, by the substantial exceptions, the defendant extends the object under litigation – which is formed dynamically on civil procedure. The conclusion points towards the idea that the substantial exceptions act in a way of limiting the ex officio judicial activity on civil procedural law

  15. Substantial increase in acceleration potential of pyroelectric crystals

    International Nuclear Information System (INIS)

    Tornow, W.; Lynam, S. M.; Shafroth, S. M.

    2010-01-01

    We report on a substantial increase in the acceleration potential achieved with a LiTaO 3 pyroelectric crystal. With a single 2.5 cm diameter and 2.5 cm long z-cut crystal without electric field-enhancing nanotip we produced positive ion beams with maximal energies between 300 and 310 keV during the cooling phase when the crystal was exposed to 5 mTorr of deuterium gas. These values are about a factor of 2 larger than previously obtained with single pyroelectric crystals.

  16. RELIGIOUS DEMOCRATIZATION IN INDONESIA: STRENGTHENING THE PRO-CEDURAL AND SUBSTANTIAL RELIGIOUS DEMOCRACY

    Directory of Open Access Journals (Sweden)

    Eka Hendry AR.

    2013-06-01

    Full Text Available Inspired by the book of Michael Mann about the dark side of democracy that discusses the paradox between the ideality of democratic values and empirical realities of violence in the name of freedom (democracy, this paper begins with the exposure of the paradox, such as the rise of the violent conflict between groups of people (both ethnic and religious-based and the high prevalence of violence between religious groups in Indonesia. Even worse, a very wrenching violence involves state actors (rulers. This paper tries to understand the roots of the paradox, with a look at how the relationship between state and religion and the religious community trend of Indonesia (especially Muslims. The author argues that the democratization of religion is a solution to the issues. To answer what kind of religious democracy lives in Indonesia, the author analyzes through a religious procedural (or constitutional democratic dimension and religious substantial democratic dimension. The phenomenon of disobedience of law and system and the euphoria of law-making that reflects “intolerance” in several places in Indonesia display the fundamental issue in the religious procedural democracy. Whereas in the context of religious substantial democracy, the prevailing trend of religion that serves as a political and economic vehicle and ignores religion as a substantial aspect of the behavior of the Indonesian society has resulted in the marginalization of religious position and function. Then, the infiltration of the model of political Islam has also led to alienation of the character of the Islamic society of Indonesia, from a democratic pattern to a revival (radical one. In this light, the author needs to present a strategy to encourage religious democracy in Indonesia, structurally through formulating the ideal relation model between state and religion and culturally through a substantial pattern of religion embedded with the character of Indonesian religious

  17. The chemical physics of surfaces

    CERN Document Server

    Morrison, Stanley Roy

    1990-01-01

    Even more importantly, some authors who have contributed substantially to an area may have been overlooked. For this I apologize. I have, however, not attempted to trace techniques or observa­ tions historically, so there is no implication (unless specified) that the authors referred to were or were not the originators of a given method or observation. I would like to acknowledge discussions with co-workers at SFU for input relative to their specialties, to acknowledge the help of students who have pointed out errors and difficulties in the earlier presentation, and to acknowledge the infinite patience of my wife Phyllis while I spent my sabbatical and more in libraries and punching computers. S. Roy Morrison 0 1 Contents Notation XV 1. Introduction 1 1. 1. Surface States and Surface Sites . 1 1. 1. 1. The Chemical versus Electronic Representation of the Surface. 1 1. 1. 2. The Surface State on the Band Diagram 4 1. 1. 3. The Fermi Energy in the Surface State Model. 6 1. 1. 4. Need for Both Surface...

  18. Fast algorithm for the rendering of three-dimensional surfaces

    Science.gov (United States)

    Pritt, Mark D.

    1994-02-01

    It is often desirable to draw a detailed and realistic representation of surface data on a computer graphics display. One such representation is a 3D shaded surface. Conventional techniques for rendering shaded surfaces are slow, however, and require substantial computational power. Furthermore, many techniques suffer from aliasing effects, which appear as jagged lines and edges. This paper describes an algorithm for the fast rendering of shaded surfaces without aliasing effects. It is much faster than conventional ray tracing and polygon-based rendering techniques and is suitable for interactive use. On an IBM RISC System/6000TM workstation it renders a 1000 X 1000 surface in about 7 seconds.

  19. Newtonian liquid jet impaction on a high-speed moving surface

    International Nuclear Information System (INIS)

    Keshavarz, B.; Green, S.I.; Davy, M.H.; Eadie, D.T.

    2011-01-01

    Highlights: ► We studied experimentally the interaction of a liquid jet with a moving surface. ► Decreasing the Reynolds number reduced the incidence of splash. ► The Weber number had a much smaller impact on splash than the Reynolds number. ► The jet impingement angle had only a small effect on the splash. ► Increasing the surface roughness substantially decreased the splash threshold. - Abstract: In the railroad industry a friction modifying agent may be applied to the rail or wheel in the form of a liquid jet. In this mode of application the interaction between the high-speed liquid jet and a fast moving surface is important. Seven different Newtonian liquids with widely varying shear viscosities were tested to isolate the effect of viscosity from other fluid properties. Tests were also done on five surfaces of different roughness heights to investigate the effects of surface roughness. High-speed video imaging was employed to scrutinize the interaction between the impacting jet and the moving surface. For all surfaces, decreasing the Reynolds number reduced the incidence of splash and consequently enhanced the transfer efficiency. At the elevated Weber numbers of the testing, the Weber number had a much smaller impact on splash than the Reynolds number. The ratio of the surface velocity to the jet velocity has only a small effect on the splash, whereas increasing the roughness-height-to-jet-diameter ratio substantially decreased the splash threshold.

  20. The structure of reconstructed chalcopyrite surfaces

    Science.gov (United States)

    Thinius, Sascha; Islam, Mazharul M.; Bredow, Thomas

    2018-03-01

    Chalcopyrite (CuFeS2) surfaces are of major interest for copper exploitation in aqueous solution, called leaching. Since leaching is a surface process knowledge of the surface structure, bonding pattern and oxidation states is important for improving the efficiency. At present such information is not available from experimental studies. Therefore a detailed computational study of chalcopyrite surfaces is performed. The structures of low-index stoichiometric chalcopyrite surfaces {hkl} h, k, l ∈ {0, 1, 2} have been studied with density functional theory (DFT) and global optimization strategies. We have applied ab initio molecular dynamics (MD) in combination with simulated annealing (SA) in order to explore possible reconstructions via a minima hopping (MH) algorithm. In almost all cases reconstruction involving substantial rearrangement has occurred accompanied by reduction of the surface energy. The analysis of the change in the coordination sphere and migration during reconstruction reveals that S-S dimers are formed on the surface. Further it was observed that metal atoms near the surface move toward the bulk forming metal alloys passivated by sulfur. The obtained surface energies of reconstructed surfaces are in the range of 0.53-0.95 J/m2.

  1. Substantiating the Incurred but not Reported Reserve

    Directory of Open Access Journals (Sweden)

    Georgeta Vintilã

    2009-12-01

    Full Text Available In order to handle past and future liability taken by insurance contracts concluded, any insurance company must constitute and maintain technical reserves. Substantiating technical reserves is done through actuarial methods and its over-evaluation or under-evaluation influence solvency and financial performance of the insurance companies, in the sense of reducing solvency through over-evaluating reserves and, respectively, influencing profit (hence of outstanding tax through under-evaluating reserves. An important reserve for insurance companies is represented by the incurred but not reported reserve, as it allows the estimation of the liability the company may confront in the future, generated by events occurred in the past, which are not currently known in the present but will be reported in the future.

  2. Pichia pastoris Mut(S) strains are prone to misincorporation of O-methyl-L-homoserine at methionine residues when methanol is used as the sole carbon source.

    Science.gov (United States)

    Schotte, Peter; Dewerte, Isabelle; De Groeve, Manu; De Keyser, Saskia; De Brabandere, Veronique; Stanssens, Patrick

    2016-06-07

    Over the last few decades the methylotrophic yeast Pichia pastoris has become a popular host for a wide range of products such as vaccines and therapeutic proteins. Several P. pastoris engineered strains and mutants have been developed to improve the performance of the expression system. Yield and quality of a recombinant product are important parameters to monitor during the host selection and development process but little information is published regarding quality differences of a product produced by different P. pastoris strains. We compared titer and quality of several Nanobodies(®) produced in wild type and Mut(S) strains. Titer in fed-batch fermentation was comparable between all strains for each Nanobody but a significant difference in quality was observed. Nanobodies expressed in Mut(S) strains contained a product variant with a Δ-16 Da mass difference that was not observed in wild type strains. This variant showed substitution of methionine residues due to misincorporation of O-methyl-L-homoserine, also called methoxine. Methoxine is likely synthesized by the enzymatic action of O-acetyl homoserine sulfhydrylase and we confirmed that Nanobodies produced in the corresponding knock-out strain contained no methoxine variants. We could show the incorporation of methoxine during biosynthesis by its addition to the culture medium. We showed that misincorporation of methoxine occurs particularly in P. pastoris Mut(S) strains. This reduction in product quality could outweigh the advantages of using Mut strains, such as lower oxygen and methanol demand, heat formation and in some cases improved expression. Methoxine incorporation in recombinant proteins is likely to occur when an excess of methanol is present during fermentation but can be avoided when the methanol feed rate protocol is carefully designed.

  3. Exploration of Venus' Deep Atmosphere and Surface Environment

    Science.gov (United States)

    Glaze, L. S.; Amato, M.; Garvin, J. B.; Johnson, N. M.

    2017-01-01

    Venus formed in the same part of our solar system as Earth, apparently from similar materials. Although both planets are about the same size, their differences are profound. Venus and Earth experienced vastly different evolutionary pathways resulting in unexplained differences in atmospheric composition and dynamics, as well as in geophysical processes of the planetary surfaces and interiors. Understanding when and why the evolutionary pathways of Venus and Earth diverged is key to understanding how terrestrial planets form and how their atmospheres and surfaces evolve. Measurements made in situ, within the near-surface or surface environment, are critical to addressing unanswered questions. We have made substantial progress modernizing and maturing pressure vessel technologies to enable science operations in the high temperature and pressure near-surface/surfaceenvironment of Venus.

  4. Stem cell responses to plasma surface modified electrospun polyurethane scaffolds.

    Science.gov (United States)

    Zandén, Carl; Hellström Erkenstam, Nina; Padel, Thomas; Wittgenstein, Julia; Liu, Johan; Kuhn, H Georg

    2014-07-01

    The topographical effects from functional materials on stem cell behavior are currently of interest in tissue engineering and regenerative medicine. Here we investigate the influence of argon, oxygen, and hydrogen plasma surface modification of electrospun polyurethane fibers on human embryonic stem cell (hESC) and rat postnatal neural stem cell (NSC) responses. The plasma gases were found to induce three combinations of fiber surface functionalities and roughness textures. On randomly oriented fibers, plasma treatments lead to substantially increased hESC attachment and proliferation as compared to native fibers. Argon plasma was found to induce the most optimal combination of surface functionality and roughness for cell expansion. Contact guided migration of cells and alignment of cell processes were observed on aligned fibers. Neuronal differentiation around 5% was found for all samples and was not significantly affected by the induced variations of surface functional group distribution or individual fiber topography. In this study the influence of argon, oxygen, and hydrogen plasma surface modification of electrospun polyurethane fibers on human embryonic stem cell and rat postnatal neural stem cell (NSC) responses is studied with the goal of clarifying the potential effects of functional materials on stem cell behavior, a topic of substantial interest in tissue engineering and regenerative medicine. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. 26 CFR 1.6662-4 - Substantial understatement of income tax.

    Science.gov (United States)

    2010-04-01

    ... arrangement does not have as its principal purpose the avoidance or evasion of Federal income tax solely as a... 26 Internal Revenue 13 2010-04-01 2010-04-01 false Substantial understatement of income tax. 1... (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Additions to the Tax, Additional Amounts, and Assessable...

  6. Electron mobility on the surface of liquid Helium: influence of surface level atoms and depopulation of lowest subbands

    International Nuclear Information System (INIS)

    Grigoriev, P. D.; Dyugaev, A. M.; Lebedeva, E. V.

    2008-01-01

    The temperature dependence of electron mobility is examined. We calculate the contribution to the electron scattering rate from the surface level atoms (SLAs), proposed in [10]. This contribution is substantial at low temperatures T < 0.5, when the He vapor concentration is exponentially small. We also study the effect of depopulation of the lowest energy subband, which leads to an increase in the electron mobility at high temperature. The results explain certain long-standing discrepancies between the existing theory and experiment on electron mobility on the surface of liquid helium

  7. Surface photovoltage studies of p-type AlGaN layers after reactive-ion etching

    Science.gov (United States)

    McNamara, J. D.; Phumisithikul, K. L.; Baski, A. A.; Marini, J.; Shahedipour-Sandvik, F.; Das, S.; Reshchikov, M. A.

    2016-10-01

    The surface photovoltage (SPV) technique was used to study the surface and electrical properties of Mg-doped, p-type AlxGa1-xN (0.06 GaN:Mg thin films and from the predictions of a thermionic model for the SPV behavior. In particular, the SPV of the p-AlGaN:Mg layers exhibited slower-than-expected transients under ultraviolet illumination and delayed restoration to the initial dark value. The slow transients and delayed restorations can be attributed to a defective surface region which interferes with normal thermionic processes. The top 45 nm of the p-AlGaN:Mg layer was etched using a reactive-ion etch which caused the SPV behavior to be substantially different. From this study, it can be concluded that a defective, near-surface region is inhibiting the change in positive surface charge by allowing tunneling or hopping conductivity of holes from the bulk to the surface, or by the trapping of electrons traveling to the surface by a high concentration of defects in the near-surface region. Etching removes the defective layer and reveals a region of presumably higher quality, as evidenced by substantial changes in the SPV behavior.

  8. 40 CFR Appendix C to Part 112 - Substantial Harm Criteria

    Science.gov (United States)

    2010-07-01

    ... to Part 112 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) WATER PROGRAMS OIL POLLUTION PREVENTION Pt. 112, App. C Appendix C to Part 112—Substantial Harm Criteria 1.0Introduction The..., except in the Gulf of Mexico. In the Gulf of Mexico, it means the area shoreward of the lines of...

  9. On the thermodynamics of biomolecule surface transformations.

    Science.gov (United States)

    Federici, Stefania; Oliviero, Giulio; Maiolo, Daniele; Depero, Laura E; Colombo, Italo; Bergese, Paolo

    2012-06-01

    Biological surface science is receiving great and renewed attention owing the rising interest in applications of nanoscience and nanotechnology to biological systems, with horizons that range from nanomedicine and biomimetic photosynthesis to the unexpected effects of nanomaterials on health and environment. Biomolecule surface transformations are among the fundamental aspects of the field that remain elusive so far and urgently need to be understood to further the field. Our recent findings indicate that surface thermodynamics can give a substantial contribution toward this challenging goal. In the first part of the article, we show that biomolecule surface transformations can be framed by a general and simple thermodynamic model. Then, we explore its effectiveness by addressing some typical cases, including ligand-receptor surface binding, protein thin film machines, nanomechanical aspects of the biomolecule-nanoparticle interface and nanomechanical biosensors. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Surface plasmon polariton amplification in semiconductor-graphene-dielectric structure

    Energy Technology Data Exchange (ETDEWEB)

    Dadoenkova, Yuliya S. [Ulyanovsk State University, Ulyanovsk (Russian Federation); Novgorod State University, Veliky Novgorod (Russian Federation); Donetsk Institute for Physics and Technology, Donetsk (Ukraine); Moiseev, Sergey G. [Ulyanovsk State University, Ulyanovsk (Russian Federation); Kotelnikov Institute of Radio Engineering and Electronics, Russian Academy of Sciences, Ulyanovsk (Russian Federation); Abramov, Aleksei S. [Ulyanovsk State University, Ulyanovsk (Russian Federation); Kadochkin, Aleksei S.; Zolotovskii, Igor O. [Ulyanovsk State University, Ulyanovsk (Russian Federation); Institute of Nanotechnologies of Microelectronics of the Russian Academy of Sciences, 32A Leninskiy Prosp., 119991, Moscow (Russian Federation); Fotiadi, Andrei A. [Ulyanovsk State University, Ulyanovsk (Russian Federation); Universite de Mons (Belgium)

    2017-05-15

    A mechanism of amplification of surface plasmon polaritons due to the transfer of electromagnetic energy from a drift current wave into a far-infrared surface wave propagating along a semiconductor-dielectric boundary in waveguide geometry is proposed. A necessary condition of the interaction of these waves is phase matching condition, i. e., when the phase velocity of the surface wave approaches the drift velocity of charge carriers. It is shown that in the spectral region of the surface plasmon polariton slowing-down its amplification coefficient can reach values substantially exceeding the ohmic loss coefficient of the surface wave in the structure. (copyright 2017 by WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  11. 20 CFR 416.1080 - Notice of right to hearing on proposed finding of substantial failure.

    Science.gov (United States)

    2010-04-01

    ... agency has substantially failed to meet our standards unless the State submits a written request for a... finding of substantial failure. 416.1080 Section 416.1080 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Determinations of Disability...

  12. TECHNICAL AND ECONOMIC SUBSTANTIATION OF PROJECTS OF THE AFFORDABLE HOUSING CONSTRUCTION

    Directory of Open Access Journals (Sweden)

    KRAVCHUNOVSKA T. S.

    2015-12-01

    Full Text Available Summary. Problem statement. The development of inhabitant locality of Ukraine is characterized by significant differences in levels of socio and economic development. An excessive concentration of population and industry in large cities, inefficient, slow development of most medium and small cities, towns and villages, considerable territorial disproportions of economic development of the country, considerable shortcomings in the territorial organization of society are observed. At the same time the tendency of the total area of increasing of inhabitant locality. The lands are used inefficiently. Among the most common problems of inhabitant locality is the housing problem. When making a decision on the design of affordable housing construction is one of the most important stages in the development of the technique and economic substantiation of the project is determination of the advisability and effectiveness of construction. The substantiation of advisability and effectiveness of affordable housing based on the definition of technical and economic indicators of projects, one of the most important among them is the cost, in its calculation is necessary to take into account the influence of organizational and technological factors, reflecting the features of construction in condition of compacted construction. Purpose. Development of methodical recommendations on substantiation of cost construction of affordable housing in the conditions of compacted construction. Conclusion. To provide a processing and the analysis of data is necessary development of the applied software on the basis of the developed block scheme of justification of cost construction of affordable housing.

  13. Laser surface wakefield in a plasma column

    International Nuclear Information System (INIS)

    Gorbunov, L.M.; Mora, P.; Ramazashvili, R.R.

    2003-01-01

    The structure of the wakefield in a plasma column, produced by a short intense laser pulse, propagating through a gas affected by tunneling ionization is investigated. It is shown that besides the usual plasma waves in the bulk part of the plasma column [see Andreev et al., Phys. Plasmas 9, 3999 (2002)], the laser pulse also generates electromagnetic surface waves propagating along the column boundary. The length of the surface wake wave substantially exceeds the length of the plasma wake wave and its electromagnetic field extends far outside the plasma column

  14. Substantiation of the road toll for heavy transport vehicles

    OpenAIRE

    Burmaka, N.; Chernykh, A.

    2010-01-01

    The existing and possible additional sources of developing state and local road funds of Ukraine have been considered. The formula for calculating monthly road toll for heavy transport vehicles has been proposed. This formula includes the payment rate per every kilometer of distance, the vehicle capacity utilization factor and the run with the load. The payment rate per every kilometer of distance for transport vehicles depending on the allowed total weight has been substantiated. The given r...

  15. 20 CFR 404.1680 - Notice of right to hearing on proposed finding of substantial failure.

    Science.gov (United States)

    2010-04-01

    ... agency has substantially failed to meet our standards unless the State submits a written request for a... finding of substantial failure. 404.1680 Section 404.1680 Employees' Benefits SOCIAL SECURITY..., following the mandatory performance support period and the 3-month adjustment period, a State agency again...

  16. Surface effects on converse piezoelectricity of crystals.

    Science.gov (United States)

    Molayem, Mohammad; Springborg, Michael; Kirtman, Bernard

    2017-09-20

    The contribution of surface units to bulk properties are often neglected in theoretical and computational studies of crystalline systems. We demonstrate that this assumption has to be made with caution in the case of (electric field) polarization. As a generalization of an earlier work on quasi-one-dimensional systems [Springborg, et al., Phys. Rev. B: Condens. Matter Mater. Phys., 2010, 82, 165442], it is shown that the polarization for 2D and 3D systems contains a surface contribution that can, in principle, take any value (within physical limits) and has consequences for converse piezoelectric responses. Subsequently, we determine the surface effects quantitatively for a group of ferroelectric perovskite structures. Our results indicate that such contributions can be substantial.

  17. Surface Forces Apparatus measurements of interactions between rough and reactive calcite surfaces.

    Science.gov (United States)

    Dziadkowiec, Joanna; Javadi, Shaghayegh; Bratvold, Jon Einar; Nilsen, Ola; Røyne, Anja

    2018-05-28

    Nm-range forces acting between calcite surfaces in water affect macroscopic properties of carbonate rocks and calcite-based granular materials, and are significantly influenced by calcite surface recrystallization. We suggest that the repulsive mechanical effects related to nm-scale surface recrystallization of calcite in water could be partially responsible for the observed decrease of cohesion in calcitic rocks saturated with water. Using the Surface Forces Apparatus (SFA), we simultaneously followed the calcite reactivity and measured the forces in water in two surface configurations: between two rough calcite surfaces (CC), or between rough calcite and a smooth mica surface (CM). We used nm-scale rough, polycrystalline calcite films prepared by Atomic Layer Deposition (ALD). We measured only repulsive forces in CC in CaCO 3 -saturated water, which was related to roughness and possibly to repulsive hydration effects. Adhesive or repulsive forces were measured in CM in CaCO 3 -saturated water depending on calcite roughness, and the adhesion was likely enhanced by electrostatic effects. The pull-off adhesive force in CM became stronger with time and this increase was correlated with a decrease of roughness at contacts, which parameter could be estimated from the measured force-distance curves. That suggested a progressive increase of real contact areas between the surfaces, caused by gradual pressure-driven deformation of calcite surface asperities during repeated loading-unloading cycles. Reactivity of calcite was affected by mass transport across nm to µm-thick gaps between the surfaces. Major roughening was observed only for the smoothest calcite films, where gaps between two opposing surfaces were nm-thick over µm-sized areas, and led to force of crystallization that could overcome confining pressures of the order of MPa. Any substantial roughening of calcite caused a significant increase of the repulsive mechanical force contribution.

  18. Flow Structure and Surface Topology on a UCAV Planform

    Science.gov (United States)

    Elkhoury, Michel; Yavuz, Metin; Rockwell, Donald

    2003-11-01

    Flow past a X-45 UCAV planform involves the complex generation and interaction of vortices, their breakdown and occurrence of surface separation and stall. A cinema technique of high-image-density particle image velocimetry, in conjunction with dye visualization, allows characterization of the time-averaged and instantaneous states of the flow, in terms of critical points of the near-surface streamlines. These features are related to patterns of surface normal vorticity and velocity fluctuation. Spectral analysis of the naturally occurring unsteadiness of the flow allows definition of the most effective frequencies for small-amplitude perturbation of the wing, which leads to substantial alterations of the aforementioned patterns of flow structure and topology adjacent to the surface.

  19. 75 FR 27504 - Substantial Product Hazard List: Hand-Held Hair Dryers

    Science.gov (United States)

    2010-05-17

    ... immersion during their use. Section 15(a) of the CPSA defines ``substantial product hazard'' to include, a....'' Hand-held hair dryers routinely contain open-coil heating elements that are, in essence, uninsulated..., bathtub, or lavatory). The proposed rule would define ``hand-held hair dryer'' as ``an electrical...

  20. Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.

    Science.gov (United States)

    Schumacher, Dominik; Lemke, Oliver; Helma, Jonas; Gerszonowicz, Lena; Waller, Verena; Stoschek, Tina; Durkin, Patrick M; Budisa, Nediljko; Leonhardt, Heinrich; Keller, Bettina G; Hackenberger, Christian P R

    2017-05-01

    The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

  1. Influence of organic surface coatings on the sorption of anticonvulsants on mineral surfaces.

    Science.gov (United States)

    Qu, Shen; Cwiertny, David M

    2013-10-01

    Here, we explore the role that sorption to mineral surfaces plays in the fate of two commonly encountered effluent-derived pharmaceuticals, the anticonvulsants phenytoin and carbamazepine. Adsorption isotherms and pH-edge experiments are consistent with electrostatics governing anticonvulsant uptake on metal oxides typically found in soil and aquifer material (e.g., Si, Al, Fe, Mn, and Ti). Appreciable, albeit limited, adsorption was observed only for phenytoin, which is anionic above pH 8.3, on the iron oxides hematite and ferrihydrite. Adsorption increased substantially in the presence of cationic and anionic surfactants, species also commonly encountered in wastewater effluent. For carbamazepine, we propose the enhanced uptake results entirely from hydrophobic interactions with apolar tails of surfactant surface coatings. For phenytoin, adsorption also arises from the ability of surfactants to alter the net charge of the mineral surface and thereby further enhance favorable electrostatic interactions with its anionic form. Collectively, our results demonstrate that although pristine mineral surfaces are likely not major sinks for phenytoin and carbamazepine in the environment, their alteration with organic matter, particularly surfactants, can considerably increase their ability to retain these emerging pollutants in subsurface systems.

  2. Density-functional calculations of the surface tension of liquid Al and Na

    Science.gov (United States)

    Stroud, D.; Grimson, M. J.

    1984-01-01

    Calculations of the surface tensions of liquid Al and Na are described using the full ionic density functional formalism of Wood and Stroud (1983). Surface tensions are in good agreement with experiment in both cases, with results substantially better for Al than those found previously in the gradient approximation. Preliminary minimization with respect to surface profile leads to an oscillatory profile superimposed on a nearly steplike ionic density disribution; the oscillations have a wavellength of about a hardsphere diameter.

  3. Stormwater Priority Pollutants Versus Surface Water Quality Criteria

    DEFF Research Database (Denmark)

    Eriksson, Eva; Ledin, Anna; Baun, Anders

    2011-01-01

    Stormwater in urban areas comprises of a substantial part of the urban water cycle, dominating the flow in many small urban streams, and the pollution levels are sizeable. No stormwater quality criteria were found here and no European or national emission limit values exist. Stormwater pollutants...... however are present in levels exceeding most of the regulated surface water quality criteria and environmental quality standards. Therefore catchment characterisation is needed to chose suitable treatment prior to discharge into receiving surface waters, as the mixing may be insufficient in small streams....

  4. 75 FR 64683 - Liability for Termination of Single-Employer Plans; Treatment of Substantial Cessation of Operations

    Science.gov (United States)

    2010-10-20

    ... Termination of Single-Employer Plans; Treatment of Substantial Cessation of Operations AGENCY: Pension Benefit... cessations of operations by employers that maintain single-employer plans. DATES: Comments must be submitted... 4062(e), which provides for reporting of and liability for certain substantial cessations of operations...

  5. conserving surface-nesting seabirds at the prince edward islands

    African Journals Online (AJOL)

    South Africa's subantarctic Prince Edward Islands support substantial proportions of the global populations of a number of surface-nesting seabirds. Populations of most of these have decreased at the islands since the 1980s and 12 of 16 species are regarded as Threatened or Near Threatened regionally or internationally.

  6. Depression cathode structure for cathode ray tubes having surface smoothness and method for producing same

    International Nuclear Information System (INIS)

    Rychlewski, T.V.

    1984-01-01

    Depression cathode structures for cathode ray tubes are produced by dispensing liquid cathode material into the depression of a metallic supporting substrate, removing excess cathode material by passing a doctor blade across the substrate surface and over the depression, and drying the cathode layer to a substantially immobile state. The cathode layer may optionally be further shaped prior to substantially complete drying thereof

  7. Clozapine-induced dysphagia with secondary substantial weight loss.

    Science.gov (United States)

    Osman, Mugtaba; Devadas, Vekneswaran

    2016-08-19

    Dysphagia is listed as a 'rare' side effect following clozapine treatment. In this case report, we describe how significant clozapine-induced dysphagia has led to significant reduction of nutritional intake with subsequent substantial weight loss. An 18-year-old single man with an established diagnosis of treatment-resistant paranoid schizophrenia recovered well on a therapeutic dose of clozapine. However, he was noted to lose weight significantly (up to 20% of his original weight) as the dose was uptitrated. This was brought about by development of dysphagia, likely to be due to clozapine. Addition of nutritional supplementary liquids and initiation of a modified behavioural dietary/swallowing programme, while repeatedly mastering the Mendelsohn manoeuvre technique, alleviated the swallowing difficulties and restored his weight. 2016 BMJ Publishing Group Ltd.

  8. Modeling surface roughness scattering in metallic nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Moors, Kristof, E-mail: kristof@itf.fys.kuleuven.be [KU Leuven, Institute for Theoretical Physics, Celestijnenlaan 200D, B-3001 Leuven (Belgium); IMEC, Kapeldreef 75, B-3001 Leuven (Belgium); Sorée, Bart [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium); Physics Department, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen (Belgium); KU Leuven, Electrical Engineering (ESAT) Department, Kasteelpark Arenberg 10, B-3001 Leuven (Belgium); Magnus, Wim [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium); Physics Department, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen (Belgium)

    2015-09-28

    Ando's model provides a rigorous quantum-mechanical framework for electron-surface roughness scattering, based on the detailed roughness structure. We apply this method to metallic nanowires and improve the model introducing surface roughness distribution functions on a finite domain with analytical expressions for the average surface roughness matrix elements. This approach is valid for any roughness size and extends beyond the commonly used Prange-Nee approximation. The resistivity scaling is obtained from the self-consistent relaxation time solution of the Boltzmann transport equation and is compared to Prange-Nee's approach and other known methods. The results show that a substantial drop in resistivity can be obtained for certain diameters by achieving a large momentum gap between Fermi level states with positive and negative momentum in the transport direction.

  9. Do substantial BMI reduction episodes among Swedish schoolchildren have any impact on their final height?

    Science.gov (United States)

    Nilsen, Bente B; Yngve, Agneta; Werner, Bo

    2018-02-06

    This study investigated whether substantial body mass index (BMI) reductions in Swedish schoolchildren aged seven years to 19 years, caused by disease, healthy or unhealthy behaviour, had any impact on their final height. We used height and weight data on 6572 subjects from two nationally representative longitudinal samples of Swedish children born in 1973 and 1981. These provided information on their final height and any BMI reduction episodes. Of the 6572 subjects (50.9% boys), among individuals with information on final height, 1118 had a BMI reduction of 5% and BMI reduction of 10% or more. On a group level, there was no statistically significant difference in the final height of individuals with BMI reductions of 10% or more and those without. The findings were independent of age and the subject's BMI at the start of the reduction episode. However, there were a number of cases where a substantial BMI reduction probably had an impact on the subject's final height. Our study found no evidence that a substantial BMI reduction had any impact on final height on a group level, but further analyses of specific case studies are necessary to determine whether substantial BMI reduction might have an impact on final height. ©2018 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  10. Venting temperature determines surface chemistry of magnetron sputtered TiN films

    Energy Technology Data Exchange (ETDEWEB)

    Greczynski, G. [Thin Film Physics Division, Department of Physics (IFM), Linköping University, SE-581 83 Linköping (Sweden); Materials Chemistry, RWTH Aachen University, Kopernikusstr. 10, D-52074 Aachen (Germany); Mráz, S.; Schneider, J. M. [Materials Chemistry, RWTH Aachen University, Kopernikusstr. 10, D-52074 Aachen (Germany); Hultman, L. [Thin Film Physics Division, Department of Physics (IFM), Linköping University, SE-581 83 Linköping (Sweden)

    2016-01-25

    Surface properties of refractory ceramic transition metal nitride thin films grown by magnetron sputtering are essential for resistance towards oxidation necessary in all modern applications. Here, typically neglected factors, including exposure to residual process gases following the growth and the venting temperature T{sub v}, each affecting the surface chemistry, are addressed. It is demonstrated for the TiN model materials system that T{sub v} has a substantial effect on the composition and thickness-evolution of the reacted surface layer and should therefore be reported. The phenomena are also shown to have impact on the reliable surface characterization by x-ray photoelectron spectroscopy.

  11. COMPOSITIONAL AND SUBSTANTIAL STRUCTURE OF THE MEDICAL DOCUMENT: FORMATION STAGES

    Directory of Open Access Journals (Sweden)

    Romashova Olga Vladimirovna

    2015-03-01

    Full Text Available The article deals with the compositional and substantial structure of the ambulatory medical record, or "case history", which has being formed for a long time. The author allocates the three main periods in the formation of this medical document: the first period (the beginning of the 19th century – 1920s is connected with the origin and formation; the second period (1920-1980s is marked by emergence of the normative legal acts regulating registration and maintaining; the third period (1980s – up to the present is associated with the cancellation of regulations and the introduction of the new order of the Ministry of Health of the USSR that changed the document's form and name. It is determined that the composition of the case history consists of the title page and the main part. The following processes take place in the course of ambulatory medical record's formation: strengthening formalization, increase in the number of pattern text fragments, increase in the text's volume, and the implementation of bigger number of functions. The author reveals the main (informative and cumulative, accounting and additional (scientific, controlling, legal, financial functions of the document. The implementation of these functions is reflected in the compositional and substantial structure of the document text and is conditioned by a number of extralinguistic factors.

  12. Effective surface passivation of InP nanowires by atomic-layer-deposited Al2O3 with POx interlayer

    NARCIS (Netherlands)

    Black, L.E.; Cavalli, A.; Verheijen, M.A.; Haverkort, J.E.M.; Bakkers, E.P.A.M.; Kessels, W.M.M.

    2017-01-01

    III/V semiconductor nanostructures have significant potential in device applications, but effective surface passivation is critical due to their large surface-to-volume ratio. For InP such passivation has proven particularly difficult, with substantial depassivation generally observed following

  13. Surface tension confined liquid cryogen cooler

    Science.gov (United States)

    Castles, Stephen H. (Inventor); Schein, Michael E. (Inventor)

    1989-01-01

    A cryogenic cooler is provided for use in craft such as launch, orbital, and space vehicles subject to substantial vibration, changes in orientation, and weightlessness. The cooler contains a small pore, large free volume, low density material to restrain a cryogen through surface tension effects during launch and zero-g operations and maintains instrumentation within the temperature range of 10 to 140 K. The cooler operation is completely passive, with no inherent vibration or power requirements.

  14. Motion of a cylinder adjacent to a free-surface: flow patterns and loading

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Q.; Lin, J.C. [Lehigh Univ., Bethlehem, PA (United States). Dept. of Mechanical Engineering and Mechanics; Unal, M.F.; Rockwell, D.

    2000-06-01

    The flow structure and loading due to combined translatory and sinusoidal motion of a cylinder adjacent to a free-surface are characterized using a cinema technique of high-image-density particle image velocimetry and simultaneous force measurements. The instantaneous patterns of vorticity and streamline topology are interpreted as a function of degree of submergence beneath the free-surface. The relative magnitudes of the peak vorticity and the circulation of vortices formed from the upper and lower surfaces of the cylinder, as well as vortex formation from the free-surface, are remarkably affected by the nominal submergence. The corresponding streamline topology, interpreted in terms of foci, saddle points, and multiple separation and reattachment points also exhibit substantial changes with submergence. All of these features affect the instantaneous loading of the cylinder. Calculation of instantaneous moments of vorticity and the incremental changes in these moments during the cylinder motion allow identification of those vortices that contribute most substantially to the instantaneous lift and drag. Furthermore, the calculated moments are in general accord with the time integrals of the measured lift and drag acting on the cylinder for sufficiently large submergence. (orig.)

  15. Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

    Science.gov (United States)

    Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

    2018-03-13

    Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

  16. 76 FR 32215 - Agency Information Collection Activities; Proposed Collection; Comment Request; Substantiation...

    Science.gov (United States)

    2011-06-03

    ... of such disease in the United States [(U.S.)], describes the role of a nutrient or dietary ingredient... substantiation for their claims in the scientific literature. The time it takes to assemble the necessary... based on emerging science, where conducting literature searches and understanding the literature takes...

  17. Process for cleaning radioactively contaminated metal surfaces

    International Nuclear Information System (INIS)

    Mihram, R.G.; Snyder, G.A.

    1975-01-01

    A process is described for removing radioactive scale from a ferrous metal surface, including the steps of initially preconditioning the surface by contacting it with an oxidizing solution (such as an aqueous solution of an alkali metal permanganate or hydrogen peroxide), then, after removal or decomposition of the oxidizing solution, the metallic surface is contacted with a cleaning solution which is a mixture of a mineral acid and a complexing agent (such as sulfuric acid and oxalic acid), and which preferably contains a corrosion inhibitor. A final step in the process is the treatment of the spent cleaning solution containing radioactive waste materials in solution by adding a reagent selected from the group consisting of calcium hydroxide or potassium permanganate and an alkali metal hydroxide to thereby form easily recovered metallic compounds containing substantially all of the dissolved metals and radioactivity. (auth)

  18. Plasma processing of large curved surfaces for superconducting rf cavity modification

    Directory of Open Access Journals (Sweden)

    J. Upadhyay

    2014-12-01

    Full Text Available Plasma-based surface modification of niobium is a promising alternative to wet etching of superconducting radio frequency (SRF cavities. We have demonstrated surface layer removal in an asymmetric nonplanar geometry, using a simple cylindrical cavity. The etching rate is highly correlated with the shape of the inner electrode, radio-frequency (rf circuit elements, gas pressure, rf power, chlorine concentration in the Cl_{2}/Ar gas mixtures, residence time of reactive species, and temperature of the cavity. Using variable radius cylindrical electrodes, large-surface ring-shaped samples, and dc bias in the external circuit, we have measured substantial average etching rates and outlined the possibility of optimizing plasma properties with respect to maximum surface processing effect.

  19. 21 CFR 101.43 - Substantial compliance of food retailers with the guidelines for the voluntary nutrition labeling...

    Science.gov (United States)

    2010-04-01

    ... FOR HUMAN CONSUMPTION FOOD LABELING Specific Nutrition Labeling Requirements and Guidelines § 101.43 Substantial compliance of food retailers with the guidelines for the voluntary nutrition labeling of raw fruit... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Substantial compliance of food retailers with the...

  20. Substantial injuries influence ranking position in young elite athletes of athletics, cross-country skiing and orienteering.

    Science.gov (United States)

    von Rosen, P; Heijne, A

    2018-04-01

    The relationship between injury and performance in young athletes is scarcely studied. The aim of this study was therefore to explore the association between injury prevalence and ranking position among adolescent elite athletes. One hundred and sixty-two male and female adolescent elite athletes (age range 15-19), competing in athletics (n = 59), cross-country skiing (n = 66), and orienteering (n = 37), were monitored weekly over 22-47 weeks using a web-based injury questionnaire. Ranking lists were collected. A significant (P = .003) difference was found in the seasonal substantial injury prevalence across the ranked athletes over the season, where the top-ranked (median 3.6%, 25-75th percentiles 0%-14.3%) and middle-ranked athletes (median 2.3%, 25-75th percentiles 0%-10.0%) had a lower substantial injury prevalence compared to the low-ranked athletes (median 11.3%, 25-75th percentiles 2.5%-27.1%), during both preseason (P = .002) and competitive season (P = .031). Athletes who improved their ranking position (51%, n = 51) reported a lower substantial injury prevalence (median 0%, 25-75th percentiles 0%-10.0%) compared to those who decreased (49%, n = 49) their ranking position (md 6.7%, 25-75th percentiles 0%-22.5%). In the top-ranked group, no athlete reported substantial injury more than 40% of all data collection time points compared to 9.6% (n = 5) in the middle-ranked, and 17.3% (n = 9) in the low-ranked group. Our results provide supporting evidence that substantial injuries, such as acute and overuse injuries leading to moderate or severe reductions in training or sports performance, influence ranking position in adolescent elite athletes. The findings are crucial to stakeholders involved in adolescent elite sports and support the value of designing effective preventive interventions for substantial injuries. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Acid-base properties of the surface of the α-Al2O3 suspension

    Science.gov (United States)

    Ryazanov, M. A.; Dudkin, B. N.

    2009-12-01

    The distribution of the acid-base centers on the surface of α-Al2O3 suspension particles was studied by potentiometric titration, and the corresponding p K spectra were constructed. It was inferred that the double electric layer created by the supporting electrolyte substantially affected the screening of the acid-base centers on the particle surface of the suspension.

  2. Neighborhood-level social processes and substantiated cases of child maltreatment.

    Science.gov (United States)

    Molnar, Beth E; Goerge, Robert M; Gilsanz, Paola; Hill, Andrea; Subramanian, S V; Holton, John K; Duncan, Dustin T; Beatriz, Elizabeth D; Beardslee, William R

    2016-01-01

    Child maltreatment is a preventable public health problem. Research has demonstrated that neighborhood structural factors (e.g. poverty, crime) can influence the proportion of a neighborhood's children who are victims of maltreatment. A newer strategy is the identification of potentially modifiable social processes at the neighborhood level that can also influence maltreatment. Toward this end, this study examines neighborhood-level data (maltreatment cases substantiated by Illinois' child protection agency, 1995-2005, social processes measured by the Project on Human Development in Chicago Neighborhoods, U.S. Census data, proportions of neighborhoods on public assistance, and crime data) that were linked across clusters of contiguous, relatively homogenous Chicago, IL census tracts with respect to racial/ethnic and socioeconomic composition. Our analysis-an ecological-level, repeated cross-sectional design utilizing random-intercept logit models-with a sensitivity analysis using spatial models to control for spatial autocorrelation-revealed consistent associations between neighborhood social processes and maltreatment. Neighborhoods higher in collective efficacy, intergenerational closure, and social networks, and lower in disorder had lower proportions of neglect, physical abuse, and sexual abuse substantiated cases, controlling for differences in structural factors. Higher collective efficacy and social network size also predicted a lower proportion of substance-exposed infants. This research indicates that strategies to mobilize neighborhood-level protective factors may decrease child maltreatment more effectively than individual and family-focused efforts alone. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Greenland meltwater storage in firn limited by near-surface ice formation

    DEFF Research Database (Denmark)

    Machguth, Horst; MacFerrin, Mike; van As, Dirk

    2016-01-01

    above sea level), firn has undergone substantial densification, while at lower elevations, where melt is most abundant, porous firn has lost most of its capability to retain meltwater. Here, the formation of near-surface ice layers renders deep pore space difficult to access, forcing meltwater to enter...

  4. Surface alloy formation by adsorption of holmium on Ag/Mo(112) bimetallic surfaces

    Science.gov (United States)

    Kołaczkiewicz, Jan; Oleksy, Czesław

    2018-03-01

    Work function change measurements, low energy electron diffraction (LEED) and density functional theory (DFT) are used to determine the structures formed on Ag/Mo(112) bimetallic surfaces upon deposition of 0.5 monolayer (ML) of holmium. As the bimetallic surfaces, we have chosen the Mo(112) substrate covered with 1 or 2 ML of Ag. Such surfaces have the same symmetry as the Mo(112) face but different electronic properties. LEED experiment indicates that the c(2 × 2) structure is formed on (1 ML Ag)/Mo(112) bimetallic surface upon deposition of 0.5 ML of Ho. DFT calculations show that a type of Ag-Ho surface alloy is formed, with Ho atoms 0.6 Å below the distorted layer of Ag. This is neither a substitutional nor a subsurface alloy. It is found that the adsorption structure formed on the (2 ML Ag)/Mo(112) bimetallic surface depends on the annealing temperature. After deposition of 0.5 ML of Ho at 300 K, the LEED pattern of p(2 × 2) symmetry is observed. Annealing of the overlayer at 640 K irreversibly changes the p(2 × 2) pattern into a pattern of c(2 × 2) type. The results of DFT computations show that the c(2 × 2) structure of the Ag-Ho surface alloy is energetically most favorable. In this structure, 0.5 ML of Ho is between the two monolayers of Ag, and the symmetry of the topmost layer is changed. The work function change calculated for the c(2 × 2) structure is in a good agreement with the measured value (0.22 eV). The results show that adsorption of Ho on the Ag/Mo(112) bimetallic surfaces is substantially different than on the clean Mo(112).

  5. Substantial Churn In Health Insurance Offerings By Small Employers, 2014-15.

    Science.gov (United States)

    Vistnes, Jessica P; Rohde, Frederick; Miller, G Edward; Cooper, Philip F

    2017-09-01

    New data for 2014-15 from the Medical Expenditure Panel Survey-Insurance Component longitudinal survey show substantial churn in insurance offers by small employers (those with fifty or fewer workers), with 14.6 percent of employers that offered insurance in 2014 having dropped it in 2015 and 5.5 percent of those that did not offer it adding coverage. Project HOPE—The People-to-People Health Foundation, Inc.

  6. Modeled heating and surface erosion comparing motile (gas borne) and stationary (surface coating) inert particle additives

    International Nuclear Information System (INIS)

    Buckingham, A.C.; Siekhaus, W.J.

    1982-01-01

    The unsteady, non-similar, chemically reactive, turbulent boundary layer equations are modified for gas plus dispersed solid particle mixtures, for gas phase turbulent combustion reactions and for heterogeneous gas-solid surface erosive reactions. The exterior (ballistic core) edge boundary conditions for the solutions are modified to include dispersed particle influences on core propellant combustion-generated turbulence levels, combustion reactants and products, and reaction-induced, non-isentropic mixture states. The wall surface (in this study it is always steel) is considered either bare or coated with a fixed particle coating which is conceptually non-reactive, insulative, and non-ablative. Two families of solutions are compared. These correspond to: (1) consideration of gas-borne, free-slip, almost spontaneously mobile (motile) solid particle additives which influence the turbulent heat transfer at the uncoated steel surface and, in contrast, (2) consideration of particle-free, gas phase turbulent heat transfer to the insulated surface coated by stationary particles. Significant differences in erosive heat transfer are found in comparing the two families of solutions over a substantial range of interior ballistic flow conditions. The most effective influences on reducing erosive heat transfer appear to favor mobile, gas-borne particle additives

  7. Pt-Si Bifunctional Surfaces for CO and Methanol Electro-Oxidation

    DEFF Research Database (Denmark)

    Permyakova, Anastasia A.; Han, Binghong; Jensen, Jens Oluf

    2015-01-01

    and storage. Here we report on Pt-Si bulk samples prepared by arc-melting, for the first time, with high activities toward the electro-oxidation of CO and methanol. Increasing the Si concentration on the surface was correlated with the shifts of onset oxidation potentials to lower values and higher activities...... for CO and methanol electro-oxidation. It is proposed that the reaction on the Pt-Si catalyst could follow a Langmuir-Hinshelwood type of mechanism, where substantially enhanced catalytic activity is attributed to the fine-tuning of the surface Pt-Si atomic structure....

  8. Particle surface area and bacterial activity in recirculating aquaculture systems

    DEFF Research Database (Denmark)

    Pedersen, Per Bovbjerg; von Ahnen, Mathis; Fernandes, Paulo

    2017-01-01

    Suspended particles in recirculating aquaculture systems (RAS) provide surface area that can be colonized by bacteria. More particles accumulate as the intensity of recirculation increases thus potentially increasing the bacterial carrying capacity of the systems. Applying a recent, rapid, culture...... but may provide significant surface area. Hence, the study substantiates that particles in RAS provide surface area supporting bacterial activity, and that particles play a key role in controlling the bacterial carrying capacity at least in less intensive RAS. Applying fast, culture-independent techniques......-independent fluorometric detection method (Bactiquant®) for measuring bacterial activity, the current study explored the relationship between total particle surface area (TSA, derived from the size distribution of particles >5 μm) and bacterial activity in freshwater RAS operated at increasing intensity of recirculation...

  9. Mars Surface Environmental Issues

    Science.gov (United States)

    Charles, John

    2002-01-01

    Planetary exploration by astronauts will require extended periods of habitation on a planet's surface, under the influence of environmental factors that are different from those of Earth and the spacecraft that delivered the crew to the planet. Human exploration of Mars, a possible near-term planetary objective, can be considered a challenging scenario. Mission scenarios currently under consideration call for surface habitation periods of from 1 to 18 months on even the earliest expeditions. Methods: Environmental issues associated with Mars exploration have been investigated by NASA and the National Space Biomedical Research Institute (NSBRI) as part of the Bioastronautics Critical Path Roadmap Project (see http ://criticalpath.jsc.nasa.gov). Results: Arrival on Mars will immediately expose the crew to gravity only 38% of that at Earth's surface in possibly the first prolonged exposure to gravity other than the 1G of Earth's surface and the zero G of weightless space flight, with yet unknown effects on crew physiology. The radiation at Mars' surface is not well documented, although the planet's bulk and even its thin atmosphere may moderate the influx of galactic cosmic radiation and energetic protons from solar flares. Secondary radiation from activated components of the soil must also be considered. Ultrafine and larger respirable and nonrespirable particles in Martian dust introduced into the habitat after surface excursions may induce pulmonary inflammation exacerbated by the additive reactive and oxidizing nature of the dust. Stringent decontamination cannot eliminate mechanical and corrosive effects of the dust on pressure suits and exposed machinery. The biohazard potential of putative indigenous Martian microorganisms may be assessed by comparison with analog environments on Earth. Even in their absence, human microorganisms, if not properly controlled, can be a threat to the crew's health. Conclusions: Mars' surface offers a substantial challenge to the

  10. Sculpting ion channel functional expression with engineered ubiquitin ligases

    Science.gov (United States)

    Kanner, Scott A; Morgenstern, Travis

    2017-01-01

    The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K+ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr t1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels. PMID:29256394

  11. A novel

    NARCIS (Netherlands)

    K.L.S. Chatalic (Kristell); J. Veldhoven-Zweistra (Joke); M. Bolkestein (Michiel); S. Hoeben (Sander); G.A. Koning (Gerben); O.C. Boerman (Otto); M. de Jong (Marion); W.M. van Weerden (Wytske)

    2015-01-01

    textabstractProstate-specific membrane antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single-domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high

  12. Surface energy of explosive nanoparticles

    Science.gov (United States)

    Pineau, Nicolas; Bidault, Xavier; Soulard, Laurent

    2017-06-01

    Recent experimental studies show that nanostructuration has a substantial impact on the detonation of high explosives: a nanostructured one leads to smaller nanodiamonds than a microstructured one. Whether it comes from a higher surface energy or from porosity, the origin of these different behaviors must be investigated. The surface energy of TATB nanoparticles with a radius from 2 nm upto 60 nm has been determined by means of ReaxFF-based simulations. Then, using the Rankine-Hugoniot relations and the equation of states of the bulk material, the contribution of this excess energy to the heating of a shock-compressed nanostructured (and porous) material is evaluated and compared to the thermal effect due to its porosity collapse. A maximum temperature increase of 50 K is found for 4-nm nanoparticles, which remains negligible when compared to the few hundred degrees induced by the compaction work.

  13. Substantiation for Approaches to Treatment of Latent Autoimmune Diabetes in Adults

    Directory of Open Access Journals (Sweden)

    T.M. Tykhonova

    2014-10-01

    Conclusions. Analysis of carbohydrate metabolism on the manifestation stage and over time development of latent autoimmune diabetes in adults as well as reduction of β-cells insulin-producing function associated with autoimmune insulitis and progressing while the development of this form of disease, substantiate the rational for insulin administration as this form of diabetes has been diagnosed. If patients with latent autoimmune diabetes in adults have metabolic syndrome clusters it is quite reasonable to add metformin to insulin.

  14. Some problems associated with radiolabeling surface antigens on helminth parasites: a brief review

    Energy Technology Data Exchange (ETDEWEB)

    Hayunga, E.G. (Division of Tropical Public Health, Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD (USA)); Murrell, K.D. (Agricultural Research Service, Beltsville, MD (USA))

    1982-06-01

    Recent developments in technology have facilitated substantial advances in the characterization of surface antigens from a wide variety of both normal and neoplastic cells. However, the immunochemistry of parasites has lagged behind. Efforts to apply conventional radiolabeling methods to helminths have not always been successful. Experimental work with Schistosoma mansoni is reviewed to illustrate common problems encountered in surface labeling studies. These findings should provide insight for the future investigation of other helminth species.

  15. Some problems associated with radiolabeling surface antigens on helminth parasites: a brief review

    International Nuclear Information System (INIS)

    Hayunga, E.G.; Murrell, K.D.

    1982-01-01

    Recent developments in technology have facilitated substantial advances in the characterization of surface antigens from a wide variety of both normal and neoplastic cells. However, the immunochemistry of parasites has lagged behind. Efforts to apply conventional radiolabeling methods to helminths have not always been successful. Experimental work with Schistosoma mansoni is reviewed to illustrate common problems encountered in surface labeling studies. These findings should provide insight for the future investigation of other helminth species. (Auth.)

  16. Improvement of Polypropylene Biological Interactions by using Superhydrophobic Surface Modification

    Directory of Open Access Journals (Sweden)

    E. Shirani

    2018-03-01

    Full Text Available The significance of producing superhydrophobic surfaces through modification of surface chemistry and structure is in preventing or delaying biofilm formation. This is done to improve biocompatibility and chemical and biological properties of the surface by creating micro-nano multilevel rough structure; and to decrease surface free energy by Fault Tolerant Control Strategy (FTCS . Here, we produced a superhydrophobic surface through TiO2 coating and flurosilanization methods. Then, in order to evaluate the physicochemical properties of the modified surfaces, they were characterized by Scanning Electron Microscope (SEM, Fourier Transform Infrared Spectroscopy (FTIR, Contact Angle (CA, cell viability assay (using Hela and MCF-7 cancer cell lines as well as non-cancerous human fibroblast cells by MTT, Bovine Serum Abumin (BSA protein adsorption using Bradford and bacterial adhesion assay (Staphylococcus aureus and Staphylococcus epidermidis using microtiter. Results showed that contact angle and surface energey of superhydrophobic modified surface increased to 150° and decreased to 5.51 mj/m2, respectively due to physicochemical modifications of the surface. In addition, the results showed a substantial reduction in protein adsorption and bacterial cell adhesion in superhydrophobic surface.

  17. Investigation of the biofouling properties of several algae on different textured chemical modified silicone surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Jihai [Key Laboratory of Marine Materials and Related Technologies, Zhejiang Key Laboratory of Marine Materials and Protective Technologies, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Zhao, Wenjie, E-mail: zhaowj@nimte.ac.cn [Key Laboratory of Marine Materials and Related Technologies, Zhejiang Key Laboratory of Marine Materials and Protective Technologies, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201 (China); Peng, Shusen; Zeng, Zhixiang; Zhang, Xin [Key Laboratory of Marine Materials and Related Technologies, Zhejiang Key Laboratory of Marine Materials and Protective Technologies, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201 (China); Wu, Xuedong, E-mail: xdwu@nimte.ac.cn [Key Laboratory of Marine Materials and Related Technologies, Zhejiang Key Laboratory of Marine Materials and Protective Technologies, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201 (China); Xue, Qunji [Key Laboratory of Marine Materials and Related Technologies, Zhejiang Key Laboratory of Marine Materials and Protective Technologies, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201 (China)

    2014-08-30

    Highlights: • Engineered pillars, pits and grooves spaced 3–12 μm apart were fabricated on siloxane modified acrylic resin films. • The effect of feature size, geometry, and wettability on the settlement of different algae was evaluated. • The feature size and geometry displayed a substantial correlation with the antifouling properties. • A comparatively physical fouling deterrent mechanism was analyzed. - Abstract: Engineered pillars, pits and grooves spaced 3, 6, 9 and 12 μm apart were fabricated on siloxane modified acrylic resin films. The effect of feature size, geometry, and wettability on the settlement of different algae was evaluated. These films showed various antifouling performances to Ulothrix, Closterium and Navicula. For Navicula (length: 10–12 μm), the feature size and geometry displayed a substantial correlation with the antifouling properties. The film with pillars spaced 3 μm reduced Navicula settlement by 73% compared to the control surface. For Closterium (length: 45–55 μm), their responses were governed by the same underlying thermodynamic principles as wettability, the largest reduction in Closterium, 81%, was obtained on the surface with grooves spaced 12 μm apart. For Ulothrix (length: 5–8 mm), the surface also showed the best antifouling performance, the reduction ratio of the settlement on the surface with grooves spaced 12 μm apart could even reach 92%. At last, physical fouling deterrent mechanisms for the films with various textures were analyzed in detail. The feature size and geometry display a substantial correlation with the antifouling properties when the size of fouling algae is close to the textures. With the increasing size for algae, antifouling performance was getting better on surface with pillars or grooves because the algae are bridged between two or more features other than stabilizing its entire mass on one single feature or able to settle between features.

  18. Investigation of the biofouling properties of several algae on different textured chemical modified silicone surfaces

    International Nuclear Information System (INIS)

    Xu, Jihai; Zhao, Wenjie; Peng, Shusen; Zeng, Zhixiang; Zhang, Xin; Wu, Xuedong; Xue, Qunji

    2014-01-01

    Highlights: • Engineered pillars, pits and grooves spaced 3–12 μm apart were fabricated on siloxane modified acrylic resin films. • The effect of feature size, geometry, and wettability on the settlement of different algae was evaluated. • The feature size and geometry displayed a substantial correlation with the antifouling properties. • A comparatively physical fouling deterrent mechanism was analyzed. - Abstract: Engineered pillars, pits and grooves spaced 3, 6, 9 and 12 μm apart were fabricated on siloxane modified acrylic resin films. The effect of feature size, geometry, and wettability on the settlement of different algae was evaluated. These films showed various antifouling performances to Ulothrix, Closterium and Navicula. For Navicula (length: 10–12 μm), the feature size and geometry displayed a substantial correlation with the antifouling properties. The film with pillars spaced 3 μm reduced Navicula settlement by 73% compared to the control surface. For Closterium (length: 45–55 μm), their responses were governed by the same underlying thermodynamic principles as wettability, the largest reduction in Closterium, 81%, was obtained on the surface with grooves spaced 12 μm apart. For Ulothrix (length: 5–8 mm), the surface also showed the best antifouling performance, the reduction ratio of the settlement on the surface with grooves spaced 12 μm apart could even reach 92%. At last, physical fouling deterrent mechanisms for the films with various textures were analyzed in detail. The feature size and geometry display a substantial correlation with the antifouling properties when the size of fouling algae is close to the textures. With the increasing size for algae, antifouling performance was getting better on surface with pillars or grooves because the algae are bridged between two or more features other than stabilizing its entire mass on one single feature or able to settle between features

  19. Effect of the coefficient of friction of a running surface on sprint time in a sled-towing exercise.

    Science.gov (United States)

    Linthorne, Nicholas P; Cooper, James E

    2013-06-01

    This study investigated the effect of the coefficient of friction of a running surface on an athlete's sprint time in a sled-towing exercise. The coefficients of friction of four common sports surfaces (a synthetic athletics track, a natural grass rugby pitch, a 3G football pitch, and an artificial grass hockey pitch) were determined from the force required to tow a weighted sled across the surface. Timing gates were then used to measure the 30-m sprint time for six rugby players when towing a sled of varied weight across the surfaces. There were substantial differences between the coefficients of friction for the four surfaces (micro = 0.21-0.58), and in the sled-towing exercise the athlete's 30-m sprint time increased linearly with increasing sled weight. The hockey pitch (which had the lowest coefficient of friction) produced a substantially lower rate of increase in 30-m sprint time, but there were no significant differences between the other surfaces. The results indicate that although an athlete's sprint time in a sled-towing exercise is affected by the coefficient offriction of the surface, the relationship relationship between the athlete's rate of increase in 30-m sprint time and the coefficient of friction is more complex than expected.

  20. Substantial Humic Acid Adsorption to Activated Carbon Air Cathodes Produces a Small Reduction in Catalytic Activity.

    Science.gov (United States)

    Yang, Wulin; Watson, Valerie J; Logan, Bruce E

    2016-08-16

    Long-term operation of microbial fuel cells (MFCs) can result in substantial degradation of activated carbon (AC) air-cathode performance. To examine a possible role in fouling from organic matter in water, cathodes were exposed to high concentrations of humic acids (HA). Cathodes treated with 100 mg L(-1) HA exhibited no significant change in performance. Exposure to 1000 mg L(-1) HA decreased the maximum power density by 14% (from 1310 ± 30 mW m(-2) to 1130 ± 30 mW m(-2)). Pore blocking was the main mechanism as the total surface area of the AC decreased by 12%. Minimization of external mass transfer resistances using a rotating disk electrode exhibited only a 5% reduction in current, indicating about half the impact of HA adsorption was associated with external mass transfer resistance and the remainder was due to internal resistances. Rinsing the cathodes with deionized water did not restore cathode performance. These results demonstrated that HA could contribute to cathode fouling, but the extent of power reduction was relatively small in comparison to large mass of humics adsorbed. Other factors, such as biopolymer attachment, or salt precipitation, are therefore likely more important contributors to long-term fouling of MFC cathodes.

  1. Technique of research of severe accidents and substantiation of safety of nuclear systems

    International Nuclear Information System (INIS)

    Ivanov, E.A.; Tchenov, S.V.

    2001-01-01

    Work is devoted to development of possible ways of solution of the problems of nuclear safety substantiation. We believe that safety in severe accidents is one of significant factors, which restrict value of nuclear industry in future power production. In connection with it we can conclude followed items: -) Substantiation of safety in severe accidents in nuclear system should be built on a deterministic way of guaranteed exception of heavy consequences; -) It is easy that this aim can be achieved by modeling in functions of common type; -) Main purpose of this work is to show that it is possible to estimate physical allowed state of system in emergency and find of trajectory of heaviest scenarios by optimization procedure; and -) In this work we have developed new method and computer code purposed for study of accident conditions of water cooled un-managed nuclear systems such as cooling ponds of spent fuel, experimental facilities etc. (authors)

  2. Surface characterization of insulin protofilaments and fibril polymorphs using tip-enhanced Raman spectroscopy (TERS).

    Science.gov (United States)

    Kurouski, Dmitry; Deckert-Gaudig, Tanja; Deckert, Volker; Lednev, Igor K

    2014-01-07

    Amyloid fibrils are β-sheet-rich protein aggregates that are strongly associated with a variety of neurodegenerative maladies, such as Alzheimer's and Parkinson's diseases. Even if the secondary structure of such fibrils is well characterized, a thorough understanding of their surface organization still remains elusive. Tip-enhanced Raman spectroscopy (TERS) is one of a few techniques that allow the direct characterization of the amino acid composition and the protein secondary structure of the amyloid fibril surface. Herein, we investigated the surfaces of two insulin fibril polymorphs with flat (flat) and left-twisted (twisted) morphology. It was found that the two differ substantially in both amino acid composition and protein secondary structure. For example, the amounts of Tyr, Pro, and His differ, as does the number of carboxyl groups on the respective surfaces, whereas the amounts of Phe and of positively charged amino and imino groups remain similar. In addition, the surface of protofilaments, the precursors of the mature flat and twisted fibrils, was investigated using TERS. The results show substantial differences with respect to the mature fibrils. A correlation of amino acid frequencies and protein secondary structures on the surface of protofilaments and on flat and twisted fibrils allowed us to propose a hypothetical mechanism for the propagation to specific fibril polymorphs. This knowledge can shed a light on the toxicity of amyloids and define the key factors responsible for fibril polymorphism. Finally, this work demonstrates the potential of TERS for the surface characterization of amyloid fibril polymorphs. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Temperature-dependent surface density of alkylthiol monolayers on gold nanocrystals

    Science.gov (United States)

    Liu, Xuepeng; Lu, Pin; Zhai, Hua; Wu, Yucheng

    2018-03-01

    Atomistic molecular dynamics (MD) simulations are performed to study the surface density of passivating monolayers of alkylthiol chains on gold nanocrystals at temperatures ranging from 1 to 800 K. The results show that the surface density of alkylthiol monolayer reaches a maximum value at near room temperature (200-300 K), while significantly decreases with increasing temperature in the higher temperature region (> 300 {{K}}), and slightly decreases with decreasing temperature at low temperature (< 200 {{K}}). We find that the temperature dependence of surface ligand density in the higher temperature region is attributed to the substantial ligand desorption induced by the thermal fluctuation, while that at low temperature results from the reduction in entropy caused by the change in the ordering of passivating monolayer. These results are expected helpful to understand the temperature-dependent surface coverage of gold nanocrystals.

  4. Properties, production and applications of camelid single-domain antibody fragments

    NARCIS (Netherlands)

    Harmsen, M.M.; Haard, de H.J.

    2007-01-01

    Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms

  5. Control of grinding polygonal surfaces

    Directory of Open Access Journals (Sweden)

    Юрій Володимирович Петраков

    2017-12-01

    Full Text Available Grinding of non-round surfaces, in particular polygonal surfaces of dies, is characterized by substantial non stationary. At different sections of the profile, the change in the main characteristic (Material Removal Rate – MRR process reaches tens of times. To stabilize the grinding process, it is recommended to control the spindle speed of the workpiece CNC grinding machine. Created software that allows to design the control program on the basis of mathematical model of the system. The determination of MRR is realized automatically in the simulation of the grinding process which uses the algorithm developed for solving problems in geometric interaction of the workpiece and the wheel. In forming the control program is possible takes into account the limitations on the maximum circumferential force of cutting, and the maximum allowable acceleration of the machine spindle. Practice has shown that full stabilization is not obtained, even though the performance is increased more than 2 times, while ensuring the quality of the surface. The developed block diagram of the grinding process can serve as a basis for further improvement in the solution of dynamic problems.

  6. 26 CFR 301.6229(c)(2)-1T - Substantial omission of income (temporary).

    Science.gov (United States)

    2010-04-01

    ....6229(c)(2)-1T Substantial omission of income (temporary). (a) Partnership return—(1) General rule. (i) If any partnership omits from the gross income stated in its return an amount properly includible... omitted from gross income if information sufficient to apprise the Commissioner of the nature and amount...

  7. Substantially parallel flux uncluttered rotor machines

    Science.gov (United States)

    Hsu, John S.

    2012-12-11

    A permanent magnet-less and brushless synchronous system includes a stator that generates a magnetic rotating field when sourced by polyphase alternating currents. An uncluttered rotor is positioned within the magnetic rotating field and is spaced apart from the stator. An excitation core is spaced apart from the stator and the uncluttered rotor and magnetically couples the uncluttered rotor. The brushless excitation source generates a magnet torque by inducing magnetic poles near an outer peripheral surface of the uncluttered rotor, and the stator currents also generate a reluctance torque by a reaction of the difference between the direct and quadrature magnetic paths of the uncluttered rotor. The system can be used either as a motor or a generator

  8. A comparison of UV surface brightness and HI surface densities for spiral galaxies

    International Nuclear Information System (INIS)

    Federman, S.R.; Strom, C.

    1990-01-01

    Shaya and Federman (1987) suggested that the ambient ultraviolet flux at 1000 A permeating a spiral galaxy controls the neutral hydrogen (HI) surface density in the galaxy. They found that the atomic envelopes surrounding small molecular clouds, because of their great number, provide the major contribution to the HI surface density over the stellar disk. The increase in HI surface density with later Hubble types was ascribed to the stronger UV fields from more high-mass stars in later Hubble types. These hypotheses are based on the observations of nearby diffuse interstellar clouds, which show a sharp atomic-to-molecular transition (Savage et al. 1977), and on the theoretical framework introduced by Federman, Glassgold, and Kwan (1979). Atomic envelopes around interstellar clouds in the solar neighborhood arise when a steady state is reached between photodissociation of H2 and the formation of H2 on grains. The photodissociation process involves photons with wavelengths between 912 A and 1108 A. Shaya and Federman used H-alpha flux as an approximate measure for the far UV flux and made their comparisons based on averages over Hubble type. Here, researchers compare, on an individual basis, UV data obtained with space-borne and balloon-borne instruments for galaxies with measurements of HI surface density (Warmels 1988a, b). The comparisons substantiate the conclusion of Shaya and Federman that the far UV field controls the HI content of spiral galaxies

  9. The Impact of Family Engagement and Child Welfare Services on Maltreatment Re-reports and Substantiated Re-reports.

    Science.gov (United States)

    Fuller, Tamara; Zhang, Saijun

    2017-08-01

    Despite decades of debate about the most effective ways to intervene with families reported to child protective services (CPS), little evidence exists regarding the types of services or approach that reduce children's risk of additional maltreatment. The current study used data collected during a statewide experimental evaluation of CPS to examine the impact of numerous service variables, family engagement, and family characteristics on the risk of maltreatment re-reports and substantiated re-reports among families initially reported for neglect and risk of harm. The sample included 4,868 families with screened-in reports that were randomly assigned to receive either an investigation or an assessment. The results of the Cox regression analyses found that service duration, intensity, and breadth were unrelated to maltreatment re-report or substantiated re-reports, but caseworker ratings of the service-need match were associated with both. The provision of domestic violence services was related to decreased risk of maltreatment re-reports. Increased levels of family engagement were associated with lowered risk of both maltreatment re-reports and substantiated re-reports. Once the effects of services, engagement, and family characteristics were taken into account, CPS response pathway (investigation or assessment) had no relationship to maltreatment re-reports or substantiated re-reports.

  10. Information Technology & Applications Corporation v. United States: An Interested Party's "Substantial Chance" at APA Standing

    National Research Council Canada - National Science Library

    Slicker, Christina

    2003-01-01

    .... Building on CICA's "interested party" definition with Information Technology's refinement of "substantial chance" rule, the Federal Circuit has effectively translated "APA standing" into the language...

  11. Dynamic bioactive stimuli-responsive polymeric surfaces

    Science.gov (United States)

    Pearson, Heather Marie

    This dissertation focuses on the design, synthesis, and development of antimicrobial and anticoagulant surfaces of polyethylene (PE), polypropylene (PP), and poly(tetrafluoroethylene) (PTFE) polymers. Aliphatic polymeric surfaces of PE and PP polymers functionalized using click chemistry reactions by the attachment of --COOH groups via microwave plasma reactions followed by functionalization with alkyne moieties. Azide containing ampicillin (AMP) was synthesized and subsequently clicked into the alkyne prepared PE and PP surfaces. Compared to non-functionalized PP and PE surfaces, the AMP clicked surfaces exhibited substantially enhanced antimicrobial activity against Staphylococcus aureus bacteria. To expand the biocompatibility of polymeric surface anticoagulant attributes, PE and PTFE surfaces were functionalized with pH-responsive poly(2-vinyl pyridine) (P2VP) and poly(acrylic acid) (PAA) polyelectrolyte tethers terminated with NH2 and COOH groups. The goal of these studies was to develop switchable stimuli-responsive polymeric surfaces that interact with biological environments and display simultaneous antimicrobial and anticoagulant properties. Antimicrobial AMP was covalently attached to --COOH terminal ends of protected PAA, while anticoagulant heparin (HEP) was attached to terminal --NH2 groups of P2VP. When pH 5.5, they collapse while the PAA segments extend. Such surfaces, when exposed to Staphylococcus aureus, inhibit bacterial growth due to the presence of AMP, as well as are effective anticoagulants due to the presence of covalently attached HEP. Comparison of these "dynamic" pH responsive surfaces with "static" surfaces terminated with AMP entities show significant enhancement of longevity and surface activity against microbial film formation. The last portion of this dissertation focuses on the covalent attachment of living T1 and Φ11 bacteriophages (phages) on PE and PTFE surface. This was accomplished by carbodiimide coupling between --COOH

  12. Nonabelian Jacobian of projective surfaces geometry and representation theory

    CERN Document Server

    Reider, Igor

    2013-01-01

    The Jacobian of a smooth projective curve is undoubtedly one of the most remarkable and beautiful objects in algebraic geometry. This work is an attempt to develop an analogous theory for smooth projective surfaces - a theory of the nonabelian Jacobian of smooth projective surfaces. Just like its classical counterpart, our nonabelian Jacobian relates to vector bundles (of rank 2) on a surface as well as its Hilbert scheme of points. But it also comes equipped with the variation of Hodge-like structures, which produces a sheaf of reductive Lie algebras naturally attached to our Jacobian. This constitutes a nonabelian analogue of the (abelian) Lie algebra structure of the classical Jacobian. This feature naturally relates geometry of surfaces with the representation theory of reductive Lie algebras/groups. This work’s main focus is on providing an in-depth study of various aspects of this relation. It presents a substantial body of evidence that the sheaf of Lie algebras on the nonabelian Jacobian is an effic...

  13. Method of driving liquid flow at or near the free surface using magnetic microparticles

    Science.gov (United States)

    Snezhko, Oleksiy [Woodridge, IL; Aronson, Igor [Darien, IL; Kwok, Wai-Kwong [Evanston, IL; Belkin, Maxim V [Woodridge, IL

    2011-10-11

    The present invention provides a method of driving liquid flow at or near a free surface using self-assembled structures composed of magnetic particles subjected to an external AC magnetic field. A plurality of magnetic particles are supported at or near a free surface of liquid by surface tension or buoyancy force. An AC magnetic field traverses the free surface and dipole-dipole interaction between particles produces in self-assembled snake structures which oscillate at the frequency of the traverse AC magnetic field. The snake structures independently move across the free surface and may merge with other snake structures or break up and coalesce into additional snake structures experiencing independent movement across the liquid surface. During this process, the snake structures produce asymmetric flow vortices across substantially the entirety of the free surface, effectuating liquid flow across the free surface.

  14. The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development

    DEFF Research Database (Denmark)

    Hviid, Lars

    2010-01-01

    There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs) such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive-determinants of clinical outcome of P. falciparum malaria. The evide...... of VSAs, and how vaccines based on this type of antigens fit into the current global strategy to reduce, eliminate and eventually eradicate the burden of malaria....

  15. Biomechanical modeling constrained surface-based image registration for prostate MR guided TRUS biopsy

    NARCIS (Netherlands)

    Ven, W.J.M. van de; Hu, Y.; Barentsz, J.O.; Karssemeijer, N.; Barratt, D.; Huisman, H.J.

    2015-01-01

    Adding magnetic resonance (MR)-derived information to standard transrectal ultrasound (TRUS) images for guiding prostate biopsy is of substantial clinical interest. A tumor visible on MR images can be projected on ultrasound (US) by using MR-US registration. A common approach is to use surface-based

  16. Substantial nitrogen pollution embedded in international trade

    Science.gov (United States)

    Oita, Azusa; Malik, Arunima; Kanemoto, Keiichiro; Geschke, Arne; Nishijima, Shota; Lenzen, Manfred

    2016-02-01

    Anthropogenic emissions of reactive nitrogen to the atmosphere and water bodies can damage human health and ecosystems. As a measure of a nation’s contribution to this potential damage, a country’s nitrogen footprint has been defined as the quantity of reactive nitrogen emitted during the production, consumption and transportation of commodities consumed within that country, whether those commodities are produced domestically or internationally. Here we use global emissions databases, a global nitrogen cycle model, and a global input-output database of domestic and international trade to calculate the nitrogen footprints for 188 countries as the sum of emissions of ammonia, nitrogen oxides and nitrous oxide to the atmosphere, and of nitrogen potentially exportable to water bodies. Per-capita footprints range from under 7 kg N yr-1 in some developing countries to over 100 kg N yr-1 in some wealthy nations. Consumption in China, India, the United States and Brazil is responsible for 46% of global emissions. Roughly a quarter of the global nitrogen footprint is from commodities that were traded across country borders. The main net exporters have significant agricultural, food and textile exports, and are often developing countries, whereas important net importers are almost exclusively developed economies. We conclude that substantial local nitrogen pollution is driven by demand from consumers in other countries.

  17. Main results of substantiation of the ecological safety of the Novovoronezh NPP-2 design

    International Nuclear Information System (INIS)

    Kopytov, I.I.; Kocher'yan, V.M.; Leonov, S.V.; Chionov, V.G.; Ehrnestova, L.S.

    2005-01-01

    Paper presents the results of the efforts to determine both the actual (hydrochemical, hydrobiological, geobotanical, soil, radiological) and the predicted parameters of the region ecology derived when substantiating the ecological safety of the Novovoronezh NPP-2 design [ru

  18. Studies of surface states in zinc oxide nanopowders

    Science.gov (United States)

    Peters, Raul Mugabe

    The surface of ZnO semiconductor nanosystems is a key performance-defining factor in numerous applications. In this work we present experimental results for the surface defect-related properties of ZnO nanoscale systems. Surface photovoltage spectroscopy was used to determine the defect level energies within the band gap, the conduction vs. valence band nature of the defect-related transitions, and to probe key dynamic parameters of the surface on a number of commercially available ZnO nanopowders. In our experimental setup, surface photovoltage characterization is conducted in high vacuum in tandem with in situ oxygen remote plasma treatments. Surface photovoltage investigations of the as-received and plasma-processed samples revealed a number of common spectral features related to surface states. Furthermore, we observed significant plasma-induced changes in the surface defect properties. Ex situ positron annihilation and photoluminescence measurements were performed on the studied samples and correlated with surface photovoltage results. The average positron lifetimes were found to be substantially longer than in a bulk single crystalline sample, which is consistent with the model of grains with defect-rich surface and subsurface layers. Compression of the powders into pellets yielded reduction of the average positron lifetimes. Surface photovoltage, positron annihilation, and photoluminescence spectra consistently showed sample-to-sample differences due to the variation in the overall quality of the nanopowders, which partially obscures observation of the scaling effects. However, the results demonstrated that our approach is efficient in detecting specific surface states in nanoscale ZnO specimens and in elucidating their nature.

  19. Internal photoemission from plasmonic nanoparticles: comparison between surface and volume photoelectric effects

    DEFF Research Database (Denmark)

    Uskov, Alexander; Protsenko, Igor E.; Ikhsanov, Renat S.

    2014-01-01

    in the surface mechanism, which leads to a substantial (by similar to 5 times) increase of the internal photoelectron emission rate from a nanoparticle compared to the case when such a discontinuity is absent. For a plasmonic nanoparticle, a comparison of the two photoeffect mechanisms was undertaken...... for the first time which showed that the surface photoeffect can in the general case be larger than the volume one, which agrees with the results obtained for a flat metal surface first formulated by Tamm and Schubin in their pioneering development of a quantum-mechanical theory of photoeffect in 1931....... In accordance with our calculations, this possible predominance of the surface effect is based on two factors: (i) effective cooling of hot carriers during their propagation from the volume of the nanoparticle to its surface in the scenario of the volume mechanism and (ii) strengthening of the surface mechanism...

  20. Synthesis and surface immobilization of antibacterial hybrid silver-poly(l-lactide) nanoparticles

    Science.gov (United States)

    Taheri, Shima; Baier, Grit; Majewski, Peter; Barton, Mary; Förch, Renate; Landfester, Katharina; Vasilev, Krasimir

    2014-08-01

    Infections associated with medical devices are a substantial healthcare problem. Consequently, there has been increasing research and technological efforts directed toward the development of coatings that are capable of preventing bacterial colonization of the device surface. Herein, we report on novel hybrid silver loaded poly(L-lactic acid) nanoparticles (PLLA-AgNPs) with narrowly distributed sizes (17 ± 3 nm) prepared using a combination of solvent evaporation and mini-emulsion technology. These particles were then immobilized onto solid surfaces premodified with a thin layer of allylamine plasma polymer (AApp). The antibacterial efficacy of the PLLA-AgNPs nanoparticles was studied in vitro against both gram-positive (Staphylococcus epidermidis) and gram-negative (Escherichia coli) bacteria. The minimal inhibitory concentration values against Staphylococcus epidermidis and Escherichia coli were 0.610 and 1.156 μg · mL-1, respectively. The capacity of the prepared coatings to prevent bacterial surface colonization was assessed in the presence of Staphylococcus epidermidis, which is a strong biofilm former that causes substantial problems with medical device associated infections. The level of inhibition of bacterial growth was 98%. The substrate independent nature and the high antibacterial efficacy of coatings presented in this study may offer new alternatives for antibacterial coatings for medical devices.

  1. Synthesis and surface immobilization of antibacterial hybrid silver-poly(l-lactide) nanoparticles

    International Nuclear Information System (INIS)

    Taheri, Shima; Majewski, Peter; Vasilev, Krasimir; Baier, Grit; Landfester, Katharina; Barton, Mary; Förch, Renate

    2014-01-01

    Infections associated with medical devices are a substantial healthcare problem. Consequently, there has been increasing research and technological efforts directed toward the development of coatings that are capable of preventing bacterial colonization of the device surface. Herein, we report on novel hybrid silver loaded poly(L-lactic acid) nanoparticles (PLLA-AgNPs) with narrowly distributed sizes (17 ± 3 nm) prepared using a combination of solvent evaporation and mini-emulsion technology. These particles were then immobilized onto solid surfaces premodified with a thin layer of allylamine plasma polymer (AApp). The antibacterial efficacy of the PLLA-AgNPs nanoparticles was studied in vitro against both gram-positive (Staphylococcus epidermidis) and gram-negative (Escherichia coli) bacteria. The minimal inhibitory concentration values against Staphylococcus epidermidis and Escherichia coli were 0.610 and 1.156 μg · mL −1 , respectively. The capacity of the prepared coatings to prevent bacterial surface colonization was assessed in the presence of Staphylococcus epidermidis, which is a strong biofilm former that causes substantial problems with medical device associated infections. The level of inhibition of bacterial growth was 98%. The substrate independent nature and the high antibacterial efficacy of coatings presented in this study may offer new alternatives for antibacterial coatings for medical devices. (paper)

  2. Method for the substantial reduction of quenching effects in luminescence spectrometry

    Science.gov (United States)

    Demas, J.N.; Jones, W.M.; Keller, R.A.

    1987-06-26

    Method for reducing quenching effects in analytical luminescence measurements. Two embodiments of the present invention are described which relate to a form of time resolution based on the amplitudes and phase shifts of modulated emission signals. In the first embodiment, the measured modulated emission signal is substantially independent of sample quenching at sufficiently high frequencies. In the second embodiment, the modulated amplitude and the phase shift between the emission signal and the excitation source are simultaneously measured. Using either method, the observed modulated amplitude may be reduced to its unquenched value. 3 figs.

  3. Collaborative Research: Fundamental Studies of Plasma Control Using Surface Embedded Electronic Devices

    International Nuclear Information System (INIS)

    Overzet, Lawrence J.; Raja, L.

    2015-01-01

    The research program was collaborative between the researchers at the University of Texas at Dallas and the University of Texas at Austin. The primary subject of this program was to investigate the possibility of active control of secondary electron emission (SEE) from surfaces in contact with plasmas and thereby actively control plasmas. Very few studies of ion-induced electron emission (IIEE) from semiconductors exist, and those that do exist primarily used high-energy ion beams in the experiments. Furthermore, those few studies took extreme measures to ensure that the measurements were performed on atomically clean surfaces because of the surface sensitivity of the IIEE process. Even a small exposure to air can change the IIEE yield significantly. In addition, much of the existing data for IIEE from semiconductors was obtained in the 1950s and '60s, when semiconductor materials were first being refined. As a result, nearly all of that data is for p-type Ge and Si. Before this investigation, experimental data on n-type materials was virtually non-existent. While the basic theory assumed that IIEE yields ought to be substantially independent of doping type and concentration, recent measurements of near atmospheric pressure plasmas and of breakdown suggested otherwise. These indirect measurements were made on surfaces that were not atomically clean and seemed to indicate that deep sub-surface changes to the bulk conduction band electron density could lead to substantial variations in the IIEE yield. Exactly in contradiction to the generally accepted theory. Insufficient direct data existed to settle the matter. We performed both experimental measurements and theoretical calculations of IIEE yields from both Si and Ge in order to help clarify whether or not conduction band electrons substantially change the IIEE yield. We used three wafers of each material to carry out the investigation: a heavily doped p-type, an intrinsic and a heavily doped n-type wafer. There

  4. Collaborative Research: Fundamental Studies of Plasma Control Using Surface Embedded Electronic Devices

    Energy Technology Data Exchange (ETDEWEB)

    Overzet, Lawrence J. [Univ. of Texas, Dallas, TX (United States); Raja, L. [Univ. of Texas, Austin, TX (United States)

    2015-06-06

    The research program was collaborative between the researchers at the University of Texas at Dallas and the University of Texas at Austin. The primary subject of this program was to investigate the possibility of active control of secondary electron emission (SEE) from surfaces in contact with plasmas and thereby actively control plasmas. Very few studies of ion-induced electron emission (IIEE) from semiconductors exist, and those that do exist primarily used high-energy ion beams in the experiments. Furthermore, those few studies took extreme measures to ensure that the measurements were performed on atomically clean surfaces because of the surface sensitivity of the IIEE process. Even a small exposure to air can change the IIEE yield significantly. In addition, much of the existing data for IIEE from semiconductors was obtained in the 1950s and ‘60s, when semiconductor materials were first being refined. As a result, nearly all of that data is for p-type Ge and Si. Before this investigation, experimental data on n-type materials was virtually non-existent. While the basic theory assumed that IIEE yields ought to be substantially independent of doping type and concentration, recent measurements of near atmospheric pressure plasmas and of breakdown suggested otherwise. These indirect measurements were made on surfaces that were not atomically clean and seemed to indicate that deep sub-surface changes to the bulk conduction band electron density could lead to substantial variations in the IIEE yield. Exactly in contradiction to the generally accepted theory. Insufficient direct data existed to settle the matter. We performed both experimental measurements and theoretical calculations of IIEE yields from both Si and Ge in order to help clarify whether or not conduction band electrons substantially change the IIEE yield. We used three wafers of each material to carry out the investigation: a heavily doped p-type, an intrinsic and a heavily doped n-type wafer. There

  5. Band Structure and Fermi Surface of Cu2Sb by the LMTO Method

    DEFF Research Database (Denmark)

    Jan, J. P.; Skriver, Hans Lomholt

    1977-01-01

    The linear muffin-tin orbital (LMTO) method of bandstructure calculation has been applied to the simple tetragonal compound Cu2Sb. The d bands of Cu lie substantially below the Fermi level, and the Fermi surface is a recognizable distortion of the free-electron model. The Fermi surface has sheets......-orbit splitting, and of another closed sheet. Earlier de Haas-van Alphen results are explained semiquantitatively by the model, which also accounts for open orbits seen in high-field magnetoresistance experiments....

  6. Videolaryngoscopes differ substantially in illumination of the oral cavity: A manikin study

    Directory of Open Access Journals (Sweden)

    Barbe MA Pieters

    2016-01-01

    Full Text Available Background and Aims: Insufficient illumination of the oral cavity during endotracheal intubation may result in suboptimal conditions. Consequently, suboptimal illumination and laryngoscopy may lead to potential unwanted trauma to soft tissues of the pharyngeal mucosa. We investigated illumination of the oral cavity by different videolaryngoscopes (VLS in a manikin model. Methods: We measured light intensity from the mouth opening of a Laerdal intubation trainer comparing different direct and indirect VLS at three occasions, resembling optimal to less-than-optimal intubation conditions; at the photographer′s dark room, in an operating theatre and outdoors in bright sunlight. Results: Substantial differences in luminance were detected between VLS. The use of LED light significantly improved light production. All VLS produced substantial higher luminance values in a well-luminated environment compared to the dark photographer′s room. The experiments outside-in bright sunlight-were interfered with by direct sunlight penetration through the synthetic material of the manikin, making correct measurement of luminance in the oropharynx invalid. Conclusion: Illumination of the oral cavity differs widely among direct and indirect VLS. The clinician should be aware of the possibility of suboptimal illumination of the oral cavity and the potential risk this poses for the patient.

  7. Adsorption of oxazole and isoxazole on BNNT surface: A DFT study

    Energy Technology Data Exchange (ETDEWEB)

    Kaur, Jasleen, E-mail: jasleen.chem@gmail.com; Singla, Preeti, E-mail: preetisingla21@gmail.com; Goel, Neetu, E-mail: neetugoel@pu.ac.in

    2015-02-15

    Highlights: • The adsorption of oxazole and isoxazole over (6,0) and (5,5) BNNTs is studied. • The adsorption energies and structural changes are inductive of covalent adsorption. • The solvent plays an important role in improving the adsorption properties. • The adsorption witnesses substantial changes in electronic properties of the BNNT. • The functionalization of the BNNTs open up their applicability as drug carrier. - Abstract: The adsorption behavior of oxazole and isoxazole heterocycles over the (6,0) zigzag and (5,5) armchair boron nitride nanotube (BNNT) has been studied within the formalism of density functional theory (DFT). The adsorption energies, the frontier molecular orbital (FMO) analysis and the structural changes at the adsorption site are indicative of covalent adsorption on the zigzag BNNT surface, while the adsorption is physical in nature on the armchair BNNT surface. The role of solvent in improving the adsorption properties over the BNNT surface is elucidated by reoptimizing the structures in aqueous phase. The solvation energy is indicative of remarkable increase in the solubility of BNNTs after adsorption of heterocyclic rings. The Density of states (DOS) Plots, natural bond orbital (NBO) analysis and the quantum molecular descriptors (QMD) are witness to the substantial changes in the electronic properties of the BNNT systems following the attachment of these heterocycles with the tube surface. The study envisages the functionalization of the BNNT as well as its applicability as carrier of the drugs containing heterocyclic rings oxazole and isoxazole with marked sensitivity to the type of adsorbate and the adsorbent.

  8. 78 FR 889 - Finding of Substantial Inadequacy of Implementation Plan; Call for California State...

    Science.gov (United States)

    2013-01-07

    ... Resources Defense Council; and Physicians for Social Responsibility--Los Angeles, (``environmental and... ENVIRONMENTAL PROTECTION AGENCY 40 CFR Part 52 [EPA-R09-OAR-2012-0721; FRL-9767-3] Finding of Substantial Inadequacy of Implementation Plan; Call for California State Implementation Plan Revision; South...

  9. Will surface winds weaken in response to global warming?

    Science.gov (United States)

    Ma, Jian; Foltz, Gregory R.; Soden, Brian J.; Huang, Gang; He, Jie; Dong, Changming

    2016-12-01

    The surface Walker and tropical tropospheric circulations have been inferred to slow down from historical observations and model projections, yet analysis of large-scale surface wind predictions is lacking. Satellite measurements of surface wind speed indicate strengthening trends averaged over the global and tropical oceans that are supported by precipitation and evaporation changes. Here we use corrected anemometer-based observations to show that the surface wind speed has not decreased in the averaged tropical oceans, despite its reduction in the region of the Walker circulation. Historical simulations and future projections for climate change also suggest a near-zero wind speed trend averaged in space, regardless of the Walker cell change. In the tropics, the sea surface temperature pattern effect acts against the large-scale circulation slow-down. For higher latitudes, the surface winds shift poleward along with the eddy-driven mid-latitude westerlies, resulting in a very small contribution to the global change in surface wind speed. Despite its importance for surface wind speed change, the influence of the SST pattern change on global-mean rainfall is insignificant since it cannot substantially alter the global energy balance. As a result, the precipitation response to global warming remains ‘muted’ relative to atmospheric moisture increase. Our results therefore show consistency between projections and observations of surface winds and precipitation.

  10. Influence of random roughness on the adhesion between metal surfaces due to capillary condensation

    OpenAIRE

    van Zwol, P. J.; Palasantzas, G.; De Hosson, J. Th. M.

    2007-01-01

    The capillary force was measured by atomic force microscopy between a gold coated sphere mounted on a cantilever and gold surfaces with different roughnesses. For smooth surfaces the capillary adhesive force surpasses in magnitude any dispersion, e.g., van der Waals/Casimir and/or electrostatic forces. A substantial decrease in the capillary force was observed by increasing the roughness ampltitude a few nanometers in the range of 1-10 nm. From these measurements two limits can be defined: a ...

  11. Magnetosphere, exosphere, and surface of Mercury

    International Nuclear Information System (INIS)

    Cheng, A.F.; Krimigis, S.M.; Johnson, R.E.; Lanzerotti, L.J.

    1987-01-01

    It is presently suggested in light of the atomic Na exosphere discovered for Mercury that this planet, like the Jupiter moon Io, is capable of maintaining a heavy ion magnetosphere. Na(+) ions from the exosphere are in this scenario accelerated to keV energies en route to making substantial contributions to the mass and energy budgets of the magnetosphere. Since Mercury's Na supply to the exosphere is primarily internal, it would appear that Mercury is losing its semivolatiles and that this process will proceed by way of photosputtering, which maintains an adequate Na-ejection rate from the planet's surface. 39 references

  12. Parallel force assay for protein-protein interactions.

    Science.gov (United States)

    Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

  13. Confidence Intervals: From tests of statistical significance to confidence intervals, range hypotheses and substantial effects

    Directory of Open Access Journals (Sweden)

    Dominic Beaulieu-Prévost

    2006-03-01

    Full Text Available For the last 50 years of research in quantitative social sciences, the empirical evaluation of scientific hypotheses has been based on the rejection or not of the null hypothesis. However, more than 300 articles demonstrated that this method was problematic. In summary, null hypothesis testing (NHT is unfalsifiable, its results depend directly on sample size and the null hypothesis is both improbable and not plausible. Consequently, alternatives to NHT such as confidence intervals (CI and measures of effect size are starting to be used in scientific publications. The purpose of this article is, first, to provide the conceptual tools necessary to implement an approach based on confidence intervals, and second, to briefly demonstrate why such an approach is an interesting alternative to an approach based on NHT. As demonstrated in the article, the proposed CI approach avoids most problems related to a NHT approach and can often improve the scientific and contextual relevance of the statistical interpretations by testing range hypotheses instead of a point hypothesis and by defining the minimal value of a substantial effect. The main advantage of such a CI approach is that it replaces the notion of statistical power by an easily interpretable three-value logic (probable presence of a substantial effect, probable absence of a substantial effect and probabilistic undetermination. The demonstration includes a complete example.

  14. Substantial population structure of Plasmodium vivax in Thailand facilitates identification of the sources of residual transmission.

    Directory of Open Access Journals (Sweden)

    Veerayuth Kittichai

    2017-10-01

    Full Text Available Plasmodium vivax transmission in Thailand has been substantially reduced over the past 10 years, yet it remains highly endemic along international borders. Understanding the genetic relationship of residual parasite populations can help track the origins of the parasites that are reintroduced into malaria-free regions within the country.A total of 127 P. vivax isolates were genotyped from two western provinces (Tak and Kanchanaburi and one eastern province (Ubon Ratchathani of Thailand using 10 microsatellite markers. Genetic diversity was high, but recent clonal expansion was detected in all three provinces. Substantial population structure and genetic differentiation of parasites among provinces suggest limited gene flow among these sites. There was no haplotype sharing among the three sites, and a reduced panel of four microsatellite markers was sufficient to assign the parasites to their provincial origins.Significant parasite genetic differentiation between provinces shows successful interruption of parasite spread within Thailand, but high diversity along international borders implies a substantial parasite population size in these regions. The provincial origin of P. vivax cases can be reliably determined by genotyping four microsatellite markers, which should be useful for monitoring parasite reintroduction after malaria elimination.

  15. Substantial population structure of Plasmodium vivax in Thailand facilitates identification of the sources of residual transmission.

    Science.gov (United States)

    Kittichai, Veerayuth; Koepfli, Cristian; Nguitragool, Wang; Sattabongkot, Jetsumon; Cui, Liwang

    2017-10-01

    Plasmodium vivax transmission in Thailand has been substantially reduced over the past 10 years, yet it remains highly endemic along international borders. Understanding the genetic relationship of residual parasite populations can help track the origins of the parasites that are reintroduced into malaria-free regions within the country. A total of 127 P. vivax isolates were genotyped from two western provinces (Tak and Kanchanaburi) and one eastern province (Ubon Ratchathani) of Thailand using 10 microsatellite markers. Genetic diversity was high, but recent clonal expansion was detected in all three provinces. Substantial population structure and genetic differentiation of parasites among provinces suggest limited gene flow among these sites. There was no haplotype sharing among the three sites, and a reduced panel of four microsatellite markers was sufficient to assign the parasites to their provincial origins. Significant parasite genetic differentiation between provinces shows successful interruption of parasite spread within Thailand, but high diversity along international borders implies a substantial parasite population size in these regions. The provincial origin of P. vivax cases can be reliably determined by genotyping four microsatellite markers, which should be useful for monitoring parasite reintroduction after malaria elimination.

  16. EFFECTS OF A SAND RUNNING SURFACE ON THE KINEMATICS OF SPRINTING AT MAXIMUM VELOCITY

    Directory of Open Access Journals (Sweden)

    P E Alcaraz

    2011-05-01

    Full Text Available Performing sprints on a sand surface is a common training method for improving sprint-specific strength. For maximum specificity of training the athlete’s movement patterns during the training exercise should closely resemble those used when performing the sport. The aim of this study was to compare the kinematics of sprinting at maximum velocity on a dry sand surface to the kinematics of sprinting on an athletics track. Five men and five women participated in the study, and flying sprints over 30 m were recorded by video and digitized using biomechanical analysis software. We found that sprinting on a sand surface was substantially different to sprinting on an athletics track. When sprinting on sand the athletes tended to ‘sit’ during the ground contact phase of the stride. This action was characterized by a lower centre of mass, a greater forward lean in the trunk, and an incomplete extension of the hip joint at take-off. We conclude that sprinting on a dry sand surface may not be an appropriate method for training the maximum velocity phase in sprinting. Although this training method exerts a substantial overload on the athlete, as indicated by reductions in running velocity and stride length, it also induces detrimental changes to the athlete’s running technique which may transfer to competition sprinting.

  17. Electron beam irradiating process for rendering rough or topographically irregular surface substrates smooth; and coated substrates produced thereby

    International Nuclear Information System (INIS)

    Nablo, S.V.

    1979-01-01

    This disclosure involves a novel process for instantaneous electron-beam curing of very thin low viscosity, solventless coating upon rough, irregular or textured surfaces of a substrate such as paper or the like. Through rather critical timing and energy adjustment procedures, the coating firmly adheres to the surface before the coating can conform to the roughness or texture contour or substantially penetrate into the surface. By this method a solidified very smooth outer surface is provided for the substrate that is particularly used for metalization and other finished layerings. (author)

  18. Measurement and modification of first-wall surface composition in the Oak Ridge Tokamak (ORMAK)

    International Nuclear Information System (INIS)

    Clausing, R.E.; Emerson, L.C.; Heatherly, L.; Colchin, R.J.; Twichell, J.C.

    1975-01-01

    Impurities coming into the plasma from the walls of present-day toroidal plasma confinement devices modify plasma behavior substantially. Small fractions of high-Z ions in the plasma greatly decrease plasma temperatures and increase plasma energy losses. Impurities from the ''first-wall'' in ORMAK were studied. Auger electron spectroscopy, soft x-ray appearance potential spectroscopy, and other surface sensitive techniques were used to characterize the surface composition of the first wall and to develop methods to remove carbon and oxygen. Oxygen glow discharge cleaning has been shown, in the laboratory, to be an effective way of removing carbon from gold films (simulated ORMAK linear material) and the use of oxygen discharge cleaning in ORMAK has resulted in a decrease in plasma contamination, a 50 percent increase in plasma current and an accompanying increase in plasma temperature. In spite of these improvements the walls of ORMAK are far from clean. Substantial amounts of carbon, oxygen, iron and other elements remain. (auth)

  19. Plasma drift towards a plane equipotential surface

    International Nuclear Information System (INIS)

    Carlqvist, P.

    1984-03-01

    Recently Alfven has qualitatively described how a collisionless plasma drifts in crossed electric and magnetic fields towards an infinite conducting plate of constant potential. In the present note we quantitatively study three models which are closely related to Alfven's model. It is found that when the plasma comes sufficiently close to a plane equipotential surface (conducting plate) it is deflected approximately along the surface. The deflection is not caused by pressure effects but rather by the electric and magnetic fields. Small fluxes of ions and electrons also cross the plane equipotential surface. These fluxes account for an electric current in the plasma which induces a magnetic field in the same direction as the total magnetic field assumed to be homogeneous. It is shown that if the Alfven number, M(sub)A, is much smaller than unity in the volume considered the magnetic field induced by plasma currents is small compared to the total magnetic field. However, if M(sub)A is of the order of unity or larger the total magnetic field is to a substantial degree generated by plasma currents. (Author)

  20. 26 CFR 1.162-17 - Reporting and substantiation of certain business expenses of employees.

    Science.gov (United States)

    2010-04-01

    ... Individuals and Corporations § 1.162-17 Reporting and substantiation of certain business expenses of employees.... The term “ordinary and necessary business expenses” means only those expenses which are ordinary and..., reimbursements, or otherwise, exceeds the ordinary and necessary business expenses paid or incurred by the...

  1. Boosted Dark Matter Quarrying at Surface Neutrino Detectors arXiv

    CERN Document Server

    Kim, Doojin; Park, Jong-Chul; Shin, Seodong

    We propose the idea of "Earth Shielding" to reject cosmic-ray backgrounds, in the search for boosted dark matter at surface neutrino detectors, resulting in the enhancement of the signal-to-background ratio. The identification of cosmic-originating rare signals, especially lacking features, at surface detectors is often considered hopeless due to a vast amount of cosmic-ray-induced background, hence underground experiments are better motivated to avoid such a challenge. We claim that surface detectors can attain remarkable sensitivities to even featureless signals, once restricting to events coming through the Earth from the opposite side of the detector location for the signals leaving appreciable tracks from which the source direction is inferred. By doing so, potential backgrounds in the signal region of interest can be substantially suppressed. To validate our claim, we study experimental reaches at several surface experiments such as SBN Program (MicroBooNE, ICARUS, and SBND) and ProtoDUNE for elastic bo...

  2. Polypropylene non-woven fabric membrane via surface modification with biomimetic phosphorylcholine in Ce(IV)/HNO{sub 3} redox system

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Jie; Shi Qiang; Luan Shifang; Song Lingjie; Yang Huawei [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Stagnaro, Paola [Istituto per Io Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, Via de Marini 6, 16149 Genova (Italy); Yin Jinghua, E-mail: yinjh@ciac.jl.cn [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China)

    2012-10-01

    Surface modification of polypropylene non-woven fabric membrane (NWF) for improving its hemocompatibility was developed by grafting a biomimic monomer, 2-methacryloyloxyethyl phosphorycholine (MPC). The NWF membrane surface was first activated by potassium peroxydisulfate to form hydroxyl groups, and then grafted with MPC using ceric (IV) ammonium nitrate as the redox initiator. The surface chemical changes before and after modification were confirmed by Fourier transform infrared spectroscopy with an ATR unit (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS); the water contact angle results showed the gradual changes in wettability from hydrophobic to hydrophilic surface. Meanwhile, the hemocompatibility of these samples was also evaluated by protein adsorption and platelet adhesion. These experimental results exhibited that the introduction of poly(MPC) onto the NWF membrane surfaces substantially improved their hemocompatibility. The feasibility and simplicity of this procedure may lead to potential applications of NWF membranes in biomedical separation and blood purification. - Graphical abstract: 2-methacryloyloxyethyl phosphorycholine (MPC), was grafted onto non-woven fabric (NWF) membrane surface by Ce(IV)/HNO{sub 3} redox system. The protein adsorption and platelet adhesion were substantially suppressed by the introduction of poly(MPC). Highlights: Black-Right-Pointing-Pointer MPC was successfully grafted onto NWF PP membrane surface. Black-Right-Pointing-Pointer Obviously enhanced hemocompatibility was acquired by the modified samples. Black-Right-Pointing-Pointer A facile redox grafting was adopted in the whole process.

  3. Comparative influence of land and sea surfaces on the Sahelian drought: a numerical study

    Directory of Open Access Journals (Sweden)

    Arona Diedhiou

    Full Text Available The aim of this work is to compare the relative impact of land and sea surface anomalies on Sahel rainfall and to describe the associated anomalies in the atmospheric general circulation. This sensitivity study was done with the Météo-France climate model: ARPEGE. The sensitivity to land surface conditions consists of changes in the management of water and heat exchanges by vegetation cover and bare soil. The sensitivity to ocean surfaces consists in forcing the lower boundary of the model with worldwide composite sea surface temperature (SST anomalies obtained from the difference between 4 dry Sahel years and 4 wet Sahel years observed since 1970. For each case, the spatiotemporal variability of the simulated rainfall anomaly and changes in the modelled tropical easterly jet (TEJ and African easterly jet (AEJ are discussed. The global changes in land surface evaporation have caused a rainfall deficit over the Sahel and over the Guinea Coast. No significant changes in the simulated TEJ and an enhancement of the AEJ are found; at the surface, the energy budget and the hydrological cycle are substantially modified. On the other hand, SST anomalies induce a negative rainfall anomaly over the Sahel and a positive rainfall anomaly to the south of this area. The rainfall deficit due to those anomalies is consistent with previous diagnostic and sensitivity studies. The TEJ is weaker and the AEJ is stronger than in the reference. The composite impact of SST and land surfaces anomalies is also analyzed: the simulated rainfall anomaly is similar to the observed mean African drought patterns. This work suggests that large-scale variations of surface conditions may have a substantial influence on Sahel rainfall and shows the importance of land surface parameterization in climate change modelling. In addition, it points out the interest in accurately considering the land and sea surfaces conditions in sensitivity studies on Sahel rainfall.

  4. Comparative influence of land and sea surfaces on the Sahelian drought: a numerical study

    Directory of Open Access Journals (Sweden)

    A. Diedhiou

    1996-01-01

    Full Text Available The aim of this work is to compare the relative impact of land and sea surface anomalies on Sahel rainfall and to describe the associated anomalies in the atmospheric general circulation. This sensitivity study was done with the Météo-France climate model: ARPEGE. The sensitivity to land surface conditions consists of changes in the management of water and heat exchanges by vegetation cover and bare soil. The sensitivity to ocean surfaces consists in forcing the lower boundary of the model with worldwide composite sea surface temperature (SST anomalies obtained from the difference between 4 dry Sahel years and 4 wet Sahel years observed since 1970. For each case, the spatiotemporal variability of the simulated rainfall anomaly and changes in the modelled tropical easterly jet (TEJ and African easterly jet (AEJ are discussed. The global changes in land surface evaporation have caused a rainfall deficit over the Sahel and over the Guinea Coast. No significant changes in the simulated TEJ and an enhancement of the AEJ are found; at the surface, the energy budget and the hydrological cycle are substantially modified. On the other hand, SST anomalies induce a negative rainfall anomaly over the Sahel and a positive rainfall anomaly to the south of this area. The rainfall deficit due to those anomalies is consistent with previous diagnostic and sensitivity studies. The TEJ is weaker and the AEJ is stronger than in the reference. The composite impact of SST and land surfaces anomalies is also analyzed: the simulated rainfall anomaly is similar to the observed mean African drought patterns. This work suggests that large-scale variations of surface conditions may have a substantial influence on Sahel rainfall and shows the importance of land surface parameterization in climate change modelling. In addition, it points out the interest in accurately considering the land and sea surfaces conditions in sensitivity studies on Sahel rainfall.

  5. Extremely short duration high intensity interval training substantially improves insulin action in young healthy males

    DEFF Research Database (Denmark)

    Babraj, John A; Vollaard, Niels B J; Keast, Cameron

    2009-01-01

    BACKGROUND: Traditional high volume aerobic exercise training reduces cardiovascular and metabolic disease risk but involves a substantial time commitment. Extremely low volume high-intensity interval training (HIT) has recently been demonstrated to produce improvements to aerobic function...... cycle sprints per session). Aerobic performance (250-kJ self-paced cycling time trial), and glucose, insulin and NEFA responses to a 75-g oral glucose load (oral glucose tolerance test; OGTT) were determined before and after training. RESULTS: Following 2 weeks of HIT, the area under the plasma glucose......, to substantially improve insulin action in young sedentary subjects is remarkable. This novel time-efficient training paradigm can be used as a strategy to reduce metabolic risk factors in young and middle aged sedentary populations who otherwise would not adhere to time consuming traditional aerobic exercise...

  6. Work function modifications of graphite surface via oxygen plasma treatment

    Science.gov (United States)

    Duch, J.; Kubisiak, P.; Adolfsson, K. H.; Hakkarainen, M.; Golda-Cepa, M.; Kotarba, A.

    2017-10-01

    The surface modification of graphite by oxygen plasma was investigated experimentally (X-ray diffraction, nanoparticle tracking analysis, laser desorption ionization mass spectrometry, thermogravimetry, water contact angle) and by molecular modelling (Density Functional Theory). Generation of surface functional groups (mainly sbnd OHsurf) leads to substantial changes in electrodonor properties and wettability gauged by work function and water contact angle, respectively. The invoked modifications were analyzed in terms of Helmholtz model taking into account the theoretically determined surface dipole moment of graphite-OHsurf system (μ = 2.71 D) and experimentally measured work function increase (from 0.75 to 1.02 eV) to determine the sbnd OH surface coverage (from 0.70 to 1.03 × 1014 groups cm-2). Since the plasma treatment was confined to the surface, the high thermal stability of the graphite material was preserved as revealed by the thermogravimetric analysis. The obtained results provide a suitable quantitative background for tuning the key operating parameters of carbon electrodes: electronic properties, interaction with water and thermal stability.

  7. Improving the engine power of a catalytic Janus-sphere micromotor by roughening its surface.

    Science.gov (United States)

    Longbottom, Brooke W; Bon, Stefan A F

    2018-03-15

    Microspheres with catalytic caps have become a popular model system for studying self-propelled colloids. Existing experimental studies involve predominantly "smooth" particle surfaces. In this study we determine the effect of irregular surface deformations on the propulsive mechanism with a particular focus on speed. The particle surfaces of polymer microspheres were deformed prior to depositing a layer of platinum which resulted in the formation of nanoscopic pillars of catalyst. Self-propulsion was induced upon exposure of the micromotors to hydrogen peroxide, whilst they were dispersed in water. The topological surface features were shown to boost speed (~2×) when the underlying deformations are small (nanoscale), whilst large deformations afforded little difference despite a substantial apparent catalytic surface area. Colloids with deformed surfaces were more likely to display a mixture of rotational and translational propulsion than their "smooth" counterparts.

  8. People attending pulmonary rehabilitation demonstrate a substantial engagement with technology and willingness to use telerehabilitation: a survey

    Directory of Open Access Journals (Sweden)

    Zachariah Seidman

    2017-07-01

    Conclusion: People attending metropolitan pulmonary rehabilitation, maintenance exercise classes and support groups had substantial technology engagement, with high device access and use, and good self-rated technology competence. The majority of participants were willing to use telerehabilitation, especially if they were regular users of technology devices. [Seidman Z, McNamara R, Wootton S, Leung R, Spencer L, Dale M, Dennis S, McKeough Z (2017 People attending pulmonary rehabilitation demonstrate a substantial engagement with technology and willingness to use telerehabilitation: a survey. Journal of Physiotherapy 63: 175–181

  9. Substantial equivalence--an appropriate paradigm for the safety assessment of genetically modified foods?

    International Nuclear Information System (INIS)

    Kuiper, Harry A.; Kleter, Gijs A.; Noteborn, Hub P.J.M.; Kok, Esther J.

    2002-01-01

    Safety assessment of genetically modified food crops is based on the concept of substantial equivalence, developed by OECD and further elaborated by FAO/WHO. The concept embraces a comparative approach to identify possible differences between the genetically modified food and its traditional comparator, which is considered to be safe. The concept is not a safety assessment in itself, it identifies hazards but does not assess them. The outcome of the comparative exercise will further guide the safety assessment, which may include (immuno)toxicological and biochemical testing. Application of the concept of substantial equivalence may encounter practical difficulties: (i) the availability of near-isogenic parental lines to compare the genetically modified food with; (ii) limited availability of methods for the detection of (un)intended effects resulting from the genetic modification; and (iii) limited information on natural variations in levels of relevant crop constituents. In order to further improve the methodology for identification of unintended effects, new 'profiling' methods are recommended. Such methods will allow for the screening of potential changes in the modified host organism at different integration levels, i.e. at the genome level, during gene expression and protein translation, and at the level of cellular metabolism

  10. Surface-based prostate registration with biomechanical regularization

    Science.gov (United States)

    van de Ven, Wendy J. M.; Hu, Yipeng; Barentsz, Jelle O.; Karssemeijer, Nico; Barratt, Dean; Huisman, Henkjan J.

    2013-03-01

    Adding MR-derived information to standard transrectal ultrasound (TRUS) images for guiding prostate biopsy is of substantial clinical interest. A tumor visible on MR images can be projected on ultrasound by using MRUS registration. A common approach is to use surface-based registration. We hypothesize that biomechanical modeling will better control deformation inside the prostate than a regular surface-based registration method. We developed a novel method by extending a surface-based registration with finite element (FE) simulation to better predict internal deformation of the prostate. For each of six patients, a tetrahedral mesh was constructed from the manual prostate segmentation. Next, the internal prostate deformation was simulated using the derived radial surface displacement as boundary condition. The deformation field within the gland was calculated using the predicted FE node displacements and thin-plate spline interpolation. We tested our method on MR guided MR biopsy imaging data, as landmarks can easily be identified on MR images. For evaluation of the registration accuracy we used 45 anatomical landmarks located in all regions of the prostate. Our results show that the median target registration error of a surface-based registration with biomechanical regularization is 1.88 mm, which is significantly different from 2.61 mm without biomechanical regularization. We can conclude that biomechanical FE modeling has the potential to improve the accuracy of multimodal prostate registration when comparing it to regular surface-based registration.

  11. Theoretical-and-Methodological Substantiation of Multilingual Model Activity in Kazakhstan Higher School Education System

    Science.gov (United States)

    Ospanova, Bikesh Revovna; Azimbayeva, Zhanat Amantayevna; Timokhina, Tatyana Vladimirovna; Seydakhmetova, Zergul Koblandiyevna

    2016-01-01

    The need of implementing the model of professional development in training an expert in the conditions of multilingualism is considered. The possibility of using the multilingual approach in the context of present day education with the use of innovative technologies of training is substantiated, the definition of "multilingual…

  12. Substantiated childhood maltreatment and young adulthood cannabis use disorders: A pre-birth cohort study.

    Science.gov (United States)

    Abajobir, Amanuel Alemu; Najman, Jake Moses; Williams, Gail; Strathearn, Lane; Clavarino, Alexandra; Kisely, Steve

    2017-10-01

    This study investigates the association between exposure to prospectively-substantiated childhood maltreatment between 0 and 14 years of age and lifetime cannabis use, abuse and dependence reported at 21 years. Data were taken from 2526 (51.6% female) participants in the Mater Hospital-University of Queensland Study of Pregnancy, a pre-birth, prospective, cohort study. Prospectively-substantiated cases of childhood maltreatment, reported to the government child protection agencies between 0 and 14 years of age, were linked to CIDI DSM-IV self-report data from the 21-year follow-up. Exposure to any childhood maltreatment, and childhood neglect in particular, predicted subsequent cannabis abuse with adjusted odds ratios (AORs) of 1.79 and 2.62, respectively. Any childhood maltreatment, physical abuse, emotional abuse and neglect predicted cannabis dependence with AORs of 2.47, 2.81, 2.44 and 2.68, respectively. The associations for an early age of onset of cannabis abuse and dependence were significant and consistent for maltreated children. In addition, frequency of maltreatment substantiations predicted cannabis abuse, dependence and an early age of onset of these disorders. The AORs for cannabis ever use without any DSM-IV cannabis disorder were 1.78 for any maltreatment and 2.15 for emotional abuse. Any childhood maltreatment and neglect predicted lifetime ever cannabis use, as well as cannabis use disorder. There was little evidence for any interaction between gender and different forms of childhood maltreatment and its association with cannabis use disorders. Physical abuse, emotional abuse and neglect, as well as multiple episodes of maltreatment independently predicted cannabis use disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Calculated electronic structure of chromium surfaces and chromium monolayers on iron

    International Nuclear Information System (INIS)

    Victora, R.H.; Falicov, L.M.

    1985-01-01

    A self-consistent calculation of the magnetic and electronic properties of the chromium (100) and (110) surfaces and of a chromium monolayer on the (100) and (110) iron surfaces is presented. It is found that (i) the (100) chromium surface is ferromagnetic with a greatly enhanced spin polarization (3.00 electrons); (ii) a substantial enhancement of the spin imbalance exists several (>5) layers into the bulk; (iii) the (110) chromium surface is antiferromagnetic with a large (2.31) spin imbalance; (iv) the (100) chromium monolayer on ferromagnetic iron is ferromagnetic, with a huge spin imbalance (3.63), and aligned antiferromagnetically with respect to the bulk iron; (v) the (110) chromium monolayer on ferromagnetic iron is also ferromagnetic, with a spin imbalance of 2.25 and antiferromagnetically aligned to the iron. The spin imbalance of chromium on iron (100) is possibly the largest of any transition-metal system

  14. Effect of surface contaminants on the light emission spectrum of LiF TLDs

    International Nuclear Information System (INIS)

    Abhold, M.E.

    1987-01-01

    Recent results show the differences between the light emissions spectra from LiF Thermoluminescent Dosimeters (TLDs) for gamma vs. alpha irradiations to be due to contaminants on the surface of the TLD. The light emission spectrum for thermal neutron irradiations was observed to be identical to that for a Cs-137 gamma irradiation in Harshaw TLD-100. Further experiments with surface treatments on TLD-100 indicate trace contaminants introduced by the standard methanol cleansing rinse in reagent grade methanol to have a substantial effect on the light emission spectrum for Am-241 alpha irradiations

  15. A review of measurement and modelling results of particle atmosphere-surface exchange

    DEFF Research Database (Denmark)

    Pryor, Sara; Gallagher, M.; Sievering, H.

    2008-01-01

    Atmosphere-surface exchange represents one mechanism by which atmospheric particle mass and number size distributions are modified. Deposition velocities (upsilon(d)) exhibit a pronounced dependence on surface type, due in part to turbulence structure (as manifest in friction velocity), with minima...... agreement between models and observations is found over less-rough surfaces though those data also imply substantially higher surface collection efficiencies than were originally proposed and are manifest in current models. We review theorized dependencies for particle fluxes, describe and critique model...... of approximately 0.01 and 0.2 cm s(-1) over grasslands and 0.1-1 cm s(-1) over forests. However, as noted over 20 yr ago, observations over forests generally do not support the pronounced minimum of deposition velocity (upsilon(d)) for particle diameters of 0.1-2 mu m as manifest in theoretical predictions. Closer...

  16. Plasma surface interactions at the JET X-point tiles

    International Nuclear Information System (INIS)

    Martinelli, A.P.; Behrisch, R.; Coad, J.P.; Kock, L. de

    1989-01-01

    Operation with a magnetic divertor, which leads to a zero poloidal field inside the volume of the discharge vessel (the X-point) has led to substantial improvements in confinement time in JET. In this mode the diverted plasma is conducted to a large number of graphite tiles (X-point tiles) near the top of the vessel. The power handling capability of these tiles limits the maximum additional heating power to the discharge. The study of the surface modifications of the X-point tiles of JET is therefore of interest both to correlate the magnetic configuration and plasma particle and energy fluxes with the surface modifications, and also to get information about the erosion and deposition at these wall areas. (author) 5 refs., 4 figs

  17. Holograms for power-efficient excitation of optical surface waves

    Science.gov (United States)

    Ignatov, Anton I.; Merzlikin, Alexander M.

    2018-02-01

    A method for effective excitation of optical surface waves based on holography principles has been proposed. For a particular example of excitation of a plasmonic wave in a dielectric layer on metal the efficiency of proposed volume holograms in the dielectric layer has been analyzed in comparison with optimized periodic gratings in the dielectric layer. Conditions when the holograms are considerably more efficient than the gratings have been found out. In addition, holograms recorded in two iterations have been proposed and studied. Such holograms are substantially more efficient than the optimized periodic gratings for all incidence angles of an exciting Gaussian beam. The proposed method is universal: it can be extended for efficient excitation of different types of optical surface waves and optical waveguide modes.

  18. A coupled inversion of pressure and surface displacement

    International Nuclear Information System (INIS)

    Vasco, D.W.; Karasaki, Kenzi; Kishida, Kiyoshi

    2001-01-01

    A coupled inversion of transient pressure observations and surface displacement measurements provides an efficient technique for estimating subsurface permeability variations. The methodology has the advantage of utilizing surface observations, which are typically much less expensive than measurements requiring boreholes. Furthermore, unlike many other geophysical observables, the relationship between surface deformation and reservoir pore fluid volume changes is relatively well understood. Our treatment enables us to partition the estimation problem into a sequence of three linear sub-problems. An application of the approach to a set of tilt and borehole pressure data from the Raymond field site in California illustrates it's efficiency and utility. The observations are associated with a well test in which fluid is withdrawn from a shallow fracture zone. During the test thirteen tiltmeters recorded the movement of the ground surface. Simultaneously, nine transducers measured pressure changes in boreholes intersecting the fracture system. We are able to image a high permeability, north trending channel located within the fracture zone. The existence and orientation of this high permeability feature is substantiated by a semi-quantitative analysis of some 4,000 transient pressure curves. (author)

  19. Surface chemistry and bonding configuration of ultrananocrystalline diamond surfaces and their effects on nanotribological properties

    International Nuclear Information System (INIS)

    Sumant, A. V.; Grierson, D. S.; Carpick, R. W.; Gerbi, J. E.; Carlisle, J. A.; Auciello, O.

    2007-01-01

    We present a comprehensive study of surface composition and nanotribology for ultrananocrystalline diamond (UNCD) surfaces, including the influence of film nucleation on these properties. We describe a methodology to characterize the underside of the films as revealed by sacrificial etching of the underlying substrate. This enables the study of the morphology and composition resulting from the nucleation and initial growth of the films, as well as the characterization of nanotribological properties which are relevant for applications including micro-/nanoelectromechanical systems. We study the surface chemistry, bonding configuration, and nanotribological properties of both the topside and the underside of the film with synchrotron-based x-ray absorption near-edge structure spectroscopy to identify the bonding state of the carbon atoms, x-ray photoelectron spectroscopy to determine the surface chemical composition, Auger electron spectroscopy to further verify the composition and bonding configuration, and quantitative atomic force microscopy to study the nanoscale topography and nanotribological properties. The films were grown on SiO 2 after mechanically polishing the surface with detonation synthesized nanodiamond powder, followed by ultrasonication in a methanol solution containing additional nanodiamond powder. The sp 2 fraction, morphology, and chemistry of the as-etched underside are distinct from the topside, exhibiting a higher sp 2 fraction, some oxidized carbon, and a smoother morphology. The nanoscale single-asperity work of adhesion between a diamond nanotip and the as-etched UNCD underside is far lower than for a silicon-silicon interface (59.2±2 vs 826±186 mJ/m 2 , respectively). Exposure to atomic hydrogen dramatically reduces nanoscale adhesion to 10.2±0.4 mJ/m 2 , at the level of van der Waals' interactions and consistent with recent ab initio calculations. Friction is substantially reduced as well, demonstrating a direct link between the

  20. Substantially Evolutionary Theorizing in Designing Software-Intensive Systems

    Directory of Open Access Journals (Sweden)

    Petr Sosnin

    2018-04-01

    Full Text Available Useful inheritances from scientific experience open perspective ways for increasing the degree of success in designing of systems with software. One such way is a search and build applied theory that takes into account the nature of design and the specificity of software engineering. This paper presents a substantially evolutionary approach to creating the project theories, the application of which leads to positive effects that are traditionally expected from theorizing. Any implementation of the approach is based on a reflection by designers of an operational space of designing onto a semantic memory of a question-answer type. One of the results of such reflection is a system of question-answer nets, the nodes of which register facts of interactions of designers with accessible experience. A set of such facts is used by designers for creating and using the theory that belongs to the new subclass of Grounded Theories. This sub-class is oriented on organizationally behavioral features of a project’s work based on design thinking, automated mental imagination, and thought experimenting that facilitate increasing the degree of controlled intellectualization in the design process and, correspondingly, increasing the degree of success in the development of software-intensive systems.

  1. Surface modification of silica nanoparticles by UV-induced graft polymerization of methyl methacrylate.

    Science.gov (United States)

    Kim, Sooyeon; Kim, Eunhye; Kim, Sungsoo; Kim, Woosik

    2005-12-01

    In this study we modified the surface of silica nanoparticles with methyl methacrylate by UV-induced graft polymerization. It is a surface-initiated polymerization reaction induced by ultraviolet irradiation. The resulting organic-inorganic nanocomposites were near-monodisperse and fabricated without homopolymerization of the monomer. Substantial increase in mean particle size was observed by SEM image analysis after UV-induced grafting of methyl methacrylate onto pure silica particles. FT-Raman spectroscopy and X-ray photoelectron spectroscopy studies of these materials revealed the successful grafting of methyl methacrylate onto the silica surface. The formation of a covalent bond between the grafted PMMA chains and silica surface was indicated by FT-Raman spectra. Thermogravimetric analysis of the PMMA-grafted silica particles indicated the polymer contents in good agreement with SEM photographs.

  2. Polyethylene imine/graphene oxide layer-by-layer surface functionalization for significantly improved limit of detection and binding kinetics of immunoassays on acrylate surfaces.

    Science.gov (United States)

    Miyazaki, Celina M; Mishra, Rohit; Kinahan, David J; Ferreira, Marystela; Ducrée, Jens

    2017-10-01

    Antibody immobilization on polymeric substrates is a key manufacturing step for microfluidic devices that implement sample-to-answer automation of immunoassays. In this work, a simple and versatile method to bio-functionalize poly(methylmethacrylate) (PMMA), a common material of such "Lab-on-a-Chip" systems, is proposed; using the Layer-by-Layer (LbL) technique, we assemble nanostructured thin films of poly(ethylene imine) (PEI) and graphene oxide (GO). The wettability of PMMA surfaces was significantly augmented by the surface treatment with (PEI/GO) 5 film, with an 81% reduction of the contact angle, while the surface roughness increased by 600%, thus clearly enhancing wettability and antibody binding capacity. When applied to enzyme-linked immunosorbent assays (ELISAs), the limit of detection of PMMA surface was notably improved from 340pgmL -1 on commercial grade polystyrene (PS) and 230pgmL -1 on plain PMMA surfaces to 130pgmL -1 on (PEI/GO) 5 treated PMMA. Furthermore, the accelerated antibody adsorption kinetics on the LbL films of GO allowed to substantially shorten incubation times, e.g. for anti-rat IgG adsorption from 2h down to 15min on conventional and treated surfaces, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. The major-element composition of Mercury's surface from MESSENGER X-ray spectrometry.

    Science.gov (United States)

    Nittler, Larry R; Starr, Richard D; Weider, Shoshana Z; McCoy, Timothy J; Boynton, William V; Ebel, Denton S; Ernst, Carolyn M; Evans, Larry G; Goldsten, John O; Hamara, David K; Lawrence, David J; McNutt, Ralph L; Schlemm, Charles E; Solomon, Sean C; Sprague, Ann L

    2011-09-30

    X-ray fluorescence spectra obtained by the MESSENGER spacecraft orbiting Mercury indicate that the planet's surface differs in composition from those of other terrestrial planets. Relatively high Mg/Si and low Al/Si and Ca/Si ratios rule out a lunarlike feldspar-rich crust. The sulfur abundance is at least 10 times higher than that of the silicate portion of Earth or the Moon, and this observation, together with a low surface Fe abundance, supports the view that Mercury formed from highly reduced precursor materials, perhaps akin to enstatite chondrite meteorites or anhydrous cometary dust particles. Low Fe and Ti abundances do not support the proposal that opaque oxides of these elements contribute substantially to Mercury's low and variable surface reflectance.

  4. The modification of nanocomposite hybrid polymer surfaces by exposure to oxygen containing plasmas

    Science.gov (United States)

    Figueiredo, Ashley; Zimmermann, Katherine; Augustine, Brian; Hughes, Chris; Chusuei, Charles

    2006-11-01

    The wetting properties of the surfaces of the nanocomposite hybrid polymer poly[(propylmethacryl-heptaisobutyl- polyhedral oligomeric silsequioxane)-co-(methylmethacrylate)] (POSS-PMMA)has been studied before and after exposure to plasmas containing oxygen. The contact angle of water droplets on the surface showed a substantial decrease after plasma exposure indicating an increase in the hydrophilicity of the surface. A model was developed in which the plasma preferentially removed organic material including both the PMMA backbone and isobutyl groups from the corners of the POSS cages leaving behind a surface characterized by the silicon oxide-like POSS material. Measurements of surface concentrations of oxygen, silicon, and carbon by x-ray photoelectron spectroscopy (XPS) showed an increase in the amount of oxygen and silicon compared to carbon and the appropriate chemical shifts were observed in the XPS data to support the model of Si-O enrichment on the surface. Variable angle spectroscopic ellipsometry (VASE) and atomic force microscopy (AFM) measurements also supported the model and these results will be presented.

  5. Surface concrete decontamination equipment developed by Pacific Northwest Laboratory

    International Nuclear Information System (INIS)

    Halter, J.M.; Sullivan, R.G.; Bevan, J.L.

    1982-08-01

    This report documents a project that the Pacific Northwest Laboratory conducted to identify and develop techniques for removing contaminated concrete surfaces. A major problem associated with nuclear facility decontamination and decommissioning is how to economically demolish and dispose of contaminated concrete. Removing only the contaminated portion of the concrete can substantially reduce costs. Evaluation of various methods for removing concrete surfaces shows that several techniques presently used require excessive manpower, time, and energy. Many times more material is removed than necessary, increasing the quantity of waste that must be handled under controlled conditions. These evaluations generated the basic criteria for developing a suitable concrete removal technique: provide a convenient method for cleaning surfaces (such as those contaminated by a small spill); reduce the contaminated waste volume that has to be placed into controlled storage; remove surfaces quickly; and minimize personal exposure to potentially harmful radiation or toxic materials. Removal to 1/4 to 1/2 in. of contaminated surface layer is sufficient for cleanup of most facilities. Two unique decontamination methods have been developed: the concrete spaller and the water cannon. The concrete spaller is the most efficient technique: it removes the concrete surface faster than the water cannons and at a lower cost (as little as $3.00/ft 2 of concrete surface). However, the .458 magnum water cannon may be well suited for small or hard-to-reach locations

  6. Human Mars Surface Mission Nuclear Power Considerations

    Science.gov (United States)

    Rucker, Michelle A.

    2018-01-01

    A key decision facing Mars mission designers is how to power a crewed surface field station. Unlike the solar-powered Mars Exploration Rovers (MER) that could retreat to a very low power state during a Martian dust storm, human Mars surface missions are estimated to need at least 15 kilowatts of electrical (kWe) power simply to maintain critical life support and spacecraft functions. 'Hotel' loads alone for a pressurized crew rover approach two kWe; driving requires another five kWe-well beyond what the Curiosity rover’s Radioisotope Power System (RPS) was designed to deliver. Full operation of a four-crew Mars field station is estimated at about 40 kWe. Clearly, a crewed Mars field station will require a substantial and reliable power source, beyond the scale of robotic mission experience. This paper explores the applications for both fission and RPS nuclear options for Mars.

  7. The validation of ocean surface heat fluxes in AMIP

    International Nuclear Information System (INIS)

    Gleckler, P.J.; Randall, D.A.

    1993-09-01

    Recent intercomparisons of Atmospheric General Circulation Models (AGCMS) constrained with sea-surface temperatures have shown that while there are substantial differences among various models (with each other and available observations), overall the differences between them have been decreasing. The primary goal of AMIP is to enable a systematic intercomparison and validation of state-of-the- art AGCMs by supporting in-depth diagnosis of and interpretation of the model results. Official AMIP simulations are 10 years long, using monthly mean Sea-Surface Temperatures (SSTs) and sea ice conditions which are representative of the 1979--1988 decade. Some model properties are also dictated by the design of AMIP such as the solar constant, the atmospheric CO 2 concentration, and the approximate horizontal resolution. In this paper, some of the preliminary results of AMIP Subproject No. 5 will be summarized. The focus will be on the intercomparison and validation of ocean surface heat fluxes of the AMIP simulations available thus far

  8. Analysis of cutting force signals by wavelet packet transform for surface roughness monitoring in CNC turning

    Science.gov (United States)

    García Plaza, E.; Núñez López, P. J.

    2018-01-01

    On-line monitoring of surface finish in machining processes has proven to be a substantial advancement over traditional post-process quality control techniques by reducing inspection times and costs and by avoiding the manufacture of defective products. This study applied techniques for processing cutting force signals based on the wavelet packet transform (WPT) method for the monitoring of surface finish in computer numerical control (CNC) turning operations. The behaviour of 40 mother wavelets was analysed using three techniques: global packet analysis (G-WPT), and the application of two packet reduction criteria: maximum energy (E-WPT) and maximum entropy (SE-WPT). The optimum signal decomposition level (Lj) was determined to eliminate noise and to obtain information correlated to surface finish. The results obtained with the G-WPT method provided an in-depth analysis of cutting force signals, and frequency ranges and signal characteristics were correlated to surface finish with excellent results in the accuracy and reliability of the predictive models. The radial and tangential cutting force components at low frequency provided most of the information for the monitoring of surface finish. The E-WPT and SE-WPT packet reduction criteria substantially reduced signal processing time, but at the expense of discarding packets with relevant information, which impoverished the results. The G-WPT method was observed to be an ideal procedure for processing cutting force signals applied to the real-time monitoring of surface finish, and was estimated to be highly accurate and reliable at a low analytical-computational cost.

  9. Pharmaceutical manufacturing facility discharges can substantially increase the pharmaceutical load to U.S. wastewaters

    Science.gov (United States)

    Scott, Tia-Marie; Phillips, Patrick J.; Kolpin, Dana W.; Colella, Kaitlyn M.; Furlong, Edward T.; Foreman, William T.; Gray, James L.

    2018-01-01

    Discharges from pharmaceutical manufacturing facilities (PMFs) previously have been identified as important sources of pharmaceuticals to the environment. Yet few studies are available to establish the influence of PMFs on the pharmaceutical source contribution to wastewater treatment plants (WWTPs) and waterways at the national scale. Consequently, a national network of 13 WWTPs receiving PMF discharges, six WWTPs with no PMF input, and one WWTP that transitioned through a PMF closure were selected from across the United States to assess the influence of PMF inputs on pharmaceutical loading to WWTPs. Effluent samples were analyzed for 120 pharmaceuticals and pharmaceutical degradates. Of these, 33 pharmaceuticals had concentrations substantially higher in PMF-influenced effluent (maximum 555,000 ng/L) compared to effluent from control sites (maximum 175 ng/L). Concentrations in WWTP receiving PMF input are variable, as discharges from PMFs are episodic, indicating that production activities can vary substantially over relatively short (several months) periods and have the potential to rapidly transition to other pharmaceutical products. Results show that PMFs are an important, national-scale source of pharmaceuticals to the environment.

  10. Further substantial improvement of interventional diagnosis and treatment via portal vein system

    International Nuclear Information System (INIS)

    Yang Weizhu; Chen Yongde

    2006-01-01

    Along with the development of interventional appliances and proficiency of operational skills, the interventional diagnosis and treatment via hepatic portal vein system have achieved great progress and improvement. However, in order to further exploit the advantages of interventional diagnosis and treatment, the review of the anatomical structures, normal aberrance of portal venous system were needed. Getting familiar with pathologic condition to discover the new interventional appliances and embolic agents, and then in term of conduct the research on a very tough substantial base in a down-to-earth manner were important. (authors)

  11. Leaf surface structures enable the endemic Namib desert grass Stipagrostis sabulicola to irrigate itself with fog water.

    Science.gov (United States)

    Roth-Nebelsick, A; Ebner, M; Miranda, T; Gottschalk, V; Voigt, D; Gorb, S; Stegmaier, T; Sarsour, J; Linke, M; Konrad, W

    2012-08-07

    The Namib grass Stipagrostis sabulicola relies, to a large degree, upon fog for its water supply and is able to guide collected water towards the plant base. This directed irrigation of the plant base allows an efficient and rapid uptake of the fog water by the shallow roots. In this contribution, the mechanisms for this directed water flow are analysed. Stipagrostis sabulicola has a highly irregular surface. Advancing contact angle is 98° ± 5° and the receding angle is 56° ± 9°, with a mean of both values of approximately 77°. The surface is thus not hydrophobic, shows a substantial contact angle hysteresis and therefore, allows the development of pinned drops of a substantial size. The key factor for the water conduction is the presence of grooves within the leaf surface that run parallel to the long axis of the plant. These grooves provide a guided downslide of drops that have exceeded the maximum size for attachment. It also leads to a minimum of inefficient drop scattering around the plant. The combination of these surface traits together with the tall and upright stature of S. sabulicola contributes to a highly efficient natural fog-collecting system that enables this species to thrive in a hyperarid environment.

  12. Leaf surface structures enable the endemic Namib desert grass Stipagrostis sabulicola to irrigate itself with fog water

    Science.gov (United States)

    Roth-Nebelsick, A.; Ebner, M.; Miranda, T.; Gottschalk, V.; Voigt, D.; Gorb, S.; Stegmaier, T.; Sarsour, J.; Linke, M.; Konrad, W.

    2012-01-01

    The Namib grass Stipagrostis sabulicola relies, to a large degree, upon fog for its water supply and is able to guide collected water towards the plant base. This directed irrigation of the plant base allows an efficient and rapid uptake of the fog water by the shallow roots. In this contribution, the mechanisms for this directed water flow are analysed. Stipagrostis sabulicola has a highly irregular surface. Advancing contact angle is 98° ± 5° and the receding angle is 56° ± 9°, with a mean of both values of approximately 77°. The surface is thus not hydrophobic, shows a substantial contact angle hysteresis and therefore, allows the development of pinned drops of a substantial size. The key factor for the water conduction is the presence of grooves within the leaf surface that run parallel to the long axis of the plant. These grooves provide a guided downslide of drops that have exceeded the maximum size for attachment. It also leads to a minimum of inefficient drop scattering around the plant. The combination of these surface traits together with the tall and upright stature of S. sabulicola contributes to a highly efficient natural fog-collecting system that enables this species to thrive in a hyperarid environment. PMID:22356817

  13. 77 FR 65169 - Extension of Certain Timber Sale Contracts; Finding of Substantial Overriding Public Interest

    Science.gov (United States)

    2012-10-25

    ... that are in breach, or (6) contracts when the purchaser's processing facility has not operated during... DEPARTMENT OF AGRICULTURE Forest Service Extension of Certain Timber Sale Contracts; Finding of Substantial Overriding Public Interest AGENCY: Forest Service, USDA. ACTION: Notice of contract extensions...

  14. Characterization of pigment-leached antifouling coatings using BET surface area measurements and mercury porosimetry

    DEFF Research Database (Denmark)

    Kiil, Søren; Dam-Johansen, Kim

    2007-01-01

    of antifouling coating behaviour because the active binder surface area and porosity of the leached layer are substantially increased. A similar effect was not observed for a coating with a mixture of ZnO and TiO2 pigments. The two experimental methods are expected to be useful for practical analysis of leaching...

  15. Design of Sb2S3 nanorod-bundles: imperfect oriented attachment

    Science.gov (United States)

    Lu, Qifei; Zeng, Haibo; Wang, Zhenyang; Cao, Xueli; Zhang, Lide

    2006-05-01

    The large scale formation of uniform Sb2S3 nanorod-bundles has been achieved via a simple and mild hydrothermal approach with the assistance of polyvinylpyrrolidone. By closely inspecting the growth process and the crystallographic analysis of as-synthesized products, conclusive evidence has been provided to show that the growth mechanism of such nanorod-bundles is imperfect oriented attachment. The anisotropic adsorption of polyvinylpyrrolidone at the different surfaces of Sb2S3 nanocrystals assists the one-dimensional preferential growth; it is just the misorientations that result in the nanorod-based superstructures. Moreover, the hydrothermal treatment time plays a crucial role, and can be used as the parameter to control the size and morphology of the bundles. This simple approach promises future large-scale controlled synthesis of various nanobody-based superstructures for many important applications in nanotechnology.

  16. Lack of Substantial Post-Cessation Weight Increase in Electronic Cigarettes Users

    Directory of Open Access Journals (Sweden)

    Cristina Russo

    2018-03-01

    Full Text Available Minimization of post-cessation weight gain in quitters is important, but existing approaches (e.g., antismoking medications shows only limited success. We investigated changes in body weight in smokers who quit or reduced substantially their cigarette consumption by switching to electronic cigarettes (ECs use. Body weight and smoking/vaping history were extracted from medical records of smokers and ex-smokers to match three study groups: (1 regular EC users on at least two consecutive follow-up visits; (2 regular smokers (and not using ECs; (3 subjects who reported sustained smoking abstinence after completing a cessation program. Review of their medical records was conducted at two follow-up visits at 6- (F/U 6m and 12-months (F/U 12m. A total of 86 EC users, 93 regular smokers, and 44 quitters were studied. In the EC users study group, cigarettes/day use decreased from 21.1 at baseline to 1.8 at F/U 12m (p < 0.0001. Dual usage was reported by approximately 50% of EC users. Both within factor (time, p < 0.0001 and between factor (study groups, p < 0.0001 produced significant effect on weight (% change from baseline, with a significant 4.8% weight gain from baseline in the quitters study group at F/U 12m. For the EC users, weight gain at F/U 12m was only 1.5% of baseline. There was no evidence of post-cessation weight increase in those who reduced substantially cigarette consumption by switching to ECs (i.e., dual users and only modest post-cessation weight increase was reported in exclusive EC users at F/U 12m. By reducing weight gain and tobacco consumption, EC-based interventions may promote an overall improvement in quality of life.

  17. Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Karunatilaka, Krishanthi S; Filman, David J; Hogle, James M

    2017-02-01

    By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion. When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productive-infection events. Copyright © 2017 American Society for Microbiology.

  18. Electropolished Titanium Implants with a Mirror-Like Surface Support Osseointegration and Bone Remodelling

    Directory of Open Access Journals (Sweden)

    Cecilia Larsson Wexell

    2016-01-01

    Full Text Available This work characterises the ultrastructural composition of the interfacial tissue adjacent to electropolished, commercially pure titanium implants with and without subsequent anodisation, and it investigates whether a smooth electropolished surface can support bone formation in a manner similar to surfaces with a considerably thicker surface oxide layer. Screw-shaped implants were electropolished to remove all topographical remnants of the machining process, resulting in a thin spontaneously formed surface oxide layer and a smooth surface. Half of the implants were subsequently anodically oxidised to develop a thickened surface oxide layer and increased surface roughness. Despite substantial differences in the surface physicochemical properties, the microarchitecture and the composition of the newly formed bone were similar for both implant surfaces after 12 weeks of healing in rabbit tibia. A close spatial relationship was observed between osteocyte canaliculi and both implant surfaces. On the ultrastructural level, the merely electropolished surface showed the various stages of bone formation, for example, matrix deposition and mineralisation, entrapment of osteoblasts within the mineralised matrix, and their morphological transformation into osteocytes. The results demonstrate that titanium implants with a mirror-like surface and a thin, spontaneously formed oxide layer are able to support bone formation and remodelling.

  19. Ultrastructural changes of photodegradation of wood surfaces exposed to UV

    International Nuclear Information System (INIS)

    Kuo, M.L.; Hu, N.

    1991-01-01

    Red pine sapwood transverse and radial surfaces were exposed to ultraviolet (UV) light for 3 to 40 days. Effect of UV irradiation on ultrastructural changes of cell walls were studied by scanning (SEM) and transmission (TEM) electron microscopy. SEM study of transverse sections showed that during initial stages of UV irradiation, lignin in cell corners and in the compound middle lamellae was preferentially degraded and that the radial middle lamellae substained a greater rate of UV degradation than did the tangential middle lamellae. Massive cell wall degradation, as indicated by cell wall thinning, did not occur until surfaces were exposed to UV light for more than 10 days. TEM study of radial cell wall surfaces indicated that lignin lining the warty layer was removed by UV irradiation in 3 days and that warts were destroyed by a UV irradiation in 7 days. UV irradiation of cell wall surfaces produced a substantial amount of water-soluble degradation products. After 30 days of UV irradiation, the S3 layer was totally removed and revealed the very fragile S2 layer. (author)

  20. Surface determinants of low density lipoprotein uptake by endothelial cells

    International Nuclear Information System (INIS)

    Goeroeg, P.; Pearson, J.D.

    1984-01-01

    The surface sialic acid content of aortic endothelial cells in vitro was substantially lower in sparse cultures than at confluence. Binding of LDL to endothelial cells did not change at different culture densities and was unaffected by brief pretreatment with neuraminidase to partially remove surface sialic acid residues. In contrast, internalisation of LDL declined by a factor of 3 between low density cell cultures and confluent monolayers; neuraminidase pretreatment increased LDL uptake and the effect was most marked (>10-fold) at confluence. Pretreatment with cationised ferritin, which removed most of the surface sialic acid residues as well as glycosaminoglycans, increased LDL internalisation by up to 20-fold, again with most effect on confluent monolayers. Thus LDL uptake is inversely correlated with sialic acid content. We conclude that changes in the surface density of sialic acid (and possibly other charged) residues significantly modulate endothelial LDL uptake, and suggest that focal increases in LDL accumulation during atherogenesis may be related to alterations in endothelial endocytic properties at sites of increased cell turnover or damage. (author)

  1. Fluid flow near the surface of earth's outer core

    Science.gov (United States)

    Bloxham, Jeremy; Jackson, Andrew

    1991-01-01

    This review examines the recent attempts at extracting information on the pattern of fluid flow near the surface of the outer core from the geomagnetic secular variation. Maps of the fluid flow at the core surface are important as they may provide some insight into the process of the geodynamo and may place useful constraints on geodynamo models. In contrast to the case of mantle convection, only very small lateral variations in core density are necessary to drive the flow; these density variations are, by several orders of magnitude, too small to be imaged seismically; therefore, the geomagnetic secular variation is utilized to infer the flow. As substantial differences exist between maps developed by different researchers, the possible underlying reasons for these differences are examined with particular attention given to the inherent problems of nonuniqueness.

  2. 26 CFR 1.6662-5T - Substantial and gross valuation misstatements under chapter 1 (temporary).

    Science.gov (United States)

    2010-04-01

    ..., DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Additions to the Tax, Additional... persons). (iii) Property. For purposes of this section, the term property refers to both tangible and... adjustments. For rules relating to the penalty imposed with respect to a substantial or gross valuation...

  3. 75 FR 69454 - Emergency Homeowners' Loan Program: Notice of Allocation of Funding for Substantially Similar...

    Science.gov (United States)

    2010-11-12

    ... assistance and provide security for such repayment by recordation of a HUD mortgage as a junior lien on the... current first lien mortgage loan payments for which the homeowner remains responsible. IV. Submission of.... Nondiscrimination and Civil Rights Requirements States operating existing programs that provide substantially...

  4. What would happen to Superstorm Sandy under the influence of a substantially warmer Atlantic Ocean?

    Science.gov (United States)

    Lau, William K. M.; Shi, J. J.; Tao, W. K.; Kim, K. M.

    2016-01-01

    Based on ensemble numerical simulations, we find that possible responses of Sandy-like superstorms under the influence of a substantially warmer Atlantic Ocean bifurcate into two groups. In the first group, storms are similar to present-day Sandy from genesis to extratropical transition, except they are much stronger, with peak Power Destructive Index (PDI) increased by 50-80%, heavy rain by 30-50%, and maximum storm size (MSS) approximately doubled. In the second group, storms amplify substantially over the interior of the Atlantic warm pool, with peak PDI increased by 100-160%, heavy rain by 70-180%, and MSS more than tripled compared to present-day Superstorm Sandy. These storms when exiting the warm pool, recurve northeastward out to sea, subsequently interact with the developing midlatitude storm by mutual counterclockwise rotation around each other and eventually amplify into a severe Northeastern coastal storm, making landfall over the extreme northeastern regions from Maine to Nova Scotia.

  5. Substantiating the Expediency of Costs on Introduction of the System of Economic Security of Enterprise

    Directory of Open Access Journals (Sweden)

    Melikhova Tetiana O.

    2018-01-01

    Full Text Available The article substantiates expediency of introduction of the system of economic security of enterprise on the basis of money flows. The methods proposed in the long-term period stipulate the use as sources of financing: accumulated conditional economic costs, accumulated conditional gross and net money flow; and as costs: cumulative advanced costs and the advanced value of fixed assets. The article suggests methods for calculation of: gross index of conditional yield; gross conditional income of project; gross conditional profitability; gross conditional profitability of money flow. The managerial decision on introduction of the system of economic security of enterprise in the short-term production period has been substantiated on the basis of calculations of the following indicators: gross profitability of annual depreciation; annual gross economic effect; index of conditional yield of the gross money flow; differences between the conditional gross cash flow and the depreciation deductions associated with the implementation of the enterprise’s economic security system.

  6. Argonne-Utah studies of 224Ra endosteal surface dosimetry

    International Nuclear Information System (INIS)

    Schlenker, R.A.; Smith, J.M.

    1985-01-01

    The activities of 212 Pb relative to 224 Ra and of 222 Rn relative to 226 Ra were measured in bone surface deposits 24 h after injection of radium into beagles. The fractional retention of 220 Rn atoms was measured in vitro with hydrated and dehydrated bone samples to determine the effect of water content on the escape of radon from bone surfaces. The experimental data suggest that substantial 224 Ra daughter-product disequilibrium exists in bone surface deposits. Estimates for the lower and upper limits on the fractional retention of 220 Rn in vivo are 0.05 and 0.25, respectively. The average bone surface activity of 212 Pb relative to 224 Ra ranged from 0.34 to 0.71 for four dogs, with the majority of the values toward the lower end of the range. Only a small portion of the deposited 212 Pb came from lead in the injection solution despite near equilibrium between 224 Ra and its daughters at the time of injection. The retention data indicate that the endosteal tissue dose rate in the dogs at one day was actually one-third to about one-half that which would be calculated assuming equilibrium of 224 Ra daughter products in bone surface deposits. 11 references, 2 figures, 7 tables

  7. Argonne-Utah studies of 224Ra endosteal surface dosimetry

    International Nuclear Information System (INIS)

    Schlenker, R.A.; Smith, J.M.

    1984-01-01

    The activities of 212 Pb relative to 224 Ra and of 222 Rn relative to 226 Ra were measured in bone surface deposits 24 h after radium injection into beagles. The fractional retention of 220 Rn atoms was measured in vitro with hydrated and dehydrated bone samples to determine the effect of water content on the escape of radon from bone surfaces. The experimental data suggest that substantial 224 Ra daughter product disequilibrium exists in bone surface deposits. Estimates for the lower and upper limits on the fractional retention of 220 Rn in vivo are 0.05 and 0.25, respectively. The average bone surface activity of 212 Pb relative to 224 Ra ranged from 0.34 to 0.71 for four dogs, with the majority of the values toward the low end of the range. Only a small portion of the deposited 212 Pb came from lead in the injection solution despite near equilibrium between 224 Ra and its daughters at the time of injection. The retention data indicate that the endosteal tissue dose rate in the dogs at one day was actually one-third to about one-half that which would be calculated assuming equilibrium of 224 Ra daughter products in bone surface deposits. 11 references, 2 figures, 7 tables

  8. Argonne-Utah studies of 224Ra endosteal surface dosimetry

    International Nuclear Information System (INIS)

    Schlenker, R.A.; Smith, J.M.

    1986-01-01

    The activities of 212 Pb relative to 224 Ra and of 222 Rn relative to 226 Ra were measured in bone surface deposits 24 h after radium injection into beagles. The fractional retention of 220 Rn atoms was measured in vitro with hydrated and dehydrated bone samples to determine the effect of water content on the escape of radon from bone surfaces. The experimental data suggest that substantial 224 Ra daughter product disequilibrium exists in bone surface deposits. Estimates for the lower and upper limits on the fractional retention of 220 Rn in vivo are 0.05 and 0.25, respectively. The average bone surface activity of 212 Pb relative to 224 Ra ranged from 0.34 to 0.71 for four dogs, with the majority of the values toward the low end of the range. Only a small portion of the deposited 212 Pb came from lead in the injection solution despite near equilibrium between 224 Ra and its daughters at the time of injection. The retention data indicate that the endosteal tissue dose rate in the dogs at one day was actually one-third to about one-half that which would be calculated assuming equilibrium of 224 Ra daughter products in bone surface deposits. (orig.)

  9. Applying the Coupled-Cluster Ansatz to Solids and Surfaces in the Thermodynamic Limit

    Science.gov (United States)

    Gruber, Thomas; Liao, Ke; Tsatsoulis, Theodoros; Hummel, Felix; Grüneis, Andreas

    2018-04-01

    Modern electronic structure theories can predict and simulate a wealth of phenomena in surface science and solid-state physics. In order to allow for a direct comparison with experiment, such ab initio predictions have to be made in the thermodynamic limit, substantially increasing the computational cost of many-electron wave-function theories. Here, we present a method that achieves thermodynamic limit results for solids and surfaces using the "gold standard" coupled cluster ansatz of quantum chemistry with unprecedented efficiency. We study the energy difference between carbon diamond and graphite crystals, adsorption energies of water on h -BN, as well as the cohesive energy of the Ne solid, demonstrating the increased efficiency and accuracy of coupled cluster theory for solids and surfaces.

  10. Effect of nonequipotentiality of magnetic surfaces on the transport processes in a tokamak

    International Nuclear Information System (INIS)

    Kovrizhnykh, L.M.; Shasharina, S.G.

    1987-01-01

    The effect of deflection of equipotential from the magnetic surfaces on the transport processes in a tokamak is considered. The values of radial and poloidal electric fields are determined self-consistently, particle and heat fluxes are calculated with regard to these fields. It is shown that in some cases the effect of the poloidal electric field on the current values is very substantial

  11. Linear-time general decoding algorithm for the surface code

    Science.gov (United States)

    Darmawan, Andrew S.; Poulin, David

    2018-05-01

    A quantum error correcting protocol can be substantially improved by taking into account features of the physical noise process. We present an efficient decoder for the surface code which can account for general noise features, including coherences and correlations. We demonstrate that the decoder significantly outperforms the conventional matching algorithm on a variety of noise models, including non-Pauli noise and spatially correlated noise. The algorithm is based on an approximate calculation of the logical channel using a tensor-network description of the noisy state.

  12. To what extent can isotopes help substantiate natural attenuation of chlorinated ethenes?

    DEFF Research Database (Denmark)

    Badin, A.; Broholm, Mette Martina; Hunkeler, D.

    to which isotopes could help substantiate natural attenuation of chlorinated ethenes at the field scale. Our work hence aims at exploring the latter based on data acquired in 2006 in a site located in Denmark which is contaminated with PCE and its end-products TCE, cDCE, and VC. Previous work on this site...... has enabled to demonstrate that PCE and TCE were undergoing reductive dechlorination while cDCE would be at least partially degraded through reductive dechlorination1. However, the magnitude of the contaminants attenuation by biodegradation was not evaluated. Based on simple modeling including...

  13. Mineralization of bacterial cell mass on a photocatalytic surface in air

    International Nuclear Information System (INIS)

    Jacoby, W.A.; Maness, P.C.; Wolfrum, E.J.; Blake, D.M.; Fennell, J.A.

    1998-01-01

    Whole cells deposited on a titanium dioxide-coated surface have been oxidized in air to carbon dioxide via photocatalysis. This paper provides the first evidence that the organic matter in whole cells can be completely oxidized. Three experimental techniques were employed to monitor this reaction: scanning electron microscopy, 14 C radioisotope labeling experiments establish that the carbon content of E. coli is oxidized to form carbon dioxide with substantial closure of the mass balance. The batch reactor experiments corroborate the mass balance and provide a preliminary indication of the rate of the oxidation reaction. These results provide evidence that a photocatalytic surface used for disinfection can also be self-cleaning in an air-solid system

  14. 34 CFR 692.41 - What standards may a State use to determine substantial financial need?

    Science.gov (United States)

    2010-07-01

    ... PARTNERSHIP PROGRAM Leveraging Educational Assistance Partnership Program How Does A State Select Students... establishes that are approved by the Secretary. A State may define substantial financial need in terms of... “independent student” as defined under section 480(d) of the HEA. However, for good cause shown, the Secretary...

  15. The effect of differing support surfaces on the efficacy of chest compressions using a resuscitation manikin model.

    Science.gov (United States)

    Tweed, M; Tweed, C; Perkins, G D

    2001-11-01

    External chest compression (ECC) efficacy is influenced by factors including the surface supporting the patient. Air-filled support surfaces are deflated for cardiopulmonary resuscitation, with little evidence to substantiate this. We investigated the effect that differing support surfaces had on ECC efficacy using a CPR manikin model. Four participants carried out four cycles of ECC with an assistant ventilating. The subjects were blinded to the seven support surfaces and the order was randomised. For each participant/surface combination, ECC variables and the participants' perceptions were measured. Participants produced effective ECC with the manikin on the floor (mean proportion correct, 94.5%; mean depth, 42.5 mm). Compared with the floor: the proportion of correct ECC was less for the overlay inflated (PCPR.

  16. Enhanced photothermal lens using a photonic crystal surface

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Yunfei; Liu, Longju [Department of Electrical and Computer Engineering, 2128 Coover Hall, Iowa State University, Ames,Iowa 50011 (United States); Zhao, Xiangwei [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,Southeast University, Nanjing, Jiangsu 211189 (China); Lu, Meng, E-mail: menglu@iastate.edu [Department of Electrical and Computer Engineering, 2128 Coover Hall, Iowa State University, Ames,Iowa 50011 (United States); Department of Mechanical Engineering, Iowa State University, Ames, Iowa 50011 (United States)

    2016-08-15

    A photonic crystal (PC)-enhanced photothermal lens (PTL) is demonstrated for the detection of optically thin light absorption materials. The PC-enhanced PTL system is based on a pump-probe scheme consisting of a PC surface, pump laser beam, and probe laser beam. Heated by the pump beam, light absorption materials on the PC surface generate the PTL and cause a substantial change to the guided-mode resonance supported by the PC structure. The change of the PC resonance is detected using the probe laser beam by measuring its reflectivity from the PC surface. When applied to analyze dye molecules deposited on the PC substrate, the developed system is capable of enhancing the PTL signal by 10-fold and reducing the lowest distinguishable concentration by 8-fold, in comparison to measuring without utilizing the PC resonance. The PC-enhanced PTL was also used to detect gold nanoparticles on the PC surface and exhibited a 20-fold improvement of the lowest distinguishable concentration. The PC-enhanced PTL technology offers a potential tool to obtain the absorption signatures of thin films in a broad spectral range with high sensitivity and inexpensive instrumentation. As a result, this technology will enable a broad range of applications of photothermal spectroscopy in chemical analysis and biomolecule sensing.

  17. Surface modification of promising cerium oxide nanoparticles for nanomedicine applications

    KAUST Repository

    Nanda, Himansu Sekhar

    2016-11-14

    Cerium oxide nanoparticles (CNPs) or nanoceria have emerged as a potential nanomedicine for the treatment of several diseases such as cancer. CNPs have a natural tendency to aggregate or agglomerate in their bare state, which leads to sedimentation in a biological environment. Since the natural biological environment is essentially aqueous, nanoparticle surface modification using suitable biocompatible hydrophilic chemical moieties is highly desirable to create effective aqueous dispersions. In this report, (6-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-hexyl)triethoxysilane was used as a functional, biocompatible organosilane to modify the surface of CNPs to produce promising nanoparticles which open substantial therapeutic avenues. The surface modified nanoparticles were produced in situ via an ammonia-induced ethylene glycol-assisted precipitation method and were characterized using complimentary characterization techniques. The interaction between the functional moiety and the nanoparticle was studied using powerful cross polarization/magic angle sample spinning solid state nuclear magnetic resonance spectroscopy. The surface-modified nanoparticles were extremely small and demonstrated a significant improvement in aqueous dispersibility. Moreover, the existence of a strong ionic coordination between the functional moiety and the surface of the nanoparticle was realised, indicating that the surface modified nanoceria are stable and that the nanoparticles should demonstrate an enhanced circulation time in a biological environment. The surface modification approach should be promising for the production of CNPs for nanomedicine applications. © The Royal Society of Chemistry.

  18. Supercritical processing as a route to high internal surface areas and permanent microporosity in metal-organic framework materials.

    Science.gov (United States)

    Nelson, Andrew P; Farha, Omar K; Mulfort, Karen L; Hupp, Joseph T

    2009-01-21

    Careful processing of four representative metal-organic framework (MOF) materials with liquid and supercritical carbon dioxide (ScD) leads to substantial, or in some cases spectacular (up to 1200%), increases in gas-accessible surface area. Maximization of surface area is key to the optimization of MOFs for many potential applications. Preliminary evidence points to inhibition of mesopore collapse, and therefore micropore accessibility, as the basis for the extraordinarily efficacious outcome of ScD-based activation.

  19. Fibre laser nitriding of titanium and its alloy in open atmosphere for orthopaedic implant applications: Investigations on surface quality, microstructure and tribological properties

    DEFF Research Database (Denmark)

    Chan, Chi-Wai; Lee, Seunghwan; Smith, Graham C.

    2017-01-01

    surfaces, such as the tapered surfacein a femoral stemor the ball-shaped surface in a femoral head. To tackle this problem, a direct laser nitridingprocess in open atmosphere was performed on commercially pure titanium (grade 2, TiG2) and Ti6Al4V alloy(grade 5, TiG5) using a continuous-wave (CW) fibre...... distribution of wear debris, were carefully characterized and compared. The experimental resultsshowed that TiG2 and TiG5 reacted differently with the laser radiation at 1.06 μm wavelength in laser nitridingas evidenced by substantial differences in the microstructure, and surface colour and morphology....... Furthermore,both friction andwear properties were strongly affected by the hardness and microstructure of titaniumsamplesand direct laser nitriding led to substantial improvements in their wear resistant properties. Between the twotypes of titanium samples, bare TiG2 showed higher friction forces and wear...

  20. Substantial Fatigue Similarity of a New Small-Scale Test Rig to Actual Wheel-Rail System

    NARCIS (Netherlands)

    Naeimi, M.; Li, Z.; Petrov, R.H.; Dollevoet, R.P.B.J.; Sietsma, J.; Wu, J.

    2014-01-01

    The substantial similarity of fatigue mechanism in a new test rig for rolling contact fatigue (RCF) has been investigated. A new reduced-scale test rig is designed to perform controlled RCF tests in wheel-rail materials. The fatigue mechanism of the rig is evaluated in this study using a combined

  1. Study on municipal road cracking and surface deformation based on image recognition

    Science.gov (United States)

    Yuan, Haitao; Wang, Shuai; Tan, Jizong

    2017-05-01

    In recent years, the digital image recognition technology of concrete structure cracks and deformation of binocular vision technology detection of civil engineering structure have made substantial development. As a result, people's understanding of the road engineering structure cracking and surface deformation recognition gives rise to a new situation. For the research on digital image concrete structure cracking and masonry structure surface deformation recognition technology, the key is to break through in the method, and to improve the traditional recognition technology and mode. Only in this way can we continuously improve the security level of the highway, to adapt to the new requirements of the development of new urbanization and modernization. This thesis focuses on and systematically analyzes the digital image road engineering structure cracking and key technologies of surface deformation recognition and its engineering applications. In addition, we change the concrete structure cracking and masonry structure surface deformation recognition pattern, and realize the breakthrough and innovation of the road structure safety testing means and methods.

  2. Monte Carlo reactor calculation with substantially reduced number of cycles

    International Nuclear Information System (INIS)

    Lee, M. J.; Joo, H. G.; Lee, D.; Smith, K.

    2012-01-01

    A new Monte Carlo (MC) eigenvalue calculation scheme that substantially reduces the number of cycles is introduced with the aid of coarse mesh finite difference (CMFD) formulation. First, it is confirmed in terms of pin power errors that using extremely many particles resulting in short active cycles is beneficial even in the conventional MC scheme although wasted operations in inactive cycles cannot be reduced with more particles. A CMFD-assisted MC scheme is introduced as an effort to reduce the number of inactive cycles and the fast convergence behavior and reduced inter-cycle effect of the CMFD assisted MC calculation is investigated in detail. As a practical means of providing a good initial fission source distribution, an assembly based few-group condensation and homogenization scheme is introduced and it is shown that efficient MC eigenvalue calculations with fewer than 20 total cycles (including inactive cycles) are possible for large power reactor problems. (authors)

  3. Visualization and targeted disruption of protein interactions in living cells

    Science.gov (United States)

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

  4. Parallel force assay for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Daniela Aschenbrenner

    Full Text Available Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

  5. Structures of TorsinA and its disease-mutant complexed with an activator reveal the molecular basis for primary dystonia

    Energy Technology Data Exchange (ETDEWEB)

    Demircioglu, F. Esra; Sosa, Brian A.; Ingram, Jessica; Ploegh, Hidde L.; Schwartz, Thomas U.

    2016-08-04

    The most common cause of early onset primary dystonia, a neuromuscular disease, is a glutamate deletion (ΔE) at position 302/303 of TorsinA, a AAA+ ATPase that resides in the endoplasmic reticulum. While the function of TorsinA remains elusive, the ΔE mutation is known to diminish binding of two TorsinA ATPase activators: lamina-associated protein 1 (LAP1) and its paralog, luminal domain like LAP1 (LULL1). Using a nanobody as a crystallization chaperone, we obtained a 1.4 Å crystal structure of human TorsinA in complex with LULL1. This nanobody likewise stabilized the weakened TorsinAΔE-LULL1 interaction, which enabled us to solve its structure at 1.4 Å also. A comparison of these structures shows, in atomic detail, the subtle differences in activator interactions that separate the healthy from the diseased state. This information may provide a structural platform for drug development, as a small molecule that rescues TorsinAΔE could serve as a cure for primary dystonia.

  6. Role of surface energy and nano-roughness in the removal efficiency of bacterial contamination by nonwoven wipes from frequently touched surfaces

    Science.gov (United States)

    Edwards, Nicholas W. M.; Best, Emma L.; Connell, Simon D.; Goswami, Parikshit; Carr, Chris M.; Wilcox, Mark H.; Russell, Stephen J.

    2017-12-01

    Healthcare associated infections (HCAIs) are responsible for substantial patient morbidity, mortality and economic cost. Infection control strategies for reducing rates of transmission include the use of nonwoven wipes to remove pathogenic bacteria from frequently touched surfaces. Wiping is a dynamic process that involves physicochemical mechanisms to detach and transfer bacteria to fibre surfaces within the wipe. The purpose of this study was to determine the extent to which systematic changes in fibre surface energy and nano-roughness influence removal of bacteria from an abiotic polymer surface in dry wiping conditions, without liquid detergents or disinfectants. Nonwoven wipe substrates composed of two commonly used fibre types, lyocell (cellulosic) and polypropylene, with different surface energies and nano-roughnesses, were manufactured using pilot-scale nonwoven facilities to produce samples of comparable structure and dimensional properties. The surface energy and nano-roughness of some lyocell substrates were further adjusted by either oxygen (O2) or hexafluoroethane (C2F6) gas plasma treatment. Static adpression wiping of an inoculated surface under dry conditions produced removal efficiencies of between 9.4% and 15.7%, with no significant difference (p < 0.05) in the relative removal efficiencies of Escherichia coli, Staphylococcus aureus or Enterococcus faecalis. However, dynamic wiping markedly increased peak wiping efficiencies to over 50%, with a minimum increase in removal efficiency of 12.5% and a maximum increase in removal efficiency of 37.9% (all significant at p < 0.05) compared with static wiping, depending on fibre type and bacterium. In dry, dynamic wiping conditions, nonwoven wipe substrates with a surface energy closest to that of the contaminated surface produced the highest E. coli removal efficiency, while the associated increase in fibre nano-roughness abrogated this trend with S. aureus and E. faecalis.

  7. Surface Properties of TNOs: Preliminary Statistical Analysis

    Science.gov (United States)

    Antonieta Barucci, Maria; Fornasier, S.; Alvarez-Cantal, A.; de Bergh, C.; Merlin, F.; DeMeo, F.; Dumas, C.

    2009-09-01

    An overview of the surface properties based on the last results obtained during the Large Program performed at ESO-VLT (2007-2008) will be presented. Simultaneous high quality visible and near-infrared spectroscopy and photometry have been carried out on 40 objects with various dynamical properties, using FORS1 (V), ISAAC (J) and SINFONI (H+K bands) mounted respectively at UT2, UT1 and UT4 VLT-ESO telescopes (Cerro Paranal, Chile). For spectroscopy we computed the spectral slope for each object and searched for possible rotational inhomogeneities. A few objects show features in their visible spectra such as Eris, whose spectral bands are displaced with respect to pure methane-ice. We identify new faint absorption features on 10199 Chariklo and 42355 Typhon, possibly due to the presence of aqueous altered materials. The H+K band spectroscopy was performed with the new instrument SINFONI which is a 3D integral field spectrometer. While some objects show no diagnostic spectral bands, others reveal surface deposits of ices of H2O, CH3OH, CH4, and N2. To investigate the surface properties of these bodies, a radiative transfer model has been applied to interpret the entire 0.4-2.4 micron spectral region. The diversity of the spectra suggests that these objects represent a substantial range of bulk compositions. These different surface compositions can be diagnostic of original compositional diversity, interior source and/or different evolution with different physical processes affecting the surfaces. A statistical analysis is in progress to investigate the correlation of the TNOs’ surface properties with size and dynamical properties.

  8. Effect of Surface Hydration on Antifouling Properties of Mixed Charged Polymers.

    Science.gov (United States)

    Leng, Chuan; Huang, Hao; Zhang, Kexin; Hung, Hsiang-Chieh; Xu, Yao; Li, Yaoxin; Jiang, Shaoyi; Chen, Zhan

    2018-05-07

    Interfacial water structure on a polymer surface in water (or surface hydration) is related to the antifouling activity of the polymer. Zwitterionic polymer materials exhibit excellent antifouling activity due to their strong surface hydration. It was proposed to replace zwitterionic polymers using mixed charged polymers because it is much easier to prepare mixed charged polymer samples with much lower costs. In this study, using sum frequency generation (SFG) vibrational spectroscopy, we investigated interfacial water structures on mixed charged polymer surfaces in water, and how such structures change while exposing to salt solutions and protein solutions. The 1:1 mixed charged polymer exhibits excellent antifouling property while other mixed charged polymers with different ratios of the positive/negative charges do not. It was found that on the 1:1 mixed charged polymer surface, SFG water signal is dominated by the contribution of the strongly hydrogen bonded water molecules, indicating strong hydration of the polymer surface. The responses of the 1:1 mixed charged polymer surface to salt solutions are similar to those of zwitterionic polymers. Interestingly, exposure to high concentrations of salt solutions leads to stronger hydration of the 1:1 mixed charged polymer surface after replacing the salt solution with water. Protein molecules do not substantially perturb the interfacial water structure on the 1:1 mixed charged polymer surface and do not adsorb to the surface, showing that this mixed charged polymer is an excellent antifouling material.

  9. Evaluation of Wear on Macro-Surface Textures Generated by ns Fiber Laser

    Science.gov (United States)

    Harish, V.; Soundarapandian, S.; Vijayaraghavan, L.; Bharatish, A.

    2018-03-01

    The demand for improved performance and long term reliability of mechanical systems dictate the use of advanced materials and surface engineering techniques. A small change in the surface topography can lead to substantial improvements in the tribological behaviour of the contact surfaces. One way of altering the surface topography is by surface texturing by introducing dimples or channels on the surfaces. Surface texturing is already a successful technique which finds a wide area of applications ranging from heavy industries to small scale devices. This paper reports the effect of macro texture shapes generated using a nanosecond fiber laser on wear of high carbon chromium steel used in large size bearings having rolling contacts. Circular and square shaped dimples were generated on the surface to assess the effect of sliding velocities on friction coefficient. Graphite was used as solid lubricant to minimise the effect of wear on textured surfaces. The laser parameters such as power, scan speed and passes were optimised to obtain macro circular and square dimples which was characterised using a laser confocal microscope. The friction coefficients of the circular and square dimples were observed to lie in the same range due to minimum wear on the surface. On the contrary, at medium and higher sliding velocities, square dimples exhibited lower friction coefficient values compared to circular dimples. The morphology of textured specimen was characterised using Scanning Electron Microscope.

  10. Surface and bulk 4f-photoemission spectra of CeIn3 and CeSn3

    International Nuclear Information System (INIS)

    Kim, H.; Tjernberg, O.; Chiaia, G.; Kumigashira, H.; Takahashi, T.; Duo, L.; Sakai, O.; Kasaya, M.; Lindau, I.

    1997-01-01

    Resonant photoemission spectroscopy was performed on CeIn 3 and CeSn 3 at the 4d-4f and 3d-4f core thresholds. Using the different surface sensitivity between the two photon energies, surface and bulk 4f-photoemission spectra were derived for both compounds. With the noncrossing approximation of the Anderson impurity model, the 4d-4f resonant spectra together with the surface and bulk spectra were self-consistently analyzed to obtain the microscopic parameters such as the 4f-electron energy and the hybridization strength with conduction electrons. The result shows a substantial difference in these parameters between the surface and the bulk, indicating that it is important to take into account the surface effect in analyzing photoemission spectra of Ce compounds. It is also found that the 4f surface core-level shift is different between CeIn 3 and CeSn 3 . copyright 1997 The American Physical Society

  11. Strategies for creating antifouling surfaces using selfassembled poly(ethylene glycol) thiol molecules

    DEFF Research Database (Denmark)

    Lokanathan, Arcot R.

    2011-01-01

    of microbial species, but then the environment is also teeming with pathogenic microbes that pose serious threat to human health. Hence the success of human survival not only depends on exploiting the useful microbes but also on our ability to defend ourselves against the pathogenic ones. Microbes...... have substantial impact on human health, as many bacterial infections are caused by or involve biofilms. Biofilm infections are for example often associated with medical implants, as artificial surfaces in the human body provide a safe haven where biofilms can form. The food industry daily combats...... polymers for making non-adhesive coatings. The work presented in this thesis involves grafting PEG chains onto surfaces using different modifications of the ‘grafting to’ technique. The main aim of studies presented in this thesis was to develop surfaces which would prevent bacteria from forming biofilm...

  12. Surface deposition from radioactive plumes

    International Nuclear Information System (INIS)

    Garland, J.A.

    1980-01-01

    Accidents involving nuclear plants may release radioactive particles and gases to the atmosphere. Dry deposition of particles has been investigated mainly in the laboratory and a general understanding of the transfer mechanisms has been established. However there is apparently a substantial discrepancy between the few field observations of dry deposition of particles and laboratory measurements, particularly for 0.1 - 1 μm particles for which laboratory work shows very small deposition rates. In addition there are few estimates of deposition rates for forest and some other kinds of terrain. The most important gas in the context of a nuclear accident is I-131 and the behaviour of this gas at grass surfaces has received much attention. However smaller quantities of other gases and vapours may be released and the surface absorption of these species may require further investigation. In addition there is little knowledge of the behaviour of gases over many types of surface. The rate of deposition of particles and gases is influenced by many parameters including wind speed and the temperature stratification of the lower atmosphere. Conditions which give poor atmospheric dispersion usually give lower deposition velocities. Transfer to man depends on the availability of deposited materials on crops and grass. A wide range of isotopes including iodine and several metallic fission products are lost with a half life for residence on grass ranging from a few days to a few tens days, depending on climatic conditions

  13. An unknown oxidative metabolism substantially contributes to soil CO2 emissions

    Directory of Open Access Journals (Sweden)

    T. Shahzad

    2013-02-01

    Full Text Available The respiratory release of CO2 from soils is a major determinant of the global carbon cycle. It is traditionally considered that this respiration is an intracellular metabolism consisting of complex biochemical reactions carried out by numerous enzymes and co-factors. Here we show that the endoenzymes released from dead organisms are stabilised in soils and have access to suitable substrates and co-factors to permit function. These enzymes reconstitute an extracellular oxidative metabolism (EXOMET that may substantially contribute to soil respiration (16 to 48% of CO2 released from soils in the present study. EXOMET and respiration from living organisms should be considered separately when studying effects of environmental factors on the C cycle because EXOMET shows specific properties such as resistance to high temperature and toxic compounds.

  14. Thermal behavior of horizontally mixed surfaces on Mars

    Science.gov (United States)

    Putzig, Nathaniel E.; Mellon, Michael T.

    2007-11-01

    Current methods for deriving thermal inertia from spacecraft observations of planetary brightness temperature generally assume that surface properties are uniform for any given observation or co-located set of observations. As a result of this assumption and the nonlinear relationship between temperature and thermal inertia, sub-pixel horizontal heterogeneity may yield different apparent thermal inertia at different times of day or seasons. We examine the effects of horizontal heterogeneity on Mars by modeling the thermal behavior of various idealized mixed surfaces containing differing proportions of either dust, sand, duricrust, and rock or slope facets at different angles and azimuths. Latitudinal effects on mixed-surface thermal behavior are also investigated. We find large (several 100 J m -2 K -1 s -1/2) diurnal and seasonal variations in apparent thermal inertia even for small (˜10%) admixtures of materials with moderately contrasting thermal properties or slope angles. Together with similar results for layered surfaces [Mellon, M.T., Putzig, N.E., 2007. Lunar Planet. Sci. XXXVIII. Abstract 2184], this work shows that the effects of heterogeneity on the thermal behavior of the martian surface are substantial and may be expected to result in large variations in apparent thermal inertia as derived from spacecraft instruments. While our results caution against the over-interpretation of thermal inertia taken from median or average maps or derived from single temperature measurements, they also suggest the possibility of using a suite of apparent thermal inertia values derived from single observations over a range of times of day and seasons to constrain the heterogeneity of the martian surface.

  15. Semiclassical multi-phonon theory for atom-surface scattering: Application to the Cu(111) system.

    Science.gov (United States)

    Daon, Shauli; Pollak, Eli

    2015-05-07

    The semiclassical perturbation theory of Hubbard and Miller [J. Chem. Phys. 80, 5827 (1984)] is further developed to include the full multi-phonon transitions in atom-surface scattering. A practically applicable expression is developed for the angular scattering distribution by utilising a discretized bath of oscillators, instead of the continuum limit. At sufficiently low surface temperature good agreement is found between the present multi-phonon theory and the previous one-, and two-phonon theory derived in the continuum limit in our previous study [Daon, Pollak, and Miret-Artés, J. Chem. Phys. 137, 201103 (2012)]. The theory is applied to the measured angular distributions of Ne, Ar, and Kr scattered from a Cu(111) surface. We find that the present multi-phonon theory substantially improves the agreement between experiment and theory, especially at the higher surface temperatures. This provides evidence for the importance of multi-phonon transitions in determining the angular distribution as the surface temperature is increased.

  16. The Effect of Water Repellent Surface Impregnation on Durability of Cement-Based Materials

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2017-01-01

    Full Text Available In many cases, service life of reinforced concrete structures is severely limited by chloride penetration until the steel reinforcement or by carbonation of the covercrete. Water repellent treatment on the surfaces of cement-based materials has often been considered to protect concrete from these deteriorations. In this paper, three types of water repellent agents have been applied on the surface of concrete specimens. Penetration profiles of silicon resin in treated concrete have been determined by FT-IR spectroscopy. Water capillary suction, chloride penetration, carbonation, and reinforcement corrosion in both surface impregnated and untreated specimens have been measured. Results indicate that surface impregnation reduced the coefficient of capillary suction of concrete substantially. An efficient chloride barrier can be established by deep impregnation. Water repellent surface impregnation by silanes also can make the process of carbonation action slow. In addition, it also has been concluded that surface impregnation can provide effective corrosion protection to reinforcing steel in concrete with migrating chloride. The improvement of durability and extension of service life for reinforced concrete structures, therefore, can be expected through the applications of appropriate water repellent surface impregnation.

  17. Design Options to Reduce Development Cost of First Generation Surface Reactors

    International Nuclear Information System (INIS)

    Poston, David I.; Marcille, Thomas F.

    2006-01-01

    Low-power surface reactors have the potential to have the lowest development cost of any space reactor application, primarily because system alpha (mass/kg) is not of utmost importance and mission lifetimes do not have to be a decade or more. Even then, the development cost of a surface reactor can vary substantially depending on the performance requirements (e.g. mass, power, lifetime, reliability) and technical development risk deemed acceptable by the end-user. It is important for potential users to be aware of these relationships before they determine their future architecture (i.e. decide what they need). Generally, the greatest potential costs of a space reactor program are a nuclear-powered ground test and extensive material development campaigns, so it is important to consider options that can minimize the need for or complexity of such tasks. The intended goal of this paper is to inform potential surface reactor users of the potential sensitivities of surface reactor development cost to design requirements, and areas where technical risk can be traded with development cost

  18. Ni3Si surface-film formation caused by radiation-induced segregation

    International Nuclear Information System (INIS)

    Potter, D.I.; Rehn, L.E.; Okamoto, P.R.; Wiedersich, H.

    1977-01-01

    Several advanced alloys being considered for reactor applications contain the ordered γ' phase Ni 3 X in which the X component is frequently Al, Si and/or Ti. These alloys are precipitation hardened, and their strength depends upon the volume fraction, size, and spatial distribution of the coherent γ' precipitate. The investigation shows that a substantial Ni 3 Si precipitate film forms on the surface of irradiated specimens of solid-solution as well as two-phase Ni-Si alloys

  19. Thermal radiation characteristics and direct evidence of tungsten cooling on the way to nanostructure formation on its surface

    Energy Technology Data Exchange (ETDEWEB)

    Takamura, S., E-mail: takamura@aitech.ac.jp [Faculty of Engineering, Aichi Institute of Technology, Yakusa-cho, Toyota 470-0392 (Japan); Miyamoto, T. [Faculty of Engineering, Aichi Institute of Technology, Yakusa-cho, Toyota 470-0392 (Japan); Ohno, N. [Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan)

    2013-07-15

    The physical properties of tungsten with nanostructure on its surface are investigated focusing on the thermal radiation and cooling characteristics. First, direct evidence of substantial W surface cooling has been clearly shown with use of a very thin thermocouple inserted into W target, which solves an uncertainty associated with a radiation thermometer. Second, the above measurements of W surface temperature make it possible to estimate quantitatively the total emissivity from which we may evaluate the radiative power through the Stefan–Boltzmann equation, which is very important for mitigation evaluation of a serious plasma heat load to the plasma-facing component.

  20. Thermal radiation characteristics and direct evidence of tungsten cooling on the way to nanostructure formation on its surface

    International Nuclear Information System (INIS)

    Takamura, S.; Miyamoto, T.; Ohno, N.

    2013-01-01

    The physical properties of tungsten with nanostructure on its surface are investigated focusing on the thermal radiation and cooling characteristics. First, direct evidence of substantial W surface cooling has been clearly shown with use of a very thin thermocouple inserted into W target, which solves an uncertainty associated with a radiation thermometer. Second, the above measurements of W surface temperature make it possible to estimate quantitatively the total emissivity from which we may evaluate the radiative power through the Stefan–Boltzmann equation, which is very important for mitigation evaluation of a serious plasma heat load to the plasma-facing component