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Sample records for fab display selection

  1. Generation and selection of immunized Fab phage display library against human B cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    Yongmei Shen; Xiaochun Yang; Ningzheng Dong; Xiaofang Xie; Xia Bai; Yizhen Shi

    2007-01-01

    The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the K light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×107. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (VH) and variable light domains (VL) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

  2. Display and selection of chicken IgA Fab fragments

    NARCIS (Netherlands)

    Wieland, W.H.; Orzaéz, D.; Lammers, A.; Parmentier, H.K.; Schots, A.

    2006-01-01

    Passive immune therapy is regaining interest to prevent and cure infectious diseases both in human and veterinary medicine. Therefore, systems are required that enable efficient targeted selection of antibodies originating from virtually any animal species. Here, a system for the selection of chicke

  3. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-Ping Wu; Bing Xiao; Tian-Mo Wan; Ya-Li Zhang; Zhen-Shu Zhang; Dian-Yuan Zhou; Zhuo-Sheng Lai; Chun-Fang Gao

    2001-01-01

    AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

  4. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  5. An improved single-chain Fab platform for efficient display and recombinant expression.

    Science.gov (United States)

    Koerber, James T; Hornsby, Michael J; Wells, James A

    2015-01-30

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

  6. Yeast mating for combinatorial Fab library generation and surface display

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Jane M.; Lou, Jianlong; Coleman, James R.; Siegel, Robert W.; Marks, James D.; Feldhaus, Michael

    2004-04-23

    Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv (scFv) antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.

  7. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  8. In vitro Fab display: a cell-free system for IgG discovery.

    Science.gov (United States)

    Stafford, Ryan L; Matsumoto, Marissa L; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R; Baliga, Ramesh; Murray, Christopher J; Thanos, Christopher D; Hallam, Trevor J; Sato, Aaron K

    2014-04-01

    Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

  9. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  10. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments.

    Science.gov (United States)

    Blaise, Lydia; Wehnert, Anita; Steukers, Mieke P G; van den Beucken, Twan; Hoogenboom, Hennie R; Hufton, Simon E

    2004-11-24

    Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288-294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.

  11. Construction and selection of human Fab antibody phage display library of extracellular domain of HER 2%人源性抗HER2胞外段Fab噬菌体抗体库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    张为家; 刘孝荣; 李官成; 贺智敏

    2011-01-01

    目的:构建全人源抗HER2胞外段(HER7. ECD)噬菌体Fab抗体库,从中筛选出特异性的抗体,并对其进行鉴定.方法:体外致敏并用EB病毒(EBV)转化HER2高表达乳腺癌患者的外周血单核细胞(PBMC),用PCR分别扩增重链Fd和轻链k/λ基因.经Sac I/Xba I和Xho/SpeI双酶切,依次克隆入噬菌体载体pComb3中,并电转化大肠杆菌XL1Blue,以辅助噬菌体VCSM13进行超感染,构建抗HER2 ECD人源化Fab噬菌体抗体库.以纯化HER2 ECD蛋白为抗原进行3轮固相淘选,富集抗HER2 ECD的抗体,并随机挑选克隆进行ELISA,获得的阳性克隆进一步以Western blot鉴定其抗原结合活性,对其中活性最高的克隆进行DNA测序.结果:构建了容量为2. 5 X 107的抗HER2 ECD的Fab噬菌体抗体库,并筛选获得了4株与HE2 ECD特异性结合的阳性克隆,Western blot分析显示其与HER?-ECD能较好的结合.选取结合活性最高的阳性克隆进行DNA序列分析,结果显示其重链可变区、轻链可变区分别与人胚系免疫球蛋白基因有高度的同源性.结论:成功构建了全人源抗HER2 ECD噬菌体抗体库,并筛选出抗HER2 ECD特异性较强的噬菌体克隆,为获得新的有临床应用价值的HER2 ECD抗体提供了实验基础.%AIM: To construct the fully humanized antiextracellular domain (ECD) of HER2.Fab fragment phage library, select antibodies against HER2 ECD specifically and identify its characteristics.METHODS: Peripheral blood monouclear cells (PBMCs) of breast cancer patients with HER2-overexpressing were immunized in vitro with purification protein of recombinant HEF2 ECD and were then transformed by Epstein-Barr virus (EBV).After total RNA was extracted, the heavy chain Fd and k/λ light chain were am plified by RT-PCR.Following restrictive digestion with Sac I/ Xba I and Xho I/Spe I, the light chain κ/λ genes and heavy chain genes Fd were inserted into the phagemid vector pComb3 successively and then electroporated into E.coil XL1-Blue

  12. A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties.

    Directory of Open Access Journals (Sweden)

    Felix Unverdorben

    Full Text Available Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD from streptococcal protein G (SpGC3 as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions.Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv and bispecific single-chain diabody (scDb. Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics.The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

  13. Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

    Science.gov (United States)

    Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

    2015-11-01

    In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

  14. Purificação e caracterização do fragmento Fab anti-digoxina obtido pela técnica de phage display.

    OpenAIRE

    2016-01-01

    A digoxina é um dos medicamentos indicados para o tratamento de falência cardíaca. Possui janela terapêutica estreita, sendo responsável por casos de intoxicação. O único antídoto disponível para a desintoxicação é o anticorpo policlonal DigiFab®, no formato Fab. O seu uso é eficaz, porém de custo elevado. Clones bacterianos produtores de fragmento Fab monoclonal anti-digoxina foram obtidos previamente pelo nosso grupo, pela técnica de phage display. Neste trabalho as variantes Fab dos 4 clo...

  15. A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain

    DEFF Research Database (Denmark)

    Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik

    2016-01-01

    molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel...

  16. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    Science.gov (United States)

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  17. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Syed A Ali

    Full Text Available Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

  18. Methods for Selecting Phage Display Antibody Libraries.

    Science.gov (United States)

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  19. Single chain Fab (scFab fragment

    Directory of Open Access Journals (Sweden)

    Brenneis Mariam

    2007-03-01

    Full Text Available Abstract Background The connection of the variable part of the heavy chain (VH and and the variable part of the light chain (VL by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd and the light chain (LC, resulting in the formation of a single chain Fab fragment (scFab, can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC connecting the constant part 1 of the heavy chain (CH1 and the constant part of the light chain (CL were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of

  20. Establishment of bacteria display technology for Fab antibody library screening%筛选Fab抗体库的细菌展示技术的建立

    Institute of Scientific and Technical Information of China (English)

    徐黎明; 尹成凯; 任桂萍; 田辉; 王雪苯; 丁良君; 李德山

    2011-01-01

    目的:利用NlpA蛋白的前六个氨基酸(CDQSSS)将抗体锚定在细菌内膜建立筛选Fabs抗体库的展示技术,为今后抗体的研发工作奠定基础.方法:从pNAD质粒中克隆出NlpA leader(含有CDQSSS序列)基因序列,利用相应的酶切位点将该序列插入pComb3表达载体中构建成用于展示Fab的重组质粒pBFD.将从pEAI质粒克隆得到的anti-human IL-1β抗体的重链Fab和全长轻链分别插入到NlpA leader和pelB leader( pComb3载体自带的果胶酶基因前导肽)的下游.将pBFD-Fab转入到E.coli DH5α中诱导表达,原生质球制备后,采用梯度浓度的抗原进行孵育,最后经流式细胞术(FCM)检测抗体展示情况并且分选阳性群体,利用质粒提取的方法来替代PCR方法拯救阳性基因,转化E.coli DH5α,利用FCM再次检测该群体展示的抗体与抗原结合情况.结果:所展示的anti-hlL-1β Fab抗体依次与抗原和FITC标记的抗原特异性抗体孵育后,用FCM实时检测,结果显示出很强的荧光信号并且表现出抗原浓度依赖性.拯救出的pBFD-Fab-原生质球的FCM检测结果与首次展示的FCM结果一致,该系统能够稳定的展示抗体.结论:经过该细菌展示系统展示的Fab抗体能够有效的折叠,与相应的抗原具有很好的特异性结合能力.成功改进该展示技术的基因拯救方法,避免了基因突变和链置换的发生.此外还证明了该展示技术具有很好的稳定性.本实验成功构建了筛选Fab抗体库的细菌展技术.%AIM:To establish bacterial display technology for the purpose of Fab antibody library screening, by u-sing six amino acids (CDQSSS) of the amino termimus of NlpA protein to anchore antibodies to the periplasmic side of the bacterial inner membrane. METHODS: The NlpA Leader sequences (encoding CDQSSS) was amplified from pNAD plasmid. The PCR product was subcloned into pComb3 expression vector to generate Fab display vector pBFD. The heavy chains of the Fab gene

  1. Produção e caracterização da porção Fab do anticorpo anti-digoxina utilizando a tecnologia de phage display.

    OpenAIRE

    Viviane Midori Murata

    2012-01-01

    A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxi...

  2. 人源Fab抗体库的构建和抗hPRLR抗体的筛选鉴定%Screening and identification of human Fab antibody against hPRLR from large phage-display library originated from breast cancer patients

    Institute of Scientific and Technical Information of China (English)

    屈芫; 魏钦俊; 姚俊; 鲁雅洁; 王天明; 曹新; 冯振卿

    2012-01-01

    cWe aim to get specific Fab antibody against human prolactin receptor (hPRLR) from the human Fab antibody library constructed by phage display technology. Human lymphocytes were collected from peripheral blood of 24 breast cancer patients. And then the total RNA was extracted and reversely transcribed to cDNA. Genes of light and heavy chains of human antibody were amplified by RT-PCR to construct anti-hPRLR immunized human antibody library. After three rounds of panning and one round of crossed-panning with against his-hPRLR fusion protein, BSA-polypeptide (epitopes of hPRLR) and GST-hPRLR fusion protein, positive clones were chosen by Phage-ELISA and DNA sequencing. Then the positive clones were transformed into E. coli Top 10' and induced to express antibody protein. The results indicated that the human Fab phage-display library consisting of l.0×l09 clones were successfully constructed, and six clones were selected after four rounds. One of them named FabG2 expressed protein correctly. ELISA and Western blot analysis showed that FabG2 could bind hPRLR specifically. We concluded that the hPRLR specific Fab antibody selected from large phage-display library could be used as candidates for therapy agent of breast cancer which over-expresses hPRLR.%目的 构建人源Fab噬菌体抗体库,筛选抗hPRLR抗体片段并进行初步鉴定.方法 从乳腺癌患者外周血提取总RNA,通过RT-PCR扩增人抗体轻链和重链基因,构建抗hPRLR人源Fab抗体库.分别以His-hPRLR融合蛋白、BSA-hPRLR表位多肽融合蛋白和GST-hPRLR融合蛋白作为抗原包板,经过3轮循环的吸附一洗脱一扩增的筛选及1轮交叉筛选,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,将筛选到的阳性克隆Fabc2转化至Top10’受体菌,诱导表达可溶性Fab抗体,通过Western blot和ELISA进行特异性的鉴定.结果 构建的人源Fab库容为1.0×109,4轮的筛选,获得6株能与hPRLR结合的人源抗体克隆.选取的Fabc2能够进行

  3. Increased Fab thermoresistance via VH-targeted directed evolution.

    Science.gov (United States)

    Entzminger, Kevin C; Johnson, Jennifer L; Hyun, Jeongmin; Lieberman, Raquel L; Maynard, Jennifer A

    2015-10-01

    Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2-3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts.

  4. Response terminated displays unload selective attention

    Directory of Open Access Journals (Sweden)

    Zachary Joseph Jackson Roper

    2013-12-01

    Full Text Available Perceptual load theory successfully replaced the early versus late selection debate by appealing to adaptive control over the efficiency of selective attention. Early selection is observed unless perceptual load (p-Load is sufficiently low to grant attentional ‘spill-over‘ to task-irrelevant stimuli. Many studies exploring load theory have used limited display durations that perhaps impose artificial limits on encoding processes. We extended the exposure duration in a classic p-Load task to alleviate temporal encoding demands that may otherwise tax mnemonic consolidation processes. If the load effect arises from perceptual demands alone, then freeing-up available mnemonic resources by extending the exposure duration should have little effect. The results of Experiment 1 falsify this prediction. We observed a reliable flanker effect under high p-Load, response-terminated displays. Next, we orthogonally manipulated exposure duration and task-relevance. Counter-intuitively, we found that the likelihood of observing the flanker effect under high p-Load resides with the duration of the task-relevant array, not the flanker itself. We propose that stimulus and encoding demands interact to produce the load effect. Our account clarifies how task parameters differentially impinge upon cognitive processes to produce attentional ‘spill-over’ by appealing to visual short-term memory as an additional processing bottleneck when stimuli are briefly presented.

  5. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3.

    Science.gov (United States)

    Rauchenberger, Robert; Borges, Eric; Thomassen-Wolf, Elisabeth; Rom, Eran; Adar, Rivka; Yaniv, Yael; Malka, Michael; Chumakov, Irina; Kotzer, Sarit; Resnitzky, Dalia; Knappik, Achim; Reiffert, Silke; Prassler, Josef; Jury, Karin; Waldherr, Dirk; Bauer, Susanne; Kretzschmar, Titus; Yayon, Avner; Rothe, Christine

    2003-10-03

    The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A., and Virnekäs, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.

  6. FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    OpenAIRE

    Ali, Syed A.; Yik Wei Chew

    2015-01-01

    Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditio...

  7. GTP-specific fab fragment-based GTPase activity assay.

    Science.gov (United States)

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri

    2015-03-17

    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  8. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  9. Beyond Fab Four

    Science.gov (United States)

    Babichev, E.; Charmousis, C.; Langlois, D.; Saito, R.

    2015-12-01

    We show that the two additional Lagrangians that appear in theories beyond Horndeski can be reexpressed in terms of simple generalizations of the 'John' and 'Paul' terms of the Fab Four theories. We find that these extended Fab Four satisfy the same properties of self-tuning as the original Fab Four.

  10. Beyond Fab Four

    CERN Document Server

    Babichev, E; Langlois, D; Saito, R

    2015-01-01

    We show that the two additional Lagrangians that appear in theories beyond Horndeski can be reexpressed in terms of simple generalizations of the "John" and "Paul" terms of the Fab Four theories. We find that these extended Fab Four satisfy the same properties of self-tuning as the original Fab Four.

  11. 用噬菌体蛋白Ⅸ展示抗HBsAg分子Fab段%Generation of Fab-phage antibody using pⅨ phage display

    Institute of Scientific and Technical Information of China (English)

    张达矜; 王琰; 化冰; 刘晓琳

    2004-01-01

    目的用丝状噬菌体外壳蛋白Ⅸ(pⅨ)展示抗体Fab段,并与外壳蛋白Ⅲ(pⅢ)展示效果进行比较. 方法采用PCR方法钓取噬菌体蛋白Ⅸ基因,构建pⅨ展示抗乙肝表面抗原Fab的表达载体,制备噬菌体抗体,鉴定其抗原结合活性和Fab呈现水平,观察了噬菌体洗脱回收效率,并与pⅢ展示进行比较. 结果噬菌体pⅨ可以展示有功能活性的抗体Fab段,但对Fab的展示率低于pⅢ,展示率的高低可能与pⅨ展示的外源蛋白分子量大小有关."结合-洗脱-回收"实验显示,酸洗脱法回收的Fab-pⅢ噬菌体的感染活性大为下降,而对Fab-pⅨ噬菌体无影响. 结论 pⅨ成功展示Fab段,讨论了pⅨ展示体系可能具有的优越性.

  12. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  13. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  14. 抗呼吸道合胞病毒Fab噬菌体抗体库的构建及初步筛选%Construction and preliminary panning of Fab phage display antibody library against respiratory syncytial virus

    Institute of Scientific and Technical Information of China (English)

    汪治华; 张国成; 李安茂; 周南; 陈一; 李小青; 苏字飞; 邓阳彬; 王治静

    2008-01-01

    Objective To construct a human phage display antibody library,which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic.This can provide a platform for human antibody preparation and diagnosis,prophylaxis and therapy of RSV infection in children.Methods Peripheral blood lymphocytes were isolated from 52 children with RSV infection,cDNA was synthesized from the total RNA of lymphocytes.The light and heavy chain Fd (VH-CH1)fragments of immunoglobulin gene were amplified by RT-PCR.The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue.The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs.The plasmids extracted from amplified E.coil were digested with restriction endonucleases Sac 1,Xba I,Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes.RSV virions were utilized as antigens to screen Fab antibodies.Results By recombination of light and heavy chain genes,an immune Fab phage display antibody library against RSV containing 2.08×107 different clones was constructed,in which 70% clones had light chains and heavy chain Fd genes.The capacity of Fab phage antibody gene library was 1.46×107 and the titre of the original Fab antibody library was about 1.06 x 1012 pfu/mL The antibody library gained an enrichment in different degrees after the preliminary panning.Condusions Utilizing the technology of phage display,an immune Fab phage display antibody library against RSV was successfully constructed in this study,which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis,therapy and prophylaxis of RSV infection in children

  15. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

    Science.gov (United States)

    Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

    2015-05-15

    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.

  16. SELECTION OF NEW EPITOPES FROM MONOVALENT DISPLAYED PHAGE OCTAPEPTIDE LIBRARY

    Institute of Scientific and Technical Information of China (English)

    李全喜; 王琰; 李竞; 王雅明; 徐建军; 王力民; 董志伟

    1998-01-01

    A library of 2×l07 random oetspaptides was constructed by use of phegemid-based monovaient phage display system. The randomly synthesized degenerated oilgodeoxyribonucleotides (oligos) were fused to the truncated gⅢ (p210-p408). Sequeraze analysis of 11 randomly chosen clones suggested that the degenerated inserts and its deduced amino acid (an) sequences are randomly distributed. The library was used to select binding paptides to the morroeloncl antlhody (mAb) 9E10, which recognizes a continuous decapaptide epitope of denatured human c-myc protein. After four to five rounds of panning, most of the eluted clones could bind to 9E10. Sequerlce analysis of the selected positive clones indlcated that the binding sequences could fall into two chsses, one class (clone 1) shares a consensus motif, ISE x x L, with c-mire decapeprider and the sequences of the other class are entirely different. The binding of both classes to 9E10 could be specifically lnhlhited by froe c-myc deeapeptide. The immunogenlcitF cff the phage peptide was further investigsted h5, construction of multivalent displayed phage peptides and immunization of animals with or without adjuvant. ELISA and competitive ELISA showed that anti-serum from both mice and rabbit immunized with either done could bind to the original antigen, c-myc decapeptide. These results denote that in spite of the dissimilarity of the selected psptides with c-myc decapeptide, they are capable of inducing similar immune respones in vivo, thus actually mimicking the antigen epitope.

  17. Content dependent selection of image enhancement parameters for mobile displays

    Science.gov (United States)

    Lee, Yoon-Gyoo; Kang, Yoo-Jin; Kim, Han-Eol; Kim, Ka-Hee; Kim, Choon-Woo

    2011-01-01

    Mobile devices such as cellular phones and portable multimedia player with capability of playing terrestrial digital multimedia broadcasting (T-DMB) contents have been introduced into consumer market. In this paper, content dependent image quality enhancement method for sharpness and colorfulness and noise reduction is presented to improve perceived image quality on mobile displays. Human visual experiments are performed to analyze viewers' preference. Relationship between the objective measures and the optimal values of image control parameters are modeled by simple lookup tables based on the results of human visual experiments. Content dependent values of image control parameters are determined based on the calculated measures and predetermined lookup tables. Experimental results indicate that dynamic selection of image control parameters yields better image quality.

  18. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

    Directory of Open Access Journals (Sweden)

    Antti Tullila

    2017-05-01

    Full Text Available Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab library.

  19. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

    Science.gov (United States)

    Tullila, Antti; Nevanen, Tarja K.

    2017-01-01

    Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

  20. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  1. Novel Schiff-base-derived FabH inhibitors with dioxygenated rings as antibiotic agents.

    Science.gov (United States)

    Zhou, Yang; Du, Qian-Ru; Sun, Jian; Li, Jing-Ran; Fang, Fei; Li, Dong-Dong; Qian, Yong; Gong, Hai-Bin; Zhao, Jing; Zhu, Hai-Liang

    2013-03-01

    Fatty acid biosynthesis plays a vital role in bacterial survival and several key enzymes involved in this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. Of these promising targets, β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is the most attractive target that could trigger the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Designing small molecules with FabH inhibitory activity displays great significance for developing antibiotic agents, which should be highly selective, nontoxic and broad-spectrum. In this manuscript, a series of novel Schiff base compounds were designed and synthesized, and their biological activities were evaluated as potential inhibitors. Among these 21 new compounds, (E)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)hexadecan-1-amine (10) showed the most potent antibacterial activity with a MIC value of 3.89-7.81 μM(-1) against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with an IC(50) value of 1.6 μM. Docking simulation was performed to position compound 10 into the E. coli FabH active site to determine the probable binding conformation.

  2. FabIO

    DEFF Research Database (Denmark)

    Bergbäck Knudsen, Erik; Sørensen, Henning O.; Wright, Jonathan P.

    2013-01-01

    FabIO is a Python module written for easy and transparent reading of raw two-dimensional data from various X-ray detectors. The module provides a function for reading any image and returning a fabioimage object which contains both metadata (header information) and the raw data. All fabioimage...

  3. Fab Four Neutron Stars

    CERN Document Server

    Maselli, Andrea; Minamitsuji, Masato; Berti, Emanuele

    2016-01-01

    Horndeski's theory of gravity is the most general scalar-tensor theory with a single scalar whose equations of motion contain at most second-order derivatives. A subsector of Horndeski's theory known as "Fab Four" gravity allows for dynamical self-tuning of the quantum vacuum energy, and therefore it has received particular attention in cosmology as a possible alternative to the $\\Lambda$CDM model. Here we study compact stars in Fab Four gravity, which includes as special cases general relativity ("George"), Einstein-dilaton-Gauss-Bonnet gravity ("Ringo"), theories with a nonminimal coupling with the Einstein tensor ("John") and theories involving the double-dual of the Riemann tensor ("Paul"). We generalize and extend previous results in theories of the John class and we show that there are no viable compact star solutions in theories of the Paul class.

  4. Phage display selects for amylases with improved low pH starch-binding

    NARCIS (Netherlands)

    Verhaert, RMD; Beekwilder, J; Olsthoorn, R; Quax, WJ; Duin, Jan van

    2002-01-01

    Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display w

  5. Phage display selects for amylases with improved low pH starch-binding

    NARCIS (Netherlands)

    Verhaert, RMD; Beekwilder, J; Olsthoorn, R; Quax, WJ; Duin, Jan van

    2002-01-01

    Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display

  6. Autostereoscopic display concept with time-sequential wavelength-selective filter-barrier

    Science.gov (United States)

    Jurk, Silvio; Kuhlmey, Mathias; Bartmann, Roland; Duckstein, Bernd; de la Barré, René

    2016-03-01

    A spatially multiplexed autostereoscopic 3D display design with a strip barrier consisting of RGB-color filters is presented. The wavelength selective filter barrier emits the light from a display area larger than that of common autostereoscopic barrier displays. However, such construction is still used rather rarely. The time sequential operation mode is a supplemental option. Wavelength selective filter barrier arrangements exhibit characteristics different from common barrier displays with similar barrier pitch and ascent. In particular these constructions show strong angular luminance dependency under barrier inclination specified by correspondent slant angle. In time sequential implementation it is important to avoid that quick eye or eyelid movement lead to visible color artifacts. Those circumstances limit the possibility to find well working and usable display designs superior to usual barrier displays. The newly introduced design is usable as a multi user display as well as a single user system with user adaptive control. In case of tracked single user mode the adaption in x-z-direction is continuously. The design has been modelled with simulation software developed for that purpose. The modelling of wavelength-selective barriers was used to calculate the light ray distribution properties of that arrangement. For the experimental verification of the image separation and evaluation of image quality, commercially available display components were combined for a display demonstrator.

  7. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties.

    Science.gov (United States)

    Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

    2013-01-01

    This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.

  8. Time-Sequential Working Wavelength-Selective Filter for Flat Autostereoscopic Displays

    Directory of Open Access Journals (Sweden)

    René de la Barré

    2017-02-01

    Full Text Available A time-sequential working, spatially-multiplexed autostereoscopic 3D display design consisting of a fast switchable RGB-color filter array and a fast color display is presented. The newly-introduced 3D display design is usable as a multi-user display, as well as a single-user system. The wavelength-selective filter barrier emits the light from a larger aperture than common autostereoscopic barrier displays with similar barrier pitch and ascent. Measurements on a demonstrator with commercial display components, simulations and computational evaluations have been carried out to describe the proposed wavelength-selective display design in static states and to show the weak spots of display filters in commercial displays. An optical modelling of wavelength-selective barriers has been used for instance to calculate the light ray distribution properties of that arrangement. In the time-sequential implementation, it is important to avoid that quick eye or eyelid movement leads to visible color artifacts. Therefore, color filter cells, switching faster than conventional LC display cells, must distribute directed light from different primaries at the same time, to create a 3D presentation. For that, electric tunable liquid crystal Fabry–Pérot color filters are presented. They switch on-off the colors red, green and blue in the millisecond regime. Their active areas consist of a sub-micrometer-thick nematic layer sandwiched between dielectric mirrors and indium tin oxide (ITO-electrodes. These cells shall switch narrowband light of red, green or blue. A barrier filter array for a high resolution, glasses-free 3D display has to be equipped with several thousand switchable filter elements having different color apertures.

  9. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  10. Meleagrin, a new FabI inhibitor from Penicillium chryosogenum with at least one additional mode of action.

    Directory of Open Access Journals (Sweden)

    Chang Ji Zheng

    Full Text Available Bacterial enoyl-acyl carrier protein reductase (FabI is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria.

  11. Recombinant human antibody fragment against tetanus toxoid produced by phage display

    Science.gov (United States)

    Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

    2014-01-01

    Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

  12. Selective display of multiple patterns encoded with different oriented ripples using femtosecond laser

    Science.gov (United States)

    Li, Jiawen; Li, Guoqiang; Hu, Yanlei; Zhang, Chenchu; Li, Xiaohong; Chu, Jiaru; Huang, Wenhao

    2015-08-01

    Uniform oriented ripples were induced on stainless steel surface by polarized femtosecond laser. Multiple patterns were successfully encoded with the different oriented ripples on the surface without spatial overlap. Each pattern can be selectively displayed with structural color while the others cannot be in the meantime when white light is irradiated on the surface from different directions. The influence of ripples with different periods for selecting pattern is also investigated. The ripple period is crucial to the color effects. This approach can be used in the fields of color display, identifying codes and anti-counterfeiting patterns.

  13. Triclosan can select for an AdeIJK-overexpressing mutant of Acinetobacter baumannii ATCC 17978 that displays reduced susceptibility to multiple antibiotics.

    Science.gov (United States)

    Fernando, Dinesh M; Xu, Wayne; Loewen, Peter C; Zhanel, George G; Kumar, Ayush

    2014-11-01

    In order to determine if triclosan can select for mutants of Acinetobacter baumannii ATCC 17978 that display reduced susceptibilities to antibiotics, we isolated a triclosan-resistant mutant, A. baumannii AB042, by serial passaging of A. baumannii ATCC 17978 in growth medium supplemented with triclosan. The antimicrobial susceptibility of AB042 was analyzed by the 2-fold serial dilution method. Expression of five different resistance-nodulation-division (RND) pump-encoding genes (adeB, adeG, adeJ, A1S_2818, and A1S_3217), two outer membrane porin-encoding genes (carO and oprD), and the MATE family pump-encoding gene abeM was analyzed using quantitative reverse transcriptase (qRT) PCR. A. baumannii AB042 exhibited elevated resistance to multiple antibiotics, including piperacillin-tazobactam, doxycycline, moxifloxacin, ceftriaxone, cefepime, meropenem, doripenem, ertapenem, ciprofloxacin, aztreonam, tigecycline, and trimethoprim-sulfamethoxazole, in addition to triclosan. Genome sequencing of A. baumannii AB042 revealed a (116)G→V mutation in fabI, the gene encoding the target enzyme for triclosan. Expression analysis of efflux pumps showed overexpression of the AdeIJK pump, and sequencing of adeN, the gene that encodes the repressor of the adeIJK operon, revealed a 73-bp deletion which would cause a premature termination of translation, resulting in an inactive truncated AdeN protein. This work shows that triclosan can select for mutants of A. baumannii that display reduced susceptibilities to multiple antibiotics from chemically distinct classes in addition to triclosan resistance. This multidrug resistance can be explained by the overexpression of the AdeIJK efflux pump.

  14. Selection of a breast cancer subpopulation-specific antibody using phage display on tissue sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla J

    2015-01-01

    of breast cancer may provide crucial knowledge for the development of individualized therapy. However, this process is challenged by the availability of biomarkers able to identify subpopulations specifically. Here, we demonstrate an approach for phage display selection of recombinant antibody fragments...

  15. Selection and characterization of a unique phage display-derived antibody against dermatan sulfate.

    NARCIS (Netherlands)

    Lensen, J.F.M.; Wijnhoven, T.J.M.; Kuik, L.H.; Versteeg, E.M.M.; Hafmans, T.G.M.; Rops, A.L.; Pavao, M.S.; Vlag, J. van der; Heuvel, L.P.W.J. van den; Berden, J.H.M.; Kuppevelt, A.H.M.S.M. van

    2006-01-01

    Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) family and is primarily located in the extracellular matrix. Using a modified phage display procedure, we selected 2 different antibodies against DS of which one antibody, LKN1, was specific for DS. LKN1 was especially reactive with

  16. Characterization of nanobodies binding human fibrinogen selected by E. coli display.

    Science.gov (United States)

    Salema, Valencio; López-Guajardo, Ana; Gutierrez, Carlos; Mencía, Mario; Fernández, Luis Ángel

    2016-09-20

    Abnormal levels of fibrinogen (Fib) in blood plasma are associated with several pathological conditions and hence methods for its detection in blood and body fluids are essential. Nanobodies (Nbs) or (VHHs) are single domain antibodies derived from camelids with excellent biophysical and antigen-binding properties, showing great promise in diagnostics and therapy. In this work, we select and characterize high affinity Nbs binding human Fib employing an E. coli cell surface display system based on the fusion of an immune library of VHH domains with the β-domain of Intimin. Bacteria displaying high-affinity Nbs against Fib were selected using magnetic cell sorting (MACS). Specific binding of the selected clones to Fib was confirmed by flow cytometry of E. coli bacteria, as well as by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) with the purified Nbs. E. coli display also provided an excellent estimation of the affinity of the selected Nbs by flow cytometry analysis under equilibrium conditions, with equilibrium constant (KD) values very similar to those obtained by SPR analysis. Finally, pairwise epitope-scouting studies revealed that the selected Nbs bound distinct epitopes on Fib. The selected Nbs are promising diagnostic tools for determination of human Fib levels. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. "Fab 13": The Learning Factory.

    Science.gov (United States)

    Crooks, Steven M.; Eucker, Tom R.

    2001-01-01

    Describes how situated learning theory was employed in the design of Fab 13, a four-day simulation-based learning experience for manufacturing professionals at Intel Corporation. Presents a conceptual framework for understanding situated learning and discusses context, content, anchored instruction, facilitation, scaffolding, collaborating,…

  18. "Fab 13": The Learning Factory.

    Science.gov (United States)

    Crooks, Steven M.; Eucker, Tom R.

    2001-01-01

    Describes how situated learning theory was employed in the design of Fab 13, a four-day simulation-based learning experience for manufacturing professionals at Intel Corporation. Presents a conceptual framework for understanding situated learning and discusses context, content, anchored instruction, facilitation, scaffolding, collaborating,…

  19. Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

    Directory of Open Access Journals (Sweden)

    Christina Monerat Toledo-Machado

    2015-01-01

    Full Text Available ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL. The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.

  20. A DNA-based system for selecting and displaying the combined result of two input variables

    DEFF Research Database (Denmark)

    Liu, Huajie; Wang, Jianbang; Song, S

    2015-01-01

    Oligonucleotide-based technologies for biosensing or bio-regulation produce huge amounts of rich high-dimensional information. There is a consequent need for flexible means to combine diverse pieces of such information to form useful derivative outputs, and to display those immediately. Here we...... demonstrate this capability in a DNA-based system that takes two input numbers, represented in DNA strands, and returns the result of their multiplication, writing this as a number in a display. Unlike a conventional calculator, this system operates by selecting the result from a library of solutions rather...

  1. A competitive interaction theory of attentional selection and decision making in brief, multielement displays.

    Science.gov (United States)

    Smith, Philip L; Sewell, David K

    2013-07-01

    We generalize the integrated system model of Smith and Ratcliff (2009) to obtain a new theory of attentional selection in brief, multielement visual displays. The theory proposes that attentional selection occurs via competitive interactions among detectors that signal the presence of task-relevant features at particular display locations. The outcome of the competition, together with attention, determines which stimuli are selected into visual short-term memory (VSTM). Decisions about the contents of VSTM are made by a diffusion-process decision stage. The selection process is modeled by coupled systems of shunting equations, which perform gated where-on-what pathway VSTM selection. The theory provides a computational account of key findings from attention tasks with near-threshold stimuli. These are (a) the success of the MAX model of visual search and spatial cuing, (b) the distractor homogeneity effect, (c) the double-target detection deficit, (d) redundancy costs in the post-stimulus probe task, (e) the joint item and information capacity limits of VSTM, and (f) the object-based nature of attentional selection. We argue that these phenomena are all manifestations of an underlying competitive VSTM selection process, which arise as a natural consequence of our theory. PsycINFO Database Record (c) 2013 APA, all rights reserved.

  2. Whole genome phage display selects for proline-rich Boi polypeptides against Bem1p.

    Science.gov (United States)

    Hertveldt, Kirsten; Robben, Johan; Volckaert, Guido

    2006-08-01

    Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second "src homology region 3'' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1([Asn142-Ile551]), which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained approximately 7.7 x 10(7) independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules.

  3. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  4. Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

    Science.gov (United States)

    Crivianu-Gaita, Victor; Thompson, Michael

    2015-08-15

    Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors.

  5. Selection of single chain variable fragments specific for the human-inducible costimulator using ribosome display.

    Science.gov (United States)

    Pan, Yangbin; Mao, Weiping; Liu, Xuanxuan; Xu, Chong; He, Zhijuan; Wang, Wenqian; Yan, Hao

    2012-11-01

    We applied a ribosome display technique to a mouse single chain variable fragment (scFv) library to select scFvs specific for the inducible costimulator (ICOS). mRNA was isolated from the spleens of BALB/c mice immunized with ICOS protein. Heavy and κ chain genes (VH and κ) were amplified separately by reverse transcriptase polymerase chain reaction, and the anti-ICOS VH/κ chain ribosome display library was constructed with a special flexible linker by overlap extension PCR. The VH/κ chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. Then, antibody-ribosome-mRNA complexes were produced and panned against ICOS protein under appropriate conditions. However, in order to isolate specific scFvs for ICOS, negative selection using CD28 was carried out before three rounds of positive selection on ICOS. After three rounds of panning, the selected scFv DNAs were cloned into pET43.1a and detected by SDS-PAGE. Then, enzyme-linked immunosorbent assay showed that we successfully constructed a native ribosome display library, and among seven clones, clone 5 had the highest affinity for the ICOS and low for the CD28. Anti-ICOS scFvs are assessed for binding specificity and affinity and may provide the potential for development of the humanized and acute and chronic allograft rejection.

  6. The use of scFv-displaying yeast in mammalian cell surface selections.

    Science.gov (United States)

    Wang, Xin Xiang; Shusta, Eric V

    2005-09-01

    Yeast surface display has proven to be a powerful tool for the directed evolution of immunological proteins when soluble ligands are available (Cho, B.K., Kieke, M.C., Boder, E.T., Wittrup, K.D., Kranz, D.M., 1998. A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179; Boder, E.T., Midelfort, K.S., Wittrup, K.D., 2000. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. U. S. A. 97, 10701; Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M., Wittrup, K.D., 2000. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754; Esteban, O., Zhao, H., 2004. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81). This investigation extends the utility of this display platform by demonstrating its capacity for use in cell panning selections. This was accomplished by employing a model single-chain antibody (scFv)-hapten system that allowed for detailed investigation of the factors governing panning success. Yeast displaying anti-fluorescein scFv (4-4-20) exhibited specific interactions with the fluoresceinated endothelial cells and could be recovered from large backgrounds of irrelevant yeast in just three rounds. Successful selections required as few as 1700 fluorescein ligands per cell, and a three-round enrichment ratio of 10(6) was possible. These results indicate that yeast surface display is a viable option for use in cell or tissue-based selections.

  7. Selection of functional T cell receptor mutants from a yeast surface-display library.

    Science.gov (United States)

    Kieke, M C; Shusta, E V; Boder, E T; Teyton, L; Wittrup, K D; Kranz, D M

    1999-05-11

    The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

  8. Triclosan Resistance in a Bacterial Fish Pathogen, Aeromonas salmonicida subsp. salmonicida, is Mediated by an Enoyl Reductase, FabV.

    Science.gov (United States)

    Khan, Raees; Lee, Myung Hwan; Joo, Hae-Jin; Jung, Yong-Hoon; Ahmad, Shabir; Choi, Jin-Hee; Lee, Seon-Woo

    2015-04-01

    Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX(8)K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.

  9. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  10. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display.

    Science.gov (United States)

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming

    2015-10-01

    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  11. Semiconductor industry wafer fab exhaust management

    CERN Document Server

    Sherer, Michael J

    2005-01-01

    Given the myriad exhaust compounds and the corresponding problems that they can pose in an exhaust management system, the proper choice of such systems is a complex task. Presenting the fundamentals, technical details, and general solutions to real-world problems, Semiconductor Industry: Wafer Fab Exhaust Management offers practical guidance on selecting an appropriate system for a given application. Using examples that provide a clear understanding of the concepts discussed, Sherer covers facility layout, support facilities operations, and semiconductor process equipment, followed by exhaust types and challenges. He reviews exhaust point-of-use devices and exhaust line requirements needed between process equipment and the centralized exhaust system. The book includes information on wet scrubbers for a centralized acid exhaust system and a centralized ammonia exhaust system and on centralized equipment to control volatile organic compounds. It concludes with a chapter devoted to emergency releases and a separ...

  12. Selection of a breast cancer subpopulation-specific antibody using phage display on tissue sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla J

    2015-01-01

    Breast cancer tumors are composed of heterogeneous cell populations. These populations display a high variance in morphology, growth and metastatic propensity. They respond differently to therapeutic interventions, and some may be more prone to cause recurrence. Studying individual subpopulations...... stem cells. We isolated an antibody fragment LH 7, which in immunohistochemistry experiments demonstrates specific binding to breast cancer subpopulations. The selection of antibody fragments directly on small defined areas within a larger section of malignant tissue is a novel approach by which...... has the potential to provide new insight and treatment strategies for breast cancer....

  13. Phage Display: Selecting Straws Instead of a Needle from a Haystack

    Directory of Open Access Journals (Sweden)

    Mojca Lunder

    2011-01-01

    Full Text Available An increasing number of peptides with specific binding affinity to various protein and even non-protein targets are being discovered from phage display libraries. The power of this method lies in its ability to efficiently and rapidly identify ligands with a desired target property from a large population of phage clones displaying diverse surface peptides. However, the search for the needle in the haystack does not always end successfully. False positive results may appear. Thus instead of specific binders phage with no actual affinity toward the target are recovered due to their propagation advantages or binding to other components of the screening system, such as the solid phase, capturing reagents, contaminants in the target sample or blocking agents, rather than the target. Biopanning experiments on different targets performed in our laboratory revealed some previously identified and many new target-unrelated peptide sequences, which have already been frequently described and published, but not yet recognized as target-unrelated. Distinguishing true binders from false positives is an important step toward phage display selections of greater integrity. This article thoroughly reviews and discusses already identified and new target-unrelated peptides and suggests strategies to avoid their isolation.

  14. Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

    Science.gov (United States)

    Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

    2015-11-01

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

  15. Selection of trkB-binding peptides from a phage-displayed random peptide library

    Institute of Scientific and Technical Information of China (English)

    马仲才; 吴晓兰; 曹明媚; 潘卫; 朱分禄; 陈景山; 戚中田

    2003-01-01

    Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 υg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXφXXφXXC (X represents the random amino acids, φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.

  16. Targeting Phosphatidylserine on Apoptotic Cells with Phages and Peptides Selected from a Bacteriophage Display Library

    Directory of Open Access Journals (Sweden)

    Ruping Shao

    2007-11-01

    Full Text Available Phosphatidylserine (PS is a well-characterized biomarker for apoptosis. Ligands that bind to PS can be used for noninvasive imaging of therapy-induced cell death, particularly apoptosis. In this study, we screened a random 12-mer peptide phage library on liposomes prepared from PS. One clone displaying the peptide SVSVGMKPSPRP (designated as PS3-10 bound to PS approximately 4-fold better than its binding to phosphatidylcholine and 18-fold better than to bovine serum albumin in a solid-phase binding assay. In addition, the binding of the corresponding PS3-10 peptide to PS was significantly higher than that of a scrambled peptide. PS3-10 phages, but not a control 4-2-2 phage, bound to aged red blood cells that had PS exposed on their surface. Binding of PS3-10 phages and PS3-10 peptide to TRAIL-induced apoptotic DLD1 cells was 3.2 and 5.4 times higher than their binding to untreated viable cells, respectively. Significantly, immunohistochemical staining confirmed selective binding of PS3-10 phages to apoptotic cells. Our data suggest that panning of phage display libraries may allow the selection of suitable peptide ligands for apoptotic cells and that PS3-10 peptide may serve as a template for further development of molecular probes for in vitro and in vivo imaging of apoptosis.

  17. Selection of novel nickel-binding peptides from flagella displayed secondary peptide library.

    Science.gov (United States)

    Dong, Jie; Liu, Chuan; Zhang, Jie; Xin, Zhong-Tao; Yang, Guang; Gao, Bo; Mao, Can-Quan; Liu, Nong-Le; Wang, Fang; Shao, Ning-Sheng; Fan, Ming; Xue, Yan-Ning

    2006-08-01

    Nickel (Ni) performs its biological or toxic functions in nickel-protein coordination form. Novel Ni-binding peptides were isolated from a random dodecapeptide library displayed on the flagella of Escherichia coli against immobilized ions. On the basis of isolated sequences rich in histidine residues, two secondary libraries were constructed respectively. By consequent selection, more Ni-chelating peptides were identified and the consensus motif RHXHR (where X was always H) was deduced. The result suggested that not only histidine, but also arginine, play an important role in Ni-binding. Furthermore, two selected clones (1035 and 2022) were chosen for further identification. They exhibited similar relative binding affinity, which was about nine times that of the original library derived clones and statistically much more significant than the positive control with polyhistidine insert. Free nickel ions could almost completely inhibit the binding of the clones 1035 and 2022 to immobilized nickel, implicating that the peptides were able to chelate nickel ions. These studies reveal that bacterial surface displayed peptide libraries may have promising future potential for the development of metal bioadsorbents. Furthermore, novel Ni-binding peptides may provide lead molecules for Ni-chelation and applications thereof.

  18. Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display.

    Science.gov (United States)

    Soares, Tatiane Sanches; Soares Torquato, Ricardo Jose; Alves Lemos, Francisco Jose; Tanaka, Aparecida Sadae

    2013-01-01

    Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. The aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1-P4'). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. The results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC(50) of 1.1 nM) and chymotrypsin-like enzymes (IC(50) of 11.6 nM), respectively. The variants T23 (LLGGL) and T149 (GGVWR) inhibited both larval chymotrypsin-like (IC(50) of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC(50) of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. The HiTI variants T6 and T23 are good candidates for the development as a larvicide to control the vector.

  19. Selection of a peptide mimicking neutralization epitope of hepatitis E virus with phage peptide display technology

    Institute of Scientific and Technical Information of China (English)

    Ying Gu; Jun Zhang; Ying-Bing Wang; Shao-Wei Li; Hai-Jie Yang; Wen-Xin Luo; Ning-Shao Xia

    2004-01-01

    AIM: To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3.METHODS: 8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E. coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor.RESULTS: Twenty-one positive monoclonal phages (10for 8CL1, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 8C11 (N′-His-Pro-Thr-LeuLeu-Arg-Ile-C′, named 8C11A) and 8H3 (N′-Ser-Ile-LeuPro- Tyr-Pro-Tyr-C′, named 8H3A) were then synthesized and cloned to the HBcAg vector, then expressed in E. coli.The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemosynthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor.CONCLUSION: These results implicate that conformational neutralizing epitope can be partially modeled by a short

  20. Selective lead adsorption by recombinant Escherichia coli displaying a lead-binding peptide.

    Science.gov (United States)

    Nguyen, Thuong T L; Lee, Hae Ryong; Hong, Soon Ho; Jang, Ji-Ryang; Choe, Woo-Seok; Yoo, Ik-Keun

    2013-02-01

    A highly specific lead-binding peptide ThrAsnThrLeuSerAsnAsn was displayed on Escherichia coli, and lead adsorption characteristics of the recombinant bacteria were investigated. Cell surface-displayed peptide was expressed under the control of an arabinose promoter using outer membrane protein C (OmpC(t)) as an anchoring motif. The optimal induction period and arabinose concentration for the expression of peptide-fused OmpC(t) were determined to be 2 h and 0.001 g/L, respectively. Selective adsorption of Pb(2+) onto recombinant cells was verified with individual or combinatory use of four metal ions, Pb(2+), Ni(2+), Co(2+), and Cu(2+); the amount of bound Pb(2+) onto the biosorbents was significantly higher than the other metal ions. The adsorption isotherm of recombinant cells for Pb(2+) followed the Langmuir isotherm with a maximum adsorption loading (q (max)) of 526 μmol/g dry cell weight.

  1. Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library.

    Science.gov (United States)

    Padmanaban, Guruprasath; Park, Hyekyung; Choi, Ji Suk; Cho, Yong-Woo; Kang, Woong Chol; Moon, Chan-Il; Kim, In-San; Lee, Byung-Heon

    2014-10-10

    Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd=57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd=125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide-peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.

  2. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

    Science.gov (United States)

    Ericson, Megan E.; Frank, Matthew W.

    2016-01-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

  3. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

    Science.gov (United States)

    Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

    2016-12-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    Science.gov (United States)

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

  5. Structure of the omalizumab Fab.

    Science.gov (United States)

    Jensen, Rasmus K; Plum, Melanie; Tjerrild, Luna; Jakob, Thilo; Spillner, Edzard; Andersen, Gregers Rom

    2015-04-01

    Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcℇRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cℇ2-4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.

  6. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

    Energy Technology Data Exchange (ETDEWEB)

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-02-23

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.

  7. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

    2011-09-09

    Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  8. Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma.

    Science.gov (United States)

    Oelsner, Sarah; Friede, Miriam E; Zhang, Congcong; Wagner, Juliane; Badura, Susanne; Bader, Peter; Ullrich, Evelyn; Ottmann, Oliver G; Klingemann, Hans; Tonn, Torsten; Wels, Winfried S

    2017-02-01

    Natural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. To enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z). Exposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γ(null) mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. Our data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-02-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.

  10. High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

    Institute of Scientific and Technical Information of China (English)

    Biplab Bose; Navin Khanna; Subrat K Acharya; Subrata Sinha

    2005-01-01

    AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS: We cloned the VH and VL genes of this mouse antibody; and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. Coli.RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal.This chimeric antibody fragment was further expressed in different strains of E> coli to increase the yield.CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.

  11. Affinity peptide developed by phage display selection for targeting gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Wen-Jie Zhang; Yan-Xia Sui; Arun Budha; Jian-Bao Zheng; Xue-Jun Sun; Ying-Chun Hou; Thomas D Wang; Shao-Ying Lu

    2012-01-01

    AIM:To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer.METHODS:A peptide screen was performed by biopanning the PhD-12 phage display library,clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues.Tumortargeted binding of selected peptides was confirmed by bound phage counts,enzyme-linked immunosorbent assay,competitive inhibition,fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues.RESULTS:Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normalappearing gastric mucosa.After the third round of positive screening,the peptide sequence AADNAKTKSFPV (AAD) appeared in 25% (12/48) of the analyzed phages.For the control peptide,these values were 6.8 ± 2.3,5.1 ± 1.7,3.5 ± 2.1,4.6 ± 1.9 and 1.1 ± 0.5,respectively.The values for AAD peptide were statistically significant (P < 0.01) for gastric cancer as compared with other histological classifications and control peptide.CONCLUSION:A novel peptide is discovered to have a specific binding activity to gastric cancer,and can be used to distinguish neoplastic from normal gastric mucosa,demonstrating the potential for early cancer detection on endoscopy.

  12. Phage-displayed peptides selected for binding to Campylobacter jejuni are antimicrobial.

    Science.gov (United States)

    Bishop-Hurley, Sharon L; Rea, Philippa J; McSweeney, Christopher S

    2010-10-01

    In developed countries, Campylobacter jejuni is a leading cause of zoonotic bacterial gastroenteritis in humans with chicken meat implicated as a source of infection. Campylobacter jejuni colonises the lower gastrointestinal tract of poultry and during processing is spread from the gastrointestinal tract onto the surface of dressed carcasses. Controlling or eliminating C.jejuni on-farm is considered to be one of the best strategies for reducing human infection. Molecules on the cell surface of C.jejuni interact with the host to facilitate its colonisation and persistence in the gastrointestinal tract of poultry. We used a subtractive phage-display protocol to affinity select for peptides binding to the cell surface of a poultry isolate of C.jejuni with the aim of finding peptides that could be used to control this microorganism in chickens. In total, 27 phage peptides, representing 11 unique clones, were found to inhibit the growth of C.jejuni by up to 99.9% in vitro. One clone was bactericidal, reducing the viability of C.jejuni by 87% in vitro. The phage peptides were highly specific. They completely inhibited the growth of two of the four poultry isolates of C.jejuni tested with no activity detected towards other Gram-negative and Gram-positive bacteria.

  13. From shunting inhibition to dynamic normalization: Attentional selection and decision-making in brief visual displays.

    Science.gov (United States)

    Smith, Philip L; Sewell, David K; Lilburn, Simon D

    2015-11-01

    Normalization models of visual sensitivity assume that the response of a visual mechanism is scaled divisively by the sum of the activity in the excitatory and inhibitory mechanisms in its neighborhood. Normalization models of attention assume that the weighting of excitatory and inhibitory mechanisms is modulated by attention. Such models have provided explanations of the effects of attention in both behavioral and single-cell recording studies. We show how normalization models can be obtained as the asymptotic solutions of shunting differential equations, in which stimulus inputs and the activity in the mechanism control growth rates multiplicatively rather than additively. The value of the shunting equation approach is that it characterizes the entire time course of the response, not just its asymptotic strength. We describe two models of attention based on shunting dynamics, the integrated system model of Smith and Ratcliff (2009) and the competitive interaction theory of Smith and Sewell (2013). These models assume that attention, stimulus salience, and the observer's strategy for the task jointly determine the selection of stimuli into visual short-term memory (VSTM) and the way in which stimulus representations are weighted. The quality of the VSTM representation determines the speed and accuracy of the decision. The models provide a unified account of a variety of attentional phenomena found in psychophysical tasks using single-element and multi-element displays. Our results show the generality and utility of the normalization approach to modeling attention.

  14. Spatial variation in pollinator-mediated selection on phenology, floral display and spur length in the orchid Gymnadenia conopsea.

    Science.gov (United States)

    Chapurlat, Elodie; Ågren, Jon; Sletvold, Nina

    2015-12-01

    Spatial variation in plant-pollinator interactions may cause variation in pollinator-mediated selection on floral traits, but to establish this link conclusively experimental studies are needed. We quantified pollinator-mediated selection on flowering phenology and morphology in four populations of the fragrant orchid Gymnadenia conopsea, and compared selection mediated by diurnal and nocturnal pollinators in two of the populations. Variation in pollinator-mediated selection explained most of the among-population variation in the strength of directional and correlational selection. Pollinators mediated correlational selection on pairs of display traits, and on one display trait and spur length, a trait affecting pollination efficiency. Only nocturnal pollinators selected for longer spurs, and mediated stronger selection on the number of flowers compared with diurnal pollinators in one population. The two types of pollinators caused correlational selection on different pairs of traits and selected for different combinations of spur length and number of flowers. The results demonstrate that spatial variation in interactions with pollinators may result in differences in directional and correlational selection on floral traits in a plant with a semi-generalized pollination system, and suggest that differences in the relative importance of diurnal and nocturnal pollinators can cause variation in selection.

  15. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S. (GSK)

    2010-07-20

    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  16. A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

    Science.gov (United States)

    Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

    2014-03-01

    Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer.

  17. FAB (Functionally Alert Behavior Strategies) to Improve Self-Control

    Science.gov (United States)

    Pagano, John

    2015-01-01

    This paper describes the FAB (Functionally Alert Behavior) Strategies approach to improve behavior in children and adolescents with complex behavioral challenges. FAB Strategies include evidence-based environmental adaptations, sensory modulation, positive behavioral support, and physical self-regulation strategies. FAB Strategies can be used by…

  18. Rapportage LTO FAB II 2008 : Functionele Agro Biodiversiteit

    NARCIS (Netherlands)

    Scheele, J.; Gurp, van H.; Alebeek, van F.A.N.; Belder, den E.; Elderson, J.

    2009-01-01

    Doel van het LTO FAB II project is een gebruiksklaar FAB concept te ontwikkelen voor een aantal ziekten en plagen in een aantal gewassen die op eenvoudige wijze door telers benut kan worden en voor de toepasser kostenneutraal zijn. Uitgangspunt in het FAB project is een evenwichtige balans tussen de

  19. Selection of scFvs specific for the HepG2 cell line using ribosome display

    Indian Academy of Sciences (India)

    Lei Zhou; Wei-Ping Mao; Juan Fen; Hong-Yun Liu; Chuan-Jing Wei; Wen-Xiu Li; Feng-Yun Zhou

    2009-06-01

    The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.

  20. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    Science.gov (United States)

    Rhiel, Laura; Krah, Simon; Günther, Ralf; Becker, Stefan; Kolmar, Harald; Hock, Björn

    2014-01-01

    We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  1. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    Directory of Open Access Journals (Sweden)

    Laura Rhiel

    Full Text Available We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  2. Novel Zn2+-chelating peptides selected from a fimbria-displayed random peptide library

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Schembri, Mark; Klemm, Per

    2001-01-01

    The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces by virtue of the Fim...... of biomatrices for bioremediation purposes or in the development of sensors for detection of heavy metals....

  3. FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

    OpenAIRE

    Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.; Rock, Charles O.

    2014-01-01

    Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were...

  4. Production of single chain Fab (scFab fragments in Bacillus megaterium

    Directory of Open Access Journals (Sweden)

    Dübel Stefan

    2007-11-01

    Full Text Available Abstract Background The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab antibody format combining properties of single chain Fv (scFv and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. Results The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. Conclusion B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.

  5. Structure-based design,synthesis of novel inhibitors of Mycobacterium tuberculosis FabH as potential anti-tuberculosis agents

    Institute of Scientific and Technical Information of China (English)

    Xue Hui Zhang; Hong Yu; Wu Zhong; Li Li Wang; Song Li

    2009-01-01

    Mycobacterium tuberculosis FabH,an essential enzyme in mycolic acids biosynthetic pathway,is an attractive target for novel anti-tuberculosis agents.Structure-based design,synthesis of novel inhibitors of mrFabH was reported in this paper.A novel scaffold structure was designed,and 12 candidate compounds that displayed favorable binding with the active site were identified and synthesized.

  6. mRNA display selection of an optimized MDM2-binding peptide that potently inhibits MDM2-p53 interaction.

    Directory of Open Access Journals (Sweden)

    Hirokazu Shiheido

    Full Text Available p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins.

  7. Selection of Immunogenic Peptide Mimics of Male Worm Origin of Schistosoma Japonicum using Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    陈欲晓; 易新元; 曾宪芳; 王林纤; 唐连飞; 章洁; McreynoldsLarry

    2004-01-01

    To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide hbrary of 12 - mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54 % of egg reduction rate in mice, revealing a significant difference ( P < 0. 001 ) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display ptide library can elicit partial protection against this pathogen.

  8. Crystal Structure of the Fab Fragment of an Anti-factor IX Antibody 10C12

    Institute of Scientific and Technical Information of China (English)

    SHI Xiao-Li; ZENG Tu; HUANG Ming-Dong

    2008-01-01

    10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C 12.

  9. Various Energy-Saving Approaches to a TFT-LCD Panel Fab

    Directory of Open Access Journals (Sweden)

    Cheng-Kuang Chang

    2016-09-01

    Full Text Available This study employs the developed simulation software for the energy use of the high-tech fabrication plant (hereafter referred as a fab to examine six energy-saving approaches for the make-up air unit (MAU of a TFT-LCD (thin-film transistor liquid-crystal display fab. The studied approaches include: (1 Approach 1: adjust the set point of dry bulb temperature and relative humidity in the cleanroom; (2 Approach 2: lower the flow rate of supply air volume in the MAU; (3 Approach 3: use a draw-through type instead of push through type MAU; (4 Approach 4: combine the two stage cooling coils in MAU to a single stage coil; (5 Approach 5: reduce the original MAU exit temperature from 16.5 °C to 14.5 °C; and (6 Approach 6: avoid an excessive increase in pressure drop over the filter by replacing the HEPA filter more frequently. The simulated results are further compared to the measured data of the studied TFT-LCD fab in Taiwan. The simulated results showed that Approach 1 exhibits more significant influence on annual power consumption than the other approaches. The advantage/disadvantage of each approach is elaborated. The impact of the six approaches on the annual power consumption of the fab is also discussed.

  10. Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

    DEFF Research Database (Denmark)

    Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette

    2003-01-01

    During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...

  11. Identification of peptide sequences that selectively bind to pentaerythritol trinitrate hemisuccinate-a surrogate of PETN, via phage display technology.

    Science.gov (United States)

    Kubas, George; Rees, William; Caguiat, Jonathan; Asch, David; Fagan, Diana; Cortes, Pedro

    2017-03-01

    The present research investigates the identification of amino acid sequences that selectively bind to a pentaerythritol tetranitrate (PETN) explosive surrogate. Through the use of a phage display technique and enzyme-linked immunosorbent assays (ELISA), a peptide library was tested against pentaerythritol trinitrate hemisuccinate (PETNH), a surrogate of PETN, to screen for those with amino acids having affinity toward the explosive. The results suggest that the library contains peptides selective to PETNH. Following three rounds of panning, clones were picked and tested for specificity toward PETNH. ELISA results from these samples show that each phage clone has some level of selectivity for binding to PETNH. The peptides from these clones have been sequenced and shown to contain certain common amino acid segments among them. This work represents a technological platform for identifying amino-acid sequences selective toward any bio-chem analyte of interest.

  12. Using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen 85, a tuberculosis biomarker.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available BACKGROUND: Current diagnostic methods for tuberculosis (TB, a major global health challenge that kills nearly two million people annually, are time-consuming and inadequate. During infection a number of bacterial molecules that play a role in the infective process are released and have been proposed as biomarkers for early TB diagnosis. Antigen 85 (Ag85 is the most abundant secreted TB protein, and a potential target for this diagnostic approach. One of the bottlenecks in the direct detection of such bacterial targets is the availability of robust, sensitive, specific antibodies. METHODS: Using Ag85 as a model, we describe a method to select antibodies against any potential target using a novel combination of phage and yeast display that exploits the advantage of each approach. RESULTS: The efficiency of this approach was attested to by the 111 specific antibodies identified in initial screens. These were assessed for binding to the different Ag85 subunits, affinity, and activity in sandwich assays. CONCLUSIONS: The novelty of this approach lies in the possibility of screening the entire output of a phage antibody selection in a single experiment by yeast display. This can be considered analogous to carrying out a million ELISAs. The monoclonal antibodies (mAbs identified in this way show high binding affinity and selectivity for the antigens and offer an advantage over traditional mAbs produced by relatively expensive and time consuming techniques. This approach has wide applicability, and the affinity of selected antibodies can be significantly improved, if required.

  13. Summarized Costs, Placement Of Quality Stars, And Other Online Displays Can Help Consumers Select High-Value Health Plans.

    Science.gov (United States)

    Greene, Jessica; Hibbard, Judith H; Sacks, Rebecca M

    2016-04-01

    Starting in 2017, all state and federal health insurance exchanges will present quality data on health plans in addition to cost information. We analyzed variations in the current design of information on state exchanges to identify presentation approaches that encourage consumers to take quality as well as cost into account when selecting a health plan. Using an online sample of 1,025 adults, we randomly assigned participants to view the same comparative information on health plans, displayed in different ways. We found that consumers were much more likely to select a high-value plan when cost information was summarized instead of detailed, when quality stars were displayed adjacent to cost information, when consumers understood that quality stars signified the quality of medical care, and when high-value plans were highlighted with a check mark or blue ribbon. These approaches, which were equally effective for participants with higher and lower numeracy, can inform the development of future displays of plan information in the exchanges.

  14. Toward a Code for the Interactions of Zinc Fingers with DNA: Selection of Randomized Fingers Displayed on Phage

    Science.gov (United States)

    Choo, Yen; Klug, Aaron

    1994-11-01

    We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few α-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.

  15. Selection of binding targets in parasites using phage-display and aptamer libraries in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Renata Rosito Tonelli

    2013-01-01

    Full Text Available Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques.The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen-host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment, were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite-host interaction.In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic

  16. Monitoring strategy to match the advanced fabs

    Science.gov (United States)

    Ackmann, Paul W.

    2004-06-01

    The reduction in feature size below the exposure wavelength, the requirement for high yields, the expectation for consistent cycletime and shipment to mix, all mean that the reticle industry must be like advanced wafer fabrication centers. Due to the lower output of write tools versus steppers, and the fact that a reticle is a lot of one instead of 25 or 50 wafers as well as the need to match ship data to Fab ramp, the reticle line monitoring strategy must be optimized for small sample size. The use of tool time and alternative inspection strategies can lead to the early detection of problems. Because every reticle is a customer specific design, the monitoring strategy takes on a new look compared to the Fab. We have organized the AMTC to resemble a wafer fab. We have a dedicated Integration group that works with the customers and technologists, to monitor the needs of the customers and then drive the development programs that improve reticle capability. We have dedicated yield team to identify and classify the yield loss mechanisms and define probable causes. The teams then work with the Process owners to fix the source of yield loss and track the corrective actions. All sources of variations must be modeled and then sources of errors reduced to levels below the tool specification. The manufacturing organization has all the process and tool experts to focus on Pilot Line and Development tasks to meet the advance needs of our customers. With the organization in place we can then develop the methods based on Reticle and Fab manufacturing to best control the line and provide development with manufacturing cycle times.

  17. Mask qualification strategies in a wafer fab

    Science.gov (United States)

    Jaehnert, Carmen; Kunowski, Angela

    2007-02-01

    Having consistent high quality photo masks is one of the key factors in lithography in the wafer fab. Combined with stable exposure- and resist processes, it ensures yield increases in production and fast learning cycles for technology development and design evaluation. Preventive controlling of incoming masks and quality monitoring while using the mask in production is essential for the fab to avoid yield loss or technical problems caused by mask issues, which eventually result in delivery problems to the customer. In this paper an overview of the procedures used for mask qualification and production release, for both logic and DRAM, at Infineon Dresden is presented. Incoming qualification procedures, such as specification checks, incoming inspection, and inline litho process window evaluation, are described here. Pinching and electrical tests, including compatibility tests for mask copies for high volume products on optimized litho processes, are also explained. To avoid mask degradation over lifetime, re-inspection checks are done for re-qualification while using the mask in production. The necessity of mask incoming inspection and re-qualification, due to the repeater printing from either the processing defects of the original mask or degrading defects of being used in the fab (i.e. haze, ESD, and moving particles, etc.), is demonstrated. The need and impact of tight mask specifications, such as CD uniformity signatures and corresponding electrical results, are shown with examples of mask-wafer CD correlation.

  18. Immunodiagnosis of human neurocysticercosis using a synthetic peptide selected by phage-display.

    Science.gov (United States)

    Hell, R C R; Amim, P; de Andrade, H M; de Avila, R A M; Felicori, L; Oliveira, A G; Oliveira, C A; Nascimento, E; Tavares, C A P; Granier, C; Chávez-Olórtegui, C

    2009-04-01

    The usefulness of a synthetic peptide in the serodiagnosis of Taenia solium human neurocysticercosis (NC) has been evaluated. Phage-displayed peptides were screened with human antibodies to scolex protein antigen from cysticercus cellulosae (SPACc). One clone was found to interact specifically with anti-SPACc IgGs. The corresponding synthetic peptide was found to be recognized in ELISA by NC patient's sera. The study was carried out with sera from 28 confirmed NC patients, 13 control sera and 73 sera from patients suffering from other infectious diseases. A 93% sensibility and a 94.3% specificity was achieved. Figures of 89% and 31.4% of sensibility and specificity were obtained in a SPACc-based ELISA. Immunoblotting of SPACc with anti-peptide antibodies revealed a single band of approximately 45 kDa in 1D and four 45 kDa isoforms in 2D-gel electrophoresis. A strong and specific immunostaining in the fibers beneath the suckers, at the base of the rostellum, and in the tissue surrounding the scolex of cysticerci was observed by immunomicroscopy. Our results show that a peptide-based immunodiagnostic of neurocisticercosis can be envisioned.

  19. Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

    OpenAIRE

    Hoang, T.T.; Schweizer, H P

    1997-01-01

    The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemop...

  20. Environmentally selected aphid variants in clonality context display differential patterns of methylation in the genome.

    Directory of Open Access Journals (Sweden)

    Claude Pasquier

    Full Text Available Heritability of acquired phenotypic traits is an adaptive evolutionary process that appears more complex than the basic allele selection guided by environmental pressure. In insects, the trans-generational transmission of epigenetic marks in clonal and/or sexual species is poorly documented. Aphids were used as a model to explore this feature because their asexual phase generates a stochastic and/or environment-oriented repertoire of variants. The a priori unchanged genome in clonal individuals prompts us to hypothesize whether covalent methyl DNA marks might be associated to the phenotypic variability and fitness selection. The full differential transcriptome between two environmentally selected clonal variants that originated from the same founder mother was mapped on the entire genomic scaffolds, in parallel with the methyl cytosine distribution. Data suggest that the assortments of heavily methylated DNA sites are distinct in these two clonal phenotypes. This might constitute an epigenetic mechanism that confers the robust adaptation of insect species to various environments involving clonal reproduction.

  1. A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor.Methods Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide.Results Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation.Conclusion RBP could effectively rescue the promoted differentiation of resistin overxepressed 3T3-L1 preadipocyte.

  2. The novel anxiolytic ELB139 displays selectivity to recombinant GABA(A) receptors different from diazepam.

    Science.gov (United States)

    Rabe, Holger; Kronbach, Christiane; Rundfeldt, Chris; Lüddens, Hartmut

    2007-03-01

    A chemically heterogeneous group of compounds acts at the benzodiazepine (BZ) recognition site of the diverse gamma-aminobutyric acid type A (GABA(A)) receptor complexes which can assemble from more than 16 known subunits. Most 1,4-BZs like diazepam recognize all GABA(A)/BZ receptors containing the alpha1-3 or alpha5 together with any beta and the gamma2 subunit. Other compounds differentiate less, e.g. Ro15-4513, that additionally recognizes alpha4- and a6-containing receptors, or differentiate more, e.g. zolpidem, that recognizes preferentially alpha1-containing receptors. Here we describe the functional properties of 1-(4-chloro-phenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on (ELB139) in the presence and absence of the BZ receptor antagonist flumazenil (Ro15-1788) on recombinant alphaibeta2gamma2 (i=1-5) receptor subtypes expressed in HEK 293 cells. The properties were measured with the whole-cell variation of the patch-clamp technique and compared to those of diazepam. Like the latter, ELB139 did not potentiate GABA-induced currents in alpha4-containing receptors, but it displays functional subtype specificity between alpha1, alpha2, alpha3, and alpha5beta2gamma2 receptors with highest potency in alpha3-containing receptors but highest efficacy in alpha1- or alpha2-containing receptors, respectively. ELB139 acted as a partial agonist on these receptor subtypes reaching 40-50% of the efficacy of diazepam.

  3. Identification and Function Reasearch of fabA and fabB of Sinorhizobium meliloti%苜蓿中华根瘤菌fabA和fabB基因功能的鉴定

    Institute of Scientific and Technical Information of China (English)

    胡喆; 马金成; 蒋晶晶; 王海洪

    2013-01-01

    在大肠杆菌(Escherichia coli)脂肪酸合成酶体系中,fabA基因编码有双功能的3-羟基脂酰ACP脱水异构酶,其异构产物能被fabB基因编码的3-酮基脂酰ACP合成酶Ⅰ延伸,合成不饱和脂肪酸,该FabA-FabB途径被认为是缺氧条件下不饱和脂肪酸合成的经典途径.生物信息学分析发现,苜蓿中华根瘤菌(Sinorhizobium meliloti)的SmFabA与EcFabA相似性达到60.6%,具有相同的保守活性位点和两个保守的α螺旋结构;SmFabB与EcFabB相似性达到61.1%,具有相同的Cys-His-His活性中心.用携带SmfabA和SmfabB的质粒载体遗传互补大肠杆菌湿度敏感突变株CY57和CY242,在添加三氯森(TCL)抑制烯脂酰ACP还原酶活性的条件下,转化子能在42℃恢复生长,且放射性薄层层析能检测到转化子中不饱和脂肪酸棕榈油酸(A9C16:1)和十八碳烯酸(△11C18:1)的合成.体外重建脂肪酸合成反应表明,SmFabA能催化羟脂酰ACP的脱水反应且能够使反-2-癸烯酰ACP异构化,SmFabB能催化不同链长的脂酰ACP和丙二酸单酰ACP的聚合反应.另外,未得到SmFabA和SmFabB的突变株,表明SmFabA和SmFabB可能是苜蓿中华根瘤菌脂肪酸合成酶系中必不可少的关键蛋白.上述结果证实了苜蓿中华根瘤菌fabA和fabB两个基因在不饱和脂肪酸合成中的功能.

  4. The prohormone 19-norandrostenedione displays selective androgen receptor modulator (SARM) like properties after subcutaneous administration.

    Science.gov (United States)

    Diel, P; Friedel, A; Geyer, H; Kamber, M; Laudenbach-Leschowsky, U; Schänzer, W; Schleipen, B; Thevis, M; Vollmer, G; Zierau, O

    2008-04-01

    One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (4-estrene-3,17-dione, NOR), which is, after oral administration, readily metabolised to nortestosterone, also known as nandrolone (durabolin). In this study we have characterised molecular mechanisms of its action determined its tissue specific androgenic and anabolic potency after subcutaneous (s.c.) administration and investigated potential adverse effects. Receptor binding tests demonstrate that NOR binds with high selectivity to the AR. The potency of NOR to transactivate androgen receptor (AR) dependent reporter gene expression was 10 times lower as compared to dihydrotestosterone (DHT). In vivo experiments in orchiectomised rats demonstrated that s.c. treatment with NOR resulted only in a stimulation of the weight of the levator ani muscle; the prostate and seminal vesicle weights remained completely unaffected. Like testosterone, administration of NOR resulted in a stimulation of AR and myostatin mRNA expression in the gastrocnemius muscle. NOR does not affect prostate proliferation, the liver weight and the expression of the tyrosine aminotransferase gene (TAT) in the liver. Summarizing these data it is obvious that NOR, if administrated s.c. and in contrast to its metabolite nandrolone, highly selectively stimulates the growth of the skeletal muscle but has only weak androgenic properties. This observation may have relevance with respect to therapeutic aspects but also doping prevention.

  5. Integrated fab process for metal oxide EUV photoresist

    Science.gov (United States)

    Grenville, Andrew; Anderson, Jeremy T.; Clark, Benjamin L.; De Schepper, Peter; Edson, Joseph; Greer, Michael; Jiang, Kai; Kocsis, Michael; Meyers, Stephen T.; Stowers, Jason K.; Telecky, Alan J.; De Simone, Danilo; Vandenberghe, Geert

    2015-03-01

    Inpria is developing directly patternable, metal oxide hardmasks as robust, high-resolution photoresists for EUV lithography. Targeted formulations have achieved 13nm half-pitch at 35 mJ/cm2 on an ASML's NXE:3300B scanner. Inpria's second-generation materials have an absorbance of 20/μm, thereby enabling an equivalent photon shot noise compared to conventional resists at a dose lower by a factor of 4X. These photoresists have ~40:1 etch selectivity into a typical carbon underlayer, so ultrathin 20nm films are possible, mitigating pattern collapse. In addition to lithographic performance, we review progress in parallel advances required to enable the transition from lab to fab for such a metal oxide photoresist. This includes considerations and data related to: solvent compatibility, metals cross-contamination, coat uniformity, stability, outgassing, and rework.

  6. Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

    Institute of Scientific and Technical Information of China (English)

    Zuanning Yuan; Minge Du; Yiwen Chen; Fei Dou

    2013-01-01

    Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli-gomers and constructed a naïve human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe-cifical y recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked immunosorbent assay demonstrated this antibody bound specifical y to human amyloid-beta 42 te-tramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc-cessful y constructed a human phage display library and screened a single-domain antibody that specifical y recognized amyloid-beta 42 oligomers.

  7. Collecting duct-derived cells display mesenchymal stem cell properties and retain selective in vitro and in vivo epithelial capacity.

    Science.gov (United States)

    Li, Joan; Ariunbold, Usukhbayar; Suhaimi, Norseha; Sunn, Nana; Guo, Jinjin; McMahon, Jill A; McMahon, Andrew P; Little, Melissa

    2015-01-01

    We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC-like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC-like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC-like cells selectively integrated into the aquaporin 2-positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC-like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC-like cells, we further examined Hoxb7(+) fractions within the kidney across postnatal development, identifying a neonatal interstitial GFP(lo) (Hoxb7(lo)) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4(GCE/+):R26(tdTomato/+) mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair.

  8. Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

    Science.gov (United States)

    Zhao, Ning; Schmitt, Margaret A; Fisk, John D

    2016-04-01

    Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

  9. Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

    Institute of Scientific and Technical Information of China (English)

    JIE CAO; PING ZHAO; X1AO HUI MIAO; LAN JUAN ZHAO; LI JUN XUE; ZHONG TIAN QI

    2003-01-01

    The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.

  10. Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment.

    Science.gov (United States)

    Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2016-01-01

    The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma.

  11. A novel peptide, selected from phage display library of random peptides, can efficiently target into human breast cancer cell

    Institute of Scientific and Technical Information of China (English)

    DONG Jian; LIU WeiQing; JIANG AiMei; ZHANG KeJian; CHEN MingQing

    2008-01-01

    To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate ex-tra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of pep-tide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by cocultur-ing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the peptide to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC (the shifted sequence of CASPSGALRSC), and after coculturing them with different cell lines, their targeting ca-pacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of peptides was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.

  12. The cosmology of the Fab-Four

    CERN Document Server

    Copeland, Edmund J; Saffin, Paul M

    2012-01-01

    We have recently proposed a novel self tuning mechanism to alleviate the famous cosmological constant problem, based on the general scalar tensor theory proposed by Horndeski. The self-tuning model ends up consisting of four geometric terms in the action, with each term containing a free potential function of the scalar field; the four together being labeled as the Fab-Four. In this paper we begin the important task of deriving the cosmology associated with the Fab-Four Lagrangian. Performing a phase plane analysis of the system we are able to obtain a number of fixed points for the system, with some remarkable new solutions emerging from the trade-off between the various potentials. As well as obtaining inflationary solutions we also find conventional radiation/matter-like solutions, but in regimes where the energy density is dominated by a cosmological constant, and where we do not have any explicit forms of radiation or matter. Stability conditions for matter solutions are obtained and we show how it is po...

  13. Een inventarisatie naar de bekendheid van Functionele Agrobiodiversiteit (FAB) en de mogelijkheden om FAB met andere agroranden te combineren

    NARCIS (Netherlands)

    Geerts, R.H.E.M.; Meerburg, B.G.

    2011-01-01

    Op dit moment zijn er regio's waar het FAB concept al breed wordt toegepast en goed is ingebed (West-Brabant/Zeeland), waar het in opmars is (Flevoland en Zuid-Holland) en enkele regio's waar het FAB gedachtegoed slechts matig aanwezig is (Groningen / Drenthe).

  14. Type 1 cannabinoid receptor ligands display functional selectivity in a cell culture model of striatal medium spiny projection neurons.

    Science.gov (United States)

    Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Dupré, Denis J; Denovan-Wright, Eileen M

    2014-09-05

    Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gβγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.

  15. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

    Science.gov (United States)

    Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

    2003-09-01

    The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

  16. K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

    Science.gov (United States)

    Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

    2017-03-11

    Amino-acid mutations of Gly(12) (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. HIV-2 neutralization by intact V3-specific Fab fragments

    Directory of Open Access Journals (Sweden)

    Sourial Samer

    2008-08-01

    Full Text Available Abstract The V3 region of both HIV-1 gp120 and HIV-2 gp125 surface glycoprotein has been described as a target for neutralizing antibodies. In this study a conformation-sensitive (3C4 and a linear site-specific (7C8 anti-HIV-2 V3 monoclonal antibody (mAb were characterized. The neutralization capacity of the purified mAbs and their respective papain-generated Fab fragments was analyzed. The Fabs were further characterized by sequence analysis. Our results demonstrate that neither purified mAbs were capable of neutralizing HIV-2, while intact Fab fragments from both mAbs blocked in vitro infection of HIV-2 isolates. Moreover, the conformation sensitive 3C4 Fab neutralized both subtype A and B HIV-2 isolates and SIVsm. Sequence analysis of the hypervariable regions of 3C4 Fab and 7C8 Fab revealed that the third CDR of the heavy chain (CDRH3 of the antibodies was not as long as many of the previously characterized neutralizing antibodies. Our findings suggest that whole 7C8 and 3C4 mAbs are sterically hindered from neutralizing HIV-2, whereas the smaller size of Fab fragments enables access to the V3 region on the virion surface.

  18. Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

    2017-01-24

    The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

  19. How Fabulous Is Fab 5 Cosmology?

    CERN Document Server

    Linder, Eric V

    2013-01-01

    Extended gravity origins for cosmic acceleration can solve some fine tuning issues and have useful characteristics, but generally have little to say regarding the cosmological constant problem. Fab 5 gravity can be ghost free and stable, have attractor solutions in the past and future, and possess self tuning that solves the original cosmological constant problem. Here we show however it does not possess all these qualities at the same time. We also demonstrate that the self tuning is so powerful that it not only cancels the cosmological constant but also all other energy density, and we derive the scalings of its approach to a renormalized de Sitter cosmology. While this strong cancellation is bad for the late universe, it greatly eases early universe inflation.

  20. Structure-activity studies of the inhibition of FabI, the enoyl reductase from Escherichia coli, by triclosan: kinetic analysis of mutant FabIs.

    Science.gov (United States)

    Sivaraman, Sharada; Zwahlen, Jacque; Bell, Alasdair F; Hedstrom, Lizbeth; Tonge, Peter J

    2003-04-22

    Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.

  1. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    Science.gov (United States)

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  2. Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies.

    Directory of Open Access Journals (Sweden)

    Valencio Salema

    Full Text Available Screening of antibody (Ab libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM. In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs from camelids (nanobodies or VHH on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS. We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM(EHEC. We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM(EHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM(EHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

  3. Selective intestinal malabsorption of vitamin B12 displays recessive Mendelian inheritance: Assignment of a locus to chromosome 10 by linkage

    Energy Technology Data Exchange (ETDEWEB)

    Aminoff, M.; Tahvanainen, E.; Chapelle, A. de la [Univ. of Helsinki (Finland)] [and others

    1995-10-01

    Juvenile megaloblastic anemia caused by selective intestinal malabsorption of vitamin B12 has been considered a distinct condition displaying autosomal recessive inheritance. It appears to have a worldwide distribution, and comparatively high incidences were reported 30 years ago in Finland and Norway. More recently, the Mendelian inheritance of the condition has been questioned because almost no new cases have occurred in these populations. Here we report linkage studies assigning a recessive-gene locus for the disease to chromosome 10 in previously diagnosed multiplex families from Finland and Norway, proving the Mendelian mode of inheritance. The locus is tentatively assigned to the 6-cM interval between markers D10S548 and D10S466, with a multipoint maximum lod score (Z{sub max}) of 5.36 near marker D10S1477. By haplotype analysis, the healthy sibs in these families did not appear to constitute any examples of nonpenetrance. We hypothesize that the paucity of new cases in these populations is due either to a dietary effect on the gene penetrance that has changed with time, or to a drop in the birth rate in subpopulations showing enrichment of the mutation, or to both of these causes. 38 refs., 4 figs., 2 tabs.

  4. A Case Study of a High School Fab Lab

    Science.gov (United States)

    Lacy, Jennifer E.

    This dissertation examines making and design-based STEM education in a formal makerspace. It focuses on how the design and implementation of a Fab Lab learning environment and curriculum affect how instructors and students see themselves engaging in science, and how the Fab Lab relates to the social sorting practices that already take place at North High School. While there is research examining design-based STEM education in informal and formal learning environments, we know little about how K-12 teachers define STEM in making activities when no university or museum partnership exists. This study sought to help fill this gap in the research literature. This case study of a formal makerspace followed instructors and students in one introductory Fab Lab course for one semester. Additional observations of an introductory woodworking course helped build the case and set it into the school context, and provided supplementary material to better understand the similarities and differences between the Fab Lab course and a more traditional design-based learning course. Using evidence from observational field notes, participant interviews, course materials, and student work, I found that the North Fab Lab relies on artifacts and rhetoric symbolic of science and STEM to set itself apart from other design-based courses at North High School. Secondly, the North Fab Lab instructors and students were unable to explain how what they were doing in the Fab Lab was science, and instead relied on vague and unsupported claims related to interdisciplinary STEM practices and dated descriptions of science. Lastly, the design and implementation of the Fab Lab learning environment and curriculum and its separation from North High School's low tech, design-based courses effectively reinforced social sorting practices and cultural assumptions about student work and intelligence.

  5. Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

    Science.gov (United States)

    Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

    2016-04-01

    The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

  6. A Human Anti-Toll Like Receptor 4 Fab Fragment Inhibits Lipopolysaccharide-Induced Pro-Inflammatory Cytokines Production in Macrophages.

    Science.gov (United States)

    Wang, Maorong; Zheng, Wenkai; Zhu, Xuhui; Xu, Jing; Cai, Binggang; Zhang, Yiqing; Zheng, Feng; Zhou, Linfu; Yang, Zhiguo; Zhang, Xin; Wang, Changjun; Nie, Shinan; Zhu, Jin

    2016-01-01

    The results of clinical and experimental studies suggest that endotoxin/toll-like receptor 4 (TLR4)-mediated proinflammatory and profibrotic signaling activation is critical in the development of hepatic fibrosis. However, studies examining the role of specific TLR4 inhibitor are still lacking. The present study was aimed to prepare a human anti-TLR4 Fab fragment, named hTLR4-Fab01, and to explore its immune activity. We screened the positive clone of anti-human TLR4 phagemid from a human phage-display antibody library using recombinant TLR4 protein, which was used as template cDNA for the amplification of variable regions of the heavy (VH) chain and light chain (VL), then coupled with highly conserved regions of the heavy chain domain 1 (CH1) and the light chain (CL), respectively. Thus, the prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was constructed and transformed into Escherichia coli (E. coli) BL21. The characteristic of hTLR4-Fab01 was examined by SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay. Further, our data demonstrate that hTLR4-Fab01 could specifically bind to TLR4, and its treatment obviously attenuated the proinflammatory effect, characterized by less LPS-induced TNF-α, IL-1, IL-6 and IL-8 production in human macrophages. In conclusion, we have successfully prepared the hTLR4-Fab01 with efficient activity for blocking LPS-induced proinflammatory cytokines production, suggesting that the hTLR4-Fab01 may be a potential candidate for the treatment of hepatic fibrosis.

  7. An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

    Science.gov (United States)

    Demissie, Robel D; Kabre, Pauline; Tuntland, Micheal L; Fung, Leslie W-M

    2016-04-01

    Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

  8. Implementation of high-resolution reticle inspection in wafer fabs

    Science.gov (United States)

    Dayal, Aditya; Bergmann, Nathan M.; Sanchez, Peter

    2003-05-01

    Many advanced wafer fabs are currently fabricating devices with 130nm or smaller design rules. To meet the challenges at these sub-wavelength technology nodes, fabs are using a variety of resolution enhancement techniques (RETs) in lithography and exploring new methods of processing, inspecting and requalifying photomasks. The acceleration of the lithography roadmap imposes more stringent requirements on mask qualification and requalification to ensure that device yields are not compromised: mask inspection tools of today need to find smaller defects on reticles against considerably more complicated patterns or tighter critical dimensions (CDs). In this paper we describe the early stages of implementation and proliferation of advanced reticle inspection tools at high volume manufacturing wafer fabs. The fabs run incoming multi-surface contamination inspections on masks sent from the mask shop (Intel Mask Operations, IMO), and follow them up with periodic inspections/review to make sure any new contaminant or damage does not go undetected. When necessary, images of defects are electronically presented to engineers at IMO for review. Reticle requalification with these inspection tools reduces or eliminates the need for print test verification. We describe the tools and procedure used to streamline reticle requalification at the fabs and improve the feedback loop between the fabs and the mask shop.

  9. Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating.

    Science.gov (United States)

    Baek, Du-San; Kim, Yong-Sung

    2014-03-28

    Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 10(9) by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~10(7). The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

  10. Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment

    Institute of Scientific and Technical Information of China (English)

    Jun LI; Xue-hao WANG; Xiao-ming WANG; Zhao-lai CHEN

    2006-01-01

    Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido) benzyl]-l,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab'. Methods: B43 was cleaved using a simple 2-step method.First, stable F(ab')2 was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a sitespecific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. Results: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. 67Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

  11. 抗内毒素Fab'的制备%Production of Fab' of the chicken egg yolk antibody against lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    马思远; 张雅萍

    2007-01-01

    目的 研究抗内毒素卵黄免疫球蛋白(IgY)的活性片断Fab',探讨防治内毒素血症的新途径.方法 用内毒素(LPS)作为抗原免疫25周龄德国罗曼鸡,改良水溶法提取抗内毒素IgY,胃蛋白酶切后提取Fab'片断,光密度法测抗内毒素Fab'的浓度和含量、ELISA检测抗内毒素Fab'效价、SDS-聚丙烯酰胺凝胶电泳检测其分子量及纯度. 结果 抗内毒素Fab'含量为4.2 mg/mL蛋黄液,效价为1∶51 200,纯度为92%,相对分子质量为44 000. 结论 抗内毒素Fab'产量大、效价高、特异性强.

  12. Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

    Science.gov (United States)

    Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

    2007-10-01

    Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

  13. Handbook of display technology

    CERN Document Server

    Castellano, Joseph A

    1992-01-01

    This book presents a comprehensive review of technical and commercial aspects of display technology. It provides design engineers with the information needed to select proper technology for new products. The book focuses on flat, thin displays such as light-emitting diodes, plasma display panels, and liquid crystal displays, but it also includes material on cathode ray tubes. Displays include a large number of products from televisions, auto dashboards, radios, and household appliances, to gasoline pumps, heart monitors, microwave ovens, and more.For more information on display tech

  14. Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Yan-Da Lai

    2016-02-01

    Full Text Available Vascular endothelial growth factor (VEGF is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume on Day 21 vs. 435% with Avastin. This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

  15. Self-tuning and the derivation of the Fab Four

    CERN Document Server

    Charmousis, Christos; Padilla, Antonio; Saffin, Paul M

    2011-01-01

    We have recently proposed a special class of scalar tensor theories known as the Fab Four. These arose from attempts to analyse the cosmological constant problem within the context of Horndeski's most general scalar tensor theory. The Fab Four together give rise to a model of self-tuning, with the relevant solutions evading Weinberg's no-go theorem by relaxing the condition of Poincare invariance in the scalar sector. The Fab Four are made up of four geometric terms in the action with each term containing a free potential function of the scalar field. In this paper we rigorously derive this model from the general model of Horndeski, proving that the Fab Four represents the only classical scalar tensor theory of this type that has any hope of tackling the cosmological constant problem. We present the full equations of motion for this theory, and give an heuristic argument to suggest that one might be able to keep radiative corrections under control. We also give the Fab Four in terms of the potentials presente...

  16. Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

    Science.gov (United States)

    Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

    2015-12-01

    Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

  17. Automated simulation and evaluation of autostereoscopic multiview 3D display designs by time-sequential and wavelength-selective filter barrier

    Science.gov (United States)

    Kuhlmey, Mathias; Jurk, Silvio; Duckstein, Bernd; de la Barré, René

    2015-09-01

    A novel simulation tool has been developed for spatial multiplexed 3D displays. Main purpose of our software is the 3D display design with optical image splitter in particular lenticular grids or wavelength-selective barriers. As a result of interaction of image splitter with ray emitting displays a spatial light-modulator generating the autostereoscopic image representation was modeled. Based on the simulation model the interaction of optoelectronic devices with the defined spatial planes is described. Time-sequential multiplexing enables increasing the resolution of such 3D displays. On that reason the program was extended with an intermediate data cumulating component. The simulation program represents a stepwise quasi-static functionality and control of the arrangement. It calculates and renders the whole display ray emission and luminance distribution on viewing distance. The degree of result complexity will increase by using wavelength-selective barriers. Visible images at the viewer's eye positon were determined by simulation after every switching operation of optical image splitter. The summation and evaluation of the resulting data is processed in correspondence to the equivalent time sequence. Hereby the simulation was expanded by a complex algorithm for automated search and validation of possible solutions in the multi-dimensional parameter space. For the multiview 3D display design a combination of ray-tracing and 3D rendering was used. Therefore the emitted light intensity distribution of each subpixel will be evaluated by researching in terms of color, luminance and visible area by using different content distribution on subpixel plane. The analysis of the accumulated data will deliver different solutions distinguished by standards of evaluation.

  18. Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library.

    Science.gov (United States)

    Ohtani, Maki; Hikima, Jun-ichi; Jung, Tae-Sung; Kondo, Hidehiro; Hirono, Ikuo; Takeyama, Haruko; Aoki, Takashi

    2013-02-01

    Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.

  19. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    Science.gov (United States)

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

  20. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fab fragment specific... Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fab fragment specific) immunological test system is a device that...

  1. 20 CFR 30.316 - How does the FAB issue a final decision on a claim?

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How does the FAB issue a final decision on a... Adjudicatory Process Hearings and Final Decisions on Claims § 30.316 How does the FAB issue a final decision on... waives any objections to all or part of the recommended decision, the FAB may issue a final...

  2. DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Nobuhide Doi

    Full Text Available The G protein-coupled receptors (GPCRs, which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II type-1 receptor (hAT1R as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.

  3. Antigen binding of human IgG Fabs mediate ERK-associated proliferation of human breast cancer cells.

    Science.gov (United States)

    Wen, Yue-Jin; Mancino, Anne; Pashov, Anastas; Whitehead, Tracy; Stanley, Joseph; Kieber-Emmons, Thomas

    2005-02-01

    Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.

  4. Pollution prevention opportunity assessment for MicroFab and SiFab facilities at Sandia National Laboratories.

    Energy Technology Data Exchange (ETDEWEB)

    Gerard, Morgan Evan

    2011-12-01

    This Pollution Prevention Opportunity Assessment (PPOA) was conducted for the MicroFab and SiFab facilities at Sandia National Laboratories/New Mexico in Fiscal Year 2011. The primary purpose of this PPOA is to provide recommendations to assist organizations in reducing the generation of waste and improving the efficiency of their processes and procedures. This report contains a summary of the information collected, the analyses performed, and recommended options for implementation. The Sandia National Laboratories Environmental Management System (EMS) and Pollution Prevention (P2) staff will continue to work with the organizations to implement the recommendations.

  5. Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics.

    Science.gov (United States)

    Vázquez, Hilda; Olvera, Felipe; Alagón, Alejandro; Sevcik, Carlos

    2013-12-15

    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

  6. Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis.

    Science.gov (United States)

    Li, Meng; Meng, Qiu; Fu, Huihui; Luo, Qixia; Gao, Haichun

    2016-11-15

    As type II fatty acid synthesis is essential for the growth of Escherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of the cis-3-decenoyl-ACP (cis-3-C10-ACP). In our previous study, we presented evidence to suggest that this may not be the case in Shewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB in S. oneidensis In fabB(+) or desA(+) (encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of both fabB and desA genes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of the fabF1 gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator for acpP, an acyl carrier protein gene immediately upstream of fabF1 There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1.

  7. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  8. 20 CFR 30.319 - May a claimant request reconsideration of a final decision of the FAB?

    Science.gov (United States)

    2010-04-01

    ... final decision of the FAB? 30.319 Section 30.319 Employees' Benefits OFFICE OF WORKERS' COMPENSATION... reconsideration of a final decision of the FAB? (a) A claimant may request reconsideration of a final decision of the FAB by filing a written request with the FAB within 30 days from the date of issuance of...

  9. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    Science.gov (United States)

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

  10. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

    Energy Technology Data Exchange (ETDEWEB)

    Obmolova, Galina, E-mail: gobmolov@its.jnj.com; Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L., E-mail: gobmolov@its.jnj.com [Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477 (United States)

    2014-07-23

    The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

  11. Building blocks X-FAB SOI 0.18 μm

    Science.gov (United States)

    Cizel, J.-B.; Ahmad, S.; Callier, S.; Cornat, R.; Dulucq, F.; Fleury, J.; Martin-Chassard, G.; Raux, L.; de La Taille, C.; Thienpont, D.

    2015-02-01

    This work has been done in order to study a new technology provided by X-FAB named xt018. It is an SOI (Silicon On Insulator) technology with a minimal gate length of 180 nm. Building blocks have been done to test the advantages and drawbacks of this technology compared to the one currently used (AMS SiGe 0.35 μm). These building blocks have been designed to fit in an existing experience housed by the CALICE collaboration: the read-out chip for the Electromagnetic CALorimeter (ECAL) of the foreseen International Linear Collider (ILC). Performances will be compared to those of the SKIROC2 chip designed by the OMEGA laboratory, trying to fit the same requirements. The chip is being manufactured and will be back for measurements in December, the displayed results are only simulation results and thus the conclusions concerning the performances of these building blocks are subject to change.

  12. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    Science.gov (United States)

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  13. The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors.

    Science.gov (United States)

    Houimel, Mehdi

    2014-08-01

    A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74-92% and 90-96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3-IGKV1, IGHV3-IGVL1, and IGHV3-IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.

  14. Potential of Peptides as Inhibitors and Mimotopes: Selection of Carbohydrate-Mimetic Peptides from Phage Display Libraries

    Directory of Open Access Journals (Sweden)

    Teruhiko Matsubara

    2012-01-01

    Full Text Available Glycoconjugates play various roles in biological processes. In particular, oligosaccharides on the surface of animal cells are involved in virus infection and cell-cell communication. Inhibitors of carbohydrate-protein interactions are potential antiviral drugs. Several anti-influenza drugs such as oseltamivir and zanamivir are derivatives of sialic acid, which inhibits neuraminidase. However, it is very difficult to prepare a diverse range of sugar derivatives by chemical synthesis or by the isolation of natural products. In addition, the pathogenic capsular polysaccharides of bacteria are carbohydrate antigens, for which a safe and efficacious method of vaccination is required. Phage-display technology has been improved to enable the identification of peptides that bind to carbohydrate-binding proteins, such as lectins and antibodies, from a large repertoire of peptide sequences. These peptides are known as “carbohydrate-mimetic peptides (CMPs” because they mimic carbohydrate structures. Compared to carbohydrate derivatives, it is easy to prepare mono- and multivalent peptides and then to modify them to create various derivatives. Such mimetic peptides are available as peptide inhibitors of carbohydrate-protein interactions and peptide mimotopes that are conjugated with adjuvant for vaccination.

  15. Production of a PEGylated Fab' of the anti-LINGO-1 Li33 antibody and assessment of its biochemical and functional properties in vitro and in a rat model of remyelination.

    Science.gov (United States)

    Pepinsky, R Blake; Walus, Lee; Shao, Zhaohui; Ji, Benxiu; Gu, Sheng; Sun, Yaping; Wen, Dingyi; Lee, Xinhua; Wang, Qin; Garber, Ellen; Mi, Sha

    2011-02-16

    The use of LINGO-1 antagonists to promote repair of damaged myelin is an emerging therapeutic opportunity for treatment of CNS diseases caused by demyelination such as multiple sclerosis. The Li33 anti-LINGO-1 antibody is a potent inducer of myelination in vitro and in vivo, but aggregation issues prevented the engineering of an optimal development candidate. PEGylated Li33 Fab' is one of several versions of the Li33 antibody that is being investigated in an attempt to identify the most favorable anti-LINGO-1 antibody design. For targeted PEGylation, a Li33 Fab' construct was engineered with a single unpaired cysteine in the heavy-chain hinge sequence. The Fab' was expressed in CHO cells, purified, and PEGylated with 20 kDa methoxy-poly(ethylene glycol) maleimide using a reaction strategy optimized to improve the yield of the PEG-Fab'. Biochemical analysis of the Li33 PEG-Fab' verified the selectivity of the PEGylation reaction. The in vitro and in vivo attributes of the PEG-Fab' were benchmarked against a Li33 full antibody. Both the Li33 PEG-Fab' and intact antibody bound LINGO-1 with nanomolar affinity, promoted myelination in an in vitro signaling assay, and promoted the repair of damaged myelin in the rat lysolecithin model. These studies extend our understanding of the biological activity of the Li33 mAb and validate the use of an anti-LINGO-1 PEG-Fab' for treatment of CNS diseases caused by demyelination.

  16. Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli

    Directory of Open Access Journals (Sweden)

    Malgorzata Korycka-Machala

    2017-01-01

    Full Text Available The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use β-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5′-triphosphate. This fact leads to the conclusion that NAD+-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD+ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide inhibited the activity of M. tuberculosis NAD+-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage DNA LigT4 was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM.

  17. Design, synthesis and antibacterial activities of 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiol derivatives containing Schiff base formation as FabH inhibitory.

    Science.gov (United States)

    Zhang, Fei; Wen, Qing; Wang, She-Feng; Shahla Karim, Baloch; Yang, Yu-Shun; Liu, Jia-Jia; Zhang, Wei-Ming; Zhu, Hai-Liang

    2014-01-01

    A series of novel schiff base derivatives (H(1)-H(20)) containing pyrazine and triazole moiety have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of β-ketoacyl-acyl carrier protein synthase III (FabH). These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Bacillus amyloliquefaciens and selected compounds among them were tested for their Escherichia coli FabH inhibitory activity. Based on the biological data, compound H(17) showed the most potent antibacterial activity with MIC values of 0.39-1.56μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC50 of 5.2μM, being better than the positive control Kanamycin B with IC50 of 6.3μM. Furthermore, docking simulation was performed to position compound H(17) into the E. coli FabH active site to determine the probable binding conformation. This study indicated that compound H(17) has demonstrated significant E. coli FabH inhibitory activity as a potential antibacterial agent and provides valuable information for the design of E. coli FabH inhibitors.

  18. Construction of a human na(i)ve Fab library and screening of phage antibody against arginine vasopressin%人源Fab噬菌体抗体库的构建与抗精氨酸加压素抗体的筛选

    Institute of Scientific and Technical Information of China (English)

    董越华; 胡占东; 公倩; 李常颖; 畅继武; 朱铁虹

    2012-01-01

    Objective: To construct a naive human Fab phage display library, screen and identify arginine vasopressin Fab antibody from the library. Methods: Total RNA was extracted from peripheral blood lymphocytes of 18 healthy donors, and the light chain and heavy chain Fd genes were amplified by RT-PCR. Then the amplification products were sequentially cloned into phagemid vector pComb3XSS to construct a human Fab phage antibody library. The insertion of the light chain or heavy chain Fd genes were identified by cutting with endonucleases and PCR amplification. Arginine vasopressin was used as target antigen to pan the original Fab antibody library. After five rounds of panning were carried out,fifty randomly selected clones were assayed by phage-ELISA analysis. The positive clones were analyzed by DNA sequencing. Results: A large human Fab phage antibody library consisting of 2.4 × 108 members was successfully constructed. After having been panned by AVP,we obtained six positive clones which had specificity and binding reactivity towards AVP. The C4 clone was analyzed and showed that its heavy chain belonged to IgG subvariety and its light chain to X family. Conclusion : We successfully constructed a large human Fab phage antibody library and isolated the specific human anti-AVP Fab antibodies,which provided a solid foundation for the establishment of rapid detection method of arginine vasopressin in future research.%目的:构建人源天然Fab噬菌体抗体库,筛选抗精氨酸加压素特异性抗体并进行初步鉴定.方法:从18位健康成人的外周血淋巴细胞,提取总RNA.利用RT-PCR扩增人Fab抗体基因片段,将其克隆至噬菌粒载体pComb3XSS内,构建人源天然Fab噬菌体抗体库.以固相化的精氨酸加压素为靶抗原对抗体库进行五轮筛选后,随机挑取50个单克隆进行phage-ELISA检测,阳性克隆行DNA测序分析.结果:成功构建库容为2.4×108的噬菌体抗体库,从中筛选到6株阳性克隆能够与精

  19. Characterization of molecular mechanisms controlling fabAB transcription in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Herbert P Schweizer

    Full Text Available BACKGROUND: The FabAB pathway is one of the unsaturated fatty acid (UFA synthesis pathways for Pseudomonas aeruginosa. It was previously noted that this operon was upregulated in biofilms and repressed by exogenous UFAs. Deletion of a 30 nt fabA upstream sequence, which is conserved in P. aeruginosa, P. putida, and P. syringae, led to a significant decrease in fabA transcription, suggesting positive regulation by an unknown positive regulatory mechanism. METHODS/PRINCIPAL FINDINGS: Here, genetic and biochemical approaches were employed to identify a potential fabAB activator. Deletion of candidate genes such as PA1611 or PA1627 was performed to determine if any of these gene products act as a fabAB activator. However, none of these genes were involved in the regulation of fabAB transcription. Use of mariner-based random mutagenesis to screen for fabA activator(s showed that several genes encoding unknown functions, rpoN and DesA may be involved in fabA regulation, but probably via indirect mechanisms. Biochemical attempts performed did fail to isolate an activator of fabAB operon. CONCLUSION/SIGNIFICANCE: The data suggest that fabA expression might not be regulated by protein-binding, but by a distinct mechanism such as a regulatory RNA-based mechanism.

  20. Human urocortin II, a new CRF-related peptide, displays selective CRF(2)-mediated action on gastric transit in rats.

    Science.gov (United States)

    Million, Mulugeta; Maillot, Céline; Saunders, Paul; Rivier, Jean; Vale, Wylie; Taché, Yvette

    2002-01-01

    Human urocortin (hUcn) II is a new member of the corticotropin-releasing factor (CRF) family that selectively binds to the CRF(2) receptor. We investigated the CRF receptors involved in mediating the effects of hUcn II and human/rat CRF (h/rCRF) on gut transit. Gastric emptying, 4 h after a solid meal, and distal colonic transit (bead expulsion time) were monitored simultaneously in conscious rats. CRF antagonists were given subcutaneously 30 min before intravenous injection of peptides or partial restraint (for 90 min). hUcn II (3 or 10 microg/kg i.v.) inhibited gastric emptying (by 45% and 55%, respectively) and did not influence distal colonic transit. The CRF(2) peptide antagonist astressin(2)-B blocked hUcn II action. h/rCRF, rat Ucn, and restraint delayed gastric emptying while accelerating distal colonic transit. The gastric response to intravenous h/rCRF and restraint was blocked by the CRF(2) antagonist but not by the CRF(1) antagonist CP-154,526, whereas the colonic response was blocked only by CP-154,526. None of the CRF antagonists influenced postprandial gut transit. These data show that intravenous h/rCRF and restraint stress-induced delayed gastric emptying involve CRF(2) whereas stimulation of distal colonic transit involves CRF(1). The distinct profile of hUcn II, only on gastric transit, is linked to its CRF(2) selectivity.

  1. Phage display in vivo selection and its application%噬菌体展示体内筛选技术及其应用进展

    Institute of Scientific and Technical Information of China (English)

    俞杨; 王自正

    2008-01-01

    Phage display technology is a kind of method which display peptides or proteins of high diversity on the surface of phage coat pmteins.This technology establish a linkage between genotype and phenotype,which makes functional screen of peptides or proteins convenient.Peptides or proteins screened from phage display library have high specificity and strong affinity,and play an important roles in basic research and clinical diagnosis and therapy.With the development of phage display,it has been used in the in vivo selection.It has characteristics of high throughput selection so that it can reveal molecular profiles and the differences of molecular profiles expressed on the vascular surface of different organs.Studies of in vivo selection can provide experimental evidences for the targeting diagnosis and therapy.%噬菌体展示技术是将高度多样性的多肽或蛋白展示于噬菌体衣壳蛋白表面的一项技术.这一技术将展示分子的基因型和表型联系在一起,极大地方便了多肽或蛋白分子的功能性筛选.从噬菌体展示文库中筛选出的多肽或蛋白具有高度的特异性和极强的亲和力,在生物医学基础研究和临床诊断治疗中具有重要的应用价值.随着噬菌体展示技术的不断发展,已将其用于活体的体内筛选.体内筛选技术发挥了高通量筛选的特点,能够在活体水平研究血管表面的分子表达状况,分析不同器官血管表面分子的表达差异,从而为临床靶向性诊断和治疗提供实验依据.

  2. Molecular modeling and simulation of FabG, an enzyme involved in the fatty acid pathway of Streptococcus pyogenes.

    Science.gov (United States)

    Shafreen, Rajamohmed Beema; Pandian, Shunmugiah Karutha

    2013-09-01

    Streptococcus pyogenes (SP) is the major cause of pharyngitis accompanied by strep throat infections in humans. 3-keto acyl reductase (FabG), an important enzyme involved in the elongation cycle of the fatty acid pathway of S. pyogenes, is essential for synthesis of the cell-membrane, virulence factors and quorum sensing-related mechanisms. Targeting SPFabG may provide an important aid for the development of drugs against S. pyogenes. However, the absence of a crystal structure for FabG of S. pyogenes limits the development of structure-based drug designs. Hence, in the present study, a homology model of FabG was generated using the X-ray crystallographic structure of Aquifex aeolicus (PDB ID: 2PNF). The modeled structure was refined using energy minimization. Furthermore, active sites were predicted, and a large dataset of compounds was screened against SPFabG. The ligands were docked using the LigandFit module that is available from Discovery Studio version 2.5. From this list, 13 best hit ligands were chosen based on the docking score and binding energy. All of the 13 ligands were screened for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. From this, the two best descriptors, along with one descriptor that lay outside the ADMET plot, were selected for molecular dynamic (MD) simulation. In vitro testing of the ligands using biological assays further substantiated the efficacy of the ligands that were screened based on the in silico methods.

  3. Crystal structure of human TWEAK in complex with the Fab fragment of a neutralizing antibody reveals insights into receptor binding.

    Directory of Open Access Journals (Sweden)

    Alfred Lammens

    Full Text Available The tumor necrosis factor-like weak inducer of apoptosis (TWEAK is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.

  4. Spherical Cows in the Sky with Fab Four

    CERN Document Server

    Kaloper, Nemanja

    2013-01-01

    We explore spherically symmetric static solutions in a subclass of unitary scalar-tensor theories of gravity, called the `Fab Four' models. The weak field large distance solutions may be phenomenologically viable, but only if the Gauss-Bonnet term is negligible. Only in this limit will the Vainshtein mechanism work consistently. Further, classical constraints and unitarity bounds constrain the models quite tightly. Nevertheless, in the limits where the range of individual terms at large scales is respectively Kinetic Braiding, Horndeski, and Gauss-Bonnet, the horizon scale effects may occur while the theory satisfies Solar system constraints and, marginally, unitarity bounds. On the other hand, to bring the cutoff down to below a millimeter constrains all the couplings scales such that `Fab Fours' can't be heard outside of the Solar system.

  5. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    Science.gov (United States)

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

  6. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    Science.gov (United States)

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

  7. Preparation of F(ab')2 fragments of immunoglobulin G.

    Science.gov (United States)

    Killion, J J; Holtgrewe, E M

    1983-11-01

    We describe a simple protocol for the preparation of F(ab')2 fragments of immunoglobulin G, based upon the known Fc- binding properties of protein A-Sepharose. The fragment preparations of xenogeneic and allogeneic anti-IgG were noncytotoxic to intact target cells, and were able to block the cytotoxicity of intact antibody. This method should therefore be useful for functional studies not requiring biochemical homogeneity.

  8. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    Science.gov (United States)

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

  9. Strategy optimization for mask rule check in wafer fab

    Science.gov (United States)

    Yang, Chuen Huei; Lin, Shaina; Lin, Roger; Wang, Alice; Lee, Rachel; Deng, Erwin

    2015-07-01

    Photolithography process is getting more and more sophisticated for wafer production following Moore's law. Therefore, for wafer fab, consolidated and close cooperation with mask house is a key to achieve silicon wafer success. However, generally speaking, it is not easy to preserve such partnership because many engineering efforts and frequent communication are indispensable. The inattentive connection is obvious in mask rule check (MRC). Mask houses will do their own MRC at job deck stage, but the checking is only for identification of mask process limitation including writing, etching, inspection, metrology, etc. No further checking in terms of wafer process concerned mask data errors will be implemented after data files of whole mask are composed in mask house. There are still many potential data errors even post-OPC verification has been done for main circuits. What mentioned here are the kinds of errors which will only occur as main circuits combined with frame and dummy patterns to form whole reticle. Therefore, strategy optimization is on-going in UMC to evaluate MRC especially for wafer fab concerned errors. The prerequisite is that no impact on mask delivery cycle time even adding this extra checking. A full-mask checking based on job deck in gds or oasis format is necessary in order to secure acceptable run time. Form of the summarized error report generated by this checking is also crucial because user friendly interface will shorten engineers' judgment time to release mask for writing. This paper will survey the key factors of MRC in wafer fab.

  10. Disulfide cross-linked Fab-aggregates: preparation and biodistribution.

    Science.gov (United States)

    Dalkara, S; Petrov, A; Trubetskoy, V S; Khaw, B A; Torchilin, V P

    1998-01-01

    The high-molecular-weight soluble aggregates of Fab fragments of murine antibodies against cardiac myosin were prepared as a potential long-circulating and low immunogenic pharmaceutical carriers by conjugation of thiolated Fab and Fab modified with succinimidyl 3-(2-pyridyldithio)propionate. The clearance time and biodistribution of 111In-radiolabeled aggregates were studied in normal and nude-mice bearing human breast tumor implant and in rabbits with experimental myocardial infarction. The aggregates had a prolonged circulation time (half clearance time ca. 3-5 h) and ability to concentrate in the tumor and in the necrotic area of infarcted myocardium. Similar tumor-to-normal and infarct-to-normal accumulation ratios (ca. 3 h in both cases) suggest that combination of long circulation with impaired filtration in necrotic tissues is responsible for this accumulation rather than a specific interaction. The aggregates prepared may serve as long-circulating drug carriers able to deliver pharmaceuticals into areas with affected and leaky vasculature.

  11. Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection

    Science.gov (United States)

    Johansson, Magnus; Zhang, Jingji; Ehrenberg, Måns

    2012-01-01

    Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg2+ concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon–anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell. PMID:22190491

  12. Projection displays

    Science.gov (United States)

    Chiu, George L.; Yang, Kei H.

    1998-08-01

    Projection display in today's market is dominated by cathode ray tubes (CRTs). Further progress in this mature CRT projector technology will be slow and evolutionary. Liquid crystal based projection displays have gained rapid acceptance in the business market. New technologies are being developed on several fronts: (1) active matrix built from polysilicon or single crystal silicon; (2) electro- optic materials using ferroelectric liquid crystal, polymer dispersed liquid crystals or other liquid crystal modes, (3) micromechanical-based transducers such as digital micromirror devices, and grating light valves, (4) high resolution displays to SXGA and beyond, and (5) high brightness. This article reviews the projection displays from a transducer technology perspective along with a discussion of markets and trends.

  13. A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

    Science.gov (United States)

    Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

    2015-01-01

    Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

  14. Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus.

    Science.gov (United States)

    Liu, Pei; Han, Lei; Wang, Fei; Petrenko, Valery A; Liu, Aihua

    2016-08-15

    Staphylococcus aureus (S. aureus) is one of the most ubiquitous pathogens in public healthcare worldwide. It holds great insterest in establishing robust analytical method for S. aureus. Herein, we report a S. aureus-specific recognition element, isolated from phage monoclone GQTTLTTS, which was selected from f8/8 landscape phage library against S. aureus in a high-throughput way. By functionalizing cysteamine (CS)-stabilized gold nanoparticles (CS-AuNPs) with S. aureus-specific pVIII fusion protein (fusion-pVIII), a bifunctional nanoprobe (CS-AuNPs@fusion-pVIII) for S. aureus was developed. In this strategy, the CS-AuNPs@fusion-pVIII could be induced to aggregate quickly in the presence of target S. aureus, resulting in a rapid colorimetric response of gold nanoparticles. More importantly, the as-designed probe exhibited excellent selectivity over other bacteria. Thus, the CS-AuNPs@fusion-pVIII could be used as the indicator of target S. aureus. This assay can detect as low as 19CFUmL(-1)S. aureus within 30min. Further, this approach can be applicable to detect S. aureus in real water samples. Due to its sensitivity, specificity and rapidness, this proposed method is promising for on-site testing of S. aureus without using any costly instruments.

  15. Assessment of Digoxin-Specific Fab Fragment Dosages in Digoxin Poisoning.

    Science.gov (United States)

    Nordt, Sean Patrick; Clark, Richard F; Machado, Carol; Cantrell, F Lee

    2016-01-01

    Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from $4818 to as high as $50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.

  16. Structure-directed construction of a high-performance version of the enzyme FabG from the photosynthetic microorganism Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Liu, Yinghui; Feng, Yanbin; Cao, Xupeng; Li, Xia; Xue, Song

    2015-10-07

    PhaB (acetoacetyl-CoA reductase) catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA in polyhydroxybutyrate (PHB) synthesis and FabG (3-ketoacyl-acyl-carrier-protein reductase) catalyzes the β-ketoacyl-ACP to yield (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis. Both of them have been classified into the same group EC 1.1.1. PhaB is limited with substrate specificities, while FabG was considered as a potential PhaB due to broad substrate selectivity despite of low activity. Here, X-ray crystal structures of FabG and PhaB from the photosynthetic microorganism Synechocystis sp. PCC 6803 were resolved. Based on them, a high-performance FabG on acyl-CoA directed by structural evolution was constructed that may serve as a critical enzyme to partition carbon flow from fatty acid synthesis to PHA. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

    OpenAIRE

    Feng, Youjun; Cronan, John E.

    2011-01-01

    Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

  18. Selection of Peptide Inhibitor to Matrix Metalloproteinase-2 Using Phage Display and Its Effects on Pancreatic Cancer Cell lines PANC-1 and CFPAC-1

    Directory of Open Access Journals (Sweden)

    Gao Lu, Maqing Zheng, Yunxia Zhu, Min Sha, Yue Wu, Xiao Han

    2012-01-01

    Full Text Available Despite tremendous advances in cancer treatment and survival rates, pancreatic cancer remains one of the most deadly afflictions and the fourth leading cause of cancer deaths in the world. Matrix Metalloproteinases (MMPs are thought to be involved in cancer progression. Matrix metalloproteinase (MMP-2 is known to play a pivotal role in tumor invasion, metastasis and angiogenesis, and validated to be the anticancer target. Inhibition of MMP-2 activity is able to reduce the cancer cell invasion and suppress tumor growth in vivo. Two novel peptides, M204C4 and M205C4, which could specially inhibit MMP-2 activity, were identified by a phage display library screening. We showed that M204C4 and M205C4 inhibited the activity of MMP-2 in a dose dependent manner in vitro. Two peptides reduced MMP-2 mediated invasion of the pancreatic cancer cell lines PANC-1 and CFPAC-1, but not affected the expression and release of MMP-2. Furthermore, these two peptides could suppress tumor growth in vivo. Our results indicated that two peptides selected by phase display technology may be used as anticancer drugs in the future.

  19. Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo

    DEFF Research Database (Denmark)

    Ank, Nina; West, Hans; Bartholdy, Christina;

    2006-01-01

    Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN......-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN...... had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between...

  20. Lambda Interferon (IFN-gamma), a Type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo

    DEFF Research Database (Denmark)

    Ank, Nina; West, Hans; Bartholdy, C.;

    2006-01-01

    Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN......-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN...... had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between...

  1. Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

    Science.gov (United States)

    Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

    2014-03-01

    Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

  2. Military display performance parameters

    Science.gov (United States)

    Desjardins, Daniel D.; Meyer, Frederick

    2012-06-01

    The military display market is analyzed in terms of four of its segments: avionics, vetronics, dismounted soldier, and command and control. Requirements are summarized for a number of technology-driving parameters, to include luminance, night vision imaging system compatibility, gray levels, resolution, dimming range, viewing angle, video capability, altitude, temperature, shock and vibration, etc., for direct-view and virtual-view displays in cockpits and crew stations. Technical specifications are discussed for selected programs.

  3. Evolutional selection of a combinatorial phage library displaying randomly-rearranged various single domains of immunoglobulin (Ig-binding proteins (IBPs with four kinds of Ig molecules

    Directory of Open Access Journals (Sweden)

    Jia Jian-An

    2008-08-01

    Full Text Available Abstract Background Protein A, protein G and protein L are three well-defined immunoglobulin (Ig-binding proteins (IBPs, which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting. Results We used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D and PA(A respectively, B2 domain of protein G (PG and B3 domain of protein L (PL for affinity selection with human IgG (hIgG, human IgM (hIgM, human IgA (hIgA and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A-PG and PA(A-PL were obtained in hIgG (hIgG1-Fc and hIgM (hIgA post-selection populations respectively. In addition, the linking peptides among all PA(A-PG and PA(A-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A-PG and PA(A-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively. Conclusion In this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A-PG and PA(A-PL, were obtained, and demonstrate the novel Ig binding properties.

  4. Apoptosis function on tumor cell by SEA-Fab' coupled protein%SWA-Fab'对肿瘤细胞的凋亡作用

    Institute of Scientific and Technical Information of China (English)

    舒晓刚; 王国斌

    2005-01-01

    目的研究SEA-Fab'对肿瘤细胞的凋亡作用及单克隆抗体于肿瘤定向治疗作用.方法实验分为三组:SEA-Fab'、SEA及对照组,三组肿瘤细胞分别用SEA-Fab'、SEA及PBMC+Walker-256细胞处理,在24~72 h内观察肿瘤细胞的凋亡情况.结果SEA-Fab'组、SEA组的肿瘤细胞指数分别为(34.6%~68.9%)和(15.5%~31.9%)高于对照组(5.5%~12.5%),差异存在显著性,SEA-Fab'组高于SEA组差异显著.结论SEA-Fab'在激活T细胞和杀死肿瘤细胞效果好于SEA,将来有可能利用单克隆抗体来作定向靶位治疗.%[Objective] To Study the anti-tumor effect of the SEA-Fab' coupled protein and the possibility of themonoclonal antibody Fab's targeted in immunotherapy of human tumor. [Methods] The experiment group was treat-ed by SEA-Fab' and SEA, the contrast group was treated by PBMC+Walker-256 cell. The apoptotic index was ob-served in 24~72 h. [Results] The poptotic index was significantly higher in the SEA-Fab's (34.6~68.9%) and SEAgroups (15.5~31.9%) compared with the PBMC+Wallker-256 cell group (5.5~12.8%). There was significant differ-ence between SEA-Fab group and SEA group (P<0.01). [Conclusion] SEA-Fab' is more effective to activate Tcells and kill tumor cell than SEA, there is ossibility of The monoclonal antibody targeted in immunotherapy of hu-man tumor.

  5. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis

    OpenAIRE

    Jeffrey D. Nanson; Himiari, Zainab; Swarbrick, Crystall M. D.; Forwood, Jade K.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II f...

  6. Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutathione peroxidase (GPX) plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening. Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immunosorbent assay(ELISA) analysis with 4 rounds of selection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester(GSH-s-DNP-Bu) and S-2,4-dinitriphenyl t-hexyl ester(GSH-s-DNP-He). Nevertheless, several studies need to be conducted to determine whether scFv-B8 and scFv-H6 possess GPX activity. To enhance the speed of the selection, selenocysteine(Sec, the catalytic group of GPX) was incorporated directly into the phages, scFv-B8 and scFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-scFv with GPX activity.

  7. Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

    Science.gov (United States)

    Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

    2017-07-12

    Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

  8. Rational optimization of drug-target residence time: Insights from inhibitor binding to the S. aureus FabI enzyme-product complex

    Science.gov (United States)

    Chang, Andrew; Schiebel, Johannes; Yu, Weixuan; Bommineni, Gopal R.; Pan, Pan; Baxter, Michael V.; Khanna, Avinash; Sotriffer, Christoph A.; Kisker, Caroline; Tonge, Peter J.

    2013-01-01

    Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI) - an important target for the development of new anti-staphylococcal drugs - as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme. PMID:23697754

  9. Crystal structure determination of anti-DNA Fab A52.

    Science.gov (United States)

    Stanfield, Robyn L; Eilat, Dan

    2014-08-01

    A52 is a murine monoclonal antibody isolated from autoimmune New Zealand Black/New Zealand White F1 mice that recognizes single and double stranded DNA. This mouse strain spontaneously develops systemic lupus erythematosus-like symptoms and has served as a model for that disease for many years. The 1.62 Å crystal structure of the A52 Fab fragment reveals an H3 complementarity determining region with four closely spaced arginine residues, creating a positively charged surface to accommodate bound DNA.

  10. Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    Institute of Scientific and Technical Information of China (English)

    Chen LI; Feng ZHANG; Hong LIN; Zhong-can WANG; Xin-jian LIU; Zhen-qing FENG; Jin ZHU; Xiao-hong GUAN

    2011-01-01

    Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting,indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.Results: A recombinant vector was constructed. The Fab was expressed in soluble form In E coll Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL.The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

  11. Functionally Approached Body (FAB) Strategies for Young Children Who Have Behavioral and Sensory Processing Challenges

    Science.gov (United States)

    Pagano, John

    2005-01-01

    Functionally Approached Body (FAB) Strategies offer a clinical approach to help parents of young children with behavioral and sensory processing strategies. This article introduces the FAB Strategies, clinical strategies developed by the author for understanding and addressing young children's behavioral and sensory processing challenges. The FAB…

  12. The Emerging Importance of IgG Fab Glycosylation in Immunity.

    Science.gov (United States)

    van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

    2016-02-15

    Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity.

  13. 75 FR 21353 - Intel Corporation, Fab 20 Division, Including On-Site Leased Workers From Volt Technical...

    Science.gov (United States)

    2010-04-23

    ... Employment and Training Administration Intel Corporation, Fab 20 Division, Including On-Site Leased Workers... for Worker Adjustment Assistance on March 10, 2010, applicable to workers of Intel Corporation, Fab 20... the Hillsboro, Oregon location of Intel Corporation, Fab 20 Division. The Department has...

  14. Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

    Science.gov (United States)

    Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

    2013-10-01

    A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

  15. 20 CFR 30.320 - Can a claim be reopened after the FAB has issued a final decision?

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can a claim be reopened after the FAB has... AMENDED Adjudicatory Process Reopening Claims § 30.320 Can a claim be reopened after the FAB has issued a final decision? (a) At any time after the FAB has issued a final decision pursuant to § 30.316,...

  16. Construction and diversity analysis of a murine IgE phage surface display library

    Institute of Scientific and Technical Information of China (English)

    LIZONGDONG; MINGYEH

    1997-01-01

    To make further investigation of the IgE antibody repertoire in Trichosanthin (TCS) allergic responses,a murine IgE phage surface display library was constructed (3.0×105 independent clones).We first constructed the Vε cDNA library (4.6×105 independent clones) and Vκ cDNA library (3.0×105 independent clones).Then,the Vε and Vκgene segments were amplified from both libraries by PCR respectively,and assembled into Fab fragment by SOE PCR.The phage library containing Fabs was thus constructed.The diversity of Vε from this library was analyzed and proved.Fab clones with high specificity to TCS have been screened out.

  17. Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

    Science.gov (United States)

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

    2015-01-01

    A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

  18. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    Science.gov (United States)

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  19. Human Engineering Design Criteria for Modern Control/Display Components and Standard Parts. Addendum. Recommended Modifications to MIL-STD-1472B; Selected Sections on Controls, Displays and Related Hardware

    Science.gov (United States)

    1980-05-01

    right " turn signal indication", etc. d. External vehicle identification/mode lights, e.g.: rear caution/stop/backup/turn lights, vehicle perimeter...Windshield washer/wiper, turn - signal , horn, speedoimnter, fuel, oi-l and tempera - ture displays, etc.). 242 A MF I RECOMMIENDED MODIFICATIONI RAT

  20. Selection of peptide ligands for the antimucin core antibody C595 using phage display technology: definition of candidate epitopes for a cancer vaccine.

    Science.gov (United States)

    Laing, P; Tighe, P; Kwiatkowski, E; Milligan, J; Price, M; Sewell, H

    1995-06-01

    Aims-To further define the specificity of the antimucin core antibody C595 by fitting it with a family of hexapeptide ligands by immunoselection of filamentous bacteriophage from a gene III display library of approximately 6.4 x 10(7) random hexapeptides.Methods-Three rounds of immuno-selection were used to enrich for C595 binding phage. DNA sequencing revealed the hexapeptides expressed. Bacteriophage and corresponding synthetic hexapeptides were used in ELISA assay to determine binding affinities.Results-Twenty nine clones from this selected population were analysed. Seven contained the natural epitope RPAP, encoded by two different DNA sequences; 17/29 contained the motif RLPP. In all, 28/29 clones contained the motif RXXP and one clone (RVRPAP) contained the motif RXXP in two peptidic registers; 24/28 clones (6/8 DNA sequences) contained a hydrophobic residue (V or I) at position 1 relative to the RXXP motif. In addition the proximity of RXXP to glycine (position 5) suggests that this contributes in the natural epitope to antibody/antigen binding, which was not detected by chemical synthetic methods. One clone, KSKAGV, bears no obvious relationship to the natural epitope and therefore qualifies as a weakly binding mimotope.Conclusions-This approach has rapidly defined the specificity of this antibody in unprecedented detail, and provides a more comprehensive molecular basis for exploring the immune recognition of the MUC1 mucin by the C595 antibody. Importantly, the novel but related epitopes seen provide peptide specificities and a strategy which may prove useful in generating cancer vaccine candidates.

  1. Tumor cell-collagen interactions: Identification and semi-quantitative evaluation of selectively-expressed genes by combination of differential display- and multiplex-PCR

    Directory of Open Access Journals (Sweden)

    Sirchia Rosalia

    2003-01-01

    Full Text Available It is widely acknowledged that the presence of extracellular matrix components as substrates can drastically modulate the phenotype and gene expression of cultured cells, including tumor cells. A number of published reports indicated that substrates made from two peculiar collagen species, i.e. type V and OF/LB, which are abnormally deposited in the stroma of primary ductal infiltrating carcinoma (d.i.c. of the breast “in vivo,” were able to exert marked and opposite effects on “in vitro” viability, growth and invasiveness of the 8701-BC cell line, isolated from d.i.c.-affected breast epithelium. To complement such functional data on the effect of cell-collagen interactions with information at molecular level, we have utilized a combination of differential display- and semi-quantitative multiplex-PCR techniques with the aim of detecting variations in the expression levels of selected genes by cells maintained in either culture condition. Here we report some prototypical data on the identification and semi-quantitation of three of the differentially-amplified PCR products found, i.e. HSP2A and MSF-B which are up-regulated in cells grown onto OF/LB collagen substrate, and SRCAP which is prominently down-regulated in the presence of type V collagen substrate. This protocol represents a powerful tool for evaluating changes in the levels and patterns of gene expression which can be theoretically adapted to any experimental model system.

  2. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Directory of Open Access Journals (Sweden)

    Peter Durand Skottrup

    Full Text Available Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18 by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15, shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48. Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  3. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Science.gov (United States)

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  4. sup 111 In-antimyosin Fab scintigraphy in cardiovascular diseases; Multicenter clinical trial

    Energy Technology Data Exchange (ETDEWEB)

    Kawai, Chuichi; Matsumori, Akira; Endo, Keigo (Kyoto Univ. (Japan). Faculty of Medicine); Nishimura, Tsunehiko

    1990-12-01

    In a multicenter study, a total of 380 patients with myocardial infarction, myocarditis and cardiomyopathy underwent {sup 111}In-antimyosin Fab myocardial imaging. {sup 111}In-antimyosin Fab was administered intravenously and myocardial images were obtained 48 hours later. Only 3 patients developed mild adverse effects. Human antimouse antibodies were detected in 7 patients. Positive scans in patients with myocardial infarction were seen in 92/119 (77%) within 2 weeks after the onset of myocardial infarction, in 58/71 (82%) at 3-4 weeks, in 20/22 (91%) at 4-8 weeks and 17/31 (55%) thereafter. The location of myocardial damage delineated by {sup 111}In-antimyosin Fab imaging was concordant with the infarct location by ECG and coronary angiography. In patients with myocarditis, {sup 111}In-antimyosin Fab uptake was positive in 7/12 (58%) within 8 weeks and 6/17 (35%) thereafter. Positive {sup 111}In-antimyosin Fab scans were seen in 12/36 (33%) in dilated cardiomyopathy and in 17/19 (89%) in hypertrophic cardiomyopathy. Although the mechanism of persistently positive {sup 111}In-antimyosin Fab images in the subacute to chronic stage of myocardial infarction and myocarditis remains to be clarified, {sup 111}In-antimyosin Fab may be useful for the detection of the disease and in evaluating the prognosis of patients with cardiomyopathy. (author).

  5. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

    Science.gov (United States)

    Barbas, C F; Björling, E; Chiodi, F; Dunlop, N; Cababa, D; Jones, T M; Zebedee, S L; Persson, M A; Nara, P L; Norrby, E

    1992-01-01

    A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies. PMID:1384050

  6. Modification of fibrin network ultrastructure by Fab fragments specific for different domain of fibrinogen.

    Science.gov (United States)

    Cierniewski, C S; Janiak, A; Wyroba, E

    1986-01-01

    Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils.

  7. 'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

    Science.gov (United States)

    Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

    2016-04-01

    A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli.

  8. Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, V. V., E-mail: vvo@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Lapuk, V. A. [Russian Academy of Sciences, Zelinskii Institute of Organic Chemistry (Russian Federation); Shtykova, E. V.; Stepina, N. D.; Dembo, K. A.; Sokolova, A. V.; Amarantov, S. V. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Timofeev, V. P. [Russian Academy of Sciences, Engelhardt Institute of Molecular Biology (Russian Federation); Ziganshin, R. Kh. [Russian Academy of Sciences, Shemyakin Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Varlamova, E. Yu. [Russian Academy of Medical Sciences, Hematology Research Center (Russian Federation)

    2008-05-15

    The structural features of the Fab fragments of monoclonal (Waldenstroem's disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that for Fab-RF (21{+-}2 and 11{+-}1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.

  9. The community FabLab platform: applications and implications in biomedical engineering.

    Science.gov (United States)

    Stephenson, Makeda K; Dow, Douglas E

    2014-01-01

    Skill development in science, technology, engineering and math (STEM) education present one of the most formidable challenges of modern society. The Community FabLab platform presents a viable solution. Each FabLab contains a suite of modern computer numerical control (CNC) equipment, electronics and computing hardware and design, programming, computer aided design (CAD) and computer aided machining (CAM) software. FabLabs are community and educational resources and open to the public. Development of STEM based workforce skills such as digital fabrication and advanced manufacturing can be enhanced using this platform. Particularly notable is the potential of the FabLab platform in STEM education. The active learning environment engages and supports a diversity of learners, while the iterative learning that is supported by the FabLab rapid prototyping platform facilitates depth of understanding, creativity, innovation and mastery. The product and project based learning that occurs in FabLabs develops in the student a personal sense of accomplishment, self-awareness, command of the material and technology. This helps build the interest and confidence necessary to excel in STEM and throughout life. Finally the introduction and use of relevant technologies at every stage of the education process ensures technical familiarity and a broad knowledge base needed for work in STEM based fields. Biomedical engineering education strives to cultivate broad technical adeptness, creativity, interdisciplinary thought, and an ability to form deep conceptual understanding of complex systems. The FabLab platform is well designed to enhance biomedical engineering education.

  10. Hepatic targeting and hypocholesterolemic effect of lactosaminated Fab against low density lipoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Bernini, F.; Bocan, T.M.A.; Via, D.P.; Gotto, A.M. Jr.; Smith, L.C.

    1986-03-01

    Lactosaminated Fab (lac-Fab) specific for human LDL induces plasma clearance and uptake of circulating (/sup 125/-I)-iodo-LDL in rat, a process mediated by galactose receptors of the liver. This study demonstrates that lac-Fab is a specific carrier of LDL to the liver parenchymal cells and exhibits hypocholesterolemic activity in vivo. Rats were injected with fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-LDL (diI-LDL) or 6 mg of LDL plus tracer amounts of (/sup 125/I)-iodo-LDL. After 10-20 min, the animals received 3-10 mg of lac-Fab. Histologic examination of the liver sections showed the uptake of diI-LDL in the parenchymal cells, as compared to diI-acetyl-LDL which was localized in sinusoidal cells. More than 85% of human LDL disappeared within 2.5 hr after lac-Fab injection, reducing plasma cholesterol from 133.0 +/- 12.6 mg/dl to 66.4 +/- 8.0 mg/dl, the basal value in the rat. In control rats, only about 20% of radioactivity and cholesterol disappeared at 2.5 hr. HDL levels were unaffected. The authors conclude that lac-Fab is a specific carrier of LDL to hepatocytes and can lower plasma LDL-cholesterol in vivo. Lac-Fab specific for other antigens may act as specific carriers of molecule or macromolecules to hepatocytes.

  11. Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

    Science.gov (United States)

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

    2014-01-15

    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.

  12. 20 CFR 30.318 - Can the FAB consider objections to HHS's reconstruction of a radiation dose or to the guidelines...

    Science.gov (United States)

    2010-04-01

    ... at 42 CFR part 82, is binding on the FAB. The FAB reviewer may determine, however, that objections... CFR part 81, is also binding on the FAB (see § 30.213). However, since OWCP applies this methodology... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can the FAB consider objections to...

  13. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  14. Direct-write scanning probe lithography: towards a desktop fab

    Science.gov (United States)

    Giam, Louise R.; Senesi, Andrew J.; Liao, Xing; Wong, Lu Shin; Chai, Jinan; Eichelsdoerfer, Daniel J.; Shim, Wooyoung; Rasin, Boris; He, Shu; Mirkin, Chad A.

    2011-06-01

    Massively parallel scanning-probe based methods have been used to address the challenges of nanometer to millimeter scale printing for a variety of materials and mark a step towards the realization of a "desktop fab." Such tools enable simple, flexible, high-throughput, and low-cost nano- and microscale patterning, which allow researchers to rapidly synthesize and study systems ranging from nanoparticle synthesis to biological processes. We have developed a novel scanning probe-based cantilever-free printing method termed polymer pen lithography (PPL), which uses an array of elastomeric tips to transfer materials (e.g. alkanethiols, proteins, polymers) in a direct-write manner onto a variety of surfaces. This technique takes the best attributes of dip-pen nanolithography (DPN) and eliminates many of the disadvantages of contact printing. Various related techniques such as beam pen lithography (BPL), scanning probe block copolymer lithography (SPBCL), and hard-tip, soft spring lithography (HSL) are also discussed.

  15. Dynamic plasmonic colour display

    Science.gov (United States)

    Duan, Xiaoyang; Kamin, Simon; Liu, Na

    2017-02-01

    Plasmonic colour printing based on engineered metasurfaces has revolutionized colour display science due to its unprecedented subwavelength resolution and high-density optical data storage. However, advanced plasmonic displays with novel functionalities including dynamic multicolour printing, animations, and highly secure encryption have remained in their infancy. Here we demonstrate a dynamic plasmonic colour display technique that enables all the aforementioned functionalities using catalytic magnesium metasurfaces. Controlled hydrogenation and dehydrogenation of the constituent magnesium nanoparticles, which serve as dynamic pixels, allow for plasmonic colour printing, tuning, erasing and restoration of colour. Different dynamic pixels feature distinct colour transformation kinetics, enabling plasmonic animations. Through smart material processing, information encoded on selected pixels, which are indiscernible to both optical and scanning electron microscopies, can only be read out using hydrogen as a decoding key, suggesting a new generation of information encryption and anti-counterfeiting applications.

  16. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  17. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were us...

  18. Universal Numeric Segmented Display

    CERN Document Server

    Azad, Md Abul kalam; Kamruzzaman, S M

    2010-01-01

    Segmentation display plays a vital role to display numerals. But in today's world matrix display is also used in displaying numerals. Because numerals has lots of curve edges which is better supported by matrix display. But as matrix display is costly and complex to implement and also needs more memory, segment display is generally used to display numerals. But as there is yet no proposed compact display architecture to display multiple language numerals at a time, this paper proposes uniform display architecture to display multiple language digits and general mathematical expressions with higher accuracy and simplicity by using a 18-segment display, which is an improvement over the 16 segment display.

  19. SpiderFab: Architecture for On-Orbit Construction of Kilometer-Scale Apertures Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The SpiderFab effort has investigated the value proposition and technical feasibility of radically changing the way we build and deploy spacecraft in order to escape...

  20. Preparation and Purification of IgY and Fab' Against Human Rotavirus%抗轮状病毒IgY和Fab'的分离与纯化

    Institute of Scientific and Technical Information of China (English)

    孙淑清; 段春燕; 胡彦涛; 孟岩

    2006-01-01

    抗轮状病毒IgY可用两步盐析结合凝胶过滤从蛋黄中分离出来,用SDS-PAGE检测其纯度可达到95%以上.纯的IgY经胃蛋白酶分解得到的抗体片断(Fab'),经SDS-PAGE和MALDI质谱法测定,其纯度达到99%以上.结果表明,所设计的分离抗轮状病毒IgY和Fab'的方法简单、有效.经ELISA法检测,Fab'的活性仍保持在IgY原始活性的70%以上.

  1. Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

    OpenAIRE

    Bondt, Albert; Wuhrer, Manfred; Kuijper, Martijn; Hazes, Mieke; Dolhain, Radboud

    2016-01-01

    Background Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from i...

  2. Anti-tumor Effect and Mechanism of SEA-Fab' Coupled Protein on Gastric Tumor

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The anti-tumor effect and mechanism of SEA-Fab' coupled protein on gastric tumor was studied. The target cell Walker-256 was treated with SEA-Fab' synthesized chemically or SEA respectively for 24 h, 36 h or 72 h. PBMC+Walke-256 cells served as controls. The apoptotic index of SEA-Fab' against effector cells was detected. In the mouse gastric cancer models (n=60), SEAFab', SEA and normal saline was injected in experimental group, SEA group and control group respectively. The occurrence and weight of tumor was observed. The results showed that the apoptotic index was significantly higher in the SEA-Fab' (34.6 %-68.9 %) and SEA group (15.5 %-31.9 %) than in PBMC+Walker-256 group (5.5 %-12.8 %) with the difference being significant (P<0.01). And there was significant difference between SEA-Fab' group and SEA group (P <0. 01). The tumor weight in SEA-Fab', SEA and control groups was 3. 64±0. 53 g, 0. 78±0.26 g and 0.49 ±0.17 g respectively with the difference being statistically significant between the SEAFab' group, SEA group and the control group (P<0.01). In the SEA-Fab's and SEA groups,there were CD4+ T and CD8+ T cell infiltrates, but in the cotnrol group, no or few T lymphocytes were seen in the mouse tumor tissue. It was concluded that SEA-Fab' was more effective to activate T lymphocytes to kill the tumor cells than SEA used alone. It was feasibility by using the monoclonal antibody as carrier to perform the targeted immunotherapy of gatric tumor.

  3. Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

    Science.gov (United States)

    Schofield, Desmond M; Templar, Alex; Newton, Joseph; Nesbeth, Darren N

    2016-07-01

    Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016.

  4. Immunoglobulin fragments, F(ab')2, that are cytotoxic to enzyme-treated cells.

    Science.gov (United States)

    Holtgrewe, E M; Killion, J J

    1984-07-01

    Bivalent immunoglobulin fragments of IgG, F(ab')2, prepared from normal murine sera were found to be cytotoxic to neuraminidase-treated cells. The fragments were cytotoxic to both allogenic and syngeneic targets (with respect to the source of the sera), suggesting that the antigen bound by the F(ab')2 is not related to the major histocompatibility locus of mice (H-2).

  5. Functional characterization of triclosan-resistant enoyl-acyl-carrier protein reductase (FabV in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Yong-Heng Huang

    2016-11-01

    Full Text Available Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR, FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholera fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acylhomoserine lactones (AHLs production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore to the fabV mutant strain the ability to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity.

  6. Functional Characterization of Triclosan-Resistant Enoyl-acyl-carrier Protein Reductase (FabV) in Pseudomonas aeruginosa

    Science.gov (United States)

    Huang, Yong-Heng; Lin, Jin-Shui; Ma, Jin-Cheng; Wang, Hai-Hong

    2016-01-01

    Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR), FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholerae fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acyl-homoserine lactones (AHLs) production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore the fabV mutant strain to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity. PMID:27965638

  7. Matching the decay half-life with the biological half-life: ImmunoPET imaging with (44)Sc-labeled cetuximab Fab fragment.

    Science.gov (United States)

    Chakravarty, Rubel; Goel, Shreya; Valdovinos, Hector F; Hernandez, Reinier; Hong, Hao; Nickles, Robert J; Cai, Weibo

    2014-12-17

    Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with (44)Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after (44)Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of (44)Sc-CHX-A″-DTPA-Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using (44)Sc-CHX-A″-DTPA-Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.

  8. Structural characterisation of the fatty acid biosynthesis enzyme FabF from the pathogen Listeria monocytogenes

    Science.gov (United States)

    Soares da Costa, Tatiana P.; Nanson, Jeffrey D.; Forwood, Jade K.

    2017-01-01

    Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes, as the number of drug resistant strains continues to increase. A promising target is the β-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.35 Å crystal structure of FabF from L. monocytogenes, providing an excellent platform for the rational design of novel inhibitors. By comparing the structure of L. monocytogenes FabF with other published bacterial FabF structures in complex with known inhibitors and substrates, we highlight conformational changes within the active site, which will need to be accounted for during drug design and virtual screening studies. This high-resolution structure of FabF represents an important step in the development of new classes of antimicrobial agents targeting FabF for the treatment of listeriosis. PMID:28045020

  9. Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

    Science.gov (United States)

    Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Parrish, Colin R; Hafenstein, Susan

    2016-11-01

    Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM.

  10. 68Ga-labelled phage-display selected peptides as tracers for positron emission tomography imaging of Staphylococcus aureus biofilm-associated infections: Selection, radiolabelling and preliminary biological evaluation

    DEFF Research Database (Denmark)

    Nielsen, Karin M.; Kyneb, Majbritt H.; Alstrup, Aage Kristian Olsen

    2016-01-01

    stable in product formulation, whereas 68Ga-A8-K-DOTA was unstable. The S. aureus binding of 68Ga-A11-GSGK-DOTA and 68Ga-A9-K-DOTA was observed in vitro, though blocking of the binding was not possible by excess of cold peptide. The 68Ga-A9-K-DOTA was degraded slowly in vitro, while the combined in vivo......Introduction: In humans, Staphylococcus aureus is a major cause of skin and deep-sited infections, often associated with the formation of biofilms. The purpose of the study was to identify and 68Ga-label phage-display peptides selected against S. aureus biofilm and evaluate their potential......-N,N′,N″,N‴-tetraacetic acid (DOTA) conjugated via a lysine linker (K), and for A11 also a glycine-serine-glycine spacer (GSG). The 68Ga-labelling of A8-K-DOTA, A9-K-DOTA, and A11-GSGK-DOTA were optimized in regards to: pre-purification methods of generator eluates, labelling solution, time, and temperature, as well...

  11. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    Science.gov (United States)

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability).

  12. ANTITUMOR EFFECTS OF PINGYANGMYCIN CONJUGATED WITH Fab' FRAGMENT OF MONOCLONAL ANTIBODY%平阳霉素与单克隆抗体Fab'片段偶联物的抗肿瘤作用

    Institute of Scientific and Technical Information of China (English)

    刘小云; 刘秀均; 李毅; 王维刚; 甄永苏

    2000-01-01

    目的研制一种以单抗Fab'片段为基础的抗肿瘤导向药物.方法制备单抗3A5 Fab'片段及其与平阳霉素(PYM)偶联物Fab'-PYM后,测定Fab'-PYM与肿瘤细胞的免疫反应性、偶联物中PYM的抑菌活性、对肿瘤细胞的杀伤作用和体内抑瘤作用.结果 Fab'及Fab'-PYM保持了与靶细胞C26的免疫反应性;偶联物中PYM的抑菌活性为游离PYM的15%;Fab'-PYM对C26细胞的杀伤作用强于PYM;对非靶细胞KB的杀伤作用与PYM相似;ip和iv给药,Fab'-PYM对小鼠皮下接种的肠癌26生长抑制作用均强于3A5-PYM和PYM.结论 Fab'-PYM具有比PYM及3A5-PYM更强的体内外抗肿瘤作用.

  13. Fab Four: When John and George play gravitation and cosmology

    CERN Document Server

    Bruneton, Jean-Philippe; Kanfon, Antonin; Hees, Aurélien; Schlögel, Sandrine; Füzfa, André

    2012-01-01

    Scalar-tensor theories of gravitation have recently regained a great interest after the discovery of the Chameleon mechanism and of the Galileon models. The former allows, in principle, to reconcile the presence of cosmological scalar fields with the constraints from experiments at the Solar System scale. The latter open up the possibility of building inflationary models that, among other things, do not need ad hoc potentials. Further generalizations have finally led to the most general tensor-scalar theory, recently dubbed the "Fab Four", with only first and second order derivatives of the fields in the equations of motion and that self-tune to a vanishing cosmological constant. This model has a very rich phenomenology that needs to be explored and confronted with experimental data in order to constrain a very large parameter space. In this paper, we present some results regarding a subset of the theory named "John", which corresponds to a non-minimal derivative coupling between the scalar field and the Eins...

  14. Fab Four: When John and George Play Gravitation and Cosmology

    Directory of Open Access Journals (Sweden)

    J.-P. Bruneton

    2012-01-01

    Full Text Available Scalar-tensor theories of gravitation attract again a great interest since the discovery of the Chameleon mechanism and of the Galileon models. The former allows reconciling the presence of a scalar field with the constraints from Solar System experiments. The latter leads to inflationary models that do not need ad hoc potentials. Further generalizations lead to a tensor-scalar theory, dubbed the “Fab Four,” with only first and second order derivatives of the fields in the equations of motion that self-tune to a vanishing cosmological constant. This model needs to be confronted with experimental data in order to constrain its large parameter space. We present some results regarding a subset of this theory named “John,” which corresponds to a nonminimal derivative coupling between the scalar field and the Einstein tensor in the action. We show that this coupling gives rise to an inflationary model with very unnatural initial conditions. Thus, we include the term named “George,” namely, a nonminimal, but nonderivative, coupling between the scalar field and Ricci scalar. We find a more natural inflationary model, and, by performing a post-Newtonian analysis, we derive the set of equations that constrain the parameter space with data from experiments in the Solar System.

  15. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  16. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  17. Population Pharmacokinetics of an Indian F(ab'2 Snake Antivenom in Patients with Russell's Viper (Daboia russelii Bites.

    Directory of Open Access Journals (Sweden)

    Geoffrey K Isbister

    Full Text Available There is limited information on antivenom pharmacokinetics. This study aimed to investigate the pharmacokinetics of an Indian snake antivenom in humans with Russell's viper bites.Patient data and serial blood samples were collected from patients with Russell's viper (Daboia russelii envenoming in Sri Lanka. All patients received Indian F(ab'2 snake antivenom manufactured by VINS Bioproducts Ltd. Antivenom concentrations were measured with sandwich enzyme immunoassays. Timed antivenom concentrations were analysed using MONOLIXvs4.2. One, two and three compartment models with zero order input and first order elimination kinetics were assessed. Models were parameterized with clearance (CL, intercompartmental clearance (Q, central compartment volume (V and peripheral compartment volume (VP. Between-subject-variability (BSV on relative bioavailability (F was included to account for dose variations. Covariates effects (age, sex, weight, antivenom batch, pre-antivenom concentrations were explored by visual inspection and in model building. There were 75 patients, median age 57 years (40-70 y and 64 (85% were male. 411 antivenom concentration data points were analysed. A two compartment model with zero order input, linear elimination kinetics and a combined error model best described the data. Inclusion of BSV on F and weight as a covariate on V improved the model. Inclusion of pre-antivenom concentrations or different batches on BSV of F did not. Final model parameter estimates were CL,0.078 L h(-1, V,2.2L, Q,0.178 L h(-1 and VP,8.33L. The median half-life of distribution was 4.6 h (10-90%iles:2.6-7.1 h and half-life of elimination, 140 h (10th-90th percentilesx:95-223h.Indian F(ab'2 snake antivenom displayed biexponential disposition pharmacokinetics, with a rapid distribution half-life and more prolonged elimination half-life.

  18. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.

    Science.gov (United States)

    Paul, Suparna; Connor, Judy; Nesspor, Tom; Haytko, Peter; Boakye, Ken; Chiu, Mark L; Jiang, Haiyan

    2016-05-01

    Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

  19. RetroFab: A Design Tool for Retrofitting Physical Interfaces using Actuators, Sensors and 3D Printing

    OpenAIRE

    2016-01-01

    We present RetroFab, an end-to-end design and fabrication environment that allows non-experts to retrofit physical interfaces. Our approach allows for changing the layout and behavior of physical interfaces. Unlike customizing software interfaces, physical interfaces are often challenging to adapt because of their rigidity. With RetroFab, a new physical interface is designed that serves as a proxy interface for the legacy controls that are now operated by actuators. RetroFab makes this concep...

  20. Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot.

    Science.gov (United States)

    Zhang, Qian; Lin, Hong; Sui, Jianxin; Wang, Jingxue; Cao, Limin

    2015-01-01

    In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry.

  1. 3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

    Science.gov (United States)

    2013-07-01

    FabI, but share low sequence identity and are poorly inhibited by triclosan.25,26 S. pneumoniae and P. aeruginosa contain FabK,24 and Vibrio cholerae ,27...Massengo-Tiasse, R. P.; Cronan, J. E. Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase. J. Biol. Chem. 2008, 283, 1308...of enoyl- (acyl-carrier protein) reductase, FabV, from Vibrio fischeri. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2012, 68, 78−80. (27

  2. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    Science.gov (United States)

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-01

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  3. Management of Tissue Loss After Agkistrodon Snakebite: Appropriate Use of Crotalidae-Fab Antivenin.

    Science.gov (United States)

    Larson, Kenneth W; Schaefer, Keith R; Austin, Cindy; Norton, Rhy; Finley, Phillip J

    2016-01-01

    Although initially created for the treatment of rattlesnake (genus: Crotalus) bites, Crotalidae-Fab antivenin is used to treat many different pit viper envenomations. However, the efficacy of Crotalidae-Fab in preventing tissue loss from copperhead (Agkistrodon contortrix) or cottonmouth (Agkistrodon piscivorus) snakebites remains unclear. Recent reports show that Agkistrodon-related bites rarely require treatment beyond simple observation and pain control. The purpose of this study was to examine the amount of tissue loss in patients who received Crotalidae-Fab compared with those who did not after an Agkistrodon bite. After institutional review board approval, a retrospective study was completed at a Level 1 trauma center. Between 2009 and 2013, a total of 57 snakebites were identified. Of the 57 bites, the snake species was documented in 36 cases including 31 copperheads, 1 cottonmouth, and 4 rattlesnakes. The other 21 bites were from unknown or nonvenomous species. Of the 32 Agkistrodon-related bites, 15 patients received Crotalidae-Fab (average of 3 vials administered) and 17 did not receive Crotalidae-Fab. None of the 32 patients, regardless of treatment option, had tissue loss or required surgical interventions. Only 1 patient received Crotalidae-Fab and debridement of a vesicle associated with the bite. No clinically significant differences were observed between the groups. These findings support previous literature that failed to show added benefit of Crotalidae-Fab treatment for Agkistrodon bites beyond patient comfort and pain control. Evaluation of current protocols for Agkistrodon envenomations is warranted. Snakebite wound education in trauma physicians and nurses may decrease unnecessary use of antivenom medication.

  4. Improved Binding Activity of Antibodies against Major Histocompatibility Complex Class I Chain-Related Gene A by Phage Display Technology for Cancer-Targeted Therapy

    Directory of Open Access Journals (Sweden)

    Achara Phumyen

    2012-01-01

    Full Text Available Major histocompatibility complex class I chain-related gene A (MICA is an NKG2D ligand that is over-expressed under cellular stress including cancer transformation and viral infection. High expression of MICA in cancer tissues or patients' sera is useful for prognostic or follow-up markers in cancer patients. In this study, phage display technology was employed to improve antigen-binding activities of anti-MICA monoclonal antibodies (WW2G8, WW6B7, and WW9B8. The 12 amino acid residues in the complementarity determining regions (CDRs on the V domain of the heavy chain CDR3 (HCDR3 of these anti-MICA antibodies were modified by PCR-random mutagenesis, and phages displaying mutated anti-MICA Fab were constructed. After seven rounds of panning, five clones of phages displaying mutant anti-MICA Fab which exhibited 3–7-folds higher antigen-binding activities were isolated. Two clones of the mutants (phage-displayed mutant Fab WW9B8.1 and phage-displayed mutant Fab WW9B8.21 were confirmed to have antigen-binding specificity for cell surface MICA proteins by flow cytometry. These phage clones are able to recognize MICA in a native form according to positive results obtained by indirect ELISA and flow cytometry. Thus, these phage particles could be potentially used for further development of nanomedicine specifically targeting cancer cells expressing MICA proteins.

  5. A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA.

    Science.gov (United States)

    Moynié, Lucile; Hope, Anthony G; Finzel, Kara; Schmidberger, Jason; Leckie, Stuart M; Schneider, Gunter; Burkart, Michael D; Smith, Andrew D; Gray, David W; Naismith, James H

    2016-01-16

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50=5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.

  6. Colorimetric evaluation of display performance

    Science.gov (United States)

    Kosmowski, Bogdan B.

    2001-08-01

    The development of information techniques, using new technologies, physical phenomena and coding schemes, enables new application areas to be benefited form the introduction of displays. The full utilization of the visual perception of a human operator, requires the color coding process to be implemented. The evolution of displays, from achromatic (B&W) and monochromatic, to multicolor and full-color, enhances the possibilities of information coding, creating however a need for the quantitative methods of display parameter assessment. Quantitative assessment of color displays, restricted to photometric measurements of their parameters, is an estimate leading to considerable errors. Therefore, the measurements of a display's color properties have to be based on spectral measurements of the display and its elements. The quantitative assessment of the display system parameters should be made using colorimetric systems like CIE1931, CIE1976 LAB or LUV. In the paper, the constraints on the measurement method selection for the color display evaluation are discussed and the relations between their qualitative assessment and the ergonomic conditions of their application are also presented. The paper presents the examples of using LUV colorimetric system and color difference (Delta) E in the optimization of color liquid crystal displays.

  7. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

    Science.gov (United States)

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

  8. Anti-fouling properties of Fab' fragments immobilized on silane-based adlayers

    Science.gov (United States)

    Crivianu-Gaita, Victor; Romaschin, Alexander; Thompson, Michael

    2015-12-01

    Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab') in biosensors. One Fab' linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab' fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab' fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab'-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection.

  9. Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry

    Science.gov (United States)

    Wecksler, Aaron T.; Kalo, Matt S.; Deperalta, Galahad

    2015-12-01

    A proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes.

  10. Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

    Energy Technology Data Exchange (ETDEWEB)

    Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.; Horne, David A.; Williams, John C., E-mail: jcwilliams@coh.org [Beckman Research Institute of City of Hope, 1710 Flower Street, Duarte, CA 91010 (United States)

    2016-05-23

    An overview of cyclization strategies of a Fab-binding peptide to maximize affinity. Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex.

  11. Reorienting the Fab domains of trastuzumab results in potent HER2 activators.

    Directory of Open Access Journals (Sweden)

    Justin M Scheer

    Full Text Available The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab'(2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

  12. Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

    Science.gov (United States)

    Shatz, Whitney; Hass, Philip E; Mathieu, Mary; Kim, Hok Seon; Leach, Kim; Zhou, Michelle; Crawford, Yongping; Shen, Amy; Wang, Kathryn; Chang, Debby P; Maia, Mauricio; Crowell, Susan R; Dickmann, Leslie; Scheer, Justin M; Kelley, Robert F

    2016-09-01

    We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

  13. Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

    Science.gov (United States)

    Nanson, Jeffrey D; Forwood, Jade K

    2015-01-01

    Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

  14. Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Nanson

    Full Text Available Ketoacyl-acyl carrier protein reductases (FabG are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP linked thioesters within the bacterial type II fatty acid synthesis (FASII pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG, the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

  15. Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies.

    Science.gov (United States)

    Lipovsek, Dasa; Lippow, Shaun M; Hackel, Benjamin J; Gregson, Melissa W; Cheng, Paul; Kapila, Atul; Wittrup, K Dane

    2007-05-11

    The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from

  16. Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards & Labeling Programs for Copy Machines, External Power Supplies, LED Displays, Residential Gas Cooktops and Televisions

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Nina [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Zhou, Nan [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Fridley, David [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2012-03-01

    This report presents a technical review of international minimum energy performance standards (MEPS), voluntary and mandatory energy efficiency labels and test procedures for five products being considered for new or revised MEPS in China: copy machines, external power supply, LED displays, residential gas cooktops and flat-screen televisions. For each product, an overview of the scope of existing international standards and labeling programs, energy values and energy performance metrics and description and detailed summary table of criteria and procedures in major test standards are presented.

  17. Targeting human prostate cancer with (111) In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, S.; Rij, C.M. van; Franssen, G.M.; Fracasso, G.; Helfrich, W.; Eek, A.; Oyen, W.J.G.; Colombatti, M.; Boerman, O.C.

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing sub

  18. Targeting human prostate cancer with In-111-labeled D2B IgG, F(ab ')(2) and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, Susanne; van Rij, Catharina M.; Franssen, Gerben M.; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J.; Colombatti, Marco; Boerman, Otto C.

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab)(2) and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing su

  19. Treatment of digitalis intoxication with emphasis on the clinical use of digoxin immune Fab.

    Science.gov (United States)

    Allen, N M; Dunham, G D

    1990-10-01

    Many studies and cases of digitalis intoxication have been reported since the time of William Withering's first publication in 1785. Recognition and management of digitalis toxicity is challenging. Before digoxin immune Fab was commercially available, treatment consisted of managing the signs and symptoms of toxicity until the digitalis was eliminated. Digoxin immune Fab offers a safe, effective, and specific method of quickly reversing digitalis toxicity. Factors that must be considered with the clinical use of this agent include the dosage calculation, administration technique, postdose monitoring, pharmacokinetics, mechanism of action, interference with commercially available digoxin assays, partial neutralizing dosing, rebound of free digoxin, and indications for use. For severe, life-threatening toxicity, digoxin immune Fab is the treatment of choice.

  20. Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

    Energy Technology Data Exchange (ETDEWEB)

    Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.; Greenberg, Harry B.; Harrison, Stephen C.; Dormitzer, Philip R.; (Stanford-MED); (CH-Boston)

    2009-06-17

    Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

  1. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.

    Science.gov (United States)

    van der Neut Kolfschoten, Marijn; Schuurman, Janine; Losen, Mario; Bleeker, Wim K; Martínez-Martínez, Pilar; Vermeulen, Ellen; den Bleker, Tamara H; Wiegman, Luus; Vink, Tom; Aarden, Lucien A; De Baets, Marc H; van de Winkel, Jan G J; Aalberse, Rob C; Parren, Paul W H I

    2007-09-14

    Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

  2. 3D printing in social education: Eki-Fab and student PBL

    Science.gov (United States)

    Makino, Masato; Saito, Azusa; Kodama, Mai; Takamatsu, Kyuuichiro; Tamate, Hideaki; Sakai, Kazuyuki; Wada, Masato; Khosla, Ajit; Kawakami, Masaru; Furukawa, Hidemitsu

    2017-04-01

    Additive manufacturing or 3D printer is one of the most innovative material processing methods. We are considering that human resources for 3D printing would be needed in the future. To educate the abilities of the digital fabrication, we have the public digital fabrication space "Eki-Fab" for junior and high school students and Project Based Learning (PBL) class for undergraduate students. Eki-Fab is held on every Saturday at the Yonezawa train station. In the "Eki-Fab", anybody can study the utilizing of 3D printer and modeling technics under the instruction of staff in Yamagata University. In the PBL class, we have the class every Thursday. The students get the techniques of the digital fabrication through the PBL.

  3. Yeast display of antibody fragments: a discovery and characterization platform

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Michael; Siegel, Robert W.

    2004-07-01

    This review will focus on some of the novel attributes of the yeast surface display platform for the discovery and characterization of novel affinity reagents, optimization of those reagents, and novel uses of the platform. This is not intended to serve as an exhaustive review on the broader topic of general scFv technologies (see Winter et al., 1994; Smith and Petrenko, 1997; Bradbury et al., 2003) Furthermore, the scFv format of antibodies are easily manipulated through molecular cloning into a number of other formats such IgG, Fab, diabodies and such, for use in down steam applications and the reader is encouraged to read ?IgG?, ?Fab?, or your favorite format whenever scFv is seen in this review. This review is presented in 5 parts; (1) description of yeast display and its components, (2) library types and construction methods, (3) screening approaches for non-immune libraries and benefits, (4) screening approaches for directed evolution, kinetic on and off rates and (5) epitope complementation binning of clones.

  4. 20 CFR 30.908 - How will the FAB evaluate new medical evidence submitted to challenge the impairment...

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How will the FAB evaluate new medical... Medical Evidence of Impairment § 30.908 How will the FAB evaluate new medical evidence submitted to... impairment evaluation that differs from the impairment evaluation relied upon by the district office, the...

  5. The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F-ab and F-c fragments

    NARCIS (Netherlands)

    Vermeer, AWP; Norde, W; van Amerongen, A

    2000-01-01

    The unfolding and further denaturation of IgG and its F-ab and F-c fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the F-ab and F-c units was ret

  6. Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma

    Institute of Scientific and Technical Information of China (English)

    Jin-Liang Xing; Xiang-Min Yang; Xi-Ying Yao; Fei Song; Zhi-Nan Chen

    2004-01-01

    AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E.coli respectively and refold them into chimeric Fab (cFab) antibody.METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then,the competent E. colicells were transformed by the recombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis. The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting,ELISA and HPLC.RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively. Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 μg/mL. The renatured cFab could specifically bind to related antigen with high affinity.CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.

  7. High affinity antibodies against Lex and sialyl Lex from a phage display library.

    Science.gov (United States)

    Dinh, Q; Weng, N P; Kiso, M; Ishida, H; Hasegawa, A; Marcus, D M

    1996-07-15

    Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.

  8. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

    Science.gov (United States)

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  9. Research advances on Fabs antibodies%Fab类抗体的研究进展

    Institute of Scientific and Technical Information of China (English)

    刘美君; 高向东; 徐晨

    2014-01-01

    In genetic engineering antibodies,Fabs antibodies have many advantages,such as lower relative molecular mass, specific tissue distribution and lower immunogenicity, which have made Fabs antibodies the research hotspot for the past few decades. Besides the widely used enzymatic generation, multiple expression systems which include E. coli. and insects are used in the generation of Fabs antibodies. E. coli. expression system is most thoroughly explored and has been used in the preparation of clinical Fabs drugs. There are six Fab drugs that have been approved by FDA, such as certolizumabpegol and many Fabs are still in clinical research. This review discusses the recent research progress of Fabs drugs, including Fabs′ structure and characteristics, the generation and expression of Fabs, strategies to increase production and clinical applications, with the emphasis on generation, expression and clinical applications.%在基因工程抗体中,Fab类(Fabs)抗体有许多优势,如相对分子质量小、组织分布特异性强和免疫原性低等,使Fab类抗体成为近几十年来的研究热点。除了应用较广泛的体外酶消化法外,多种表达系统(如大肠杆菌和昆虫等)皆用于Fab类抗体的获得,其中大肠杆菌表达的系统研究最为彻底,且已经应用于制备Fab类临床药物。目前已经有赛妥珠单抗等6种Fab类抗体药物通过了美国FDA申请并上市,并有多种Fab类药物正处于临床试验阶段。本文分别对Fab类抗体的基本结构及特点、Fab类抗体的生成、表达和提高产量的策略、临床应用的研究进展进行介绍。

  10. International Foot and Ankle Biomechanics Community (i-FAB: past, present and beyond

    Directory of Open Access Journals (Sweden)

    Rosenbaum Dieter

    2009-06-01

    Full Text Available Abstract The International Foot and Ankle Biomechanics Community (i-FAB is an international collaborative activity which will have an important impact on the foot and ankle biomechanics community. It was launched on July 2nd 2007 at the foot and ankle session of the International Society of Biomechanics (ISB meeting in Taipei, Taiwan. i-FAB is driven by the desire to improve our understanding of foot and ankle biomechanics as it applies to health, disease, and the design, development and evaluation of foot and ankle surgery, and interventions such as footwear, insoles and surfaces.

  11. High quality mask storage in an advanced Logic-Fab

    Science.gov (United States)

    Jähnert, Carmen; Fritsche, Silvio

    2012-02-01

    High efficient mask logistics as well as safe and high quality mask storage are essential requirements within an advanced lithography area of a modern logic waferfab. Fast operational availability of the required masks at the exposure tool with excellent mask condition requires a safe mask handling, safeguarding of high mask quality over the whole mask usage time without any quality degradation and an intelligent mask logistics. One big challenge is the prevention of haze on high advanced phase shift masks used in a high volume production line for some thousands of 248nm or 193nm exposures. In 2008 Infineon Dresden qualified a customer specific developed semi-bare mask storage system from DMSDynamic Micro Systems in combination with a high advanced mask handling and an interconnected complex logistic system. This high-capacity mask storage system DMS M1900.22 for more than 3000 masks with fully automated mask and box handling as well as full-blown XCDA purge has been developed and adapted to the Infineon Lithotoollandscape using Nikon and SMIF reticle cases. Advanced features for ESD safety and mask security, mask tracking via RFID and interactions with the exposure tools were developed and implemented. The stocker is remote controlled by the iCADA-RSM system, ordering of the requested mask directly from the affected exposure tool allows fast access. This paper discusses the advantages and challenges for this approach as well as the practical experience gained during the implementation of the new system which improves the fab performance with respect to mask quality, security and throughput. Especially the realization of an extremely low and stable humidity level in addition with a well controlled air flow at each mask surface, preventing masks from haze degradation and particle contamination, turns out to be a notable technical achievement. The longterm stability of haze critical masks has been improved significantly. Relevant environmental parameters like

  12. AGILE integration into APC for high mix logic fab

    Science.gov (United States)

    Gatefait, M.; Lam, A.; Le Gratiet, B.; Mikolajczak, M.; Morin, V.; Chojnowski, N.; Kocsis, Z.; Smith, I.; Decaunes, J.; Ostrovsky, A.; Monget, C.

    2015-09-01

    mix logic Fab) in term of product and technology portfolio AGILE corrects for up to 120nm of product topography error on process layer with less than 50nm depth of focus Based on tool functionalities delivered by ASML and on high volume manufacturing requirement, AGILE integration is a real challenge. Regarding ST requirements "Automatic AGILE" functionality developed by ASML was not a turnkey solution and a dedicated functionality was needed. A "ST homemade AGILE integration" has been fully developed and implemented within ASML and ST constraints. This paper describes this integration in our Advanced Process Control platform (APC).

  13. 20 CFR 30.317 - Can the FAB request a further response from the claimant or return a claim to the district office?

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can the FAB request a further response from....317 Can the FAB request a further response from the claimant or return a claim to the district office? At any time before the issuance of its final decision, the FAB may request that the claimant...

  14. 20 CFR 30.312 - What will the FAB do if the claimant objects to the recommended decision but does not request a...

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false What will the FAB do if the claimant objects....312 What will the FAB do if the claimant objects to the recommended decision but does not request a... period of time allotted in § 30.310 but does not request a hearing, the FAB will consider any...

  15. Displaying gray shades in liquid crystal displays

    Indian Academy of Sciences (India)

    T N Ruckmongathan

    2003-08-01

    Quality of image in a display depends on the contrast, colour, resolution and the number of gray shades. A large number of gray shades is necessary to display images without any contour lines. These contours are due to limited number of gray shades in the display causing abrupt changes in grayness of the image, while the original image has a gradual change in brightness. Amplitude modulation has the capability to display a large number of gray shades with minimum number of time intervals [1,2]. This paper will cover the underlying principle of amplitude modulation, some variants and its extension to multi-line addressing. Other techniques for displaying gray shades in passive matrix displays are reviewed for the sake of comparison.

  16. Invisible Display in Aluminum

    DEFF Research Database (Denmark)

    Prichystal, Jan Phuklin; Hansen, Hans Nørgaard; Bladt, Henrik Henriksen

    2005-01-01

    for an integrated display in a metal surface is often ruled by design and functionality of a product. The integration of displays in metal surfaces requires metal removal in order to clear the area of the display to some extent. The idea behind an invisible display in Aluminum concerns the processing of a metal...

  17. Quench-condensing superconducting thin films using the Fab on a Chip approach

    Science.gov (United States)

    Han, Han; Imboden, Matthias; Del Corro, Pablo; Stark, Thomas; Lally, Richard; Pardo, Flavio; Bolle, Cristian; Bishop, David

    Micro-electromechanical systems (MEMS) being manufactured in a macroscopic fab inspires the idea of getting the process further down to fabricate even smaller structures, namely nano-structures, using MEMS. The Fab on a Chip concept was proposed based on such ideas. By implementing the final-step, additive fabrication approach, manufacturing, characterization and experiments of nano-structures are integrated in-situ. Due to the miniature size of MEMS, the thickness precision is significantly improved while the power consumption is significantly depressed, making the quench-condensation of very thin films well controlled and easily achievable. Among various types of nano-structures, quench-condensed superconducting thin films are of great interest for physicists. Here we present such experiments done on superconducting thin films quench-condensed using the Fab on a Chip. We show that we are able to fabricate very thin films with its thickness precisely controlled, and the base temperature kept under ~3K during the process. The resistivity data demonstrates the high purity and uniformity of the film, as well as the annealing effect when cycling to higher temperatures. Based on the tremendous results obtained from the superconducting thin films, more complex nano-circuits can be fabricated and investigated using the Fab on a Chip, enabling a new approach for novel condensed matter physics experiments. This research is funded by the NSF through their CMMI division. This research is funded by the NSF through their CMMI division.

  18. Integrated MEMS mass sensor and atom source for a ``Fab on a Chip''

    Science.gov (United States)

    Han, Han; Imboden, Matthias; Stark, Thomas; Bishop, David

    2014-03-01

    ``Fab on a Chip'' is a new concept suggesting that the semiconductor fabrication facility can be integrated into a single silicon chip for nano-manufacturing. Such a chip contains various MEMS devices which can work together, operating in a similar way as a conventional fab does, to fabricate nano-structures. Here we present two crucial ``Fab on a chip'' components: the MEMS mass sensor and atomic evaporation source. The mass sensor is essentially a parallel plate capacitor with one suspended plate. When incident atoms deposit on the suspended plate, the mass change of the plate can be measured by detecting the resonant frequency shift. Using the mass sensor, a mass resolution of 3 fg is achieved. The MEMS evaporation source consists of a polysilicon plate suspended by two electrical leads with constrictions. By resistively heating the plate, this device works as a tunable atom flux source. By arranging many of these devices into an array, one can build a multi-element atom evaporator. The mass sensor and atom source are integrated so that the mass sensor is used to monitor and characterize the atomic flux. A material source and a sensor to monitor the fabrication are two integral components for our ``Fab on a Chip.''

  19. 75 FR 9438 - Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung Electronics Corporation...

    Science.gov (United States)

    2010-03-02

    ... Employment and Training Administration Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung..., applicable to workers of Samsung Austin Semiconductor, LLC, a subsidiary of Samsung Electronics Corporation... Systems, Inc. were employed on-site at the Austin, Texas location of Samsung Austin Semiconductor, LLC,...

  20. Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

    NARCIS (Netherlands)

    A. Bondt (Albert); M. Wuhrer (Manfred); T.M. Kuijper (Martijn); J.M.W. Hazes (Mieke); R.J.E.M. Dolhain (Radboud)

    2016-01-01

    textabstractBackground: Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods:

  1. Part I, FAB evaluation & application trials AFUE measurements: Part II, Integrated heating system (IHS) development

    Energy Technology Data Exchange (ETDEWEB)

    Leigh, R.W. [Brookhaven National Lab., Upton, NY (United States); Fisher, L. [BNL Consultant, Colrain, MA (United States)

    1996-07-01

    An oil burner/boiler efficiency test stand has been set up in the BNL oil heat laboratory which can measure the Annual Fuel Utilization Efficiency (AFUE) of burner/boiler combinations in accordance with ASHRAE and DOE standards. Measurements include both steady state efficiencies and heat-up and cool-down characteristics so that cycling effects can be included in an estimate of seasonal average performance. In addition to AFUE measurements, the direct conversion of fuel energy content to enthalpy increase in the boiler water is monitored. The system is largely automated, with most control functions under computer control and data taken electronically and permanently recorded on disks for future reference. To date, a retention-head burner and a fan atomized burner (FAB) have been tested in a steel boiler, the latter operating at two different fuel flow rates. The results are presented below, and verify that the very tight construction of the FAB`s fan results in a significant decrease in off-cycle sensible heat losses. Tests were also performed on a center-flue water heater fired with a conventional retention-head burner and with an FAB. The tests conformed to DOE standard procedures for hot water heaters, and the results are discussed below.

  2. Cyclization strategies of meditopes: affinity and diffraction studies of meditope-Fab complexes.

    Science.gov (United States)

    Bzymek, Krzysztof P; Ma, Yuelong; Avery, Kendra A; Horne, David A; Williams, John C

    2016-06-01

    Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic `pocket' and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope-Fab complex.

  3. Orienterende Fast Atom Bombardment (FAB) experimenten met de VG-70-SQ massaspectrometer

    NARCIS (Netherlands)

    Hove GJ ten; Boer AC den; Burgers PC; Jong APJM de

    1988-01-01

    Eerste orienterende metingen met fast atom bombardment (FAB) ionisatietechniek zijn uitgevoerd. De techniek werd toegepast bij de analyse van korte-keten polypeptiden (n=2-5), cyclosporine, NADP en microperoxidase. Onderzocht werd de invloed van de aard van de matrix (glycerol, thioglycerol) op

  4. Impact of Fab Lab Tulsa on Student Self-Efficacy toward STEM Education

    Science.gov (United States)

    Dubriwny, Nicholas; Pritchett, Nathan; Hardesty, Michelle; Hellman, Chan M.

    2016-01-01

    Student self-confidence is important to any attempt to increase interest and achievement in Science, Technology, Engineering, and Math (STEM) education. This study presents a longitudinal examination of Fab Lab Tulsa's impact on attitude and self-efficacy toward STEM education among middle-school aged students. Paired samples t-test showed a…

  5. Insight into screening immunoglobulin gene combinatorial libraries in a phage display vector: a tale of two antibodies.

    Science.gov (United States)

    Kakinuma, A; Portolano, S; Chazenbalk, G; Rapoport, B; McLachlan, S M

    1997-01-01

    Combinatorial libraries of immunoglobulin genes in "phage display" vectors are a powerful tool for obtaining antigen-specific antibody fragments. To date, this approach has been used to isolate abundant, but not rare, human autoantibodies of IgG class. We have compared the relative efficiencies of panning pComb3 libraries made from intrathyroidal plasma cells for abundant human autoantibodies to thyroid peroxidase (TPO) and rare autoantibodies to the thyrotropin receptor (TSHR). TPO-specific Fab were readily obtained from a library using three different forms of recombinant antigen, (i) purified TPO, (ii) impure TPO in culture medium and, (iii) TPO expressed on the surface of CHO cells. In contrast, TSHR-specific Fab were not isolated. This was the case despite repeated pannings of six libraries from three optimal patients (IgG/kappa and IgG/lambda libraries for each patient). Both purified recombinant TSHR and CHO cells expressing TSHR on their surface were used. Library enrichment was observed on some screenings. However, Fab expressed by individual clones or from enriched libraries were not specific as determined by (i) binding to purified, radio-labeled antigen, (ii) FACS analysis of TSHR on intact CHO cells and, (iii) inhibition of radiolabeled TSH binding. Remarkably, in screening for both TPO- and TSHR-specific Fab, neither library enrichment nor the retention of cDNA inserts of the correct size correlated with obtaining Fab with the antigenic specificity sought. Indeed, excellent enrichment could be observed with conditioned medium from untransfected cells. Our data suggest that the key to isolating rare antibodies from phage display libraries is not the creation of vast libraries of greater diversity or even the development of more stable vectors. Rather, success in this endeavor appears to require reducing the "noise" of non-specific clones in a moderately sized library.

  6. Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

    Science.gov (United States)

    Caballero, A; Foletti, D; Bierdeman, M; Tang, A; Arana, A; Hasa-Moreno, A; Sangalang, E; O'Callaghan, R J

    2014-06-01

    Abstract Purpose: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. Methods: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. Results: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). Conclusions: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

  7. The tumor-inhibitory effectiveness of a novel anti-Trop2 Fab conjugate in pancreatic cancer.

    Science.gov (United States)

    Mao, Yuan; Wang, Xiaoying; Zheng, Feng; Wang, Changjun; Tang, Qi; Tang, Xiaojun; Xu, Ning; Zhang, Huiling; Zhang, Dawei; Xiong, Lin; Liang, Jie; Zhu, Jin

    2016-04-26

    Human trophoblastic cell surface antigen 2 (Trop2) has been reported to act oncogenically. In this study, one-step quantitative real-time polymerase chain reaction (qPCR) test and immunohistochemistry (IHC) analysis with were employed to evaluate the relationship between Trop2 expression and the clinicopathological features of patients with PC. Then a novel anti-Trop2 Fab antibody was conjugated with Doxorubicin (DOX) to form Trop2Fab-DOX, an antibody-drug conjugate. This Trop2Fab-DOX conjugate was characterized by cell ELISA and immunofluorescence assay. MTT and wound healing analyses were used to evaluate the inhibitory effect of Trop2Fab-DOX on PC cell growth in vitro, while xenograft nude mice model was established to examine the tumor-inhibitory effects of PC in vivo. High Trop2 expression was observed in PC tissues and Trop2 expression was associated with several malignant attributes of PC patients, including overall survival. Trop2Fab-DOX can bind to the Trop2-expressing PC cells and provide an improved releasing type of DOX. In addition, Trop2Fab-DOX inhibited the proliferation and suppressed the migration of PC cells in a dose-dependent manner in vitro, while inhibited the growth of PC xenografts in vivo. Trop2 is a specific marker for PC, and a novel Trop2Fab-DOX ADC has a potent antitumor activity.

  8. Docking and molecular dynamics studies on triclosan derivatives binding to FabI.

    Science.gov (United States)

    Yang, Xuyun; Lu, Junrui; Ying, Ming; Mu, Jiangbei; Li, Peichun; Liu, Yue

    2017-01-01

    FabI, enoyl-ACP reductase (ENR), is the rate-limiting enzyme in the last step for fatty acids biosynthesis in many bacteria. Triclosan (TCL) is a commercial bactericide, and as a FabI inhibitor, it can depress the substrate (trans-2-enoyl-ACP) binding with FabI to hinder the fatty acid synthesis. The structure-activity relationship between TCL derivatives and FabI protein has already been acknowledged, however, their combination at the molecular level has never been investigated. This paper uses the computer-aided approaches, such as molecular docking, molecular dynamics simulation, and binding free energy calculation based on the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method to illustrate the interaction rules of TCL derivatives with FabI and guide the development of new derivatives. The consistent data of the experiment and corresponding activity demonstrates that electron-withdrawing groups on side chain are better than electron-donating groups. 2-Hydroxyl group on A ring, promoting the formation of hydrogen bond, is vital for bactericidal effect; and the substituents at 4-position of A ring, 2'-position and 4'-position of B ring benefit antibacterial activity due to forming a hydrogen bond or stabilizing the conformation of active pocket residues of receptor. While the substituents at 3'-position and 5'-position of B ring destroy the π-π stacking interaction of A ring and NAD(+) which depresses the antibacterial activity. This study provides a new sight for designing novel TCL derivatives with superior antibacterial activity.

  9. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  10. Construction, Expression and in vitro Biological Effects of Idiotype Ig Fab Fragment of B-Chronic Lymphocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    Feng WANG; Ping LEI; Ping HU; Lijuan ZHU; Huifeng ZHU; Yue ZHANG; Jing YANG; Guanxin SHEN

    2008-01-01

    Summary: The purpose of this study was to construct expression vectors of idiotype (Id) Smlg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id,and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by 1PTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ, in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. Coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.

  11. Plasmid selection in Escherichia coli using an endogenous essential gene marker

    Directory of Open Access Journals (Sweden)

    Good Liam

    2008-08-01

    Full Text Available Abstract Background Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent. Results The new cloning vector, pFab, enabled selection by triclosan at 1 μM. Interestingly, pFab out-performed the parent pUC19-ampicillin system in cell growth, plasmid stability and plasmid yield. Also, pFab was toxic to host cells in a way that was reversed by triclosan. Therefore, pFab and triclosan are toxic when used alone but in combination they enhance growth and plasmid production through a gene-inhibitor interaction. Conclusion The fabI-triclosan model system provides an alternative plasmid selection method based on essential gene over-expression, without the use of antibiotic-resistance genes and conventional antibiotics.

  12. FabSim: facilitating computational research through automation on large-scale and distributed e-infrastructures

    CERN Document Server

    Groen, Derek; Suter, James; Hetherington, James; Zasada, Stefan; Coveney, Peter

    2015-01-01

    We present FabSim, a toolkit developed to simplify a range of computational tasks for researchers in diverse disciplines. FabSim is flexible, adaptable, and allows users to perform a wide range of tasks with ease. It also provides a systematic way to automate the use of resourcess, including HPC and distributed resources, and to make tasks easier to repeat by recording contextual information. To demonstrate this, we present three use cases where FabSim has enhanced our research productivity. These include simulating cerebrovascular bloodflow, modelling clay-polymer nanocomposites across multiple scales, and calculating ligand-protein binding affinities.

  13. Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

    Science.gov (United States)

    Zhang, Ying; Cui, Weidong; Wecksler, Aaron T.; Zhang, Hao; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2016-07-01

    Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

  14. A new type of pseudothrombocytopenia: EDTA-mediated agglutination of platelets bearing Fab fragments of a chimaeric antibody.

    Science.gov (United States)

    Christopoulos, C G; Machin, S J

    1994-07-01

    In vitro agglutination of platelets leading to low automated platelet counts was observed in EDTA-anticoagulated blood from human volunteers receiving infusions of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb-IIIa. This pseudothrombocytopenia depended on the presence of chimaeric Fab on the platelet surface and was not seen when sodium citrate was used as anticoagulent. Preliminary evidence suggests that this phenomenon might be mediated by immunoglobulin G reactive with the human component of the chimaeric Fab. It is important to exclude pseudothrombocytopenia when low automated platelet counts are reported in association with the administration of chimaeric anti-platelet antibodies.

  15. Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Xin-Yuan Yi; Xian-Ping Li; Dong-Ming Zhou; McReynolds Larry; Xian-Fang Zeng

    2005-01-01

    AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice.METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced.Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P = 0.001), 14.5% (P = 0.074) worm reduction and 61.2% (P = 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA.CONCLUSION: Specific peptides mimic antigenic

  16. Core–shell magnetic nanoparticles display synergistic antibacterial effects against Pseudomonas aeruginosa and Staphylococcus aureus when combined with cathelicidin LL-37 or selected ceragenins

    Science.gov (United States)

    Niemirowicz, Katarzyna; Piktel, Ewelina; Wilczewska, Agnieszka Z; Markiewicz, Karolina H; Durnaś, Bonita; Wątek, Marzena; Puszkarz, Irena; Wróblewska, Marta; Niklińska, Wiesława; Savage, Paul B; Bucki, Robert

    2016-01-01

    Core–shell magnetic nanoparticles (MNPs) are promising candidates in the development of new treatment methods against infections, including those caused by antibiotic-resistant pathogens. In this study, the bactericidal activity of human antibacterial peptide cathelicidin LL-37, synthetic ceragenins CSA-13 and CSA-131, and classical antibiotics vancomycin and colistin, against methicillin-resistant Staphylococcus aureus Xen 30 and Pseudomonas aeruginosa Xen 5, was assessed alone and in combination with core–shell MNPs. Fractional inhibitory concentration index and fractional bactericidal concentration index were determined by microdilution methods. The potential of combined therapy using nanomaterials and selected antibiotics was confirmed using chemiluminescence measurements. Additionally, the ability of tested agents to prevent bacterial biofilm formation was evaluated using crystal violet staining. In most conditions, synergistic or additive effects were observed when combinations of core–shell MNPs with ceragenins or classical antibiotics were used. Our study revealed that a mixture of membrane-active agents such as LL-37 peptide or ceragenin CSA-13 with MNPs potentialized their antibacterial properties and might be considered as a method of delaying and overcoming bacterial drug resistance. PMID:27799768

  17. The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants.

    Science.gov (United States)

    Medová, Michaela; Pochon, Benoît; Streit, Bruno; Blank-Liss, Wieslawa; Francica, Paola; Stroka, Deborah; Keogh, Adrian; Aebersold, Daniel M; Blaukat, Andree; Bladt, Friedhelm; Zimmer, Yitzhak

    2013-11-01

    The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results show a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five of the eight cell lines (IC50 2-43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biologic functions, such as cellular morphology, MET-dependent cell motility, and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small-molecule inhibitor with selective activity towards mutated MET variants. ©2013 AACR.

  18. 双功能螯合剂BAT与鼠IgG1 Fab'片段的点特异性交联方法的研究%Site-Specific Conjugation of Bifunctional Chelator BAT to Mouse IgG1 Fab' Fragment

    Institute of Scientific and Technical Information of China (English)

    李君; 王学浩; 成峰; 王晓明; 陈兆雷; 沈喜平

    2005-01-01

    目的报道一种用双功能螯合剂BAT与鼠单克隆抗体Fab'片段行点特异性交联的新方法.方法通过位于Fab'铰链区、远离抗原结合位点的活性巯基(-SH)直接与BAT相交联.用简单的两步法制备B43单抗的Fab'片段.首先单抗用胃蛋白酶处理,产生稳定的F(ab')2 片段,然后 F(ab')2经半胱氨酸还原后产生Fab'片段,最后Fab'片段直接与BAT交联.结果每个Fab'分子平均含1.8个-SH基团,74%的Fab'片段可与BAT生成交联物,平均每个Fab'分子携带1.28个BAT.经检测,F(ab')2、Fab' 及 Fab'-BAT 均保持着良好的免疫活性.结论该交联法简单、高效,为双功能螯合剂BAT与鼠单抗Fab'片段的交联提供了一个新途径.

  19. A Compressive Superresolution Display

    KAUST Repository

    Heide, Felix

    2014-06-22

    In this paper, we introduce a new compressive display architecture for superresolution image presentation that exploits co-design of the optical device configuration and compressive computation. Our display allows for superresolution, HDR, or glasses-free 3D presentation.

  20. Lunar Sample Display Locations

    Data.gov (United States)

    National Aeronautics and Space Administration — NASA provides a number of lunar samples for display at museums, planetariums, and scientific expositions around the world. Lunar displays are open to the public....

  1. Imaging of low-grade bone infection with a technetium-99m labelled monoclonal anti-NCA-90 Fab' fragment in patients with previous joint surgery

    Energy Technology Data Exchange (ETDEWEB)

    Ivaneeviae, V.; Sandrock, D.; Munz, D.L. [Clinic for Nuclear Medicine, University Hospital Charite, Humboldt University of Berlin (Germany); Perka, C.; Hasart, O. [Orthopaedic Clinic, University Hospital Charite, Humboldt University of Berlin (Germany)

    2002-04-01

    We analysed 38 scans in 30 consecutive patients (age range, 30-85 years; median age, 62 years) referred for suspected low-grade bone infection. There were 17 patients (21 scans) with total hip arthroplasty (THA), six with total knee arthroplasty (TKA), three who had undergone hip or knee surgery for trauma and five (seven scans) with resected hips and no endoprostheses (Girdlestone situations); There were no patients with rheumatoid arthritis as the underlying disease. Results were verified by means of bacteriological cultures, histopathological findings and/or follow-up and compared with the respective Zimmerli scores, which were used for clinical assessment of inflammatory activity. In one patient, the final diagnosis could not be established. One, 5 and 24 h after intravenous injection of up to 1.1 GBq of MN3 Fab', whole-body and planar scans were performed using a dual-head gamma camera. Scans were analysed visually and semiquantitatively adopting an arbitrary score ranging from 0 to 3. There were 13 true positive, 14 true negative and 10 false positive outcomes, yielding an overall sensitivity of 100%, an overall specificity of 58%, an accuracy of 73% and positive and negative predictive values of 57% and 100%, respectively. In patients with THA or TKA, accuracy was 81% and 80%, respectively, while it dropped to 43% in patients with Girdlestone situations owing to a high proportion of false positive findings (4/7) in this subgroup. Scintigraphic score was 1 in all of the false positive and in 11/13 true positive findings. The two remaining true positive findings displayed scintigraphic scores of 2 and 3, respectively. Scintigraphic and Zimmerli scores were loosely correlated (Spearman {rho}=0.38, P<0.05). Infection was excluded in 22/24 investigations with Zimmerli scores of <6. In this group, there were 13 scintigraphically true negative, nine false positive outcomes, and just two true positive outcomes. In 11/12 investigations with Zimmerli scores of 6

  2. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    Energy Technology Data Exchange (ETDEWEB)

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong; (Houston)

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  3. Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

    Science.gov (United States)

    Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

    1990-01-01

    A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

  4. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  5. Activités insecticides de Striga hermonthica (Del.) Benth (Scrophulariaceae) sur Callosobrichus maculatus (Fab.) (Coleoptera : Bruchidae)

    OpenAIRE

    Nacoulma OG.; Ouedraogo AP.; Kiendrebeogo M.

    2006-01-01

    Insecticidal activities of Striga hermonthica (Del.) Benth (Scrophulariacecae) on Callobruchus maculatus (Fab.) (Coleptera Bruchidae). This paper deals with insecticidal potentialities of Striga hermonthica (Del.) (Scrophulariaceae) in protection of cowpea Vigna unguculata (L.) Walp against Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae) during storage. Crude acetone extract at 0,5% w/w (100 mg of extract for 20 g of grain) exhibits 48% of ovicidal effect and then reduces by half emer...

  6. Improving Assessment of Work Related Mental Health Function Using the Work Disability Functional Assessment Battery (WD-FAB).

    Science.gov (United States)

    Marfeo, Elizabeth E; Ni, Pengsheng; McDonough, Christine; Peterik, Kara; Marino, Molly; Meterko, Mark; Rasch, Elizabeth K; Chan, Leighton; Brandt, Diane; Jette, Alan M

    2017-05-05

    Purpose To improve the mental health component of the Work Disability Functional Assessment Battery (WD-FAB), developed for the US Social Security Administration's (SSA) disability determination process. Specifically our goal was to expand the WD-FAB scales of mood & emotions, resilience, social interactions, and behavioral control to improve the depth and breadth of the current scales and expand the content coverage to include aspects of cognition & communication function. Methods Data were collected from a random, stratified sample of 1695 claimants applying for the SSA work disability benefits, and a general population sample of 2025 working age adults. 169 new items were developed to replenish the WD-FAB scales and analyzed using factor analysis and item response theory (IRT) analysis to construct unidimensional scales. We conducted computer adaptive test (CAT) simulations to examine the psychometric properties of the WD-FAB. Results Analyses supported the inclusion of four mental health subdomains: Cognition & Communication (68 items), Self-Regulation (34 items), Resilience & Sociability (29 items) and Mood & Emotions (34 items). All scales yielded acceptable psychometric properties. Conclusions IRT methods were effective in expanding the WD-FAB to assess mental health function. The WD-FAB has the potential to enhance work disability assessment both within the context of the SSA disability programs as well as other clinical and vocational rehabilitation settings.

  7. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

    Science.gov (United States)

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans

    2016-01-01

    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

  8. SWARM INTELLIGENCE BASED DYNAMIC REAL-TIME SCHEDULING APPROACH FOR SEMICONDUCTOR WAFER FAB

    Institute of Scientific and Technical Information of China (English)

    Li Li; Fei Qiao; Wu Qidi

    2005-01-01

    Based on the analysis of collective activities of ant colonies, the typical example of swarm intelligence, a new approach to construct swarm intelligence based multi-agent-system (SMAS) for dynamic real-time scheduling for semiconductor wafer fab is proposed. The relevant algorithm,pheromone-based dynamic real-time scheduling algorithm (PBDR), is given. MIMAC test bed data set mini-fab is used to compare PBDR with FIFO (first in first out), SRPT(shortest remaining processing time) and CR(critical ratio) under three different release rules, i.e. deterministic rule, Poisson rule and CONWIP (constant WIP). It is shown that PBDR is prior to FIFO, SRPT and CR with better performance of cycle time, throughput, and on-time delivery, especially for on-time delivery performance.

  9. The FermiFab Toolbox for Fermionic Many-Particle Quantum Systems

    CERN Document Server

    Mendl, Christian B

    2011-01-01

    This paper introduces the FermiFab toolbox for many-particle quantum systems. It is mainly concerned with the representation of (symbolic) fermionic wavefunctions and the calculation of corresponding reduced density matrices (RDMs). The toolbox transparently handles the inherent antisymmetrization of wavefunctions and incorporates the creation/annihilation formalism. Thus, it aims at providing a solid base for a broad audience to use fermionic wavefunctions with the same ease as matrices in Matlab, say. Leveraging symbolic computation, the toolbox can greatly simply tedious pen-and-paper calculations for concrete quantum mechanical systems, and serves as "sandbox" for theoretical hypothesis testing. FermiFab (including full source code) is freely available as a plugin for both Matlab and Mathematica.

  10. Development of tools to study personal weight control strategies: OxFAB taxonomy

    OpenAIRE

    Hartmann‐Boyce, Jamie; Aveyard, Paul; Koshiaris, Constantinos; Jebb, Susan A

    2016-01-01

    Objective To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts. Methods The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think‐aloud interviews were...

  11. Development of tools to study personal weight control strategies: OxFAB taxonomy.

    OpenAIRE

    Hartmann-Boyce, J; Aveyard, P; Koshiaris, C; Jebb, SA

    2016-01-01

    To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts.The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think-aloud interviews were conducted to test ...

  12. Fab the coming revolution on your desktop : from personal computers to personal fabrication

    CERN Document Server

    Gershenfeld, Neil

    2005-01-01

    What if you could someday put the manufacturing power of an automobile plant on your desktop? According to Neil Gershenfeld, the renowned MIT scientist and inventor, the next big thing is personal fabrication-the ability to design and produce your own products, in your own home, with a machine that combines consumer electronics and industrial tools. Personal fabricators are about to revolutionize the world just as personal computers did a generation ago, and Fab shows us how.

  13. A status analysis of current digital marketing: a case study of Kauneusstudio FAB

    OpenAIRE

    Tran, Trong

    2015-01-01

    The subject of this thesis is a small company's current digital marketing status. This study was con-ducted in order for the owners of the beauty and hair salon Kauneusstudio FAB to improve their understanding of their customers’ behavior online and the significance of each digital channel they are using in the present marketing strategy. The goal of this study is to provide information for the company to recognize the strengths and the development points of the current digital marketing stra...

  14. Aerial image measurement technique for automated reticle defect disposition (ARDD) in wafer fabs

    Science.gov (United States)

    Zibold, Axel M.; Schmid, Rainer M.; Stegemann, B.; Scheruebl, Thomas; Harnisch, Wolfgang; Kobiyama, Yuji

    2004-08-01

    The Aerial Image Measurement System (AIMS)* for 193 nm lithography emulation has been brought into operation successfully worldwide. A second generation system comprising 193 nm AIMS capability, mini-environment and SMIF, the AIMS fab 193 plus is currently introduced into the market. By adjustment of numerical aperture (NA), illumination type and partial illumination coherence to match the conditions in 193 nm steppers or scanners, it can emulate the exposure tool for any type of reticles like binary, OPC and PSM down to the 65 nm node. The system allows a rapid prediction of wafer printability of defects or defect repairs, and critical features, like dense patterns or contacts on the masks without the need to perform expensive image qualification consisting of test wafer exposures followed by SEM measurements. Therefore, AIMS is a mask quality verification standard for high-end photo masks and established in mask shops worldwide. The progress on the AIMS technology described in this paper will highlight that besides mask shops there will be a very beneficial use of the AIMS in the wafer fab and we propose an Automated Reticle Defect Disposition (ARDD) process. With smaller nodes, where design rules are 65 nm or less, it is expected that smaller defects on reticles will occur in increasing numbers in the wafer fab. These smaller mask defects will matter more and more and become a serious yield limiting factor. With increasing mask prices and increasing number of defects and severability on reticles it will become cost beneficial to perform defect disposition on the reticles in wafer production. Currently ongoing studies demonstrate AIMS benefits for wafer fab applications. An outlook will be given for extension of 193 nm aerial imaging down to the 45 nm node based on emulation of immersion scanners.

  15. Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

    Science.gov (United States)

    Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

    2016-01-01

    Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

  16. Functional capacity of immunoglobulin G preparations and the F(ab')2 split product.

    OpenAIRE

    Steele, R W

    1989-01-01

    Five immunoglobulin G preparations, including one 5S F(ab')2 split product, were compared for activity against common bacterial, viral, and protozoan pathogens. Standard assays were used to quantitate antibodies to tetanus, diphtheria, cytomegalovirus, herpes simplex virus types 1 and 2, rubella virus, and Toxoplasma gondii. Opsonization and killing of bacteria were examined by chemiluminescence methods using Streptococcus pneumoniae types 5, 12F, and 14 and Staphylococcus aureus. Antibodies ...

  17. Family of Advanced Beyond Line-of-Sight Terminals (FAB-T)

    Science.gov (United States)

    2015-12-01

    Conferencing (PNVC) function; the Telemetry , Tracking & Control for the Milstar and AEHF constellations, for Nuclear Command, Control, & Communications...Coexistences: 4 ​Footnotes: 1/ For FAB-T, access to privileged Tracking Telemetry and Control (TT&C) capabilities and resource controller capabilities is...DIACAP - DoD Information Assurance Certification & Accreditation Process DIRECT - Defense IEMATS Replacement Command and Control Terminal DMU - Dual

  18. Invisible Display in Aluminum

    DEFF Research Database (Denmark)

    Prichystal, Jan Phuklin; Hansen, Hans Nørgaard; Bladt, Henrik Henriksen

    2005-01-01

    Bang & Olufsen a/s has been working with ideas for invisible integration of displays in metal surfaces. Invisible integration of information displays traditionally has been possible by placing displays behind transparent or semitransparent materials such as plastic or glass. The wish for an integ...... be obtained by shining light from the backside of the workpiece. When there is no light from the backside, the front surface seems totally untouched. This was achieved by laser ablation with ultra-short pulses.......Bang & Olufsen a/s has been working with ideas for invisible integration of displays in metal surfaces. Invisible integration of information displays traditionally has been possible by placing displays behind transparent or semitransparent materials such as plastic or glass. The wish...... for an integrated display in a metal surface is often ruled by design and functionality of a product. The integration of displays in metal surfaces requires metal removal in order to clear the area of the display to some extent. The idea behind an invisible display in Aluminum concerns the processing of a metal...

  19. [Digitoxin poisoning: reversing ventricular fibrillation with Fab fragments of anti-digoxin antibody].

    Science.gov (United States)

    Domart, Y; Bismuth, C; Schermann, J M; Abuaf, N; Pontal, P G; Baud, F; Bolo, A; Gailliot, M; Fournier, P E

    1982-12-25

    Purified Fab fragments of ovine anti-digoxin antibodies (Wellcome Foundation) were used to treat a patient who attempted suicide by absorbing 10 mg of digitoxin (serum concentration 265 micrograms/l). The poor prognosis, as assessed clinically and from serum potassium levels (7.5 mEq/l), seemed to warrant such a treatment. The weak (6.85%) cross-reactivity elicited in vitro between the anti-digoxin antibodies and digitoxin was compensated by increasing the doses, but improvement was observed with 3.6 g, i.e. about half the effective dosage initially considered. The criteria of effectiveness were clinical, electrocardiographic (reversal of the ventricular fibrillation), biochemical (simultaneous and opposite changes in extra- and intracellular potassium levels, suggesting that ATPase inhibition by digitalis is a reversible process) and toxicological: there was an increase in digitoxin serum levels suggesting displacement of the drug from tissue sites to plasma and other extracellular compartments where the Fab fragments are distributed, and Fab-bound digitoxin appeared fairly rapidly in the urine, which suggested shunting of the normal hepatic metabolic pathway.

  20. Productivity improvement through automated operation of reticle defect inspection tools in a wafer fab environment

    Science.gov (United States)

    Holfeld, Christian; Wagner, Heiko; Tchikoulaeva, Anna; Loebeth, Steffen; Melzig, Stephan; Zhang, Yulin; Tanabe, Shinichi; Katoh, Takenori; Moriizumi, Koichi

    2013-04-01

    Traditionally, product development for reticle defect inspection mostly addressed operational requirements of the mask shops with highly individualized manufacturing. As a result, limited automation capability was available as compared to the standards in wafer production. Wafer fabs are guided by completely different conditions. Thousands of active reticles exist in a single fab requiring frequent re-inspections without interruption of wafer exposures. This requires high throughput of inspection tools, smart management of tool fleet, sophisticated scheduling and in-time execution of reticle inspections linked to the wafer manufacturing. The paper reports about the successful implementation of fully automated reticle defect inspection in a high-volume advanced logic fab. Automation scenarios - created based on existing SEMI standards - included inspection scheduling, reticle transport and inspection tool operation. A considerable productivity gain for the operation of Lasertec MATRICS X700 series inspection tools was obtained. Based on the learning throughout implementation, the requirements to the automation capability and tool operation as well as adjustments to working procedures are discussed.

  1. Fabricating quench condensed lead thin film circuits using MEMS Fab on a Chip technology

    Science.gov (United States)

    Imboden, Matthias; Han, Han; Del Corro, Pablo; Pardo, Flavio; Bolle, Cristian; Bishop, David

    2015-03-01

    We have developed a MEMS Fab on a Chip consisting of micro-sources, mass sensors, heaters/thermometers, shutters and a dynamic stencil. The fab only occupies a volume of a few cubic millimeters and consumes milliwatts of power, and hence can be operated in a cryostat. Thin film patterns of arbitrary shapes using multiple materials can be manufactured, while strongly suppressing thermal annealing effects. We demonstrate deposition of quench condensed lead films with fractions of a monolayer thickness control. Furthermore, using low deposition rates it is estimated that the surface temperature of the target heats by only 1.7 K. We study the effects of growing quench condensed films with different evaporation rates to demonstrate thermal annealing effects which occur during deposition. We measure the minimum conduction thickness (insulator to metal transition) as well as the superconducting transition temperature as a function of film thickness in order to shed light on growth of amorphous films and the transition to nanocluster formations. The Fab on a Chip will allow us to build nanocircuits made of ultra-thin materials. Annealing and doping is controlled and measurements occur in situ, without exposing the fabricated circuits to thermal fluctuations or foreign contaminants. This enables new types of experiments based on quantum circuits which cannot be fabricated using standard lithography techniques.

  2. Precise construction of oligonucleotide-Fab fragment conjugate for homogeneous immunoassay using HaloTag technology.

    Science.gov (United States)

    Päkkilä, Henna; Peltomaa, Riikka; Lamminmäki, Urpo; Soukka, Tero

    2015-03-01

    The use of oligonucleotide-protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide-protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.

  3. LHCb Event display

    CERN Document Server

    Trisovic, Ana

    2014-01-01

    The LHCb Event Display was made for educational purposes at the European Organization for Nuclear Research, CERN in Geneva, Switzerland. The project was implemented as a stand-alone application using C++ and ROOT, a framework developed by CERN for data analysis. This paper outlines the development and architecture of the application in detail, as well as the motivation for the development and the goals of the exercise. The application focuses on the visualization of events recorded by the LHCb detector, where an event represents a set of charged particle tracks in one proton-proton collision. Every particle track is coloured by its type and can be selected to see its essential information such as mass and momentum. The application allows students to save this information and calculate the invariant mass for any pair of particles. Furthermore, the students can use additional calculating tools in the application and build up a histogram of these invariant masses. The goal for the students is to find a $D^0$ par...

  4. Polyplanar optic display

    Energy Technology Data Exchange (ETDEWEB)

    Veligdan, J.; Biscardi, C.; Brewster, C.; DeSanto, L. [Brookhaven National Lab., Upton, NY (United States). Dept. of Advanced Technology; Beiser, L. [Leo Beiser Inc., Flushing, NY (United States)

    1997-07-01

    The Polyplanar Optical Display (POD) is a unique display screen which can be used with any projection source. This display screen is 2 inches thick and has a matte black face which allows for high contrast images. The prototype being developed is a form, fit and functional replacement display for the B-52 aircraft which uses a monochrome ten-inch display. The new display uses a 100 milliwatt green solid state laser (532 nm) as its optical source. In order to produce real-time video, the laser light is being modulated by a Digital Light Processing (DLP{trademark}) chip manufactured by Texas Instruments, Inc. A variable astigmatic focusing system is used to produce a stigmatic image on the viewing face of the POD. In addition to the optical design, the authors discuss the electronic interfacing to the DLP{trademark} chip, the opto-mechanical design and viewing angle characteristics.

  5. OLED displays and lighting

    CERN Document Server

    Koden, Mitsuhiro

    2017-01-01

    Organic light-emitting diodes (OLEDs) have emerged as the leading technology for the new display and lighting market. OLEDs are solid-state devices composed of thin films of organic molecules that create light with the application of electricity. OLEDs can provide brighter, crisper displays on electronic devices and use less power than conventional light-emitting diodes (LEDs) or liquid crystal displays (LCDs) used today. This book covers both the fundamentals and practical applications of flat and flexible OLEDs.

  6. Scalable Resolution Display Walls

    KAUST Repository

    Leigh, Jason

    2013-01-01

    This article will describe the progress since 2000 on research and development in 2-D and 3-D scalable resolution display walls that are built from tiling individual lower resolution flat panel displays. The article will describe approaches and trends in display hardware construction, middleware architecture, and user-interaction design. The article will also highlight examples of use cases and the benefits the technology has brought to their respective disciplines. © 1963-2012 IEEE.

  7. JAVA Stereo Display Toolkit

    Science.gov (United States)

    Edmonds, Karina

    2008-01-01

    This toolkit provides a common interface for displaying graphical user interface (GUI) components in stereo using either specialized stereo display hardware (e.g., liquid crystal shutter or polarized glasses) or anaglyph display (red/blue glasses) on standard workstation displays. An application using this toolkit will work without modification in either environment, allowing stereo software to reach a wider audience without sacrificing high-quality display on dedicated hardware. The toolkit is written in Java for use with the Swing GUI Toolkit and has cross-platform compatibility. It hooks into the graphics system, allowing any standard Swing component to be displayed in stereo. It uses the OpenGL graphics library to control the stereo hardware and to perform the rendering. It also supports anaglyph and special stereo hardware using the same API (application-program interface), and has the ability to simulate color stereo in anaglyph mode by combining the red band of the left image with the green/blue bands of the right image. This is a low-level toolkit that accomplishes simply the display of components (including the JadeDisplay image display component). It does not include higher-level functions such as disparity adjustment, 3D cursor, or overlays all of which can be built using this toolkit.

  8. Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans

    OpenAIRE

    Faustoferri, R.C.; Hubbard, C.J.; Santiago, B.; Buckley, A.A.; Seifert, T.B.; Quivey, R.G.

    2014-01-01

    SMU.1745c, encoding a putative transcriptional regulator of the MarR family, maps to a location proximal to the fab gene cluster in Streptococcus mutans. Deletion of the SMU.1745c (fabTSm) coding region resulted in a membrane fatty acid composition comprised of longer-chained, unsaturated fatty acids (UFA), compared with the parent strain. Previous reports have indicated a role for FabT in regulation of genes in the fab gene cluster in other organisms, through binding to a palindromic DNA seq...

  9. Standardizing visual display quality

    NARCIS (Netherlands)

    Besuijen, Ko; Spenkelink, Gerd P.J.

    1998-01-01

    The current ISO 9241–3 standard for visual display quality and the proposed user performance tests are reviewed. The standard is found to be more engineering than ergonomic and problems with system configuration, software applications, display settings, user behaviour, wear and physical environment

  10. Polyplanar optical display electronics

    Energy Technology Data Exchange (ETDEWEB)

    DeSanto, L.; Biscardi, C. [Brookhaven National Lab., Upton, NY (United States). Dept. of Advanced Technology

    1997-07-01

    The Polyplanar Optical Display (POD) is a unique display screen which can be used with any projection source. The prototype ten inch display is two inches thick and has a matte black face which allows for high contrast images. The prototype being developed is a form, fit and functional replacement display for the B-52 aircraft which uses a monochrome ten-inch display. In order to achieve a long lifetime, the new display uses a 100 milliwatt green solid-state laser (10,000 hr. life) at 532 nm as its light source. To produce real-time video, the laser light is being modulated by a Digital Light Processing (DLP{trademark}) chip manufactured by Texas Instruments. In order to use the solid-state laser as the light source and also fit within the constraints of the B-52 display, the Digital Micromirror Device (DMD{trademark}) circuit board is removed from the Texas Instruments DLP light engine assembly. Due to the compact architecture of the projection system within the display chassis, the DMD{trademark} chip is operated remotely from the Texas Instruments circuit board. The authors discuss the operation of the DMD{trademark} divorced from the light engine and the interfacing of the DMD{trademark} board with various video formats (CVBS, Y/C or S-video and RGB) including the format specific to the B-52 aircraft. A brief discussion of the electronics required to drive the laser is also presented.

  11. Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

    Directory of Open Access Journals (Sweden)

    Olga Kyrchanova

    2016-07-01

    Full Text Available Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

  12. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-01

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  13. Cholestatic liver disease after rituximab and adalimumab and the possible role of cross-reacting antibodies to Fab 2 fragments.

    Directory of Open Access Journals (Sweden)

    Joerg Latus

    Full Text Available BACKGROUND: Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA-associated granulomatosis with polyangiitis (GPA who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. METHODS: Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. RESULTS: Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients' sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. CONCLUSION: This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects

  14. Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

    Science.gov (United States)

    Kyrchanova, Olga; Mogila, Vladic; Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

    2016-07-01

    Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

  15. Highly Selective Targeting of Hepatic Stellate Cells for Liver Fibrosis Treatment Using a d-Enantiomeric Peptide Ligand of Fn14 Identified by Mirror-Image mRNA Display.

    Science.gov (United States)

    Huang, Luying; Xie, Jing; Bi, Qiuyan; Li, Zhuoxuan; Liu, Sha; Shen, Qing; Li, Chong

    2017-05-01

    Although liver fibrosis is a major public health issue, there is still no effective drug therapy in the clinic. Fibroblast growth factor-inducible 14 (Fn14), a membrane receptor highly specifically expressed in activated hepatic stellate cells (HSCs), is the key driver of liver fibrosis, and thus, it has a great potential as a novel target for the development of effective treatment. Here, we identified a d-enantiomeric peptide ligand of Fn14 through mirror-image mRNA display. This included the chemical synthesis of a d-enantiomer of the target protein (extracellular domain of Fn14), identification of an l-peptide ligand of d-Fn14 using a constructed mRNA peptide library, and identification of a d-enantiomer of the l-peptide, which is a ligand of the natural Fn14 for reasons of symmetry. The obtained d-peptide ligand showed strong binding to Fn14 while maintaining high proteolytic resistance. As a targeting moiety, this d-peptide successfully mediated high selectivity of activated HSCs for liposomal vehicles compared to that of other major cell types in the liver and significantly enhanced the accumulation of liposomes in the liver fibrosis region of a carbon tetrachloride-induced mouse model. Moreover, in combination with curcumin as an encapsulated load, a liposomal formulation conjugated with this d-peptide showed powerful inhibition of the proliferation of activated HSCs and reduced the liver fibrosis to a significant extent in vivo. This Fn14-targeting strategy may represent a promising approach to targeted drug delivery for liver fibrosis treatment. Meanwhile, the mirror-image mRNA display can provide a new arsenal for the development of d-peptide-based therapeutics against a variety of human diseases.

  16. Helmet-Mounted Displays (HMD)

    Data.gov (United States)

    Federal Laboratory Consortium — The Helmet-Mounted Display labis responsible for monocular HMD day display evaluations; monocular HMD night vision performance processes; binocular HMD day display...

  17. Visual merchandising window display

    Directory of Open Access Journals (Sweden)

    Opris (Cas. Stanila M.

    2013-12-01

    Full Text Available Window display plays a major part in the selling strategies; it does not only include the simple display of goods, nowadays it is a form of art, also having the purpose of sustaining the brand image. This article wants to reveal the tools that are essential in creating a fabulous window display. Being a window designer is not an easy job, you have to always think ahead trends, to have a sense of colour, to know how to use light to attract customers in the store after only one glance at the window. The big store window displays are theatre scenes: with expensive backgrounds, special effects and high fashion mannequins. The final role of the displays is to convince customers to enter the store and trigger the purchasing act which is the final goal of the retail activity.

  18. Defense display market assessment

    Science.gov (United States)

    Desjardins, Daniel D.; Hopper, Darrel G.

    1998-09-01

    This paper addresses the number, function and size of principal military displays and establishes a basis to determine the opportunities for technology insertion in the immediate future and into the next millennium. Principal military displays are defined as those occupying appreciable crewstation real-estate and/or those without which the platform could not carry out its intended mission. DoD 'office' applications are excluded from this study. The military displays market is specified by such parameters as active area and footprint size, and other characteristics such as luminance, gray scale, resolution, angle, color, video capability, and night vision imaging system (NVIS) compatibility. Funded, future acquisitions, planned and predicted crewstation modification kits, and form-fit upgrades are taken into account. This paper provides an overview of the DoD niche market, allowing both government and industry a necessary reference by which to meet DoD requirements for military displays in a timely and cost-effective manner. The aggregate DoD market for direct-view and large-area military displays is presently estimated to be in excess of 242,000. Miniature displays are those which must be magnified to be viewed, involve a significantly different manufacturing paradigm and are used in helmet mounted displays and thermal weapon sight applications. Some 114,000 miniature displays are presently included within Service weapon system acquisition plans. For vendor production planning purposes it is noted that foreign military sales could substantially increase these quantities. The vanishing vendor syndrome (VVS) for older display technologies continues to be a growing, pervasive problem throughout DoD, which consequently must leverage the more modern display technologies being developed for civil- commercial markets.

  19. Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

    Science.gov (United States)

    Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts.

  20. PREPARATION AND IDENTIFICATION OF ANTI-PROGESTERONE Fab'-SBP%抗孕酮Fab'-大豆过氧化物酶交联物的制备和鉴定

    Institute of Scientific and Technical Information of China (English)

    龚振明; 李蓓; 陈鲁勇; 沈明泉

    2003-01-01

    用硫酸铵沉淀法对兔抗孕酮血清进行粗提后用DEAE Sepharose Fast Fl0W对IgG进行层析纯化,然后用胃蛋白酶切除Fc片段而制备F(ab')2,用β-巯基乙醇还原F(ab')2后得到Fab',再将Fab'与热稳定性较高的大豆过氧化物酶(SBP)偶联制得抗孕酮Fab'-SBP.将抗孕酮Fab'-SBP用于二级竞争酶联免疫法测定孕酮有以下优点:(1)减少因Fc片段与固相载体非特异性吸附而引起的高背景干扰;(2)由于Fab'和SBP在很温和的条件下偶联,可使偶联产物保持较高的免疫活性和酶活性.

  1. Panoramic projection avionics displays

    Science.gov (United States)

    Kalmanash, Michael H.

    2003-09-01

    Avionics projection displays are entering production in advanced tactical aircraft. Early adopters of this technology in the avionics community used projection displays to replace or upgrade earlier units incorporating direct-view CRT or AMLCD devices. Typical motivation for these upgrades were the alleviation of performance, cost and display device availability concerns. In these systems, the upgraded (projection) displays were one-for-one form / fit replacements for the earlier units. As projection technology has matured, this situation has begun to evolve. The Lockheed-Martin F-35 is the first program in which the cockpit has been specifically designed to take advantage of one of the more unique capabilities of rear projection display technology, namely the ability to replace multiple small screens with a single large conformal viewing surface in the form of a panoramic display. Other programs are expected to follow, since the panoramic formats enable increased mission effectiveness, reduced cost and greater information transfer to the pilot. Some of the advantages and technical challenges associated with panoramic projection displays for avionics applications are described below.

  2. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    Science.gov (United States)

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  3. Deletion of fabN in Enterococcus faecalis results in unsaturated fatty acid auxotrophy and decreased release of inflammatory cytokines.

    Science.gov (United States)

    Diederich, Ann-Kristin; Duda, Katarzyna A; Romero-Saavedra, Felipe; Engel, Regina; Holst, Otto; Huebner, Johannes

    2016-05-01

    The Gram-positive bacterium Enterococcus faecalis can cause life-threatening infections and is resistant to several commonly used antibiotics. The type II fatty acid pathway in bacteria is discussed as a potential target for antimicrobial therapy. However, it was shown that inhibition or deletion of its enzymes can be rescued in Gram-positive bacteria by supplementation with fatty acids. Here we show that by deletion of the fabN gene, which is essential for unsaturated fatty acid (UFA) synthesis in E. faecalis, growth is impaired but can be rescued by supplementation with oleic acid or human serum. Nonetheless, we demonstrate alterations of the UFA profile after supplementation with oleic acid in the ΔfabN mutant using a specific glycolipid. In addition, we demonstrate that cytokine release in vitro is almost abolished after stimulation of mouse macrophages by the mutant in comparison to the wild type. The results indicate that fabN is not a suitable target for antimicrobials as UFA auxotrophy can be overcome. However, deletion of fabN resulted in a decreased inflammatory response indicating that fabN and resulting UFA synthesis are relevant for virulence.

  4. 抗HBs Fab-IFNα融合蛋白的制备与初步鉴定%Preparation and preliminary identification of anti HBs Fab-IFNa fusion protein

    Institute of Scientific and Technical Information of China (English)

    陆慧琦; 宋杰; 叶伟民; 韩焕兴

    2011-01-01

    目的 构建抗HBsAg的pComb Fab-IFNα载体,原核表达具双重生物活性的抗HBs Fab-IFNα融合蛋白.方法 以pBAD-Fab和pBAD IFNα作为模板,分别扩增Fd、λ和IENα基因,经相应的限制性内切酶酶切后分3次克隆人pComBHss质粒,转化XL-1 Blue大肠埃希菌.限制性酶切和测序鉴定重组质粒,免疫蛋向印迹(Western blotting)和斑点印迹(Dot blotting)鉴定融合蛋白的表达及抗原结合活性.结果 重组载体的酶切、电泳及测序表明抗HBs Fab-IFNα基因克隆正确.表达产物经12%SDS-PAGE电泳、转印,Western blotting显示该融合蛋白分子量约为65kD,Dot blotting显示其与HBsAg具有结合能力.细胞病变抑制法测定IFNα生物学活性为7.8×104~5.1×105U/ml.结论 该原核系统成功表达了抗HBs Fab-IFNα融合蛋白,表明其既具有抗HBsAg结合能力,又具备IFNα的生物活性,为进一步的系统表达和应用研究提供了条件.%Objective To construct the anti HBsAg pComb Fab-IFNe vector, and to express the fusion protein consisting of IFN and anti-HBs Fab in prokaryon. Methods Using pBAD-IFN plasmid and pBAD-Fab plasmid as template, the anti-HBs Fd, and IFN were amplified separately with corresponding endonuclease sites by polymerase chain reaction (PCR). Each PCR product was digested with specific endonuclease and inserted into pComBHss vector, and then transformed into XL-1 Blue. The recombinant plasmid was isolated by miniprep for restriction analysis and sequencing. Fusion protein was identified by Western blotting and Dot blotting. Results The recombinant plasmid was confirmed by restriction electrophoresis and sequencing. Aliquots of human anti-HBs Fab-IFNα were concentrated and size fractionated by 12% SDS-PAGE and stained with Coomassie. The proteins were transferred to nitrocellulose, incubated with HRP- conjugated goat anti-human IgG Fab and Rah anti-human IFN. The molecular weight of the fusion protein was about 65kD. Anti HBs Fab-IFNa fusion

  5. Small - Display Cartography

    DEFF Research Database (Denmark)

    Nissen, Flemming; Hvas, Anders; Münster-Swendsen, Jørgen

    This report comprises the work carried out in the work-package of small display cartography. The work-package has aimed at creating a general framework for the small-display cartography. A solid framework facilitates an increased use of spatial data in mobile devices - thus enabling, together...... with the rapidly evolving positioning techniques, a new category of position-dependent, map-based services to be introduced. The report consists of the following parts: Part I: Categorization of handheld devices, Part II: Cartographic design for small-display devices, Part III: Study on the GiMoDig Client ? Portal...... Service Communication and finally, Part IV: Concluding remarks and topics for further research on small-display cartography. Part II includes a separate Appendix D consisting of a cartographic design specification. Part III includes a separate Appendix C consisting of a schema specification, a separate...

  6. Flexible displays, rigid designs?

    DEFF Research Database (Denmark)

    Hornbæk, Kasper

    2015-01-01

    Rapid technological progress has enabled a wide range of flexible displays for computing devices, but the user experience--which we're only beginning to understand--will be the key driver for successful designs....

  7. Liquid Crystal Airborne Display

    Science.gov (United States)

    1977-08-01

    81/2X 11- 10 -9 .8 display using a large advertising alphanimeric ( TCI ) has been added to the front of the optical box used in the F-4 aircraft for HUD...properties over a wide range of tempera - tures, including normal room temperature. What are Liquid Crystals? Liquid crystals have been classified in three...natic fanctions and to present data needed for the semi- automatic and manual control of system functions. Existing aircraft using CRT display

  8. Raster graphics display library

    Science.gov (United States)

    Grimsrud, Anders; Stephenson, Michael B.

    1987-01-01

    The Raster Graphics Display Library (RGDL) is a high level subroutine package that give the advanced raster graphics display capabilities needed. The RGDL uses FORTRAN source code routines to build subroutines modular enough to use as stand-alone routines in a black box type of environment. Six examples are presented which will teach the use of RGDL in the fastest, most complete way possible. Routines within the display library that are used to produce raster graphics are presented in alphabetical order, each on a separate page. Each user-callable routine is described by function and calling parameters. All common blocks that are used in the display library are listed and the use of each variable within each common block is discussed. A reference on the include files that are necessary to compile the display library is contained. Each include file and its purpose are listed. The link map for MOVIE.BYU version 6, a general purpose computer graphics display system that uses RGDL software, is also contained.

  9. The FermiFab toolbox for fermionic many-particle quantum systems

    Science.gov (United States)

    Mendl, Christian B.

    2011-06-01

    This paper introduces the FermiFab toolbox for many-particle quantum systems. It is mainly concerned with the representation of (symbolic) fermionic wavefunctions and the calculation of corresponding reduced density matrices (RDMs). The toolbox transparently handles the inherent antisymmetrization of wavefunctions and incorporates the creation/annihilation formalism. Thus, it aims at providing a solid base for a broad audience to use fermionic wavefunctions with the same ease as matrices in Matlab, say. Leveraging symbolic computation, the toolbox can greatly simply tedious pen-and-paper calculations for concrete quantum mechanical systems, and serves as "sandbox" for theoretical hypothesis testing. FermiFab (including full source code) is freely available as a plugin for both Matlab and Mathematica. Program summaryProgram title:FermiFab Catalogue identifier: AEIN_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEIN_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Special license provided by the author No. of lines in distributed program, including test data, etc.: 1 165 461 No. of bytes in distributed program, including test data, etc.: 15 557 308 Distribution format: tar.gz Programming language: MATLAB 7.9, Mathematica 7.0, C Computer: PCs, Sun Solaris workstation Operating system: Any platform supporting MATLAB or Mathematica; tested with Windows (32 and 64 bit) and Sun Solaris. RAM: Case dependent Classification: 4.15 Nature of problem: Representation of fermionic wavefunctions, computation of RDMs (reduced density matrices) and handing of the creation/annihilation operator formalism. Solution method: Mapping of Slater determinants to bitfields, implementation of the creation/annihilation and RDM formalism by bit operations. Running time: Depends on the problem size; several seconds for the provided demonstration files.

  10. PREPARATION OF IMMUNOGEN AND PURIFICA¬TION OF HIGH AFFINITY AND SPECIFICITY FAB FRAGMENT OF ANTI-DIGOXIN POLYCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    M. Pour-Amir

    2000-01-01

    Full Text Available In this study we produced and purified a high titer of specific and high affin¬ity Fab fragments of anti-digoxin antibody. Immunization of rabbits with a conju¬gate of the cardiac glycoside digoxin, coupled by a periodate oxidation method to the amino group of lysine in bovine serum albumin resulted in the production of this type of high titer digoxin-specific antibodies with exceptionally high affinity (109 L/mol and specificity in immune response. Increase in titer was found in steps of purification ending up with the highest titer for Fab fragment to be at 1.75 ug of purified Fab (for 50% binding of I25I-digoxin. High specificity for antigenic determinants of the steroid nucleus of digoxin was observed such that much less cross-reaction with digoxin (2.3% and no cross-reaction with ouabaine, estradiol, Cortisol, progesterone and testosterone were detected.

  11. Critical Stage Rule-Based Real Time Dispatch(RTD)System in Highly-Mixed-Products (HMP) FAB

    Institute of Scientific and Technical Information of China (English)

    YUXiao-hua; XIANGYu-qun

    2005-01-01

    An improving utilization and efficiency of critical equipments in semiconductor wafer fabrication facilities are concerned. Semiconductor manufacturing FAB is one of the most complicated and cost sensitive environments. A good dispatching tool will make big difference in equipment utilization and FAB output as a whole. The equipment in this paper is In-Line DUV Scanner.There are many factors impacting utilization and output on this equipment group. In HMP environment one of the issues is changing of reticule in this area and idle counts due to load unbalance between equipments. Here we'll introduce a rule-based RTD system which aiming at decreasing the number of recipe change and idle counts among a group of scanner equipment in a high-mixedproducts FAB.

  12. Critical Stage Rule-Based Real Time Dispatch(RTD) System in Highly-Mixed-Products (HMP) FAB

    Institute of Scientific and Technical Information of China (English)

    YU Xiao-hua; XIANG Yu-qun

    2005-01-01

    An improving utilization and efficiency of critical equipments in semiconductor wafer fabrication facilities are concerned. Semiconductor manufacturing FAB is one of the most omplicated and cost sensitive environments. A good dispatching tool will make big difference in equipment utilization and FAB output as a whole. The equipment in this paper is In-Line DUV Scanner.There are many factors impacting utilization and output on this equipment group. In HMP environment one of the issues is changing of reticule in this area and idle counts due to load unbalance between equipments. Here we'll introduce a rule-based RTD system which aiming at decreasing the number of recipe change and idle counts among a group of scanner equipment in a high-mixedproducts FAB.

  13. Antibacterial FabH Inhibitors with Mode of Action Validated in Haemophilus influenzae by in Vitro Resistance Mutation Mapping.

    Science.gov (United States)

    McKinney, David C; Eyermann, Charles J; Gu, Rong-Fang; Hu, Jun; Kazmirski, Steven L; Lahiri, Sushmita D; McKenzie, Andrew R; Shapiro, Adam B; Breault, Gloria

    2016-07-01

    Fatty acid biosynthesis is essential to bacterial growth in Gram-negative pathogens. Several small molecules identified through a combination of high-throughput and fragment screening were cocrystallized with FabH (β-ketoacyl-acyl carrier protein synthase III) from Escherichia coli and Streptococcus pneumoniae. Structure-based drug design was used to merge several scaffolds to provide a new class of inhibitors. After optimization for Gram-negative enzyme inhibitory potency, several compounds demonstrated antimicrobial activity against an efflux-negative strain of Haemophilus influenzae. Mutants resistant to these compounds had mutations in the FabH gene near the catalytic triad, validating FabH as a target for antimicrobial drug discovery.

  14. Display of nuclear medicine imaging studies

    CERN Document Server

    Singh, B; Samuel, A M

    2002-01-01

    Nuclear medicine imaging studies involve evaluation of a large amount of image data. Digital signal processing techniques have introduced processing algorithms that increase the information content of the display. Nuclear medicine imaging studies require interactive selection of suitable form of display and pre-display processing. Static imaging study requires pre-display processing to detect focal defects. Point operations (histogram modification) along with zoom and capability to display more than one image in one screen is essential. This album mode of display is also applicable to dynamic, MUGA and SPECT data. Isometric display or 3-D graph of the image data is helpful in some cases e.g. point spread function, flood field data. Cine display is used on a sequence of images e.g. dynamic, MUGA and SPECT imaging studies -to assess the spatial movement of tracer with time. Following methods are used at the investigator's discretion for inspection of the 3-D object. 1) Display of orthogonal projections, 2) Disp...

  15. A study on EUV reticle surface molecular contamination under different storage conditions in a HVM foundry fab

    Science.gov (United States)

    Singh, SherJang; Yatzor, Brett; Taylor, Ron; Wood, Obert; Mangat, Pawitter

    2017-03-01

    The prospect of EUVL (Extreme Ultraviolet Lithography) insertion into HVM (High Volume Manufacturing) has never been this promising. As technology is prepared for "lab to fab" transition, it becomes important to comprehend challenges associated with integrating EUVL infrastructure within existing high volume chip fabrication processes in a foundry fab. The existing 193nm optical lithography process flow for reticle handling and storage in a fab atmosphere is well established and in-fab reticle contamination concerns are mitigated with the reticle pellicle. However EUVL reticle pellicle is still under development and if available, may only provide protection against particles but not molecular contamination. HVM fab atmosphere is known to be contaminated with trace amounts of AMC's (Atmospheric Molecular Contamination). If such contaminants are organic in nature and get absorbed on the reticle surface, EUV photon cause photo-dissociation resulting into carbon generation which is known to reduce multilayer reflectivity and also degrades exposure uniformity. Chemical diffusion and aggregation of other ions is also reported under the e-beam exposure of a EUV reticle which is known to cause haze issues in optical lithography. Therefore it becomes paramount to mitigate absorbed molecular contaminant concerns on EUVL reticle surface. In this paper, we have studied types of molecular contaminants that are absorbed on an EUVL reticle surface under HVM fab storage and handling conditions. Effect of storage conditions (gas purged vs atmospheric) in different storage pods (Dual pods, Reticle Clamshells) is evaluated. Absorption analysis is done both on ruthenium capping layer as well as TaBN absorber. Ru surface chemistry change as a result of storage is also studied. The efficacy of different reticle cleaning processes to remove absorbed contaminant is evaluated as well.

  16. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Sixiang [University of Wisconsin, Materials Science Program, Madison, WI (United States); Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D. [University of Wisconsin, Department of Radiology, Madison, WI (United States); Graves, Stephen A.; Nickles, Robert J. [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); Liu, Bai; Wong, Hing C. [Altor BioScience, Miramar, FL (United States); Cai, Weibo [University of Wisconsin, Materials Science Program, Madison, WI (United States); University of Wisconsin, Department of Radiology, Madison, WI (United States); University of Wisconsin, Department of Medical Physics, Madison, WI (United States); University of Wisconsin Carbone Cancer Center, Madison, WI (United States); University of Wisconsin, Departments of Radiology and Medical Physics, Madison, WI (United States)

    2015-07-15

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and {sup 64}Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of {sup 64}Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of {sup 64}Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. {sup 64}Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management. (orig.)

  17. Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino-1-(3-pyridyl-1-butanone (NNK-Specific Monoclonal Antibody and Recombinant F(ab

    Directory of Open Access Journals (Sweden)

    Lawrence K. Silbart

    2013-03-01

    Full Text Available Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs such as 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

  18. The Ultimate Display

    CERN Document Server

    Fluke, C J

    2016-01-01

    Astronomical images and datasets are increasingly high-resolution and multi-dimensional. The vast majority of astronomers perform all of their visualisation and analysis tasks on low-resolution, two-dimensional desktop monitors. If there were no technological barriers to designing the ultimate stereoscopic display for astronomy, what would it look like? What capabilities would we require of our compute hardware to drive it? And are existing technologies even close to providing a true 3D experience that is compatible with the depth resolution of human stereoscopic vision? We consider the CAVE2 (an 80 Megapixel, hybrid 2D and 3D virtual reality environment directly integrated with a 100 Tflop/s GPU-powered supercomputer) and the Oculus Rift (a low- cost, head-mounted display) as examples at opposite financial ends of the immersive display spectrum.

  19. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    Science.gov (United States)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  20. Nurturing Creativity and Innovation Through FabKids: A Case Study

    Science.gov (United States)

    Beyers, Ronald Noel

    2010-10-01

    This paper will report on a case study that was conducted involving Grade 10 learners who were exposed to a high-tech rapid-prototyping environment of a Fabrication Laboratory as part of a FabKids experience. This project must be viewed in the context of a global shortage of key skills placing a higher priority on the initiation and development of a pipeline to attract youth into science, engineering and technology careers. Creativity and innovation feature high on the skills agenda but more importantly preliminary results indicate that learners from a broad range of schools were able to operate effectively in this post constructivist environment. Participants had to apply their knowledge, skill, attitudes and values in order to produce a solution to the challenges provided. The fundamentals of the design process of investigate, design, make, evaluate and communicate were emphasized where the FabKids had to draw on their own collective knowledge using a range of technologies available to them.

  1. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    Science.gov (United States)

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

  2. Refreshing Refreshable Braille Displays.

    Science.gov (United States)

    Russomanno, Alexander; O'Modhrain, Sile; Gillespie, R Brent; Rodger, Matthew W M

    2015-01-01

    The increased access to books afforded to blind people via e-publishing has given them long-sought independence for both recreational and educational reading. In most cases, blind readers access materials using speech output. For some content such as highly technical texts, music, and graphics, speech is not an appropriate access modality as it does not promote deep understanding. Therefore blind braille readers often prefer electronic braille displays. But, these are prohibitively expensive. The search is on, therefore, for a low-cost refreshable display that would go beyond current technologies and deliver graphical content as well as text. And many solutions have been proposed, some of which reduce costs by restricting the number of characters that can be displayed, even down to a single braille cell. In this paper, we demonstrate that restricting tactile cues during braille reading leads to poorer performance in a letter recognition task. In particular, we show that lack of sliding contact between the fingertip and the braille reading surface results in more errors and that the number of errors increases as a function of presentation speed. These findings suggest that single cell displays which do not incorporate sliding contact are likely to be less effective for braille reading.

  3. Virtual Auditory Displays

    Science.gov (United States)

    2000-01-01

    timbre , intensity, distance, room modeling, radio communication Virtual Environments Handbook Chapter 4 Virtual Auditory Displays Russell D... musical note “A” as a pure sinusoid, there will be 440 condensations and rarefactions per second. The distance between two adjacent condensations or...and complexity are pitch, loudness, and timbre respectively. This distinction between physical and perceptual measures of sound properties is an

  4. Virtual acoustic displays

    Science.gov (United States)

    Wenzel, Elizabeth M.

    1991-01-01

    A 3D auditory display can potentially enhance information transfer by combining directional and iconic information in a quite naturalistic representation of dynamic objects in the interface. Another aspect of auditory spatial clues is that, in conjunction with other modalities, it can act as a potentiator of information in the display. For example, visual and auditory cues together can reinforce the information content of the display and provide a greater sense of presence or realism in a manner not readily achievable by either modality alone. This phenomenon will be particularly useful in telepresence applications, such as advanced teleconferencing environments, shared electronic workspaces, and monitoring telerobotic activities in remote or hazardous situations. Thus, the combination of direct spatial cues with good principles of iconic design could provide an extremely powerful and information-rich display which is also quite easy to use. An alternative approach, recently developed at ARC, generates externalized, 3D sound cues over headphones in realtime using digital signal processing. Here, the synthesis technique involves the digital generation of stimuli using Head-Related Transfer Functions (HRTF's) measured in the two ear-canals of individual subjects. Other similar approaches include an analog system developed by Loomis, et. al., (1990) and digital systems which make use of transforms derived from normative mannikins and simulations of room acoustics. Such an interface also requires the careful psychophysical evaluation of listener's ability to accurately localize the virtual or synthetic sound sources. From an applied standpoint, measurement of each potential listener's HRTF's may not be possible in practice. For experienced listeners, localization performance was only slightly degraded compared to a subject's inherent ability. Alternatively, even inexperienced listeners may be able to adapt to a particular set of HRTF's as long as they provide adequate

  5. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    Directory of Open Access Journals (Sweden)

    Elena Gustchina

    Full Text Available A series of mini-antibodies (monovalent and bivalent Fabs targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066 broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062 non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN363 or 3-H has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen

  6. Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

    Science.gov (United States)

    Zhang, Huimin; Zheng, Beiwen; Gao, Rongsui; Feng, Youjun

    2015-09-01

    The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid

  7. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

    Full Text Available Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

  8. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    Science.gov (United States)

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  9. FabSim: Facilitating computational research through automation on large-scale and distributed e-infrastructures

    Science.gov (United States)

    Groen, Derek; Bhati, Agastya P.; Suter, James; Hetherington, James; Zasada, Stefan J.; Coveney, Peter V.

    2016-10-01

    We present FabSim, a toolkit developed to simplify a range of computational tasks for researchers in diverse disciplines. FabSim is flexible, adaptable, and allows users to perform a wide range of tasks with ease. It also provides a systematic way to automate the use of resources, including HPC and distributed machines, and to make tasks easier to repeat by recording contextual information. To demonstrate this, we present three use cases where FabSim has enhanced our research productivity. These include simulating cerebrovascular bloodflow, modelling clay-polymer nanocomposites across multiple scales, and calculating ligand-protein binding affinities. Catalogue identifier: AFAO_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AFAO_v1_0.html Program obtainable from: CPC Programme Library, Queen's University, Belfast, N. Ireland Licensing provisions: BSD 3-Clause No. of lines in distributed program, including test data, etc.: 268282 No. of bytes in distributed program, including test data, etc.: 2791792 Distribution format: tar.gz Programming language: Python. Computer: PC or Mac. Operating system: Unix, OSX. RAM: 1 Gbytes Classification: 3, 4, 6.5. External routines: NumPy, SciPy, Fabric (1.5 or newer), PyYaml Nature of problem: Running advanced computations using remote resources is an activity that requires considerable time and human attention. These activities, such as organizing data, configuring software and setting up individual runs often vary slightly each time they are performed. To lighten this burden, we required an approach that introduced little burden of its own to set up and adapt, beyond which very substantial productivity ensues. Solution method: We present a toolkit which helps to simplify and automate the activities which surround computational science research. FabSim is aimed squarely at the experienced computational scientist, who can use the command line interface and a system of modifiable content to quickly automate sets of

  10. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity.

    Science.gov (United States)

    Graille, M; Stura, E A; Corper, A L; Sutton, B J; Taussig, M J; Charbonnier, J B; Silverman, G J

    2000-05-09

    Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

  11. Image Descriptors for Displays

    Science.gov (United States)

    1975-03-01

    hypothetical televison display. The viewing distance is 4 picture heights, and the bandwidth limitation has been set by the U.S. Monochrome Standards...significantly influence the power spectrum over most of the video frequency range. A large dc component and a small random component provide another scene... influences . It was Illuminated with natural light to a brightness of over 300 ft-L. The high brightness levels were chosen so as to nearly reproduce the

  12. Refrigerated display cabinets; Butikskyla

    Energy Technology Data Exchange (ETDEWEB)

    Fahlen, Per

    2000-07-01

    This report summarizes experience from SP research and assignments regarding refrigerated transport and storage of food, mainly in the retail sector. It presents the fundamentals of heat and mass transfer in display cabinets with special focus on indirect systems and secondary refrigerants. Moreover, the report includes a brief account of basic food hygiene and the related regulations. The material has been compiled for educational purposes in the Masters program at Chalmers Technical University.

  13. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies.

    Science.gov (United States)

    Quintero-Hernández, Veronica; Juárez-González, Victor R; Ortíz-León, Mauricio; Sánchez, Rosalba; Possani, Lourival D; Becerril, Baltazar

    2007-02-01

    The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

  14. Application of {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Hiroomi [Kyoto Prefectural Univ. of Medicine (Japan)

    1999-06-01

    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with {sup 99m}Tc, preventing depression of the antigen-binding activity. {sup 99m}Tc-labeled monoclonal antibody A7, {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7, {sup 99m}Tc-labeled normal mouse IgG and {sup 99m}Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  15. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  16. [Peptide phage display in biotechnology and biomedicine].

    Science.gov (United States)

    Kuzmicheva, G A; Belyavskaya, V A

    2016-07-01

    To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

  17. Fab'-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis.

    Science.gov (United States)

    Laroui, Hamed; Viennois, Emilie; Xiao, Bo; Canup, Brandon S B; Geem, Duke; Denning, Timothy L; Merlin, Didier

    2014-07-28

    Patients suffering from inflammatory bowel disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab'-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab'-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab'-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab'-bearing NPs loaded with TNFα siRNA than without the Fab'-bearing. Grafting the Fab'-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab'-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages.

  18. Photomask film degradation effects in the wafer fab: how to detect and monitor over time

    Science.gov (United States)

    Whittey, John; Hess, Carl; Garcia, Edgardo; Wagner, Mark; Duffy, Brian

    2012-11-01

    As a result of repeated cleanings and exposure effects such as chrome migration or MoSi oxidation some photomasks in the semiconductor fabs exhibit changes in critical dimension uniformity (CDU) over time. Detecting these effects in a timely manner allows for better risk management and process control in manufacturing. By monitoring changes in film reflectance intensity due to the various degradation mechanisms it is possible to predict when they may begin to influence across chip line width variations (ACLV). By accurately predicting the magnitude of these changes it is possible for semiconductor manufacturers to replace the photomasks before they have an impact on yields. This paper looks at possible causes of CDU variations on reticles during use and how this information might be used to improve or monitor reticle CDU changes over time.

  19. Military market for flat panel displays

    Science.gov (United States)

    Desjardins, Daniel D.; Hopper, Darrel G.

    1997-07-01

    This paper addresses the number, function and size of primary military displays and establishes a basis to determine the opportunities for technology insertion in the immediate future and into the next millennium. The military displays market is specified by such parameters as active area and footprint size, and other characteristics such as luminance, gray scale, resolution, color capability and night vision imaging system capability. A select grouping of funded, future acquisitions, planned and predicted cockpit kits, and form-fit-function upgrades are taken into account. It is the intent of this paper to provide an overview of the DoD niche market, allowing both government and industry a timely reference to insure meeting DoD requirements for flat-panel displays on schedule and in a cost-effective manner. The aggregate DoD market for direct view displays is presently estimated to be in excess of 157,000. Helmet/head mounted displays will add substantially to this total. The vanishing vendor syndrome for older display technologies is becoming a growing, pervasive problem throughout DoD, which consequently just leverage the more modern display technologies being developed for civil-commercial markets.

  20. Precarious manhood and displays of physical aggression.

    Science.gov (United States)

    Bosson, Jennifer K; Vandello, Joseph A; Burnaford, Rochelle M; Weaver, Jonathan R; Arzu Wasti, S

    2009-05-01

    The results of three experiments demonstrate that physically aggressive displays are part of men's cultural script for restoring threatened gender status. In Studies 1 and 2, challenges to men's gender status elicited heightened physically aggressive displays, including punching a pad with greater force and selecting an aggressive boxing activity over a nonaggressive puzzle activity. Study 3 established that a public display of aggressive readiness reduced men's anxiety-related cognitions in the wake of a gender threat. This suggests that aggressive displays may function to downregulate negative affect when manhood has been threatened. The discussion considers past research on gender and physical aggression in light of the authors' thesis that manhood, relative to womanhood, is culturally defined as a precarious status that must be actively, even aggressively, defended.

  1. White constancy method for mobile displays

    Science.gov (United States)

    Yum, Ji Young; Park, Hyun Hee; Jang, Seul Ki; Lee, Jae Hyang; Kim, Jong Ho; Yi, Ji Young; Lee, Min Woo

    2014-03-01

    In these days, consumer's needs for image quality of mobile devices are increasing as smartphone is widely used. For example, colors may be perceived differently when displayed contents under different illuminants. Displayed white in incandescent lamp is perceived as bluish, while same content in LED light is perceived as yellowish. When changed in perceived white under illuminant environment, image quality would be degraded. Objective of the proposed white constancy method is restricted to maintain consistent output colors regardless of the illuminants utilized. Human visual experiments are performed to analyze viewers'perceptual constancy. Participants are asked to choose the displayed white in a variety of illuminants. Relationship between the illuminants and the selected colors with white are modeled by mapping function based on the results of human visual experiments. White constancy values for image control are determined on the predesigned functions. Experimental results indicate that propsed method yields better image quality by keeping the display white.

  2. LABELING McAb 3H11 AND ITS Fab FRAGMENT WITH 211At AND THEIR IMMUNOREACTIVITIES AND INJURY EFFECTS ON HUMAN GASTRIC CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    刘宁; 罗德元; 等

    1995-01-01

    An antigastric cancer monoclonal antibody,3H11,and its Fab fragment,were labeled using p-(211At)-astatobenzoic acid(pAtBA) intermediate,in the yields of more than 30%.The results of in vitro experiments show that 211 At-3H11 and 211At-3H11 Fab are stable,and have specific immunoreactivities and cytotoxic effects to human gastric cancer cell M85,The cytotoxic effects are dependent on the concentration of 211At-3H11 or 211 At-3H11 Fab and obviously stronger than that of Na211At.

  3. Efecto de la temperatura, humedad y dieta sobre el desarrollo de Gnathocerus cornutus Fab. (Coleopera: Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Núñez Fernando

    1989-06-01

    Full Text Available The influence of temperature, relative humídíty and diet composítíon in the duration and velocity of the development cicle of Gnathoccnis comutus Fab., was studied. Each of two diets (Aand Bl was tried in presence of 5 different combinations of temperature and humídíty. Optimal conditions fOI development were found to be: 28°C and 85-90% R.H.. in which the complete cycle lasts 54 days. the larval stage is the critical one in the development in which a htgh variation among individuals in both, velocity and span of development, are notorious. The cycle duration is virtually the same for both diets, but in the diet A. a better adaptability, expressed as a proportion of descendents, was observed. Life tables and surviving curves were made.
    Se estudió la influencia de la temperatura. la humedad relativa y la composición de la dieta sobre la duración del ciclo de desarrollo y la velocidad del mismo en Gnathocerus comutus Fab, Para el efecto. cada una de las dos dietas (A y B] se ensayó en presencia de 5 combinaciones distintas de temperatura y humedad. Se encontró que las condiciones óptimas para el desarrollo. son: 28°C y 85-90% H.R.. en las cuales. el ciclo completo se desenvuelve en 54 días. El estado crítico en cuanto al desarrollo. es el larval en el cual es notoria una alta variación entre los individuos en velocidad y tiempo de desarrollo. La duración del ciclo es virtualmente la misma en ambas dietas. pero en la A se observa una mejor adaptabilidad expresada en proporctón de descendientes. Se construyeron tablas de vida y curvas de supervivencia.

  4. The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model

    Directory of Open Access Journals (Sweden)

    Ji-Guo Su

    2015-12-01

    Full Text Available Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM. Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF of the residues, as well as the MSF in the internal distance (MSFID between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein.

  5. Generation, characterization, and docking studies of DNA-hydrolyzing recombinant F(ab) antibodies.

    Science.gov (United States)

    Zein, Haggag S; El-Sehemy, Ahmed A; Fares, Mohamed O; ElHefnawi, Mahmoud; da Silva, Jaime A Teixeira; Miyatake, Kazutaka

    2011-01-01

    Previously we established a series of catalytic antibodies (catAbs) capable of hydrolyzing DNA prepared by hybridoma technology. A group of these catAbs exhibited high reactivity and substrate specificity. To determine the molecular basis for these catAbs, we cloned, sequenced, and expressed the variable regions of this group of antibodies as functional F(ab) fragments. The nucleotide and deduced amino acid sequences of the expressed light chain (Vκ) germline gene assignments confidently belonged to germline family Vκ1A, gene bb1.1 and GenBank accession number EF672207 while heavy chain variable region V(H) genes belonged to V(H) 1/V(H) J558, gene V130.3 and GenBank accession number EF672221. A well-established expression system based on the pARA7 vector was examined for its ability to produce catalytically active antibodies. Recombinant F(ab) (rF(ab) ) fragments were purified and their hydrolyzing activity was analyzed against supercoiled pUC19 plasmid DNA (scDNA). The study of rF(ab) provides important information about the potential catalytic activities of antibodies whose structure allows us to understand their basis of catalysis. Molecular surface analysis and docking studies were performed on the molecular interactions between the antibodies and poly(dA9), poly(dG9), poly(dT9), and poly(dC9) oligomers. Surface analysis identified the important sequence motifs at the binding sites, and different effects exerted by arginine and tyrosine residues at different positions in the light and heavy chains. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies. These studies may define important features of DNA catAbs.

  6. Book Display as Adult Service

    Directory of Open Access Journals (Sweden)

    Matthew S. Moore

    1997-03-01

    Full Text Available 無Book display as an adult service is defined as choosing and positioning adult books from the collection to increase their circulation. The author contrasts bookstore arrangement for sales versus library arrangement for access. The paper considers the library-as-a-whole as a display, examines the right size for an in-library display, and discusses mass displays, end-caps, on-shelf displays, and the Tiffany approach. The author proposes that an effective display depends on an imaginative, unifying theme, and that book displays are part of the joy of libraries.

  7. Handbook of Visual Display Technology

    CERN Document Server

    Cranton, Wayne; Fihn, Mark

    2012-01-01

    The Handbook of Visual Display Technology is a unique work offering a comprehensive description of the science, technology, economic and human interface factors associated with the displays industry. An invaluable compilation of information, the Handbook will serve as a single reference source with expert contributions from over 150 international display professionals and academic researchers. All classes of display device are covered including LCDs, reflective displays, flexible solutions and emissive devices such as OLEDs and plasma displays, with discussion of established principles, emergent technologies, and particular areas of application. The wide-ranging content also encompasses the fundamental science of light and vision, image manipulation, core materials and processing techniques, display driving and metrology.

  8. Acrobatic courtship display coevolves with brain size in manakins (Pipridae).

    Science.gov (United States)

    Lindsay, Willow R; Houck, Justin T; Giuliano, Claire E; Day, Lainy B

    2015-01-01

    Acrobatic display behaviour is sexually selected in manakins (Pipridae) and can place high demands on many neural systems. Manakin displays vary across species in terms of behavioural complexity, differing in number of unique motor elements, production of mechanical sounds, cooperation between displaying males, and construction of the display site. Historically, research emphasis has been placed on neurological specializations for vocal aspects of courtship, and less is known about the control of physical, non-vocal displays. By examining brain evolution in relation to extreme acrobatic feats such as manakin displays, we can vastly expand our knowledge of how sexual selection acts on motor behaviour. We tested the hypothesis that sexual selection for complex motor displays has selected for larger brains across the Pipridae. We found that display complexity positively predicts relative brain weight (adjusted for body size) after controlling for phylogeny in 12 manakin species and a closely related flycatcher. This evidence suggests that brain size has evolved in response to sexual selection to facilitate aspects of display such as motor, sensorimotor, perceptual, and cognitive abilities. We show, for the first time, that sexual selection for acrobatic motor behaviour can drive brain size evolution in avian species and, in particular, a family of suboscine birds.

  9. Simplified Night Sky Display System

    Science.gov (United States)

    Castellano, Timothy P.

    2010-01-01

    A document describes a simple night sky display system that is portable, lightweight, and includes, at most, four components in its simplest configuration. The total volume of this system is no more than 10(sup 6) cm(sup 3) in a disassembled state, and weighs no more than 20 kilograms. The four basic components are a computer, a projector, a spherical light-reflecting first surface and mount, and a spherical second surface for display. The computer has temporary or permanent memory that contains at least one signal representing one or more images of a portion of the sky when viewed from an arbitrary position, and at a selected time. The first surface reflector is spherical and receives and reflects the image from the projector onto the second surface, which is shaped like a hemisphere. This system may be used to simulate selected portions of the night sky, preserving the appearance and kinesthetic sense of the celestial sphere surrounding the Earth or any other point in space. These points will then show motions of planets, stars, galaxies, nebulae, and comets that are visible from that position. The images may be motionless, or move with the passage of time. The array of images presented, and vantage points in space, are limited only by the computer software that is available, or can be developed. An optional approach is to have the screen (second surface) self-inflate by means of gas within the enclosed volume, and then self-regulate that gas in order to support itself without any other mechanical support.

  10. Latest development of display technologies

    Science.gov (United States)

    Gao, Hong-Yue; Yao, Qiu-Xiang; Liu, Pan; Zheng, Zhi-Qiang; Liu, Ji-Cheng; Zheng, Hua-Dong; Zeng, Chao; Yu, Ying-Jie; Sun, Tao; Zeng, Zhen-Xiang

    2016-09-01

    In this review we will focus on recent progress in the field of two-dimensional (2D) and three-dimensional (3D) display technologies. We present the current display materials and their applications, including organic light-emitting diodes (OLEDs), flexible OLEDs quantum dot light emitting diodes (QLEDs), active-matrix organic light emitting diodes (AMOLEDs), electronic paper (E-paper), curved displays, stereoscopic 3D displays, volumetric 3D displays, light field 3D displays, and holographic 3D displays. Conventional 2D display devices, such as liquid crystal devices (LCDs) often result in ambiguity in high-dimensional data images because of lacking true depth information. This review thus provides a detailed description of 3D display technologies.

  11. Selection of Triptolide Ligands from a Random Phage Display Library and Primary Verification of Their Combination%雷公藤内酯醇靶蛋白的筛选及其结合的初步验证

    Institute of Scientific and Technical Information of China (English)

    杨旭光; 徐晓煜; 李家璜; 姚其正; 朱彤阳; 华子春; 郑伟娟

    2012-01-01

    Triptolide is an extract from the Chinese herb Tripterygium wilfordii Hook f. We screened out a peptide from a C7 phage display library and presumed it was a potential peptide ligand for triptolide. The specificity of the selected clone for triptolide was confirmed by ELISA and immunoprecipi-tation. After DNA sequencing, a BLAST search was carried out and 77 matching sequences was retrieved. The most promising candidate is human steroidogenic factor-1 (hSF-1), a member of the nuclear receptor family that controls the synthesis of steroid hormones by regulating steroidogenic enzyme genes. We purified the ligand binding domain (LBD) of hSF-1 and then used it for further experiments. Fluorescence spectrum experiments showed that triptolide could cause fluorescence quenching of hSF-1-LBD, isothermal titration calorimetric(ITC) measurements showed a enthalpydriven interaction between triptolide and hSF-1-LBD, and a dose-dependent interaction between them was observed by surface plasmon resonance(SPR). All these results confirmed the specific interaction between hSF-1 and triptolide.%采用噬菌体展示肽库技术筛选雷公藤内酯醇的靶蛋白,得到了一个肽段,并通过酶联免疫吸附(ELISA)和免疫共沉淀验证了该片段对雷公藤内酯醇的结合特异性.用Basic Local Alignment Search Tool (BLAST)进行序列对比后,找到了77个匹配序列,其中最为匹配的序列是人类类固醇生成因子-1(hSF-1),因此hSF-1可能是雷公藤内酯醇的一个潜在受体.在大肠杆菌中表达纯化了hSF-1的配体结合域(LBD),荧光光谱实验表明雷公藤内酯醇对hSF-1-LBD有荧光淬灭作用、等温滴定量热(ITC)实验表明雷公藤内酯醇与hSF-1-LBD发生焓驱动的特异性结合,表面等离子体共振(SPR)实验表明雷公藤内酯醇可以与hSF-1-LBD剂量依赖性结合,这些都证实了hSF-1与雷公藤内酯醇存在特异性相互作用.

  12. Methods and apparatus for transparent display using scattering nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Chia Wei; Qiu, Wenjun; Zhen, Bo; Shapira, Ofer; Soljacic, Marin

    2017-06-14

    Transparent displays enable many useful applications, including heads-up displays for cars and aircraft as well as displays on eyeglasses and glass windows. Unfortunately, transparent displays made of organic light-emitting diodes are typically expensive and opaque. Heads-up displays often require fixed light sources and have limited viewing angles. And transparent displays that use frequency conversion are typically energy inefficient. Conversely, the present transparent displays operate by scattering visible light from resonant nanoparticles with narrowband scattering cross sections and small absorption cross sections. More specifically, projecting an image onto a transparent screen doped with nanoparticles that selectively scatter light at the image wavelength(s) yields an image on the screen visible to an observer. Because the nanoparticles scatter light at only certain wavelengths, the screen is practically transparent under ambient light. Exemplary transparent scattering displays can be simple, inexpensive, scalable to large sizes, viewable over wide angular ranges, energy efficient, and transparent simultaneously.

  13. Methods and apparatus for transparent display using scattering nanoparticles

    Science.gov (United States)

    Hsu, Chia Wei; Qiu, Wenjun; Zhen, Bo; Shapira, Ofer; Soljacic, Marin

    2016-05-10

    Transparent displays enable many useful applications, including heads-up displays for cars and aircraft as well as displays on eyeglasses and glass windows. Unfortunately, transparent displays made of organic light-emitting diodes are typically expensive and opaque. Heads-up displays often require fixed light sources and have limited viewing angles. And transparent displays that use frequency conversion are typically energy inefficient. Conversely, the present transparent displays operate by scattering visible light from resonant nanoparticles with narrowband scattering cross sections and small absorption cross sections. More specifically, projecting an image onto a transparent screen doped with nanoparticles that selectively scatter light at the image wavelength(s) yields an image on the screen visible to an observer. Because the nanoparticles scatter light at only certain wavelengths, the screen is practically transparent under ambient light. Exemplary transparent scattering displays can be simple, inexpensive, scalable to large sizes, viewable over wide angular ranges, energy efficient, and transparent simultaneously.

  14. Review of Display Technologies Focusing on Power Consumption

    Directory of Open Access Journals (Sweden)

    María Rodríguez Fernández

    2015-08-01

    Full Text Available This paper provides an overview of the main manufacturing technologies of displays, focusing on those with low and ultra-low levels of power consumption, which make them suitable for current societal needs. Considering the typified value obtained from the manufacturer’s specifications, four technologies—Liquid Crystal Displays, electronic paper, Organic Light-Emitting Display and Electroluminescent Displays—were selected in a first iteration. For each of them, several features, including size and brightness, were assessed in order to ascertain possible proportional relationships with the rate of consumption. To normalize the comparison between different display types, relative units such as the surface power density and the display frontal intensity efficiency were proposed. Organic light-emitting display had the best results in terms of power density for small display sizes. For larger sizes, it performs less satisfactorily than Liquid Crystal Displays in terms of energy efficiency.

  15. Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen.

    Science.gov (United States)

    Matsuoka, Daiko; Mizutani, Nobuaki; Sae-Wong, Chutha; Yoshino, Shin

    2014-09-01

    Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28-30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1-10) with papain were also intranasally administered 15min before each OVA challenge. The results showed that treatment with O1-10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1-10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.

  16. Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

    Science.gov (United States)

    Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

    2014-11-01

    The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.

  17. Active site modification of the β-ketoacyl-ACP synthase FabF3 of Streptomyces coelicolor affects the fatty acid chain length of the CDA lipopeptides.

    Science.gov (United States)

    Lewis, Richard A; Nunns, Laura; Thirlway, Jenny; Carroll, Kathleen; Smith, Colin P; Micklefield, Jason

    2011-02-14

    Using site directed mutagenesis we altered an active site residue (Phe107) of the enzyme encoded by fabF3 (SCO3248) in the Streptomyces coelicolor gene cluster required for biosynthesis of the calcium dependent antibiotics (CDAs), successfully generating two novel CDA derivatives comprising truncated (C4) lipid side chains and confirming that fabF3 encodes a KAS-II homologue that is involved in determining CDA fatty acid chain length.

  18. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  19. Recognition of epoxy with phage displayed peptides.

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2013-07-01

    The development of a general approach for non-destructive chemical and biological functionalization of epoxy could expand opportunities for both fundamental studies and creating various device platforms. Epoxy shows unique electrical, mechanical, chemical and biological compatibility and has been widely used for fabricating a variety of devices. Phage display has emerged as a powerful method for selecting peptides that possess enhanced selectivity and binding affinity toward a variety of targets. In this letter, we demonstrate for the first time a powerful yet benign approach for identifying binding motifs to epoxy via comprehensively screened phage displayed peptides. Our results show that the epoxy can be selectively recognized with peptide-displaying phages. Further, along with the development of epoxy-based microstructures; recognition of the epoxy with phage displayed peptides can be specifically localized in these microstructures. We anticipate that these results could open up exciting opportunities in the use of peptide-recognized epoxy in fundamental biochemical recognition studies, as well as in applications ranging from analytical devices, hybrid materials, surface and interface, to cell biology. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    Science.gov (United States)

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  1. Transcription of the Escherichia coli fatty acid synthesis operon fabHDG is directly activated by FadR and inhibited by ppGpp.

    Science.gov (United States)

    My, Laetitia; Rekoske, Brian; Lemke, Justin J; Viala, Julie P; Gourse, Richard L; Bouveret, Emmanuelle

    2013-08-01

    In Escherichia coli, FadR and FabR are transcriptional regulators that control the expression of fatty acid degradation and unsaturated fatty acid synthesis genes, depending on the availability of fatty acids. In this report, we focus on the dual transcriptional regulator FadR. In the absence of fatty acids, FadR represses the transcription of fad genes required for fatty acid degradation. However, FadR is also an activator, stimulating transcription of the products of the fabA and fabB genes responsible for unsaturated fatty acid synthesis. In this study, we show that FadR directly activates another fatty acid synthesis promoter, PfabH, which transcribes the fabHDG operon, indicating that FadR is a global regulator of both fatty acid degradation and fatty acid synthesis. We also demonstrate that ppGpp and its cofactor DksA, known primarily for their role in regulation of the synthesis of the translational machinery, directly inhibit transcription from the fabH promoter. ppGpp also inhibits the fadR promoter, thereby reducing transcription activation of fabH by FadR indirectly. Our study shows that both ppGpp and FadR have direct roles in the control of fatty acid promoters, linking expression in response to both translation activity and fatty acid availability.

  2. Resistance Mechanisms and the Future of Bacterial Enoyl-Acyl Carrier Protein Reductase (FabI) Antibiotics.

    Science.gov (United States)

    Yao, Jiangwei; Rock, Charles O

    2016-03-01

    Missense mutations leading to clinical antibiotic resistance are a liability of single-target inhibitors. The enoyl-acyl carrier protein reductase (FabI) inhibitors have one intracellular protein target and drug resistance is increased by the acquisition of single-base-pair mutations that alter drug binding. The spectrum of resistance mechanisms to FabI inhibitors suggests criteria that should be considered during the development of single-target antibiotics that would minimize the impact of missense mutations on their clinical usefulness. These criteria include high-affinity, fast on/off kinetics, few drug contacts with residue side chains, and no toxicity. These stringent criteria are achievable by structure-guided design, but this approach will only yield pathogen-specific drugs. Single-step acquisition of resistance may limit the clinical application of broad-spectrum, single-target antibiotics, but appropriately designed pathogen-specific antibiotics have the potential to overcome this liability.

  3. Bright color reflective displays with interlayer reflectors

    Science.gov (United States)

    Kitson, Stephen; Geisow, Adrian; Rudin, John; Taphouse, Tim

    2011-08-01

    A good solution to the reflective display of color has been a major challenge for the display industry, with very limited color gamuts demonstrated to date. Conventional side-by-side red, green and blue color filters waste two-thirds of incident light. The alternative of stacking cyan, magenta and yellow layers is also challenging -- a 10% loss per layer compounds to nearly 50% overall. Here we demonstrate an architecture that interleaves absorbing-to-clear shutters with matched wavelength selective reflectors. This increases color gamut by reducing losses and more cleanly separating the color channels, and gives much wider choice of electro-optic colorants.

  4. Enterococcus faecalis endocarditis severity in rabbits is reduced by IgG Fabs interfering with aggregation substance.

    Directory of Open Access Journals (Sweden)

    Patrick M Schlievert

    Full Text Available BACKGROUND: Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue. Our previous research determined that enterococcal aggregation substance (AS is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS+ E. faecalis endocarditis. METHODOLOGY/PRINCIPAL FINDINGS: When challenged with AS+ E. faecalis, 10 rabbits actively immunized against AS+ E. faecalis developed more significant vegetations than 9 animals immunized against AS⁻E. faecalis, and 9/10 succumbed compared to 2/9 (p<0.005, suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group immediately before challenge with AS+E. faecalis, reduced total vegetation (endocarditis lesion microbial counts (7.9 x 10⁶ versus 2.0 x 10⁵, p = 0.02 and size (40 mg versus 10, p = 0.05. In vitro, the Fabs prevented enterococcal aggregation. CONCLUSIONS/SIGNIFICANCE: The data confirm the role of AS in infective endocarditis formation and suggest that use of Fabs against AS will provide partial protection from AS+E. faecalis illness.

  5. Phage display: concept, innovations, applications and future.

    Science.gov (United States)

    Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

    2010-01-01

    Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.

  6. Multifunctional g3p-peptide tag for current phage display systems.

    Science.gov (United States)

    Beckmann, C; Haase, B; Timmis, K N; Tesar, M

    1998-03-15

    We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid [Tesar, M., Beckmann, C., Röttgen, P., Haase, B., Faude, U., Timmis, K., 1995. Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunology 1, 53-54]. In this study we report mapping of the antigenic epitope of the mAb 10C3, by means of short overlapping peptide-sequences [Frank, R., Overwin, H., 1996. Spot synthesis. In: Morris, G.E. (Ed.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, pp. 149-169.] comprising the C-terminal half of the g3-protein. A minimal recognizable peptide was found which is represented in the 11 amino acid sequence from positions 292 to 302 of g3p [Wezenbeek van, P.M.G.P., Hulsebos, T.J.M., Schoenmakers, J.G.G., 1980. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene 11, 129-148]. In order to use the antibody also for detection and purification of recombinant proteins, such as single chain antibodies, the epitope was introduced as a tag sequence into the phagemid pHEN1 [Hoogenboom, H.R., Griffith, A.D., Johnson, K., Chiswell, D.J., Hudson, P., Winter, G., 1991. Multi-subunit proteins on the surface of the filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acid Res. 19, 4133-4137; Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994. Antibody fragments from a single pot phage display library as immunochemical reagents. EMBO J. 13 (3) 692-698]. Purified single chain antibodies containing this tag were detectable down to a concentration of 2 ng ml(-1) under non-denaturing conditions (ELISA) or 4 ng per lane on immunoblots

  7. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae.

    Science.gov (United States)

    Mohedano, Maria L; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  8. Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

    Energy Technology Data Exchange (ETDEWEB)

    Niemi, Merja, E-mail: merja.niemi@joensuu.fi; Jänis, Janne [Department of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu (Finland); Jylhä, Sirpa [VTT Technical Research Centre of Finland, PO Box 1000, FIN-02044 VTT (Finland); Kallio, Johanna M.; Hakulinen, Nina [Department of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu (Finland); Laukkanen, Marja-Leena; Takkinen, Kristiina [VTT Technical Research Centre of Finland, PO Box 1000, FIN-02044 VTT (Finland); Rouvinen, Juha [Department of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu (Finland)

    2008-01-01

    The high-resolution mass-spectrometric characterization, crystallization and X-ray diffraction studies of a recombinant IgE Fab fragment in complex with bovine β-lactoglobulin are reported. A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT–ICR mass spectrometry and crystallized with bovine β-lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 Å resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 Å. The three-dimensional structure of the D1 Fab fragment–BLG complex will provide the first insight into IgE antibody–allergen interactions at the molecular level.

  9. Crystallization and preliminary X-ray analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Saito, Jun, E-mail: jun-saito@meiji.co.jp; Yamada, Mototsugu; Watanabe, Takashi; Kitagawa, Hideo; Takeuchi, Yasuo [Pharmaceutical Research Center, Meiji Seika Kaisha Ltd, 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567 (Japan)

    2006-06-01

    Enoyl-acyl carrier protein (ACP) reductases are responsible for bacterial type II fatty-acid biosynthesis and are attractive targets for developing novel antibiotics. The S. pneumoniae enoyl-ACP reductase (FabK) was crystallized and selenomethionine MAD data were collected to 2 Å resolution. The enoyl-acyl carrier protein (ACP) reductase from Streptococcus pneumoniae (FabK; EC 1.3.1.9) is responsible for catalyzing the final step in each elongation cycle of fatty-acid biosynthesis. Selenomethionine-substituted FabK was purified and crystallized by the hanging-drop vapour-diffusion method at 277 K. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.26, b = 126.70, c = 53.63 Å, β = 112.46°. Diffraction data were collected to 2.00 Å resolution using synchrotron beamline BL32B2 at SPring-8. Two molecules were estimated to be present in the asymmetric unit, with a solvent content of 45.1%.

  10. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    Science.gov (United States)

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  11. Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

    Science.gov (United States)

    Schlievert, Patrick M.; Chuang-Smith, Olivia N.; Peterson, Marnie L.; Cook, Laura C. C.; Dunny, Gary M.

    2010-01-01

    Background Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS+ E. faecalis endocarditis. Methodology/Principal Findings When challenged with AS+ E. faecalis, 10 rabbits actively immunized against AS+ E. faecalis developed more significant vegetations than 9 animals immunized against AS− E. faecalis, and 9/10 succumbed compared to 2/9 (pendocarditis lesion) microbial counts (7.9×106 versus 2.0×105, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation. Conclusions/Significance The data confirm the role of AS in infective endocarditis formation and suggest that use of Fabs against AS will provide partial protection from AS+ E. faecalis illness. PMID:20957231

  12. Independent evolution of Fc- and Fab-mediated HIV-1-specific antiviral antibody activity following acute infection

    Science.gov (United States)

    Dugast, Anne-Sophie; Stamatatos, Leonidas; Tonelli, Andrew; Suscovich, Todd J.; Licht, Anna F.; Mikell, Iliyana; Ackerman, Margaret E.; Streeck, Hendrik; Klasse, P.J.; Moore, John P.; Alter, Galit

    2014-01-01

    Fc-related antibody activities, such as antibody-dependent cellular cytotoxicity (ADCC), or more broadly, antibody-mediated cellular viral inhibition (ADCVI), play a role in curbing early SIV viral replication, are enriched in human long-term infected non-progressors, and could potentially contribute to protection from infection. However, little is known about the mechanism by which such humoral immune responses are naturally induced following infection. Here we focused on the early evolution of the functional antibody response, largely driven by the Fc portion of the antibody, in the context of the evolving binding and neutralizing antibody response, which is driven mainly by the antibody binding fragment (Fab). We show that ADCVI/ADCC-inducing responses in humans are rapidly generated following acute HIV-1 infection, peak at approximately 6 months post-infection, but decay rapidly in the setting of persistent immune activation, as Fab-related activities persistently increase. Moreover, the loss of Fc activity occurred in synchrony with a loss of HIV-specific IgG3 responses. Our data strongly suggest that Fc- and Fab-related antibody functions are modulated in a distinct manner following acute HIV infection. Vaccination strategies intended to optimally induce both sets of antiviral antibody activities may, therefore, require a fine-tuning of the inflammatory response. PMID:25043633

  13. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    Energy Technology Data Exchange (ETDEWEB)

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  14. Dual Constant Domain-Fab: A novel strategy to improve half-life and potency of a Met therapeutic antibody.

    Science.gov (United States)

    Cignetto, Simona; Modica, Chiara; Chiriaco, Cristina; Fontani, Lara; Milla, Paola; Michieli, Paolo; Comoglio, Paolo M; Vigna, Elisa

    2016-06-01

    The kinase receptor encoded by the Met oncogene is a sensible target for cancer therapy. The chimeric monovalent Fab fragment of the DN30 monoclonal antibody (MvDN30) has an odd mechanism of action, based on cell surface removal of Met via activation of specific plasma membrane proteases. However, the short half-life of the Fab, due to its low molecular weight, is a severe limitation for the deployment in therapy. This issue was addressed by increasing the Fab molecular weight above the glomerular filtration threshold through the duplication of the constant domains, in tandem (DCD-1) or reciprocally swapped (DCD-2). The two newly engineered molecules showed biochemical properties comparable to the original MvDN30 in vitro, acting as full Met antagonists, impairing Met phosphorylation and activation of downstream signaling pathways. As a consequence, Met-mediated biological responses were inhibited, including anchorage-dependent and -independent cell growth. In vivo DCD-1 and DCD-2 showed a pharmacokinetic profile significantly improved over the original MvDN30, doubling the circulating half-life and reducing the clearance. In pre-clinical models of cancer, generated by injection of tumor cells or implant of patient-derived samples, systemic administration of the engineered molecules inhibited the growth of Met-addicted tumors.

  15. 抗轮状病毒IgY和Fab的研制%Preparation of IgY and Fab against HRV

    Institute of Scientific and Technical Information of China (English)

    孙淑清; 孟岩; 段春燕; 胡彦涛

    2005-01-01

    目的对抗轮状病毒(RV)IgY和胃蛋白酶水解片断Fab进行分离与纯化.方法免疫鸡得到抗HRVIgY,用两步盐析结合凝胶过滤将其从蛋黄中分离出来,纯的IgY经胃蛋白酶水解得抗体片断Fab.结果抗HRVIgY用SDS-PAGE检测纯度可达到95%以上.抗轮状病毒(RV)IgY和抗体片断Fab经SDS-PAGE和MALDIMS法测定,其纯度达到99%以上,经ELISA法检测,Fab'的活性保持在IgY原始活性的70%以上.结论我们所设计的分离和纯化抗HRVIgY和Fab'的方法简单、有效.

  16. Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain.

    Science.gov (United States)

    Kanje, Sara; von Witting, Emma; Chiang, Samuel C C; Bryceson, Yenan T; Hober, Sophia

    2016-09-21

    Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody's binding to its target. Moreover, an emerging field for therapeutics is antibody-drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.

  17. Sexing murine embryos with an indirect immunofluorescence assay using phage antibody B9-Fab against SDM antigen.

    Science.gov (United States)

    Wang, Naidong; Yuan, Anwen; Ma, Jun; Deng, Zhibang; Xue, Liqun

    2015-06-01

    The use of serologically detectable male (SDM; also called H-Y) antigens to identify male embryos may be limited by the source of anti-SDM antibody. In the present study, novel anti-SDM B9-Fab recombinant clones (obtained by chain shuffling of an A8 original clone) were used to detect SDM antigens on murine embryos. Murine morulae and blastocysts (n=138) were flushed from the oviducts of Kunming mice and incubated with anti-SDM B9-Fab for 30 min at 37°C. With an indirect immunofluorescence assay, the membrane and inner cell mass had bright green fluorescence (presumptive males). Overall, 43.5% (60/138) were classified as presumptive males and 56.5% (78/138) as presumptive females, with 85.0 and 88.5% of these, respectively, confirmed as correct predictions (based on PCR analysis of a male-specific [Sry] sequence). We concluded that the anti-SDM B9-Fab molecule had potential for non-invasive, technically simple immunological sexing of mammalian embryos.

  18. The response regulator YycF inhibits expression of the fatty acid biosynthesis repressor FabT in Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Maria Luz Mohedano

    2016-08-01

    Full Text Available The YycFG (also known as WalRK, VicRK, MicAB or TCS02 two-component system (TCS is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  19. Structural and functional insights into the anti-BACE1 Fab fragment that recognizes the BACE1 exosite.

    Science.gov (United States)

    Gutiérrez, Lucas Joel; Andujar, Sebastián Antonio; Enriz, Ricardo Daniel; Baldoni, Héctor Armando

    2014-01-01

    A molecular modeling study giving structural, functional, and mutagenesis insights into the anti-BACE1 Fab fragment that recognizes the BACE1 exosite is reported. Our results allow extending experimental data resulting from X-ray diffraction experiments in order to examine unknown aspects for the Fab-BACE1 recognition and its binding mode. Thus, the study performed here allows extending the inherently static nature of crystallographic structures in order to gain a deeper understanding of the structural and dynamical basis at the atomic level. The characteristics and strength of the interatomic interactions involved in the immune complex formation are exhaustively analyzed. The results might explain how the anti-BACE1 Fab fragment and other BACE1 exosite binders are capable to produce an allosteric modulation of the BACE1 activity. Our site-directed mutagenesis study indicated that the functional anti-BACE1 paratope, residues Tyr32 (H1), Trp50 (H2), Arg98 (H3), Phe101 (H3), Trp104 (H3) and Tyr94 (L3), strongly dominates the binding energetics with the BACE1 exosite. The mutational studies described in this work might accelerate the development of new BACE1 exosite binders with interesting pharmacological activity.

  20. Colorimetry for CRT displays.

    Science.gov (United States)

    Golz, Jürgen; MacLeod, Donald I A

    2003-05-01

    We analyze the sources of error in specifying color in CRT displays. These include errors inherent in the use of the color matching functions of the CIE 1931 standard observer when only colorimetric, not radiometric, calibrations are available. We provide transformation coefficients that prove to correct the deficiencies of this observer very well. We consider four different candidate sets of cone sensitivities. Some of these differ substantially; variation among candidate cone sensitivities exceeds the variation among phosphors. Finally, the effects of the recognized forms of observer variation on the visual responses (cone excitations or cone contrasts) generated by CRT stimuli are investigated and quantitatively specified. Cone pigment polymorphism gives rise to variation of a few per cent in relative excitation by the different phosphors--a variation larger than the errors ensuing from the adoption of the CIE standard observer, though smaller than the differences between some candidate cone sensitivities. Macular pigmentation has a larger influence, affecting mainly responses to the blue phosphor. The estimated combined effect of all sources of observer variation is comparable in magnitude with the largest differences between competing cone sensitivity estimates but is not enough to disrupt very seriously the relation between the L and M cone weights and the isoluminance settings of individual observers. It is also comparable with typical instrumental colorimetric errors, but we discuss these only briefly.

  1. Generation and characterization of a human neutralizing bivalent antibody Fab094-DDD against rabies virus%人源特异性抗狂犬病毒二价Fab094-DDD抗体的制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    目的:利用蛋白激酶A(protein kinase A,PKA)调节亚基(R亚基)的二聚体化功能结构域(DDD)的二聚化功能,制备具有中和活性的人源特异性抗狂犬病毒二价抗体.方法:优化合成linker-C-DDD序列,设计引物扩增抗狂犬病毒抗体Fab094的Fd段和轻链可变区序列(Vκ),通过overlap PCR将Fab094的Fd段和linker-C-DDD基因重组为Fd-DDD,将其和Vκ分别克隆到真核表达载体中,共转染293Free style细胞,表达纯化该抗体.应用SDS-PAGE 、Western blot、ELISA、免疫共沉淀、亲和力测定及免疫荧光试验检测该抗体的免疫学特性,用荧光抗体病毒中和试验检测其中和活性.结果:成功构建了全人源抗狂犬病毒二价抗体Fab094-DDD的真核表达载体,获得的二价Fab094-DDD抗体与抗原保持着较好的亲和力,能特异性结合狂犬病毒,中和效价为213.2 U/mg.结论:成功制备了抗狂犬病毒二价Fab094-DDD抗体,具有较高的中和活性,为进一步研发狂犬病治疗性抗体药物奠定了基础,对于其他疾病双表位人源特异性抗体的制备也具有借鉴作用.

  2. Recombinant baculovirus displayed vaccine

    Science.gov (United States)

    Prabakaran, Mookkan; Kwang, Jimmy

    2014-01-01

    The rapid evolution of new sublineages of H5N1 influenza in Asia poses the greatest challenge in vaccine development for pre-pandemic preparedness. To overcome the antigenic diversity of H5N1 strains, multiple vaccine strains can be designed based on the distribution of neutralizing epitopes in the globular head of H5 hemagglutinin (HA). Recently, we selected two different HAs of H5N1 strains based on the neutralizing epitopes and reactivity with different neutralizing antibodies. The HAs of selected vaccine strains were individually expressed on the baculovirus envelope (bivalent-BacHA) with its native antigenic configuration. Further, oral delivery of live bivalent-BacHA elicited broadly reactive humoral, mucosal and cell-mediated immune responses and showed complete protection against antigenically distinct H5N1 strains in mice. The strategy for the vaccine strain selection, vaccine design and route of administration will provide an idea for development of a widely protective vaccine against highly pathogenic H5N1 for pre-pandemic preparedness. PMID:23941989

  3. Data Display in Qualitative Research

    Directory of Open Access Journals (Sweden)

    Susana Verdinelli PsyD

    2013-02-01

    Full Text Available Visual displays help in the presentation of inferences and conclusions and represent ways of organizing, summarizing, simplifying, or transforming data. Data displays such as matrices and networks are often utilized to enhance data analysis and are more commonly seen in quantitative than in qualitative studies. This study reviewed the data displays used by three prestigious qualitative research journals within a period of three years. The findings include the types of displays used in these qualitative journals, the frequency of use, and the purposes for using visual displays as opposed to presenting data in text.

  4. Unique interactive projection display screen

    Energy Technology Data Exchange (ETDEWEB)

    Veligdan, J.T.

    1997-11-01

    Projection systems continue to be the best method to produce large (1 meter and larger) displays. However, in order to produce a large display, considerable volume is typically required. The Polyplanar Optic Display (POD) is a novel type of projection display screen, which for the first time, makes it possible to produce a large projection system that is self-contained and only inches thick. In addition, this display screen is matte black in appearance allowing it to be used in high ambient light conditions. This screen is also interactive and can be remotely controlled via an infrared optical pointer resulting in mouse-like control of the display. Furthermore, this display need not be flat since it can be made curved to wrap around a viewer as well as being flexible.

  5. Pitfalls to avoid when using phage display for snake toxins.

    Science.gov (United States)

    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno; Andersen, Mikael Rørdam; Lohse, Brian

    2017-02-01

    Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions.

  6. Route of infection and hematological effect of Metarhizium anisopliae (Metsch.) Sorokin on Dysdercus cingulatus (Fab.) adult.

    Science.gov (United States)

    Sahayaraj, Kitherian; Borgio, Jesu Francis; Lucini, Luigi

    2014-01-01

    The primary objective of this work was to identify, under laboratory conditions, the route of infection and hemogram of Dysdercus cingulatus (Fab.) adults by Metarhizium anisopliae. The infection process in D. cingulatus by M. anisopliae involved the conidia adherence to the host cuticle and germination after 24 h post-infection, accompanied by falling of bristles. The subsequent step, within 24-48 h post-infection, comprised penetration of fungus through spiracles, root of bristles, hemolymph, and the three dorsal sacs. Subsequently, within 72-96 h post-infection, the fungus penetrated into trachea and sacs, then emerged on cuticular surface and was found to be maximum in hemolymph. A great decrease in hemocytes count was observed within 96 h from infection. The hemosomic index (HSI) decreased gradually as the incubation period increased. As far as we know, this is the first study to know the mechanism of action of M. anisopliae to D. cingulatus.

  7. Characterization of silicon photomultipliers at National Nano-Fab Center for PET-MR.

    Science.gov (United States)

    Kim, Hyoungtaek; Sul, Woo Suk; Cho, Gyuseong

    2014-10-01

    The silicon photomultipliers (SiPMs) were fabricated for magnetic resonance compatible positron emission tomography (PET) applications using customized CMOS processes at National NanoFab Center. Each micro-cell consists of a shallow n+/p well junction on a p-type epitaxial wafer and passive quenching circuit was applied. The size of the SiPM is 3 × 3 mm(2) and the pitch of each micro-cell is 65 μm. In this work, several thousands of SiPMs were packaged and tested to build a PET ring detector which has a 60 mm axial and 390 mm radial field of view. I-V characteristics of the SiPMs are shown good uniformity and breakdown voltage is around 20 V. The photon detection efficiency was measured via photon counting method and the maximum value was recorded as 16% at 470 nm. The gamma ray spectrum of a Ge-68 isotope showed nearly 10% energy resolution at 511 keV with a 3 × 3 × 20 mm(3) LYSO crystal.

  8. Imaging of cardiac allograft rejection in dogs using indium-111 monoclonal antimyosin Fab

    Energy Technology Data Exchange (ETDEWEB)

    Addonizio, L.J.; Michler, R.E.; Marboe, C.; Esser, P.E.; Johnson, L.L.; Seldin, D.W.; Gersony, W.M.; Alderson, P.O.; Rose, E.A.; Cannon, P.J.

    1987-03-01

    The acute rejection of cardiac allografts is currently diagnosed by the presence of myocyte necrosis on endomyocardial biopsy. We evaluated the efficacy of noninvasive scintigraphic imaging with indium-111-labeled anticardiac myosin Fab fragments (indium-111 antimyosin) to detect and quantify cardiac allograft rejection. Six dogs that had intrathoracic heterotopic cardiac allograft transplantation were injected with indium-111 antimyosin and planar and single photon emission computed tomographic (SPECT) images were obtained in various stages of acute and subacute rejection. Four dogs had an allograft older than 8 months and had been on long-term immunosuppressive therapy; two dogs had an allograft less than 2 weeks old and were not on immunosuppressive therapy. Count ratios comparing heterotopic with native hearts were calculated from both SPECT images and in vitro scans of excised and sectioned hearts and were compared with the degree of rejection scored by an independent histopathologic review. Indium-111 antimyosin uptake was not visible in planar or SPECT images of native hearts. Faint diffuse uptake was apparent in cardiac allografts during long-term immunosuppression and intense radioactivity was present in hearts with electrocardiographic evidence of rejection. The heterotopic to native heart count ratios in SPECT images correlated significantly with the count ratios in the excised hearts (r = 0.93) and with the histopathologic rejection score (r = 0.97). The distribution of indium-111 antimyosin activity in right and left ventricles corresponded to areas of histopathologic abnormalities.

  9. Characterization of silicon photomultipliers at National Nano-Fab Center for PET-MR

    Science.gov (United States)

    Kim, Hyoungtaek; Sul, Woo Suk; Cho, Gyuseong

    2014-10-01

    The silicon photomultipliers (SiPMs) were fabricated for magnetic resonance compatible positron emission tomography (PET) applications using customized CMOS processes at National NanoFab Center. Each micro-cell consists of a shallow n+/p well junction on a p-type epitaxial wafer and passive quenching circuit was applied. The size of the SiPM is 3 × 3 mm2 and the pitch of each micro-cell is 65 μm. In this work, several thousands of SiPMs were packaged and tested to build a PET ring detector which has a 60 mm axial and 390 mm radial field of view. I-V characteristics of the SiPMs are shown good uniformity and breakdown voltage is around 20 V. The photon detection efficiency was measured via photon counting method and the maximum value was recorded as 16% at 470 nm. The gamma ray spectrum of a Ge-68 isotope showed nearly 10% energy resolution at 511 keV with a 3 × 3 × 20 mm3 LYSO crystal.

  10. Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin.

    Science.gov (United States)

    Song, Hyung-Nam; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2015-01-01

    Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144-154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 10(4) M(-1) s(-1) and a dissociation constant of 2.28 × 10(-2) s(-1), which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease.

  11. Integration of the APC framework with AMD's Fab25 factory system

    Science.gov (United States)

    Bushman, Scott; Campbell, William J.; Miller, Michael L.

    1999-09-01

    This paper discusses the integration and development of advanced process control technologies with AMD's Fab25 factory systems using the Advance Process Control Framework. The Framework is an open software architecture that allows the integration of existing factory systems, such as the manufacturing execution systems, configurable equipment interfaces, recipe management systems, metrology tools, process tools, and add-on sensors, into a system which provides advanced process control specific functionality. The Advanced Process Control Framework project was formulated to enable effective integration of Advanced Process Control applications into a semiconductor facility to improve manufacturing productivity and product yields. The main communication link between the factory system and the Framework is the Configurable Equipment Interface. It interfaces through a specialized component in the framework, the Machine Interface, which converts the factory system communication protocol, ISIS, to the Framework protocol, CORBA. The Framework is a distributed architecture that uses CORBA as a communication protocol between specialized components. A generalized example of how the Framework is integrated into the semiconductor facility is provided, as well as a description of the overall architecture used for process control strategy development. The main development language, Tcl/Tk, provides for increased development and deployment over traditional coding methods.

  12. Augmenting digital displays with computation

    Science.gov (United States)

    Liu, Jing

    As we inevitably step deeper and deeper into a world connected via the Internet, more and more information will be exchanged digitally. Displays are the interface between digital information and each individual. Naturally, one fundamental goal of displays is to reproduce information as realistically as possible since humans still care a lot about what happens in the real world. Human eyes are the receiving end of such information exchange; therefore it is impossible to study displays without studying the human visual system. In fact, the design of displays is rather closely coupled with what human eyes are capable of perceiving. For example, we are less interested in building displays that emit light in the invisible spectrum. This dissertation explores how we can augment displays with computation, which takes both display hardware and the human visual system into consideration. Four novel projects on display technologies are included in this dissertation: First, we propose a software-based approach to driving multiview autostereoscopic displays. Our display algorithm can dynamically assign views to hardware display zones based on multiple observers' current head positions, substantially reducing crosstalk and stereo inversion. Second, we present a dense projector array that creates a seamless 3D viewing experience for multiple viewers. We smoothly interpolate the set of viewer heights and distances on a per-vertex basis across the arrays field of view, reducing image distortion, crosstalk, and artifacts from tracking errors. Third, we propose a method for high dynamic range display calibration that takes into account the variation of the chrominance error over luminance. We propose a data structure for enabling efficient representation and querying of the calibration function, which also allows user-guided balancing between memory consumption and the amount of computation. Fourth, we present user studies that demonstrate that the ˜ 60 Hz critical flicker fusion

  13. A kit to prepare (111)In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer.

    Science.gov (United States)

    Scollard, Deborah A; Chan, Conrad; Holloway, Claire M B; Reilly, Raymond M

    2011-01-01

    The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for (111)In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) ((111)In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of (111)In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer. Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37°C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency (≥ 85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of (111)In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K(a) = 0.6-9.6 × 10(7) L/mol; B(max) = 0.6-10.4 × 10(6) sites/cell). (111)In-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of (111)InCl(3) to a single kit vial and incubating for 30 min at room temperature. (111)In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for pH, radiochemical purity (RCP), appearance and sterility. Pure and

  14. A kit to prepare {sup 111}In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Scollard, Deborah A.; Chan, Conrad [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Holloway, Claire M.B. [Department of Surgery, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 1H1 (Canada); Reilly, Raymond M., E-mail: raymond.reilly@utoronto.c [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3E2 (Canada); Toronto General Research Institute, University Health Network, Toronto, ON, M5G 2M9 (Canada)

    2011-01-15

    Introduction: The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for {sup 111}In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) ({sup 111}In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of {sup 111}In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer. Methods: Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37{sup o}C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency ({>=}85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of {sup 111}In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K{sub a}=0.6-9.6x10{sup 7} L/mol; B{sub max}=0.6-10.4x10{sup 6} sites/cell). {sup 111}In-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of {sup 111}InCl{sub 3} to a single kit vial and incubating for 30 min at room temperature. {sup 111}In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for p

  15. Rapid display of radiographic images

    Science.gov (United States)

    Cox, Jerome R., Jr.; Moore, Stephen M.; Whitman, Robert A.; Blaine, G. James; Jost, R. Gilbert; Karlsson, L. M.; Monsees, Thomas L.; Hassen, Gregory L.; David, Timothy C.

    1991-07-01

    The requirements for the rapid display of radiographic images exceed the capabilities of widely available display, computer, and communications technologies. Computed radiography captures data with a resolution of about four megapixels. Large-format displays are available that can present over four megapixels. One megapixel displays are practical for use in combination with large-format displays and in areas where the viewing task does not require primary diagnosis. This paper describes an electronic radiology system that approximates the highest quality systems, but through the use of several interesting techniques allows the possibility of its widespread installation throughout hospitals. The techniques used can be grouped under three major system concepts: a local, high-speed image server, one or more physician's workstations each with one or more high-performance auxiliary displays specialized to the radiology viewing task, and dedicated, high-speed communication links between the server and the displays. This approach is enhanced by the use of a progressive transmission scheme to decrease the latency for viewing four megapixel images. The system includes an image server with storage for over 600 4-megapixel images and a high-speed link. A subsampled megapixel image is fetched from disk and transmitted to the display in about one second followed by the full resolution 4-megapixel image in about 2.5 seconds. Other system components include a megapixel display with a 6-megapixel display memory space and frame-rate update of image roam, zoom, and contrast. Plans for clinical use are presented.

  16. Military display market segment: helicopters

    Science.gov (United States)

    Desjardins, Daniel D.; Hopper, Darrel G.

    2004-09-01

    The military display market is analyzed in terms of one of its segments: helicopter displays. Parameters requiring special consideration, to include luminance ranges, contrast ratio, viewing angles, and chromaticity coordinates, are examined. Performance requirements for rotary-wing displays relative to several premier applications are summarized. Display sizes having aggregate defense applications of 5,000 units or greater and having DoD applications across 10 or more platforms, are tabulated. The issue of size commonality is addressed where distribution of active area sizes across helicopter platforms, individually, in groups of two through nine, and ten or greater, is illustrated. Rotary-wing displays are also analyzed by technology, where total quantities of such displays are broken out into CRT, LCD, AMLCD, EM, LED, Incandescent, Plasma and TFEL percentages. Custom, versus Rugged commercial, versus commercial off-the-shelf designs are contrasted. High and low information content designs are identified. Displays for several high-profile military helicopter programs are discussed, to include both technical specifications and program history. The military display market study is summarized with breakouts for the helicopter market segment. Our defense-wide study as of March 2004 has documented 1,015,494 direct view and virtual image displays distributed across 1,181 display sizes and 503 weapon systems. Helicopter displays account for 67,472 displays (just 6.6% of DoD total) and comprise 83 sizes (7.0% of total DoD) in 76 platforms (15.1% of total DoD). Some 47.6% of these rotary-wing applications involve low information content displays comprising just a few characters in one color; however, as per fixed-wing aircraft, the predominant instantiation involves higher information content units capable of showing changeable graphics, color and video.

  17. X-1 on display

    Science.gov (United States)

    1949-01-01

    A Bell Aircraft Corporation X-1 series aircraft on display at an Open House at NACA Muroc Flight Test Unit or High-Speed Flight Research Station hangar on South Base of Edwards Air Force Base, California. (The precise date of the photo is uncertain, but it is probably before 1948.) The instrumentation that was carried aboard the aircraft to gather data is on display. The aircraft data was recorded on oscillograph film that was read, calibrated, and converted into meaningful parameters for the engineers to evaluate from each research flight. In the background of the photo are several early U.S. jets. These include several Lockheed P-80 Shooting Stars, which were used as chase planes on X-1 flights; two Bell P-59 Airacomets, the first U.S. jet pursuit aircraft (fighter in later parlance); and a prototype Republic XP-84 Thunderjet. There were five versions of the Bell X-1 rocket-powered research aircraft that flew at the NACA High-Speed Flight Research Station, Edwards, California. The bullet-shaped X-1 aircraft were built by Bell Aircraft Corporation, Buffalo, N.Y. for the U.S. Army Air Forces (after 1947, U.S. Air Force) and the National Advisory Committee for Aeronautics (NACA). The X-1 Program was originally designated the XS-1 for eXperimental Sonic. The X-1's mission was to investigate the transonic speed range (speeds from just below to just above the speed of sound) and, if possible, to break the 'sound barrier.' Three different X-1s were built and designated: X-1-1, X-1-2 (later modified to become the X-1E), and X-1-3. The basic X-1 aircraft were flown by a large number of different pilots from 1946 to 1951. The X-1 Program not only proved that humans could go beyond the speed of sound, it reinforced the understanding that technological barriers could be overcome. The X-1s pioneered many structural and aerodynamic advances including extremely thin, yet extremely strong wing sections; supersonic fuselage configurations; control system requirements; powerplant

  18. Improved renal clearance and tumor targeting of {sup 99m}Tc-labeled anti-Tac monoclonal antibody Fab by chemical modifications

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Meyoung-kon; Jeong, Hyeh-Jean; Kao, Chih-Hao K.; Yao, Zhengsheng; Paik, David S.; Pie, Jae Eun; Kobayashi, Hisataka; Waldmann, Thomas A.; Carrasquillo, Jorge A.; Paik, Chang H. E-mail: cpaik@mail.cc.nih.gov

    2002-02-01

    This study was undertaken to improve the renal clearance and tumor targeting properties of {sup 99m}Tc-labeled humanized anti-Tac (HuTac) monoclonal antibody Fab fragments using two chemical approaches: 1) labeling with a renal secretion agent {sup 99m}Tc-mercaptoacetyltriglycine (MAG3) and 2) lowering its isoelectric point (pI) by acylation. HuTac Fab (3.3 mg/mL) was reacted with a trifluorophenyl ester (TFP) of {sup 99m}Tc-MAG3 alone or was additionally reacted with TFP-glycolate to reduce the pI. In Balb/c mice, {sup 99m}Tc-MAG3-Fab (pI>9.3) rapidly accumulated in the kidneys (177% injected dose [ID]/g at 15 min) and then gradually cleared out of the kidneys. In contrast, the glycolation (pI 4.6{approx}6.6) drastically reduced the renal uptake (31% ID/g) and also the whole-body retention (82% ID vs 101% for the nonglycolated) at 15 min, indicating that the glycolated {sup 99m}Tc-MAG3-Fab (pI 4.6{approx}6.6) was rapidly excreted. The glycolated remained in the blood longer than the nonglycolated (1.2% vs 0.3% ID/g at 360 min), but this effect was less drastic than the effect shown on the renal uptake. In nude mice bearing receptor-positive (ATAC4) tumors, the glycolated {sup 99m}Tc-MAG3-Fab increased the peak tumor uptake to 14.8% ID/g from 8.3% ID/g for {sup 99m}Tc-MAG3-Fab, whereas the glycolation resulted in a drastic reduction of the renal uptake at 15 min. We demonstrated that the renal clearance and the tumor targeting of Fab could be optimized by chemical modifications.

  19. Laser illuminated flat panel display

    Energy Technology Data Exchange (ETDEWEB)

    Veligdan, J.T.

    1995-12-31

    A 10 inch laser illuminated flat panel Planar Optic Display (POD) screen has been constructed and tested. This POD screen technology is an entirely new concept in display technology. Although the initial display is flat and made of glass, this technology lends itself to applications where a plastic display might be wrapped around the viewer. The display screen is comprised of hundreds of planar optical waveguides where each glass waveguide represents a vertical line of resolution. A black cladding layer, having a lower index of refraction, is placed between each waveguide layer. Since the cladding makes the screen surface black, the contrast is high. The prototype display is 9 inches wide by 5 inches high and approximately I inch thick. A 3 milliwatt HeNe laser is used as the illumination source and a vector scanning technique is employed.

  20. Miniature information displays: primary applications

    Science.gov (United States)

    Alvelda, Phillip; Lewis, Nancy D.

    1998-04-01

    Positioned to replace current liquid crystal display technology in many applications, miniature information displays have evolved to provide several truly portable platforms for the world's growing personal computing and communication needs. The technology and functionality of handheld computer and communicator systems has finally surpassed many of the standards that were originally established for desktop systems. In these new consumer electronics, performance, display size, packaging, power consumption, and cost have always been limiting factors for fabricating genuinely portable devices. The rapidly growing miniature information display manufacturing industry is making it possible to bring a wide range of highly anticipated new products to new markets.

  1. Maintenance Procedure Display: Head Mounted Display (HMD) Evaluations

    Science.gov (United States)

    Whitmore, Milrian; Litaker, Harry L., Jr.; Solem, Jody A.; Holden, Kritina L.; Hoffman, Ronald R.

    2007-01-01

    A viewgraph presentation describing maintenance procedures for head mounted displays is shown. The topics include: 1) Study Goals; 2) Near Eye Displays (HMDs); 3) Design; 4) Phase I-Evaluation Methods; 5) Phase 1 Results; 6) Improved HMD Mounting; 7) Phase 2 -Evaluation Methods; 8) Phase 2 Preliminary Results; and 9) Next Steps.

  2. Updated defense display market assessment

    Science.gov (United States)

    Desjardins, Daniel D.; Hopper, Darrel G.

    1999-08-01

    This paper addresses the number, function and size of principal military displays and establishes a basis to determine the opportunities for technology insertion in the immediate future and into the next millennium. Principal military displays are defined as those occupying appreciable crewstation real-estate and/or those without which the platform could not carry out its intended mission. DoD 'office' applications are excluded from this study. The military displays market is specified by such parameters as active area and footprint size, and other characteristics such as luminance, gray scale, resolution, angle, color, video capability, and night vision imaging system compatibility. Funded, future acquisitions, planned and predicted crewstation modification kits, and form-fit upgrades are taken into account. This paper provides an overview of the DoD niche market, allowing both government and industry a necessary reference by which to meet DoD requirements for military displays in a timely and cost-effective manner. The aggregate DoD installed base for direct-view and large-area military displays is presently estimated to be in excess of 313,000. Miniature displays are those which must be magnified to be viewed, involve a significantly different manufacturing paradigm and are used in helmet mounted displays and thermal weapon sight applications. Some 114,000 miniature displays are presently included within future weapon system acquisition plans. For vendor production planning purposes it is noted that foreign military sales could substantially increase these quantities. The vanishing vendor syndrome (VVS) for older display technologies continues to be a growing, pervasive problem throughout DoD, which consequently must leverage the more modern, especially flat panel, display technologies being developed to replace older, especially cathode ray tube, technology for civil-commercial markets. Total DoD display needs (FPD, HMD) are some 427,000.

  3. Intracellular routing in breast cancer cells of streptavidin-conjugated trastuzumab Fab fragments linked to biotinylated doxorubicin-functionalized metal chelating polymers.

    Science.gov (United States)

    Liu, Peng; Cai, Zhongli; Kang, Jae W; Boyle, Amanda J; Adams, Jarret; Lu, Yijie; Ngo Ndjock Mbong, Ghislaine; Sidhu, Sachdev; Reilly, Raymond M; Winnik, Mitchell A

    2014-03-10

    We describe the synthesis of a heterotelechelic metal-chelating polymer (Bi-MCP-Dox), a polyacrylamide with a number average degree of polymerization DPn = 50 (PDI = 1.2), with biotin (Bi) and doxorubicin (Dox) as functional chain ends and diethylenetriaminepentaacetic acid (DTPA) pendant groups as the binding sites for metal ions. We compared its behavior in cell-uptake experiments with a similar polymer (Bi-MCP) without Dox. These MCPs were complexed with trastuzumab Fab (tmFab) fragments covalently linked to streptavidin (SAv) to form tmFab-SAv-Bi-MCP-Dox and tmFab-SAv-Bi-MCP via the strong affinity between Bi and SAv. tmFab targets human epidermal growth factor receptor-2 (HER2), which is overexpressed on certain human breast cancer cells. Surface plasmon resonance (SPR) experiments with the extracellular domain (ECD) of HER2 showed that incorporation of the MCPs in these complexes had no significant effect on the association or dissociation rate with the HER2 ECD and the dissociation constants. The tmFab-complexed MCPs were subsequently labeled with (111)In (an Auger electron emitting radionuclide). Auger electrons can cause lethal DNA double strand breaks (DSBs) but only if they are emitted intracellularly and especially, in close proximity to the nucleus. To evaluate the cellular and nuclear uptake of tmFab-SAv-Bi-MCP-Dox, we incubated HER2+ SK-BR-3 human breast cancer cells with the complexes saturated with stable In(3+) and visualized their distribution by confocal fluorescence microscopy, monitoring the fluorescence of Dox. In parallel, we carried out cell fractionation studies on tmFab-SAv-Bi-MCP-Dox and on tmFab-SAv-Bi-MCP labeled with (111)In. Both radiolabeled complexes showed cell internalization and nuclear localization. We conclude that metal-chelating polymers with this composition appear to encourage internalization, nuclear uptake, and chromatin (DNA) binding of trastuzumab fragments modified with streptavidin in human breast cancer cells

  4. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD[superscript +] and triclosan

    Energy Technology Data Exchange (ETDEWEB)

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2010-11-19

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD{sup +} has been solved to a resolution of 2.1 {angstrom}. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD{sup +} reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD{sup +} cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

  5. Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using {sup 111}In-trastuzumab (Herceptin) Fab fragments

    Energy Technology Data Exchange (ETDEWEB)

    Tang Ying [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, M5S 2S2 (Canada); Wang, Judy [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Scollard, Deborah A. [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Mondal, Hridya [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Holloway, Claire [Sunnybrook and Women' s College Health Sciences Center, Toronto, Ontario, M4N 3M5 (Canada); Kahn, Harriette J. [Sunnybrook and Women' s College Health Sciences Center, Toronto, Ontario, M4N 3M5 (Canada); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada) and Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, M5S 2S2 (Canada) and Department of Medical Imaging, University of Toronto, Toronto, Ontario, M5S 3E2 (Canada)]. E-mail: raymond.reilly@utoronto.ca

    2005-01-01

    Trastuzumab (Herceptin) Fab were prepared by digestion of intact IgG with immobilized papain, derivatized with diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with {sup 111}In. The dissociation constant (K{sub d}) for binding of Fab to HER2/neu-positive SK-BR-3 human breast cancer cells was two- to threefold higher than for intact IgG (14-36 vs. 8-14 nM). The binding affinity was not significantly decreased after DTPA derivatization (K{sub d}=47 nM). {sup 111}In-trastuzumab Fab localized specifically in HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice with tumor uptake of 7.8{+-}0.7% injected dose (ID)/g and tumor/blood ratio of 25.2{+-}1.6 at 72 h postinjection compared with 2.7{+-}0.7% ID/g and 7.0{+-}0.9 for {sup 111}In-HuM195 anti-CD33 Fab (significantly different, P<.001). Small (3-5 mm in diameter) BT-474 tumors were imaged with {sup 111}In-trastuzumab Fab as early as 24 h postinjection.

  6. Deletion of the β-acetoacetyl synthase FabY in Pseudomonas aeruginosa induces hypoacylation of lipopolysaccharide and increases antimicrobial susceptibility.

    Science.gov (United States)

    Six, David A; Yuan, Yanqiu; Leeds, Jennifer A; Meredith, Timothy C

    2014-01-01

    The β-acetoacetyl-acyl carrier protein synthase FabY is a key enzyme in the initiation of fatty acid biosynthesis in Pseudomonas aeruginosa. Deletion of fabY results in an increased susceptibility of P. aeruginosa in vitro to a number of antibiotics, including vancomycin and cephalosporins. Because antibiotic susceptibility can be influenced by changes in membrane lipid composition, we determined the total fatty acid profile of the ΔfabY mutant, which suggested alterations in the lipid A region of the lipopolysaccharide. The majority of lipid A species in the ΔfabY mutant lacked a single secondary lauroyl group, resulting in hypoacylated lipid A. Adding exogenous fatty acids to the growth media restored the wild-type antibiotic susceptibility profile and the wild-type lipid A fatty acid profile. We suggest that incorporation of hypoacylated lipid A species into the outer membrane contributes to the shift in the antibiotic susceptibility profile of the ΔfabY mutant.

  7. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

    Science.gov (United States)

    Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

    2015-04-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

  8. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    Science.gov (United States)

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  9. Best-practice evaluation-methods for wafer-fab photomask-requalification inspection tools

    Science.gov (United States)

    Cho, Chan Seob; Mungmode, Ashish; Taylor, Ron; Cho, David; Koh, Hui Peng

    2014-10-01

    Requalifying semiconductor photomasks remains critically important and is increasingly challenging for 20nm and 14nm node logic reticles. Patterns are becoming more complex on the photomask, and defect sensitivity requirements are more stringent than ever before. Reticle inspection tools are equally important for effective process development and the successful ramp and sustained yield for high volume manufacturing. The inspection stages considered were: incoming inspection to match with Mask Shop Outgoing result and to detect defects generated during transport; requalification by routine cycle inspection to detect Haze and any other defects; and inspection by in-house or Mask shop at the post cleaning. There are many critical capability and capacity factors for the decision for best inspection tool and strategy for high volume manufacturing, especially objective Lens NA, wavelength, power, pixel size, throughput, full-automation inspection linked with Overhead Transport, algorithm application, engineering application function, and inspection of PSM and OMOG . These tools are expensive but deliver differentiated value in terms of performance and throughput as well as extendibility. Performing a thorough evaluation and making a technically sound choice which explores these many factors is critical for success of a fab. This paper examines the methodology for evaluating two different photomask inspection tools. The focus is on ensuring production worthiness on real and advanced product photomasks requiring accurate evaluation of sensitivity, throughput, data analysis function and engineering work function on those product photomasks. Photomasks used for data collection are production reticles, PDM(Program defect Mask), SiN spray defect Reticle which is described that evaluates how the tools would perform on a contaminated plate.

  10. Flexible Bistable Cholesteric Reflective Displays

    Science.gov (United States)

    Yang, Deng-Ke

    2006-03-01

    Cholesteric liquid crystals (ChLCs) exhibit two stable states at zero field condition-the reflecting planar state and the nonreflecting focal conic state. ChLCs are an excellent candidate for inexpensive and rugged electronic books and papers. This paper will review the display cell structure,materials and drive schemes for flexible bistable cholesteric (Ch) reflective displays.

  11. Three-dimensional display technologies.

    Science.gov (United States)

    Geng, Jason

    2013-01-01

    The physical world around us is three-dimensional (3D), yet traditional display devices can show only two-dimensional (2D) flat images that lack depth (i.e., the third dimension) information. This fundamental restriction greatly limits our ability to perceive and to understand the complexity of real-world objects. Nearly 50% of the capability of the human brain is devoted to processing visual information [Human Anatomy & Physiology (Pearson, 2012)]. Flat images and 2D displays do not harness the brain's power effectively. With rapid advances in the electronics, optics, laser, and photonics fields, true 3D display technologies are making their way into the marketplace. 3D movies, 3D TV, 3D mobile devices, and 3D games have increasingly demanded true 3D display with no eyeglasses (autostereoscopic). Therefore, it would be very beneficial to readers of this journal to have a systematic review of state-of-the-art 3D display technologies.

  12. Flat panel display - Impurity doping technology for flat panel displays

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Toshiharu [Advanced Technology Planning, Sumitomo Eaton Nova Corporation, SBS Tower 9F, 10-1, Yoga 4-chome, Setagaya-ku, 158-0097 Tokyo (Japan)]. E-mail: suzuki_tsh@senova.co.jp

    2005-08-01

    Features of the flat panel displays (FPDs) such as liquid crystal display (LCD) and organic light emitting diode (OLED) display, etc. using low temperature poly-Si (LTPS) thin film transistors (TFTs) are briefly reviewed comparing with other FPDs. The requirements for fabricating TFTs used for high performance FPDs and system on glass (SoG) are addressed. This paper focuses on the impurity doping technology, which is one of the key technologies together with crystallization by laser annealing, formation of high quality gate insulator and gate-insulator/poly-Si interface. The issues to be solved in impurity doping technology for state of the art and future TFTs are clarified.

  13. Screening of Human Antibody Fab Fragment against HBsAg and the Construction of its dsFv Form

    Directory of Open Access Journals (Sweden)

    Leili Jia, Jiyun Yu, Hongbin Song, Xuelin Liu, Weina Ma, Yuanyong Xu, Chuanfu Zhang, Shicun Dong, Qiao Li

    2008-01-01

    Full Text Available The objective of this study was to pursue the techniques involving the screening of the human antibody Fab fragment against hepatitis B virus surface antigen (HBsAg and the construction of its disulfide-stabilized Fv fragment (dsFv. The phage antibody Fab fragments against HBsAg were screened from the human combinatorial immunoglobulin library. Sequence analysis revealed that its heavy chain gene was complete, but the light chain gene was lost. To improve the affinity of the antibody by chain shuffling, a human antibody light chain gene repertoire was generated by reverse transcriptase-polymerase chain reaction (RT-PCR from the human peripheral blood lymphocytes. A phage antibody sub-library was then constructed by inserting the light chain gene repertoire into the phagmid that contained the Fd gene. Five clones with appreciably higher absorbance than that of the original clone were obtained, which indicated that the affinity of the light chain-shuffled phage antibodies was improved. Then, the mutated genes of dsFv against HBsAg were constructed by using PCR-based point mutagenesis method. Purified VH and VL proteins were folded into a 25-kDa protein, designated as anti-HBsAg dsFv. ELISA and competition ELISA revealed that the dsFv maintained the specificity of the Fab by binding to HBsAg, even through with a lower binding activity. These results have facilitated the undertaking of further functional analyses of the constructed dsFv, and may therefore provide an improved technique for the production and application of dsFvs against HBsAg.

  14. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon, E-mail: choe@salk.edu [Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 (United States)

    2006-09-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K{sup +} channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments.

  15. Pester power: snackfoods displayed at supermarket checkouts in Melbourne, Australia.

    Science.gov (United States)

    Dixon, Helen; Scully, Maree; Parkinson, Kristiina

    2006-08-01

    To establish the amount and accessibility of snack food displayed at supermarket checkouts located in Melbourne, Australia. Observational survey of 24 randomly selected supermarkets situated within a 20-kilometre radius of Melbourne's General Post Office. Individual checkouts within each store (n=257) were observed to determine the types of items that were displayed, how they were promoted, and whether they were within the reach of children. All supermarkets surveyed displayed food products at their checkouts, with most checkouts displaying chocolate (87%), gum (81%) and sweets (80%). Only 7% of checkouts had their display of foods or drinks out of the reach of children. Foods displayed at supermarket checkouts in Melbourne are predominantly energy-dense confectionery items. They are often promoted in a way that targets children and encourages parents to impulse buy for their children.

  16. A novel screening system for claudin binder using baculoviral display.

    Directory of Open Access Journals (Sweden)

    Hideki Kakutani

    Full Text Available Recent progress in cell biology has provided new insight into the claudin (CL family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE, but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.

  17. Tactile display device using an electrorheological fluid

    Science.gov (United States)

    Garner, H. Douglas

    1994-08-01

    A tactile display device utilizes an electrorheological fluid to activate a plurality of tactile dots. A voltage is selectively produced uniformly across an electrorheological fluid flowing between a common ground electrode and a plurality of conductive dot electrodes, thereby producing an increase in the fluid's viscosity to the extent that fluid flow between the two electrodes is restricted. The flow restriction produces a build-up of electrorheological fluid in a corresponding dot actuator chamber. The resulting pressure increase in the chamber displaces an elastic diaphragm fixed to a display surface to form a lump which can be perceived by the reader as one dot in a Braille character cell. A flow regulation system provides a continually pressurized flow system and provides for free flow of the electrorheological fluid through the plurality of dot actuator chambers when they are not activated. The device is adaptable to printed circuit techniques and can simultaneously display tactile dots representative of a full page of Braille characters stored on a medium such as a tape cassette or to display tactile dots representative of non-Braille data appearing on a computer monitor or contained on another data storage medium. In an alternate embodiment, the elastic diaphragm drives a plurality of spring-loaded pins provided with positive stops to maintain consistent displacements of the pins in both their actuated and nonactuated positions.

  18. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Haustraete Jurgen

    2009-08-01

    Full Text Available Abstract Background Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. Results Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. Conclusion Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression

  19. Tone compatibility between HDR displays

    Science.gov (United States)

    Bist, Cambodge; Cozot, Rémi; Madec, Gérard; Ducloux, Xavier

    2016-09-01

    High Dynamic Range (HDR) is the latest trend in television technology and we expect an in ux of HDR capable consumer TVs in the market. Initial HDR consumer displays will operate on a peak brightness of about 500-1000 nits while in the coming years display peak brightness is expected to go beyond 1000 nits. However, professionally graded HDR content can range from 1000 to 4000 nits. As with Standard Dynamic Range (SDR) content, we can expect HDR content to be available in variety of lighting styles such as low key, medium key and high key video. This raises concerns over tone-compatibility between HDR displays especially when adapting to various lighting styles. It is expected that dynamic range adaptation between HDR displays uses similar techniques as found with tone mapping and tone expansion operators. In this paper, we survey simple tone mapping methods of 4000 nits color-graded HDR content for 1000 nits HDR displays. We also investigate tone expansion strategies when HDR content graded in 1000 nits is displayed on 4000 nits HDR monitors. We conclude that the best tone reproduction technique between HDR displays strongly depends on the lighting style of the content.

  20. A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

    DEFF Research Database (Denmark)

    Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.;

    2002-01-01

    The fab1 mutant of Arabidopsis is partially deficient in activity of ß-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a c...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

  1. Data of rational process optimization for the production of a full IgG and its Fab fragment from hybridoma cells.

    Science.gov (United States)

    Röhm, Martina; Handl, Alina; König, Maria; Mavoungou, Chrystelle; Handrick, René; Schindowski, Katharina

    2016-09-01

    This data article focuses on the production of monoclonal antibodies (mAb) and their fragments Fab and F(ab')2. Here, we present the data of an optimization protocol to improve the product yield of a hybridoma cell process using a Design of Experiment (DoE) strategy. Furthermore, the data of the evaluated conditions were used to test feeding strategies in shake flasks. They were verified in controlled 2 L fed-batch bioreactor processes. Supplementing the culture medium with human insulin-like growth factor-I (IGF-I) and Pluronic F-68, as well as a nutrient rich additive for fed-batch, resulted in improved cell growth correlating with a 7 day elongated process time and a 4.5 fold higher product titer. Finally, a rapid Fab generation protocol and the respective data are presented using different papain digestion and a camelid anti-kappa light chain VHH affinity ligand.

  2. Crystallization and X-ray diffraction analysis of the β-ketoacyl-acyl carrier protein reductase FabG from Aquifex aeolicus VF5

    Energy Technology Data Exchange (ETDEWEB)

    Mao, Qilong [Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203 (United States); Duax, William L.; Umland, Timothy C., E-mail: umland@hwi.buffalo.edu [Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203 (United States); Department of Structural Biology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY (United States)

    2007-02-01

    FabG from A. aeolicus, a putative component of fatty-acid synthase II, has been overexpressed, purified and crystallized. Diffraction data have been collected to 1.8 Å resolution. The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 Å and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative β-ketoacyl-acyl carrier protein reductase. Structure–function studies of this protein are being performed as part of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.

  3. Absorbed Doses and Risk Estimates of (211)At-MX35 F(ab')2 in Intraperitoneal Therapy of Ovarian Cancer Patients

    DEFF Research Database (Denmark)

    Cederkrantz, Elin; Andersson, Håkan; Bernhardt, Peter

    2015-01-01

    100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective......, intraperitoneal (i.p.) targeted α therapy has been proposed as an adjuvant treatment for minimal residual disease after successful primary treatment. In the present study, we calculated absorbed and relative biological effect (RBE)-weighted (equivalent) doses in relevant normal tissues and estimated the effective...... dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation...

  4. Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports efficient operation of a functional reversal of β-oxidation cycle.

    Science.gov (United States)

    Vick, Jacob E; Clomburg, James M; Blankschien, Matthew D; Chou, Alexander; Kim, Seohyoung; Gonzalez, Ramon

    2015-02-01

    We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the -oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782).While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a -oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled deltaffabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli.

  5. Initial volume of a drug before it reaches the volume of distribution: pharmacokinetics of F(ab')2 antivenoms and other drugs.

    Science.gov (United States)

    Sevcik, Carlos; Salazar, Victor; Díaz, Patricia; D'Suze, Gina

    2007-10-01

    Fast disappearance of F(ab')2 antivenoms from the plasma compartment [Sevcik et al., 2004. Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')2 extrusion mechanism from blood to tissues. Toxicon 44, 731-734; Vazquez et al., 2005. Pharmacokinetics of a F(ab')2 scorpion antivenom in healthy human volunteers. Toxicon 46, 797-805] suggests a quick time course to reach its final distribution volume. An equation was developed to describe how the volume occupied by a drug in the body grows with time. As discussed in the paper this equation is free of some shortcomings of an equation developed for the same purpose by Niazi [1976. Volume of distribution as a function of time. J. Pharm. Sci. 65, 452-454]. Fluorescence microscopy showed that the rapid initial decay in plasmatic F(ab')2 concentration may be related to uptake of F(ab')2 by vascular endothelium which, in combination with accumulation in the vascular wall connective tissue, may produce an intermediate plateau in F(ab')2 V(sl)(t), which reached its final value after 10 h. The V(sl)(t) equation predicts that the plasma concentration half-time of decay has little use to estimate how a drug distributes through the body to exert its action, and predicts that, in some instances, intermediate plateaus in the time course of V(sl)(t) exist. Data from the literature showed that the kinetic considerations for V(sl)(t) also apply to clevidipine, digoxin, digitoxin, lidocaine and thiopentone.

  6. Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases.

    Science.gov (United States)

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A; Meredith, Timothy C

    2012-10-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.

  7. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    Science.gov (United States)

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy.

  8. Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin.

    Science.gov (United States)

    Kornberger, Petra; Skerra, Arne

    2014-01-01

    We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH 6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly 2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography--utilizing the Strep-tag II appended to gelonin--and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC 50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.

  9. Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.

    Science.gov (United States)

    Schlapschy, Martin; Fogarasi, Marton; Gruber, Helga; Gresch, Oliver; Schäfer, Claudia; Aguib, Yasmine; Skerra, Arne

    2009-03-01

    An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

  10. Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli.

    Science.gov (United States)

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-11-19

    The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.

  11. Visual displays and Neuro-Linguistic Programming

    Energy Technology Data Exchange (ETDEWEB)

    Brown-VanHoozer, S.A. [Argonne National Lab., Idaho Falls, ID (United States); VanHoozer, W.R. [Tranceformations Unlimited, Rigby, ID (United States)

    1994-10-01

    Advancement of computer technology is forthcoming at such a rapid pace that the research concerning the interplay of humans and computer technology is lagging far behind. One area of particular concern is the design of visual displays that are pragmatic, ``user friendly,`` and ``user assisting.`` When engineers design visual displays, they generally do so methodically and logically, but only from within their own individual perspective or ``model of the world.`` They select the human aspects which make sense to them and not necessarily to non-engineers, operators, and others. The model design is what the engineer chooses to relate, based on his or her perspective of reality. These choices limit the model design thereby excluding the users` perspective. A set of techniques which can be used to assist the designers in expanding their choices and include the users` model is Neuro-Linguistic Programming (NLP).

  12. Multiview multiperspective time multiplexed autostereoscopic display

    Science.gov (United States)

    Kupiec, Stephen A.; Markov, Vladimir B.; Hopper, Darrel G.; Saini, Gurdial

    2008-02-01

    The implementation of a time multiplexed display capable of eight simultaneously visible viewing zones will be described. The system employs a high speed digital micromirror device (DMD) to allow for the high framerate essential for flicker free display of multiple viewing zones. A combination of custom graphical processor unit (GPU) programming and a correspondingly optimized field programmable gate array (FPGA) DMD driver allows for real time interactive rendering of scenes. The rendering engine is entirely based on off the shelf with the use of a standard DVI-D interface for data transfer to the DMD interface. A rapidly switched LED light engine is employed to overcome the speed limitations of color wheel light sources, as well as providing a highly saturated color gamut. Selection of viewing zones is achieved by the use of a high-speed shutter interfaced directly to the DMD driver for precise synchronization.

  13. Ten inch Planar Optic Display

    Energy Technology Data Exchange (ETDEWEB)

    Beiser, L. [Beiser (Leo) Inc., Flushing, NY (United States); Veligdan, J. [Brookhaven National Lab., Upton, NY (United States)

    1996-04-01

    A Planar Optic Display (POD) is being built and tested for suitability as a high brightness replacement for the cathode ray tube, (CRT). The POD display technology utilizes a laminated optical waveguide structure which allows a projection type of display to be constructed in a thin (I to 2 inch) housing. Inherent in the optical waveguide is a black cladding matrix which gives the display a black appearance leading to very high contrast. A Digital Micromirror Device, (DMD) from Texas Instruments is used to create video images in conjunction with a 100 milliwatt green solid state laser. An anamorphic optical system is used to inject light into the POD to form a stigmatic image. In addition to the design of the POD screen, we discuss: image formation, image projection, and optical design constraints.

  14. ENERGY STAR Certified Displays - Deprecated

    Data.gov (United States)

    U.S. Environmental Protection Agency — This dataset is up-to-date but newer better data can be retrieved at: https://data.energystar.gov/dataset/ENERGY-STAR-Certified-Displays/xsyb-v8gs Certified models...

  15. Color speckle in laser displays

    Science.gov (United States)

    Kuroda, Kazuo

    2015-07-01

    At the beginning of this century, lighting technology has been shifted from discharge lamps, fluorescent lamps and electric bulbs to solid-state lighting. Current solid-state lighting is based on the light emitting diodes (LED) technology, but the laser lighting technology is developing rapidly, such as, laser cinema projectors, laser TVs, laser head-up displays, laser head mounted displays, and laser headlamps for motor vehicles. One of the main issues of laser displays is the reduction of speckle noise1). For the monochromatic laser light, speckle is random interference pattern on the image plane (retina for human observer). For laser displays, RGB (red-green-blue) lasers form speckle patterns independently, which results in random distribution of chromaticity, called color speckle2).

  16. Ultraminiature, Micropower Multipurpose Display Project

    Data.gov (United States)

    National Aeronautics and Space Administration — High information content electronic displays remain the most difficult element of the human-machine interface to effectively miniaturize. Mobile applications need a...

  17. Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice.

    Science.gov (United States)

    Ding, Haiming; Carlton, Michelle M; Povoski, Stephen P; Milum, Keisha; Kumar, Krishan; Kothandaraman, Shankaran; Hinkle, George H; Colcher, David; Brody, Rich; Davis, Paul D; Pokora, Alex; Phelps, Mitchell; Martin, Edward W; Tweedle, Michael F

    2013-11-20

    The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.

  18. Crystallization and X-ray diffraction analysis of the beta-ketoacyl-acyl carrier protein reductase FabG from Aquifex aeolicus VF5.

    Science.gov (United States)

    Mao, Qilong; Duax, William L; Umland, Timothy C

    2007-02-01

    The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 A and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative beta-ketoacyl-acyl carrier protein reductase. Structure-function studies of this protein are being performed as part of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.

  19. Effective color design for displays

    Science.gov (United States)

    MacDonald, Lindsay W.

    2002-06-01

    Visual communication is a key aspect of human-computer interaction, which contributes to the satisfaction of user and application needs. For effective design of presentations on computer displays, color should be used in conjunction with the other visual variables. The general needs of graphic user interfaces are discussed, followed by five specific tasks with differing criteria for display color specification - advertising, text, information, visualization and imaging.

  20. 利用体内噬菌体展示技术筛选膀胱癌特异性结合肽%Selection of peptide specifically binding to bladder carcinoma by using phage display in vivo

    Institute of Scientific and Technical Information of China (English)

    罗俊茜; 张帆; 杨晓峰; 罗芳; 刘杰昊; 庞建智; 闫三华; 张小雷

    2015-01-01

    目的:利用体内噬菌体展示技术筛选与人膀胱移行细胞癌特异性结合的小分子多肽。方法:将人膀胱移行细胞癌细胞BIU87接种于裸鼠体内,制备膀胱癌荷瘤小鼠模型,尾静脉注射噬菌体展示环七肽库,然后筛选与膀胱移行细胞癌特异性结合的含外源多肽的噬菌体,经过3轮体内筛选后,免疫组织化学法及ELISA法鉴定单克隆噬菌体对BIU87的亲和力。提取阳性单克隆噬菌体单链DNA进行测序,并推导出外源多肽氨基酸序列,化学合成多肽、制备分子探针后采用激光扫描共聚焦显微镜术及流式细胞术鉴定多肽对膀胱癌细胞和组织的特异性。结果:3轮体内筛选后,噬菌体富集率达到4.334×102倍。免疫组化结果显示,肿瘤组织中噬菌体肽的含量随着每一轮筛选呈增长趋势,且结合逐渐增强,同时由于噬菌体经肝、肾代谢,可见肝脏结合大量非特异性的噬菌体。 ELISA结果显示,随机挑选的30个单克隆噬菌体斑中,有24个阳性噬菌体,其中10个噬菌体对BIU87有较强的亲和力,对其测序并推导出3种多肽序列,重复率最高的序列CSSPIGRHC(8/10)命名为NYZL1。化学合成FITC-C6-NYZL1,通过激光扫描共聚焦显微镜术、流式细胞术均证明多肽NYZL1可以特异性结合膀胱癌细胞。结论:利用体内噬菌体展示技术筛选出了与人膀胱移行细胞癌特异性结合的小分子多肽NYZL1,为膀胱癌早期诊断和靶向治疗提供一定的理论依据。%Objective:To screen the peptide binding to human bladder carcinoma cells specifically by using phage display technology in vivo.Methods: Nude mice were inoculated with bladder carcinoma cells BIU87 for establishing tumor-bearing mice model.The Ph.D.-C7CTM Peptide Library was injected intravenously via tail vein.Then we screened Phage containing exogenous peptides binding to bladder transitional carcinoma cells