A facile method to screen inhibitors of protein-protein interactions including MDM2-p53 displayed on T7 phage.

PubMed

Ishi, Kazutomo; Sugawara, Fumio

2008-05-01

Protein-protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein-protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein-protein interactions. Because T7 phage display is an effective method for detecting protein-protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST-MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53-MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call 'reverse phage display'.

Data visualization in interactive maps and time series

NASA Astrophysics Data System (ADS)

Maigne, Vanessa; Evano, Pascal; Brockmann, Patrick; Peylin, Philippe; Ciais, Philippe

2014-05-01

State-of-the-art data visualization has nothing to do with plots and maps we used few years ago. Many opensource tools are now available to provide access to scientific data and implement accessible, interactive, and flexible web applications. Here we will present a web site opened November 2013 to create custom global and regional maps and time series from research models and datasets. For maps, we explore and get access to data sources from a THREDDS Data Server (TDS) with the OGC WMS protocol (using the ncWMS implementation) then create interactive maps with the OpenLayers javascript library and extra information layers from a GeoServer. Maps become dynamic, zoomable, synchroneaously connected to each other, and exportable to Google Earth. For time series, we extract data from a TDS with the Netcdf Subset Service (NCSS) then display interactive graphs with a custom library based on the Data Driven Documents javascript library (D3.js). This time series application provides dynamic functionalities such as interpolation, interactive zoom on different axes, display of point values, and export to different formats. These tools were implemented for the Global Carbon Atlas (http://www.globalcarbonatlas.org): a web portal to explore, visualize, and interpret global and regional carbon fluxes from various model simulations arising from both human activities and natural processes, a work led by the Global Carbon Project.

Building the interspace: Digital library infrastructure for a University Engineering Community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schatz, B.

A large-scale digital library is being constructed and evaluated at the University of Illinois, with the goal of bringing professional search and display to Internet information services. A testbed planned to grow to 10K documents and 100K users is being constructed in the Grainger Engineering Library Information Center, as a joint effort of the University Library and the National Center for Supercomputing Applications (NCSA), with evaluation and research by the Graduate School of Library and Information Science and the Department of Computer Science. The electronic collection will be articles from engineering and science journals and magazines, obtained directly from publishersmore » in SGML format and displayed containing all text, figures, tables, and equations. The publisher partners include IEEE Computer Society, AIAA (Aerospace Engineering), American Physical Society, and Wiley & Sons. The software will be based upon NCSA Mosaic as a network engine connected to commercial SGML displayers and full-text searchers. The users will include faculty/students across the midwestern universities in the Big Ten, with evaluations via interviews, surveys, and transaction logs. Concurrently, research into scaling the testbed is being conducted. This includes efforts in computer science, information science, library science, and information systems. These efforts will evaluate different semantic retrieval technologies, including automatic thesaurus and subject classification graphs. New architectures will be designed and implemented for a next generation digital library infrastructure, the Interspace, which supports interaction with information spread across information spaces within the Net.« less

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

PubMed Central

Lagerkvist, Ann Catrin; Földes-Papp, Zeno; Persson, Mats A.A.; Rigler, Rudolf

2001-01-01

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage–antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. PMID:11468349

Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library.

PubMed

Krumpe, Lauren R H; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2007-10-05

Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

Epitope selection from an uncensored peptide library displayed on avian leukosis virus.

PubMed

Khare, Pranay D; Rosales, Ana G; Bailey, Kent R; Russell, Stephen J; Federspiel, Mark J

2003-10-25

Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10(6) different members was generated from a plasmid library of approximately 5 x 10(6) diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries.

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display.

PubMed

Kügler, Jonas; Nieswandt, Simone; Gerlach, Gerald F; Meens, Jochen; Schirrmann, Thomas; Hust, Michael

2008-09-01

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes.

PubMed

Ivarsson, Ylva; Arnold, Roland; McLaughlin, Megan; Nim, Satra; Joshi, Rakesh; Ray, Debashish; Liu, Bernard; Teyra, Joan; Pawson, Tony; Moffat, Jason; Li, Shawn Shun-Cheng; Sidhu, Sachdev S; Kim, Philip M

2014-02-18

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.

jvenn: an interactive Venn diagram viewer.

PubMed

Bardou, Philippe; Mariette, Jérôme; Escudié, Frédéric; Djemiel, Christophe; Klopp, Christophe

2014-08-29

Venn diagrams are commonly used to display list comparison. In biology, they are widely used to show the differences between gene lists originating from different differential analyses, for instance. They thus allow the comparison between different experimental conditions or between different methods. However, when the number of input lists exceeds four, the diagram becomes difficult to read. Alternative layouts and dynamic display features can improve its use and its readability. jvenn is a new JavaScript library. It processes lists and produces Venn diagrams. It handles up to six input lists and presents results using classical or Edwards-Venn layouts. User interactions can be controlled and customized. Finally, jvenn can easily be embeded in a web page, allowing to have dynamic Venn diagrams. jvenn is an open source component for web environments helping scientists to analyze their data. The library package, which comes with full documentation and an example, is freely available at http://bioinfo.genotoul.fr/jvenn.

The IDL astronomy user's library

NASA Technical Reports Server (NTRS)

Landsman, W. B.

1992-01-01

IDL (Interactive Data Language) is a commercial programming, plotting, and image display language, which is widely used in astronomy. The IDL Astronomy User's Library is a central repository of over 400 astronomy-related IDL procedures accessible via anonymous FTP. The author will overview the use of IDL within the astronomical community and discuss recent enhancements at the IDL astronomy library. These enhancements include a fairly complete I/O package for FITS images and tables, an image deconvolution package and an image mosaic package, and access to IDL Open Windows/Motif widgets interface. The IDL Astronomy Library is funded by NASA through the Astrophysics Software and Research Aids Program.

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

PubMed

Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

2010-01-01

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries.

PubMed

Krumpe, Lauren R H; Atkinson, Andrew J; Smythers, Gary W; Kandel, Andrea; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2006-08-01

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.

Ligand-regulated peptide aptamers.

PubMed

Miller, Russell A

2009-01-01

The peptide aptamer approach employs high-throughput selection to identify members of a randomized peptide library displayed from a scaffold protein by virtue of their interaction with a target molecule. Extending this approach, we have developed a peptide aptamer scaffold protein that can impart small-molecule control over the aptamer-target interaction. This ligand-regulated peptide (LiRP) scaffold, consisting of the protein domains FKBP12, FRB, and GST, binds to the cell-permeable small-molecule rapamycin and the binding of this molecule can prevent the interaction of the randomizable linker region connecting FKBP12 with FRB. Here we present a detailed protocol for the creation of a peptide aptamer plasmid library, selection of peptide aptamers using the LiRP scaffold in a yeast two-hybrid system, and the screening of those peptide aptamers for a ligand-regulated interaction.

Phage display for the discovery of hydroxyapatite-associated peptides.

PubMed

Jin, Hyo-Eon; Chung, Woo-Jae; Lee, Seung-Wuk

2013-01-01

In nature, proteins play a critical role in the biomineralization process. Understanding how different peptide or protein sequences selectively interact with the target crystal is of great importance. Identifying such protein structures is one of the critical steps in verifying the molecular mechanisms of biomineralization. One of the promising ways to obtain such information for a particular crystal surface is to screen combinatorial peptide libraries in a high-throughput manner. Among the many combinatorial library screening procedures, phage display is a powerful method to isolate such proteins and peptides. In this chapter, we will describe our established methods to perform phage display with inorganic crystal surfaces. Specifically, we will use hydroxyapatite as a model system for discovery of apatite-associated proteins in bone or tooth biomineralization studies. This model approach can be generalized to other desired crystal surfaces using the same experimental design principles with a little modification of the procedures. © 2013 Elsevier Inc. All rights reserved.

Methods for Selecting Phage Display Antibody Libraries.

PubMed

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. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Tangible display systems: bringing virtual surfaces into the real world

NASA Astrophysics Data System (ADS)

Ferwerda, James A.

2012-03-01

We are developing tangible display systems that enable natural interaction with virtual surfaces. Tangible display systems are based on modern mobile devices that incorporate electronic image displays, graphics hardware, tracking systems, and digital cameras. Custom software allows the orientation of a device and the position of the observer to be tracked in real-time. Using this information, realistic images of surfaces with complex textures and material properties illuminated by environment-mapped lighting, can be rendered to the screen at interactive rates. Tilting or moving in front of the device produces realistic changes in surface lighting and material appearance. In this way, tangible displays allow virtual surfaces to be observed and manipulated as naturally as real ones, with the added benefit that surface geometry and material properties can be modified in real-time. We demonstrate the utility of tangible display systems in four application areas: material appearance research; computer-aided appearance design; enhanced access to digital library and museum collections; and new tools for digital artists.

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

PubMed

Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

2016-01-01

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

PubMed

Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

2018-04-30

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular Dynamics Visualization (MDV): Stereoscopic 3D Display of Biomolecular Structure and Interactions Using the Unity Game Engine.

PubMed

Wiebrands, Michael; Malajczuk, Chris J; Woods, Andrew J; Rohl, Andrew L; Mancera, Ricardo L

2018-06-21

Molecular graphics systems are visualization tools which, upon integration into a 3D immersive environment, provide a unique virtual reality experience for research and teaching of biomolecular structure, function and interactions. We have developed a molecular structure and dynamics application, the Molecular Dynamics Visualization tool, that uses the Unity game engine combined with large scale, multi-user, stereoscopic visualization systems to deliver an immersive display experience, particularly with a large cylindrical projection display. The application is structured to separate the biomolecular modeling and visualization systems. The biomolecular model loading and analysis system was developed as a stand-alone C# library and provides the foundation for the custom visualization system built in Unity. All visual models displayed within the tool are generated using Unity-based procedural mesh building routines. A 3D user interface was built to allow seamless dynamic interaction with the model while being viewed in 3D space. Biomolecular structure analysis and display capabilities are exemplified with a range of complex systems involving cell membranes, protein folding and lipid droplets.

Advanced Technology for Portable Personal Visualization.

DTIC Science & Technology

1992-06-01

interactive radiosity . 6 Advanced Technology for Portable Personal Visualization Progress Report January-June 1992 9 2.5 Virtual-Environment Ultrasound...the system, with support for textures, model partitioning, more complex radiosity emitters, and the replacement of model parts with objects from our...model libraries. "* Add real-time, interactive radiosity to the display program on Pixel-Planes 5. "* Move the real-time model mesh-generation to the

Targeting mammalian organelles with internalizing phage (iPhage) libraries

PubMed Central

Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2015-01-01

Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

ORF phage display to identify cellular proteins with different functions.

PubMed

Li, Wei

2012-09-01

Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages. Copyright © 2012 Elsevier Inc. All rights reserved.

The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3.

PubMed

Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Seppä, Titta; Akter, Sultana; Khan, Liton Md Ferdhos; Lamminmäki, Urpo

2014-09-19

Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins. In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection. The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.

Detection of protein-protein interactions by ribosome display and protein in situ immobilisation.

PubMed

He, Mingyue; Liu, Hong; Turner, Martin; Taussig, Michael J

2009-12-31

We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

Beyond helper phage: Using "helper cells" to select peptide affinity ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report onmore » the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Here, based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.« less

Beyond helper phage: Using "helper cells" to select peptide affinity ligands

DOE PAGES

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin; ...

2016-09-14

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report onmore » the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Here, based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.« less

Selection dynamic of Escherichia coli host in M13 combinatorial peptide phage display libraries.

PubMed

Zanconato, Stefano; Minervini, Giovanni; Poli, Irene; De Lucrezia, Davide

2011-01-01

Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness.

Comprehensive peptidomimetic libraries targeting protein-protein interactions.

PubMed

Whitby, Landon R; Boger, Dale L

2012-10-16

Transient protein-protein interactions (PPIs) are essential components in cellular signaling pathways as well as in important processes such as viral infection, replication, and immune suppression. The unknown or uncharacterized PPIs involved in such interaction networks often represent compelling therapeutic targets for drug discovery. To date, however, the main strategies for discovery of small molecule modulators of PPIs are typically limited to structurally characterized targets. Recent developments in molecular scaffolds that mimic the side chain display of peptide secondary structures have yielded effective designs, but few screening libraries of such mimetics are available to interrogate PPI targets. We initiated a program to prepare a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate PPIs (α-helix, β-turn, and β-strand). Three libraries would be built around templates designed to mimic each such secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains. When combined, the three libraries would contain a member capable of mimicking the key interaction and recognition residues of most targetable PPIs. In this Account, we summarize the results of the design, synthesis, and validation of an 8000 member α-helix mimetic library and a 4200 member β-turn mimetic library. We expect that the screening of these libraries will not only provide lead structures against α-helix- or β-turn-mediated protein-protein or peptide-receptor interactions, even if the nature of the interaction is unknown, but also yield key insights into the recognition motif (α-helix or β-turn) and identify the key residues mediating the interaction. Consistent with this expectation, the screening of the libraries against p53/MDM2 and HIV-1 gp41 (α-helix mimetic library) or the opioid receptors (β-turn mimetic library) led to the discovery of library members expected to mimic the known endogenous ligands. These efforts led to the discovery of high-affinity α-helix mimetics (K(i) = 0.7 μM) against HIV-1 gp41 as well as high-affinity and selective β-turn mimetics (K(i) = 80 nM) against the κ-opioid receptor. The results suggest that the use of such comprehensive libraries of peptide secondary structure mimetics, built around effective molecular scaffolds, constitutes a powerful method of interrogating PPIs. These structures provide small molecule modulators of PPI networks for therapeutic target validation, lead compound discovery, and the identification of modulators of biological processes for further study.

GLCF: Library

Science.gov Websites

Global Land Cover Facility About GLCF Research Publications Data & Products Gallery Library Services Contact Site Map Go Library Documents Proposal Reports Publications FAQ Display Materials Release News Archive Library * Display Materials * Documents * News Archive * Software e-link 4321

Clustering of disulfide-rich peptides provides scaffolds for hit discovery by phage display: application to interleukin-23.

PubMed

Barkan, David T; Cheng, Xiao-Li; Celino, Herodion; Tran, Tran T; Bhandari, Ashok; Craik, Charles S; Sali, Andrej; Smythe, Mark L

2016-11-23

Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity. We developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the representatives were structurally distinct from one another, but similar to other DRPs in their respective clusters. To demonstrate the utility of the clusters, phage libraries were constructed for three of the representative scaffolds and panned against interleukin-23. One library produced a peptide that bound to this target with an IC 50 of 3.3 μM. Most DRP clusters contained members that were diverse in sequence, host organism, and interacting proteins, indicating that cluster members were functionally diverse despite having similar structure. Only 20 peptide scaffolds accounted for most of the natural DRP structural diversity, providing suitable starting points for seeding phage display experiments. Through selection of the scaffold surface to vary in phage display, libraries can be designed that present sequence diversity in architecturally distinct, biologically relevant combinations of secondary structures. We supported this hypothesis with a proof-of-concept experiment in which three phage libraries were constructed and panned against the IL-23 target, resulting in a single-digit μM hit and suggesting that a collection of libraries based on the full set of 20 scaffolds increases the potential to identify efficiently peptide binders to a protein target in a drug discovery program.

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

PubMed

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

Porting and redesign of Geotool software system to Qt

NASA Astrophysics Data System (ADS)

Miljanovic Tamarit, V.; Carneiro, L.; Henson, I. H.; Tomuta, E.

2016-12-01

Geotool is a software system that allows a user to interactively display and process seismoacoustic data from International Monitoring System (IMS) station. Geotool can be used to perform a number of analysis and review tasks, including data I/O, waveform filtering, quality control, component rotation, amplitude and arrival measurement and review, array beamforming, correlation, Fourier analysis, FK analysis, event review and location, particle motion visualization, polarization analysis, instrument response convolution/deconvolution, real-time display, signal to noise measurement, spectrogram, and travel time model display. The Geotool program was originally written in C using the X11/Xt/Motif libraries for graphics. It was later ported to C++. Now the program is being ported to the Qt graphics system to be more compatible with the other software in the International Data Centre (IDC). Along with this port, a redesign of the architecture is underway to achieve a separation between user interface, control, and data model elements, in line with design patterns such as Model-View-Controller. Qt is a cross-platform application framework that will allow geotool to easily run on Linux, Mac, and Windows. The Qt environment includes modern libraries and user interfaces for standard utilities such as file and database access, printing, and inter-process communications. The Qt Widgets for Technical Applications library (QWT) provides tools for displaying standard data analysis graphics.

Raster graphics display library

NASA Technical Reports Server (NTRS)

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.

Construction of a filamentous phage display peptide library.

PubMed

Fagerlund, Annette; Myrset, Astrid Hilde; Kulseth, Mari Ann

2014-01-01

The concept of phage display is based on insertion of random oligonucleotides at an appropriate location within a structural gene of a bacteriophage. The resulting phage will constitute a library of random peptides displayed on the surface of the bacteriophages, with the encoding genotype packaged within each phage particle. Using a phagemid/helper phage system, the random peptides are interspersed between wild-type coat proteins. Libraries of phage-expressed peptides may be used to search for novel peptide ligands to target proteins. The success of finding a peptide with a desired property in a given library is highly dependent on the diversity and quality of the library. The protocols in this chapter describe the construction of a high-diversity library of phagemid vector encoding fusions of the phage coat protein pVIII with random peptides, from which a phage library displaying random peptides can be prepared.

Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

NASA Astrophysics Data System (ADS)

Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

2016-05-01

A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

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

PubMed

Tonelli, R R; Colli, W; Alves, M J M

2012-01-01

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, and 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 organisms will be discussed. Using these techniques, recent results on the interaction of Trypanosoma cruzi with the host will be highlighted focusing on members of the 85 kDa protein family, a subset of the gp85/TS superfamily.

Management Matters. Display and Promotion Ideas for Library Media Centers

ERIC Educational Resources Information Center

Pappas, Marjorie L.

2005-01-01

School library media centers should be warm and inviting places where the environment entices children to read and explore. Creative bulletin board and case displays along with other exhibits help to make the library media center an exciting place. Bulletin board displays that promote authors, books, and exciting ideas motivate children to find…

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display.

PubMed

Patel, Chirag A; Wang, Jinqing; Wang, Xinwei; Dong, Feng; Zhong, Pingyu; Luo, Peter P; Wang, Kevin C

2011-09-01

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

IViPP: A Tool for Visualization in Particle Physics

NASA Astrophysics Data System (ADS)

Tran, Hieu; Skiba, Elizabeth; Baldwin, Doug

2011-10-01

Experiments and simulations in physics generate a lot of data; visualization is helpful to prepare that data for analysis. IViPP (Interactive Visualizations in Particle Physics) is an interactive computer program that visualizes results of particle physics simulations or experiments. IViPP can handle data from different simulators, such as SRIM or MCNP. It can display relevant geometry and measured scalar data; it can do simple selection from the visualized data. In order to be an effective visualization tool, IViPP must have a software architecture that can flexibly adapt to new data sources and display styles. It must be able to display complicated geometry and measured data with a high dynamic range. We therefore organize it in a highly modular structure, we develop libraries to describe geometry algorithmically, use rendering algorithms running on the powerful GPU to display 3-D geometry at interactive rates, and we represent scalar values in a visual form of scientific notation that shows both mantissa and exponent. This work was supported in part by the US Department of Energy through the Laboratory for Laser Energetics (LLE), with special thanks to Craig Sangster at LLE.

Advances in synthetic peptides reagent discovery

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Stratis-Cullum, Dimitra N.

2013-05-01

Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Cartographic symbol library considering symbol relations based on anti-aliasing graphic library

NASA Astrophysics Data System (ADS)

Mei, Yang; Li, Lin

2007-06-01

Cartographic visualization represents geographic information with a map form, which enables us retrieve useful geospatial information. In digital environment, cartographic symbol library is the base of cartographic visualization and is an essential component of Geographic Information System as well. Existing cartographic symbol libraries have two flaws. One is the display quality and the other one is relations adjusting. Statistic data presented in this paper indicate that the aliasing problem is a major factor on the symbol display quality on graphic display devices. So, effective graphic anti-aliasing methods based on a new anti-aliasing algorithm are presented and encapsulated in an anti-aliasing graphic library with the form of Component Object Model. Furthermore, cartographic visualization should represent feature relation in the way of correctly adjusting symbol relations besides displaying an individual feature. But current cartographic symbol libraries don't have this capability. This paper creates a cartographic symbol design model to implement symbol relations adjusting. Consequently the cartographic symbol library based on this design model can provide cartographic visualization with relations adjusting capability. The anti-aliasing graphic library and the cartographic symbol library are sampled and the results prove that the two libraries both have better efficiency and effect.

Isolation and characterization of anti-SEB peptides using magnetic sorting and bacterial peptide display library technology

NASA Astrophysics Data System (ADS)

Pennington, Joseph M.; Kogot, Joshua M.; Sarkes, Deborah A.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

2012-06-01

Peptide display libraries offer an alternative method to existing antibody development methods enabling rapid isolation of highly stable reagents for detection of new and emerging biological threats. Bacterial display libraries are used to isolate new peptide reagents within 1 week, which is simpler and timelier than using competing display library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high affinity and specificity to staphylococcal enterotoxin B (SEB), a suspected food pathogen. Flow cytometry methods were used for on-cell characterization and the binding affinity (Kd) of this new peptide reagent was determined to be 56 nm with minimal cross-reactivity to other proteins. These results demonstrated that magnetic sorting for new reagents using bacterial display libraries is a rapid and effective method and has the potential for current and new and emerging food pathogen targets.

Status of MAPA (Modular Accelerator Physics Analysis) and the Tech-X Object-Oriented Accelerator Library

NASA Astrophysics Data System (ADS)

Cary, J. R.; Shasharina, S.; Bruhwiler, D. L.

1998-04-01

The MAPA code is a fully interactive accelerator modeling and design tool consisting of a GUI and two object-oriented C++ libraries: a general library suitable for treatment of any dynamical system, and an accelerator library including many element types plus an accelerator class. The accelerator library inherits directly from the system library, which uses hash tables to store any relevant parameters or strings. The GUI can access these hash tables in a general way, allowing the user to invoke a window displaying all relevant parameters for a particular element type or for the accelerator class, with the option to change those parameters. The system library can advance an arbitrary number of dynamical variables through an arbitrary mapping. The accelerator class inherits this capability and overloads the relevant functions to advance the phase space variables of a charged particle through a string of elements. Among other things, the GUI makes phase space plots and finds fixed points of the map. We discuss the object hierarchy of the two libraries and use of the code.

InCHlib - interactive cluster heatmap for web applications.

PubMed

Skuta, Ctibor; Bartůněk, Petr; Svozil, Daniel

2014-12-01

Hierarchical clustering is an exploratory data analysis method that reveals the groups (clusters) of similar objects. The result of the hierarchical clustering is a tree structure called dendrogram that shows the arrangement of individual clusters. To investigate the row/column hierarchical cluster structure of a data matrix, a visualization tool called 'cluster heatmap' is commonly employed. In the cluster heatmap, the data matrix is displayed as a heatmap, a 2-dimensional array in which the colour of each element corresponds to its value. The rows/columns of the matrix are ordered such that similar rows/columns are near each other. The ordering is given by the dendrogram which is displayed on the side of the heatmap. We developed InCHlib (Interactive Cluster Heatmap Library), a highly interactive and lightweight JavaScript library for cluster heatmap visualization and exploration. InCHlib enables the user to select individual or clustered heatmap rows, to zoom in and out of clusters or to flexibly modify heatmap appearance. The cluster heatmap can be augmented with additional metadata displayed in a different colour scale. In addition, to further enhance the visualization, the cluster heatmap can be interconnected with external data sources or analysis tools. Data clustering and the preparation of the input file for InCHlib is facilitated by the Python utility script inchlib_clust . The cluster heatmap is one of the most popular visualizations of large chemical and biomedical data sets originating, e.g., in high-throughput screening, genomics or transcriptomics experiments. The presented JavaScript library InCHlib is a client-side solution for cluster heatmap exploration. InCHlib can be easily deployed into any modern web application and configured to cooperate with external tools and data sources. Though InCHlib is primarily intended for the analysis of chemical or biological data, it is a versatile tool which application domain is not limited to the life sciences only.

Development of bacterial display peptides for use in biosensing applications

NASA Astrophysics Data System (ADS)

Stratis-Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James P.; Brown, Rebecca L.; Pellegrino, Paul M.

2012-06-01

Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.

Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

PubMed

Shahsavarian, Melody A; Le Minoux, Damien; Matti, Kalyankumar M; Kaveri, Srini; Lacroix-Desmazes, Sébastien; Boquet, Didier; Friboulet, Alain; Avalle, Bérangère; Padiolleau-Lefèvre, Séverine

2014-05-01

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library. Copyright © 2014 Elsevier B.V. All rights reserved.

Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique.

PubMed

You, Fei; Yin, Guangfu; Pu, Ximing; Li, Yucan; Hu, Yang; Huang, Zhongbin; Liao, Xiaoming; Yao, Yadong; Chen, Xianchun

2016-05-01

Functionalization of inorganic nanoparticles (NPs) play an important role in biomedical applications. A proper functionalization of NPs can improve biocompatibility, avoid a loss of bioactivity, and further endow NPs with unique performances. Modification with vairous specific binding biomolecules from random biological libraries has been explored. In this work, two 7-mer peptides with sequences of HYIDFRW and TVNFKLY were selected from a phage display random peptide library by using ferromagnetic NPs as targets, and were verified to display strong binding affinity to Fe3O4 NPs. Fourier transform infrared spectrometry, fluorescence microscopy, thermal analysis and X-ray photoelectron spectroscopy confirmed the presence of peptides on the surface of Fe3O4 NPs. Sequence analyses revealed that the probable binding mechanism between the peptide and Fe3O4 NPs might be driven by Pearson hard acid-hard base specific interaction and hydrogen bonds, accompanied with hydrophilic interactions and non-specific electrostatic attractions. The cell viability assay indicated a good cytocompatibility of peptide-bound Fe3O4 NPs. Furthermore, TVNFKLY peptide and an ovarian tumor cell A2780 specific binding peptide (QQTNWSL) were conjugated to afford a liner 14-mer peptide (QQTNWSLTVNFKLY). The binding and targeting studies showed that 14-mer peptide was able to retain both the strong binding ability to Fe3O4 NPs and the specific binding ability to A2780 cells. The results suggested that the Fe3O4-binding peptides would be of great potential in the functionalization of Fe3O4 NPs for the tumor-targeted drug delivery and magnetic hyperthermia. Copyright © 2016 Elsevier B.V. All rights reserved.

The Effect of Face-Front Display on the Circulation of Books in a Public Library.

ERIC Educational Resources Information Center

Long, Sarah P.

This study considers the theories of impulse buying in an examination of the effects on circulation of library books when books are displayed face front (with all or most of the book jacket showing) as opposed to spine front. Reviews of the literature on consumer behavior and on library displays support the hypothesis of this study, i.e., that…

Smart boards: a reemerging technology.

PubMed

Brigham, Tara J

2013-01-01

Smart boards, also known as interactive whiteboards (IWBs), are large, interactive, touch-sensitive displays that are mainly used for presentation or educational purposes. While some may not consider this an emerging technology today, changes in the design and capabilities challenge that line of thinking. This column will explain what a smart board is, provide a brief history about it, and describe where it is currently used and why it might be a technology to consider having in a library today.

Multimodal Virtual Environments: MAGIC Toolkit and Visual-Haptic Interaction Paradigms

DTIC Science & Technology

1998-01-01

2.7.3 Load/Save Options ..... 2.7.4 Information Display .... 2.8 Library Files. 2.9 Evaluation .............. 3 Visual-Haptic Interactions 3.1...Northwestern University[ Colgate , 1994]. It is possible for a user to touch one side of a thin object and be propelled out the opposite side, because...when there is a high correlation in motion and force between the visual and haptic realms. * Chapter 7 concludes with an evaluation of the application

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Display of a maize cDNA library on baculovirus infected insect cells.

PubMed

Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

2008-08-12

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Solid-Phase Synthesis of Diverse Peptide Tertiary Amides By Reductive Amination

PubMed Central

Pels, Kevin; Kodadek, Thomas

2015-01-01

The synthesis of libraries of conformationally-constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as peptide tertiary amide (PTA). PTAs are strongly biased conformationally due to allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers. PMID:25695359

Solid-phase synthesis of diverse peptide tertiary amides by reductive amination.

PubMed

Pels, Kevin; Kodadek, Thomas

2015-03-09

The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers.

Recombinant Peptides as Biomarkers for Metastatic Breast Cancer Response

DTIC Science & Technology

2007-10-01

could be specific to breast cancer tumor models has just been concluded. In vivo biopanning wsa conducted with a T7 phage -based random peptide library...peptides selected from phage -displayed libraries. 15. SUBJECT TERMS Breast cancer, phage display, molecular imaging, personalized medicine 16...recombinant peptides from phage -displayed peptide libraries can be selected that bind to receptors activated in response to therapy. These peptides in turn

Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

PubMed Central

Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

2013-01-01

Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

A method to improve visual similarity of breast masses for an interactive computer-aided diagnosis environment.

PubMed

Zheng, Bin; Lu, Amy; Hardesty, Lara A; Sumkin, Jules H; Hakim, Christiane M; Ganott, Marie A; Gur, David

2006-01-01

The purpose of this study was to develop and test a method for selecting "visually similar" regions of interest depicting breast masses from a reference library to be used in an interactive computer-aided diagnosis (CAD) environment. A reference library including 1000 malignant mass regions and 2000 benign and CAD-generated false-positive regions was established. When a suspicious mass region is identified, the scheme segments the region and searches for similar regions from the reference library using a multifeature based k-nearest neighbor (KNN) algorithm. To improve selection of reference images, we added an interactive step. All actual masses in the reference library were subjectively rated on a scale from 1 to 9 as to their "visual margins speculations". When an observer identifies a suspected mass region during a case interpretation he/she first rates the margins and the computerized search is then limited only to regions rated as having similar levels of spiculation (within +/-1 scale difference). In an observer preference study including 85 test regions, two sets of the six "similar" reference regions selected by the KNN with and without the interactive step were displayed side by side with each test region. Four radiologists and five nonclinician observers selected the more appropriate ("similar") reference set in a two alternative forced choice preference experiment. All four radiologists and five nonclinician observers preferred the sets of regions selected by the interactive method with an average frequency of 76.8% and 74.6%, respectively. The overall preference for the interactive method was highly significant (p < 0.001). The study demonstrated that a simple interactive approach that includes subjectively perceived ratings of one feature alone namely, a rating of margin "spiculation," could substantially improve the selection of "visually similar" reference images.

Peptide Transduction-Based Therapies for Prostate Cancer

DTIC Science & Technology

2004-06-01

using an M13 peptide phage display library. Initial screening of the library for transduction of tumors in vivo has identified peptides able to...marker conjugates may have to be tested. (Months 6-12, Year 1) Progress: These experiments have been initiated. Task 4. An M13 peptide phage display ... phage 12 amino acid control peptide display library (New England Biolabs, Beverly, MA ) was used. Briefly, One nude mouse bearing a human tumor line

Protein Transduction Based Therapies for Breast Cancer

DTIC Science & Technology

2006-07-01

we also have developed a method for screening for tissue-targeted transduction peptides using an M13 peptide phage display library. Using this...Instead the focus was on the ability to identify a tumor specific peptide. Task 4. An M13 peptide phage display library will be used for...cancespecific tumor lines by screening a peptide phage display library both in cell culture as well as in nude micebearing xenografts. Initial results in

T7 lytic phage-displayed peptide libraries: construction and diversity characterization.

PubMed

Krumpe, Lauren R H; Mori, Toshiyuki

2014-01-01

In this chapter, we describe the construction of T7 bacteriophage (phage)-displayed peptide libraries and the diversity analyses of random amino acid sequences obtained from the libraries. We used commercially available reagents, Novagen's T7Select system, to construct the libraries. Using a combination of biotinylated extension primer and streptavidin-coupled magnetic beads, we were able to prepare library DNA without applying gel purification, resulting in extremely high ligation efficiencies. Further, we describe the use of bioinformatics tools to characterize library diversity. Amino acid frequency and positional amino acid diversity and hydropathy are estimated using the REceptor LIgand Contacts website http://relic.bio.anl.gov. Peptide net charge analysis and peptide hydropathy analysis are conducted using the Genetics Computer Group Wisconsin Package computational tools. A comprehensive collection of the estimated number of recombinants and titers of T7 phage-displayed peptide libraries constructed in our lab is included.

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2010-07-01

cloning them in phage display vectors. We are characterizing the libraries and trying to figure out how best to screen them. We ran into the problem...auto-antibody screening project onto glass slides for screening purposes. We have abandoned this aim in that we switched our approach to a phage ...display library which does not require glass slide. We made our first comprehensive library in a T7 display vector. Task 9 (Months 24-36) We will

Touch Interaction with 3D Geographical Visualization on Web: Selected Technological and User Issues

NASA Astrophysics Data System (ADS)

Herman, L.; Stachoň, Z.; Stuchlík, R.; Hladík, J.; Kubíček, P.

2016-10-01

The use of both 3D visualization and devices with touch displays is increasing. In this paper, we focused on the Web technologies for 3D visualization of spatial data and its interaction via touch screen gestures. At the first stage, we compared the support of touch interaction in selected JavaScript libraries on different hardware (desktop PCs with touch screens, tablets, and smartphones) and software platforms. Afterward, we realized simple empiric test (within-subject design, 6 participants, 2 simple tasks, LCD touch monitor Acer and digital terrain models as stimuli) focusing on the ability of users to solve simple spatial tasks via touch screens. An in-house testing web tool was developed and used based on JavaScript, PHP, and X3DOM languages and Hammer.js libraries. The correctness of answers, speed of users' performances, used gestures, and a simple gesture metric was recorded and analysed. Preliminary results revealed that the pan gesture is most frequently used by test participants and it is also supported by the majority of 3D libraries. Possible gesture metrics and future developments including the interpersonal differences are discussed in the conclusion.

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

PubMed Central

Gagic, Dragana; Ciric, Milica; Wen, Wesley X.; Ng, Filomena; Rakonjac, Jasna

2016-01-01

Microbial surface and secreted proteins (the secretome) contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of bacteriophage, called filamentous phage, have the ability to hijack bacterial protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to “bait” of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that are implicated in colonization of host tissues and pathogenesis, as well as vaccine candidates through filamentous phage display library screening. Secretome selection aided by next-generation sequence analysis was successfully applied for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea. PMID:27092113

Successful application of the dual-vector system II in creating a reliable phage-displayed combinatorial Fab library.

PubMed

Song, Suk-yoon; Hur, Byung-ung; Lee, Kyung-woo; Choi, Hyo-jung; Kim, Sung-soo; Kang, Goo; Cha, Sang-hoon

2009-03-31

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a 1.3 x 10(7) combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a 1.5 x 10(9) combinatorial antibody complexity, was also generated in a rapid manner by combining 1.3 x 10(7) heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecule.

Defining RNA motif-aminoglycoside interactions via two-dimensional combinatorial screening and structure-activity relationships through sequencing.

PubMed

Velagapudi, Sai Pradeep; Disney, Matthew D

2013-10-15

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3×3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure-activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif-aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site. Copyright © 2013 Elsevier Ltd. All rights reserved.

Defining RNA motif–aminoglycoside interactions via two-dimensional combinatorial screening and structure–activity relationships through sequencing

PubMed Central

Velagapudi, Sai Pradeep; Disney, Matthew D.

2013-01-01

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3 × 3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure–activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif–aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site. PMID:23719281

Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.

PubMed

Yeung, Yik A; Wittrup, K Dane

2002-01-01

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.

Design and implementation of a PC-based image-guided surgical system.

PubMed

Stefansic, James D; Bass, W Andrew; Hartmann, Steven L; Beasley, Ryan A; Sinha, Tuhin K; Cash, David M; Herline, Alan J; Galloway, Robert L

2002-11-01

In interactive, image-guided surgery, current physical space position in the operating room is displayed on various sets of medical images used for surgical navigation. We have developed a PC-based surgical guidance system (ORION) which synchronously displays surgical position on up to four image sets and updates them in real time. There are three essential components which must be developed for this system: (1) accurately tracked instruments; (2) accurate registration techniques to map physical space to image space; and (3) methods to display and update the image sets on a computer monitor. For each of these components, we have developed a set of dynamic link libraries in MS Visual C++ 6.0 supporting various hardware tools and software techniques. Surgical instruments are tracked in physical space using an active optical tracking system. Several of the different registration algorithms were developed with a library of robust math kernel functions, and the accuracy of all registration techniques was thoroughly investigated. Our display was developed using the Win32 API for windows management and tomographic visualization, a frame grabber for live video capture, and OpenGL for visualization of surface renderings. We have begun to use this current implementation of our system for several surgical procedures, including open and minimally invasive liver surgery.

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

PubMed

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.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

PubMed

Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

2009-09-01

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Principles and application of antibody libraries for infectious diseases.

PubMed

Lim, Bee Nar; Tye, Gee Jun; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Lim, Theam Soon

2014-12-01

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

Old Books Bring New Life to the Brick and Mortar Library

NASA Astrophysics Data System (ADS)

Bosken, S.

2012-08-01

If all the library books and journals can be viewed on your desk top, why come to the physical library? The USNO Library tried to bring the patrons inside the library. One method was to rotate rare book displays each month. As the library holds a fabulous collection of ancient astronomy books, including Copernicus, Kepler, Galileo, and Newton, we have abundant resources. The presentation will highlight the varied displays and offer a Rare Books 101 explanation of paper, printing, binding and a behind-the-scenes look at how old books are maintained and preserved.

Phage display—A powerful technique for immunotherapy

PubMed Central

Bazan, Justyna; Całkosiński, Ireneusz; Gamian, Andrzej

2012-01-01

One of the most effective molecular diversity techniques is phage display. This technology is based on a direct linkage between phage phenotype and its encapsulated genotype, which leads to presentation of molecule libraries on the phage surface. Phage display is utilized in studying protein-ligand interactions, receptor binding sites and in improving or modifying the affinity of proteins for their binding partners. Generating monoclonal antibodies and improving their affinity, cloning antibodies from unstable hybridoma cells and identifying epitopes, mimotopes and functional or accessible sites from antigens are also important advantages of this technology. Techniques originating from phage display have been applied to transfusion medicine, neurological disorders, mapping vascular addresses and tissue homing of peptides. Phages have been applicable to immunization therapies, which may lead to development of new tools used for treating autoimmune and cancer diseases. This review describes the phage display technology and presents the recent advancements in therapeutic applications of phage display. PMID:22906939

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

PubMed

Körbelin, J; Hunger, A; Alawi, M; Sieber, T; Binder, M; Trepel, M

2017-08-01

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

Developing Novel Therapeutics Targeting Undifferentiated and Castration-Resistant Prostate Cancer Stem Cells

DTIC Science & Technology

2016-10-01

identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to identify novel PCSC-targeting chemicals. In the past...display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to...Goals of the Project (SOW): Aim 1: To perform phage display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides

Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing.

PubMed

Bidlingmaier, Scott; Ha, Kevin; Lee, Nam-Kyung; Su, Yang; Liu, Bin

2016-04-01

Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

SPAM- SPECTRAL ANALYSIS MANAGER (DEC VAX/VMS VERSION)

NASA Technical Reports Server (NTRS)

Solomon, J. E.

1994-01-01

The Spectral Analysis Manager (SPAM) was developed to allow easy qualitative analysis of multi-dimensional imaging spectrometer data. Imaging spectrometers provide sufficient spectral sampling to define unique spectral signatures on a per pixel basis. Thus direct material identification becomes possible for geologic studies. SPAM provides a variety of capabilities for carrying out interactive analysis of the massive and complex datasets associated with multispectral remote sensing observations. In addition to normal image processing functions, SPAM provides multiple levels of on-line help, a flexible command interpretation, graceful error recovery, and a program structure which can be implemented in a variety of environments. SPAM was designed to be visually oriented and user friendly with the liberal employment of graphics for rapid and efficient exploratory analysis of imaging spectrometry data. SPAM provides functions to enable arithmetic manipulations of the data, such as normalization, linear mixing, band ratio discrimination, and low-pass filtering. SPAM can be used to examine the spectra of an individual pixel or the average spectra over a number of pixels. SPAM also supports image segmentation, fast spectral signature matching, spectral library usage, mixture analysis, and feature extraction. High speed spectral signature matching is performed by using a binary spectral encoding algorithm to separate and identify mineral components present in the scene. The same binary encoding allows automatic spectral clustering. Spectral data may be entered from a digitizing tablet, stored in a user library, compared to the master library containing mineral standards, and then displayed as a timesequence spectral movie. The output plots, histograms, and stretched histograms produced by SPAM can be sent to a lineprinter, stored as separate RGB disk files, or sent to a Quick Color Recorder. SPAM is written in C for interactive execution and is available for two different machine environments. There is a DEC VAX/VMS version with a central memory requirement of approximately 242K of 8 bit bytes and a machine independent UNIX 4.2 version. The display device currently supported is the Raster Technologies display processor. Other 512 x 512 resolution color display devices, such as De Anza, may be added with minor code modifications. This program was developed in 1986.

SPAM- SPECTRAL ANALYSIS MANAGER (UNIX VERSION)

NASA Technical Reports Server (NTRS)

Solomon, J. E.

1994-01-01

The Spectral Analysis Manager (SPAM) was developed to allow easy qualitative analysis of multi-dimensional imaging spectrometer data. Imaging spectrometers provide sufficient spectral sampling to define unique spectral signatures on a per pixel basis. Thus direct material identification becomes possible for geologic studies. SPAM provides a variety of capabilities for carrying out interactive analysis of the massive and complex datasets associated with multispectral remote sensing observations. In addition to normal image processing functions, SPAM provides multiple levels of on-line help, a flexible command interpretation, graceful error recovery, and a program structure which can be implemented in a variety of environments. SPAM was designed to be visually oriented and user friendly with the liberal employment of graphics for rapid and efficient exploratory analysis of imaging spectrometry data. SPAM provides functions to enable arithmetic manipulations of the data, such as normalization, linear mixing, band ratio discrimination, and low-pass filtering. SPAM can be used to examine the spectra of an individual pixel or the average spectra over a number of pixels. SPAM also supports image segmentation, fast spectral signature matching, spectral library usage, mixture analysis, and feature extraction. High speed spectral signature matching is performed by using a binary spectral encoding algorithm to separate and identify mineral components present in the scene. The same binary encoding allows automatic spectral clustering. Spectral data may be entered from a digitizing tablet, stored in a user library, compared to the master library containing mineral standards, and then displayed as a timesequence spectral movie. The output plots, histograms, and stretched histograms produced by SPAM can be sent to a lineprinter, stored as separate RGB disk files, or sent to a Quick Color Recorder. SPAM is written in C for interactive execution and is available for two different machine environments. There is a DEC VAX/VMS version with a central memory requirement of approximately 242K of 8 bit bytes and a machine independent UNIX 4.2 version. The display device currently supported is the Raster Technologies display processor. Other 512 x 512 resolution color display devices, such as De Anza, may be added with minor code modifications. This program was developed in 1986.

YPD™, PombePD™ and WormPD™: model organism volumes of the BioKnowledge™ Library, an integrated resource for protein information

PubMed Central

Costanzo, Maria C.; Crawford, Matthew E.; Hirschman, Jodi E.; Kranz, Janice E.; Olsen, Philip; Robertson, Laura S.; Skrzypek, Marek S.; Braun, Burkhard R.; Hopkins, Kelley Lennon; Kondu, Pinar; Lengieza, Carey; Lew-Smith, Jodi E.; Tillberg, Michael; Garrels, James I.

2001-01-01

The BioKnowledge Library is a relational database and web site (http://www.proteome.com) composed of protein-specific information collected from the scientific literature. Each Protein Report on the web site summarizes and displays published information about a single protein, including its biochemical function, role in the cell and in the whole organism, localization, mutant phenotype and genetic interactions, regulation, domains and motifs, interactions with other proteins and other relevant data. This report describes four species-specific volumes of the BioKnowledge Library, concerned with the model organisms Saccharo­myces cerevisiae (YPD), Schizosaccharomyces pombe (PombePD) and Caenorhabditis elegans (WormPD), and with the fungal pathogen Candida albicans (CalPD™). Protein Reports of each species are unified in format, easily searchable and extensively cross-referenced between species. The relevance of these comprehensively curated resources to analysis of proteins in other species is discussed, and is illustrated by a survey of model organism proteins that have similarity to human proteins involved in disease. PMID:11125054

Phage display peptide libraries: deviations from randomness and correctives

PubMed Central

Ryvkin, Arie; Ashkenazy, Haim; Weiss-Ottolenghi, Yael; Piller, Chen; Pupko, Tal; Gershoni, Jonathan M

2018-01-01

Abstract Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications. PMID:29420788

WHOI and SIO (I): Next Steps toward Multi-Institution Archiving of Shipboard and Deep Submergence Vehicle Data

NASA Astrophysics Data System (ADS)

Detrick, R. S.; Clark, D.; Gaylord, A.; Goldsmith, R.; Helly, J.; Lemmond, P.; Lerner, S.; Maffei, A.; Miller, S. P.; Norton, C.; Walden, B.

2005-12-01

The Scripps Institution of Oceanography (SIO) and the Woods Hole Oceanographic Institution (WHOI) have joined forces with the San Diego Supercomputer Center to build a testbed for multi-institutional archiving of shipboard and deep submergence vehicle data. Support has been provided by the Digital Archiving and Preservation program funded by NSF/CISE and the Library of Congress. In addition to the more than 92,000 objects stored in the SIOExplorer Digital Library, the testbed will provide access to data, photographs, video images and documents from WHOI ships, Alvin submersible and Jason ROV dives, and deep-towed vehicle surveys. An interactive digital library interface will allow combinations of distributed collections to be browsed, metadata inspected, and objects displayed or selected for download. The digital library architecture, and the search and display tools of the SIOExplorer project, are being combined with WHOI tools, such as the Alvin Framegrabber and the Jason Virtual Control Van, that have been designed using WHOI's GeoBrowser to handle the vast volumes of digital video and camera data generated by Alvin, Jason and other deep submergence vehicles. Notions of scalability will be tested, as data volumes range from 3 CDs per cruise to 200 DVDs per cruise. Much of the scalability of this proposal comes from an ability to attach digital library data and metadata acquisition processes to diverse sensor systems. We are able to run an entire digital library from a laptop computer as well as from supercomputer-center-size resources. It can be used, in the field, laboratory or classroom, covering data from acquisition-to-archive using a single coherent methodology. The design is an open architecture, supporting applications through well-defined external interfaces maintained as an open-source effort for community inclusion and enhancement.

Transferring the Characteristics of Naturally Occurring and Biased Antibody Repertoires to Human Antibody Libraries by Trapping CDRH3 Sequences

PubMed Central

Venet, Sophie; Ravn, Ulla; Buatois, Vanessa; Gueneau, Franck; Calloud, Sébastien; Kosco-Vilbois, Marie; Fischer, Nicolas

2012-01-01

Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications. PMID:22937053

Screening of synthetic phage display scFv libraries yields competitive ligands of human leptin receptor.

PubMed

Molek, Peter; Vodnik, Miha; Strukelj, Borut; Bratkovič, Tomaž

2014-09-26

Initially considered the main endogenous anorexigenic factor, fat-derived leptin turned out to be a markedly pleiotropic hormone, influencing diverse physiological processes. Moreover, hyperleptinemia in obese individuals has been linked to the onset or progression of serious disorders, such as cancer, autoimmune diseases, and atherosclerosis, and antagonizing peripheral leptin's signalization has been shown to improve these conditions. To develop an antibody-based leptin antagonist we have devised a tailored panning procedure and screened two phage display libraries of single chain variable antibody fragments (scFvs) against recombinant leptin receptor. One of the scFvs was expressed in Escherichia coli and its interaction with leptin receptor was characterized in more detail. It was found to recognize a discontinuous epitope and to compete with leptin for receptor binding with IC50 and Kd values in the nanomolar range. The reported scFv represents a lead for development of leptin antagonists that may ultimately find use in therapy of various hyperleptinemia-related disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

The New Visual Displays That Are "Floating" Your Way. Building Digital Libraries

ERIC Educational Resources Information Center

Huwe, Terence K.

2005-01-01

In this column, the author describes three very experimental visual display technologies that will affect library collections and services in the near future. While each of these new display strategies is unique in its technological approach, there is a common denominator to all three: better freedom of mobility that will allow people to interact…

Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

PubMed Central

Martínez-Arteaga, Rocio; Ruano-Gallego, David; Fraile, Sofía; Margolles, Yago; Teira, Xema; Gutierrez, Carlos; Bodelón, Gustavo; Fernández, Luis Ángel

2013-01-01

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 (TirMEHEC). 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 TirMEHEC 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 TirMEHEC 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. PMID:24086454

Rapid isolation of novel FK506 binding proteins from multiple organisms using gDNA and cDNA T7 phage display.

PubMed

Piggott, Andrew M; Kriegel, Alison M; Willows, Robert D; Karuso, Peter

2009-10-01

Reverse chemical proteomics using T7 phage display is a powerful technique for identifying cellular receptors of biologically active small molecules. However, to date this method has generally been limited to cDNA libraries constructed from mRNA isolated from eukaryotes. In this paper, we describe the construction of the first prokaryotic T7 phage display libraries from randomly digested Pseudomonas stutzeri and Vibrio fischeri gDNA, as well as a plant cDNA library from Arabidopsis thaliana. We also describe the use of T7 phage display to identify novel proteins from environmental DNA samples using biotinylated FK506 as a model affinity probe.

Oligopeptide M13 Phage Display in Pathogen Research

PubMed Central

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-01-01

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline. PMID:24136040

Oligopeptide m13 phage display in pathogen research.

PubMed

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-10-16

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

Reprogramming cell fate with a genome-scale library of artificial transcription factors.

PubMed

Eguchi, Asuka; Wleklinski, Matthew J; Spurgat, Mackenzie C; Heiderscheit, Evan A; Kropornicka, Anna S; Vu, Catherine K; Bhimsaria, Devesh; Swanson, Scott A; Stewart, Ron; Ramanathan, Parameswaran; Kamp, Timothy J; Slukvin, Igor; Thomson, James A; Dutton, James R; Ansari, Aseem Z

2016-12-20

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.

Reprogramming cell fate with a genome-scale library of artificial transcription factors

PubMed Central

Eguchi, Asuka; Wleklinski, Matthew J.; Spurgat, Mackenzie C.; Heiderscheit, Evan A.; Kropornicka, Anna S.; Vu, Catherine K.; Bhimsaria, Devesh; Swanson, Scott A.; Stewart, Ron; Ramanathan, Parameswaran; Kamp, Timothy J.; Slukvin, Igor; Thomson, James A.; Dutton, James R.; Ansari, Aseem Z.

2016-01-01

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices. PMID:27930301

Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

DOE PAGES

Sun, Yue; Ban, Bhupal; Bradbury, Andrew; ...

2016-08-29

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Yue; Ban, Bhupal; Bradbury, Andrew

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins.

PubMed

Cochran, A G; Tong, R T; Starovasnik, M A; Park, E J; McDowell, R S; Theaker, J E; Skelton, N J

2001-01-31

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.

Quantitative synthesis of genetically encoded glycopeptide libraries displayed on M13 phage.

PubMed

Ng, Simon; Jafari, Mohammad R; Matochko, Wadim L; Derda, Ratmir

2012-09-21

Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.D.-7) in a two-step, one-pot reaction in <1.5 h. Unlike previous reports, we bypassed genetic engineering of phage. The glycan moiety was introduced via an oxime ligation following oxidation of an N-terminal Ser/Thr; these residues are present in the peptide libraries at 20-30% abundance. The construction of libraries was facilitated by simple characterization, which directly assessed the yield and regioselectivity of chemical reactions performed on phage. This quantification method also allowed facile yield determination of reactions in 10(9) distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries.

A user's guide for DTIZE an interactive digitizing and graphical editing computer program

NASA Technical Reports Server (NTRS)

Thomas, C. C.

1981-01-01

A guide for DTIZE, a two dimensional digitizing program with graphical editing capability, is presented. DTIZE provides the capability to simultaneously create and display a picture on the display screen. Data descriptions may be permanently saved in three different formats. DTIZE creates the picture graphics in the locator mode, thus inputting one coordinate each time the terminator button is pushed. Graphic input devices (GIN) are also used to select function command menu. These menu commands and the program's interactive prompting sequences provide a complete capability for creating, editing, and permanently recording a graphical picture file. DTIZE is written in FORTRAN IV language for the Tektronix 4081 graphic system utilizing the Plot 80 Distributed Graphics Library (DGL) subroutines. The Tektronix 4953/3954 Graphic Tablet with mouse, pen, or joystick are used as graphics input devices to create picture graphics.

High performance interactive graphics for shower reconstruction in HPC, the DELPHI barrel electromagnetic calorimeter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanescu, C.

1990-08-01

Complex software for shower reconstruction in DELPHI barrel electromagnetic calorimeter which deals, for each event, with great amounts of information, due to the high spatial resolution of this detector, needs powerful verification tools. An interactive graphics program, running on high performance graphics display system Whizzard 7555 from Megatek, was developed to display the logical steps in showers and their axes reconstruction. The program allows both operations on the image in real-time (rotation, translation and zoom) and the use of non-geometrical criteria to modify it (as the use of energy) thresholds for the representation of the elements that compound the showersmore » (or of the associated lego plots). For this purpose graphics objects associated to user parameters were defined. Instancing and modelling features of the native graphics library were extensively used.« less

The Medical Library Association Benchmarking Network: development and implementation.

PubMed

Dudden, Rosalind Farnam; Corcoran, Kate; Kaplan, Janice; Magouirk, Jeff; Rand, Debra C; Smith, Bernie Todd

2006-04-01

This article explores the development and implementation of the Medical Library Association (MLA) Benchmarking Network from the initial idea and test survey, to the implementation of a national survey in 2002, to the establishment of a continuing program in 2004. Started as a program for hospital libraries, it has expanded to include other nonacademic health sciences libraries. The activities and timelines of MLA's Benchmarking Network task forces and editorial board from 1998 to 2004 are described. The Benchmarking Network task forces successfully developed an extensive questionnaire with parameters of size and measures of library activity and published a report of the data collected by September 2002. The data were available to all MLA members in the form of aggregate tables. Utilization of Web-based technologies proved feasible for data intake and interactive display. A companion article analyzes and presents some of the data. MLA has continued to develop the Benchmarking Network with the completion of a second survey in 2004. The Benchmarking Network has provided many small libraries with comparative data to present to their administrators. It is a challenge for the future to convince all MLA members to participate in this valuable program.

The Medical Library Association Benchmarking Network: development and implementation*

PubMed Central

Dudden, Rosalind Farnam; Corcoran, Kate; Kaplan, Janice; Magouirk, Jeff; Rand, Debra C.; Smith, Bernie Todd

2006-01-01

Objective: This article explores the development and implementation of the Medical Library Association (MLA) Benchmarking Network from the initial idea and test survey, to the implementation of a national survey in 2002, to the establishment of a continuing program in 2004. Started as a program for hospital libraries, it has expanded to include other nonacademic health sciences libraries. Methods: The activities and timelines of MLA's Benchmarking Network task forces and editorial board from 1998 to 2004 are described. Results: The Benchmarking Network task forces successfully developed an extensive questionnaire with parameters of size and measures of library activity and published a report of the data collected by September 2002. The data were available to all MLA members in the form of aggregate tables. Utilization of Web-based technologies proved feasible for data intake and interactive display. A companion article analyzes and presents some of the data. MLA has continued to develop the Benchmarking Network with the completion of a second survey in 2004. Conclusions: The Benchmarking Network has provided many small libraries with comparative data to present to their administrators. It is a challenge for the future to convince all MLA members to participate in this valuable program. PMID:16636702

[Peptide phage display in biotechnology and biomedicine].

PubMed

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.

Ligand-directed profiling of organelles with internalizing phage libraries

PubMed Central

Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

2015-01-01

Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PubMed

Yee, Andrew; Tan, Fen-Lai; Ginsburg, David

2013-01-01

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Construction of High-Quality Camel Immune Antibody Libraries.

PubMed

Romão, Ema; Poignavent, Vianney; Vincke, Cécile; Ritzenthaler, Christophe; Muyldermans, Serge; Monsion, Baptiste

2018-01-01

Single-domain antibodies libraries of heavy-chain only immunoglobulins from camelids or shark are enriched for high-affinity antigen-specific binders by a short in vivo immunization. Thus, potent binders are readily retrieved from relatively small-sized libraries of 10 7 -10 8 individual transformants, mostly after phage display and panning on a purified target. However, the remaining drawback of this strategy arises from the need to generate a dedicated library, for nearly every envisaged target. Therefore, all the procedures that shorten and facilitate the construction of an immune library of best possible quality are definitely a step forward. In this chapter, we provide the protocol to generate a high-quality immune VHH library using the Golden Gate Cloning strategy employing an adapted phage display vector where a lethal ccdB gene has to be substituted by the VHH gene. With this procedure, the construction of the library can be shortened to less than a week starting from bleeding the animal. Our libraries exceed 10 8 individual transformants and close to 100% of the clones harbor a phage display vector having an insert with the length of a VHH gene. These libraries are also more economic to make than previous standard approaches using classical restriction enzymes and ligations. The quality of the Nanobodies that are retrieved from immune libraries obtained by Golden Gate Cloning is identical to those from immune libraries made according to the classical procedure.

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

PubMed

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening.

PubMed

Mazor, Yariv; Van Blarcom, Thomas; Carroll, Sean; Georgiou, George

2010-05-01

Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

PubMed

Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

2014-08-01

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

Prospective identification of parasitic sequences in phage display screens

PubMed Central

Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

2014-01-01

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

ESTminer: a Web interface for mining EST contig and cluster databases.

PubMed

Huang, Yecheng; Pumphrey, Janie; Gingle, Alan R

2005-03-01

ESTminer is a Web application and database schema for interactive mining of expressed sequence tag (EST) contig and cluster datasets. The Web interface contains a query frame that allows the selection of contigs/clusters with specific cDNA library makeup or a threshold number of members. The results are displayed as color-coded tree nodes, where the color indicates the fractional size of each cDNA library component. The nodes are expandable, revealing library statistics as well as EST or contig members, with links to sequence data, GenBank records or user configurable links. Also, the interface allows 'queries within queries' where the result set of a query is further filtered by the subsequent query. ESTminer is implemented in Java/JSP and the package, including MySQL and Oracle schema creation scripts, is available from http://cggc.agtec.uga.edu/Data/download.asp agingle@uga.edu.

Identification of UQCRB as an oxymatrine recognizing protein using a T7 phage display screen.

PubMed

Sun, Yan-Hui; Zhang, Xiao-Yuan; Xie, Wei-Qun; Liu, Guang-Jian; He, Xi-Xin; Huang, Ya-Li; Zhang, Guang-Xian; Wang, Jian; Kuang, Zao-Yuan; Zhang, Ren

2016-12-04

Sophora flavescens Aiton (Radix Sophorae Flavescentis, Kushen) is used in traditional Chinese medicine to treat chronic hepatitis B (CHB), and has the ability to clear heat and dampness from the body. Oxymatrine is one of the major bioactive compounds extracted from Sophora flavescens Aiton and constitutes more than 90% of the oxymatrine injection commonly used for CHB treatment in clinics in China. We aim to analyze the protein binding target of oxymatrine in treating CHB by screening a T7 phage display cDNA library of human CHB and examine the biochemistry of protein-ligand binding between oxymatrine and its ligands. A T7 phage cDNA library of human CHB was biopanned by affinity selection using oxymatrine as bait. The interaction of oxymatrine with its candidate binding protein was investigated by affinity assay, molecular docking, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR). A library of potential oxymatrine binding peptides was generated. Ubiquinol-cytochrome c reductase binding protein (UQCRB) was one of the candidate binding proteins of oxymatrine. UQCRB-displaying T7 phage binding numbers in the oxymatrine group were significantly higher than that in the control group, biotin group, and matrine group (p<0.05 or p<0.01). Three-dimensional structure modeling of the UQCRB with oxymatrine showed that their binding interfaces matched and oxymatrine inserted into a deeper pocket of UQCRB, which mainly involved amino acid residues Tyr21, Arg33, Tyr83, Glu84, Asp86, Pro88, and Glu91. The binding affinity constant (Kb) from SPR was 4.2mM. The Kb from ITC experiment was 3.9mM and stoichiometry was fixed as 1, which fit very well with the result of SPR. The binding of oxymatrine to UQCRB was driven by strong enthalpy forces such as hydrogen bonds and polar interactions as the heat released was about 157kcal/mol and ΔG was less than zero. In this study, using the T7 phage display system, we have identified UQCRB as a direct binding protein of oxymatrine. Furthermore, the specificity and molecular interaction of oxymatrine with UQCRB were also determined. The binding of UQCRB to oxymatrine suggests that UQCRB is a potential target of oxymatrine in treating CHB. These results provide new understanding into the mechanism of oxymatrine and insights into the strategy on the treatment of CHB. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean

PubMed Central

Wang, Bing; Swaminathan, Sivakumar; Bhattacharyya, Madan K.

2015-01-01

Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS) is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL). A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed. PMID:26709700

CH5M3D: an HTML5 program for creating 3D molecular structures.

PubMed

Earley, Clarke W

2013-11-18

While a number of programs and web-based applications are available for the interactive display of 3-dimensional molecular structures, few of these provide the ability to edit these structures. For this reason, we have developed a library written in JavaScript to allow for the simple creation of web-based applications that should run on any browser capable of rendering HTML5 web pages. While our primary interest in developing this application was for educational use, it may also prove useful to researchers who want a light-weight application for viewing and editing small molecular structures. Molecular compounds are drawn on the HTML5 Canvas element, with the JavaScript code making use of standard techniques to allow display of three-dimensional structures on a two-dimensional canvas. Information about the structure (bond lengths, bond angles, and dihedral angles) can be obtained using a mouse or other pointing device. Both atoms and bonds can be added or deleted, and rotation about bonds is allowed. Routines are provided to read structures either from the web server or from the user's computer, and creation of galleries of structures can be accomplished with only a few lines of code. Documentation and examples are provided to demonstrate how users can access all of the molecular information for creation of web pages with more advanced features. A light-weight (≈ 75 kb) JavaScript library has been made available that allows for the simple creation of web pages containing interactive 3-dimensional molecular structures. Although this library is designed to create web pages, a web server is not required. Installation on a web server is straightforward and does not require any server-side modules or special permissions. The ch5m3d.js library has been released under the GNU GPL version 3 open-source license and is available from http://sourceforge.net/projects/ch5m3d/.

CH5M3D: an HTML5 program for creating 3D molecular structures

PubMed Central

2013-01-01

Background While a number of programs and web-based applications are available for the interactive display of 3-dimensional molecular structures, few of these provide the ability to edit these structures. For this reason, we have developed a library written in JavaScript to allow for the simple creation of web-based applications that should run on any browser capable of rendering HTML5 web pages. While our primary interest in developing this application was for educational use, it may also prove useful to researchers who want a light-weight application for viewing and editing small molecular structures. Results Molecular compounds are drawn on the HTML5 Canvas element, with the JavaScript code making use of standard techniques to allow display of three-dimensional structures on a two-dimensional canvas. Information about the structure (bond lengths, bond angles, and dihedral angles) can be obtained using a mouse or other pointing device. Both atoms and bonds can be added or deleted, and rotation about bonds is allowed. Routines are provided to read structures either from the web server or from the user’s computer, and creation of galleries of structures can be accomplished with only a few lines of code. Documentation and examples are provided to demonstrate how users can access all of the molecular information for creation of web pages with more advanced features. Conclusions A light-weight (≈ 75 kb) JavaScript library has been made available that allows for the simple creation of web pages containing interactive 3-dimensional molecular structures. Although this library is designed to create web pages, a web server is not required. Installation on a web server is straightforward and does not require any server-side modules or special permissions. The ch5m3d.js library has been released under the GNU GPL version 3 open-source license and is available from http://sourceforge.net/projects/ch5m3d/. PMID:24246004

Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.

PubMed

He, Bifang; Tjhung, Katrina F; Bennett, Nicholas J; Chou, Ying; Rau, Andrea; Huang, Jian; Derda, Ratmir

2018-01-19

Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

Rapid discovery of protein interactions by cell-free protein technologies.

PubMed

He, M; Taussig, M J

2007-11-01

Cell-free transcription and translation provides an open, controllable environment for production of correctly folded, soluble proteins and allows the rapid generation of proteins from DNA without the need for cloning. Thus it is becoming an increasingly attractive alternative to conventional in vivo expression systems, especially when parallel expression of multiple proteins is required. Through novel design and exploitation, powerful cell-free technologies of ribosome display and protein in situ arrays have been developed for in vitro production and isolation of protein-binding molecules from large libraries. These technologies can be combined for rapid detection of protein interactions.

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

PubMed

Kügler, Jonas; Wilke, Sonja; Meier, Doris; Tomszak, Florian; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan; Garritsen, Henk; Hock, Björn; Toleikis, Lars; Schütte, Mark; Hust, Michael

2015-02-19

Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

VICAR - VIDEO IMAGE COMMUNICATION AND RETRIEVAL

NASA Technical Reports Server (NTRS)

Wall, R. J.

1994-01-01

VICAR (Video Image Communication and Retrieval) is a general purpose image processing software system that has been under continuous development since the late 1960's. Originally intended for data from the NASA Jet Propulsion Laboratory's unmanned planetary spacecraft, VICAR is now used for a variety of other applications including biomedical image processing, cartography, earth resources, and geological exploration. The development of this newest version of VICAR emphasized a standardized, easily-understood user interface, a shield between the user and the host operating system, and a comprehensive array of image processing capabilities. Structurally, VICAR can be divided into roughly two parts; a suite of applications programs and an executive which serves as the interfaces between the applications, the operating system, and the user. There are several hundred applications programs ranging in function from interactive image editing, data compression/decompression, and map projection, to blemish, noise, and artifact removal, mosaic generation, and pattern recognition and location. An information management system designed specifically for handling image related data can merge image data with other types of data files. The user accesses these programs through the VICAR executive, which consists of a supervisor and a run-time library. From the viewpoint of the user and the applications programs, the executive is an environment that is independent of the operating system. VICAR does not replace the host computer's operating system; instead, it overlays the host resources. The core of the executive is the VICAR Supervisor, which is based on NASA Goddard Space Flight Center's Transportable Applications Executive (TAE). Various modifications and extensions have been made to optimize TAE for image processing applications, resulting in a user friendly environment. The rest of the executive consists of the VICAR Run-Time Library, which provides a set of subroutines (image I/O, label I/O, parameter I/O, etc.) to facilitate image processing and provide the fastest I/O possible while maintaining a wide variety of capabilities. The run-time library also includes the Virtual Raster Display Interface (VRDI) which allows display oriented applications programs to be written for a variety of display devices using a set of common routines. (A display device can be any frame-buffer type device which is attached to the host computer and has memory planes for the display and manipulation of images. A display device may have any number of separate 8-bit image memory planes (IMPs), a graphics overlay plane, pseudo-color capabilities, hardware zoom and pan, and other features). The VRDI supports the following display devices: VICOM (Gould/Deanza) IP8500, RAMTEK RM-9465, ADAGE (Ikonas) IK3000 and the International Imaging Systems IVAS. VRDI's purpose is to provide a uniform operating environment not only for an application programmer, but for the user as well. The programmer is able to write programs without being concerned with the specifics of the device for which the application is intended. The VICAR Interactive Display Subsystem (VIDS) is a collection of utilities for easy interactive display and manipulation of images on a display device. VIDS has characteristics of both the executive and an application program, and offers a wide menu of image manipulation options. VIDS uses the VRDI to communicate with display devices. The first step in using VIDS to analyze and enhance an image (one simple example of VICAR's numerous capabilities) is to examine the histogram of the image. The histogram is a plot of frequency of occurrence for each pixel value (0 - 255) loaded in the image plane. If, for example, the histogram shows that there are no pixel values below 64 or above 192, the histogram can be "stretched" so that the value of 64 is mapped to zero and 192 is mapped to 255. Now the user can use the full dynamic range of the display device to display the data and better see its contents. Another example of a VIDS procedure is the JMOVIE command, which allows the user to run animations interactively on the display device. JMOVIE uses the concept of "frames", which are the individual frames which comprise the animation to be viewed. The user loads images into the frames after the size and number of frames has been selected. VICAR's source languages are primarily FORTRAN and C, with some VAX Assembler and array processor code. The VICAR run-time library is designed to work equally easily from either FORTRAN or C. The program was implemented on a DEC VAX series computer operating under VMS 4.7. The virtual memory required is 1.5MB. Approximately 180,000 blocks of storage are needed for the saveset. VICAR (version 2.3A/3G/13H) is a copyrighted work with all copyright vested in NASA and is available by license for a period of ten (10) years to approved licensees. This program was developed in 1989.

Selection of stable scFv antibodies by phage display.

PubMed

Brockmann, Eeva-Christine

2012-01-01

ScFv fragments are popular recombinant antibody formats but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and applicable also for stability selection. ScFv variants with improved stability can be selected from large randomly mutated phage displayed libraries with a specific antigen after the unstable variants have been inactivated by heat or GdmCl. Irreversible scFv denaturation, which is a prerequisite for efficient selection, is achieved by combining denaturation with reduction of the intradomain disulfide bonds. Repeated selection cycles of increasing stringency result in enrichment of stabilized scFv fragments. Procedures for constructing a randomly mutated scFv library by error-prone PCR and phage display selection for enrichment of stable scFv antibodies from the library are described here.

Marrow-Derived Antibody Library for Treatment of Neuroblastoma

DTIC Science & Technology

2013-09-01

to capture the auto-immune response reaction in neuroblastoma patients using phage display and B cell hybridoma technologies. The scope of this...project is to use NB patient-derived materials to create NB cell lines, xenograft models, NB specific phage display libraries and to identify and...the auto-immune response reaction in neuroblastoma patients using phage display and B cell hybridoma technologies. The scope of this project is to

Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries.

PubMed

Nguyen, Kieu T H; Adamkiewicz, Marta A; Hebert, Lauren E; Zygiel, Emily M; Boyle, Holly R; Martone, Christina M; Meléndez-Ríos, Carola B; Noren, Karen A; Noren, Christopher J; Hall, Marilena Fitzsimons

2014-10-01

A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Tampa Bay Study Data and Information Management System (DIMS)

NASA Astrophysics Data System (ADS)

Edgar, N. T.; Johnston, J. B.; Yates, K.; Smith, K. E.

2005-05-01

Providing easy access to data and information is an essential component of both science and management. The Tampa Bay Data and Information Management System (DIMS) catalogs and publicizes data and products which are generated through the Tampa Bay Integrated Science Study. The publicly accessible interface consists of a Web site (http://gulfsci.usgs.gov), a digital library, and an interactive map server (IMS). The Tampa Bay Study Web site contains information from scientists involved in the study, and is also the portal site for the digital library and IMS. Study information is highlighted on the Web site according to the estuarine component: geology and geomorphology, water and sediment quality, ecosystem structure and function, and hydrodynamics. The Tampa Bay Digital Library is a web-based clearinghouse for digital products on Tampa Bay, including documents, maps, spatial and tabular data sets, presentations, etc. New developments to the digital library include new search features, 150 new products over the past year, and partnerships to expand the offering of science products. The IMS is a Web-based geographic information system (GIS) used to store, analyze and display data pertaining to Tampa Bay. Upgrades to the IMS have improved performance and speed, as well as increased the number of data sets available for mapping. The Tampa Bay DIMS is a dynamic entity and will continue to evolve with the study. Beginning in 2005, the Tampa Bay Integrated Coastal Model will have a more prominent presence within the DIMS. The Web site will feature model projects and plans; the digital library will host model products and data sets; the IMS will display spatial model data sets and analyses. These tools will be used to increase communication of USGS efforts in Tampa Bay to the public, local managers, and scientists.

A highly functional synthetic phage display library containing over 40 billion human antibody clones.

PubMed

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody Clones

PubMed Central

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics. PMID:24950200

PMD2HD--a web tool aligning a PubMed search results page with the local German Cancer Research Centre library collection.

PubMed

Bohne-Lang, Andreas; Lang, Elke; Taube, Anke

2005-06-27

Web-based searching is the accepted contemporary mode of retrieving relevant literature, and retrieving as many full text articles as possible is a typical prerequisite for research success. In most cases only a proportion of references will be directly accessible as digital reprints through displayed links. A large number of references, however, have to be verified in library catalogues and, depending on their availability, are accessible as print holdings or by interlibrary loan request. The problem of verifying local print holdings from an initial retrieval set of citations can be solved using Z39.50, an ANSI protocol for interactively querying library information systems. Numerous systems include Z39.50 interfaces and therefore can process Z39.50 interactive requests. However, the programmed query interaction command structure is non-intuitive and inaccessible to the average biomedical researcher. For the typical user, it is necessary to implement the protocol within a tool that hides and handles Z39.50 syntax, presenting a comfortable user interface. PMD2HD is a web tool implementing Z39.50 to provide an appropriately functional and usable interface to integrate into the typical workflow that follows an initial PubMed literature search, providing users with an immediate asset to assist in the most tedious step in literature retrieval, checking for subscription holdings against a local online catalogue. PMD2HD can facilitate literature access considerably with respect to the time and cost of manual comparisons of search results with local catalogue holdings. The example presented in this article is related to the library system and collections of the German Cancer Research Centre. However, the PMD2HD software architecture and use of common Z39.50 protocol commands allow for transfer to a broad range of scientific libraries using Z39.50-compatible library information systems.

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library.

PubMed

Liu, Jinny L; Anderson, George P; Goldman, Ellen R

2007-11-19

Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

PubMed Central

Liu, Jinny L; Anderson, George P; Goldman, Ellen R

2007-01-01

Background Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins. PMID:18021450

DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

PubMed

Franzini, Raphael M; Neri, Dario; Scheuermann, Jörg

2014-04-15

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

Development of a bacteriophage displayed peptide library and biosensor

NASA Astrophysics Data System (ADS)

Chin, Robert C.; Salazar, Noe; Mayo, Michael W.; Villavicencio, Victor I.; Taylor, Richard B.; Chambers, James P.; Valdes, James J.

1996-04-01

A miniaturized, handheld biosensor for identification of hazardous biowarfare agents with high specificity is being developed. An innovative biological recognition system based on bacteriophage displayed peptide receptors will be utilized in conjunction with the miniature biosensor technology being developed. A bacteriophage library has been constructed to provide the artificial receptors. The library can contain millions of bacteriophage with randomly displayed peptide sequences in the phage outer protein coat which act as binding sites for the agents of interest. This library will be used to 'bio-pan' for phages that bind to a number of toxins and infectious agents and can, thus, provide an endless supply of low cost, reliable, specific, and stable artificial receptors. The biosensor instrument will utilize evanescent wave, planar waveguide, far-red dyes, diode laser and miniature circuit technologies for performance and portability.

Advanced data acquisition and display techniques for laser velocimetry

NASA Technical Reports Server (NTRS)

Kjelgaard, Scott O.; Weston, Robert P.

1991-01-01

The Basic Aerodynamics Research Tunnel (BART) has been equipped with state-of-the-art instrumentation for acquiring the data needed for code validation. This paper describes the three-component LDV and the workstation-based data-acquisition system (DAS) which has been developed for the BART. The DAS allows the use of automation and the quick integration of advanced instrumentation, while minimizing the software development time required between investigations. The paper also includes a description of a graphics software library developed to support the windowing environment of the DAS. The real-time displays generated using the graphics library help the researcher ensure the test is proceeding properly. The graphics library also supports the requirements of posttest data analysis. The use of the DAS and graphics libraries is illustrated by presenting examples of the real-time and postprocessing display graphics for LDV investigations.

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

PubMed

Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

Bacteriophage vehicles for phage display: biology, mechanism, and application.

PubMed

Ebrahimizadeh, Walead; Rajabibazl, Masoumeh

2014-08-01

The phage display technique is a powerful tool for selection of various biological agents. This technique allows construction of large libraries from the antibody repertoire of different hosts and provides a fast and high-throughput selection method. Specific antibodies can be isolated based on distinctive characteristics from a library consisting of millions of members. These features made phage display technology preferred method for antibody selection and engineering. There are several phage display methods available and each has its unique merits and application. Selection of appropriate display technique requires basic knowledge of available methods and their mechanism. In this review, we describe different phage display techniques, available bacteriophage vehicles, and their mechanism.

Evolution of phage display technology: from discovery to application.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Ahmadzadeh, Vahideh; Akbari, Bahman

2017-03-01

Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody-Ligand Interaction Using Chain Shuffling.

PubMed

Rangnoi, Kuntalee; Choowongkomon, Kiattawee; O'Kennedy, Richard; Rüker, Florian; Yamabhai, Montarop

2018-06-06

A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.

Merchandising Your Library.

ERIC Educational Resources Information Center

Sivulich, Kenneth G.

1989-01-01

Discusses library circulation figures as a reflection of the success of library services and describes merchandising techniques that have produced a 137 percent circulation increase at Queens Borough Public Library over the past seven years. Merchandising techniques such as minibranches, displays, signage, dumps, and modified shelving are…

Strategies for the construction and use of peptide and antibody libraries displayed on phages.

PubMed

Pini, Alessandro; Giuliani, Andrea; Ricci, Claudia; Runci, Ylenia; Bracci, Luisa

2004-12-01

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

Construction of Rabbit Immune Antibody Libraries.

PubMed

Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

2018-01-01

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Next-Generation Sequencing of Antibody Display Repertoires

PubMed Central

Rouet, Romain; Jackson, Katherine J. L.; Langley, David B.; Christ, Daniel

2018-01-01

In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation. PMID:29472918

Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

PubMed

Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

2018-01-01

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures.

PubMed

Thomas, William D; Golomb, Miriam; Smith, George P

2010-12-15

Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. Copyright © 2010 Elsevier Inc. All rights reserved.

Corruption of phage-display libraries by target-unrelated clones: Diagnosis and countermeasures

PubMed Central

Thomas, William D.; Golomb, Miriam; Smith, George P.

2010-01-01

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior, and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phage (TUPs), that lack the target behavior. Many TUPs are propagation-related; they have mutations conferring a growth advantage, and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. PMID:20692225

Web-based metabolic network visualization with a zooming user interface

PubMed Central

2011-01-01

Background Displaying complex metabolic-map diagrams, for Web browsers, and allowing users to interact with them for querying and overlaying expression data over them is challenging. Description We present a Web-based metabolic-map diagram, which can be interactively explored by the user, called the Cellular Overview. The main characteristic of this application is the zooming user interface enabling the user to focus on appropriate granularities of the network at will. Various searching commands are available to visually highlight sets of reactions, pathways, enzymes, metabolites, and so on. Expression data from single or multiple experiments can be overlaid on the diagram, which we call the Omics Viewer capability. The application provides Web services to highlight the diagram and to invoke the Omics Viewer. This application is entirely written in JavaScript for the client browsers and connect to a Pathway Tools Web server to retrieve data and diagrams. It uses the OpenLayers library to display tiled diagrams. Conclusions This new online tool is capable of displaying large and complex metabolic-map diagrams in a very interactive manner. This application is available as part of the Pathway Tools software that powers multiple metabolic databases including Biocyc.org: The Cellular Overview is accessible under the Tools menu. PMID:21595965

LabVIEW Task Manager v. 1.10.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vargo, Timothy D.

LabVIEW Task Manager is a debugging tool for use during code development in the National Instruments (NI) LabVIEW® IDE. While providing a dynamic & big-picture view of running code, an expandable/collapsible tree diagram displays detailed information (both static and dynamic) on all VIs in memory, belonging to a selected project/target. It allows for interacting with single or multiple selected VIs at a time, providing significant benefits while troubleshooting, and has the following features: Look & Feel similar to Windows® Task Manager; Selection of project/target; Lists all VIs in memory, grouped by class/library; Searches for and enumerates clones in memory; DropInmore » VI for including dynamically referenced clones (Clone Beacon); 'Refresh Now' (F5) re-reads all VIs in memory and adds new ones to the tree; Displays VI name, owning class/library, state, path, data size & code size; Displays VI FP Behavior, Reentrant?, Reentrancy Type, Paused? & Highlight?; Sort by any column, including by library name; Filter by item types vi, ctl, and vit/ctt; Filter out vi.lib and global VIs; Tracking of, and ability to toggle, execution highlighting on multiple selected VIs; Tracking of paused VIs with ability to Pause/Resume/TogglePause multiple selected VIs; DropIn VI for pausing on a condition; If a clone initiates a pause, a different pause symbol is used for all clones of that same reentrant original VI; Select multiple VIs and open or close their FPs or BDs; Double Click a VI from the tree to bring the BD (first choice) or FP to front, if already open; and Select multiple top-level VIs and Abort them.« less

Development and validation of novel AAV2 random libraries displaying peptides of diverse lengths and at diverse capsid positions.

PubMed

Naumer, Matthias; Ying, Ying; Michelfelder, Stefan; Reuter, Antje; Trepel, Martin; Müller, Oliver J; Kleinschmidt, Jürgen A

2012-05-01

Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

Specific ligands for classical swine fever virus screened from landscape phage display library.

PubMed

Yin, Long; Luo, Yuzi; Liang, Bo; Wang, Fei; Du, Min; Petrenko, Valery A; Qiu, Hua-Ji; Liu, Aihua

2014-09-01

Classical swine fever (CSF) is a devastating infectious disease caused by classical swine fever virus (CSFV). The screening of CSFV-specific ligands is of great significance for diagnosis and treatment of CSF. Affinity selection from random peptide libraries is an efficient approach to discover ligands with high stability and specificity. Here, we screened phage ligands for the CSFV E2 protein from f8/8 landscape phage display library by biopanning and obtained four phage clones specific for the E2 protein of CSFV. Viral blocking assays indicated that the phage clone displaying the octapeptide sequence DRATSSNA remarkably inhibited the CSFV replication in PK-15 cells at a titer of 10(10) transduction units, as evidenced by significantly decreased viral RNA copies and viral titers. The phage-displayed E2-binding peptides have the potential to be developed as antivirals for CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

Browse without a Browser at the ATRF Library | Poster

Cancer.gov

By Robin Meckley, Contributing Writer Employees at the Advanced Technology Research Facility (ATRF) asked the Scientific Library, and the library responded: print journals are now available in the ATRF Library. Employees can now browse 20 print journals, which will rotate, with one issue available at a time for each title. The library will also temporarily display some new

Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

PubMed

Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

2015-01-01

We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

User Guide for the Plotting Software for the Los Alamos National Laboratory Nuclear Weapons Analysis Tools Version 2.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cleland, Timothy James

The Los Alamos National Laboratory Plotting Software for the Nuclear Weapons Analysis Tools is a Java™ application based upon the open source library JFreeChart. The software provides a capability for plotting data on graphs with a rich variety of display options while allowing the viewer interaction via graph manipulation and scaling to best view the data. The graph types include XY plots, Date XY plots, Bar plots and Histogram plots.

A laboratory investigation of the variability of cloud reflected radiance fields

NASA Technical Reports Server (NTRS)

Mckee, T. B.; Cox, S. K.

1986-01-01

A method to determine the radiative properties of complex cloud fields was developed. A Cloud field optical simulator (CFOS) was constructed to simulate the interaction of cloud fields with visible radiation. The CFOS was verified by comparing experimental results from it with calculations performed with a Monte Carlo radiative transfer model. A software library was developed to process, reduce, and display CFOS data. The CFSOS was utilized to study the reflected radiane patterns from simulated cloud fields.

Mechanism of Tumor Metastasis Suppression by the KAI1 Gene

DTIC Science & Technology

2008-02-01

CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b . ABSTRACT U c. THIS...activated by the interaction of DARC and Kai1. Task1- b . (completed) Examine the effects of siRNA against the DARC gene on the binding and...suggesting that the N terminus of DARC is essential for binding to KAI1. Task 2- b . Screening a phage display library followed by sequencing the

37 CFR 201.14 - Warnings of copyright for use by certain libraries and archives.

Code of Federal Regulations, 2012 CFR

2012-07-01

... by certain libraries and archives. 201.14 Section 201.14 Patents, Trademarks, and Copyrights COPYRIGHT OFFICE, LIBRARY OF CONGRESS COPYRIGHT OFFICE AND PROCEDURES GENERAL PROVISIONS § 201.14 Warnings of copyright for use by certain libraries and archives. (a) Definitions. (1) A Display Warning of...

37 CFR 201.14 - Warnings of copyright for use by certain libraries and archives.

Code of Federal Regulations, 2013 CFR

2013-07-01

... by certain libraries and archives. 201.14 Section 201.14 Patents, Trademarks, and Copyrights COPYRIGHT OFFICE, LIBRARY OF CONGRESS COPYRIGHT OFFICE AND PROCEDURES GENERAL PROVISIONS § 201.14 Warnings of copyright for use by certain libraries and archives. (a) Definitions. (1) A Display Warning of...

37 CFR 201.14 - Warnings of copyright for use by certain libraries and archives.

Code of Federal Regulations, 2010 CFR

2010-07-01

... by certain libraries and archives. 201.14 Section 201.14 Patents, Trademarks, and Copyrights COPYRIGHT OFFICE, LIBRARY OF CONGRESS COPYRIGHT OFFICE AND PROCEDURES GENERAL PROVISIONS § 201.14 Warnings of copyright for use by certain libraries and archives. (a) Definitions. (1) A Display Warning of...

37 CFR 201.14 - Warnings of copyright for use by certain libraries and archives.

Code of Federal Regulations, 2011 CFR

2011-07-01

... by certain libraries and archives. 201.14 Section 201.14 Patents, Trademarks, and Copyrights COPYRIGHT OFFICE, LIBRARY OF CONGRESS COPYRIGHT OFFICE AND PROCEDURES GENERAL PROVISIONS § 201.14 Warnings of copyright for use by certain libraries and archives. (a) Definitions. (1) A Display Warning of...

Ten years of change: National Library of Medicine TOXMAP gets a new look.

PubMed

Hochstein, Colette; Gemoets, Darren; Goshorn, Jeanne

2014-01-01

The United States National Library of Medicine (NLM) TOXNET® databases < http://toxnet.nlm.nih.gov > provide broad coverage of environmental health information covering a wide variety of topics, including access to the U.S. Environment Protection Agency (EPA)'s Toxics Release Inventory (TRI) data. The NLM web-based geographic information system (GIS), TOXMAP® < http://toxmap.nlm.nih.gov/ >, provides interactive maps which show where TRI chemicals are released into the environment and links to TOXNET for information about these chemicals. TOXMAP also displays locations of Superfund sites on the EPA National Priority List, as well as information about the chemical contaminants at these sites. This column focuses on a new version of TOXMAP which brings it up to date with current web GIS technologies and user expectations.

Employing WebGL to develop interactive stereoscopic 3D content for use in biomedical visualization

NASA Astrophysics Data System (ADS)

Johnston, Semay; Renambot, Luc; Sauter, Daniel

2013-03-01

Web Graphics Library (WebGL), the forthcoming web standard for rendering native 3D graphics in a browser, represents an important addition to the biomedical visualization toolset. It is projected to become a mainstream method of delivering 3D online content due to shrinking support for third-party plug-ins. Additionally, it provides a virtual reality (VR) experience to web users accommodated by the growing availability of stereoscopic displays (3D TV, desktop, and mobile). WebGL's value in biomedical visualization has been demonstrated by applications for interactive anatomical models, chemical and molecular visualization, and web-based volume rendering. However, a lack of instructional literature specific to the field prevents many from utilizing this technology. This project defines a WebGL design methodology for a target audience of biomedical artists with a basic understanding of web languages and 3D graphics. The methodology was informed by the development of an interactive web application depicting the anatomy and various pathologies of the human eye. The application supports several modes of stereoscopic displays for a better understanding of 3D anatomical structures.

Phage display discovery of novel molecular targets in glioblastoma-initiating cells.

PubMed

Liu, J K; Lubelski, D; Schonberg, D L; Wu, Q; Hale, J S; Flavahan, W A; Mulkearns-Hubert, E E; Man, J; Hjelmeland, A B; Yu, J; Lathia, J D; Rich, J N

2014-08-01

Glioblastoma is the most common primary intrinsic brain tumor and remains incurable despite maximal therapy. Glioblastomas display cellular hierarchies with self-renewing glioma-initiating cells (GICs) at the apex. To discover new GIC targets, we used in vivo delivery of phage display technology to screen for molecules selectively binding GICs that may be amenable for targeting. Phage display leverages large, diverse peptide libraries to identify interactions with molecules in their native conformation. We delivered a bacteriophage peptide library intravenously to a glioblastoma xenograft in vivo then derived GICs. Phage peptides bound to GICs were analyzed for their corresponding proteins and ranked based on prognostic value, identifying VAV3, a Rho guanine exchange factor involved tumor invasion, and CD97 (cluster of differentiation marker 97), an adhesion G-protein-coupled-receptor upstream of Rho, as potentially enriched in GICs. We confirmed that both VAV3 and CD97 were preferentially expressed by tumor cells expressing GIC markers. VAV3 expression correlated with increased activity of its downstream mediator, Rac1 (ras-related C3 botulinum toxin substrate 1), in GICs. Furthermore, targeting VAV3 by ribonucleic acid interference decreased GIC growth, migration, invasion and in vivo tumorigenesis. As CD97 is a cell surface protein, CD97 selection enriched for sphere formation, a surrogate of self-renewal. In silico analysis demonstrated VAV3 and CD97 are highly expressed in tumors and inform poor survival and tumor grade, and more common with epidermal growth factor receptor mutations. Finally, a VAV3 peptide sequence identified on phage display specifically internalized into GICs. These results show a novel screening method for identifying oncogenic pathways preferentially activated within the tumor hierarchy, offering a new strategy for developing glioblastoma therapies.

Phage display discovery of novel molecular targets in glioblastoma-initiating cells

PubMed Central

Liu, J K; Lubelski, D; Schonberg, D L; Wu, Q; Hale, J S; Flavahan, W A; Mulkearns-Hubert, E E; Man, J; Hjelmeland, A B; Yu, J; Lathia, J D; Rich, J N

2014-01-01

Glioblastoma is the most common primary intrinsic brain tumor and remains incurable despite maximal therapy. Glioblastomas display cellular hierarchies with self-renewing glioma-initiating cells (GICs) at the apex. To discover new GIC targets, we used in vivo delivery of phage display technology to screen for molecules selectively binding GICs that may be amenable for targeting. Phage display leverages large, diverse peptide libraries to identify interactions with molecules in their native conformation. We delivered a bacteriophage peptide library intravenously to a glioblastoma xenograft in vivo then derived GICs. Phage peptides bound to GICs were analyzed for their corresponding proteins and ranked based on prognostic value, identifying VAV3, a Rho guanine exchange factor involved tumor invasion, and CD97 (cluster of differentiation marker 97), an adhesion G-protein-coupled-receptor upstream of Rho, as potentially enriched in GICs. We confirmed that both VAV3 and CD97 were preferentially expressed by tumor cells expressing GIC markers. VAV3 expression correlated with increased activity of its downstream mediator, Rac1 (ras-related C3 botulinum toxin substrate 1), in GICs. Furthermore, targeting VAV3 by ribonucleic acid interference decreased GIC growth, migration, invasion and in vivo tumorigenesis. As CD97 is a cell surface protein, CD97 selection enriched for sphere formation, a surrogate of self-renewal. In silico analysis demonstrated VAV3 and CD97 are highly expressed in tumors and inform poor survival and tumor grade, and more common with epidermal growth factor receptor mutations. Finally, a VAV3 peptide sequence identified on phage display specifically internalized into GICs. These results show a novel screening method for identifying oncogenic pathways preferentially activated within the tumor hierarchy, offering a new strategy for developing glioblastoma therapies. PMID:24832468

Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

PubMed

Henry, Kevin A

2018-01-01

Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

Performance evaluation of phage-displayed synthetic human single-domain antibody libraries: A retrospective analysis.

PubMed

Henry, Kevin A; Tanha, Jamshid

2018-05-01

Fully human synthetic single-domain antibodies (sdAbs) are desirable therapeutic molecules but their development is a considerable challenge. Here, using a retrospective analysis of in-house historical data, we examined the parameters that impact the outcome of screening phage-displayed synthetic human sdAb libraries to discover antigen-specific binders. We found no evidence for a differential effect of domain type (V H or V L ), library randomization strategy, incorporation of a stabilizing disulfide linkage or sdAb display format (monovalent vs. multivalent) on the probability of obtaining any antigen-binding human sdAbs, instead finding that the success of library screens was primarily related to properties of target antigens, especially molecular mass. The solubility and binding affinity of sdAbs isolated from successful screens depended both on properties of the sdAb libraries (primarily domain type) and the target antigens. Taking attrition of sdAbs with major manufacturability concerns (aggregation; low expression) and sdAbs that do not recognize native cell-surface antigens as independent probabilities, we calculate the overall likelihood of obtaining ≥1 antigen-binding human sdAb from a single library-target screen as ~24%. Successful library-target screens should be expected to yield ~1.3 human sdAbs on average, each with average binding affinity of ~2 μM. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

PubMed Central

Yoshida, Kimiko; Goto, Naoko; Ohnami, Shumpei; Aoki, Kazunori

2012-01-01

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:23029088

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

PubMed Central

Sotelo, Pablo H.; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

2012-01-01

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles. PMID:22692130

The Effect of a Poster, Display, and Recommended Listening List on the Circulation of Audiobooks in the Public Library.

ERIC Educational Resources Information Center

Kucalaba, Linda

Previous studies have found that the librarian's use of book displays and recommended lists are an effective means to increase circulation in the public library. Yet conflicting results were found when these merchandising techniques were used with collection materials in the nonprint format, specifically audiobooks and videos, instead of books.…

Incorporating Library Instruction in a General Education Program for College Freshmen.

ERIC Educational Resources Information Center

Fenske, Rachel F.; Clark, Susan E.

1995-01-01

Discusses steps taken in planning, implementing, evaluating, and revising library instruction in a general education course for freshmen at the University of the Pacific, and examines results of student library skill tests taken before and after having library instruction. Figures offer example test questions and tables display test averages and…

37 CFR 201.14 - Warnings of copyright for use by certain libraries and archives.

Code of Federal Regulations, 2014 CFR

2014-07-01

... by certain libraries and archives. 201.14 Section 201.14 Patents, Trademarks, and Copyrights U.S. COPYRIGHT OFFICE, LIBRARY OF CONGRESS COPYRIGHT OFFICE AND PROCEDURES GENERAL PROVISIONS § 201.14 Warnings of copyright for use by certain libraries and archives. (a) Definitions. (1) A Display Warning of...

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

PubMed

Tikunova, N V; Morozova, V V

2009-10-01

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

SalHUD--A Graphical Interface to Public Health Data in Puerto Rico.

PubMed

Ortiz-Zuazaga, Humberto G; Arce-Corretjer, Roberto; Solá-Sloan, Juan M; Conde, José G

2015-12-22

This paper describes SalHUD, a prototype web-based application for visualizing health data from Puerto Rico. Our initial focus was to provide interactive maps displaying years of potential life lost (YPLL). The public-use mortality file for year 2008 was downloaded from the Puerto Rico Institute of Statistics website. Data was processed with R, Python and EpiInfo to calculate years of potential life lost for the leading causes of death on each of the 78 municipalities in the island. Death records were classified according to ICD-10 codes. YPLL for each municipality was integrated into AtlasPR, a D3 Javascript map library. Additional Javascript, HTML and CSS programing was required to display maps as a web-based interface. YPLL for all municipalities are displayed on a map of Puerto Rico for each of the ten leading causes of death and for all causes combined, so users may dynamically explore the impact of premature mortality. This work is the first step in providing the general public in Puerto Rico with user-friendly, interactive, visual access to public health data that is usually published in numerical, text-based media.

Identification and Characterization of Strychnine-Binding Peptides Using Phage-Display Screening.

PubMed

Zhang, Fang; Wang, Min; Qiu, Zheng; Wang, Xiao-Meng; Xu, Chun-Lei; Zhang, Xia

2017-01-01

In drug development, phage display is a high-throughput method for identifying the specific cellular targets of drugs. However, insoluble small chemicals remain intractable to this technique because of the difficulty of presenting molecules to phages without occupying or destroying the limited functional groups. In the present study, we selected Strychnine (Stry) as a model compounda and sought to develope an alternative in vitro biopanning strategy against insoluble suspension. A phage library displaying random sequences of fifteen peptides was employed to screen for interactions between Stry and its cellular selective binding peptides, which are of great value to have a complete understanding of the mechanism of Stry for its antitumor activity. After four rounds of biopanning, a selection of 100 binding clones was randomly picked and subjected to modified proliferation and diffusion assays to evaluate the binding affinity of the clones. Finally, eleven clones were identified as positive binders. The corresponding peptides were synthesized and detected for their binding activities using surface plasmon resonance imaging (SPRi). Our study provides a feasible scheme for confirming the interaction of chemical compounds and cellular binding peptides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Isolation of phage-display library-derived scFv antibody specific to Listeria monocytogenes by a novel immobilized method.

PubMed

Nguyen, X-H; Trinh, T-L; Vu, T-B-H; Le, Q-H; To, K-A

2018-02-01

To select Listeria monocytogenes-specific single-chain fragment variable (scFv) antibodies from a phage-display library by a novel simple and cost-effective immobilization method. Light expanded clay aggregate (LECA) was used as biomass support matrix for biopanning of a phage-display library to select L. monocytogenes-specific scFv antibody. Four rounds of positive selection against LECA-immobilized L. monocytogenes and an additional subtractive panning against Listeria innocua were performed. The phage clones selected using this panning scheme and LECA-based immobilization method exhibited the ability to bind L. monocytogenes without cross-reactivity toward 10 other non-L. monocytogenes bacteria. One of the selected phage clones was able to specifically recognize three major pathogenic serotypes (1/2a, 1/2b and 4b) of L. monocytogenes and 11 tested L. monocytogenes strains isolated from foods. The LECA-based immobilization method is applicable for isolating species-specific anti-L. monocytogenes scFv antibodies by phage display. The isolated scFv antibody has potential use in development of immunoassay-based methods for rapid detection of L. monocytogenes in food and environmental samples. In addition, the LECA immobilization method described here could feasibly be employed to isolate specific monoclonal antibodies against any given species of pathogenic bacteria from phage-display libraries. © 2017 The Society for Applied Microbiology.

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

PubMed

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

2014-03-28

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

A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis

NASA Astrophysics Data System (ADS)

Ghosh, Eshan; Srivastava, Ashish; Baidya, Mithu; Kumari, Punita; Dwivedi, Hemlata; Nidhi, Kumari; Ranjan, Ravi; Dogra, Shalini; Koide, Akiko; Yadav, Prem N.; Sidhu, Sachdev S.; Koide, Shohei; Shukla, Arun K.

2017-12-01

Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.

Fas-associated Protein with Death Domain (FADD)-independent Recruitment of c-FLIPL to Death Receptor 5*

PubMed Central

Jin, Tai-Guang; Kurakin, Alexei; Benhaga, Nordine; Abe, Karon; Mohseni, Mehrdad; Sandra, Ferry; Song, Keli; Kay, Brian K.; Khosravi-Far, Roya

2010-01-01

Here we show a novel mechanism by which FLICE-like inhibitory protein (c-FLIP) regulates apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and one of its receptors, DR5. c-FLIP is a critical regulator of the TNF family of cytokine receptor signaling. c-FLIP has been postulated to prevent formation of the competent death-inducing signaling complex (DISC) in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. In order to identify regulators of TRAIL function, we used the intracellular death domain (DD) of DR5 as a target to screen a phage-displayed combinatorial peptide library. The DD of DR5 selected from the library a peptide that showed sequence similarity to a stretch of amino acids in the C terminus of c-FLIPL. The phage-displayed peptide selectively interacted with the DD of DR5 in in vitro binding assays. Similarly, full-length c-FLIP (c-FLIPL) and the C-terminal p12 domain of c-FLIP interacted with DR5 both in in vitro pull-down assays and in mammalian cells. This interaction was independent of TRAIL. To the contrary, TRAIL treatment released c-FLIPL from DR5, permitting the recruitment of FADD to the active DR5 signaling complex. By employing FADD-deficient Jurkat cells, we demonstrate that DR5 and c-FLIPL interact in a FADD-independent manner. Moreover, we show that a cellular membrane permeable version of the peptide corresponding to the DR5 binding domain of c-FLIP induces apoptosis in mammalian cells. Taken together, these findings indicate that c-FLIPL interacts with the DD of DR5, thus preventing death signaling by DR5 prior to the formation of an active DISC. Because TRAIL and DR5 are ubiquitously expressed, the interaction of c-FLIPL and DR5 indicates a mechanism by which tumor selective apoptosis can be achieved through protecting normal cells from undergoing death receptor-induced apoptosis. PMID:15485835

Platform for intraoperative analysis of video streams

NASA Astrophysics Data System (ADS)

Clements, Logan; Galloway, Robert L., Jr.

2004-05-01

Interactive, image-guided surgery (IIGS) has proven to increase the specificity of a variety of surgical procedures. However, current IIGS systems do not compensate for changes that occur intraoperatively and are not reflected in preoperative tomograms. Endoscopes and intraoperative ultrasound, used in minimally invasive surgery, provide real-time (RT) information in a surgical setting. Combining the information from RT imaging modalities with traditional IIGS techniques will further increase surgical specificity by providing enhanced anatomical information. In order to merge these techniques and obtain quantitative data from RT imaging modalities, a platform was developed to allow both the display and processing of video streams in RT. Using a Bandit-II CV frame grabber board (Coreco Imaging, St. Laurent, Quebec) and the associated library API, a dynamic link library was created in Microsoft Visual C++ 6.0 such that the platform could be incorporated into the IIGS system developed at Vanderbilt University. Performance characterization, using two relatively inexpensive host computers, has shown the platform capable of performing simple image processing operations on frames captured from a CCD camera and displaying the processed video data at near RT rates both independent of and while running the IIGS system.

Discovering Peptide Inhibitors of Human Squalene Synthase Through Screening the Phage-Displayed Cyclic Peptide c7c Library.

PubMed

Shiuan, David; Chen, Yue-Hao; Lin, Hwan-Kang; Huang, Kao-Jean; Tai, Da-Fu; Chang, Ding-Kwo

2016-06-01

Many drugs for the treatment of hypercholesterolemia are targeting the enzymes involved in human cholesterol biosynthesis pathway. Squalene synthase, the rate-limiting enzyme located at the downstream of cholesterol synthesis pathway, has become a better candidate to develop next-generation hypocholesterolemia drugs. In the present study, we cloned and expressed the recombinant human squalene synthase (hSQS) as the lure to isolate potential peptide inhibitors from screening the conformation-constrained phage-displayed cyclic peptide c7c library. Their binding capabilities were further estimated by ELISA. Their pharmaceutical potentials were then analyzed through molecular modeling and the ADMET property evaluations. Four ennea-peptides and nine tetra-peptides were finally synthesized to evaluate their inhibitory potentials toward hSQS. The results indicate that the ennea-peptide CLSPHSMFC, tetra-peptides SMFC, CKTE, and WHQW can effectively inhibit hSQS activities (IC50 values equal to 64, 76, 87, and 90 μM, respectively). These peptides may have potentials to develop future cholesterol-lowering therapeutics. The ligand-protein interaction analysis also reveals that the inner hydrophobic pocket could be a more critical site of hSQS.

Phage-display screening identifies LMP1-binding peptides targeting the C-terminus region of the EBV oncoprotein.

PubMed

Ammous-Boukhris, Nihel; Mosbah, Amor; Sahli, Emna; Ayadi, Wajdi; Hadhri-Guiga, Boutheina; Chérif, Ameur; Gargouri, Ali; Mokdad-Gargouri, Raja

2016-11-01

Latent membrane protein 1 (LMP1), a major oncoprotein of Epstein Barr Virus (EBV) is responsible for transforming B lymphocytes in vitro. LMP1 is overexpressed in several EBV-associated malignancies, and different approaches have been developed to reduce its level and accordingly its oncogenic function in tumor tissues. This study aimed to use phage display peptide library to obtain peptides which could specifically bind to the cytoplasmic region of LMP1 to prevent its interaction with signaling proteins. The LMP1 C-terminus region was produced in bacterial E. coli and used as target for the phage library panning. After 3 rounds, 20 phage clones were randomly selected and 8 showed high binding affinity to the recombinant C-terminus LMP1 protein. The most interesting candidates are the FO5 "QPTKDSSPPLRV" and NO4 "STTSPPAVPHNN" peptides since both bind the C-terminus LMP1 as showed by molecular docking. Furthermore, sequence alignment revealed that the FO5 peptide shared sequence similarity with the Death Receptor 4 which belongs to the tumor necrosis factor-related apoptosis-inducing receptor which plays key role in anti-tumor immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

Selection and maturation of antibodies by phage display through fusion to pIX.

PubMed

Tornetta, Mark; Reddy, Ramachandra; Wheeler, John C

2012-09-01

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM. Copyright © 2012 Elsevier Inc. All rights reserved.

The ELF in Your Library.

ERIC Educational Resources Information Center

McKimmie, Tim; Smith, Jeanette

1994-01-01

Presents an overview of the issues related to extremely low frequency (ELF) radiation from computer video display terminals. Highlights include electromagnetic fields; measuring ELF; computer use in libraries; possible health effects; electromagnetic radiation; litigation and legislation; standards and safety; and what libraries can do. (Contains…

An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

USDA-ARS?s Scientific Manuscript database

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

Use of Phage Display to Identify Novel Mineralocorticoid Receptor-Interacting Proteins

PubMed Central

Yang, Jun; Fuller, Peter J.; Morgan, James; Shibata, Hirotaka; McDonnell, Donald P.; Clyne, Colin D.

2014-01-01

The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter. PMID:25000480

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

PubMed Central

Tiller, Kathryn E.; Chowdhury, Ratul; Li, Tong; Ludwig, Seth D.; Sen, Sabyasachi; Maranas, Costas D.; Tessier, Peter M.

2017-01-01

The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies. PMID:28928732

Bridging the Gaps: Measuring Cultural Competence among Future School Library and Youth Services Library Professionals

ERIC Educational Resources Information Center

Hill, Renee Franklin; Kumasi, Kafi

2011-01-01

School library and youth services professionals must develop and display a strong sense of cultural competence to effectively serve their patrons. Cultural competence is defined here as one's ability to understand the needs of populations different from their own. This paper reports on the perceptions of school library and youth services students…

Instructional Materials Centers; Selected Readings.

ERIC Educational Resources Information Center

Pearson, Neville P.; Butler, Lucius

Revolutionary innovation in the traditional school library has produced "the media center", where--in addition to books--films, television, tapes, and multimedia displays are available to increase student learning. This book represents a collection of eighty-three articles from library journals dealing with library science in its modern…

Merchandising Techniques and Libraries.

ERIC Educational Resources Information Center

Green, Sylvie A.

1981-01-01

Proposes that libraries employ modern booksellers' merchandising techniques to improve circulation of library materials. Using displays in various ways, the methods and reasons for weeding out books, replacing worn book jackets, and selecting new books are discussed. Suggestions for learning how to market and 11 references are provided. (RBF)

[Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

PubMed

Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

2013-06-01

To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Phage display as a technology delivering on the promise of peptide drug discovery.

PubMed

Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

2013-12-01

Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reclassification and Documentation in a Medium-sized Medical Center Library: The MTST System in the Simultaneous Production of Catalog Cards and a Computer Stored Record

PubMed Central

Love, Erika; Butzin, Diane; Robinson, Robert E.; Lee, Soo

1971-01-01

A project to recatalog and reclassify the book collection of the Bowman Gray School of Medicine Library utilizing the Magnetic Tape/Selectric Typwriter system for simultaneous catalog card production and computer stored data acquisition marks the beginning of eventual computerization of all library operations. A keyboard optical display system will be added by late 1970. Major input operations requiring the creation of “hard copy” will continue via the MTST system. Updating, editing and retrieval operations as well as input without hard copy production will be done through the “on-line” keyboard optical display system. Once the library's first data bank, the book catalog, has been established the computer may be consulted directly for library holdings from any optical display terminal throughout the medical center. Three basic information retrieval operations may be carried out through “on-line” optical display terminals. Output options include the reproduction of part or all of a given document, or the generation of statistical data, which are derived from two Acquisition Code lines. The creation of a central bibliographic record of Bowman Gray Faculty publications patterned after the cataloging program is presently under way. The cataloging and computer storage of serial holdings records will begin after completion of the reclassification project. All acquisitions added to the collection since October 1967 are computer-stored and fully retrievable. Reclassification of older titles will be completed in early 1971. PMID:5542915

Combinatorial Libraries As a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens.

PubMed

Fleeman, Renee; LaVoi, Travis M; Santos, Radleigh G; Morales, Angela; Nefzi, Adel; Welmaker, Gregory S; Medina-Franco, José L; Giulianotti, Marc A; Houghten, Richard A; Shaw, Lindsey N

2015-04-23

Mixture based synthetic combinatorial libraries offer a tremendous enhancement for the rate of drug discovery, allowing the activity of millions of compounds to be assessed through the testing of exponentially fewer samples. In this study, we used a scaffold-ranking library to screen 37 different libraries for antibacterial activity against the ESKAPE pathogens. Each library contained between 10000 and 750000 structural analogues for a total of >6 million compounds. From this, we identified a bis-cyclic guanidine library that displayed strong antibacterial activity. A positional scanning library for these compounds was developed and used to identify the most effective functional groups at each variant position. Individual compounds were synthesized that were broadly active against all ESKAPE organisms at concentrations <2 μM. In addition, these compounds were bactericidal, had antibiofilm effects, showed limited potential for the development of resistance, and displayed almost no toxicity when tested against human lung cells and erythrocytes. Using a murine model of peritonitis, we also demonstrate that these agents are highly efficacious in vivo.

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Engineering M13 for phage display.

PubMed

Sidhu, S S

2001-09-01

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.

Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library.

PubMed

Buzatti, Andréia; Fernandez, Arnielis Diaz; Arenal, Amilcar; Pereira, Erlán; Monteiro, Alda Lucia Gomes; Molento, Marcelo Beltrão

2018-05-24

The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.

Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library.

PubMed

Wu, Qian; Wang, Xiaodong; Gu, Yong; Zhang, Xiao; Qin, Yao; Chen, Heng; Xu, Xinyu; Yang, Tao; Zhang, Mei

2016-07-01

Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed, Histidine-Doped Master Library.

PubMed

Könning, Doreen; Zielonka, Stefan; Sellmann, Carolin; Schröter, Christian; Grzeschik, Julius; Becker, Stefan; Kolmar, Harald

2016-04-01

In recent years, engineering of pH-sensitivity into antibodies as well as antibody-derived fragments has become more and more attractive for biomedical and biotechnological applications. Herein, we report the isolation of the first pH-sensitive IgNAR variable domain (vNAR), which was isolated from a yeast-displayed, semi-synthetic master library. This strategy enables the direct identification of pH-dependent binders from a histidine-enriched CDR3 library. Displayed vNAR variants contained two histidine substitutions on average at random positions in their 12-residue CDR3 loop. Upon screening of seven rounds against the proof-of-concept target EpCAM (selection for binding at pH 7.4 and decreased binding at pH 6.0), a single clone was obtained that showed specific and pH-dependent binding as characterized by yeast surface display and biolayer interferometry. Potential applications for such pH-dependent vNAR domains include their employment in tailored affinity chromatography, enabling mild elution protocols. Moreover, utilizing a master library for the isolation of pH-sensitive vNAR variants may be a generic strategy to obtain binding entities with prescribed characteristics for applications in biotechnology, diagnostics, and therapy.

Identification of chondrocyte-binding peptides by phage display.

PubMed

Cheung, Crystal S F; Lui, Julian C; Baron, Jeffrey

2013-07-01

As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. Copyright © 2013 Orthopaedic Research Society.

Constitutional dynamic self-sensing in a zinc(II)/polyiminofluorenes system.

PubMed

Giuseppone, Nicolas; Lehn, Jean-Marie

2004-09-22

The interaction of an external effector, ZnII ions, with a constitutional dynamic library of fluorescent polyiminofluorenes leads to component exchange, which generates an entity responding by a change in emission to the effector that has induced its formation. The overall coupled system displays a tuning of optical signal, resulting from two synergistic processes: adaptative constitutional reorganization and self-sensing. In broader terms, this work highlights the perspectives opened by constitutional dynamic chemistry toward the design of smart materials, capable of expressing different latent properties in response to environmental conditions.

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

NASA Technical Reports Server (NTRS)

Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

2000-01-01

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

PhD7Faster: predicting clones propagating faster from the Ph.D.-7 phage display peptide library.

PubMed

Ru, Beibei; 't Hoen, Peter A C; Nie, Fulei; Lin, Hao; Guo, Feng-Biao; Huang, Jian

2014-02-01

Phage display can rapidly discover peptides binding to any given target; thus, it has been widely used in basic and applied research. Each round of panning consists of two basic processes: Selection and amplification. However, recent studies have showed that the amplification step would decrease the diversity of phage display libraries due to different propagation capacity of phage clones. This may induce phages with growth advantage rather than specific affinity to appear in the final experimental results. The peptides displayed by such phages are termed as propagation-related target-unrelated peptides (PrTUPs). They would mislead further analysis and research if not removed. In this paper, we describe PhD7Faster, an ensemble predictor based on support vector machine (SVM) for predicting clones with growth advantage from the Ph.D.-7 phage display peptide library. By using reduced dipeptide composition (ReDPC) as features, an accuracy (Acc) of 79.67% and a Matthews correlation coefficient (MCC) of 0.595 were achieved in 5-fold cross-validation. In addition, the SVM-based model was demonstrated to perform better than several representative machine learning algorithms. We anticipate that PhD7Faster can assist biologists to exclude potential PrTUPs and accelerate the finding of specific binders from the popular Ph.D.-7 library. The web server of PhD7Faster can be freely accessed at http://immunet.cn/sarotup/cgi-bin/PhD7Faster.pl.

Advances in phage display technology for drug discovery.

PubMed

Omidfar, Kobra; Daneshpour, Maryam

2015-06-01

Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand-target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery. This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs. Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.

Structure based re-design of the binding specificity of anti-apoptotic Bcl-xL

PubMed Central

Chen, T. Scott; Palacios, Hector; Keating, Amy E.

2012-01-01

Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Re-designed proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to re-engineer the multi-specific anti-apoptotic protein Bcl-xL to remove its interactions with many of its binding partners, making it a high affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-xL sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-xL variants, we successfully identified a protein with ~1,000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best re-designed protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems. PMID:23154169

Biased selection of propagation-related TUPs from phage display peptide libraries.

PubMed

Zade, Hesam Motaleb; Keshavarz, Reihaneh; Shekarabi, Hosna Sadat Zahed; Bakhshinejad, Babak

2017-08-01

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

Plug into PR. NJLA Public Relations Handbook.

ERIC Educational Resources Information Center

New Jersey Library Association, Trenton.

A guide to using public relations techniques to promote both everyday services and special events at libraries, this handbook describes and suggests ways to use library displays; in-house printing; the local media, e.g., radio spots and cable television; library programs; marketing and promotion; and fundraising, including the formation of a…

Continuous microfluidic assortment of interactive ligands (CMAIL)

NASA Astrophysics Data System (ADS)

Hsiao, Yi-Hsing; Huang, Chao-Yang; Hu, Chih-Yung; Wu, Yen-Yu; Wu, Chung-Hsiun; Hsu, Chia-Hsien; Chen, Chihchen

2016-08-01

Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 105 CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 109 individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

Bibliographic Displays in OPACs and Web Catalogs: How Well Do They Comply with Display Guidelines?

ERIC Educational Resources Information Center

Cherry, Joan M.

1998-01-01

Evaluation of data from assessments of full bibliographic displays in academic library OPACs (online public access catalogs) and World Wide Web catalogs against a checklist of desirable features found that OPAC displays scored 58% and Web displays scored 60%. Discusses weaknesses, focusing on those found in the majority of the displays…

Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing.

PubMed

Li, Chenghua; Feng, Weida; Qiu, Lihua; Xia, Changge; Su, Xiurong; Jin, Chunhua; Zhou, Tingting; Zeng, Yuan; Li, Taiwu

2012-08-01

MicroRNAs (miRNAs) constitute a family of small RNA species which have been demonstrated to be one of key effectors in mediating host-pathogen interaction. In this study, two haemocytes miRNA libraries were constructed with deep sequenced by illumina Hiseq2000 from healthy (L1) and skin ulceration syndrome Apostichopus japonicus (L2). The high throughput solexa sequencing resulted in 9,579,038 and 7,742,558 clean data from L1 and L2, respectively. Sequences analysis revealed that 40 conserved miRNAs were found in both libraries, in which let-7 and mir-125 were speculated to be clustered together and expressed accordingly. Eighty-six miRNA candidates were also identified by reference genome search and stem-loop structure prediction. Importantly, mir-31 and mir-2008 displayed significant differential expression between the two libraries according to FPKM model, which might be considered as promising targets for elucidating the intrinsic mechanism of skin ulceration syndrome outbreak in the species. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

High-Throughput Method for Ranking the Affinity of Peptide Ligands Selected from Phage Display Libraries

PubMed Central

González-Techera, A.; Umpiérrez-Failache, M.; Cardozo, S.; Obal, G.; Pritsch, O.; Last, J. A.; Gee, S. J.; Hammock, B. D.; González-Sapienza, G.

2010-01-01

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major “bottleneck” of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred “en masse” from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide–BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide–BAP protein can find direct application as a tracer reagent. PMID:18393454

Identification of antibiotics using small molecule variable ligand display on gold nanoparticles.

PubMed

Bresee, Jamee; Maier, Keith E; Melander, Christian; Feldheim, Daniel L

2010-10-28

Here we describe the use of simple 1-pot thiol exchange reactions to generate a library of mixed ligand-coated gold nanoparticles that was screened for antibiotic activity. A library of 120 nanoparticle conjugates was assembled and antibiotic activity toward E. coli was determined and found to depend upon the combination of thiols assembled onto the nanoparticles. The most active conjugate displayed 99.9% growth inhibition at 0.5 μM.

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

PubMed

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

2014-08-01

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

A new helper phage for improved monovalent display of Fab molecules.

PubMed

Beaber, John W; Tam, Eric M; Lao, Llewelyn S; Rondon, Isaac J

2012-02-28

Phage display technology is a powerful tool for the identification of novel antibodies for drug discovery. Phage display libraries have been constructed with massive diversity, but their use may be hindered by limited antibody display levels when rescued with the M13KO7 helper phage. Variants of M13KO7 have been constructed previously that increase the levels of display of rescued phage, but all produce phage that display multiple copies of the antibody fragment on their surface and have reduced titer and infectivity. In this study, we describe a new helper phage, XP5, which increased the display level of Fab molecules more than two-fold compared to phage rescued with M13KO7. XP5 uses a combination of ribosome binding site spacing alterations and rare codon clusters to reduce the expression of pIII from the helper phage. This reduction in pIII expression leads to an increase in the incorporation of pIII-Fab fusions during phage rescue. The rescued phage displayed a single copy of the Fab molecule, preventing any avidity effects during the selection process. This also suggests that the percentage of the population of phage displaying a Fab molecule is increased when rescued with XP5. Additionally, the phage titers and infectivity are comparable to libraries rescued with M13KO7. After two rounds of panning we observed a nearly 5-fold increase in the number of antigen binding Fab molecules compared to panning conducted with the same library rescued with M13KO7. The nature of the mutations in XP5 makes it a universal substitute for M13KO7 in pIII-based phage display, compatible with most phagemids and bacterial strains. Copyright © 2011 Elsevier B.V. All rights reserved.

Elaboration of Programmes, Programmes, Plans and Budgets of the National Libraries in Socialist Countries (37th Session, Liverpool, August 1971).

ERIC Educational Resources Information Center

Kanevsky, B. P.

Long-term and short-term planning and programming and budgeting are an important part of the every-day activity of the national libraries in the socialist countries. The libraries extensively apply various types of planning ranging from current annual plans to complicated prognoses for 15-20 years ahead. These libraries display increasing…

Display technologies: application for the discovery of drug and gene delivery agents

PubMed Central

Sergeeva, Anna; Kolonin, Mikhail G.; Molldrem, Jeffrey J.; Pasqualini, Renata; Arap, Wadih

2007-01-01

Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed. PMID:17123658

Browse without a Browser at the ATRF Library | Poster

Cancer.gov

By Robin Meckley, Contributing Writer Employees at the Advanced Technology Research Facility (ATRF) asked the Scientific Library, and the library responded: print journals are now available in the ATRF Library. Employees can now browse 20 print journals, which will rotate, with one issue available at a time for each title. The library will also temporarily display some new books each week. ATRF employees may indicate their interest in these books by signing the wait lists.

AOIPS data base management systems support for GARP data sets

NASA Technical Reports Server (NTRS)

Gary, J. P.

1977-01-01

A data base management system is identified, developed to provide flexible access to data sets produced by GARP during its data systems tests. The content and coverage of the data base are defined and a computer-aided, interactive information storage and retrieval system, implemented to facilitate access to user specified data subsets, is described. The computer programs developed to provide the capability were implemented on the highly interactive, minicomputer-based AOIPS and are referred to as the data retrieval system (DRS). Implemented as a user interactive but menu guided system, the DRS permits users to inventory the data tape library and create duplicate or subset data sets based on a user selected window defined by time and latitude/longitude boundaries. The DRS permits users to select, display, or produce formatted hard copy of individual data items contained within the data records.

Mimotope mapping as a complementary strategy to define allergen IgE-epitopes: peach Pru p 3 allergen as a model.

PubMed

Pacios, Luis F; Tordesillas, Leticia; Cuesta-Herranz, Javier; Compes, Esther; Sánchez-Monge, Rosa; Palacín, Arantxa; Salcedo, Gabriel; Díaz-Perales, Araceli

2008-04-01

Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76 I77 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.

Discovery of novel SERCA inhibitors by virtual screening of a large compound library.

PubMed

Elam, Christopher; Lape, Michael; Deye, Joel; Zultowsky, Jodie; Stanton, David T; Paula, Stefan

2011-05-01

Two screening protocols based on recursive partitioning and computational ligand docking methodologies, respectively, were employed for virtual screens of a compound library with 345,000 entries for novel inhibitors of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA), a potential target for cancer chemotherapy. A total of 72 compounds that were predicted to be potential inhibitors of SERCA were tested in bioassays and 17 displayed inhibitory potencies at concentrations below 100 μM. The majority of these inhibitors were composed of two phenyl rings tethered to each other by a short link of one to three atoms. Putative interactions between SERCA and the inhibitors were identified by inspection of docking-predicted poses and some of the structural features required for effective SERCA inhibition were determined by analysis of the classification pattern employed by the recursive partitioning models. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

A CMOS microdisplay with integrated controller utilizing improved silicon hot carrier luminescent light sources

NASA Astrophysics Data System (ADS)

Venter, Petrus J.; Alberts, Antonie C.; du Plessis, Monuko; Joubert, Trudi-Heleen; Goosen, Marius E.; Janse van Rensburg, Christo; Rademeyer, Pieter; Fauré, Nicolaas M.

2013-03-01

Microdisplay technology, the miniaturization and integration of small displays for various applications, is predominantly based on OLED and LCoS technologies. Silicon light emission from hot carrier electroluminescence has been shown to emit light visibly perceptible without the aid of any additional intensification, although the electrical to optical conversion efficiency is not as high as the technologies mentioned above. For some applications, this drawback may be traded off against the major cost advantage and superior integration opportunities offered by CMOS microdisplays using integrated silicon light sources. This work introduces an improved version of our previously published microdisplay by making use of new efficiency enhanced CMOS light emitting structures and an increased display resolution. Silicon hot carrier luminescence is often created when reverse biased pn-junctions enter the breakdown regime where impact ionization results in carrier transport across the junction. Avalanche breakdown is typically unwanted in modern CMOS processes. Design rules and process design are generally tailored to prevent breakdown, while the voltages associated with breakdown are too high to directly interact with the rest of the CMOS standard library. This work shows that it is possible to lower the operating voltage of CMOS light sources without compromising the optical output power. This results in more efficient light sources with improved interaction with other standard library components. This work proves that it is possible to create a reasonably high resolution microdisplay while integrating the active matrix controller and drivers on the same integrated circuit die without additional modifications, in a standard CMOS process.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Liu, Yang; Zu, Xiangyang

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.more » 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.« less

True Stories of Censorship Battles in America's Libraries

ERIC Educational Resources Information Center

Nye, Valerie, Ed.; Barco, Kathy, Ed.

2012-01-01

Intellectual freedom is a core value of librarianship, but fighting to keep controversial materials on the shelves can sometimes feel like a lonely battle. And not all censorship controversies involve the public objecting to a book in the collection--libraries are venues for displays and meetings, and sometimes library staff themselves are tempted…

A Tour of the Stacks--HyperCard for Libraries.

ERIC Educational Resources Information Center

Ertel, Monica; Oros, Jane

1989-01-01

Description of HyperCard, a software package that runs on Macintosh microcomputers, focuses on its use in the Apple Computer, Inc., Library as a user guide to the library. Examples of screen displays are given, and a list of resources is included to help use and understand HyperCard more completely. (LRW)

Principal component analysis as a tool for library design: a case study investigating natural products, brand-name drugs, natural product-like libraries, and drug-like libraries.

PubMed

Wenderski, Todd A; Stratton, Christopher F; Bauer, Renato A; Kopp, Felix; Tan, Derek S

2015-01-01

Principal component analysis (PCA) is a useful tool in the design and planning of chemical libraries. PCA can be used to reveal differences in structural and physicochemical parameters between various classes of compounds by displaying them in a convenient graphical format. Herein, we demonstrate the use of PCA to gain insight into structural features that differentiate natural products, synthetic drugs, natural product-like libraries, and drug-like libraries, and show how the results can be used to guide library design.

Principal Component Analysis as a Tool for Library Design: A Case Study Investigating Natural Products, Brand-Name Drugs, Natural Product-Like Libraries, and Drug-Like Libraries

PubMed Central

Wenderski, Todd A.; Stratton, Christopher F.; Bauer, Renato A.; Kopp, Felix; Tan, Derek S.

2015-01-01

Principal component analysis (PCA) is a useful tool in the design and planning of chemical libraries. PCA can be used to reveal differences in structural and physicochemical parameters between various classes of compounds by displaying them in a convenient graphical format. Herein, we demonstrate the use of PCA to gain insight into structural features that differentiate natural products, synthetic drugs, natural product-like libraries, and drug-like libraries, and show how the results can be used to guide library design. PMID:25618349

M13 bacteriophage coat proteins engineered for improved phage display.

PubMed

Sidhu, Sachdev S; Feld, Birte K; Weiss, Gregory A

2007-01-01

This chapter describes a method for increasing levels of protein fusions displayed on the surfaces of M13 bacteriophage particles. By introducing mutations into the anchoring M13 coat protein, protein display levels can be increased by up to two orders of magnitude. Experimental methods are presented for the design, construction, and screening of phage-displayed libraries for improved protein display.

Role of messenger RNA-ribosome complex in complementary DNA display.

PubMed

Naimuddin, Mohammed; Ohtsuka, Isao; Kitamura, Koichiro; Kudou, Motonori; Kimura, Shinnosuke

2013-07-15

In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of a cardiac specific protein transduction domain by in vivo biopanning using a M13 phage peptide display library in mice.

PubMed

Zahid, Maliha; Phillips, Brett E; Albers, Sean M; Giannoukakis, Nick; Watkins, Simon C; Robbins, Paul D

2010-08-17

A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

PubMed

Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

2016-12-01

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

Somatostatin displayed on filamentous phage as a receptor-specific agonist

PubMed Central

Rousch, Mat; Lutgerink, Jan T; Coote, James; de Bruïne, Adriaan; Arends, Jan-Willem; Hoogenboom, Hennie R

1998-01-01

In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand. PMID:9776337

An improved yeast transformation method for the generation of very large human antibody libraries.

PubMed

Benatuil, Lorenzo; Perez, Jennifer M; Belk, Jonathan; Hsieh, Chung-Ming

2010-04-01

Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

Website creation and resource management: developing collaborative strategies for asynchronous interaction with library users.

PubMed

Hopkins, Mark E; Summers-Ables, Joy E; Clifton, Shari C; Coffman, Michael A

2011-06-01

To make electronic resources available to library users while effectively harnessing intellectual capital within the library, ultimately fostering the library's use of technology to interact asynchronously with its patrons (users). The methods used in the project included: (1) developing a new library website to facilitate the creation, management, accessibility, maintenance and dissemination of library resources; and (2) establishing ownership by those who participated in the project, while creating effective work allocation strategies through the implementation of a content management system that allowed the library to manage cost, complexity and interoperability. Preliminary results indicate that contributors to the system benefit from an increased understanding of the library's resources and add content valuable to library patrons. These strategies have helped promote the manageable creation and maintenance of electronic content in accomplishing the library's goal of interacting with its patrons. Establishment of a contributive system for adding to the library's electronic resources and electronic content has been successful. Further work will look at improving asynchronous interaction, particularly highlighting accessibility of electronic content and resources. © 2010 The authors. Health Information and Libraries Journal © 2010 Health Libraries Group.

Recombinant phage probes for Listeria monocytogenes

NASA Astrophysics Data System (ADS)

Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

2007-10-01

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

Construction of naïve camelids VHH repertoire in phage display-based library.

PubMed

Sabir, Jamal S M; Atef, Ahmed; El-Domyati, Fotouh M; Edris, Sherif; Hajrah, Nahid; Alzohairy, Ahmed M; Bahieldin, Ahmed

2014-04-01

Camelids have unique antibodies, namely HCAbs (VHH) or commercially named Nanobodies(®) (Nb) that are composed only of a heavy-chain homodimer. As libraries based on immunized camelids are time-consuming, costly and likely redundant for certain antigens, we describe the construction of a naïve camelid VHHs library from blood serum of non-immunized camelids with affinity in the subnanomolar range and suitable for standard immune applications. This approach is rapid and recovers VHH repertoire with the advantages of being more diverse, non-specific and devoid of subpopulations of specific antibodies, which allows the identification of binders for any potential antigen (or pathogen). RNAs from a number of camelids from Saudi Arabia were isolated and cDNAs of the diverse vhh gene were amplified; the resulting amplicons were cloned in the phage display pSEX81 vector. The size of the library was found to be within the required range (10(7)) suitable for subsequent applications in disease diagnosis and treatment. Two hundred clones were randomly selected and the inserted gene library was either estimated for redundancy or sequenced and aligned to the reference camelid vhh gene (acc. No. ADE99145). Results indicated complete non-specificity of this small library in which no single event of redundancy was detected. These results indicate the efficacy of following this approach in order to yield a large and diverse enough gene library to secure the presence of the required version encoding the required antibodies for any target antigen. This work is a first step towards the construction of phage display-based biosensors useful in disease (e.g., TB or tuberculosis) diagnosis and treatment. Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Synthetic antibodies: concepts, potential and practical considerations.

PubMed

Miersch, S; Sidhu, S S

2012-08-01

The last 100 years of enquiry into the fundamental basis of humoral immunity has resulted in the identification of antibodies as key molecular sentinels responsible for the in vivo surveillance, neutralization and clearance of foreign substances. Intense efforts aimed at understanding and exploiting their exquisite molecular specificity have positioned antibodies as a cornerstone supporting basic research, diagnostics and therapeutic applications [1]. More recently, efforts have aimed to circumvent the limitations of developing antibodies in animals by developing wholly in vitro techniques for designing antibodies of tailored specificity. This has been realized with the advent of synthetic antibody libraries that possess diversity outside the scope of natural immune repertoires and are thus capable of yielding specificities not otherwise attainable. This review examines the convergence of technologies that have contributed to the development of combinatorial phage-displayed antibody libraries. It further explores the practical concepts that underlie phage display, antibody diversity and the methods used in the generation of and selection from phage-displayed synthetic antibody libraries, highlighting specific applications in which design approaches gave rise to specificities that could not easily be obtained with libraries based upon natural immune repertories. Copyright © 2012 Elsevier Inc. All rights reserved.

Complementary DNA libraries: an overview.

PubMed

Ying, Shao-Yao

2004-07-01

The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.

Digital Images over the Internet: Rome Reborn at the Library of Congress.

ERIC Educational Resources Information Center

Valauskas, Edward J.

1994-01-01

Describes digital images of incunabula from the Library of the Vatican that are available over the Internet based on an actual exhibit that was displayed at the Library of Congress. Viewers, i.e., compression routines created to efficiently send color images, are explained; and other digital exhibits are described. (Contains three references.)…

Using Pamphlets With Disadvantaged Adults. Public Library Training Institutes Library Service Guide Number Three.

ERIC Educational Resources Information Center

Schmidt, Susan K.

Pamphlets are a useful way of presenting alternative sources of information to disadvantaged adults. Pamphlets are easy to handle and to read, inexpensive, and provide current information of various topics of interest. This brief guide lists sources of free or inexpensive pamphlets and describes various methods for their display in the library,…

99mTc-HYNIC-TNF analogs (WH701) derived from phage display peptide libraries for imaging TNF-receptor-positive ovarian carcinoma: preclinical evaluation

NASA Astrophysics Data System (ADS)

Xiang, Yan; Xia, Jinsong; Wu, H.; Li, H. F.

2002-04-01

Radiolabeled bioactive peptides which bind specifically to surface receptors over expressed in tumor cells are considered as alternatives for tumor detection with ECT. In this investigation, 99mTc-hydrazinonicotinyl - TNF analogs (WH701) was labeled using ethylenediaminediacetic acid (EDDA) as coligand (a number of TNF analogs had been selected and synthesized using random phage-display peptides library in our lab) and Pharmacokinetics and feasibility studies were performed.

Selection of specific interactors from phage display library based on sea lamprey variable lymphocyte receptor sequences.

PubMed

Wezner-Ptasinska, Magdalena; Otlewski, Jacek

2015-12-01

Variable lymphocyte receptors (VLRs) are non-immunoglobulin components of adaptive immunity in jawless vertebrates. These proteins composed of leucine-rich repeat modules offer some advantages over antibodies in target binding and therefore are attractive candidates for biotechnological applications. In this paper we report the design and characterization of a phage display library based on a previously proposed dVLR scaffold containing six LRR modules [Wezner-Ptasinska et al., 2011]. Our library was designed based on a consensus approach in which the randomization scheme reflects the frequencies of amino acids naturally occurring in respective positions responsible for antigen recognition. We demonstrate general applicability of the scaffold by selecting dVLRs specific for lysozyme and S100A7 protein with KD values in the micromolar range. The dVLR library could be used as a convenient alternative to antibodies for effective isolation of high affinity binders.

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

PubMed

Tullila, Antti; Nevanen, Tarja K

2017-05-31

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.

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

PubMed Central

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

Import Manipulate Plot RELAP5/MOD3 Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, K. R.

1999-10-05

XMGR5 was derived from an XY plotting tool called ACE/gr, which is copyrighted by Paul J. Turner and in the public domain. The interactive version of ACE/GR is xmgr, and includes a graphical interface to the X-windows system. Enhancements to xmgr have been developed which import, manipualate, and plot data from RELAP/MOD3, MELCOR, FRAPCON, and SINDA codes, and NRC databank files. capabilities, include two-phase property table lookup functions, an equation interpreter, arithmetic library functions, and units conversion. Plot titles, labels, legends, and narrative can be displayed using Latin or Cyrillic alphabets.

Biodiscovery of aluminum binding peptides

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

2013-05-01

Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool

PubMed Central

2013-01-01

Background System-wide profiling of genes and proteins in mammalian cells produce lists of differentially expressed genes/proteins that need to be further analyzed for their collective functions in order to extract new knowledge. Once unbiased lists of genes or proteins are generated from such experiments, these lists are used as input for computing enrichment with existing lists created from prior knowledge organized into gene-set libraries. While many enrichment analysis tools and gene-set libraries databases have been developed, there is still room for improvement. Results Here, we present Enrichr, an integrative web-based and mobile software application that includes new gene-set libraries, an alternative approach to rank enriched terms, and various interactive visualization approaches to display enrichment results using the JavaScript library, Data Driven Documents (D3). The software can also be embedded into any tool that performs gene list analysis. We applied Enrichr to analyze nine cancer cell lines by comparing their enrichment signatures to the enrichment signatures of matched normal tissues. We observed a common pattern of up regulation of the polycomb group PRC2 and enrichment for the histone mark H3K27me3 in many cancer cell lines, as well as alterations in Toll-like receptor and interlukin signaling in K562 cells when compared with normal myeloid CD33+ cells. Such analyses provide global visualization of critical differences between normal tissues and cancer cell lines but can be applied to many other scenarios. Conclusions Enrichr is an easy to use intuitive enrichment analysis web-based tool providing various types of visualization summaries of collective functions of gene lists. Enrichr is open source and freely available online at: http://amp.pharm.mssm.edu/Enrichr. PMID:23586463

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.

PubMed

Chen, Edward Y; Tan, Christopher M; Kou, Yan; Duan, Qiaonan; Wang, Zichen; Meirelles, Gabriela Vaz; Clark, Neil R; Ma'ayan, Avi

2013-04-15

System-wide profiling of genes and proteins in mammalian cells produce lists of differentially expressed genes/proteins that need to be further analyzed for their collective functions in order to extract new knowledge. Once unbiased lists of genes or proteins are generated from such experiments, these lists are used as input for computing enrichment with existing lists created from prior knowledge organized into gene-set libraries. While many enrichment analysis tools and gene-set libraries databases have been developed, there is still room for improvement. Here, we present Enrichr, an integrative web-based and mobile software application that includes new gene-set libraries, an alternative approach to rank enriched terms, and various interactive visualization approaches to display enrichment results using the JavaScript library, Data Driven Documents (D3). The software can also be embedded into any tool that performs gene list analysis. We applied Enrichr to analyze nine cancer cell lines by comparing their enrichment signatures to the enrichment signatures of matched normal tissues. We observed a common pattern of up regulation of the polycomb group PRC2 and enrichment for the histone mark H3K27me3 in many cancer cell lines, as well as alterations in Toll-like receptor and interlukin signaling in K562 cells when compared with normal myeloid CD33+ cells. Such analyses provide global visualization of critical differences between normal tissues and cancer cell lines but can be applied to many other scenarios. Enrichr is an easy to use intuitive enrichment analysis web-based tool providing various types of visualization summaries of collective functions of gene lists. Enrichr is open source and freely available online at: http://amp.pharm.mssm.edu/Enrichr.

BiNChE: a web tool and library for chemical enrichment analysis based on the ChEBI ontology.

PubMed

Moreno, Pablo; Beisken, Stephan; Harsha, Bhavana; Muthukrishnan, Venkatesh; Tudose, Ilinca; Dekker, Adriano; Dornfeldt, Stefanie; Taruttis, Franziska; Grosse, Ivo; Hastings, Janna; Neumann, Steffen; Steinbeck, Christoph

2015-02-21

Ontology-based enrichment analysis aids in the interpretation and understanding of large-scale biological data. Ontologies are hierarchies of biologically relevant groupings. Using ontology annotations, which link ontology classes to biological entities, enrichment analysis methods assess whether there is a significant over or under representation of entities for ontology classes. While many tools exist that run enrichment analysis for protein sets annotated with the Gene Ontology, there are only a few that can be used for small molecules enrichment analysis. We describe BiNChE, an enrichment analysis tool for small molecules based on the ChEBI Ontology. BiNChE displays an interactive graph that can be exported as a high-resolution image or in network formats. The tool provides plain, weighted and fragment analysis based on either the ChEBI Role Ontology or the ChEBI Structural Ontology. BiNChE aids in the exploration of large sets of small molecules produced within Metabolomics or other Systems Biology research contexts. The open-source tool provides easy and highly interactive web access to enrichment analysis with the ChEBI ontology tool and is additionally available as a standalone library.

Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

PubMed

McElhiney, J; Lawton, L A; Porter, A J

2000-12-01

Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

Phage display peptide libraries in molecular allergology: from epitope mapping to mimotope-based immunotherapy.

PubMed

Luzar, J; Štrukelj, B; Lunder, M

2016-11-01

Identification of allergen epitopes is a key component in proper understanding of the pathogenesis of type I allergies, for understanding cross-reactivity and for the development of mimotope immunotherapeutics. Phage particles have garnered recognition in the field of molecular allergology due to their value not only in competitive immunoscreening of peptide libraries but also as immunogenic carriers of allergen mimotopes. They integrate epitope discovery technology and immunization functions into a single platform. This article provides an overview of allergen mimotopes identified through the phage display technique. We discuss the contribution of phage display peptide libraries in determining dominant B-cell epitopes of allergens, in developing mimotope immunotherapy, in understanding cross-reactivity, and in determining IgE epitope profiles of individual patients to improve diagnostics and individualize immunotherapy. We also discuss the advantages and pitfalls of the methodology used to identify and validate the mimotopes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed

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.

Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

PubMed Central

Noppe, Wim; Plieva, Fatima; Galaev, Igor Yu; Pottel, Hans; Deckmyn, Hans; Mattiasson, Bo

2009-01-01

Background Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library. Results Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages. Conclusion Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection. PMID:19292898

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

PubMed

Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

2015-03-31

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Automating Small Libraries.

ERIC Educational Resources Information Center

Swan, James

1996-01-01

Presents a four-phase plan for small libraries strategizing for automation: inventory and weeding, data conversion, implementation, and enhancements. Other topics include selecting a system, MARC records, compatibility, ease of use, industry standards, searching capabilities, support services, system security, screen displays, circulation modules,…

Creating Library Interiors: Planning and Design Considerations.

ERIC Educational Resources Information Center

Jones, Plummer Alston, Jr.; Barton, Phillip K.

1997-01-01

Examines design considerations for public library interiors: access; acoustical treatment; assignable and nonassignable space; building interiors: ceilings, clocks, color, control, drinking fountains; exhibit space: slotwall display, floor coverings, floor loading, furniture, lighting, mechanical systems, public address, copying machines,…

Filamentous Phage: Structure and Biology.

PubMed

Rakonjac, Jasna; Russel, Marjorie; Khanum, Sofia; Brooke, Sam J; Rajič, Marina

2017-01-01

Ff filamentous phage (fd, M13 and f1) of Escherichia coli have been the workhorse of phage display technology for the past 30 years. Dominance of Ff over other bacteriophage in display technology stems from the titres that are about 100-fold higher than any other known phage, efficacious transformation ensuring large library size and superior stability of the virion at high temperatures, detergents and pH extremes, allowing broad range of biopanning conditions in screening phage display libraries. Due to the excellent understanding of infection and assembly requirements, Ff phage have also been at the core of phage-assisted continual protein evolution strategies (PACE). This chapter will give an overview of the Ff filamentous phage structure and biology, emphasizing those properties of the Ff phage life cycle and virion that are pertinent to phage display applications.

Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections.

PubMed

Hamidon, Nurul Hamizah; Suraiya, Siti; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd Nor; Lim, Theam Soon

2018-03-01

B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10 9 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage-display

PubMed Central

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K.

2012-01-01

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display. PMID:22985966

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.

PubMed

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K

2012-11-23

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious, and time-consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13 the N-terminal Forkhead-associated (FHA1) domain of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be nonfunctional due to misfolding in the bacterial periplasm. To overcome this limitation, we constructed a library of FHA1 variants by mutagenic PCR and isolated functional variants after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1 strand was discovered to be essential for phage display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermally stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20-25mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage display. Copyright © 2012 Elsevier Ltd. All rights reserved.

Report of the Ad Hoc Committee on Religious and Cultural Celebrations in the Library.

ERIC Educational Resources Information Center

Rathemacher, Andree; Grubman, Sheila Black; Lahiri, Amar; Gilton, Donna; Sharif, Mohammed

The charge of the University of Rhode Island's Ad Hoc Committee on Religious and Cultural Celebrations in the Library was to: investigate all opportunities for the library to educate the campus community about religious and cultural holidays; consider all the major religions of the world and the possibility of having displays for the symbols of…

Therapeutic Antibodies by Phage Display.

PubMed

Shim, Hyunbo

2016-01-01

Antibody phage display is a major technological platform for the generation of fully human antibodies for therapeutic purposes. The in vitro binder selection by phage display allows researchers to have more extensive control over binding parameters and facilitates the isolation of clinical candidate antibodies with desired binding and/or functional profiles. Since the invention of antibody phage display in late 1980s, significant technological advancements in the design, construction, and selection of the antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. In this review, the background and details of antibody phage display technology, and representative antibody libraries with natural or synthetic sequence diversity and different construction strategies are described. The generation, optimization, functional and biophysical properties, and preclinical and clinical developments of some of the phage display-derived therapeutic antibodies approved for use in patients or in late-stage clinical trials are also discussed. With evolving novel disease targets and therapeutic strategies, antibody phage display is expected to continue to play a central role in the development of the next generation of therapeutic antibodies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Liquid Array Platform For the Multiplexed Analysis of Synthetic Molecule-Protein Interactions

PubMed Central

Doran, Todd M.; Kodadek, Thomas

2014-01-01

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative comprised of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships and for certain types of serological analyses. PMID:24245981

HYDRA Hyperspectral Data Research Application Tom Rink and Tom Whittaker

NASA Astrophysics Data System (ADS)

Rink, T.; Whittaker, T.

2005-12-01

HYDRA is a freely available, easy to install tool for visualization and analysis of large local or remote hyper/multi-spectral datasets. HYDRA is implemented on top of the open source VisAD Java library via Jython - the Java implementation of the user friendly Python programming language. VisAD provides data integration, through its generalized data model, user-display interaction and display rendering. Jython has an easy to read, concise, scripting-like, syntax which eases software development. HYDRA allows data sharing of large datasets through its support of the OpenDAP and OpenADDE server-client protocols. The users can explore and interrogate data, and subset in physical and/or spectral space to isolate key areas of interest for further analysis without having to download an entire dataset. It also has an extensible data input architecture to recognize new instruments and understand different local file formats, currently NetCDF and HDF4 are supported.

ShelXle: a Qt graphical user interface for SHELXL.

PubMed

Hübschle, Christian B; Sheldrick, George M; Dittrich, Birger

2011-12-01

ShelXle is a graphical user interface for SHELXL [Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122], currently the most widely used program for small-molecule structure refinement. It combines an editor with syntax highlighting for the SHELXL-associated .ins (input) and .res (output) files with an interactive graphical display for visualization of a three-dimensional structure including the electron density (F(o)) and difference density (F(o)-F(c)) maps. Special features of ShelXle include intuitive atom (re-)naming, a strongly coupled editor, structure visualization in various mono and stereo modes, and a novel way of displaying disorder extending over special positions. ShelXle is completely compatible with all features of SHELXL and is written entirely in C++ using the Qt4 and FFTW libraries. It is available at no cost for Windows, Linux and Mac-OS X and as source code.

Interactive robot control system and method of use

NASA Technical Reports Server (NTRS)

Abdallah, Muhammad E. (Inventor); Sanders, Adam M. (Inventor); Platt, Robert (Inventor); Reiland, Matthew J. (Inventor); Linn, Douglas Martin (Inventor)

2012-01-01

A robotic system includes a robot having joints, actuators, and sensors, and a distributed controller. The controller includes command-level controller, embedded joint-level controllers each controlling a respective joint, and a joint coordination-level controller coordinating motion of the joints. A central data library (CDL) centralizes all control and feedback data, and a user interface displays a status of each joint, actuator, and sensor using the CDL. A parameterized action sequence has a hierarchy of linked events, and allows the control data to be modified in real time. A method of controlling the robot includes transmitting control data through the various levels of the controller, routing all control and feedback data to the CDL, and displaying status and operation of the robot using the CDL. The parameterized action sequences are generated for execution by the robot, and a hierarchy of linked events is created within the sequence.

Therapeutic Potential of Shark Anti-ICOSL VNAR Domains is Exemplified in a Murine Model of Autoimmune Non-Infectious Uveitis.

PubMed

Kovaleva, Marina; Johnson, Katherine; Steven, John; Barelle, Caroline J; Porter, Andrew

2017-01-01

Induced costimulatory ligand (ICOSL) plays an important role in the activation of T cells through its interaction with the inducible costimulator, ICOS. Suppression of full T cell activation can be achieved by blocking this interaction and has been shown to be an effective means of ameliorating disease in models of autoimmunity and inflammation. In this study, we demonstrated the ability of a novel class of anti-ICOSL antigen-binding single domains derived from sharks (VNARs) to effectively reduce inflammation in a murine model of non-infectious uveitis. In initial selections, specific VNARs that recognized human ICOSL were isolated from an immunized nurse shark phage display library and lead domains were identified following their performance in a series of antigen selectivity and in vitro bioassay screens. High potency in cell-based blocking assays suggested their potential as novel binders suitable for further therapeutic development. To test this hypothesis, surrogate anti-mouse ICOSL VNAR domains were isolated from the same phage display library and the lead VNAR clone selected via screening in binding and ICOS/ICOSL blocking experiments. The VNAR domain with the highest potency in cell-based blocking of ICOS/ICOSL interaction was fused to the Fc portion of human IgG1 and was tested in vivo in a mouse model of interphotoreceptor retinoid-binding protein-induced uveitis. The anti-mICOSL VNAR Fc, injected systemically, resulted in a marked reduction of inflammation in treated mice when compared with untreated control animals. This approach inhibited disease progression to an equivalent extent to that seen for the positive corticosteroid control, cyclosporin A, reducing both clinical and histopathological scores. These results represent the first demonstration of efficacy of a VNAR binding domain in a relevant clinical model of disease and highlight the potential of VNARs for the treatment of auto-inflammatory conditions.

Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications

PubMed Central

Chevrel, Anne; Urvoas, Agathe; de la Sierra-Gallay, Ines Li; Aumont-Nicaise, Magali; Moutel, Sandrine; Desmadril, Michel; Perez, Franck; Gautreau, Alexis; van Tilbeurgh, Herman; Minard, Philippe; Valerio-Lepiniec, Marie

2015-01-01

A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes. PMID:26182430

Taking the world for a spin: teaching spatial and data visualization with a digital globe

NASA Astrophysics Data System (ADS)

Teplitzky, S.; Powell, S.

2016-12-01

In the summer of 2016, the Earth Sciences & Map Library at the University of California, Berkeley, purchased a Magic Planet digital globe in a collaboration between the library and the departments of Geography and Earth & Planetary Science. This 30" diameter 3D display supplements and expands the library's instruction and outreach activities in GIS, data visualization and modeling. Faculty and graduate students were surveyed regarding their interest in using the globe for teaching and research projects. Based on this feedback, librarians developed a basic training plan for using the globe in the classroom, as well as an assessment tool to rate the effectiveness of instruction with the digital globe. Student and faculty responses at the end of fall semester (2016) will be evaluated for future plans to increase the variety of data sets and animations available to view on the globe. Curriculum and guides for visualizing custom and interactive data sets will be developed and made available based on current researcher and student interests. We are excited about partnering with our departments and engaging our students in the possibilities of 3d visualization, and look forward to sharing lessons learned.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farajpour, Zahra; Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir; Kazemi, Bahram

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

'SON-GO-KU' : a dream of automated library

NASA Astrophysics Data System (ADS)

Sato, Mamoru; Kishimoto, Juji

In the process of automating libraries, the retrieval of books through the browsing of shelves is being overlooked. The telematic library is a document based DBMS which can deliver the content of books by simulating the browsing process. The retrieval actually simulates the process a person would use in selecting a book in a real library, where a visual presentation using a graphic display is substituted. The characteristics of prototype system "Son-Go-Ku" for such retrieval implemented in 1988 are mentioned.

Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang Yongmin; IgE Therapeutics, Inc., San Diego, CA 92121-2233; Barankiewicz, Teresa J.

2007-07-27

Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes onmore » either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.« less

Molecular Determinants of Estrogen Receptor Alpha Stability

DTIC Science & Technology

2008-07-01

presence of E2. This question can be addressed by a T7 phage display screen using a breast cancer cell library and DNA-bound ERα in the presence of...conformation of ERα induced by 27HC versus E2. To accomplish this, we performed combinatorial phage display using a modified M13 phage display screen

Structure-Guided Screening for Functionally Selective D2 Dopamine Receptor Ligands from a Virtual Chemical Library.

PubMed

Männel, Barbara; Jaiteh, Mariama; Zeifman, Alexey; Randakova, Alena; Möller, Dorothee; Hübner, Harald; Gmeiner, Peter; Carlsson, Jens

2017-10-20

Functionally selective ligands stabilize conformations of G protein-coupled receptors (GPCRs) that induce a preference for signaling via a subset of the intracellular pathways activated by the endogenous agonists. The possibility to fine-tune the functional activity of a receptor provides opportunities to develop drugs that selectively signal via pathways associated with a therapeutic effect and avoid those causing side effects. Animal studies have indicated that ligands displaying functional selectivity at the D 2 dopamine receptor (D 2 R) could be safer and more efficacious drugs against neuropsychiatric diseases. In this work, computational design of functionally selective D 2 R ligands was explored using structure-based virtual screening. Molecular docking of known functionally selective ligands to a D 2 R homology model indicated that such compounds were anchored by interactions with the orthosteric site and extended into a common secondary pocket. A tailored virtual library with close to 13 000 compounds bearing 2,3-dichlorophenylpiperazine, a privileged orthosteric scaffold, connected to diverse chemical moieties via a linker was docked to the D 2 R model. Eighteen top-ranked compounds that occupied both the orthosteric and allosteric site were synthesized, leading to the discovery of 16 partial agonists. A majority of the ligands had comparable maximum effects in the G protein and β-arrestin recruitment assays, but a subset displayed preference for a single pathway. In particular, compound 4 stimulated β-arrestin recruitment (EC 50 = 320 nM, E max = 16%) but had no detectable G protein signaling. The use of structure-based screening and virtual libraries to discover GPCR ligands with tailored functional properties will be discussed.

Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

PubMed

Scott, Benjamin M; Matochko, Wadim L; Gierczak, Richard F; Bhakta, Varsha; Derda, Ratmir; Sheffield, William P

2014-01-01

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of "designer serpins" with novel reactivity and/or specificity.

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

PubMed Central

Scott, Benjamin M.; Matochko, Wadim L.; Gierczak, Richard F.; Bhakta, Varsha; Derda, Ratmir; Sheffield, William P.

2014-01-01

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity. PMID:24427287

The Effect of Prime Display Location on Public Library Circulation of Selected Adult Titles.

ERIC Educational Resources Information Center

Goldhor, Herbert

A study of the effects on public library circulation of putting a group of selected adult titles in a prime physical location is reported. It is hypothesized that public library circulation of these titles will be significantly greater when they are collected and placed in a prime location than when they are scattered on the shelves of even an…

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

PubMed

Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

2011-10-28

Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

PubMed

Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H

2000-04-01

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Discovery and application of peptides that bind to proteins and solid state inorganic materials

NASA Astrophysics Data System (ADS)

Stearns, Linda A.

A series of three projects was undertaken on the theme of peptide-based molecular recognition. In the first project, a messenger RNA (mRNA) display selection was carried out against the II-VI semiconductors zinc sulfide (ZnS), zinc selenide (ZnSe), and cadmium sulfide (CdS). Sequence analysis of 18-mer semiconductor-binding peptides (SBPs) following four rounds of selection indicated that the amino acid sequences were enriched in polar residues compared to the naive library, suggesting that hydrogen-bonding interactions are a dominant mode of interaction between the SBPs and their cognate inorganic surfaces. Select peptides were expressed as fusions of the green fluorescent protein (GFP) to visualize their recognition of semiconductor crystals. Interpretation of the results was complicated by a high fluorescence background that was observed with certain control GFP fusions. Additional experiments, including cross-specificity binding assays, are needed to characterize the peptides that were isolated in this selection. A second project described the practical application of a known inorganic-binding and nucleating peptide. Peptide A3, which was previously isolated by phage display, was chemically conjugated to a short DNA strand using the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). The resulting peptide-DNA conjugate was hybridized to ten complementary single-stranded capture probes extending outward from the surface of an origami DNA nanotube. A gold precursor solution was added to initiate nucleation and growth of gold nanoparticles at the site of the peptide. Transmission electron microscopy (TEM) was used to visualize the gold nanoparticle-decorated nanostructures. This approach holds immense promise for organizing compositionally-diverse materials at the nanoscale. In a third project, a novel non-iterative approach to mRNA display called covalent capture was demonstrated. Using human transferrin as a target protein, peptides with low-nanomolar affinity were isolated from a combinatorial library of one trillion distinct 12-mer peptide sequences by using UV light to covalently crosslink the peptides to a photoreactive arm that was displayed on the protein surface. The best peptide isolated from this screen exhibited a binding affinity constant (Kd) of 3 nM, which is equivalent to some of the best peptides isolated after many rounds of traditional bead-based selection. The approach itself is general and could be applied to many different types of problems in molecular biology.

Development of a DICOM library

NASA Astrophysics Data System (ADS)

Kim, Dongsun; Shin, Dongkyu M.; Kim, Dongyoun M.

2001-08-01

Object-oriented DICOM decoding library was developed as a type of DLL for MS-Windows environment development. It supports all DICOM standard Transfer Syntaxes, multi-frame images, RLE decoding and window level adjusting. Image library for medical application was also developed as a type of DLL and ActiveX Control using proposed DICOM library. It supports display of DICOM image, cine mode and basic manipulations. For an application of a proposed image library, a couple of DICOM viewers were developed. One can be used as an off-line DICOM Workstation, and the other can be used for browsing the local DICOM files.

The Chair Tables the Motion: An American Libraries Report on Contemporary Furniture.

ERIC Educational Resources Information Center

Brawner, Lee B.; And Others

1988-01-01

A series of articles reviews current trends in library furniture, including designs to accommodate computer equipment and workstations, children's furniture, shelving, and display aids. Design requirements and evaluation criteria are discussed, and a directory of furniture suppliers is provided. (CLB)

Identification and immunogenicity of immunodominant mimotopes of outer membrane protein U (OmpU) of Vibrio mimicus from phage display peptide library.

PubMed

Cen, Junyu; Liu, Xueqin; Li, Jinnian; Zhang, Ming; Wang, Wei

2013-01-01

Vibrio mimicus (V. mimicus) is the causative agent of ascites disease in aquatic animals. Outer membrane protein U (OmpU) is an important antigen of V. mimicus, but its protective epitopes are still unclear. A random 12-mer phage-displayed peptide library was used to screen and identify immunodominant mimotopes of the OmpU protein in V. mimicus by panning against purified OmpU-specific polyclonal antibody. Then the immunogenicity and immunoprotection in fish of these mimotopes was evaluated. Nine positive phage clones presented seven different 12- peptide sequences and more than 50% of them carried a consensus core motif of DSSK-P. These positive clones reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by OmpU protein. Intraperitoneal injection of seven positive phage clones into fish induced a specific antibody response to OmpU protein. The fish immunized respectively with the positive phage clones C17, C24, C60 and C66 obtained 100% immunoprotective effect against experimental V. mimicus challenge. Taken together, these mimotopes presented by clone C17, C24, C60 and C66 were immunodominant mimotopes of the OmpU protein and exhibited a more appropriate candidate as epitope-based vaccine against V. mimicus infection in aquatic animals. Copyright © 2012 Elsevier Ltd. All rights reserved.

Data visualization and analysis tools for the MAVEN mission

NASA Astrophysics Data System (ADS)

Harter, B.; De Wolfe, A. W.; Putnam, B.; Brain, D.; Chaffin, M.

2016-12-01

The Mars Atmospheric and Volatile Evolution (MAVEN) mission has been collecting data at Mars since September 2014. We have developed new software tools for exploring and analyzing the science data. Our open-source Python toolkit for working with data from MAVEN and other missions is based on the widely-used "tplot" IDL toolkit. We have replicated all of the basic tplot functionality in Python, and use the bokeh and matplotlib libraries to generate interactive line plots and spectrograms, providing additional functionality beyond the capabilities of IDL graphics. These Python tools are generalized to work with missions beyond MAVEN, and our software is available on Github. We have also been exploring 3D graphics as a way to better visualize the MAVEN science data and models. We have constructed a 3D visualization of MAVEN's orbit using the CesiumJS library, which not only allows viewing of MAVEN's orientation and position, but also allows the display of selected science data sets and their variation over time.

COSMIC monthly progress report

NASA Technical Reports Server (NTRS)

1994-01-01

Activities of the Computer Software Management and Information Center (COSMIC) are summarized for the month of May 1994. Tables showing the current inventory of programs available from COSMIC are presented and program processing and evaluation activities are summarized. Nine articles were prepared for publication in the NASA Tech Brief Journal. These articles (included in this report) describe the following software items: (1) WFI - Windowing System for Test and Simulation; (2) HZETRN - A Free Space Radiation Transport and Shielding Program; (3) COMGEN-BEM - Composite Model Generation-Boundary Element Method; (4) IDDS - Interactive Data Display System; (5) CET93/PC - Chemical Equilibrium with Transport Properties, 1993; (6) SDVIC - Sub-pixel Digital Video Image Correlation; (7) TRASYS - Thermal Radiation Analyzer System (HP9000 Series 700/800 Version without NASADIG); (8) NASADIG - NASA Device Independent Graphics Library, Version 6.0 (VAX VMS Version); and (9) NASADIG - NASA Device Independent Graphics Library, Version 6.0 (UNIX Version). Activities in the areas of marketing, customer service, benefits identification, maintenance and support, and dissemination are also described along with a budget summary.

Building a Digital Library for Multibeam Data, Images and Documents

NASA Astrophysics Data System (ADS)

Miller, S. P.; Staudigel, H.; Koppers, A.; Johnson, C.; Cande, S.; Sandwell, D.; Peckman, U.; Becker, J. J.; Helly, J.; Zaslavsky, I.; Schottlaender, B. E.; Starr, S.; Montoya, G.

2001-12-01

The Scripps Institution of Oceanography, the UCSD Libraries and the San Diego Supercomputing Center have joined forces to establish a digital library for accessing a wide range of multibeam and marine geophysical data, to a community that ranges from the MGG researcher to K-12 outreach clients. This digital library collection will include 233 multibeam cruises with grids, plots, photographs, station data, technical reports, planning documents and publications, drawn from the holdings of the Geological Data Center and the SIO Archives. Inquiries will be made through an Ocean Exploration Console, reminiscent of a cockpit display where a multitude of data may be displayed individually or in two or three-dimensional projections. These displays will provide access to cruise data as well as global databases such as Global Topography, crustal age, and sediment thickness, thus meeting the day-to-day needs of researchers as well as educators, students, and the public. The prototype contains a few selected expeditions, and a review of the initial approach will be solicited from the user community during the poster session. The search process can be focused by a variety of constraints: geospatial (lat-lon box), temporal (e.g., since 1996), keyword (e.g., cruise, place name, PI, etc.), or expert-level (e.g., K-6 or researcher). The Storage Resource Broker (SRB) software from the SDSC manages the evolving collection as a series of distributed but related archives in various media, from shipboard data through processing and final archiving. The latest version of MB-System provides for the systematic creation of standard metadata, and for the harvesting of metadata from multibeam files. Automated scripts will be used to load the metadata catalog to enable queries with an Oracle database management system. These new efforts to bridge the gap between libraries and data archives are supported by the NSF Information Technology and National Science Digital Library (NSDL) programs, augmented by UC funds, and closely coordinated with Digital Library for Earth System Education (DLESE) activities.

The Computer as a Tool for Learning

PubMed Central

Starkweather, John A.

1986-01-01

Experimenters from the beginning recognized the advantages computers might offer in medical education. Several medical schools have gained experience in such programs in automated instruction. Television images and graphic display combined with computer control and user interaction are effective for teaching problem solving. The National Board of Medical Examiners has developed patient-case simulation for examining clinical skills, and the National Library of Medicine has experimented with combining media. Advances from the field of artificial intelligence and the availability of increasingly powerful microcomputers at lower cost will aid further development. Computers will likely affect existing educational methods, adding new capabilities to laboratory exercises, to self-assessment and to continuing education. PMID:3544511

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

PubMed

Solforosi, Laura; Mancini, Nicasio; Canducci, Filippo; Clementi, Nicola; Sautto, Giuseppe Andrea; Diotti, Roberta Antonia; Clementi, Massimo; Burioni, Roberto

2012-07-01

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Antibody phage display: overview of a powerful technology that has quickly translated to the clinic.

PubMed

Kotlan, Beatrix; Glassy, Mark C

2009-01-01

Antibody-based immunologic reagents are useful for identifying, isolating, or eliminating cells with particular characteristics related to different diseases. Phage display is a highly valuable technique for antibody selection related to this purpose. In brief, a diverse group of antibody genes prepared from a patient or generated in vitro are inserted into a phagemid vector or the phage genome so that when the protein is expressed, it becomes anchored on the surface of the phage by fusion to a coat protein. A diverse library of recombinant antibodies is generated in this way and can then be exposed or panned on the antigen of interest, typically, this being a molecule associated with a particular pathological condition. Phage that carry proteins or peptides bind preferentially to the target and can thus be isolated from the library. The viruses that are recovered in this way also carry the gene for the binding moiety facilitating its over-expression or manipulation. Recent reviews highlight key milestones in the development of antibody libraries and their screening by phage display, and the impact of these technologies on drug discovery seems assured.

Binding of leachable components of polymethyl methacrylate (PMMA) and peptide on modified SPR chip

NASA Astrophysics Data System (ADS)

Szaloki, M.; Vitalyos, G.; Harfalvi, J.; Hegedus, Cs

2013-12-01

Many types of polymers are often used in dentistry, which may cause allergic reaction, mainly methyl methacrylate allergy due to the leachable, degradable components of polymerized dental products. The aim of this study was to investigate the interaction between the leachable components of PMMA and peptides by Fourier-transform Surface Plasmon Resonance (FT SPR). In our previous work binding of oligopeptides (Ph.D.-7 and Ph.D.-12 Peptide Library Kit) was investigated to PMMA surface by phage display technique. It was found that oligopeptides bounded specifically to PMMA surface. The most common amino acids were leucine and proline inside the amino acids sequences of DNA of phages. The binding of haptens, as formaldehyde and methacrylic acid, to frequent amino acids was to investigate on the modified gold SPR chip. Self assembled monolayer (SAM) modified the surface of gold chip and ensured the specific binding between the haptens and amino acids. It was found that amino acids bounded to modified SPR gold and the haptens bounded to amino acids by creating multilayer on the chip surface. By the application of phage display and SPR modern bioanalytical methods the interaction between allergens and peptides can be investigated.

High Tech and Library Access for People with Disabilities.

ERIC Educational Resources Information Center

Roatch, Mary A.

1992-01-01

Describes tools that enable people with disabilities to access print information, including optical character recognition, synthetic voice output, other input devices, Braille access devices, large print displays, television and video, TDD (Telecommunications Devices for the Deaf), and Telebraille. Use of technology by libraries to meet mandates…

LS/2000--The Integrated Library System from OCLC.

ERIC Educational Resources Information Center

Olson, Susan

1984-01-01

Discusses design features of the Online Catalog of LS/2000, OCLC's enhanced version of Integrated Library System. This minicomputer-based system provides bibliographic file maintenance, circulation control, and online catalog searching. Examples of available displays--holdings, full MARC, work forms, keyword entry, index selection, brief citation,…

Advances in Projection Technology for On-Line Instruction.

ERIC Educational Resources Information Center

Davis, H. Scott; Miller, Marsha

This document consists of supplemental information designed to accompany a presentation on the application of projection technology, including video projectors and liquid crystal display (LCD) devices, in the online catalog library instruction program at the Indiana State University libraries. Following an introductory letter, the packet includes:…

Marketing Books in the School Library.

ERIC Educational Resources Information Center

Langhorne, Mary Jo

1987-01-01

Circulation increased and student attitudes toward paperback books in a junior high school library media center collection improved when chain bookstore marketing techniques were used, i.e., topical arrangement; high visibility; clear, colorful labels; books shelved face out; and moveable display racks. These techniques are recommended with some…

Six Online Periodical Databases: A Librarian's View.

ERIC Educational Resources Information Center

Willems, Harry

1999-01-01

Compares the following World Wide Web-based periodical databases, focusing on their usefulness in K-12 school libraries: EBSCO, Electric Library, Facts on File, SIRS, Wilson, and UMI. Search interfaces, display options, help screens, printing, home access, copyright restrictions, database administration, and making a decision are discussed. A…

Selection of peptides binding to metallic borides by screening M13 phage display libraries.

PubMed

Ploss, Martin; Facey, Sandra J; Bruhn, Carina; Zemel, Limor; Hofmann, Kathrin; Stark, Robert W; Albert, Barbara; Hauer, Bernhard

2014-02-10

Metal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials. In this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles. This study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

Wide screening of phage-displayed libraries identifies immune targets in planta.

PubMed

Rioja, Cristina; Van Wees, Saskia C; Charlton, Keith A; Pieterse, Corné M J; Lorenzo, Oscar; García-Sánchez, Susana

2013-01-01

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7) different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies.

PubMed

Romao, Ema; Morales-Yanez, Francisco; Hu, Yaozhong; Crauwels, Maxine; De Pauw, Pieter; Hassanzadeh, Gholamreza Ghassanzadeh; Devoogdt, Nick; Ackaert, Chloe; Vincke, Cecile; Muyldermans, Serge

2016-01-01

The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system.

PubMed

Li, Jingquan; Xu, Yongping; Wang, Xitao; Li, Yuan; Wang, Lili; Li, Xiaoyu

2016-06-01

The purpose of this study was to construct a single-chain variable fragment (scFv) antibody from chicken egg yolk immunoglobulin (IgY) by means of genetic engineering and subsequent panning for a specific antibody against Staphylococcus aureus. We amplified the scFv using blood and spleen obtained from 100-day-old Roman chickens immunized with inactivated S. aureus and subsequently constructed a T7 phage display antibody library using phage display technology. Four non-repeated blood scFv and 6 spleen scFv were obtained following 3 rounds of panning of the T7 phage display antibody library, enzyme-linked immunosorbent assay and sequencing. These 10 scFv were cloned into the prokaryotic expression vector pCold I with expression induced at a low temperature. Four soluble proteins were obtained. Among them, soluble protein SFV6 derived from the spleen showed good reactivity against S. aureus using indirect ELISA and produced a particularly strong antibacterial effect in vitro. We were successful in isolating a highly specific scFv antibody against S. aureus from the spleen phage display library. This study provides a simple and rapid method for the quick preparation of a large number of antibodies against S. aureus and provides the foundation for the positioning of antibodies in the organism and the study of the antibacterial mechanism through which the antibody functions. Copyright © 2016 Elsevier B.V. All rights reserved.

MINDS: A microcomputer interactive data system for 8086-based controllers

NASA Technical Reports Server (NTRS)

Soeder, J. F.

1985-01-01

A microcomputer interactive data system (MINDS) software package for the 8086 family of microcomputers is described. To enhance program understandability and ease of code maintenance, the software is written in PL/M-86, Intel Corporation's high-level system implementation language. The MINDS software is intended to run in residence with real-time digital control software to provide displays of steady-state and transient data. In addition, the MINDS package provides classic monitor capabilities along with extended provisions for debugging an executing control system. The software uses the CP/M-86 operating system developed by Digital Research, Inc., to provide program load capabilities along with a uniform file structure for data and table storage. Finally, a library of input and output subroutines to be used with consoles equipped with PL/M-86 and assembly language is described.

New library building: Mercer University School of Medicine, Macon, Georgia.

PubMed Central

Rankin, J A; Bernard, G R

1984-01-01

The Mercer University School of Medicine (MUSM) enrolled its charter class in 1982. The curriculum is problem-based and adaptable to the learning needs of each student. MUSM is housed in a new building designed to support this unique educational program. Its library is an example of a comparatively small, but fully functional, medical school library. The planning process, design, and layout of the new library facility are described. Among its unique features are an integrated print and non-print collection, current periodical display space, and extensive use of task lighting. PMID:6733330

Uniform amplification of phage display libraries in monodisperse emulsions.

PubMed

Matochko, Wadim L; Ng, Simon; Jafari, Mohammad R; Romaniuk, Joseph; Tang, Sindy K Y; Derda, Ratmir

2012-09-01

In this paper, we describe a complete experimental setup for the uniform amplification of libraries of phage. Uniform amplification, which multiplies every phage clone by the same amount irrespective of the growth rate of the clone is essential for phage-display screening. Amplification of phage libraries in a common solution is often non-uniform: it favors fast-growing clones and eliminates those that grow slower. This competition leads to elimination of many useful binding clones, and it is a major barrier to identification of ligands for targets with multiple binding sites such as cells, tissues, or mixtures of proteins. Uniform amplification is achieved by encapsulating individual phage clones into isolated compartments (droplets) of identical volume. Each droplet contains culture medium and an excess of host (Escherichia coli). Here, we describe microfluidics devices that generate mono-disperse droplet-based compartments, and optimal conditions for amplification of libraries of different size. We also describe the detailed synthesis of a perfluoro surfactant, which gives droplets exceptional stability. Droplets stabilized by this compound do not coalesce after many hours in shaking culture. We identified a commercially available compound (Krytox), which destabilizes these droplets to recover the amplified libraries. Overall, uniform amplification is a sequence of three simple steps: (1) encapsulation of mixture of phage and bacteria in droplets using microfluidics; (2) incubation of droplets in a shaking culture; (3) destabilization of droplets to harvest the amplified phage. We anticipate that this procedure can be easily adapted in any academic or industrial laboratory that uses phage display. Copyright © 2012 Elsevier Inc. All rights reserved.

Diagnostic value of protein chips constructed by lung-cancer-associated markers selected by the T7 phage display library.

PubMed

Li, Hong-Mei; Guo, Kang; Yu, Zhuang; Feng, Rui; Xu, Ping

2015-07-01

Traditional diagnostic technology with tumor biomarkers is inefficient, expensive and requires a large number of serum samples. The purpose of this study was to construct human lung cancer protein chips with new lung cancer biomarkers screened by the T7-phage display library, and improve the early diagnosis rate of lung cancer. A T7-phage cDNA display library was constructed of fresh samples from 30 lung cancer patients. With biopanning and high-throughput screening, we gained the immunogenic phage clones from the cDNA library. The insert of selected phage was blasted at GeneBank for alignment to find the exact or the most similar known genes. Protein chips were then constructed and used to assay their expression level in lung cancer serum from 217 cases of lung cancer groups:80 cases of benign lung disease and 220 healthy controls. After four rounds of Biopanning and two rounds of enzyme-linked immunosorbent assay, 12 phage monoclonal samples were selected from 2880 phage monoclonal samples. After blasting at GeneBank, six similar genes were used to construct diagnostic protein chips. The protein chips were then used to assay expression level in lung cancer serum. The expression level of six genes in lung cancer groups was significantly higher than those in the other two groups (P < 0.05). In this study, we successfully constructed diagnostic protein chips with biomarkers selected from the lung cancer T7-phage cDNA library, which can be used for the early screening of lung cancer patients.

Phage display of engineered binding proteins.

PubMed

Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

2014-01-01

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

Testing the Effectiveness of Interactive Multimedia for Library-User Education

ERIC Educational Resources Information Center

Markey, Karen; Armstrong, Annie; De Groote, Sandy; Fosmire, Michael; Fuderer, Laura; Garrett, Kelly; Georgas, Helen; Sharp, Linda; Smith, Cheri; Spaly, Michael; Warner, Joni E.

2005-01-01

A test of the effectiveness of interactive multimedia Web sites demonstrates that library users' topic knowledge was significantly greater after visiting the sites than before. Library users want more such sites about library services, their majors, and campus life generally. Librarians describe the roles they want to play on multimedia production…

Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment.

PubMed

Stewart, Alex; Harrison, Joseph S; Regula, Lauren K; Lai, Jonathan R

2013-04-08

Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464-5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1-3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high-affinity interaction involving an antibody from the VH1-69 germline segment.

CD-ROM and Libraries.

ERIC Educational Resources Information Center

Murphy, Brower

1985-01-01

The Compact Disc-Read Only Memory (CD-ROM) data format is explained and illustrated, noting current and potential applications. The "5-inch" compact laserdisc is described and photographs of an IBM PC/Hitachi CD-ROM system adopted by Library Corporation to support its MARC database--BiblioFile--are presented. Screen displays for…

Teen Area, Solon Branch, Cuyahoga County Public Library, Solon, Ohio.

ERIC Educational Resources Information Center

Voice of Youth Advocates, 2003

2003-01-01

Describes the teen area of the Solon Branch library in Cuyahoga County (Ohio). Highlights include the collection; catalog computers; hours and teen traffic; planning the space, including extra display units of bulletin boards; teen programming, including monthly programs and a summer reading program; and the teen advisory group. (LRW)

The Integrated Library System (ILS): User Manual.

DTIC Science & Technology

1981-07-01

high-frequency words which in themselves carry little meaning, e.g., the words "and,", "for," or "Mr." and are therefore not useful as searching...children boysbratsgirls,kids,tots boys brats,males girls females The synonyms displayed for children are: boys.bzats,qrls,kids,tots. The synonyms...displayed for boys are: brats.childrenboys. The synonyms displayed for girls are: children,females. The synonyms displayed for kids are: children. The

IDSP- INTERACTIVE DIGITAL SIGNAL PROCESSOR

NASA Technical Reports Server (NTRS)

Mish, W. H.

1994-01-01

The Interactive Digital Signal Processor, IDSP, consists of a set of time series analysis "operators" based on the various algorithms commonly used for digital signal analysis work. The processing of a digital time series to extract information is usually achieved by the application of a number of fairly standard operations. However, it is often desirable to "experiment" with various operations and combinations of operations to explore their effect on the results. IDSP is designed to provide an interactive and easy-to-use system for this type of digital time series analysis. The IDSP operators can be applied in any sensible order (even recursively), and can be applied to single time series or to simultaneous time series. IDSP is being used extensively to process data obtained from scientific instruments onboard spacecraft. It is also an excellent teaching tool for demonstrating the application of time series operators to artificially-generated signals. IDSP currently includes over 43 standard operators. Processing operators provide for Fourier transformation operations, design and application of digital filters, and Eigenvalue analysis. Additional support operators provide for data editing, display of information, graphical output, and batch operation. User-developed operators can be easily interfaced with the system to provide for expansion and experimentation. Each operator application generates one or more output files from an input file. The processing of a file can involve many operators in a complex application. IDSP maintains historical information as an integral part of each file so that the user can display the operator history of the file at any time during an interactive analysis. IDSP is written in VAX FORTRAN 77 for interactive or batch execution and has been implemented on a DEC VAX-11/780 operating under VMS. The IDSP system generates graphics output for a variety of graphics systems. The program requires the use of Versaplot and Template plotting routines and IMSL Math/Library routines. These software packages are not included in IDSP. The virtual memory requirement for the program is approximately 2.36 MB. The IDSP system was developed in 1982 and was last updated in 1986. Versaplot is a registered trademark of Versatec Inc. Template is a registered trademark of Template Graphics Software Inc. IMSL Math/Library is a registered trademark of IMSL Inc.

Electron lithography STAR design guidelines. Part 1: The STAR user design manual

NASA Technical Reports Server (NTRS)

Trotter, J. D.; Newman, W.

1982-01-01

The STAR system developed by NASA enables any user with a logic diagram to design a semicustom digital MOS integrated circuit. The system is comprised of a library of standard logic cells and computer programs to place, route, and display designs implemented with cells from the library. Library cells of the CMOS metal gate and CMOS silicon gate technologies were simulated using SPICE, and the results are shown and compared.

LINCS Canvas Browser: interactive web app to query, browse and interrogate LINCS L1000 gene expression signatures.

PubMed

Duan, Qiaonan; Flynn, Corey; Niepel, Mario; Hafner, Marc; Muhlich, Jeremy L; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Chen, Edward Y; Golub, Todd R; Sorger, Peter K; Subramanian, Aravind; Ma'ayan, Avi

2014-07-01

For the Library of Integrated Network-based Cellular Signatures (LINCS) project many gene expression signatures using the L1000 technology have been produced. The L1000 technology is a cost-effective method to profile gene expression in large scale. LINCS Canvas Browser (LCB) is an interactive HTML5 web-based software application that facilitates querying, browsing and interrogating many of the currently available LINCS L1000 data. LCB implements two compacted layered canvases, one to visualize clustered L1000 expression data, and the other to display enrichment analysis results using 30 different gene set libraries. Clicking on an experimental condition highlights gene-sets enriched for the differentially expressed genes from the selected experiment. A search interface allows users to input gene lists and query them against over 100 000 conditions to find the top matching experiments. The tool integrates many resources for an unprecedented potential for new discoveries in systems biology and systems pharmacology. The LCB application is available at http://www.maayanlab.net/LINCS/LCB. Customized versions will be made part of the http://lincscloud.org and http://lincs.hms.harvard.edu websites. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing.

PubMed

Liu, Gary W; Livesay, Brynn R; Kacherovsky, Nataly A; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C; Salipante, Stephen J; Pun, Suzie H

2015-08-19

Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.

Production of Recombinant Human scFv Against Tetanus Toxin Heavy Chain by Phage Display Technology.

PubMed

Khalili, Ehsan; Lakzaei, Mostafa; Rasaee, Mohhamad Javad; Aminian, Mahdi

2015-10-01

Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of antibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.

Detection of Cystic Fibrosis Serological Biomarkers Using a T7 Phage Display Library.

PubMed

Talwar, Harvinder; Hanoudi, Samer Najeeb; Geamanu, Andreea; Kissner, Dana; Draghici, Sorin; Samavati, Lobelia

2017-12-18

Cystic fibrosis (CF) is an autosomal recessive disorder affecting the cystic fibrosis transmembrane conductance regulator (CFTR). CF is characterized by repeated lung infections leading to respiratory failure. Using a high-throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients. This library was biopanned to obtain 1070 potential antigens. A microarray platform was constructed and immunoscreened with sera from healthy (n = 49), lung cancer (LC) (n = 31) and CF (n = 31) subjects. We built 1,000 naïve Bayes models on the training sets. We selected the top 20 frequently significant clones ranked with student t-test discriminating CF antigens from healthy controls and LC at a False Discovery Rate (FDR) < 0.01. The performances of the models were validated on an independent validation set. The mean of the area under the receiver operating characteristic (ROC) curve for the classifiers was 0.973 with a sensitivity of 0.999 and specificity of 0.959. Finally, we identified CF specific clones that correlate highly with sweat chloride test, BMI, and FEV1% predicted values. For the first time, we show that CF specific serological biomarkers can be identified through immunocreenings of a T7 phage display library with high accuracy, which may have utility in development of molecular therapy.

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

PubMed

Yang, Xiao; Lennard, Katherine R; He, Chang; Walker, Mark C; Ball, Andrew T; Doigneaux, Cyrielle; Tavassoli, Ali; van der Donk, Wilfred A

2018-04-01

In this article we describe the production and screening of a genetically encoded library of 10 6 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

PubMed Central

Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels

2006-01-01

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566

An IBM-compatible program for interactive three-dimensional gravity modeling

NASA Astrophysics Data System (ADS)

Broome, John

1992-04-01

G3D is a 3-D interactive gravity modeling program for IBM-compatible microcomputers. The program allows a model to be created interactively by defining multiple tabular bodies with horizontal tops and bottoms. The resulting anomaly is calculated using Plouff's algorithm at up to 2000 predefined random or regularly located points. In order to display the anomaly as a color image, the point data are interpolated onto a regular grid and quantized into discrete intervals. Observed and residual gravity field images also can be generated. Adjustments to the model are made using a graphics cursor to move, insert, and delete body points or whole bodies. To facilitate model changes, planview body outlines can be overlain on any of the gravity field images during editing. The model's geometry can be displayed in planview or along a user-defined vertical section. G3D is written in Microsoft® FORTRAN and utilizes the Halo-Professional® (or Halo-88®) graphics subroutine library. The program is written for use on an IBM-compatible microcomputer equipped with hard disk, numeric coprocessor, and VGA, Number Nine Revolution (Halo-88® only), or TIGA® compatible graphics cards. A mouse or digitizing tablet is recommended for cursor positioning. Program source code, a user's guide, and sample data are available as Geological Survey of Canada Open File (G3D: A Three-dimensional Gravity Modeling Program for IBM-compatible Microcomputers).

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peabody, David S.; Chackerian, Bryce; Ashley, Carlee

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 referredmore » 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.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

PubMed Central

Yu, Hai-bo; Zou, Bei-yan; Wang, Xiao-liang; Li, Min

2016-01-01

Aim: hERG potassium channels display miscellaneous interactions with diverse chemical scaffolds. In this study we assessed the hERG inhibition in a large compound library of diverse chemical entities and provided data for better understanding of the mechanisms underlying promiscuity of hERG inhibition. Methods: Approximately 300 000 compounds contained in Molecular Library Small Molecular Repository (MLSMR) library were tested. Compound profiling was conducted on hERG-CHO cells using the automated patch-clamp platform–IonWorks Quattro™. Results: The compound library was tested at 1 and 10 μmol/L. IC50 values were predicted using a modified 4-parameter logistic model. Inhibitor hits were binned into three groups based on their potency: high (IC50<1 μmol/L), intermediate (1 μmol/L< IC50<10 μmol/L), and low (IC50>10 μmol/L) with hit rates of 1.64%, 9.17% and 16.63%, respectively. Six physiochemical properties of each compound were acquired and calculated using ACD software to evaluate the correlation between hERG inhibition and the properties: hERG inhibition was positively correlative to the physiochemical properties ALogP, molecular weight and RTB, and negatively correlative to TPSA. Conclusion: Based on a large diverse compound collection, this study provides experimental evidence to understand the promiscuity of hERG inhibition. This study further demonstrates that hERG liability compounds tend to be more hydrophobic, high-molecular, flexible and polarizable. PMID:26725739

Old Books Bring New Life to the Brick and Mortar Library

DTIC Science & Technology

2012-08-01

ancient astronomy books, including Copernicus, Kepler, Galileo, and Newton, we have abundant resources. The presentation will highlight the varied...library. One method was to rotate rare book displays each month. As the library holds a fabulous collection of ancient astronomy books, including Copernicus...800 and a ticket to Europe to purchase important astronomy books. It is a sanctuary for some. For me, it is a lonely place and I wanted more people to

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.

PubMed

Makeyev, E V; Kolb, V A; Spirin, A S

1999-02-12

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.

Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

PubMed

Huang, Renhua; Fang, Pete; Kay, Brian K

2012-09-01

Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

Interactive Learning Modules: Enabling Near Real-Time Oceanographic Data Use In Undergraduate Education

NASA Astrophysics Data System (ADS)

Kilb, D. L.; Fundis, A. T.; Risien, C. M.

2012-12-01

The focus of the Education and Public Engagement (EPE) component of the NSF's Ocean Observatories Initiative (OOI) is to provide a new layer of cyber-interactivity for undergraduate educators to bring near real-time data from the global ocean into learning environments. To accomplish this, we are designing six online services including: 1) visualization tools, 2) a lesson builder, 3) a concept map builder, 4) educational web services (middleware), 5) collaboration tools and 6) an educational resource database. Here, we report on our Fall 2012 release that includes the first four of these services: 1) Interactive visualization tools allow users to interactively select data of interest, display the data in various views (e.g., maps, time-series and scatter plots) and obtain statistical measures such as mean, standard deviation and a regression line fit to select data. Specific visualization tools include a tool to compare different months of data, a time series explorer tool to investigate the temporal evolution of select data parameters (e.g., sea water temperature or salinity), a glider profile tool that displays ocean glider tracks and associated transects, and a data comparison tool that allows users to view the data either in scatter plot view comparing one parameter with another, or in time series view. 2) Our interactive lesson builder tool allows users to develop a library of online lesson units, which are collaboratively editable and sharable and provides starter templates designed from learning theory knowledge. 3) Our interactive concept map tool allows the user to build and use concept maps, a graphical interface to map the connection between concepts and ideas. This tool also provides semantic-based recommendations, and allows for embedding of associated resources such as movies, images and blogs. 4) Education web services (middleware) will provide an educational resource database API.

Science Outreach through Art: A Journal Article Cover Gallery

ERIC Educational Resources Information Center

McCullough, Ian

2015-01-01

Research faculty journal covers were used to create a gallery in the Science & Technology branch library at the University of Akron. The selection, presentation, and promotion process is shared along with copyright considerations and a review of galleries used for library outreach. The event and display was a great success attracting faculty…

Makeover Madness: Tips for Revamping your Young Adult Area.

ERIC Educational Resources Information Center

Bolan, Kimberly; Wemett, Lisa C.

1999-01-01

Presents tips from a project that made modest but significant improvements to the young adult areas and services of 11 rural libraries in the Pioneer Library System in upstate New York. Discusses floor plans, layout and location; furniture and fixtures; collections and displays; technology; and staff. Illustrates changes with before-and-after…

Management of Library Pedagogical Development: From Models to Statistics.

ERIC Educational Resources Information Center

Herron, David

Pedagogical research and empirical studies show that student learning is encouraged by a number of general characteristics displayed by teachers (Ramsden, 1992). One of them is enthusiasm for the subject and for teaching it. User-education in research libraries is often highly repetitive and contact time with students is limited. So the question…

Speaking up for Equity Takes Courage--But the Standards Have Your Back

ERIC Educational Resources Information Center

Lechtenberg, Kate; Phillips, Jeanie

2018-01-01

The National School Library Standards require school librarians to make equity a value that permeates the entire school library community. Creating displays to celebrate diversity is not enough. We cannot allow ourselves to approach diversity as a "social good," in which isolated programs serve marginalized students without challenging…

User Experience on Display

ERIC Educational Resources Information Center

Schmidt, Aaron

2010-01-01

What do a pink door, reading glasses, a website, and a Beetle Kill Pine ceiling have in common? Yes, readers will find them all in this collection of great design in libraries. But the reason they're featured is their important commonality: they were intentionally and thoughtfully placed in a library to meet people's needs or otherwise engage…

A Library Network for the Geosciences.

ERIC Educational Resources Information Center

Olsen, Wallace C.

The concept paper prepared by the American Geological Institute (AGI) Committee on Geoscience Information is evaluated and areas which need more detailed plans if the geoscience community is to be persuaded of the need for a library network are discussed. For example: the concept plan does not display adequate awareness or concern for the role of…

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

PubMed

Schirrmann, Thomas; Hust, Michael

2010-01-01

Recombinant antibodies as therapeutics offer new opportunities for the treatment of many tumor diseases. To date, 18 antibody-based drugs are approved for cancer treatment and hundreds of anti-tumor antibodies are under development. The first clinically approved antibodies were of murine origin or human-mouse chimeric. However, since murine antibody domains are immunogenic in human patients and could result in human anti-mouse antibody (HAMA) responses, currently mainly humanized and fully human antibodies are developed for therapeutic applications.Here, in vitro antibody selection technologies directly allow the selection of human antibodies and the corresponding genes from human antibody gene libraries. Antibody phage display is the most common way to generate human antibodies and has already yielded thousands of recombinant antibodies for research, diagnostics and therapy. Here, we describe methods for the construction of human scFv gene libraries and the antibody selection.

Online Catalog Screen Displays. A Series of Discussions. Report of a Conference Sponsored by the Council on Library Resources (Austin, Texas, March 10-13, 1985).

ERIC Educational Resources Information Center

Williams, Joan Frye, Ed.

Papers presented and summaries of discussions at a 3-day conference which focused on screen displays for online catalogs are included in this report. Papers presented were: (1) "Suggested Guidelines for Screen Layouts and Design of Online Catalogs" (Joseph R. Matthews); (2) "Displays in Database Search Systems" (Fran Spigai);…

Versatile microbial surface-display for environmental remediation and biofuels production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Cindy H.; Mulchandani, Ashok; Chen, wilfred

2008-02-14

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

PubMed

Lim, Theam Soon; Chan, Soo Khim

2016-01-01

Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display.

PubMed

Hacker, David E; Hoinka, Jan; Iqbal, Emil S; Przytycka, Teresa M; Hartman, Matthew C T

2017-03-17

Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.

Documentation of a graphical display program for the saturated- unsaturated transport (SUTRA) finite-element simulation model

USGS Publications Warehouse

Souza, W.R.

1987-01-01

This report documents a graphical display program for the U. S. Geological Survey finite-element groundwater flow and solute transport model. Graphic features of the program, SUTRA-PLOT (SUTRA-PLOT = saturated/unsaturated transport), include: (1) plots of the finite-element mesh, (2) velocity vector plots, (3) contour plots of pressure, solute concentration, temperature, or saturation, and (4) a finite-element interpolator for gridding data prior to contouring. SUTRA-PLOT is written in FORTRAN 77 on a PRIME 750 computer system, and requires Version 9.0 or higher of the DISSPLA graphics library. The program requires two input files: the SUTRA input data list and the SUTRA simulation output listing. The program is menu driven and specifications for individual types of plots are entered and may be edited interactively. Installation instruction, a source code listing, and a description of the computer code are given. Six examples of plotting applications are used to demonstrate various features of the plotting program. (Author 's abstract)

Identification of host factors potentially involved in RTM-mediated resistance during potyvirus long distance movement.

PubMed

Sofer, Luc; Cabanillas, Daniel Garcia; Gayral, Mathieu; Téplier, Rachèle; Pouzoulet, Jérôme; Ducousso, Marie; Dufin, Laurène; Bréhélin, Claire; Ziegler-Graff, Véronique; Brault, Véronique; Revers, Frédéric

2017-07-01

The long distance movement of potyviruses is a poorly understood step of the viral cycle. Only factors inhibiting this process, referred to as "Restricted TEV Movement" (RTM), have been identified in Arabidopsis thaliana. On the virus side, the potyvirus coat protein (CP) displays determinants required for long-distance movement and for RTM-based resistance breaking. However, the potyvirus CP was previously shown not to interact with the RTM proteins. We undertook the identification of Arabidopsis factors which directly interact with either the RTM proteins or the CP of lettuce mosaic virus (LMV). An Arabidopsis cDNA library generated from companion cells was screened with LMV CP and RTM proteins using the yeast two-hybrid system. Fourteen interacting proteins were identified. Two of them were shown to interact with CP and the RTM proteins suggesting that a multiprotein complex could be formed between the RTM proteins and virions or viral ribonucleoprotein complexes. Co-localization experiments in Nicotiana benthamiana showed that most of the viral and cellular protein pairs co-localized at the periphery of chloroplasts which suggests a putative role for plastids in this process.

Identification and analysis of unsatisfactory psychosocial work situations: a participatory approach employing video-computer interaction.

PubMed

Hanse, J J; Forsman, M

2001-02-01

A method for psychosocial evaluation of potentially stressful or unsatisfactory situations in manual work was developed. It focuses on subjective responses regarding specific situations and is based on interactive worker assessment when viewing video recordings of oneself. The worker is first video-recorded during work. The video is then displayed on the computer terminal, and the filmed worker clicks on virtual controls on the screen whenever an unsatisfactory psychosocial situation appears; a window of questions regarding psychological demands, mental strain and job control is then opened. A library with pictorial information and comments on the selected situations is formed in the computer. The evaluation system, called PSIDAR, was applied in two case studies, one of manual materials handling in an automotive workshop and one of a group of workers producing and testing instrument panels. The findings indicate that PSIDAR can provide data that are useful in a participatory ergonomic process of change.

Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL.

PubMed

Schieferdecker, Aneta; Voigt, Mareike; Riecken, Kristoffer; Braig, Friederike; Schinke, Thorsten; Loges, Sonja; Bokemeyer, Carsten; Fehse, Boris; Binder, Mascha

2014-08-30

Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2006-07-01

display vectors to allow for both lytic and filamentous versions of the library. For lytic phage display, we chose the T7 -based vector T7Select®10-3b... phage , which do not display a FLAG epitope (data not shown). These data suggest that peptides can be displayed on the surface of T7 using this system...Auto-Antibodies as Breast Cancer Biomarkers To identify auto-antibodies that could be used as breast cancer biomarkers, we are generating a phage

A specific RAGE-binding peptide biopanning from phage display random peptide library that ameliorates symptoms in amyloid β peptide-mediated neuronal disorder.

PubMed

Cai, Cuizan; Dai, Xiaoyong; Zhu, Yujie; Lian, Mengyang; Xiao, Fei; Dong, Fangyuan; Zhang, Qihao; Huang, Yadong; Zheng, Qing

2016-01-01

Alzheimer's disease (AD) is an age-related neurodegenerative disorder in which amyloid β (Aβ) peptide accumulates in the brain. The receptor for advanced glycation end product (RAGE) is a cellular binding site for Aβ peptide and mediates amyloid β-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a specific high-affinity RAGE inhibitor (APDTKTQ named RP-1) from a phage display library. RP-1 bound to RAGE and inhibited Aβ peptide-induced cellular stress in human neuroblastoma SH-SYSY cells in vitro. Three amino acids in RP-1 are identical to those in the Aβ peptide. RP-1 shows high homology to the 16-23 (KLVFFAED) regions in Aβ peptide and high-affinity RAGE. Functional analyses indicated that RP-1 significantly reduced the level of reactive oxygen species (ROS) and ROS products and that it enhanced catalase and glutathione peroxidase (GPx) activity. Furthermore, it inactivated caspase3 and caspase9 and inhibited the upregulation of RAGE, nuclear factor-κB (NF-κB), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein expression. In addition, RP-1 activated the PI3K/AKT signaling pathway, inhibiting the interaction between Bax and Bcl-2. Our data suggest that RP-1 is a potent RAGE blocker that effectively controls the progression of Aβ peptide-mediated brain disorders and that it may have potential as a disease-modifying agent for AD.

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations

PubMed Central

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W.; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-01-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. PMID:24792385

Creating stable stem regions for loop elongation in Fcabs - insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations.

PubMed

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-09-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. Copyright © 2014. Published by Elsevier B.V.

Measuring patrons' technology habits: an evidence-based approach to tailoring library services

PubMed Central

Wu, Jin; Chatfield, Amy J.; Hughes, Annie M.; Kysh, Lynn; Rosenbloom, Megan Curran

2014-01-01

Librarians continually integrate new technologies into library services for health sciences students. Recently published data are lacking about student ownership of technological devices, awareness of new technologies, and interest in using devices and technologies to interact with the library. A survey was implemented at seven health sciences libraries to help answer these questions. Results show that librarian assumptions about awareness of technologies are not supported, and student interest in using new technologies to interact with the library varies widely. Collecting this evidence provides useful information for successfully integrating technologies into library services. PMID:24860272

Measuring patrons' technology habits: an evidence-based approach to tailoring library services.

PubMed

Wu, Jin; Chatfield, Amy J; Hughes, Annie M; Kysh, Lynn; Rosenbloom, Megan Curran

2014-04-01

Librarians continually integrate new technologies into library services for health sciences students. Recently published data are lacking about student ownership of technological devices, awareness of new technologies, and interest in using devices and technologies to interact with the library. A survey was implemented at seven health sciences libraries to help answer these questions. Results show that librarian assumptions about awareness of technologies are not supported, and student interest in using new technologies to interact with the library varies widely. Collecting this evidence provides useful information for successfully integrating technologies into library services.

Using Phage Display to Create Recombinant Antibodies.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

A variety of phage display technologies have been developed since the approach was first described for antibodies. The most widely used approaches incorporate antibody sequences into the minor coat protein pIII of the nonlytic filamentous phage fd or M13. Libraries of variable gene sequences, encoding either scFv or Fab fragments, are made by incorporating sequences into phagemid vectors. The phagemid is packaged into phage particles with the assistance of a helper phage to produce the antibody display phage. This protocol describes a method for creating a phagemid library. The multiple cloning site (MCS) of the pBluescript KS(-) phagemid vector is replaced by digestion with the restriction enzyme BssHII, followed by the insertion of four overlapping oligonucleotides to create a new MCS within the vector. Next, the 3' portion of gene III (from M13mp18) is amplified and combined with an antibody sequence using overlap extension PCR. This product is inserted into the phagemid vector to create pPDS. Two helper plasmids are also created from the modified pBluescript vector: pLINK provides the linker between the heavy and light chains, and pFABC provides the CH1 domain of the heavy chain. An antibody cDNA library is constructed from the RNA of interest and ligated into pPDS. The phagemid library is electroporated into Escherichia coli cells along with the VCS-M13 helper phage. © 2017 Cold Spring Harbor Laboratory Press.

Single-Domain Antibodies: Rugged Recognition Element

DTIC Science & Technology

2007-01-01

and spiny dogfish shark (60 million representatives), which are displayed on filamentous phage M13 . We selected the llama- derived library for binding...subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE

Biomolecular Recognition of Semiconductors and Magnetic Materials to Assemble Nanoparticle Heterostructures

DTIC Science & Technology

2002-01-01

shown that engineered viruses can recognize specific semiconductor surfaces through the selection by combinatorial phage display method. These specific... phage display libraries. The screening method selected for binding affinity of a population of random peptides displayed as part of the pIII minor coat...shorter spacing than expected distance ( M13 phage length: 880 nm) corresponds to the length scale imposed by the phage which formed the tilted

IceT users' guide and reference.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moreland, Kenneth D.

2011-01-01

The Image Composition Engine for Tiles (IceT) is a high-performance sort-last parallel rendering library. In addition to providing accelerated rendering for a standard display, IceT provides the unique ability to generate images for tiled displays. The overall resolution of the display may be several times larger than any viewport that may be rendered by a single machine. This document is an overview of the user interface to IceT.

Pace's Maxims for Homegrown Library Projects. Coming Full Circle

ERIC Educational Resources Information Center

Pace, Andrew K.

2005-01-01

This article discusses six maxims by which to run library automation. The following maxims are discussed: (1) Solve only known problems; (2) Avoid changing data to fix display problems; (3) Aut viam inveniam aut faciam; (4) If you cannot make it yourself, buy something; (5) Kill the alligator closest to the boat; and (6) Just because yours is…

Library and Information Science Research Areas: A Content Analysis of Articles from the Top 10 Journals 2007-8

ERIC Educational Resources Information Center

Aharony, Noa

2012-01-01

The current study seeks to describe and analyze journal research publications in the top 10 Library and Information Science journals from 2007-8. The paper presents a statistical descriptive analysis of authorship patterns (geographical distribution and affiliation) and keywords. Furthermore, it displays a thorough content analysis of keywords and…

N-N bond formation in Ugi processes: from nitric acid to libraries of nitramines.

PubMed

Mercalli, Valentina; Nyadanu, Aude; Cordier, Marie; Tron, Gian Cesare; Grimaud, Laurence; El Kaim, Laurent

2017-02-09

The Ugi reaction has drawn considerable attention over the years leading to numerous libraries of heterocycles and various extensions changing the nature of the components of the coupling. We report here the use of nitric acid as carboxylic acids surrogates, displaying the first aminative Ugi-type reaction leading to nitramines.

Report of Library Services and Construction Act Project # 2842, January 1 - June 30, 1967.

ERIC Educational Resources Information Center

Los Angeles Public Library, CA.

The third of the Los Angeles Public Library's semi-annual reports on its federal project for the disadvantaged includes individual staff reports from the Central Region, Bookmobile Division, Lincoln Heights, Venice, The Display Artist, and a student worker. In these reports are discussions of (1) conditions and people in the communities served,…

A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

PubMed

Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

2016-11-04

When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

An integrated vector system for cellular studies of phage display-derived peptides.

PubMed

Voss, Stephan D; DeGrand, Alec M; Romeo, Giulio R; Cantley, Lewis C; Frangioni, John V

2002-09-15

Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.

Macintosh and Photo-CD Technologies Provide Orientations to Southwestern College Library.

ERIC Educational Resources Information Center

Alexander, William J.; And Others

Due to a rapidly increasing demand for bibliographic instruction, Southwestern College in San Ysidro, California, devised an interactive English-Spanish multimedia library skills program using Macintosh and Kodak PhotoCD technologies. First, a PhotoCD containing 100 photos of library services was produced. Then, an interactive Macintosh program…

The Library as Ecosystem

ERIC Educational Resources Information Center

Walter, Scott

2008-01-01

Ecology is the study of interactions between organisms and their environment, and the academic library could be considered to be an ecosystem, i.e., a "biological organization" in which multiple species must interact, both with one another and with their environment. The metaphor of the library as ecosystem is flexible enough to be applied not…

Biomathematical Description of Synthetic Peptide Libraries

PubMed Central

Trepel, Martin

2015-01-01

Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries. PMID:26042419

Three-Dimensional Audio Client Library

NASA Technical Reports Server (NTRS)

Rizzi, Stephen A.

2005-01-01

The Three-Dimensional Audio Client Library (3DAudio library) is a group of software routines written to facilitate development of both stand-alone (audio only) and immersive virtual-reality application programs that utilize three-dimensional audio displays. The library is intended to enable the development of three-dimensional audio client application programs by use of a code base common to multiple audio server computers. The 3DAudio library calls vendor-specific audio client libraries and currently supports the AuSIM Gold-Server and Lake Huron audio servers. 3DAudio library routines contain common functions for (1) initiation and termination of a client/audio server session, (2) configuration-file input, (3) positioning functions, (4) coordinate transformations, (5) audio transport functions, (6) rendering functions, (7) debugging functions, and (8) event-list-sequencing functions. The 3DAudio software is written in the C++ programming language and currently operates under the Linux, IRIX, and Windows operating systems.

Multiplex single-molecule interaction profiling of DNA barcoded proteins

PubMed Central

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

Combining history of medicine and library instruction: an innovative approach to teaching database searching to medical students.

PubMed

Timm, Donna F; Jones, Dee; Woodson, Deidra; Cyrus, John W

2012-01-01

Library faculty members at the Health Sciences Library at the LSU Health Shreveport campus offer a database searching class for third-year medical students during their surgery rotation. For a number of years, students completed "ten-minute clinical challenges," but the instructors decided to replace the clinical challenges with innovative exercises using The Edwin Smith Surgical Papyrus to emphasize concepts learned. The Surgical Papyrus is an online resource that is part of the National Library of Medicine's "Turning the Pages" digital initiative. In addition, vintage surgical instruments and historic books are displayed in the classroom to enhance the learning experience.

[A novel protein equalizer based on single chain variable fragment display M13 phage library for nephropathy patient urine study].

PubMed

Zhao, Peng; Tao, Dingyin; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2009-05-01

A novel protein equalizer was developed with single chain variable fragment (scFv) library displaying M13 phage covalently bonded on monolithic cryogel. Due to the great number and various kinds of displayed scFv fragments, as well as strong and specific binding capacity between scFv fragments and proteins, a new protein equalizer technology is preferable in the pretreatment of complex protein samples. After the sample dissolved in phosphate buffer solution (PBS), it was repeatedly loaded onto the equalizer for five times, the bound proteins were in sequence eluted by 2 mol/L NaCl and 50 mmol/L Gly-HC1 (pH 2.5) solution, followed by digestion with thrombin. All proteins or peptides collected from each fraction were further analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (RPLC-ESI-MS/MS) with a serially coupled long microcolumn. Compared with the untreated samples, the identified protein number was increased from 142 to 396. Furthermore, from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results, it was found that the protein concentration difference was reduced obviously in the eluant of direct sample loading, and most high abundance proteins were identified in the eluant of NaCl. All these results demonstrate that the novel protein equalizer with scFv display M13 phage library immobilized on cyrogel could effectively reduce the dynamic range of proteins in complex samples, enabling the identification of more low abundance proteins.

QUICK - AN INTERACTIVE SOFTWARE ENVIRONMENT FOR ENGINEERING DESIGN

NASA Technical Reports Server (NTRS)

Schlaifer, R. S.

1994-01-01

QUICK provides the computer user with the facilities of a sophisticated desk calculator which can perform scalar, vector and matrix arithmetic, propagate conic orbits, determine planetary and satellite coordinates and perform other related astrodynamic calculations within a Fortran-like environment. QUICK is an interpreter, therefore eliminating the need to use a compiler or a linker to run QUICK code. QUICK capabilities include options for automated printing of results, the ability to submit operating system commands on some systems, and access to a plotting package (MASL)and a text editor without leaving QUICK. Mathematical and programming features of QUICK include the ability to handle arbitrary algebraic expressions, the capability to define user functions in terms of other functions, built-in constants such as pi, direct access to useful COMMON areas, matrix capabilities, extensive use of double precision calculations, and the ability to automatically load user functions from a standard library. The MASL (The Multi-mission Analysis Software Library) plotting package, included in the QUICK package, is a set of FORTRAN 77 compatible subroutines designed to facilitate the plotting of engineering data by allowing programmers to write plotting device independent applications. Its universality lies in the number of plotting devices it puts at the user's disposal. The MASL package of routines has proved very useful and easy to work with, yielding good plots for most new users on the first or second try. The functions provided include routines for creating histograms, "wire mesh" surface plots and contour plots as well as normal graphs with a large variety of axis types. The library has routines for plotting on cartesian, polar, log, mercator, cyclic, calendar, and stereographic axes, and for performing automatic or explicit scaling. The lengths of the axes of a plot are completely under the control of the program using the library. Programs written to use the MASL subroutines can be made to output to the Calcomp 1055 plotter, the Hewlett-Packard 2648 graphics terminal, the HP 7221, 7475 and 7550 pen plotters, the Tektronix 40xx and 41xx series graphics terminals, the DEC VT125/VT240 graphics terminals, the QMS 800 laser printer, the Sun Microsystems monochrome display, the Ridge Computers monochrome display, the IBM/PC color display, or a "dumb" terminal or printer. Programs using this library can be written so that they always use the same type of plotter or they can allow the choice of plotter type to be deferred until after program execution. QUICK is written in RATFOR for use on Sun4 series computers running SunOS. No source code is provided. The standard distribution medium for this program is a .25 inch streaming magnetic tape cartridge in UNIX tar format. An electronic copy of the documentation in ASCII format is included on the distribution medium. QUICK was developed in 1991 and is a copyrighted work with all copyright vested in NASA.

An in vivo library-versus-library selection of optimized protein-protein interactions.

PubMed

Pelletier, J N; Arndt, K M; Plückthun, A; Michnick, S W

1999-07-01

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Selection of stably folded proteins by phage-display with proteolysis.

PubMed

Bai, Yawen; Feng, Hanqiao

2004-05-01

To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.

Ribosome display: next-generation display technologies for production of antibodies in vitro.

PubMed

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Biological Nanoplatforms for Self-Assembled Electronics

DTIC Science & Technology

2015-03-24

as M13 , a virus that infects Escherichia coli. Approximately one billion different amino acid sequences are displayed on different viruses in the...sequence when contained within a phage M13 coat protein sequence, not chemically linked to the surface of phage MS2 VLPs. Thus, binding properties may...gallium arsenide in a bacteriophage M13 phage display library, MS2 VLPs modified with the metal binding peptides do not display the same activity

Building a Better Web Site: A Practical Guide to Interactivity for Libraries.

ERIC Educational Resources Information Center

Braun, Linda W.

1998-01-01

Describes selected commercial and academic Web sites providing interactive services (Amazon; Jones Library, Amherst, MA; Pine Crest Lower School, Ft. Lauderdale, FL; Barnes & Noble; Cal State's Information Literacy Tutorials; PBS's techknow site; K.I.D.S. Report), and argues that libraries that stop at links and policy statements miss…

Tumor-targeting peptides from combinatorial libraries*

PubMed Central

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S.

2018-01-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges infighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. PMID:27210583

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott

2010-06-25

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the {alpha}-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the designmore » of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques - yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis - to elucidate specificity determinants for binding to Bcl-x{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub L} selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub L}-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x{sub L} binders.« less

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

Shhh! No Opinions in the Library: "IndyKids" and Kids' Right to an Independent Press

ERIC Educational Resources Information Center

Vender, Amanda

2011-01-01

"Nintendo Power," "Sports Illustrated for Kids," and a biography of President Obama were on prominent display as the author entered the branch library in Forest Hills, Queens. The librarian looked skeptical when the author asked the librarian if she could leave copies of "IndyKids" newspapers on the free literature table. The branch manager…

Give 'em What They Want! Reorganizing Your Fiction Collection by Genre

ERIC Educational Resources Information Center

Dumas, Elizabeth P.

2005-01-01

As a former elementary library media specialist now in a new middle school library media center, it was frustrating for the author to not have enough time to assist students and discouraging to see the apathy that so many of the students displayed toward books and reading. Middle schoolers are busy with social activities, sports, and schoolwork.…

Free Oscilloscope Web App Using a Computer Mic, Built-In Sound Library, or Your Own Files

ERIC Educational Resources Information Center

Ball, Edward; Ruiz, Frances; Ruiz, Michael J.

2017-01-01

We have developed an online oscilloscope program which allows users to see waveforms by utilizing their computer microphones, selecting from our library of over 30 audio files, and opening any *.mp3 or *.wav file on their computers. The oscilloscope displays real-time signals against time. The oscilloscope has been calibrated so one can make…

Epitope Mapping by Phage Display.

PubMed

Moreira, Gustavo Marçal Schmidt Garcia; Fühner, Viola; Hust, Michael

2018-01-01

Among the molecules of the immune system, antibodies, particularly monoclonal antibodies (mAbs), have been shown to be interesting for many biological applications. Due to their ability to recognize only a unique part of their target, mAbs are usually very specific. These targets can have many different compositions, but the most common ones are proteins or peptides that are usually from outside the host, although self-proteins can also be targeted in autoimmune diseases, or in some types of cancer. The parts of a mAb that interact with its target compose the paratope, while the recognized parts of the target compose the epitope. Knowing the epitope is valuable for the improvement of a biological product, e.g., a diagnostic assay, a therapeutic mAb, or a vaccine, as well as for the elucidation of immune responses. The current techniques for epitope mapping rely on the presentation of the target, or parts of it, in a way that it can interact with a certain mAb. Even though there are several techniques available, each has its pros and cons. Thus, the choice for one of them is usually dependent on the preference and availability of the researcher, opening possibility for improvement, or development of alternative techniques. Phage display, for example, is a versatile technology, which allows the presentation of many different oligopeptides that can be tested against different antibodies, fitting the need for an epitope mapping approach. In this chapter, a protocol for the construction of a single-target oligopeptide phage library, as well as for the panning procedure for epitope mapping using phage display is given.

A Javascript library that uses Windows Script Host (WSH) to analyze prostate motion data fragmented across a multitude of Excel files by the Calypso 4D Localization System.

PubMed

Vali, Faisal S; Hsi, Alex; Cho, Paul; Parsai, Homayon; Garver, Elizabeth; Garza, Richard

2008-11-06

The Calypso 4D Localization System records prostate motion continuously during radiation treatment. It stores the data across thousands of Excel files. We developed Javascript (JScript) libraries for Windows Script Host (WSH) that use ActiveX Data Objects, OLE Automation and SQL to statistically analyze the data and display the results as a comprehensible Excel table. We then leveraged these libraries in other research to perform vector math on data spread across multiple access databases.

Software to model AXAF image quality

NASA Technical Reports Server (NTRS)

Ahmad, Anees

1993-01-01

This draft final report describes the work performed under this delivery order from May 1992 through June 1993. The purpose of this contract was to enhance and develop an integrated optical performance modeling software for complex x-ray optical systems such as AXAF. The GRAZTRACE program developed by the MSFC Optical Systems Branch for modeling VETA-I was used as the starting baseline program. The original program was a large single file program and, therefore, could not be modified very efficiently. The original source code has been reorganized, and a 'Make Utility' has been written to update the original program. The new version of the source code consists of 36 small source files to make it easier for the code developer to manage and modify the program. A user library has also been built and a 'Makelib' utility has been furnished to update the library. With the user library, the users can easily access the GRAZTRACE source files and build a custom library. A user manual for the new version of GRAZTRACE has been compiled. The plotting capability for the 3-D point spread functions and contour plots has been provided in the GRAZTRACE using the graphics package DISPLAY. The Graphics emulator over the network has been set up for programming the graphics routine. The point spread function and the contour plot routines have also been modified to display the plot centroid, and to allow the user to specify the plot range, and the viewing angle options. A Command Mode version of GRAZTRACE has also been developed. More than 60 commands have been implemented in a Code-V like format. The functions covered in this version include data manipulation, performance evaluation, and inquiry and setting of internal parameters. The user manual for these commands has been formatted as in Code-V, showing the command syntax, synopsis, and options. An interactive on-line help system for the command mode has also been accomplished to allow the user to find valid commands, command syntax, and command function. A translation program has been written to convert FEA output from structural analysis to GRAZTRACE surface deformation file (.dfm file). The program can accept standard output files and list files from COSMOS/M and NASTRAN finite analysis programs. Some interactive options are also provided, such as Cartesian or cylindrical coordinate transformation, coordinate shift and scale, and axial length change. A computerized database for technical documents relating to the AXAF project has been established. Over 5000 technical documents have been entered into the master database. A user can now rapidly retrieve the desired documents relating to the AXAF project. The summary of the work performed under this contract is shown.

Covalent antibody display—an in vitro antibody-DNA library selection system

PubMed Central

Reiersen, Herald; Løbersli, Inger; Løset, Geir Å.; Hvattum, Else; Simonsen, Bjørg; Stacy, John E.; McGregor, Duncan; FitzGerald, Kevin; Welschof, Martin; Brekke, Ole H.; Marvik, Ole J.

2005-01-01

The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription–translation mixture of Escherichia coli S30 lysate. Complexes of scFv–P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA–protein complexes. PMID:15653626

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

PubMed

Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

2017-09-15

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structured grid technology to enable flow simulation in an integrated system environment

NASA Astrophysics Data System (ADS)

Remotigue, Michael Gerard

An application-driven Computational Fluid Dynamics (CFD) environment needs flexible and general tools to effectively solve complex problems in a timely manner. In addition, reusable, portable, and maintainable specialized libraries will aid in rapidly developing integrated systems or procedures. The presented structured grid technology enables the flow simulation for complex geometries by addressing grid generation, grid decomposition/solver setup, solution, and interpretation. Grid generation is accomplished with the graphical, arbitrarily-connected, multi-block structured grid generation software system (GUM-B) developed and presented here. GUM-B is an integrated system comprised of specialized libraries for the graphical user interface and graphical display coupled with a solid-modeling data structure that utilizes a structured grid generation library and a geometric library based on Non-Uniform Rational B-Splines (NURBS). A presented modification of the solid-modeling data structure provides the capability for arbitrarily-connected regions between the grid blocks. The presented grid generation library provides algorithms that are reliable and accurate. GUM-B has been utilized to generate numerous structured grids for complex geometries in hydrodynamics, propulsors, and aerodynamics. The versatility of the libraries that compose GUM-B is also displayed in a prototype to automatically regenerate a grid for a free-surface solution. Grid decomposition and solver setup is accomplished with the graphical grid manipulation and repartition software system (GUMBO) developed and presented here. GUMBO is an integrated system comprised of specialized libraries for the graphical user interface and graphical display coupled with a structured grid-tools library. The described functions within the grid-tools library reduce the possibility of human error during decomposition and setup for the numerical solver by accounting for boundary conditions and connectivity. GUMBO is linked with a flow solver interface, to the parallel UNCLE code, to provide load balancing tools and solver setup. Weeks of boundary condition and connectivity specification and validation has been reduced to hours. The UNCLE flow solver is utilized for the solution of the flow field. To accelerate convergence toward a quick engineering answer, a full multigrid (FMG) approach coupled with UNCLE, which is a full approximation scheme (FAS), is presented. The prolongation operators used in the FMG-FAS method are compared. The procedure is demonstrated on a marine propeller in incompressible flow. Interpretation of the solution is accomplished by vortex feature detection. Regions of "Intrinsic Swirl" are located by interrogating the velocity gradient tensor for complex eigenvalues. The "Intrinsic Swirl" parameter is visualized on a solution of a marine propeller to determine if any vortical features are captured. The libraries and the structured grid technology presented herein are flexible and general enough to tackle a variety of complex applications. This technology has significantly enabled the capability of the ERC personnel to effectively calculate solutions for complex geometries.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.

PubMed

Sun, Dongbo; Shi, Hongyan; Chen, Jianfei; Shi, Da; Zhu, Qinghe; Zhang, Hong; Liu, Shengwang; Wang, Yunfeng; Qiu, Huaji; Feng, Li

2012-06-01

Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0×10(7) recombinant phage clones, and the titer of the amplified library was 3.6×10(9)pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described. Copyright © 2012 Elsevier B.V. All rights reserved.

Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding.

PubMed

Iera, Jaclyn A; Jenkins, Lisa M Miller; Kajiyama, Hiroshi; Kopp, Jeffrey B; Appella, Daniel H

2010-11-15

Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein's interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication. Published by Elsevier Ltd.

Artificial intelligence support for scientific model-building

NASA Technical Reports Server (NTRS)

Keller, Richard M.

1992-01-01

Scientific model-building can be a time-intensive and painstaking process, often involving the development of large and complex computer programs. Despite the effort involved, scientific models cannot easily be distributed and shared with other scientists. In general, implemented scientific models are complex, idiosyncratic, and difficult for anyone but the original scientific development team to understand. We believe that artificial intelligence techniques can facilitate both the model-building and model-sharing process. In this paper, we overview our effort to build a scientific modeling software tool that aids the scientist in developing and using models. This tool includes an interactive intelligent graphical interface, a high-level domain specific modeling language, a library of physics equations and experimental datasets, and a suite of data display facilities.

Functional Proteomics to Identify Moderators of CD8+ T-Cell Function in Melanoma

DTIC Science & Technology

2013-09-01

could then be used to develop imaging agents that are targeted to the TILs. We used an M13 phage vector, which displays a pentavalent peptide as...of the identified transcript. To directly indentify inhibitory molecules, we have proposed to use phage - display expression libraries to perform...of TCD8. 3. To determine the ligands for molecules that can modulate the TCD8 functional state. Body: A. Phage Screen Summary Phage display

Construction of a metagenomic DNA library of sponge symbionts and screening of antibacterial metabolites

NASA Astrophysics Data System (ADS)

Chen, Juan; Zhu, Tianjiao; Li, Dehai; Cui, Chengbin; Fang, Yuchun; Liu, Hongbing; Liu, Peipei; Gu, Qianqun; Zhu, Weiming

2006-04-01

To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper dise assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DH5α and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.

Tumor-targeting peptides from combinatorial libraries.

PubMed

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S

2017-02-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges in fighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. Copyright © 2017. Published by Elsevier B.V.

VSO For Dummies

NASA Astrophysics Data System (ADS)

Schwartz, Richard A.; Zarro, D.; Csillaghy, A.; Dennis, B.; Tolbert, A. K.; Etesi, L.

2009-05-01

We report on our activities to integrate VSO search and retrieval capabilities into standard data access, display, and analysis tools. In addition to its standard Web-based search form, the VSO provides an Interactive Data Language (IDL) client (vso_search) that is available through the Solar Software (SSW) package. We have incorporated this client into an IDL-widget interface program (show_synop) that allows for more simplified searching and downloading of VSO datasets directly into a user's IDL data analysis environment. In particular, we have provided the capability to read VSO datasets into a general purpose IDL package (plotman) that can display different datatypes (lightcurves, images, and spectra) and perform basic data operations such as zooming, image overlays, solar rotation, etc. Currently, the show_synop tool supports access to ground-based and space-based (SOHO, STEREO, and Hinode) observations, and has the capability to include new datasets as they become available. A user encounters two major hurdles when using the VSO: (1) Instrument-specific software (such as level-0 file readers and data-prepping procedures) may not be available in the user's local SSW distribution. (2) Recent calibration files (such as flat-fields) are not automatically distributed with the analysis software. To address these issues, we have developed a dedicated server (prepserver) that incorporates all the latest instrument-specific software libraries and calibration files. The prepserver uses an IDL-Java bridge to read and implement data processing requests from a client and return a processed data file that can be readily displayed with the show_synop/plotman package. The advantage of the prepserver is that the user is only required to install the general branch (gen) of the SSW tree, and is freed from the more onerous task of installing instrument-specific libraries and calibration files. We will demonstrate how the prepserver can be used to read, process, and overlay SOHO/EIT, TRACE, SECCHI/EUVI, and RHESSI images.

What Influences Public Library Adult Patrons to Choose the Books They Borrow.

ERIC Educational Resources Information Center

Goldhor, Herbert

This study was conducted in l978/79 to test the hypothesis that the circulation of books put in a prominent display location would increase because of the tendency of adults to select books in public libraries by browsing, rather than looking for specific titles. The circulation of all copies of a sample of ll5 titles from the adult individual…

Developing recombinant antibodies for biomarker detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.

2010-10-01

Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune librariesmore » provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.« less

Phage display screening without repetitious selection rounds.

PubMed

't Hoen, Peter A C; Jirka, Silvana M G; Ten Broeke, Bradley R; Schultes, Erik A; Aguilera, Begoña; Pang, Kar Him; Heemskerk, Hans; Aartsma-Rus, Annemieke; van Ommen, Gertjan J; den Dunnen, Johan T

2012-02-15

Phage display screenings are frequently employed to identify high-affinity peptides or antibodies. Although successful, phage display is a laborious technology and is notorious for identification of false positive hits. To accelerate and improve the selection process, we have employed Illumina next generation sequencing to deeply characterize the Ph.D.-7 M13 peptide phage display library before and after several rounds of biopanning on KS483 osteoblast cells. Sequencing of the naive library after one round of amplification in bacteria identifies propagation advantage as an important source of false positive hits. Most important, our data show that deep sequencing of the phage pool after a first round of biopanning is already sufficient to identify positive phages. Whereas traditional sequencing of a limited number of clones after one or two rounds of selection is uninformative, the required additional rounds of biopanning are associated with the risk of losing promising clones propagating slower than nonbinding phages. Confocal and live cell imaging confirms that our screen successfully selected a peptide with very high binding and uptake in osteoblasts. We conclude that next generation sequencing can significantly empower phage display screenings by accelerating the finding of specific binders and restraining the number of false positive hits. Copyright © 2011 Elsevier Inc. All rights reserved.

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

PubMed

Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

2018-06-05

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

Scaffold design of trivalent chelator heads dictates high-affinity and stable His-tagged protein labeling in vitro and in cellulo.

PubMed

Gatterdam, Karl; Joest, Eike F; Gatterdam, Volker; Tampé, Robert

2018-05-29

Small chemical/biological interaction pairs are at the forefront in tracing proteins' function and interaction at high signal-to-background ratio in cellular pathways. Pharma ventures have eager plans to develop trisNTA probes for in vitro and in vivo screening of His-tagged protein targets. However, the optimal design of scaffold, linker, and chelator head yet deserves systematic investigations to achieve highest affinity and kinetic stability for in vitro and especially cell applications. In this study, we report on a library of N-nitrilotriacetic acid (NTA) based multivalent chelator heads (MCHs) built up on linear, cyclic, and dendritic scaffolds and contrast these with regard to their binding affinity and stability for labeling of cellular His-tagged proteins. Furthermore, we assign a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we describe fundamental differences between the MCH scaffold and define a cyclic trisNTA chelator, which displays the highest affinity and kinetic stability of all reversible, low-molecular weight interaction pairs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Earthscape, a Multi-Purpose Interactive 3d Globe Viewer for Hybrid Data Visualization and Analysis

NASA Astrophysics Data System (ADS)

Sarthou, A.; Mas, S.; Jacquin, M.; Moreno, N.; Salamon, A.

2015-08-01

The hybrid visualization and interaction tool EarthScape is presented here. The software is able to display simultaneously LiDAR point clouds, draped videos with moving footprint, volume scientific data (using volume rendering, isosurface and slice plane), raster data such as still satellite images, vector data and 3D models such as buildings or vehicles. The application runs on touch screen devices such as tablets. The software is based on open source libraries, such as OpenSceneGraph, osgEarth and OpenCV, and shader programming is used to implement volume rendering of scientific data. The next goal of EarthScape is to perform data analysis using ENVI Services Engine, a cloud data analysis solution. EarthScape is also designed to be a client of Jagwire which provides multisource geo-referenced video fluxes. When all these components will be included, EarthScape will be a multi-purpose platform that will provide at the same time data analysis, hybrid visualization and complex interactions. The software is available on demand for free at france@exelisvis.com.

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

PubMed

Matsumoto, Yuki; Shindo, Yosuke; Takakusagi, Yoichi; Takakusagi, Kaori; Tsukuda, Senko; Kusayanagi, Tomoe; Sato, Hitoshi; Kawabe, Takumi; Sugawara, Fumio; Sakaguchi, Kengo

2011-12-01

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix. Copyright © 2011 Elsevier Ltd. All rights reserved.

Isolation of a peptide from Ph.D.-C7C phage display library for detection of Cry1Ab.

PubMed

Wang, Yun; Wang, Qian; Wu, Ai-Hua; Hao, Zhen-Ping; Liu, Xian-Jin

2017-12-15

Traditional ELISA methods of using animal immunity yield antibodies for detection Cry toxin. Not only is this incredibly harmful to the animals, but is also time-intensive. Here we developed a simple method to yield the recognition element. Using a critical selection strategy and immunoassay we confirmed a clone from the Ph.D-C7C phage library, which has displayed the most interesting Cry1Ab-binding characteristics examined in this study (Fig. 1). The current study indicates that isolating peptide is an alternative method for the preparation of a recognition element, and that the developed assay is a potentially useful tool for detecting Cry1Ab. Copyright © 2017. Published by Elsevier Inc.

Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

PubMed Central

Zhu, Bo; Mizoguchi, Takuro; Kojima, Takaaki; Nakano, Hideo

2015-01-01

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases. PMID:25993095

WORM - WINDOWED OBSERVATION OF RELATIVE MOTION

NASA Technical Reports Server (NTRS)

Bauer, F.

1994-01-01

The Windowed Observation of Relative Motion, WORM, program is primarily intended for the generation of simple X-Y plots from data created by other programs. It allows the user to label, zoom, and change the scale of various plots. Three dimensional contour and line plots are provided, although with more limited capabilities. The input data can be in binary or ASCII format, although all data must be in the same format. A great deal of control over the details of the plot is provided, such as gridding, size of tick marks, colors, log/semilog capability, time tagging, and multiple and phase plane plots. Many color and monochrome graphics terminals and hard copy printer/plotters are supported. The WORM executive commands, menu selections and macro files can be used to develop plots and tabular data, query the WORM Help library, retrieve data from input files, and invoke VAX DCL commands. WORM generated plots are displayed on local graphics terminals and can be copied using standard hard copy capabilities. Some of the graphics features of WORM include: zooming and dezooming various portions of the plot; plot documentation including curve labeling and function listing; multiple curves on the same plot; windowing of multiple plots and insets of the same plot; displaying a specific on a curve; and spinning the curve left, right, up, and down. WORM is written in PASCAL for interactive execution and has been implemented on a DEC VAX computer operating under VMS 4.7 with a virtual memory requirement of approximately 392K of 8 bit bytes. It uses the QPLOT device independent graphics library included with WORM. It was developed in 1988.

OsiriX: an open-source software for navigating in multidimensional DICOM images.

PubMed

Rosset, Antoine; Spadola, Luca; Ratib, Osman

2004-09-01

A multidimensional image navigation and display software was designed for display and interpretation of large sets of multidimensional and multimodality images such as combined PET-CT studies. The software is developed in Objective-C on a Macintosh platform under the MacOS X operating system using the GNUstep development environment. It also benefits from the extremely fast and optimized 3D graphic capabilities of the OpenGL graphic standard widely used for computer games optimized for taking advantage of any hardware graphic accelerator boards available. In the design of the software special attention was given to adapt the user interface to the specific and complex tasks of navigating through large sets of image data. An interactive jog-wheel device widely used in the video and movie industry was implemented to allow users to navigate in the different dimensions of an image set much faster than with a traditional mouse or on-screen cursors and sliders. The program can easily be adapted for very specific tasks that require a limited number of functions, by adding and removing tools from the program's toolbar and avoiding an overwhelming number of unnecessary tools and functions. The processing and image rendering tools of the software are based on the open-source libraries ITK and VTK. This ensures that all new developments in image processing that could emerge from other academic institutions using these libraries can be directly ported to the OsiriX program. OsiriX is provided free of charge under the GNU open-source licensing agreement at http://homepage.mac.com/rossetantoine/osirix.

What Is Librarian 2.0--New Competencies or Interactive Relations? A Library Professional Viewpoint

ERIC Educational Resources Information Center

Huvila, Isto; Holmberg, Kim; Kronqvist-Berg, Maria; Nivakoski, Outi; Widén, Gunilla

2013-01-01

Library 2.0 is a change in the way libraries interact with their users. Technological developments on the Web have had a major influence on these changes. The change also places new requirements on librarians' competencies and skills. This research investigates how librarians themselves see this change in terms of their work identity and working…

An Interaction Library for the FcεRI Signaling Network

DOE PAGES

Chylek, Lily A.; Holowka, David A.; Baird, Barbara A.; ...

2014-04-15

Antigen receptors play a central role in adaptive immune responses. Although the molecular networks associated with these receptors have been extensively studied, we currently lack a systems-level understanding of how combinations of non-covalent interactions and post-translational modifications are regulated during signaling to impact cellular decision-making. To fill this knowledge gap, it will be necessary to formalize and piece together information about individual molecular mechanisms to form large-scale computational models of signaling networks. To this end, we have developed an interaction library for signaling by the high-affinity IgE receptor, FcεRI. The library consists of executable rules for protein–protein and protein–lipid interactions.more » This library extends earlier models for FcεRI signaling and introduces new interactions that have not previously been considered in a model. Thus, this interaction library is a toolkit with which existing models can be expanded and from which new models can be built. As an example, we present models of branching pathways from the adaptor protein Lat, which influence production of the phospholipid PIP 3 at the plasma membrane and the soluble second messenger IP 3. We find that inclusion of a positive feedback loop gives rise to a bistable switch, which may ensure robust responses to stimulation above a threshold level. In addition, the library is visualized to facilitate understanding of network circuitry and identification of network motifs.« less

An Interaction Library for the FcεRI Signaling Network

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chylek, Lily A.; Holowka, David A.; Baird, Barbara A.

Antigen receptors play a central role in adaptive immune responses. Although the molecular networks associated with these receptors have been extensively studied, we currently lack a systems-level understanding of how combinations of non-covalent interactions and post-translational modifications are regulated during signaling to impact cellular decision-making. To fill this knowledge gap, it will be necessary to formalize and piece together information about individual molecular mechanisms to form large-scale computational models of signaling networks. To this end, we have developed an interaction library for signaling by the high-affinity IgE receptor, FcεRI. The library consists of executable rules for protein–protein and protein–lipid interactions.more » This library extends earlier models for FcεRI signaling and introduces new interactions that have not previously been considered in a model. Thus, this interaction library is a toolkit with which existing models can be expanded and from which new models can be built. As an example, we present models of branching pathways from the adaptor protein Lat, which influence production of the phospholipid PIP 3 at the plasma membrane and the soluble second messenger IP 3. We find that inclusion of a positive feedback loop gives rise to a bistable switch, which may ensure robust responses to stimulation above a threshold level. In addition, the library is visualized to facilitate understanding of network circuitry and identification of network motifs.« less

Mimtags: the use of phage display technology to produce novel protein-specific probes.

PubMed

Ahmed, Nayyar; Dhanapala, Pathum; Sadli, Nadia; Barrow, Colin J; Suphioglu, Cenk

2014-03-01

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β.

PubMed

Jung, Hye-Won; Park, Inae; Ghil, Sungho

2014-09-01

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.

Experimenter's laboratory for visualized interactive science

NASA Technical Reports Server (NTRS)

Hansen, Elaine R.; Klemp, Marjorie K.; Lasater, Sally W.; Szczur, Marti R.; Klemp, Joseph B.

1992-01-01

The science activities of the 1990's will require the analysis of complex phenomena and large diverse sets of data. In order to meet these needs, we must take advantage of advanced user interaction techniques: modern user interface tools; visualization capabilities; affordable, high performance graphics workstations; and interoperable data standards and translator. To meet these needs, we propose to adopt and upgrade several existing tools and systems to create an experimenter's laboratory for visualized interactive science. Intuitive human-computer interaction techniques have already been developed and demonstrated at the University of Colorado. A Transportable Applications Executive (TAE+), developed at GSFC, is a powerful user interface tool for general purpose applications. A 3D visualization package developed by NCAR provides both color shaded surface displays and volumetric rendering in either index or true color. The Network Common Data Form (NetCDF) data access library developed by Unidata supports creation, access and sharing of scientific data in a form that is self-describing and network transparent. The combination and enhancement of these packages constitutes a powerful experimenter's laboratory capable of meeting key science needs of the 1990's. This proposal encompasses the work required to build and demonstrate this capability.

Experimenter's laboratory for visualized interactive science

NASA Technical Reports Server (NTRS)

Hansen, Elaine R.; Klemp, Marjorie K.; Lasater, Sally W.; Szczur, Marti R.; Klemp, Joseph B.

1993-01-01

The science activities of the 1990's will require the analysis of complex phenomena and large diverse sets of data. In order to meet these needs, we must take advantage of advanced user interaction techniques: modern user interface tools; visualization capabilities; affordable, high performance graphics workstations; and interoperatable data standards and translator. To meet these needs, we propose to adopt and upgrade several existing tools and systems to create an experimenter's laboratory for visualized interactive science. Intuitive human-computer interaction techniques have already been developed and demonstrated at the University of Colorado. A Transportable Applications Executive (TAE+), developed at GSFC, is a powerful user interface tool for general purpose applications. A 3D visualization package developed by NCAR provides both color-shaded surface displays and volumetric rendering in either index or true color. The Network Common Data Form (NetCDF) data access library developed by Unidata supports creation, access and sharing of scientific data in a form that is self-describing and network transparent. The combination and enhancement of these packages constitutes a powerful experimenter's laboratory capable of meeting key science needs of the 1990's. This proposal encompasses the work required to build and demonstrate this capability.

Inhibitors for the Vitamin D Receptor–Coregulator Interaction

PubMed Central

Teske, Kelly A.; Yu, Olivia; Arnold, Leggy A.

2016-01-01

The vitamin D receptor (VDR) belongs to the superfamily of nuclear receptors and is activated by the endogenous ligand 1,25-dihydroxyvitamin D3. The genomic effects mediated by VDR consist of the activation and repression of gene transcription, which includes the formation of multi-protein complexes with coregulator proteins. Coregulators bind many nuclear receptors and can be categorized according their role as coactivators (gene activation) or corepressors (gene repression). Herein, different approaches to develop compounds that modulate the interaction between VDR and coregulators are summarized. This include coregulator peptides that were identified by creating phage display libraries. Subsequent modification of these peptides including the introduction of a tether or non-hydrolysable bonds resulted in the first direct VDR–coregulator inhibitors. Later, small molecules that inhibit VDR–coregulator inhibitors were identified using rational drug design and high throughput screening. Early on, allosteric inhibition of VDR–coregulator interactions was achieved with VDR antagonists that change the conformation of VDR and modulate the interactions with coregulators. A detailed discussion of their dual agonist/antagonist effects is given as well as a summary of their biological effects in cell-based assays and in vivo studies. PMID:26827948

Protein complex purification from Thermoplasma acidophilum using a phage display library.

PubMed

Hubert, Agnes; Mitani, Yasuo; Tamura, Tomohiro; Boicu, Marius; Nagy, István

2014-03-01

We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available. Copyright © 2013 Elsevier B.V. All rights reserved.

Screening phage display libraries for organ-specific vascular immunotargeting in vivo

PubMed Central

Valadon, Philippe; Garnett, Jeff D.; Testa, Jacqueline E.; Bauerle, Marc; Oh, Phil; Schnitzer, Jan E.

2006-01-01

The molecular diversity of the luminal endothelial cell surface arising in vivo from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of specific organs in vivo. We differentially screen phage libraries to select organ-targeting antibodies by using luminal endothelial cell plasma membranes isolated directly from tissue and highly enriched in natively expressed proteins exposed to the bloodstream. To obviate liver uptake of intravenously injected phage, we convert the phage-displayed antibodies into scFv-Fc fusion proteins, which then are able to rapidly target select organ(s) in vivo as visualized directly by γ-scintigraphic whole-body imaging. Mass spectrometry helps identify the antigen targets. This comprehensive strategy provides new promise for harnessing the power of phage display for mapping vascular endothelia natively in tissue and for achieving vascular targeting of specific tissues in vivo. PMID:16384919

Spatial Data Management System (SDMS)

NASA Technical Reports Server (NTRS)

Hutchison, Mark W.

1994-01-01

The Spatial Data Management System (SDMS) is a testbed for retrieval and display of spatially related material. SDMS permits the linkage of large graphical display objects with detail displays and explanations of its smaller components. SDMS combines UNIX workstations, MIT's X Window system, TCP/IP and WAIS information retrieval technology to prototype a means of associating aggregate data linked via spatial orientation. SDMS capitalizes upon and extends previous accomplishments of the Software Technology Branch in the area of Virtual Reality and Automated Library Systems.

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2007-07-01

The T7Select 10-3b system of lytic phage display is a mid-copy vector that displays between 5-15 copies on the surface of the T7 capsid. The natural... Phage are amplified on a bacterial host that carries an ampicillin-resistant plasmid expressing additional 10A capsid protein from a T7 promoter. We... phage display library of coding fragments encompassing all open reading frames of the human genome. We designed approximately 467,000 overlapping

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

PubMed

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Asymmetric triplex metallohelices with high and selective activity against cancer cells

NASA Astrophysics Data System (ADS)

Faulkner, Alan D.; Kaner, Rebecca A.; Abdallah, Qasem M. A.; Clarkson, Guy; Fox, David J.; Gurnani, Pratik; Howson, Suzanne E.; Phillips, Roger M.; Roper, David I.; Simpson, Daniel H.; Scott, Peter

2014-09-01

Small cationic amphiphilic α-helical peptides are emerging as agents for the treatment of cancer and infection, but they are costly and display unfavourable pharmacokinetics. Helical coordination complexes may offer a three-dimensional scaffold for the synthesis of mimetic architectures. However, the high symmetry and modest functionality of current systems offer little scope to tailor the structure to interact with specific biomolecular targets, or to create libraries for phenotypic screens. Here, we report the highly stereoselective asymmetric self-assembly of very stable, functionalized metallohelices. Their anti-parallel head-to-head-to-tail ‘triplex’ strand arrangement creates an amphipathic functional topology akin to that of the active sub-units of, for example, host-defence peptides and p53. The metallohelices display high, structure-dependent toxicity to the human colon carcinoma cell-line HCT116 p53++, causing dramatic changes in the cell cycle without DNA damage. They have lower toxicity to human breast adenocarcinoma cells (MDA-MB-468) and, most remarkably, they show no significant toxicity to the bacteria methicillin-resistant Staphylococcus aureus and Escherichia coli.

The energy landscape of a selective tumor-homing pentapeptide

PubMed Central

Zanuy, David; Flores-Ortega, Alejandra; Casanovas, Jordi; Curco, David; Nussinov, Ruth; Aleman, Carlos

2009-01-01

Recently, a potentially powerful strategy based on the of phage-display libraries has been presented to target tumors via homing peptides attached to nanoparticles. The Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence has been identified as a tumor-homing peptide that binds to clotted plasmas proteins present in tumor vessels and interstitium. The aim of this work consists of mapping the conformational profile of CREKA to identify the bioactive conformation. For this purpose, a conformational search procedure based on modified Simulated Annealing combined with Molecular Dynamics was applied to three systems that mimic the experimentally used conditions: (i) the free peptide; (ii) the peptide attached to a nanoparticle; and (iii) the peptide inserted in a phage display protein. In addition, the free peptide was simulated in an ionized aqueous solution environment, which mimics the ionic strength of the physiological medium. Accessible minima of all simulated systems reveal a multiple interaction pattern involving the ionized side chains of Arg, Glu and Lys, which induces a β-turn motif in the backbone observed in all simulated CREKA systems. PMID:18588341

Interactive digital image manipulation system

NASA Technical Reports Server (NTRS)

Henze, J.; Dezur, R.

1975-01-01

The system is designed for manipulation, analysis, interpretation, and processing of a wide variety of image data. LANDSAT (ERTS) and other data in digital form can be input directly into the system. Photographic prints and transparencies are first converted to digital form with an on-line high-resolution microdensitometer. The system is implemented on a Hewlett-Packard 3000 computer with 128 K bytes of core memory and a 47.5 megabyte disk. It includes a true color display monitor, with processing memories, graphics overlays, and a movable cursor. Image data formats are flexible so that there is no restriction to a given set of remote sensors. Conversion between data types is available to provide a basis for comparison of the various data. Multispectral data is fully supported, and there is no restriction on the number of dimensions. In this way multispectral data collected at more than one point in time may simply be treated as a data collected with twice (three times, etc.) the number of sensors. There are various libraries of functions available to the user: processing functions, display functions, system functions, and earth resources applications functions.

Eukaryotic ribosome display with in situ DNA recovery.

PubMed

He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

2012-01-01

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber.

PubMed

Szot-Karpińska, Katarzyna; Golec, Piotr; Leśniewski, Adam; Pałys, Barbara; Marken, Frank; Niedziółka-Jönsson, Joanna; Węgrzyn, Grzegorz; Łoś, Marcin

2016-12-21

With the advent of nanotechnology, carbon nanomaterials such as carbon nanofibers (CNF) have aroused substantial interest in various research fields, including energy storage and sensing. Further improvement of their properties might be achieved via the application of viral particles such as bacteriophages. In this report, we present a filamentous M13 bacteriophage with a point mutation in gene VII (pVII-mutant-M13) that selectively binds to the carbon nanofibers to form 3D structures. The phage-display technique was utilized for the selection of the pVII-mutant-M13 phage from the phage display peptide library. The properties of this phage make it a prospective candidate for a scaffold material for CNFs. The results for binding of CNF by mutant phage were compared with those for maternal bacteriophage (pVII-M13). The efficiency of binding between pVII-mutant-M13 and CNF is about 2 orders of magnitude higher compared to that of the pVII-M13. Binding affinity between pVII-mutant-M13 and CNF was also characterized using atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, which confirmed the specificity of the interaction of the phage pVII-mutant-M13 and the CNF; the binding occurs via the phage's ending, where the mutated pVII protein is located. No similar behavior has been observed for other carbon nanomaterials such as graphite, reduced graphene oxide, single-walled carbon nanotubes, and multiwalled carbon nanotubes. Infrared spectra confirmed differences in the interaction with CNF between the pVII-mutant-M13 and the pVII-M13. Basing on conducted research, we hypothesize that the interactions are noncovalent in nature, with π-π interactions playing the dominant role. Herein, the new bioconjugate material is introduced.

Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning.

PubMed

Chin, Chai Fung; Ler, Lian Wee; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Tye, Gee Jun; Lim, Theam Soon

2016-01-01

Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

In-Flight Performance Evaluation of Experimental Information Displays

DTIC Science & Technology

1979-05-01

Chemical Systems Laboratory Experimentation Command Aberden Proving Ground ,MD Technical Library 21010 (1) Box 22 Fort Ord, CA 93941 (1) 21 US Amy Materiel...US Army Missile R&D Command Library, Bldg 3071 Redstone Arsenal, AL 35809 (1) ATTN: ATSL-DOSL Aberdeen Proving Ground , MD US Army Yuma Proving Ground ...Systems Chief Analysis Agency Benet Weapons Laboratory ATTN: Reports Distribution LCWSL, USA ARRADCOH Aberdeen Proving Ground , MD ATTN: DRDAR-LCB-TL

Where Creativity Meets Technology: A Library-Led, Multi-Disciplinary Online Showcase for Artworks, Creative Writings, and Movies Displayed with 3D and HTML5 Technology

ERIC Educational Resources Information Center

Wong, Shun Han Rebekah

2015-01-01

This article introduces the Hong Kong Baptist University's Heritage project (http://heritage.lib.hkbu.edu.hk/), a multi-disciplinary online showcase for curriculum-related creative outputs that were produced by faculty and students of the university. Initiated and led by the University Library, this project was a collaborative effort with six…

Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries.

PubMed

Randrianjatovo-Gbalou, Irina; Rosario, Sandrine; Sismeiro, Odile; Varet, Hugo; Legendre, Rachel; Coppée, Jean-Yves; Huteau, Valérie; Pochet, Sylvie; Delarue, Marc

2018-05-21

Nucleic acid aptamers, especially RNA, exhibit valuable advantages compared to protein therapeutics in terms of size, affinity and specificity. However, the synthesis of libraries of large random RNAs is still difficult and expensive. The engineering of polymerases able to directly generate these libraries has the potential to replace the chemical synthesis approach. Here, we start with a DNA polymerase that already displays a significant template-free nucleotidyltransferase activity, human DNA polymerase theta, and we mutate it based on the knowledge of its three-dimensional structure as well as previous mutational studies on members of the same polA family. One mutant exhibited a high tolerance towards ribonucleotides (NTPs) and displayed an efficient ribonucleotidyltransferase activity that resulted in the assembly of long RNA polymers. HPLC analysis and RNA sequencing of the products were used to quantify the incorporation of the four NTPs as a function of initial NTP concentrations and established the randomness of each generated nucleic acid sequence. The same mutant revealed a propensity to accept other modified nucleotides and to extend them in long fragments. Hence, this mutant can deliver random natural and modified RNA polymers libraries ready to use for SELEX, with custom lengths and balanced or unbalanced ratios.

Identification and characterization of epitopes on Plasmodium knowlesi merozoite surface protein-142 (MSP-142) using synthetic peptide library and phage display library.

PubMed

Cheong, Fei Wen; Fong, Mun Yik; Lau, Yee Ling

2016-02-01

Plasmodium knowlesi can cause potentially life threatening human malaria. The Plasmodium merozoite surface protein-142 (MSP-142) is a potential target for malaria blood stage vaccine, and for diagnosis of malaria. Two epitope mapping techniques were used to identify the potential epitopes within P. knowlesi MSP-142. Nine and 14 potential epitopes were identified using overlapping synthetic peptide library and phage display library, respectively. Two regions on P. knowlesi MSP-142 (amino acid residues 37-95 and residues 240-289) were identified to be the potential dominant epitope regions. Two of the prominent epitopes, P10 (TAKDGMEYYNKMGELYKQ) and P31 (RCLLGFKEVGGKCVPASI), were evaluated using mouse model. P10- and P31-immunized mouse sera reacted with recombinant P. knowlesi MSP-142, with the IgG isotype distribution of IgG2b>IgG1>IgG2a>IgG3. Significant higher level of cytokines interferon-gamma and interleukin-2 was detected in P31-immunized mice. Both P10 and P31 could be the suitable epitope candidates to be used in malaria vaccine designs and immunodiagnostic assays, provided further evaluation is needed to validate the potential uses of these epitopes. Copyright © 2015 Elsevier B.V. All rights reserved.

Using a Gravity Model to Predict Circulation in a Public Library System.

ERIC Educational Resources Information Center

Ottensmann, John R.

1995-01-01

Describes the development of a gravity model based upon principles of spatial interaction to predict the circulation of libraries in the Indianapolis-Marion County Public Library (Indiana). The model effectively predicted past circulation figures and was tested by predicting future library circulation, particularly for a new branch library.…

Basics of Antibody Phage Display Technology.

PubMed

Ledsgaard, Line; Kilstrup, Mogens; Karatt-Vellatt, Aneesh; McCafferty, John; Laustsen, Andreas H

2018-06-09

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

PubMed

Shukla, Girja S; Krag, David N; Peletskaya, Elena N; Pero, Stephanie C; Sun, Yu-Jing; Carman, Chelsea L; McCahill, Laurence E; Roland, Thomas A

2013-08-01

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

Computational and biophysical approaches to protein-protein interaction inhibition of Plasmodium falciparum AMA1/RON2 complex

NASA Astrophysics Data System (ADS)

Pihan, Emilie; Delgadillo, Roberto F.; Tonkin, Michelle L.; Pugnière, Martine; Lebrun, Maryse; Boulanger, Martin J.; Douguet, Dominique

2015-06-01

Invasion of the red blood cell by Plasmodium falciparum parasites requires formation of an electron dense circumferential ring called the Moving Junction (MJ). The MJ is anchored by a high affinity complex of two parasite proteins: Apical Membrane Antigen 1 ( PfAMA1) displayed on the surface of the parasite and Rhoptry Neck Protein 2 that is discharged from the parasite and imbedded in the membrane of the host cell. Structural studies of PfAMA1 revealed a conserved hydrophobic groove localized to the apical surface that coordinates RON2 and invasion inhibitory peptides. In the present work, we employed computational and biophysical methods to identify competitive P. falciparum AMA1-RON2 inhibitors with the goal of exploring the `druggability' of this attractive antimalarial target. A virtual screen followed by molecular docking with the PfAMA1 crystal structure was performed using an eight million compound collection that included commercial molecules, the ChEMBL malaria library and approved drugs. The consensus approach resulted in the selection of inhibitor candidates. We also developed a fluorescence anisotropy assay using a modified inhibitory peptide to experimentally validate the ability of the selected compounds to inhibit the AMA1-RON2 interaction. Among those, we identified one compound that displayed significant inhibition. This study offers interesting clues to improve the throughput and reliability of screening for new drug leads.

Discovery of new antimalarial chemotypes through chemical methodology and library development.

PubMed

Brown, Lauren E; Chih-Chien Cheng, Ken; Wei, Wan-Guo; Yuan, Pingwei; Dai, Peng; Trilles, Richard; Ni, Feng; Yuan, Jing; MacArthur, Ryan; Guha, Rajarshi; Johnson, Ronald L; Su, Xin-zhuan; Dominguez, Melissa M; Snyder, John K; Beeler, Aaron B; Schaus, Scott E; Inglese, James; Porco, John A

2011-04-26

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

Exploring interaction with 3D volumetric displays

NASA Astrophysics Data System (ADS)

Grossman, Tovi; Wigdor, Daniel; Balakrishnan, Ravin

2005-03-01

Volumetric displays generate true volumetric 3D images by actually illuminating points in 3D space. As a result, viewing their contents is similar to viewing physical objects in the real world. These displays provide a 360 degree field of view, and do not require the user to wear hardware such as shutter glasses or head-trackers. These properties make them a promising alternative to traditional display systems for viewing imagery in 3D. Because these displays have only recently been made available commercially (e.g., www.actuality-systems.com), their current use tends to be limited to non-interactive output-only display devices. To take full advantage of the unique features of these displays, however, it would be desirable if the 3D data being displayed could be directly interacted with and manipulated. We investigate interaction techniques for volumetric display interfaces, through the development of an interactive 3D geometric model building application. While this application area itself presents many interesting challenges, our focus is on the interaction techniques that are likely generalizable to interactive applications for other domains. We explore a very direct style of interaction where the user interacts with the virtual data using direct finger manipulations on and around the enclosure surrounding the displayed 3D volumetric image.

Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

PubMed Central

Henry, Kevin A.; Kim, Dae Young; Kandalaft, Hiba; Lowden, Michael J.; Yang, Qingling; Schrag, Joseph D.; Hussack, Greg; MacKenzie, C. Roger; Tanha, Jamshid

2017-01-01

Human autonomous VH/VL single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged VH/VL domains. Here, we describe the design and characterization of three novel human VH/VL sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential VH/VL sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three VH/VL sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three VH/VL libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 VHs and 7 VLs in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 µM (average: 2–3 µM), but had highly variable expression yields (range: 0.1–19 mg/L). Despite our efforts to identify the most stable VH/VL scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing VH/VL sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some VH/VL sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence clearly dramatically impacts the stability of human autonomous VH/VL immunoglobulin domain folds, and sequence-stability tradeoffs must be taken into account during the design of such libraries. PMID:29375542

Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

PubMed

Miersch, Shane; Maruthachalam, Bharathikumar Vellalore; Geyer, C Ronald; Sidhu, Sachdev S

2017-05-19

We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

PubMed

Yang, Fang; Lei, Yingying; Zhou, Meiling; Yao, Qili; Han, Yichao; Wu, Xiang; Zhong, Wanshun; Zhu, Chenghang; Xu, Weize; Tao, Ran; Chen, Xi; Lin, Da; Rahman, Khaista; Tyagi, Rohit; Habib, Zeshan; Xiao, Shaobo; Wang, Dang; Yu, Yang; Chen, Huanchun; Fu, Zhenfang; Cao, Gang

2018-02-16

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

PubMed

Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

2018-04-01

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

PubMed Central

Adler, Adam S.; Bedinger, Daniel; Adams, Matthew S.; Asensio, Michael A.; Edgar, Robert C.; Leong, Renee; Leong, Jackson; Mizrahi, Rena A.; Spindler, Matthew J.; Bandi, Srinivasa Rao; Huang, Haichun; Brams, Peter; Johnson, David S.

2018-01-01

ABSTRACT Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs. PMID:29376776

Rational and Modular Design of Potent Ligands Targeting the RNA that Causes Myotonic Dystrophy 2

PubMed Central

Lee, Melissa M.; Pushechnikov, Alexei; Disney, Matthew D.

2009-01-01

Most ligands targeting RNA are identified through screening a therapeutic target for binding members of a ligand library. A potential alternative way to construct RNA binders is through rational design using information about the RNA motifs ligands prefer to bind. Herein, we describe such an approach to design modularly assembled ligands targeting the RNA that causes myotonic dystrophy type 2 (DM2), a currently untreatable disease. A previous study identified that 6′-N-5-hexynoate kanamycin A (1) prefers to bind 2×2 nucleotide, pyrimidine-rich RNA internal loops. Multiple copies of such loops were found in the RNA hairpin that causes DM2. The 1 ligand was then modularly displayed on a peptoid scaffold with varied number and spacing to target several internal loops simultaneously. Modularly assembled ligands were tested for binding to a series of RNAs and for inhibiting the formation of the toxic DM2 RNA-muscleblind protein (MBNL-1) interaction. The most potent ligand displays three 1 modules, each separated by four spacing submonomers, and inhibits the formation of the RNA-protein complex with an IC50 of 25 nM. This ligand is higher affinity and more specific for binding DM2 RNA than MBNL-1. It binds the DM2 RNA at least 20-times more tightly than related RNAs and 15-fold more tightly than MBNL-1. A related control peptoid displaying 6′-N-5-hexynoate neamine (2) is >100-fold less potent at inhibiting the RNA-protein interaction and binds to DM2 RNA >125-fold more weakly. Uptake studies into a mouse myoblast cell line also show that the most potent ligand is cell permeable. PMID:19348464

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

PubMed Central

Zhao, Wenjing; McCallum, Scott A.; Xiao, Zhongping; Zhang, Fuming; Linhardt, Robert J.

2011-01-01

Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs. PMID:21658003

Creating an Engaging Library Orientation: First Year Experience Courses at UC San Diego

ERIC Educational Resources Information Center

Goldman, Crystal; Turnbow, Dominique; Roth, Amanda; Friedman, Lia; Heskett, Karen

2016-01-01

This article focuses on the development of an engaging library orientation module for UC San Diego First Year Experience (FYE) courses. The library module included a brief in-class presentation about research concepts and library services, an online interactive library scavenger hunt given as an in-class activity, and a homework assignment where…

E-Commerce for Library Promotion and Sustainability: How Library Technicians Can Market Themselves and Their Library's Services Online

ERIC Educational Resources Information Center

von Retzlaff, Lothar

2006-01-01

As players in the delivery of services and online information, libraries are in the best position to use staff expertise and the technology they have developed to foster community awareness of social, professional and commercial interactions on the internet. Historically, libraries have been progressive institutions, undertaking constant…

IFLA General Conference, 1987. Division of Management and Technology. Library Building and Equipment Section. Information Technology Section. Papers.

ERIC Educational Resources Information Center

International Federation of Library Associations, The Hague (Netherlands).

The six papers in this collection focus on library buildings and equipment and information technology: (1) "Library Equipment in a Changing Library Environment" (Margaret Beckman, Canada); (2) "The New Building for the British Library" (Colin St. John Wilson, United Kingdom); (3) "User Interaction with Online Catalogue" (Stephen Walker, United…

Probing the Structure-Activity Relationship of the Natural Antifouling Agent Polygodial against both Micro- and Macrofoulers by Semisynthetic Modification.

PubMed

Moodie, Lindon W K; Trepos, Rozenn; Cervin, Gunnar; Larsen, Lesley; Larsen, David S; Pavia, Henrik; Hellio, Claire; Cahill, Patrick; Svenson, Johan

2017-02-24

The current study represents the first comprehensive investigation into the general antifouling activities of the natural drimane sesquiterpene polygodial. Previous studies have highlighted a high antifouling effect toward macrofoulers, such as ascidians, tubeworms, and mussels, but no reports about the general antifouling effect of polygodial have been communicated before. To probe the structural and chemical basis for antifouling activity, a library of 11 polygodial analogues was prepared by semisynthesis. The library was designed to yield derivatives with ranging polarities and the ability to engage in both covalent and noncovalent interactions, while still remaining within the drimane sesquiterpene scaffold. The prepared compounds were screened against 14 relevant marine micro- and macrofouling species. Several of the polygodial analogues displayed inhibitory activities at sub-microgram/mL concentrations. These antifouling effects were most pronounced against the macrofouling ascidian Ciona savignyi and the barnacle Balanus improvisus, with inhibitory activities observed for selected compounds comparable or superior to several commercial antifouling products. The inhibitory activity against the microfouling bacteria and microalgae was reversible and significantly less pronounced than for the macrofoulers. This study illustrates that the macro- and microfoulers are targeted by the compounds via different mechanisms.

Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

PubMed

Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

2016-01-01

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine. Copyright © 2016 Elsevier GmbH. All rights reserved.

CD68 acts as a major gateway for malaria sporozoite liver infection

PubMed Central

Cha, Sung-Jae; Park, Kiwon; Srinivasan, Prakash; Schindler, Christian W.; van Rooijen, Nico; Stins, Monique

2015-01-01

After being delivered by the bite from an infected mosquito, Plasmodium sporozoites enter the blood circulation and infect the liver. Previous evidence suggests that Kupffer cells, a macrophage-like component of the liver blood vessel lining, are traversed by sporozoites to initiate liver invasion. However, the molecular determinants of sporozoite–Kupffer cell interactions are unknown. Understanding the molecular basis for this specific recognition may lead to novel therapeutic strategies to control malaria. Using a phage display library screen, we identified a peptide, P39, that strongly binds to the Kupffer cell surface and, importantly, inhibits sporozoite Kupffer cell entry. Furthermore, we determined that P39 binds to CD68, a putative receptor for sporozoite invasion of Kupffer cells that acts as a gateway for malaria infection of the liver. PMID:26216124

Direct comparison of linear and macrocyclic compound libraries as a source of protein ligands.

PubMed

Gao, Yu; Kodadek, Thomas

2015-03-09

There has been much discussion of the potential desirability of macrocyclic molecules for the development of tool compounds and drug leads. But there is little experimental data comparing otherwise equivalent macrocyclic and linear compound libraries as a source of protein ligands. In this Letter, we probe this point in the context of peptoid libraries. Bead-displayed libraries of macrocyclic and linear peptoids containing four variable positions and 0-2 fixed residues, to vary the ring size, were screened against streptavidin and the affinity of every hit for the target was measured. The data show that macrocyclization is advantageous, but only when the ring contains 17 atoms, not 20 or 23 atoms. This technology will be useful for conducting direct comparisons between many different types of chemical libraries to determine their relative utility as a source of protein ligands.

New partner proteins containing novel internal recognition motif for human Glutaminase Interacting Protein (hGIP)

PubMed Central

Zencir, Sevil; Banerjee, Monimoy; Dobson, Melanie J.; Ayaydin, Ferhan; Fodor, Elfrieda Ayaydin; Topcu, Zeki; Mohanty, Smita

2013-01-01

Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein–protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human Glutaminase Interacting Protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. PMID:23395680

Toward a Federal Library and Information Services Network: A Proposal. A Report of the Intergovernmental Library Cooperation Project.

ERIC Educational Resources Information Center

Trezza, Alphonse F.

The current interaction among federal libraries and information centers was examined and assessed to determine ways to improve the coordination of resources and services among federal libraries and to increase cooperation between federal and non-federal libraries in order to meet national, state, and local information needs. The more than 400…

Ethnographic Methods in Academic Libraries: A Review

ERIC Educational Resources Information Center

Ramsden, Bryony

2016-01-01

Research in academic libraries has recently seen an increase in the use of ethnographic-based methods to collect data. Primarily used to learn about library users and their interaction with spaces and resources, the methods are proving particularly useful to academic libraries. The data ethnographic methods retrieve is rich, context specific, and…

Software Repository

NASA Technical Reports Server (NTRS)

Merwarth, P., D.

1983-01-01

The Common Software Module Repository (CSMR) is computerized library system with high product and service visibility to potential users. Online capabilities of system allow both librarian and user to interact with library. Librarian is responsible for maintaining information in CSMR library. User searches library to locate software modules that meet his or her current needs.

Library Programs for Teens: Mystery Theater. VOYA Guides

ERIC Educational Resources Information Center

Siwak, Karen J.

2010-01-01

It's no mystery that fun and exciting programs bring teens into the library. Theater programs provide a venue for teens to express themselves creatively, encourage their participation in library programming, and offer them the opportunity for lively interaction with peers and adults. In "Library Programs for Teens: Mystery Theater," Karen Siwak…

Interactive 3D display simulator for autostereoscopic smart pad

NASA Astrophysics Data System (ADS)

Choe, Yeong-Seon; Lee, Ho-Dong; Park, Min-Chul; Son, Jung-Young; Park, Gwi-Tae

2012-06-01

There is growing interest of displaying 3D images on a smart pad for entertainments and information services. Designing and realizing various types of 3D displays on the smart pad is not easy for costs and given time. Software simulation can be an alternative method to save and shorten the development. In this paper, we propose a 3D display simulator for autostereoscopic smart pad. It simulates light intensity of each view and crosstalk for smart pad display panels. Designers of 3D display for smart pad can interactively simulate many kinds of autostereoscopic displays interactively by changing parameters required for panel design. Crosstalk to reduce leakage of one eye's image into the image of the other eye, and light intensity for computing visual comfort zone are important factors in designing autostereoscopic display for smart pad. Interaction enables intuitive designs. This paper describes an interactive 3D display simulator for autostereoscopic smart pad.

Stochastic hyperfine interactions modeling library-Version 2

NASA Astrophysics Data System (ADS)

Zacate, Matthew O.; Evenson, William E.

2016-02-01

The stochastic hyperfine interactions modeling library (SHIML) provides a set of routines to assist in the development and application of stochastic models of hyperfine interactions. The library provides routines written in the C programming language that (1) read a text description of a model for fluctuating hyperfine fields, (2) set up the Blume matrix, upon which the evolution operator of the system depends, and (3) find the eigenvalues and eigenvectors of the Blume matrix so that theoretical spectra of experimental techniques that measure hyperfine interactions can be calculated. The optimized vector and matrix operations of the BLAS and LAPACK libraries are utilized. The original version of SHIML constructed and solved Blume matrices for methods that measure hyperfine interactions of nuclear probes in a single spin state. Version 2 provides additional support for methods that measure interactions on two different spin states such as Mössbauer spectroscopy and nuclear resonant scattering of synchrotron radiation. Example codes are provided to illustrate the use of SHIML to (1) generate perturbed angular correlation spectra for the special case of polycrystalline samples when anisotropy terms of higher order than A22 can be neglected and (2) generate Mössbauer spectra for polycrystalline samples for pure dipole or pure quadrupole transitions.

Vcs.js - Visualization Control System for the Web

NASA Astrophysics Data System (ADS)

Chaudhary, A.; Lipsa, D.; Doutriaux, C.; Beezley, J. D.; Williams, D. N.; Fries, S.; Harris, M. B.

2016-12-01

VCS is a general purpose visualization library, optimized for climate data, which is part of the UV-CDAT system. It provides a Python API for drawing 2D plots such as lineplots, scatter plots, Taylor diagrams, data colored by scalar values, vector glyphs, isocontours and map projections. VCS is based on the VTK library. Vcs.js is the corresponding JavaScript API, designed to be as close as possible to the original VCS Python API and to provide similar functionality for the Web. Vcs.js includes additional functionality when compared with VCS. This additional API is used to introspect data files available on the server and variables available in a data file. Vcs.js can display plots in the browser window. It always works with a server that reads a data file, extracts variables from the file and subsets the data. From this point, two alternate paths are possible. First the system can render the data on the server using VCS producing an image which is send to the browser to be displayed. This path works for for all plot types and produces a reference image identical with the images produced by VCS. This path uses the VTK-Web library. As an optimization, usable in certain conditions, a second path is possible. Data is packed, and sent to the browser which uses a JavaScript plotting library, such as plotly, to display the data. Plots that work well in the browser are line-plots, scatter-plots for any data and many other plot types for small data and supported grid types. As web technology matures, more plots could be supported for rendering in the browser. Rendering can be done either on the client or on the server and we expect that the best place to render will change depending on the available web technology, data transfer costs, server management costs and value provided to users. We intend to provide a flexible solution that allows for both client and server side rendering and a meaningful way to choose between the two. We provide a web-based user interface called vCdat which uses Vcs.js as its visualization library. Our paper will discuss the principles guiding our design choices for Vcs.js, present our design in detail and show a sample usage of the library.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Xiaoyong; Cai, Cuizan; Xiao, Fei

Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs havemore » been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.« less

Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

PubMed

Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

2014-09-01

Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

PubMed Central

Groves, Maria AT; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

2014-01-01

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics. PMID:24256948

Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

PubMed

Cohen-Khait, Ruth; Schreiber, Gideon

2018-04-27

Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

PubMed

Cardona-Correa, Albin; Rios-Velazquez, Carlos

2016-05-01

The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents.

Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

PubMed

Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

2007-03-01

Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodi, D. J.; Soares, A. S.; Makowski, L.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usagemore » patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.« less

Ribosome Display of Combinatorial Antibody Libraries Derived from Mice Immunized with Heat-Killed Xylella fastidiosa and the Selection of MopB-Specific Single-Chain Antibodies

PubMed Central

Azizi, Armaghan; Arora, Arinder; Markiv, Anatoliy; Lampe, David J.; Miller, Thomas A.

2012-01-01

Pierce's disease is a devastating lethal disease of Vitus vinifera grapevines caused by the bacterium Xylella fastidiosa. There is no cure for Pierce's disease, and control is achieved predominantly by suppressing transmission of the glassy-winged sharpshooter insect vector. We present a simple robust approach for the generation of panels of recombinant single-chain antibodies against the surface-exposed elements of X. fastidiosa that may have potential use in diagnosis and/or disease transmission blocking studies. In vitro combinatorial antibody ribosome display libraries were assembled from immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed X. fastidiosa. The libraries were used in a single round of selection against an outer membrane protein, MopB, resulting in the isolation of a panel of recombinant antibodies. The potential use of selected anti-MopB antibodies was demonstrated by the successful application of the 4XfMopB3 antibody in an enzyme-linked immunosorbent assay (ELISA), a Western blot assay, and an immunofluorescence assay (IFA). These immortalized in vitro recombinant single-chain antibody libraries generated against heat-killed X. fastidiosa are a resource for the Pierce's disease research community that may be readily accessed for the isolation of antibodies against a plethora of X. fastidiosa surface-exposed antigenic molecules. PMID:22327580

Public Libraries as Partners in Astronomy Outreach

NASA Astrophysics Data System (ADS)

Percy, J.

2017-09-01

Public libraries have proven to be effective partners in bringing astronomy to audiences across the large and diverse city of Toronto, Canada, and enabling astronomers - both young and old - to interact with members of our community. This article reflects on the author's experience working with public libraries, especially the Toronto Public Library (TPL), the busiest public library system in the world, to deliver over forty public presentations.

A Work Station For Control Of Changing Systems

NASA Technical Reports Server (NTRS)

Mandl, Daniel J.

1988-01-01

Touch screen and microcomputer enable flexible control of complicated systems. Computer work station equipped to produce graphical displays used as command panel and status indicator for command-and-control system. Operator uses images of control buttons displayed on touch screen to send prestored commands. Use of prestored library of commands reduces incidence of errors. If necessary, operator uses conventional keyboard to enter commands in real time to handle unforeseeable situations.

Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

NASA Astrophysics Data System (ADS)

Golec, Piotr; Karczewska-Golec, Joanna; Łoś, Marcin; Węgrzyn, Grzegorz

2012-11-01

Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

LibQUAL+ and the Community College Library

ERIC Educational Resources Information Center

Johnson, Wendell G.

2007-01-01

Community colleges can use market surveys to better understand the delivery of library services from the standpoint of the end user. One such survey, LibQUAL+, measures library service in three dimensions: 1. Affect of Service (patron interaction with library staff); 2. Information Control (availability of needed resources and ease of accessing);…

Old Skills and New Practices Mean Radical Change for Library Education

ERIC Educational Resources Information Center

Turner, Deborah; Gorichanaz, Tim

2016-01-01

Technological advances and other societal change have resulted in public libraries' increased reliance on online resources when providing access to information. However, a portion of those served by public libraries includes members of urban poor populations who may prefer to interact with information by talking. How can library educators ensure…

The Impact of the Academic Library on Student Success: Connecting the Dots

ERIC Educational Resources Information Center

Thorpe, Angie; Lukes, Ria; Bever, Diane J.; He, Yan

2016-01-01

In an age of assessment and accountability, academic libraries feel much pressure to prove their value according to new university measurements of student success. This study describes a methodology for how libraries may examine student interactions with services to assess whether library usage impacts student grade point averages (GPAs) and…

Interactive Augmented Reality System for Enhancing Library Instruction in Elementary Schools

ERIC Educational Resources Information Center

Chen, Chih-Ming; Tsai, Yen-Nung

2012-01-01

Due to limited budgets and manpower, most elementary schools in Taiwan do not plan or provide library instruction for students. Although students can use libraries, they typically lack the knowledge needed to use library resources effectively. Consequently, students have difficulty finding the books they need and can easily become overwhelmed by…

Digital Ethnography: Library Web Page Redesign among Digital Natives

ERIC Educational Resources Information Center

Klare, Diane; Hobbs, Kendall

2011-01-01

Presented with an opportunity to improve Wesleyan University's dated library home page, a team of librarians employed ethnographic techniques to explore how its users interacted with Wesleyan's current library home page and web pages in general. Based on the data that emerged, a group of library staff and members of the campus' information…

Selected pharmacokinetic issues of the use of antiepileptic drugs and parenteral nutrition in critically ill patients

PubMed Central

2010-01-01

Objectives To conduct a systematic review for the evidence supporting or disproving the reality of parenteral nutrition- antiepileptic drugs interaction, especially with respect to the plasma protein-binding of the drug. Methods The articles related to the topic were identified through Medline and PubMed search (1968-Feburary 2010) for English language on the interaction between parenteral nutrition and antiepileptic drugs; the search terms used were anti-epileptic drugs, parenteral nutrition, and/or interaction, and/or in vitro. The search looked for prospective randomized and nonrandomized controlled studies; prospective nonrandomized uncontrolled studies; retrospective studies; case reports; and in vitro studies. Full text of the articles were then traced from the Universiti Sains Malaysia (USM) library subscribed databases, including Wiley-Blackwell Library, Cochrane Library, EBSCOHost, OVID, ScienceDirect, SAGE Premier, Scopus, SpringerLINK, and Wiley InterScience. The articles from journals not listed by USM library were traced through inter library loan. Results There were interactions between parenteral nutrition and drugs, including antiepileptics. Several guidelines were designed for the management of illnesses such as traumatic brain injuries or cancer patients, involving the use of parenteral nutrition and antiepileptics. Moreover, many studies demonstrated the in vitro and in vivo parenteral nutrition -drugs interactions, especially with antiepileptics. Conclusions There was no evidence supporting the existence of parenteral nutrition-antiepileptic drugs interaction. The issue has not been studied in formal researches, but several case reports and anecdotes demonstrate this drug-nutrition interaction. However, alteration in the drug-free fraction result from parenteral nutrition-drug (i.e. antiepileptics) interactions may necessitate scrupulous reassessment of drug dosages in patients receiving these therapies. This reassessment may be particularly imperative in certain clinical situations characterized by hypoalbuminemia (e.g., burn patients). PMID:21194458

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

PubMed

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

2018-04-30

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC 50 ) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC 10 ) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification and characterization of a salivary-pellicle-binding peptide by phage display.

PubMed

Cukkemane, Nivedita; Bikker, Floris J; Nazmi, Kamran; Brand, Henk S; Veerman, Enno C I

2014-05-01

Dental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs. A library of 2×10(9) phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii. Ten unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces. Use of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii. Copyright © 2014 Elsevier Ltd. All rights reserved.

Heterobimetallic porphyrin complexes displaying triple dynamics: coupled metal motions controlled by constitutional evolution.

PubMed

Le Gac, Stéphane; Fusaro, Luca; Roisnel, Thierry; Boitrel, Bernard

2014-05-07

A bis-strap porphyrin ligand (1), with an overhanging carboxylic acid group on each side of the macrocycle, has been investigated toward the formation of dynamic libraries of bimetallic complexes with Hg(II), Cd(II), and Pb(II). Highly heteroselective metalation processes occurred in the presence of Pb(II), with Hg(II) or Cd(II) bound out-of-plane to the N-core and "PbOAc" bound to a carboxylate group of a strap on the opposite side. The resulting complexes, 1(Hg)·PbOAc and 1(Cd)·PbOAc, display three levels of dynamics. The first is strap-level (interactional dynamics), where the PbOAc moiety swings between the left and right side of the strap owing to a second sphere of coordination with lateral amide functions. The second is ligand-level (motional dynamics), where 1(Hg)·PbOAc and 1(Cd)·PbOAc exist as two degenerate states in equilibrium controlled by a chemical effector (AcO(-)). The process corresponds to a double translocation of the metal ions according to an intramolecular migration of Hg(II) or Cd(II) through the N-core, oscillating between the two equivalent overhanging carbonyl groups, coupled to an intermolecular pathway for PbOAc exchanging between the two equivalent overhanging carboxylate groups (N-core(up) ⇆ N-core(down) coupled to strap(down) ⇆ strap(up), i.e., coupled motion #1 in the abstract graphic). The third is library-level (constitutional dynamics), where a dynamic constitutional evolution of the system was achieved by the successive addition of two chemical effectors (DMAP and then AcO(-)). It allowed shifting equilibrium forward and backward between 1(Hg)·PbOAc and the corresponding homobimetallic complexes 1(Hg2)·DMAP and 1(Pb)·PbOAc. The latter displays a different ligand-level dynamics, in the form of an intraligand coupled migration of the Pb(II) ions (N-core(up) ⇆ strap(up) coupled to strap(down) ⇆ N-core(down), i.e., coupled motion #2 in the abstract graphic). In addition, the neutral "bridged" complexes 1HgPb and 1CdPb, with the metal ions on opposite sides both bound to the N-core and to a carboxylate of a strap, were structurally characterized. These results establish an unprecedented approach in supramolecular coordination chemistry, by considering the reversible interaction of a metal ion with the porphyrin N-core as a new source of self-organization processes. This work should provide new inspirations for the design of innovative adaptative materials and devices.

Redesigning of Microbial Cell Surface and Its Application to Whole-Cell Biocatalysis and Biosensors.

PubMed

Han, Lei; Zhao, Yukun; Cui, Shan; Liang, Bo

2018-06-01

Microbial cell surface display technology can redesign cell surfaces with functional proteins and peptides to endow cells some unique features. Foreign peptides or proteins are transported out of cells and immobilized on cell surface by fusing with anchoring proteins, which is an effective solution to avoid substance transfer limitation, enzyme purification, and enzyme instability. As the most frequently used prokaryotic and eukaryotic protein surface display system, bacterial and yeast surface display systems have been widely applied in vaccine, biocatalysis, biosensor, bioadsorption, and polypeptide library screening. In this review of bacterial and yeast surface display systems, different cell surface display mechanisms and their applications in biocatalysis as well as biosensors are described with their strengths and shortcomings. In addition to single enzyme display systems, multi-enzyme co-display systems are presented here. Finally, future developments based on our and other previous reports are discussed.

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production

PubMed Central

Trollope, K. M.; Görgens, J. F.

2015-01-01

The Aspergillus japonicus β-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition. PMID:26253664

LiveInventor: An Interactive Development Environment for Robot Autonomy

NASA Technical Reports Server (NTRS)

Neveu, Charles; Shirley, Mark

2003-01-01

LiveInventor is an interactive development environment for robot autonomy developed at NASA Ames Research Center. It extends the industry-standard OpenInventor graphics library and scenegraph file format to include kinetic and kinematic information, a physics-simulation library, an embedded Scheme interpreter, and a distributed communication system.

A Survey of the Usability of Digital Reference Services on Academic Health Science Library Web Sites

ERIC Educational Resources Information Center

Dee, Cheryl; Allen, Maryellen

2006-01-01

Reference interactions with patrons in a digital library environment using digital reference services (DRS) has become widespread. However, such services in many libraries appear to be underutilized. A study surveying the ease and convenience of such services for patrons in over 100 academic health science library Web sites suggests that…

Phage display as a promising approach for vaccine development.

PubMed

Aghebati-Maleki, Leili; Bakhshinejad, Babak; Baradaran, Behzad; Motallebnezhad, Morteza; Aghebati-Maleki, Ali; Nickho, Hamid; Yousefi, Mehdi; Majidi, Jafar

2016-09-29

Bacteriophages are specific antagonists to bacterial hosts. These viral entities have attracted growing interest as optimal vaccine delivery vehicles. Phages are well-matched for vaccine design due to being highly stable under harsh environmental conditions, simple and inexpensive large scale production, and potent adjuvant capacities. Phage vaccines have efficient immunostimulatory effects and present a high safety profile because these viruses have made a constant relationship with the mammalian body during a long-standing evolutionary period. The birth of phage display technology has been a turning point in the development of phage-based vaccines. Phage display vaccines are made by expressing multiple copies of an antigen on the surface of immunogenic phage particles, thereby eliciting a powerful and effective immune response. Also, the ability to produce combinatorial peptide libraries with a highly diverse pool of randomized ligands has transformed phage display into a straightforward, versatile and high throughput screening methodology for the identification of potential vaccine candidates against different diseases in particular microbial infections. These libraries can be conveniently screened through an affinity selection-based strategy called biopanning against a wide variety of targets for the selection of mimotopes with high antigenicity and immunogenicity. Also, they can be panned against the antiserum of convalescent individuals to recognize novel peptidomimetics of pathogen-related epitopes. Phage display has represented enormous promise for finding new strategies of vaccine discovery and production and current breakthroughs promise a brilliant future for the development of different phage-based vaccine platforms.

Auditory display as feedback for a novel eye-tracking system for sterile operating room interaction.

PubMed

Black, David; Unger, Michael; Fischer, Nele; Kikinis, Ron; Hahn, Horst; Neumuth, Thomas; Glaser, Bernhard

2018-01-01

The growing number of technical systems in the operating room has increased attention on developing touchless interaction methods for sterile conditions. However, touchless interaction paradigms lack the tactile feedback found in common input devices such as mice and keyboards. We propose a novel touchless eye-tracking interaction system with auditory display as a feedback method for completing typical operating room tasks. Auditory display provides feedback concerning the selected input into the eye-tracking system as well as a confirmation of the system response. An eye-tracking system with a novel auditory display using both earcons and parameter-mapping sonification was developed to allow touchless interaction for six typical scrub nurse tasks. An evaluation with novice participants compared auditory display with visual display with respect to reaction time and a series of subjective measures. When using auditory display to substitute for the lost tactile feedback during eye-tracking interaction, participants exhibit reduced reaction time compared to using visual-only display. In addition, the auditory feedback led to lower subjective workload and higher usefulness and system acceptance ratings. Due to the absence of tactile feedback for eye-tracking and other touchless interaction methods, auditory display is shown to be a useful and necessary addition to new interaction concepts for the sterile operating room, reducing reaction times while improving subjective measures, including usefulness, user satisfaction, and cognitive workload.

Free oscilloscope web app using a computer mic, built-in sound library, or your own files

NASA Astrophysics Data System (ADS)

Ball, Edward; Ruiz, Frances; Ruiz, Michael J.

2017-07-01

We have developed an online oscilloscope program which allows users to see waveforms by utilizing their computer microphones, selecting from our library of over 30 audio files, and opening any *.mp3 or *.wav file on their computers. The oscilloscope displays real-time signals against time. The oscilloscope has been calibrated so one can make accurate frequency measurements of periodic waves to within 1%. The web app is ideal for computer projection in class.

Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

A small diversity library of α-methyl amide analogs of sulindac for probing anticancer structure-activity relationships.

PubMed

Mathew, Bini; Snowden, Timothy S; Connelly, Michele C; Guy, R Kiplin; Reynolds, Robert C

2018-05-10

Non-steroidal anti-inflammatory drugs (NSAIDs) have a variety of potential indications that include management of pain and inflammation as well as chemoprevention and/or treatment of cancer. Furthermore, a specific form of ibuprofen, dexibuprofen or the S-(+) form, shows interesting neurological activities and has been proposed for the treatment of Alzheimer's disease. In a continuation of our work probing the anticancer activity of small sulindac libraries, we have prepared and screened a small diversity library of α-methyl substituted sulindac amides in the profen class. Several compounds of this series displayed promising activity compared with a lead sulindac analog. Copyright © 2018. Published by Elsevier Ltd.

Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts.

PubMed

Neveu, Julie; Regeard, Christophe; DuBow, Michael S

2011-08-01

The screening of environmental DNA metagenome libraries for functional activities can provide an important source of new molecules and enzymes. In this study, we identified 17 potential protease-producing clones from two metagenomic libraries derived from samples of surface sand from the Gobi and Death Valley deserts. Two of the proteases, DV1 and M30, were purified and biochemically examined. These two proteases displayed a molecular mass of 41.5 kDa and 45.7 kDa, respectively, on SDS polyacrylamide gels. Alignments with known protease sequences showed less than 55% amino acid sequence identity. These two serine proteases appear to belong to the subtilisin (S8A) family and displayed several unique biochemical properties. Protease DV1 had an optimum pH of 8 and an optimal activity at 55°C, while protease M30 had an optimum pH >11 and optimal activity at 40°C. The properties of these enzymes make them potentially useful for biotechnological applications and again demonstrate that metagenomic approaches can be useful, especially when coupled with the study of novel environments such as deserts.

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

PubMed Central

Moisy, Dorothée; Avilov, Sergiy V.; Jacob, Yves; Laoide, Brid M.; Ge, Xingyi; Baudin, Florence; Jestin, Jean-Luc

2012-01-01

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses. PMID:22696656

Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

PubMed

Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

2017-01-01

Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

PubMed

Gardères, Johan; Domart-Coulon, Isabelle; Marie, Arul; Hamer, Bojan; Batel, Renato; Müller, Werner E G; Bourguet-Kondracki, Marie-Lise

2016-10-01

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.