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Sample records for combinatorial fab library

  1. Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture.

    PubMed

    Bender, E; Woof, J M; Atkin, J D; Barker, M D; Bebbington, C R; Burton, D R

    1993-04-01

    The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries. PMID:8518367

  2. Recursive deconvolution of combinatorial chemical libraries.

    PubMed

    Erb, E; Janda, K D; Brenner, S

    1994-11-22

    A recursive strategy that solves for the active members of a chemical library is presented. A pentapeptide library with an alphabet of Gly, Leu, Phe, and Tyr (1024 members) was constructed on a solid support by the method of split synthesis. One member of this library (NH2-Tyr-Gly-Gly-Phe-Leu) is a native binder to a beta-endorphin antibody. A variation of the split synthesis approach is used to build the combinatorial library. In four vials, a member of the library's alphabet is coupled to a solid support. After each coupling, a portion of the resin from each of the four reaction vials was set aside and catalogued. The solid support from each vial is then combined, mixed, and redivided. The steps of (i) coupling, (ii) saving and cataloging, and (iii) randomizing were repeated until a pentapeptide library was obtained. The four pentapeptide libraries where the N-terminal amino acid is defined were screened against the beta-endorphin antibody and quantitated via an ELISA. The amino acid of the four pools that demonstrated the most binding was then coupled to the four tetrapeptide partial libraries that had been set aside and catalogued during the split synthesis. This recursive deconvolution was repeated until the best binders were deduced. Besides the anticipated native binder, two other members of the library displayed significant binding. This recursive method of deconvolution does not use a molecular tag, requires only one split synthesis, and can be applied to the deconvolution of nonlinear small-molecule combinatorial libraries and linear oligomeric combinatorial libraries, since it is based only on the procedure of the synthesis. PMID:7972077

  3. Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

    NASA Astrophysics Data System (ADS)

    Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

    1993-05-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

  4. Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries.

    PubMed Central

    Williamson, R A; Burioni, R; Sanna, P P; Partridge, L J; Barbas, C F; Burton, D R

    1993-01-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro. Images Fig. 1 Fig. 2 PMID:7683424

  5. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions. PMID:26014116

  6. Kinetically controlled phenomena in dynamic combinatorial libraries.

    PubMed

    Ji, Qing; Lirag, Rio Carlo; Miljanić, Ognjen Š

    2014-03-21

    Dynamic combinatorial libraries (DCLs) are collections of structurally related compounds that can interconvert through reversible chemical reaction(s). Such reversibility endows DCLs with adaptability to external stimuli, as rapid interconversion allows quick expression of those DCL components which best respond to the disturbing stimulus. This Tutorial Review focuses on the kinetically controlled phenomena that occur within DCLs. Specifically, it will describe dynamic chiral resolution of DCLs, their self-sorting under the influence of irreversible chemical and physical stimuli, and the autocatalytic behaviours within DCLs which can result in self-replicating systems. A brief discussion of precipitation-induced phenomena will follow and the review will conclude with the presentation of covalent organic frameworks (COFs)-porous materials whose synthesis critically depends on the fine tuning of the crystal growth and error correction rates within large DCLs. PMID:24445841

  7. Structure-based design of combinatorial mutagenesis libraries

    PubMed Central

    Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

    2015-01-01

    The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called “Structure-based Optimization of Combinatorial Mutagenesis” (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states. PMID:25611189

  8. Plasma sputtering system for deposition of thin film combinatorial libraries

    NASA Astrophysics Data System (ADS)

    Cooper, James S.; Zhang, Guanghai; McGinn, Paul J.

    2005-06-01

    The design of a plasma sputtering system for the deposition of combinatorial libraries is described. A rotating carousel is used to position shadow masks between the targets and the substrate. Multilayer films are built up by depositing sequentially through various masks. Postdeposition annealing is used to promote interdiffusion of the layered structures. Either discrete or compositional gradient libraries can be deposited in this system. Samples appropriate for characterization with a scanning electrochemical microscope or a multichannel microelectrode array system can be produced. The properties of some deposited Pt-Ru films for fuel cell applications are described.

  9. Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort.

    PubMed

    Jeschek, Markus; Gerngross, Daniel; Panke, Sven

    2016-01-01

    Rational flux design in metabolic engineering approaches remains difficult since important pathway information is frequently not available. Therefore empirical methods are applied that randomly change absolute and relative pathway enzyme levels and subsequently screen for variants with improved performance. However, screening is often limited on the analytical side, generating a strong incentive to construct small but smart libraries. Here we introduce RedLibs (Reduced Libraries), an algorithm that allows for the rational design of smart combinatorial libraries for pathway optimization thereby minimizing the use of experimental resources. We demonstrate the utility of RedLibs for the design of ribosome-binding site libraries by in silico and in vivo screening with fluorescent proteins and perform a simple two-step optimization of the product selectivity in the branched multistep pathway for violacein biosynthesis, indicating a general applicability for the algorithm and the proposed heuristics. We expect that RedLibs will substantially simplify the refactoring of synthetic metabolic pathways. PMID:27029461

  10. Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort

    PubMed Central

    Jeschek, Markus; Gerngross, Daniel; Panke, Sven

    2016-01-01

    Rational flux design in metabolic engineering approaches remains difficult since important pathway information is frequently not available. Therefore empirical methods are applied that randomly change absolute and relative pathway enzyme levels and subsequently screen for variants with improved performance. However, screening is often limited on the analytical side, generating a strong incentive to construct small but smart libraries. Here we introduce RedLibs (Reduced Libraries), an algorithm that allows for the rational design of smart combinatorial libraries for pathway optimization thereby minimizing the use of experimental resources. We demonstrate the utility of RedLibs for the design of ribosome-binding site libraries by in silico and in vivo screening with fluorescent proteins and perform a simple two-step optimization of the product selectivity in the branched multistep pathway for violacein biosynthesis, indicating a general applicability for the algorithm and the proposed heuristics. We expect that RedLibs will substantially simplify the refactoring of synthetic metabolic pathways. PMID:27029461

  11. Direct selection for a catalytic mechanism from combinatorial antibody libraries.

    PubMed Central

    Janda, K D; Lo, C H; Li, T; Barbas, C F; Wirsching, P; Lerner, R A

    1994-01-01

    Semisynthetic combinatorial antibody library methodology in the phage-display format was used to select for a cysteine residue in complementarity-determining regions. Libraries were panned with an alpha-phenethyl pyridyl disulfide that undergoes disulfide interchange. Out of 10 randomly picked clones, two contained an unpaired cysteine, one of which was studied. The antibody catalyzed the hydrolysis of the corresponding thioester where the electrophilic carbonyl occupies the three-dimensional space that was defined by the reactive sulfur atom during selection. The reaction operates by covalent catalysis. Although the steady-state rate enhancement relative to the activated thiol ester substrate is modest, hydrolysis of the acylated cysteine intermediate is remarkably efficient with a catalytic advantage of about four orders of magnitude. The results suggest that iterative mechanism-based selection procedures can recapitulate the enzymatic mechanisms refined through evolution. Images PMID:8146149

  12. Device for preparing combinatorial libraries in powder metallurgy.

    PubMed

    Yang, Shoufeng; Evans, Julian R G

    2004-01-01

    This paper describes a powder-metering, -mixing, and -dispensing mechanism that can be used as a method for producing large numbers of samples for metallurgical evaluation or electrical or mechanical testing from multicomponent metal and cermet powder systems. It is designed to make use of the same commercial powders that are used in powder metallurgy and, therefore, to produce samples that are faithful to the microstructure of finished products. The particle assemblies produced by the device could be consolidated by die pressing, isostatic pressing, laser sintering, or direct melting. The powder metering valve provides both on/off and flow rate control of dry powders in open capillaries using acoustic vibration. The valve is simple and involves no relative movement, avoiding seizure with fine powders. An orchestra of such valves can be arranged on a building platform to prepare multicomponent combinatorial libraries. As with many combinatorial devices, identification and evaluation of sources of mixing error as a function of sample size is mandatory. Such an analysis is presented. PMID:15244416

  13. Selecting agonists from single cells infected with combinatorial antibody libraries.

    PubMed

    Zhang, Hongkai; Yea, Kyungmoo; Xie, Jia; Ruiz, Diana; Wilson, Ian A; Lerner, Richard A

    2013-05-23

    We describe a system for direct selection of antibodies that are receptor agonists. Combinatorial antibody libraries in lentiviruses are used to infect eukaryotic cells that contain a fluorescent reporter system coupled to the receptor for which receptor agonist antibodies are sought. In this embodiment of the method, very large numbers of candidate antibodies expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. Following infection, cells that fluoresce are sorted and the integrated genes encoding the agonist antibodies recovered. We validated the system by illustrating its ability to generate rapidly potent antibody agonists that are complete thrombopoietin phenocopies. The system should be generalizable to any pathway where its activation can be linked to production of a selectable phenotype. PMID:23706638

  14. Simultaneous Disulfide and Boronic Acid Ester Exchange in Dynamic Combinatorial Libraries

    PubMed Central

    Diemer, Sanna L.; Kristensen, Morten; Rasmussen, Brian; Beeren, Sophie R.; Pittelkow, Michael

    2015-01-01

    Dynamic combinatorial chemistry has emerged as a promising tool for the discovery of complex receptors in supramolecular chemistry. At the heart of dynamic combinatorial chemistry are the reversible reactions that enable the exchange of building blocks between library members in dynamic combinatorial libraries (DCLs) ensuring thermodynamic control over the system. If more than one reversible reaction operates in a single dynamic combinatorial library, the complexity of the system increases dramatically, and so does its possible applications. One can imagine two reversible reactions that operate simultaneously or two reversible reactions that operate independently. Both these scenarios have advantages and disadvantages. In this contribution, we show how disulfide exchange and boronic ester transesterification can function simultaneous in dynamic combinatorial libraries under appropriate conditions. We describe the detailed studies necessary to establish suitable reaction conditions and highlight the analytical techniques appropriate to study this type of system. PMID:26378519

  15. An Indexed Combinatorial Library: The Synthesis and Testing of Insect Repellents

    NASA Astrophysics Data System (ADS)

    Miles, William H.; Gelato, Kathy A.; Pompizzi, Kristen M.; Scarbinsky, Aislinn M.; Albrecht, Brian K.; Reynolds, Elaine R.

    2001-04-01

    An indexed combinatorial library of amides was prepared by the reaction of amines and acid chlorides. A simple test for insect repellency using fruit flies (Drosophila melanogaster) allowed the determination of the most repellent sublibraries. The student-generated data were collected and analyzed to determine the most active amide(s) in the library. This experiment illustrates the fundamentals of combinatorial chemistry, a field that has undergone explosive growth in the last decade.

  16. Mimicking nature: Phosphopeptide enrichment using combinatorial libraries of affinity ligands.

    PubMed

    Batalha, Iris L; Zhou, Houjiang; Lilley, Kathryn; Lowe, Christopher R; Roque, Ana C A

    2016-07-29

    Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins (α- and β-caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides. PMID:27345211

  17. Combinatorial synthesis of thin film libraries for microwave dielectrics

    NASA Astrophysics Data System (ADS)

    Wessler, B.; Jéhanno, V.; Rossner, W.; Maier, W. F.

    2004-02-01

    The short innovation cycles in communication technology require the development and optimization of high-performance dielectrics for passive integration, utilized, e.g., in band pass filters, antennas, or dielectric resonators. Applying combinatorial methods offers the advantage to accelerate the discovery of more efficient microwave dielectrics and to investigate the effects of a variety of dopants on the dielectric properties. In the present study, libraries consisting of chemically diverse thin films are produced by chemical solution deposition. A sol-gel precursor set was developed consisting of precursor solutions for different elements, such as Ba, Ti, W, and Sr, which convert to the corresponding oxides by annealing. These precursor solutions are mixed in various ratios by pipetting robots and are then deposited on structured substrates followed by calcination and sintering. The influence of different parameters on the film quality such as the type of precursor used, their miscibility with each other, the thermal processing, and the technique used to pre-structure the wafer is discussed.

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

    PubMed Central

    Yuan, Zuanning; Du, Minge; Chen, Yiwen; Dou, Fei

    2013-01-01

    Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli-gomers and constructed a naïve human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that specifically recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked immunosorbent assay demonstrated this antibody bound specifically to human amyloid-beta 42 tetramer and nonamer, but not the monomer or high molecular weight oligomers. This study successfully constructed a human phage display library and screened a single-domain antibody that specifically recognized amyloid-beta 42 oligomers. PMID:25206631

  19. Combinatorial Library Screening Coupled to Mass Spectrometry to Identify Valuable Cyclic Peptides.

    PubMed

    Camperi, Silvia A; Giudicessi, Silvana L; Martínez-Ceron, María C; Gurevich-Messina, Juan M; Saavedra, Soledad L; Acosta, Gerardo; Cascone, Osvaldo; Erra-Balsells, Rosa; Albericio, Fernando

    2016-01-01

    Combinatorial library screening coupled to mass spectrometry (MS) analysis is a practical approach to identify useful peptides. Cyclic peptides can have high biological activity, selectivity, and affinity for target proteins, and high stability against proteolytic degradation. Here we describe two strategies to prepare combinatorial libraries suitable for MS analysis to accelerate the discovery of cyclic peptide structures. Both approaches use ChemMatrix resin and the linker 4-hydroxymethylbenzoic acid. One strategy involves the synthesis of a one-bead-two-peptides library in which each bead contains both the cyclic peptide and its linear counterpart to facilitate MS analysis. The other protocol is based on the synthesis of a cyclic depsipeptide library in which a glycolamidic ester group is incorporated by adding glycolic acid. After library screening, the ring is opened and the peptide is released simultaneously for subsequent MS analysis. © 2016 by John Wiley & Sons, Inc. PMID:27258690

  20. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity

    PubMed Central

    Kang, Seungmin; Kim, Wankyu; Shim, Hyunbo

    2015-01-01

    The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications. PMID:26484868

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

    PubMed Central

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

    2013-01-01

    This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

  2. Combinatorial bulk ceramic magnetoelectric composite libraries of strontium hexaferrite and barium titanate.

    PubMed

    Pullar, Robert C

    2012-07-01

    Bulk ceramic combinatorial libraries were produced via a novel, high-throughput (HT) process, in the form of polycrystalline strips with a gradient composition along the length of the library. Step gradient ceramic composite libraries with 10 mol % steps of SrFe12O19-BaTiO3 (SrM-BT) were made and characterized using HT methods, as a proof of principle of the combinatorial bulk ceramic process, and sintered via HT thermal processing. It was found that the SrM-BT libraries sintered at 1175 °C had the optimum morphology and density. The compositional, electrical and magnetic properties of this library were analyzed, and it was found that the SrM and BT phases did not react and remained discrete. The combinatorial synthesis method produced a relatively linear variation in composition. The magnetization of the library followed the measured compositions very well, as did the low frequency permittivity values of most compositions in the library. However, with high SrM content of ≥80 mol %, the samples became increasingly conductive, and no reliable dielectric measurements could be made. Such conductivity would also greatly inhibit any ferroelectricity and magnetoelectric coupling with these composites with high levels of the SrM hexagonal ferrite. PMID:22676556

  3. 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. PMID:21512219

  4. A Structure-Based Design Protocol for Optimizing Combinatorial Protein Libraries.

    PubMed

    Lunt, Mark W; Snow, Christopher D

    2016-01-01

    Protein variant libraries created via site-directed mutagenesis are a powerful approach to engineer improved proteins for numerous applications such as altering enzyme substrate specificity. Conventional libraries commonly use a brute force approach: saturation mutagenesis via degenerate codons that encode all 20 natural amino acids. In contrast, this chapter describes a protocol for designing "smarter" degenerate codon libraries via direct combinatorial optimization in "library space." Several case studies illustrate how it is possible to design degenerate codon libraries that are highly enriched for favorable, low-energy sequences as assessed using a standard all-atom scoring function. There is much to gain for experimental protein engineering laboratories willing to think beyond site saturation mutagenesis. In the common case that the exact experimental screening budget is not fixed, it is particularly helpful to perform a Pareto analysis to inspect favorable libraries at a range of possible library sizes. PMID:27094288

  5. Combinatorial synthesis and antibacterial evaluation of an indexed chalcone library.

    PubMed

    Ansari, Farzana Latif; Nazir, Samina; Noureen, Humaira; Mirza, Bushra

    2005-12-01

    A 120-membered chalcone library has been designed and prepared from six differently substituted acetophenones (A1-A6) and 20 benzaldehydes (B1-B20). The library was subjected to biological studies targeted against six bacterial strains. For the identification of the most-active member(s) of the library, the so-called indexed or positional-scanning method was applied. Six out of 26 sub-libraries, i.e., AL1-AL6, were synthesized by keeping the acetophenone moiety A fixed and using equimolar quantities of the 20 different benzaldehydes. The remaining 20 sub-libraries BL1-BL20 were prepared by keeping the benzaldehyde B component fixed and varying the six acetophenones (Table 1). The bactericidal activities of the resulting sub-libraries were tested and used as indices to the rows or columns of a two-dimensional matrix. Finally, parallel synthesis of 24 specific members with the highest-expected antibacterial activities, present in two sub-libraries, was carried out. These chalcones were screened again, and the results were exploited for establishing the structure-activity relationship (SAR) and the identification of the lead compound, which turned out to be 1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (A2B2) in terms of activity towards Staphylococcus aureus and Bacillus subtilis (Tables 5-7). PMID:17191962

  6. Scanning electrochemical microscope characterization of thin film combinatorial libraries for fuel cell electrode applications

    NASA Astrophysics Data System (ADS)

    Black, M.; Cooper, J.; McGinn, P.

    2005-01-01

    Pt-Ru combinatorial libraries of potential fuel cell anode catalysts are formed by sequential sputter deposition through masks onto Si wafers. Scanning electrochemical microscopy (SECM) is employed for characterization of electrocatalytic activity. Aspects of using a scanning electrochemical microscope for characterization of an array of thin film fuel cell electrode materials are discussed. It is shown that in applying SECM to library characterization, careful attention must be paid to thin film annealing, specimen topography and tip degradation in order to realize meaningful results. Results from a Pt-Ru thin film library reveal the most active members near the 50 Pt/50 Ru composition.

  7. Combinatorial Libraries of Transition Metal Oxides Using an Ab Initio High Throughput Approach

    NASA Astrophysics Data System (ADS)

    Li, Guo; Yan, Qimin; Newhouse, Paul; Zhou, Lan; Gregoire, John; Neaton, Jeffrey

    2015-03-01

    Using the results of first-principles calculations and data from the Materials Project (materialsproject.org), we have developed a simple but efficient scheme to theoretically simulate phase coexistence in experimental combinatorial libraries as a function of composition and temperature. In our approach, each experimental sample in a combinatorial library at a fixed composition is considered as a mixture of all the known compounds; and the compound concentrations are determined from calculations of their compositions and relevant thermodynamic potentials. Consequently, multiple compounds can be identified in every sample. To test our approach, we studied the pseudobinary library MnxV(1-x)Oy, and found that, together with those stable compounds predicted in a phase diagram, some of the above-convex-hull compounds, which are viewed unstable, also play a significant role in the combinatorial library. We validated our approach via comparison of calculated X-ray diffraction spectra for multiple phases and recent measurements. This work supported by DOE (the JCAP under Award number DE-SC000499 and the Molecular Foundry of LBNL), and computational resources provided by NERSC.

  8. Price-Focused Analysis of Commercially Available Building Blocks for Combinatorial Library Synthesis.

    PubMed

    Kalliokoski, Tuomo

    2015-10-12

    Combinatorial libraries are synthesized by combining smaller reagents (building blocks), the price of which is an important component of the total costs associated with the synthetic exercise. A significant portion of commercially available reagents are too expensive for large scale work. In this study, 13 commonly used reagent classes in combinatorial library synthesis (primary and secondary amines, carboxylic acids, alcohols, ketones, aldehydes, boronic acids, acyl halides, sulfonyl chlorides, isocyanates, isothiocyanates, azides and chloroformates) were analyzed with respect to the cost, physicochemical properties (molecular weight and calculated logP), chemical diversity, and 3D-likeness using a large data set. The results define the chemical space accessible under a constraint of limited financial resources. PMID:26371511

  9. Positional scanning substrate combinatorial library (PS-SCL) approach to define caspase substrate specificity.

    PubMed

    Poręba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Drąg, Marcin

    2014-01-01

    Positional scanning substrate combinatorial library (PS-SCL) is a powerful tool for studying substrate specificity of proteolytic enzymes. Here, we describe the protocol for analyzing S4-S2 pockets preferences of caspases using PS-SCL. Additionally, we describe procedures for the identification of optimal substrates sequence after PS-SCL, solid phase synthesis, and purification of selected fluorogenic substrates, as well as their kinetic analysis. PMID:24567093

  10. Late stage modification of receptors identified from dynamic combinatorial libraries.

    PubMed

    Pinkin, Nicholas K; N Power, Amanie; Waters, Marcey L

    2015-11-28

    Small molecule receptors are attractive potential sensors of post-translational modifications, including methylated lysine and methylated arginine. Using dynamic combinatorial chemistry (DCC), our lab previously identified a suite of receptors that bind to Kme3 with a range of affinities ranging from low micromolar to high nanomolar, each with a unique selectivity for Kme3 over the lower methylation states. To enable these receptors to have broad application as Kme3 sensors, we have developed a method for their late-stage modification, which we used to synthesize biotinylated derivatives of A2B, A2D, and A2G in a single step. For our most attractive receptor for applications, A2N, we needed to develop an alternative method for its selective functionalization, which we achieved by "activating" the carboxylic acids on the constituent monomer A or N by pre-functionalizing them with glycine (Gly). Using the resulting Gly-A and Gly-N monomers, we synthesized the novel A2N variants A2Gly-N, Gly-A2N, and Gly-A2Gly-N, which enabled the late stage biotinylation of A2N wherever Gly was incorporated. Finally, we performed ITC and NMR binding experiments to study the effect that carboxylate spacing has on the affinity and selectivity of A2Gly-N and Gly-A2N for KmeX guests compared to A2N. These studies revealed the proximity of the carboxylates to play a complex role in the molecular recognition event, despite their positioning on the outside of the receptor. PMID:26384269

  11. Asymmetric Proteome Equalization of the Skeletal Muscle Proteome Using a Combinatorial Hexapeptide Library

    PubMed Central

    Rivers, Jenny; Hughes, Chris; McKenna, Thérèse; Woolerton, Yvonne; Vissers, Johannes P. C.; Langridge, James I.; Beynon, Robert J.

    2011-01-01

    Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method. PMID:22205978

  12. Direct screening of solution phase combinatorial libraries encoded with externally sensitized photolabile tags

    PubMed Central

    Kottani, Rudresha; Valiulin, Roman A.; Kutateladze, Andrei G.

    2006-01-01

    Solution phase combinatorial chemistry holds an enormous promise for modern drug discovery. Much needed are direct methods to assay such libraries for binding of biological targets. An approach to encoding and screening of solution phase libraries has been developed based on the conditional photorelease of externally sensitized photolabile tags. The encoding tags are released into solution only when a sought-for binding event occurs between the ligand and the receptor, outfitted with an electron-transfer sensitizer. The released tags are analyzed in solution revealing the identity of the lead ligand or narrowing the range of potential leads. PMID:16956977

  13. Selective Chromium(VI) Ligands Identified Using Combinatorial Peptoid Libraries

    PubMed Central

    Knight, Abigail S.; Zhou, Effie Y.; Pelton, Jeffrey G.; Francis, Matthew B.

    2013-01-01

    Hexavalent chromium (Cr(VI)) is a world-wide water contaminant that is currently without cost-effective and efficient remediation strategies. This is in part due to a lack of ligands that can bind it amid an excess of innocuous ions in aqueous solution. We present herein the design and application of a peptoid-based library of ligand candidates for toxic metal ions. A selective screening process was used to identify members of the library that can bind to Cr(VI) species at neutral pH and in the presence of a large excess of spectator ions. Eleven sequences were identified, and their affinities were compared using titrations monitored with UV-Vis spectroscopy. To identify the interactions involved in coordination and specificity, we evaluated the effects of sequence substitutions and backbone variation in the highest affinity structure. Additional characterization of the complex formed between this sequence and Cr(VI) was performed using NMR spectroscopy. To evaluate the ability of the developed sequences to remediate contaminated solutions, the structures were synthesized on a solid-phase resin and incubated with environmental water samples that contained simulated levels of chromium contamination. The synthetic structures demonstrated the ability to reduce the amount of toxic chromium to levels within the range of the EPA contamination guidelines. In addition to providing some of the first selective ligands for Cr(VI), these studies highlight the promise of peptoid sequences as easily-prepared components of environmental remediation materials. PMID:24195610

  14. Design, Synthesis, and Application of OB2C Combinatorial Peptide and Peptidomimetic Libraries

    PubMed Central

    Liu, Ruiwu; Shih, Tsung-Chieh; Deng, Xiaojun; Anwar, Lara; Ahadi, Sara; Kumaresan, Pappanaicken; Lam, Kit S.

    2015-01-01

    The “one-bead two-compound” (OB2C) combinatorial library is constructed on topologically segregated trifunctional bilayer beads such that each bead has a fixed cell-capturing ligand and a random library compound co-displayed on its surface and a chemical coding tag (bar code) inside the bead. An OB2C library containing thousands to millions of compounds can be synthesized and screened concurrently within a short period of time. When live cells are incubated with such OB2C libraries, every bead will be coated with a monolayer of cells. The cell membranes of the captured cells facing the bead surface are exposed to the library compounds tethered to each bead. A specific biochemical or cellular response can be detected with an appropriate reporter system. The OB2C method enables investigators to rapidly discover synthetic molecules that not only interact with cell-surface receptors but can also stimulate or inhibit downstream cell signaling. To demonstrate this powerful method, one OB2C peptide library and two OB2C peptidomimetic libraries were synthesized and screened against Molt-4 lymphoma cells to discover “death ligands.” Apoptosis of the bead-bound cells was detected with immunocytochemistry using horseradish peroxidase (HRP)-conjugated anti-cleaved caspase-3 antibody and 3,3′-diaminobenzidine as a substrate. Two novel synthetic “death ligands” against Molt-4 cells were discovered using this OB2C library approach. PMID:25616322

  15. Design, synthesis, and application of OB2C combinatorial peptide and peptidomimetic libraries.

    PubMed

    Liu, Ruiwu; Shih, Tsung-Chieh; Deng, Xiaojun; Anwar, Lara; Ahadi, Sara; Kumaresan, Pappanaicken; Lam, Kit S

    2015-01-01

    The "one-bead two-compound" (OB2C) combinatorial library is constructed on topologically segregated trifunctional bilayer beads such that each bead has a fixed cell-capturing ligand and a random library compound co-displayed on its surface and a chemical coding tag (bar code) inside the bead. An OB2C library containing thousands to millions of compounds can be synthesized and screened concurrently within a short period of time. When live cells are incubated with such OB2C libraries, every bead will be coated with a monolayer of cells. The cell membranes of the captured cells facing the bead surface are exposed to the library compounds tethered to each bead. A specific biochemical or cellular response can be detected with an appropriate reporter system. The OB2C method enables investigators to rapidly discover synthetic molecules that not only interact with cell-surface receptors but can also stimulate or inhibit downstream cell signaling. To demonstrate this powerful method, one OB2C peptide library and two OB2C peptidomimetic libraries were synthesized and screened against Molt-4 lymphoma cells to discover "death ligands." Apoptosis of the bead-bound cells was detected with immunocytochemistry using horseradish peroxidase (HRP)-conjugated anti-cleaved caspase-3 antibody and 3,3'-diaminobenzidine as a substrate. Two novel synthetic "death ligands" against Molt-4 cells were discovered using this OB2C library approach. PMID:25616322

  16. Template-based combinatorial enumeration of virtual compound libraries for lipids

    PubMed Central

    2012-01-01

    A variety of software packages are available for the combinatorial enumeration of virtual libraries for small molecules, starting from specifications of core scaffolds with attachments points and lists of R-groups as SMILES or SD files. Although SD files include atomic coordinates for core scaffolds and R-groups, it is not possible to control 2-dimensional (2D) layout of the enumerated structures generated for virtual compound libraries because different packages generate different 2D representations for the same structure. We have developed a software package called LipidMapsTools for the template-based combinatorial enumeration of virtual compound libraries for lipids. Virtual libraries are enumerated for the specified lipid abbreviations using matching lists of pre-defined templates and chain abbreviations, instead of core scaffolds and lists of R-groups provided by the user. 2D structures of the enumerated lipids are drawn in a specific and consistent fashion adhering to the framework for representing lipid structures proposed by the LIPID MAPS consortium. LipidMapsTools is lightweight, relatively fast and contains no external dependencies. It is an open source package and freely available under the terms of the modified BSD license. PMID:23006594

  17. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  18. Composition-Dependent Phase Concentrations from First Principles: Simulating Combinatorial Libraries of Transition Metal Oxides

    NASA Astrophysics Data System (ADS)

    Li, Guo; Yan, Qimin; Zhou, Lan; Newhouse, Paul; Gregoire, John; Neaton, Jeffrey

    To identify material phases in experimental combinatorial libraries, we develop a theoretical model as a complementary approach to accelerate phase identification. In this approach, samples in a combinatorial library are simulated as mixtures in chemical equilibria. Each of these mixtures contains all the solid-state phases, which can possibly exist in the library. Using the total energies of these phases obtained in first-principle calculations, we calculate the Gibbs free energy changes in the corresponding chemical reactions, and subsequently evaluate the equilibrium concentrations of the phases in every sample according to the law of mass action. Furthermore, to test this approach, we simulate pseudobinary libraries MnxV1-xOy and CuxV1-xOy. Interestingly, we find that the composition-dependent phase concentrations calculated within our approach agree well with the experimental results measured with XRD spectroscopy. This work supported by DOE (the JCAP under Award Number DE-SC0004993 and the Molecular Foundry of LBNL), and computational resources provided by NERSC.

  19. A drop-on-demand ink-jet printer for combinatorial libraries and functionally graded ceramics.

    PubMed

    Mohebi, Mohammad Masoud; Evans, Julian R G

    2002-01-01

    A printer has been designed and built for the preparation of combinatorial libraries of ceramics and for solid freeforming of functionally graded ceramics with three-dimensionally programmable spatial variation in composition. Several ceramic suspensions (as inks) can be subjected to micromixing behind the nozzle and printed at precise positions. Both mixing and positioning are computer-controlled. The machine consists of an XY table to control the geometry, a set of electromagnetic valves that manage the mixing, a combined electromagnetic valve and sapphire nozzle that form the print head, and a computer that controls the whole system. The mixing valves can eject as little as 1 mg/s ink into the mixing chamber. The printer has been controlled, run, calibrated and tested; the composition and geometry of printed mixtures can be controlled precisely. This method for the controlled mixing of powders facilitates the advance of combinatorial methods within the materials sciences. PMID:12099843

  20. Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates.

    PubMed

    Kong, Dehui; Yeung, Wayland; Hili, Ryan

    2016-07-11

    Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acid-templated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function. PMID:27275512

  1. Decoding Split and Pool Combinatorial Libraries with Electron-Transfer Dissociation Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Sarkar, Mohosin; Pascal, Bruce D.; Steckler, Caitlin; Aquino, Claudio; Micalizio, Glenn C.; Kodadek, Thomas; Chalmers, Michael J.

    2013-07-01

    Screening of bead-based split and pool combinatorial chemistry libraries is a powerful approach to aid the discovery of new chemical compounds able to interact with, and modulate the activities of, protein targets of interest. Split and pool synthesis provides for large and well diversified chemical libraries, in this case comprised of oligomers generated from a well-defined starting set. At the end of the synthesis, each bead in the library displays many copies of a unique oligomer sequence. Because the sequence of the oligomer is not known at the time of screening, methods for decoding of the sequence of each screening "hit" are essential. Here we describe an electron-transfer dissociation (ETD) based tandem mass spectrometry approach for the decoding of mass-encoded split and pool libraries. We demonstrate that the newly described "chiral oligomers of pentenoic amides (COPAs)" yield non-sequence-specific product ions upon collisional activated dissociation; however, complete sequence information can be obtained with ETD. To aid in the decoding of libraries from MS and MS/MS data, we have incorporated 79Br/81Br isotope "tags" to differentiate N- and C-terminal product ions. In addition, we have created "Hit-Find," a software program that allows users to generate libraries in silico . The user can then search all possible members of the chemical library for those that fall within a user-defined mass error.

  2. Decoding Split and Pool Combinatorial Libraries with Electron Transfer Dissociation Tandem Mass Spectrometry

    PubMed Central

    Sarkar, Mohosin; Pascal, Bruce D.; Steckler, Caitlin; Aquino, Claudio; Micalizio, Glenn C.; Kodadek, Thomas; Chalmers, Michael J.

    2015-01-01

    Screening of bead-based split and pool combinatorial chemistry libraries is a powerful approach to aid the discovery of new chemical compounds able to interact with, and modulate the activities of, protein targets of interest. Split and pool synthesis provides for large and well diversified chemical libraries, in this case comprised of oligomers generated from a well-defined starting set. At the end of the synthesis, each bead in the library displays many copies of a unique oligomer sequence. Because the sequence of the oligomer is not known at the time of screening, methods for decoding of the sequence of each screening “hit” are essential. Here we describe an electron transfer dissociation (ETD) based tandem mass spectrometry approach for the decoding of mass-encoded split and pool libraries. We demonstrate that the newly described “chiral oligomers of pentenoic amides (COPAs)” yield non-sequence-specific product ions upon collisional activated dissociation; however, complete sequence information can be obtained with ETD. To aid in the decoding of libraries from MS and MS/MS data, we have incorporated 79Br/81Br isotope “tags” to differentiate N- and C-terminal product ions. In addition, we have created “Hit-Find,” a software program that allows users to generate libraries in silico. The user can then search all possible members of the chemical library for those that fall within a user-defined mass error. PMID:23636859

  3. Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening

    PubMed Central

    Choi, Hye-Ji; Kim, Ye-Jin; Choi, Dong-Ki; Kim, Yong-Sung

    2015-01-01

    Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A) with mutations in one CH3 domain (CH3A) on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B) with mutations in the other CH3 domain (CH3B). In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80–90%) with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display. PMID:26675656

  4. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    PubMed

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  5. Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning.

    PubMed

    Kokoszka, Malgorzata E; Kay, Brian K

    2015-01-01

    One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. PMID:25616333

  6. Biodegradable Fibrous Scaffolds with Diverse Properties by Electrospinning Candidates from a Combinatorial Macromer Library

    PubMed Central

    Metter, Robert B.; Ifkovits, Jamie L.; Hou, Kevin; Vincent, Ludovic; Hsu, Benjamin; Wang, Louis; Mauck, Robert L.; Burdick, Jason A.

    2009-01-01

    The properties of electrospun fibrous scaffolds, including degradation, mechanics and cellular interactions, are important for their use in tissue engineering applications. Although some diversity has been obtained previously in fibrous scaffolds, optimization of scaffold properties relies on iterative techniques in both polymer synthesis and processing. Here, we electrospun candidates from a combinatorial library of biodegradable and photopolymerizable poly(β-amino ester)s (PBAEs) to show that the diversity in properties found in this library is retained when processed into fibrous scaffolds. Specifically, three PBAE macromers were electrospun into scaffolds and possessed similar initial mechanical properties, but exhibited mass loss ranging from rapid (complete degradation within ∼2 weeks) to moderate (complete degradation within ∼ 3 months) to slow (only partial degradation after 3 months). These trends in mechanics and degradation mimicked what was previously observed in the bulk polymers. Although cellular adhesion was dependent on the polymer composition in films, adhesion to scaffolds that were electrospun with gelatin was similar on all formulations and controls. To further illustrate the diverse properties that are attainable in these systems, the fastest and slowest degrading polymers were electrospun together into one scaffold, but as distinct fiber populations. This dual-polymer scaffold exhibited behavior in mass loss and mechanics with time that fell between the single-polymer scaffolds. In general, this work indicates that combinatorial libraries may be an important source of information and specific polymer compositions for the fabrication of electrospun fibrous scaffolds with tunable properties. PMID:19853066

  7. Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

    SciTech Connect

    Kay, B. K.; Castagnoli, L.; Biosciences Division; Univ. of Rome

    2003-01-01

    This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

  8. Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling.

    PubMed

    Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J; Parker, Heather; Winterbourn, Christine C; Salvesen, Guy S; Drag, Marcin

    2014-02-18

    The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1-S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

  9. High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

    PubMed Central

    2015-01-01

    High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

  10. A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules

    PubMed Central

    Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

    2015-01-01

    A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis. PMID:26355961

  11. Optical tools for high-throughput screening of abrasion resistance of combinatorial libraries of organic coatings

    NASA Astrophysics Data System (ADS)

    Potyrailo, Radislav A.; Chisholm, Bret J.; Olson, Daniel R.; Brennan, Michael J.; Molaison, Chris A.

    2002-02-01

    Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8x6 arrays of coating elements that are deposited on a plastic substrate. Each coating element of the library is 10 mm in diameter and 2 to 5 micrometers thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the organic coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at single-or multiple-abrasion conditions, followed by high- throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0 to 30% haze. System precision of 0.1 to 2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. While the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied to a variety of other applications where materials ranking can be achieved using optical spectroscopic tools.

  12. Characterization of new outer membrane proteins of Pseudomonas aeruginosa using a combinatorial peptide ligand library.

    PubMed

    Ben Mlouka, Mohamed Amine; Khemiri, Arbia; Seyer, Damien; Hardouin, Julie; Chan Tchi Song, Philippe; Dé, Emmanuelle; Jouenne, Thierry; Cosette, Pascal

    2015-02-01

    Most often, the use of ProteoMiner beads has been restricted to human serum proteins for the normalization of major proteins, such as albumin. However, there are other situations of interest in which the presence of major proteins would quench the signals of low abundance polypeptides. We propose the use of these beads for investigating the envelope of the gram-negative bacterium Pseudomonas aeruginosa. Initially, we performed comparative 2D electrophoresis to qualitatively evaluate the incidence of the normalization stage. This demonstrated a significant reduction of the major membrane proteins. Thereafter, using shotgun analysis, the same protein extract was targeted by using combinatorial peptide ligand library capture. This treatment yielded 154 additional outer membrane proteins (OMPs) uncovered by the study of the crude sample. PMID:25471289

  13. Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

    PubMed Central

    Vegas, Arturo J; Veiseh, Omid; Doloff, Joshua C; Ma, Minglin; Tam, Hok Hei; Bratlie, Kaitlin; Li, Jie; Bader, Andrew R; Langan, Erin; Olejnik, Karsten; Fenton, Patrick; Kang, Jeon Woong; Hollister-Locke, Jennifer; Bochenek, Matthew A; Chiu, Alan; Siebert, Sean; Tang, Katherine; Jhunjhunwala, Siddharth; Aresta-Dasilva, Stephanie; Dholakia, Nimit; Thakrar, Raj; Vietti, Thema; Chen, Michael; Cohen, Josh; Siniakowicz, Karolina; Qi, Meirigeng; McGarrigle, James; Graham, Adam C; Lyle, Stephen; Harlan, David M; Greiner, Dale L; Oberholzer, Jose; Weir, Gordon C; Langer, Robert; Anderson, Daniel G

    2016-01-01

    The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient1–6. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response. PMID:26807527

  14. HTS by NMR of Combinatorial Libraries: A Fragment-Based Approach to Ligand Discovery

    PubMed Central

    Wu, Bainan; Zhang, Ziming; Noberini, Roberta; Barile, Elisa; Giulianotti, Marc; Pinilla, Clemencia; Houghten, Richard A.; Pasquale, Elena B.; Pellecchia, Maurizio

    2014-01-01

    SUMMARY Fragment-based ligand design (FBLD) approaches have become more widely used in drug discovery projects from both academia and industry, and are even often preferred to traditional high-throughput screening (HTS) of large collection of compounds (>105). A key advantage of FBLD approaches is that these often rely on robust biophysical methods such as NMR spectroscopy for detection of ligand binding, hence are less prone to artifacts that too often plague the results from HTS campaigns. In this article, we introduce a screening strategy that takes advantage of both the robustness of protein NMR spectroscopy as the detection method, and the basic principles of combinatorial chemistry to enable the screening of large libraries of fragments (>105 compounds) preassembled on a common backbone. We used the method to identify compounds that target protein-protein interactions. PMID:23352136

  15. Utility of Redundant Combinatorial Libraries in Distinguishing High and Low Quality Screening Hits

    PubMed Central

    2014-01-01

    Large one-bead one-compound (OBOC) combinatorial libraries can be constructed relatively easily by solid-phase split and pool synthesis. The use of resins with hydrophilic surfaces, such as TentaGel, allows the beads to be used directly in screens for compounds that bind selectively to labeled proteins, nucleic acids, or other biomolecules. However, we have found that this method, while useful, has a high false positive rate. In other words, beads that are scored as hits often display compounds that prove to be poor ligands for the target of interest when they are resynthesized and carried through validation trials. This results in a significant waste of time and resources in cases where putative hits cannot be validated without resynthesis. Here, we report that this problem can be largely eliminated through the use of redundant OBOC libraries, where more than one bead displaying the same compound is present in the screen. We show that compounds isolated more than once are likely to be high quality ligands for the target of interest, whereas compounds isolated only once have a much higher likelihood of being poor ligands. While the use of redundant libraries does limit the number of unique compounds that can be screened at one time in this format, the overall savings in time, effort, and materials makes this a more efficient route to the isolation of useful ligands for biomolecules. PMID:24749624

  16. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David

    2005-04-01

    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

  17. Identification of SNARE complex modulators that inhibit exocytosis from an alpha-helix-constrained combinatorial library.

    PubMed Central

    Blanes-Mira, Clara; Pastor, Maria T; Valera, Elvira; Fernández-Ballester, Gregorio; Merino, Jaime M; Gutierrez, Luis M; Perez-Payá, Enrique; Ferrer-Montiel, Antonio

    2003-01-01

    Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An alpha-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro -reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and L-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs. PMID:12852787

  18. The pharmacological properties of a novel MCH1 receptor antagonist isolated from combinatorial libraries

    PubMed Central

    Nagasaki, Hiroshi; Chung, Shinjae; Dooley, Colette T.; Wang, Zhiwei; Li, Chunying; Saito, Yumiko; Clark, Stewart D; Houghten, Richard A.; Civelli, Olivier

    2009-01-01

    Melanin-concentrating hormone (MCH) is a neuropeptide that exhibits potent orexigenic activity. In rodents, it exerts its actions by interacting with one receptor, MCH1 receptor which is expressed in many parts of the central nervous system (CNS). To study the physiological implications of the MCH system, we need to be able to block it locally and acutely. This necessitates the use of MCH1 receptor antagonists. While MCH1 receptor antagonists have been previously reported, they are mainly not accessible to academic research. We apply here a strategy that leads to the isolation of a high affinity and selective MCH1 receptor antagonist amenable to in vivo analyses without further chemical modifications. This antagonist, TPI 1361-17, was identified through the screening of multiple non-peptide positional scanning synthetic combinatorial libraries (PS-SCL) totaling more than eight hundred thousand compounds in conditions that allow for the identification of only high-affinity compounds. TPI 1361-17 exhibited an IC50 value of 6.1 nM for inhibition of 1 nM MCH-induced Ca2+ mobilization and completely displaced the binding of [125I] MCH to rat MCH1 receptor. TPI 1361-17 was found specific, having no affinity for a variety of other G-protein coupled receptors and channels. TPI 1361-17 was found active in vivo since it blocked MCH-induced food intake by 75 %. Our results indicate that TPI 1361-17 is a novel and selective MCH1 receptor antagonist and is an effective tool to study the physiological functions of the MCH system. These results also illustrate the successful application of combinatorial library screening to identify specific surrogate antagonists in an academic setting. PMID:19041642

  19. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  20. Double Dutch: A Tool for Designing Combinatorial Libraries of Biological Systems.

    PubMed

    Roehner, Nicholas; Young, Eric M; Voigt, Christopher A; Gordon, D Benjamin; Densmore, Douglas

    2016-06-17

    Recently, semirational approaches that rely on combinatorial assembly of characterized DNA components have been used to engineer biosynthetic pathways. In practice, however, it is not practical to assemble and test millions of pathway variants in order to elucidate how different DNA components affect the behavior of a pathway. To address this challenge, we apply a rigorous mathematical approach known as design of experiments (DOE) that can be used to construct empirical models of system behavior without testing all variants. To support this approach, we have developed a tool named Double Dutch, which uses a formal grammar and heuristic algorithms to automate the process of DOE library design. Compared to designing by hand, Double Dutch enables users to more efficiently and scalably design libraries of pathway variants that can be used in a DOE framework and uniquely provides a means to flexibly balance design considerations of statistical analysis, construction cost, and risk of homologous recombination, thereby demonstrating the utility of automating decision making when faced with complex design trade-offs. PMID:27110633

  1. 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. PMID:27296960

  2. A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches

    SciTech Connect

    Murtaugh, Megan L.; Fanning, Sean W.; Sharma, Tressa M.; Terry, Alexandra M.; Horn, James R.

    2012-09-05

    There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK{sub a} change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine 'hot-spots,' which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.

  3. Identification of avocado (Persea americana) pulp proteins by nano-LC-MS/MS via combinatorial peptide ligand libraries.

    PubMed

    Esteve, Clara; D'Amato, Alfonsina; Marina, María Luisa; García, María Concepción; Righetti, Pier Giorgio

    2012-09-01

    Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse-phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low-abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone reductase like protein. PMID:23019098

  4. Review of high-throughput techniques for detecting solid phase Transformation from material libraries produced by combinatorial methods

    NASA Technical Reports Server (NTRS)

    Lee, Jonathan A.

    2005-01-01

    High-throughput measurement techniques are reviewed for solid phase transformation from materials produced by combinatorial methods, which are highly efficient concepts to fabricate large variety of material libraries with different compositional gradients on a single wafer. Combinatorial methods hold high potential for reducing the time and costs associated with the development of new materials, as compared to time-consuming and labor-intensive conventional methods that test large batches of material, one- composition at a time. These high-throughput techniques can be automated to rapidly capture and analyze data, using the entire material library on a single wafer, thereby accelerating the pace of materials discovery and knowledge generation for solid phase transformations. The review covers experimental techniques that are applicable to inorganic materials such as shape memory alloys, graded materials, metal hydrides, ferric materials, semiconductors and industrial alloys.

  5. An exchangeable-tip scanning probe instrument for the analysis of combinatorial libraries of electrocatalysts.

    PubMed

    Rus, Eric D; Wang, Hongsen; Legard, Anna E; Ritzert, Nicole L; Van Dover, Robert Bruce; Abruña, Héctor D

    2013-02-01

    A combined scanning differential electrochemical mass spectrometer (SDEMS)-scanning electrochemical microscope (SECM) apparatus is described. The SDEMS is used to detect and spatially resolve volatile electrochemically generated species at the surface of a substrate electrode. The SECM can electrochemically probe the reactivity of the surface and also offers a convenient means of leveling the sample. It is possible to switch between these two different scanning tips and techniques without moving the sample and while maintaining potential control of the substrate electrode. A procedure for calibration of the SDEMS tip-substrate separation, based upon the transit time of electrogenerated species from the substrate to the tip is also described. This instrument can be used in the characterization of combinatorial libraries of direct alcohol fuel cell anode catalysts. The apparatus was used to analyze the products of methanol oxidation at a Pt substrate, with the SDEMS detecting carbon dioxide and methyl formate, and a PtPb-modified Pt SECM tip used for the selective detection of formic acid. As an example system, the electrocatalytic methanol oxidation activity of a sputter-deposited binary PtRu composition spread in acidic media was analyzed using the SDEMS. These results are compared with those obtained from a pH-sensitive fluorescence assay. PMID:23464226

  6. In-depth proteomic analysis of banana (Musa spp.) fruit with combinatorial peptide ligand libraries.

    PubMed

    Esteve, Clara; D'Amato, Alfonsina; Marina, María Luisa; García, María Concepción; Righetti, Pier Giorgio

    2013-01-01

    Musa ssp. is among the world's leading fruit crops. Although a strong interest on banana biochemistry exists in the scientific community, focused on metabolite composition, proteins have been scarcely investigated even if they play an important role in food allergy and stability, are a source of biologically active peptides, and can provide information about nutritional aspects of this fruit. In this work we have employed the combinatorial peptide ligand libraries after different types of protein extractions, for searching the very low-abundance proteins in banana. The use of advanced MS techniques and Musa ssp. mRNAs database in combination with the Uniprot_viridiplantae database allowed us to identify 1131 proteins. Among this huge amount of proteins we found several already known allergens such as Mus a 1, pectinesterase, superoxide dismutase, and potentially new allergens. Additionally several enzymes involved in degradation of starch granules and strictly correlated to ripening stage were identified. This is the first in-depth exploration of the banana fruit proteome and one of the largest descriptions of the proteome of any vegetable system. PMID:23161558

  7. An exchangeable-tip scanning probe instrument for the analysis of combinatorial libraries of electrocatalysts

    NASA Astrophysics Data System (ADS)

    Rus, Eric D.; Wang, Hongsen; Legard, Anna E.; Ritzert, Nicole L.; Bruce Van Dover, Robert; Abruña, Héctor D.

    2013-02-01

    A combined scanning differential electrochemical mass spectrometer (SDEMS)-scanning electrochemical microscope (SECM) apparatus is described. The SDEMS is used to detect and spatially resolve volatile electrochemically generated species at the surface of a substrate electrode. The SECM can electrochemically probe the reactivity of the surface and also offers a convenient means of leveling the sample. It is possible to switch between these two different scanning tips and techniques without moving the sample and while maintaining potential control of the substrate electrode. A procedure for calibration of the SDEMS tip-substrate separation, based upon the transit time of electrogenerated species from the substrate to the tip is also described. This instrument can be used in the characterization of combinatorial libraries of direct alcohol fuel cell anode catalysts. The apparatus was used to analyze the products of methanol oxidation at a Pt substrate, with the SDEMS detecting carbon dioxide and methyl formate, and a PtPb-modified Pt SECM tip used for the selective detection of formic acid. As an example system, the electrocatalytic methanol oxidation activity of a sputter-deposited binary PtRu composition spread in acidic media was analyzed using the SDEMS. These results are compared with those obtained from a pH-sensitive fluorescence assay.

  8. Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity.

    PubMed

    van der Veen, Bart A; Potocki-Véronèse, Gabrielle; Albenne, Cécile; Joucla, Gilles; Monsan, Pierre; Remaud-Simeon, Magali

    2004-02-27

    Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme trade mark, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes. PMID:14988004

  9. High-throughput measurements of thermochromic behavior in V(1-x)Nb(x)O(2) combinatorial thin film libraries.

    PubMed

    Barron, S C; Gorham, J M; Patel, M P; Green, M L

    2014-10-13

    We describe a high-throughput characterization of near-infrared thermochromism in V1-xNbxO2 combinatorial thin film libraries. The oxide thin film library was prepared with a VO2 crystal structure and a continuous gradient in composition with Nb concentrations in the range of less than 1% to 45%. The thermochromic phase transition from monoclinic to tetragonal was characterized by the accompanying change in near-infrared reflectance. With increasing Nb substitution, the transition temperature was depressed from 65 to 35 °C, as desirable for smart window applications. However, the magnitude of the reflectance change across the thermochromic transition was also reduced with increasing Nb film content. Data collection, handling, and analysis supporting thermochromic characterization were fully automated to achieve high throughput. Using this system, in 14 h, temperature-dependent infrared reflectances were measured at 165 arbitrary locations on a thin film combinatorial library; these measurements were analyzed for thermochromic transitions in minutes. PMID:25180465

  10. 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. PMID:25780985

  11. Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

    PubMed

    Brylinski, Michal; Waldrop, Grover L

    2014-01-01

    As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×10⁸ amino-oxazole derivatives. A subset of 9×10⁶ of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug

  12. Recombinant Human Respiratory Syncytial Virus (RSV) Monoclonal Antibody Fab is Effective Therapeutically when Introduced Directly into the Lungs of RSV-Infected Mice

    NASA Astrophysics Data System (ADS)

    Crowe, James E., Jr.; Murphy, Brian R.; Chanock, Robert M.; Williamson, R. Anthony; Barbas, Carlos F., III; Burton, Dennis R.

    1994-02-01

    Previously, recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fabs were generated by antigen selection from random combinatorial libraries displayed at the tip of filamentous phage. Two such Fabs, which exhibited high binding affinity for RSV F glycoprotein (a major protective antigen), were evaluated for therapeutic efficacy in infected mice just before or at the time of peak virus replication in the lungs. Fab 19, which neutralized RSV infectivity with high efficiency in tissue culture, was effective therapeutically when delivered directly into the lungs by intranasal instillation under anesthesia. In contrast, RSV Fab 126, which failed to neutralize virus in cell culture, did not exhibit a therapeutic effect under these conditions. The amount of Fab 19 required to effect a 5000- to 12,000-fold reduction in titer of RSV in the lungs within 24 hr was rather small. In four separate experiments, a single instillation of 12.9-50 μg of RSV Fab 19 was sufficient to achieve such a reduction in pulmonary virus in a 25g mouse. The use of Fabs instead of the whole immunoglobulin molecules from which they are derived reduced the protein content of a therapeutic dose. This is important because the protein load that can be delivered effectively into the lungs is limited. The therapeutic effect of a single treatment with Fab 19 was not sustained, so that a rebound in pulmonary virus titer occurred on the 2nd day after treatment. This rebound in pulmonary RSV titer could be prevented by treating infected mice with a single dose of Fab 19 daily for 3 days. These observations suggest that human monoclonal Fabs grown in Escherichia coli may prove useful in the treatment of serious RSV disease as well as diseases caused by other viruses where replication in vivo is limited primarily to the lumenal lining of the respiratory tract.

  13. CCLab--a multi-objective genetic algorithm based combinatorial library design software and an application for histone deacetylase inhibitor design.

    PubMed

    Fang, Guanghua; Xue, Mengzhu; Su, Mingbo; Hu, Dingyu; Li, Yanlian; Xiong, Bing; Ma, Lanping; Meng, Tao; Chen, Yuelei; Li, Jingya; Li, Jia; Shen, Jingkang

    2012-07-15

    The introduction of the multi-objective optimization has dramatically changed the virtual combinatorial library design, which can consider many objectives simultaneously, such as synthesis cost and drug-likeness, thus may increase positive rates of biological active compounds. Here we described a software called CCLab (Combinatorial Chemistry Laboratory) for combinatorial library design based on the multi-objective genetic algorithm. Tests of the convergence ability and the ratio to re-take the building blocks in the reference library were conducted to assess the software in silico, and then it was applied to a real case of designing a 5×6 HDAC inhibitor library. Sixteen compounds in the resulted library were synthesized, and the histone deactetylase (HDAC) enzymatic assays proved that 14 compounds showed inhibitory ratios more than 50% against tested 3 HDAC enzymes at concentration of 20 μg/mL, with IC(50) values of 3 compounds comparable to SAHA. These results demonstrated that the CCLab software could enhance the hit rates of the designed library and would be beneficial for medicinal chemists to design focused library in drug development (the software can be downloaded at: http://202.127.30.184:8080/drugdesign.html). PMID:22738629

  14. Comparative molecular surface analysis (CoMSA) for virtual combinatorial library screening of styrylquinoline HIV-1 blocking agents.

    PubMed

    Niedbala, Halina; Polanski, Jaroslaw; Gieleciak, Rafal; Musiol, Robert; Tabak, Dominik; Podeszwa, Barbara; Bak, Andrzej; Palka, Anna; Mouscadet, Jean-Francois; Gasteiger, Johann; Le Bret, Marc

    2006-12-01

    We used comparative molecular surface analysis to design molecules for the synthesis as part of the search for new HIV-1 integrase inhibitors. We analyzed the virtual combinatorial library (VCL) constituted from various moieties of styrylquinoline and styrylquinazoline inhibitors. Since imines can be applied in a strategy of dynamic combinatorial chemistry (DCC), we also tested similar compounds in which the -C=N- or -N=C- linker connected the heteroaromatic and aromatic moieties. We then used principal component analysis (PCA) or self-organizing maps (SOM), namely, the Kohonen neural networks to obtain a clustering plot analyzing the diversity of the VCL formed. Previously synthesized compounds of known activity, used as molecular probes, were projected onto this plot, which provided a set of promising virtual drugs. Moreover, we further modified the above mentioned VCL to include the single bond linker -C-N- or -N-C-. This allowed increasing compound stability but expanded also the diversity between the available molecular probes and virtual targets. The application of the CoMSA with SOM indicated important differences between such compounds and active molecular probes. We synthesized such compounds to verify the computational predictions. PMID:17168681

  15. Development of combinatorial chemistry methods for coatings: high-throughput screening of abrasion resistance of coatings libraries.

    PubMed

    Potyrailo, Radislav A; Chisholm, Bret J; Olson, Daniel R; Brennan, Michael J; Molaison, Chris A

    2002-10-01

    Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8 x 6 arrays of coating elements that are discretely deposited on a single plastic substrate. Each coating element of the library is 10 mm in diameter and 2-5 microm thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the coating materials undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at a single or multiple abrasion conditions followed by the high-throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0-30% haze. System precision of 0.1-2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. Although the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied for a variety of other applications for which materials ranking can be achieved using optical spectroscopic tools. PMID:12380837

  16. An apparatus for spatially resolved, temperature dependent reflectance measurements for identifying thermochromism in combinatorial thin film libraries

    NASA Astrophysics Data System (ADS)

    Barron, S. C.; Patel, M. P.; Nguyen, Nam; Nguyen, N. V.; Green, M. L.

    2015-11-01

    A metrology and data analysis protocol is described for high throughput determination of thermochromic metal-insulator phase diagrams for lightly substituted VO2 thin films. The technique exploits the abrupt change in near infrared optical properties, measured in reflection, as an indicator of the temperature- or impurity-driven metal-insulator transition. Transition metal impurities were introduced in a complementary combinatorial synthesis process for producing thin film libraries with the general composition space V 1-x-yMxM'yO2, with M and M' being transition metals and x and y varying continuously across the library. The measurement apparatus acquires reflectance spectra in the visible or near infrared at arbitrarily many library locations, each with a unique film composition, at temperatures of 1 °C-85 °C. Data collection is rapid and automated; the measurement protocol is computer controlled to automate the collection of thousands of reflectance spectra, representing hundreds of film compositions at tens of different temperatures. A straightforward analysis algorithm is implemented to extract key information from the thousands of spectra such as near infrared thermochromic transition temperatures and regions of no thermochromic transition; similarly, reflectance to the visible spectrum generates key information for materials selection of smart window materials. The thermochromic transition for 160 unique compositions on a thin film library with the general formula V 1-x-yMxM'yO2 can be measured and described in a single 20 h experiment. The resulting impurity composition-temperature phase diagrams will contribute to the understanding of metal-insulator transitions in doped VO2 systems and to the development of thermochromic smart windows.

  17. An apparatus for spatially resolved, temperature dependent reflectance measurements for identifying thermochromism in combinatorial thin film libraries.

    PubMed

    Barron, S C; Patel, M P; Nguyen, Nam; Nguyen, N V; Green, M L

    2015-11-01

    A metrology and data analysis protocol is described for high throughput determination of thermochromic metal-insulator phase diagrams for lightly substituted VO2 thin films. The technique exploits the abrupt change in near infrared optical properties, measured in reflection, as an indicator of the temperature- or impurity-driven metal-insulator transition. Transition metal impurities were introduced in a complementary combinatorial synthesis process for producing thin film libraries with the general composition space V(1-x-y)M(x)M'(y)O2, with M and M' being transition metals and x and y varying continuously across the library. The measurement apparatus acquires reflectance spectra in the visible or near infrared at arbitrarily many library locations, each with a unique film composition, at temperatures of 1 °C-85 °C. Data collection is rapid and automated; the measurement protocol is computer controlled to automate the collection of thousands of reflectance spectra, representing hundreds of film compositions at tens of different temperatures. A straightforward analysis algorithm is implemented to extract key information from the thousands of spectra such as near infrared thermochromic transition temperatures and regions of no thermochromic transition; similarly, reflectance to the visible spectrum generates key information for materials selection of smart window materials. The thermochromic transition for 160 unique compositions on a thin film library with the general formula V(1-x-y)M(x)M'(y)O2 can be measured and described in a single 20 h experiment. The resulting impurity composition-temperature phase diagrams will contribute to the understanding of metal-insulator transitions in doped VO2 systems and to the development of thermochromic smart windows. PMID:26628147

  18. Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering. Combinatorial library determination of optimal sequences.

    PubMed

    Krezel, Artur; Kopera, Edyta; Protas, Anna Maria; Poznański, Jarosław; Wysłouch-Cieszyńska, Aleksandra; Bal, Wojciech

    2010-03-17

    Previously we demonstrated for several examples that peptides having a general internal sequence R(N)-Yaa-Ser/Thr-Xaa-His-Zaa-R(C) (Yaa = Glu or Ala, Xaa = Ala or His, Zaa = Lys, R(N) and R(C) = any N- and C-terminal amino acid sequence) were hydrolyzed specifically at the Yaa-Ser/Thr peptide bond in the presence of Ni(II) ions at alkaline pH (Krezel, A., Mylonas, M., Kopera, E. and Bal, E. Acta Biochim. Polon. 2006, 53, 721-727 and references therein). Hereby we report the synthesis of a combinatorial library of CH(3)CO-Gly-Ala-(Ser/Thr)-Xaa-His-Zaa-Lys-Phe-Leu-NH(2) peptides, where Xaa residues included 17 common alpha-amino acids (except Asp, Glu, and Cys) and Zaa residues included 19 common alpha-amino acids (except Cys). The Ni(II)-dependent hydrolysis at 37 and 45 degrees C of batches of combinatorial peptide mixtures randomized at Zaa was monitored by MALDI-TOF mass spectrometry. The correctness of library-based predictions was confirmed by accurate measurements of hydrolysis rates of seven selected peptides using HPLC. The hydrolysis was strictly limited to the Ala-Ser/Thr bond in all library and individual peptide experiments. The effects of individual residues on hydrolysis rates were quantified and correlated with physical properties of their side chains according to a model of independent contributions of Xaa and Zaa residues. The principal component analysis calculations demonstrated partial molar side chain volume and the free energy of amino acid vaporization for both Xaa and Zaa residues and the amine pK(a) for Zaa residues to be the most significant empirical parameters influencing the hydrolysis rate. Therefore, efficient hydrolysis required bulky and hydrophobic residues at both variable positions Xaa and Zaa, which contributed independently to the hydrolysis rate. This relationship between the peptide sequence and the hydrolysis rate provides a basis for further research, aimed at the elucidation of the reaction mechanism and biotechnological

  19. Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides

    PubMed Central

    2015-01-01

    Cyclic peptides have great potential as therapeutic agents and research tools. However, their applications against intracellular targets have been limited, because cyclic peptides are generally impermeable to the cell membrane. It was previously shown that fusion of cyclic peptides with a cyclic cell-penetrating peptide resulted in cell-permeable bicyclic peptides that are proteolytically stable and biologically active in cellular assays. In this work, we tested the generality of the bicyclic approach by synthesizing a combinatorial library of 5.7 × 106 bicyclic peptides featuring a degenerate sequence in the first ring and an invariant cell-penetrating peptide in the second ring. Screening of the library against oncoprotein K-Ras G12V followed by hit optimization produced a moderately potent and cell-permeable K-Ras inhibitor, which physically blocks the Ras-effector interactions in vitro, inhibits the signaling events downstream of Ras in cancer cells, and induces apoptosis of the cancer cells. Our approach should be generally applicable to developing cell-permeable bicyclic peptide inhibitors against other intracellular proteins. PMID:26645887

  20. Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

    PubMed Central

    Vágner, J; Barany, G; Lam, K S; Krchnák, V; Sepetov, N F; Ostrem, J A; Strop, P; Lebl, M

    1996-01-01

    Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads. PMID:8710846

  1. A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display

    PubMed Central

    Schröter, Christian; Günther, Ralf; Rhiel, Laura; Becker, Stefan; Toleikis, Lars; Doerner, Achim; Becker, Janine; Schönemann, Andreas; Nasu, Daichi; Neuteboom, Berend; Kolmar, Harald; Hock, Björn

    2015-01-01

    There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics. PMID:25523975

  2. Generation of a Multicomponent Library of Disulfide Donor-Acceptor Architectures Using Dynamic Combinatorial Chemistry

    PubMed Central

    Drożdż, Wojciech; Kołodziejski, Michał; Markiewicz, Grzegorz; Jenczak, Anna; Stefankiewicz, Artur R.

    2015-01-01

    We describe here the generation of new donor-acceptor disulfide architectures obtained in aqueous solution at physiological pH. The application of a dynamic combinatorial chemistry approach allowed us to generate a large number of new disulfide macrocyclic architectures together with a new type of [2]catenanes consisting of four distinct components. Up to fifteen types of structurally-distinct dynamic architectures have been generated through one-pot disulfide exchange reactions between four thiol-functionalized aqueous components. The distribution of disulfide products formed was found to be strongly dependent on the structural features of the thiol components employed. This work not only constitutes a success in the synthesis of topologically- and morphologically-complex targets, but it may also open new horizons for the use of this methodology in the construction of molecular machines. PMID:26193265

  3. Preparation of pseudo-ternary library by combinatorial robot system based on wet and dry processes

    NASA Astrophysics Data System (ADS)

    Fujimoto, Kenjiro; Watanabe, Mamoru

    2005-01-01

    A fully automatic combinatorial robot system was developed for investigating inorganic materials. The system can prepare and characterize about 200 samples in 1 day. The phase relation of the pseudo-ternary LiO0.5-X-TiO2 (X: CrO1.5, FeO1.5 and NiO) system was investigated in order to determine the formation range of ramsdellite structures. A wider composition range was obtained for the ramsdellite-type compounds in the LiO0.5-CrO1.5-TiO2 system than for other compounds. It was found that the differences in the composition ranges for ramsdellite-type structures were caused by the different ionic radii of Cr3+, Fe3+ and Ni2+ in the octahedral site.

  4. Combinatorial synthesis, lead identification, and antitumor study of a chalcone-based positional-scanning library.

    PubMed

    Ullah, Ahsan; Ansari, Farzana Latif; Nazir, Samina; Mirza, Bushra

    2007-02-01

    A 175-member chalcone library was designed and synthesized from seven differently substituted acetophenones (A(1)-A(7)) and 25 differently substituted aryl or heteroaryl aldehydes (B(1)-B(25)). Potential lead compounds were identified by deconvolution of a two-dimensional library matrix via positional scanning, and the members of the most-active sub-libraries were synthesized and screened against crown-gall tumors with the aid of the potato-disc assay. The resulting hits gave rise to significant antitumor activities, with no antibacterial effect on the tumor-producing bacterium Agrobacterium tumefaciens. Two identified lead structures, (2E)-3-(2-chlorophenyl)-1-phenylprop-2-en-1-one (A(1)B(9)) and the hydroxy analogue (2E)-3-(2-chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (A(2)B(9)), are promising candidates to be developed into highly effective anticancer chemotherapeutics. PMID:17311221

  5. Site-specific Labeling of a Protein Lysine Residue By Novel Kinetic Labeling Combinatorial Libraries

    PubMed Central

    Krantz, Allen; Hanel, Arthur M; Strug, Ivona; Wilczynski, Andrzej; Wolff, Jeremy J; Huang, Wolin; Huang, Linda H; Settineri, Tina; Holmes, Darren L; Hardy, Margaret C; Bridon, Dominique P

    2014-01-01

    The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q) that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E) emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks. PMID:24757504

  6. Site-specific Labeling of a Protein Lysine Residue By Novel Kinetic Labeling Combinatorial Libraries.

    PubMed

    Krantz, Allen; Hanel, Arthur M; Strug, Ivona; Wilczynski, Andrzej; Wolff, Jeremy J; Huang, Wolin; Huang, Linda H; Settineri, Tina; Holmes, Darren L; Hardy, Margaret C; Bridon, Dominique P

    2014-01-01

    The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q) that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E) emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks. PMID:24757504

  7. Laser direct writing of combinatorial libraries of idealized cellular constructs: Biomedical applications

    NASA Astrophysics Data System (ADS)

    Schiele, Nathan R.; Koppes, Ryan A.; Corr, David T.; Ellison, Karen S.; Thompson, Deanna M.; Ligon, Lee A.; Lippert, Thomas K. M.; Chrisey, Douglas B.

    2009-03-01

    The ability to control cell placement and to produce idealized cellular constructs is essential for understanding and controlling intercellular processes and ultimately for producing engineered tissue replacements. We have utilized a novel intra-cavity variable aperture excimer laser operated at 193 nm to reproducibly direct write mammalian cells with micrometer resolution to form a combinatorial array of idealized cellular constructs. We deposited patterns of human dermal fibroblasts, mouse myoblasts, rat neural stem cells, human breast cancer cells, and bovine pulmonary artery endothelial cells to study aspects of collagen network formation, breast cancer progression, and neural stem cell proliferation, respectively. Mammalian cells were deposited by matrix assisted pulsed laser evaporation direct write from ribbons comprised of a UV transparent quartz coated with either a thin layer of extracellular matrix or triazene as a dynamic release layer using CAD/CAM control. We demonstrate that through optical imaging and incorporation of a machine vision algorithm, specific cells on the ribbon can be laser deposited in spatial coherence with respect to geometrical arrays and existing cells on the receiving substrate. Having the ability to direct write cells into idealized cellular constructs can help to answer many biomedical questions and advance tissue engineering and cancer research.

  8. The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library.

    PubMed Central

    Bunin, B A; Plunkett, M J; Ellman, J A

    1994-01-01

    A library of 192 structurally diverse 1,4-benzodiazepine derivatives containing a variety of chemical functionalities including amides, carboxylic acids, amines, phenols, and indoles was constructed from three components, 2-aminobenzophenones, amino acids, and alkylating agents, by employing Geysen's pin apparatus [Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G. & Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274]. Rigorous analytical verification of the chemical integrity and yield of a representative collection of the diverse derivatives was carried out. In addition, the library of derivatives was evaluated for binding to the cholecystokinin A receptor by employing a competitive radio-ligand binding assay. This provided detailed structure versus activity relationships that were confirmed by independent large-scale synthesis and evaluation of several of the 1,4-benzodiazepine derivatives. PMID:8197123

  9. Proteomic Analysis of Lonicera japonica Thunb. Immature Flower Buds Using Combinatorial Peptide Ligand Libraries and Polyethylene Glycol Fractionation.

    PubMed

    Zhu, Wei; Xu, Xiaobao; Tian, Jingkui; Zhang, Lin; Komatsu, Setsuko

    2016-01-01

    Lonicera japonica Thunb. flower is a well-known medicinal plant that has been widely used for the treatment of human disease. To explore the molecular mechanisms underlying the biological activities of L. japonica immature flower buds, a gel-free/label-free proteomic technique was used in combination with combinatorial peptide ligand libraries (CPLL) and polyethylene glycol (PEG) fractionation for the enrichment of low-abundance proteins and removal of high-abundance proteins, respectively. A total of 177, 614, and 529 proteins were identified in crude protein extraction, CPLL fractions, and PEG fractions, respectively. Among the identified proteins, 283 and 239 proteins were specifically identified by the CPLL and PEG methods, respectively. In particular, proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were highly enriched by CPLL and PEG fractionation compared to crude protein extraction. A total of 28 secondary metabolism-related proteins and 25 metabolites were identified in L. japonica immature flower buds. To determine the specificity of the identified proteins and metabolites for L. japonica immature flower buds, Cerasus flower buds were used, which resulted in the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds being 10-fold higher than that in Cerasus flower buds. These results suggest that proteins related to secondary metabolism might be responsible for the biological activities of L. japonica immature flower buds. PMID:26573373

  10. Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.

    PubMed

    Cruz-Teran, Carlos A; Carlin, Kevin B; Efimenko, Kirill; Genzer, Jan; Rao, Balaji M

    2016-08-30

    While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts. PMID:27490089

  11. Autocrine-Based Selection of Drugs That Target Ion Channels from Combinatorial Venom Peptide Libraries.

    PubMed

    Zhang, Hongkai; Du, Mingjuan; Xie, Jia; Liu, Xiao; Sun, Jingying; Wang, Wei; Xin, Xiu; Possani, Lourival D; Yea, Kyungmoo; Lerner, Richard A

    2016-08-01

    Animal venoms represent a rich source of pharmacologically active peptides that interact with ion channels. However, a challenge to discovering drugs remains because of the slow pace at which venom peptides are discovered and refined. An efficient autocrine-based high-throughput selection system was developed to discover and refine venom peptides that target ion channels. The utility of this system was demonstrated by the discovery of novel Kv1.3 channel blockers from a natural venom peptide library that was formatted for autocrine-based selection. We also engineered a Kv1.3 blocker peptide (ShK) derived from sea anemone to generate a subtype-selective Kv1.3 blocker with a long half-life in vivo. PMID:27197631

  12. Quantum Efficiency and Bandgap Analysis for Combinatorial Photovoltaics: Sorting Activity of Cu–O Compounds in All-Oxide Device Libraries

    PubMed Central

    2014-01-01

    All-oxide-based photovoltaics (PVs) encompass the potential for extremely low cost solar cells, provided they can obtain an order of magnitude improvement in their power conversion efficiencies. To achieve this goal, we perform a combinatorial materials study of metal oxide based light absorbers, charge transporters, junctions between them, and PV devices. Here we report the development of a combinatorial internal quantum efficiency (IQE) method. IQE measures the efficiency associated with the charge separation and collection processes, and thus is a proxy for PV activity of materials once placed into devices, discarding optical properties that cause uncontrolled light harvesting. The IQE is supported by high-throughput techniques for bandgap fitting, composition analysis, and thickness mapping, which are also crucial parameters for the combinatorial investigation cycle of photovoltaics. As a model system we use a library of 169 solar cells with a varying thickness of sprayed titanium dioxide (TiO2) as the window layer, and covarying thickness and composition of binary compounds of copper oxides (Cu–O) as the light absorber, fabricated by Pulsed Laser Deposition (PLD). The analysis on the combinatorial devices shows the correlation between compositions and bandgap, and their effect on PV activity within several device configurations. The analysis suggests that the presence of Cu4O3 plays a significant role in the PV activity of binary Cu–O compounds. PMID:24410367

  13. Quantum efficiency and bandgap analysis for combinatorial photovoltaics: sorting activity of Cu-O compounds in all-oxide device libraries.

    PubMed

    Anderson, Assaf Y; Bouhadana, Yaniv; Barad, Hannah-Noa; Kupfer, Benjamin; Rosh-Hodesh, Eli; Aviv, Hagit; Tischler, Yaakov R; Rühle, Sven; Zaban, Arie

    2014-02-10

    All-oxide-based photovoltaics (PVs) encompass the potential for extremely low cost solar cells, provided they can obtain an order of magnitude improvement in their power conversion efficiencies. To achieve this goal, we perform a combinatorial materials study of metal oxide based light absorbers, charge transporters, junctions between them, and PV devices. Here we report the development of a combinatorial internal quantum efficiency (IQE) method. IQE measures the efficiency associated with the charge separation and collection processes, and thus is a proxy for PV activity of materials once placed into devices, discarding optical properties that cause uncontrolled light harvesting. The IQE is supported by high-throughput techniques for bandgap fitting, composition analysis, and thickness mapping, which are also crucial parameters for the combinatorial investigation cycle of photovoltaics. As a model system we use a library of 169 solar cells with a varying thickness of sprayed titanium dioxide (TiO2) as the window layer, and covarying thickness and composition of binary compounds of copper oxides (Cu-O) as the light absorber, fabricated by Pulsed Laser Deposition (PLD). The analysis on the combinatorial devices shows the correlation between compositions and bandgap, and their effect on PV activity within several device configurations. The analysis suggests that the presence of Cu4O3 plays a significant role in the PV activity of binary Cu-O compounds. PMID:24410367

  14. Targeting deeper the human serum fucome by a liquid-phase multicolumn platform in combination with combinatorial peptide ligand libraries.

    PubMed

    Selvaraju, Subhashini; El Rassi, Ziad

    2014-03-01

    Combinatorial peptide ligand library (CPLL) was evaluated as an off line step to narrow the differences of protein concentration in human serum prior to the capturing of human fucome from disease-free and breast cancer sera by a multicolumn platform via lectin affinity chromatography (LAC) followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two monolithic lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) columns were utilized to capture the fucome, which was subsequently fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to tryptic digestion prior to LC-MS/MS analysis. AAL has a strong affinity towards core fucosylated N-glycans and has a weak binding towards fucose in the outer arm while LTA can bind to glycans having fucose present in the outer arm. The combined strategy consisting of the CPLL, multicolumn platform and LC-MS/MS analysis permitted the identification of the differentially expressed proteins (DEPs) in breast cancer serum yielding 58 DEPs in both the LTA and AAL fractions with 6 DEPs common to both lectins. 17 DEPs were of the low abundance type, 16 DEPs of the borderline abundance type, 4 DEPs of the medium abundance type and 15 DEPs of the high abundance type. The remaining 6 DEPs are of unknown concentration. Only proteins exhibiting 99.9% protein identification probability, 95% peptide identification probability, and a minimum of 5 unique peptides were considered in finding the DEPs via scatterplots. PMID:24556279

  15. Targeting Deeper the Human Serum Fucome by a Liquid-phase Multicolumn Platform in Combination with Combinatorial Peptide Ligand Libraries

    PubMed Central

    Selvaraju, Subhashini; Rassi, Ziad El

    2014-01-01

    Combinatorial peptide ligand library (CPLL) was evaluated as an off line step to narrow the differences of protein concentration in human serum prior to the capturing of human fucome from disease-free and breast cancer sera by a multicolumn platform via lectin affinity chromatography (LAC) followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two monolithic lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) columns were utilized to capture the fucome, which was subsequently fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to tryptic digestion prior to LC-MS/MS analysis. AAL has a strong affinity towards core fucosylated N-glycans and has a weak binding towards fucose in the outer arm while LTA can bind to glycans having fucose present in the outer arm. The combined strategy consisting of the CPLL, multicolumn platform and LC-MS/MS analysis permitted the identification of the differentially expressed proteins (DEPs) in breast cancer serum yielding 58 DEPs in both the LTA and AAL fractions with 6 DEPs common to both lectins. 17 DEPs were of the low abundance type, 16 DEPs of the borderline abundance type, 4 DEPs of the medium abundance type and 15 DEPs of the high abundance type. The remaining 6 DEPs are of unknown concentration. Only proteins exhibiting 99.9% protein identification probability, 95% peptide identification probability, and a minimum of 5 unique peptides were considered in finding the DEPs via scatterplots. PMID:24556279

  16. Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

    PubMed

    Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

    2012-02-01

    Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents. PMID:22064367

  17. Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

    PubMed

    Rhaiem, Rafik Ben; Houimel, Mehdi

    2016-07-01

    Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency

  18. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology.

    PubMed

    Iverson, Sonya V; Haddock, Traci L; Beal, Jacob; Densmore, Douglas M

    2016-01-15

    Multipart and modular DNA part libraries and assembly standards have become common tools in synthetic biology since the publication of the Gibson and Golden Gate assembly methods, yet no multipart modular library exists for use in bacterial systems. Building upon the existing MoClo assembly framework, we have developed a publicly available collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design, and expression tuning in Escherichia coli. The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library is openly available and contains promoters, ribosomal binding sites, coding sequence, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from that of previously published protocols. PMID:26479688

  19. Optimization of Combinatorial Mutagenesis

    NASA Astrophysics Data System (ADS)

    Parker, Andrew S.; Griswold, Karl E.; Bailey-Kellogg, Chris

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (stability, activity, immunogenicity, etc.). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries - a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 107 variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  20. Inhibition of Plasmodium falciparum fatty acid biosynthesis: evaluation of FabG, FabZ, and FabI as drug targets for flavonoids.

    PubMed

    Tasdemir, Deniz; Lack, Gabriela; Brun, Reto; Rüedi, Peter; Scapozza, Leonardo; Perozzo, Remo

    2006-06-01

    After the discovery of a potent natural flavonoid glucoside as a potent inhibitor of FabI, a large flavonoid library was screened against three important enzymes (i.e., FabG, FabZ, and FabI) involved in the fatty acid biosynthesis of P. falciparum. Although flavones with a simple hydroxylation pattern (compounds 4-9) showed moderate inhibitory activity toward the enzymes tested (IC50 10-100 microM), the more complex flavonoids (12-16) exhibited strong activity toward all three enzymes (IC50 0.5-8 microM). Isoflavonoids 26-28 showed moderate (IC50 7-30 microM) but selective activity against FabZ. The most active compounds were C-3 gallic acid esters of catechins (32, 33, 37, 38), which are strong inhibitors of all three enzymes (IC50 0.2-1.1 microM). Kinetic analysis using luteolin (12) and (-)-catechin gallate (37) as model compounds revealed that FabG was inhibited in a noncompetitive manner. FabZ was inhibited competitively, whereas both compounds behaved as tight-binding noncompetitive inhibitors of FabI. In addition, these polyphenols showed in vitro activity against chloroquine-sensitive (NF54) and -resistant (K1) P. falciparum strains in the low to submicromolar range. PMID:16722653

  1. Rainbow beads: a color coding method to facilitate high-throughput screening and optimization of one-bead one-compound combinatorial libraries.

    PubMed

    Luo, Juntao; Zhang, Hongyong; Xiao, Wenwu; Kumaresan, Pappanaicken R; Shi, Changying; Pan, Chong-Xian; Aina, Olulanu H; Lam, Kit S

    2008-01-01

    We have developed a new color-encoding method that facilitates high-throughput screening of one-bead one-compound (OBOC) combinatorial libraries. Polymer beads displaying chemical compounds or families of compounds are stained with oil-based organic dyes that are used as coding tags. The color dyes do not affect cell binding to the compounds displayed on the surface of the beads. We have applied such rainbow beads in a multiplex manner to discover and profile ligands against cell surface receptors. In the first application, a series of OBOC libraries with different scaffolds or motifs are each color-coded; small samples of each library are then combined and screened concurrently against live cells for cell attachment. Preferred libraries can be rapidly identified and selected for subsequent large-scale screenings for cell surface binding ligands. In a second application, beads with a series of peptide analogues (e.g., alanine scan) are color-coded, combined, and tested for binding against a specific cell line in a single-tissue culture well; the critical residues required for binding can be easily determined. In a third application, ligands reacting against a series of integrins are color-coded and used as a readily applied research tool to determine the integrin profile of any cell type. One major advantage of this straightforward and yet powerful method is that only an ordinary inverted microscope is needed for the analysis, instead of sophisticated (and expensive) fluorescent microscopes or flow cytometers. PMID:18558750

  2. Surface characteristics and protein adsorption on combinatorial binary Ti-M (Cr, Al, Ni) and Al-M (Ta, Zr) library films.

    PubMed

    Bai, Zhijun; Filiaggi, M J; Sanderson, R J; Lohstreter, L B; McArthur, M A; Dahn, J R

    2010-02-01

    Systematic studies of protein adsorption onto metallic biomaterial surfaces are generally lacking. Here, combinatorial binary library films with compositional gradients of Ti(1-x)Cr(x), Ti(1-x)Al(x), Ti(1-x)Ni(x) and Al(1-x)Ta(x), (0 library and an amorphous zone dominating along the gradient. These mirror-like films were generally found by atomic force microscopy to have a roughness of less than 8 nm, with any relative increases in roughness consistent with the development of crystalline phases. Surface chemistry by quantitative high-resolution X-ray photoelectron spectroscopy differed significantly from bulk film composition as measured by electron microprobe, with TiO(2) and Al(2)O(3) preferentially forming on the binary film surfaces. Correspondingly, protein adsorption onto these films closely correlated with their surface oxide fractions. Aluminum deposited as either a constant-composition film or as part of a binary library consistently adsorbed the least amount of albumin and fibrinogen, with alumina-enrichment of the surface oxide correlating with this adsorption. Overall, this combinatorial materials approach coupled with high-throughput surface analytical methods provides an efficient method of screening potential metallic biomaterials that may enable as well systematic studies of surface properties driving protein adsorption on these metal / metal oxide systems. PMID:19235218

  3. High-performance combinatorial algorithms

    SciTech Connect

    Pinar, Ali

    2003-10-31

    Combinatorial algorithms have long played an important role in many applications of scientific computing such as sparse matrix computations and parallel computing. The growing importance of combinatorial algorithms in emerging applications like computational biology and scientific data mining calls for development of a high performance library for combinatorial algorithms. Building such a library requires a new structure for combinatorial algorithms research that enables fast implementation of new algorithms. We propose a structure for combinatorial algorithms research that mimics the research structure of numerical algorithms. Numerical algorithms research is nicely complemented with high performance libraries, and this can be attributed to the fact that there are only a small number of fundamental problems that underlie numerical solvers. Furthermore there are only a handful of kernels that enable implementation of algorithms for these fundamental problems. Building a similar structure for combinatorial algorithms will enable efficient implementations for existing algorithms and fast implementation of new algorithms. Our results will promote utilization of combinatorial techniques and will impact research in many scientific computing applications, some of which are listed.

  4. Synthesis of solution-phase combinatorial library of 4,6-diamino-1,2-dihydro-1,3,5-triazine and identification of new leads against A16V+S108T mutant dihydrofolate reductase of Plasmodium falciparum.

    PubMed

    Vilaivan, Tirayut; Saesaengseerung, Neungruthai; Jarprung, Deanpen; Kamchonwongpaisan, Sumalee; Sirawaraporn, Worachart; Yuthavong, Yongyuth

    2003-01-17

    An efficient method to synthesize solution-phase combinatorial library of 1-aryl-4,6-diamino-1,2-dihydro-1,3,5-triazine was developed. The strategy involved an acid-catalyzed cyclocondensation between arylbiguanide hydrochlorides and carbonyl compounds in the presence of triethyl orthoacetate as water scavenger. A 96-membered combinatorial library was constructed from 6 aryl biguanides and 16 carbonyl compounds. Screening of the library by iterative deconvolution method revealed two candidate leads which are equally active against wild-type Plasmodium falciparum dihydrofolate reductase, but are about 100-fold more effective against the A16V+S108T mutant enzyme as compared to cycloguanil. PMID:12470716

  5. Identification of a potent and selective oxytocin antagonist, from screening a fully encoded differential release combinatorial chemical library.

    PubMed

    Evans, B; Pipe, A; Clark, L; Banks, M

    2001-05-21

    A library of 1,296 1,4-benzodiazepines was prepared on 160 microM Tentagel beads. Compounds are attached to the beads using orthogonally cleavable linkers. The library was first screened as pools of 30 beads where 50% of the material is released and screened. GW405212X, a selective oxytocin antagonist, was identified by picking single beads from active pools. PMID:11392541

  6. Algorithmic Strategies in Combinatorial Chemistry

    SciTech Connect

    GOLDMAN,DEBORAH; ISTRAIL,SORIN; LANCIA,GIUSEPPE; PICCOLBONI,ANTONIO; WALENZ,BRIAN

    2000-08-01

    Combinatorial Chemistry is a powerful new technology in drug design and molecular recognition. It is a wet-laboratory methodology aimed at ``massively parallel'' screening of chemical compounds for the discovery of compounds that have a certain biological activity. The power of the method comes from the interaction between experimental design and computational modeling. Principles of ``rational'' drug design are used in the construction of combinatorial libraries to speed up the discovery of lead compounds with the desired biological activity. This paper presents algorithms, software development and computational complexity analysis for problems arising in the design of combinatorial libraries for drug discovery. The authors provide exact polynomial time algorithms and intractability results for several Inverse Problems-formulated as (chemical) graph reconstruction problems-related to the design of combinatorial libraries. These are the first rigorous algorithmic results in the literature. The authors also present results provided by the combinatorial chemistry software package OCOTILLO for combinatorial peptide design using real data libraries. The package provides exact solutions for general inverse problems based on shortest-path topological indices. The results are superior both in accuracy and computing time to the best software reports published in the literature. For 5-peptoid design, the computation is rigorously reduced to an exhaustive search of about 2% of the search space; the exact solutions are found in a few minutes.

  7. Cleavage of highly structured viral RNA molecules by combinatorial libraries of hairpin ribozymes. The most effective ribozymes are not predicted by substrate selection rules.

    PubMed

    Yu, Q; Pecchia, D B; Kingsley, S L; Heckman, J E; Burke, J M

    1998-09-01

    Combinatorial libraries of hairpin ribozymes representing all possible cleavage specificities (>10(5)) were used to evaluate all ribozyme cleavage sites within a large (4.2-kilobase) and highly structured viral mRNA, the 26 S subgenomic RNA of Sindbis virus. The combinatorial approach simultaneously accounts for target site structure and dynamics, together with ribozyme folding, and the sequences that result in a ribozyme-substrate complex with maximal activity. Primer extension was used to map and rank the relative activities of the ribozyme pool against individual sites and revealed two striking findings. First, only a small fraction of potential recognition sites are effectively cleaved (activity-selected sites). Second, nearly all of the most effectively cleaved sites deviated substantially from the established consensus selection rules for the hairpin ribozyme and were not predicted by examining the sequence, or through the use of computer-assisted predictions of RNA secondary structure. In vitro selection methods were used to isolate ribozymes with increased activity against substrates that deviate from the GUC consensus sequence. trans-Acting ribozymes targeting nine of the activity-selected sites were synthesized, together with ribozymes targeting four sites with a perfect match to the cleavage site consensus (sequence-selected sites). Activity-selected ribozymes have much higher cleavage activity against the long, structured RNA molecules than do sequence-selected ribozymes, although the latter are effective in cleaving oligoribonucleotides, as predicted. These results imply that, for Sindbis virus 26 S RNA, designing ribozymes based on matches to the consensus sequence may be an ineffective strategy. PMID:9722591

  8. Optimization of combinatorial mutagenesis.

    PubMed

    Parker, Andrew S; Griswold, Karl E; Bailey-Kellogg, Chris

    2011-11-01

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (e.g., stability, activity, immunogenicity). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries--a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 10⁷ variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  9. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    SciTech Connect

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  10. Characterization of a human antibody fragment Fab and its calcium phosphate nanoparticles that inhibit rabies virus infection with vaccine.

    PubMed

    Liu, Xinjian; Lin, Hong; Tang, Qi; Li, Chen; Yang, Songtao; Wang, Zhongcan; Wang, Changjun; He, Qing; Cao, Brian; Feng, Zhenqing; Guan, Xiaohong; Zhu, Jin

    2011-01-01

    Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×10(8), was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP). PMID:21573024

  11. Neutralizing Human Fab Fragments against Measles Virus Recovered by Phage Display

    PubMed Central

    de Carvalho Nicacio, Cristina; Williamson, R. Anthony; Parren, Paul W. H. I.; Lundkvist, Åke; Burton, Dennis R.; Björling, Ewa

    2002-01-01

    Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an ⊘80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an ⊘60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H- and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between ≈2 and 7 μg ml−1. Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries. PMID:11739690

  12. Arabidopsis thaliana root cell wall proteomics: Increasing the proteome coverage using a combinatorial peptide ligand library and description of unexpected Hyp in peroxidase amino acid sequences.

    PubMed

    Nguyen-Kim, Huan; San Clemente, Hélène; Balliau, Thierry; Zivy, Michel; Dunand, Christophe; Albenne, Cécile; Jamet, Elisabeth

    2016-02-01

    Plant cell walls (CWs) contain a large proportion of polysaccharides (90-95% of CW mass) and proteins (5-10%) that play major roles in CW plasticity during development and in response to environmental cues. Here, we present CW proteomics data of Arabidopsis thaliana roots. Plants were cultivated in hydroponic conditions. CW protein (CWP) extracts were prepared and analyzed in two different ways in order to enlarge the coverage of the root CW proteome: proteins were analyzed either directly or following an affinity chromatography on a combinatorial peptide ligand library (CPLL) to reduce the concentration dynamic range. Proteins were identified by LC-MS/MS and bioinformatics. Altogether, 424 proteins having predicted signal peptides have been identified (CWPs). CPLL permitted to identify low-abundant CWPs never described before, thus enlarging the coverage of the root CW proteome. The number of oxidoreductases is particularly high and includes a large collection of class III peroxidases (CIII Prxs; 38 out of the 73 A. thaliana CIII Prxs). For the first time, hydroxyproline residues were localized at conserved positions in CIII Prx amino acid sequences. PMID:26572690

  13. Design, synthesis, and biological evaluation of the combinatorial library with a new spirodiketopiperazine scaffold. Discovery of novel potent and selective low-molecular-weight CCR5 antagonists.

    PubMed

    Habashita, Hiromu; Kokubo, Masaya; Hamano, Shin-ichi; Hamanaka, Nobuyuki; Toda, Masaaki; Shibayama, Shiro; Tada, Hideaki; Sagawa, Kenji; Fukushima, Daikichi; Maeda, Kenji; Mitsuya, Hiroaki

    2006-07-13

    We previously reported the discovery of several spirodiketopiperazine derivatives as potent CCR5 antagonists with anti-HIV activity. Herein, we describe in detail the identification of these lead compounds using a combinatorial chemistry approach. A novel spirodiketopiperazine scaffold was designed on the basis of the concept of the privileged structure of G-protein-coupled receptors (GPCRs). This new framework was obtained in acceptable yield with high purity from the readily prepared isonitrile resin through the Ugi reaction, sequential transformations, and cyclative cleavage. By measuring the inhibitory activity of each compound in the initial library against the intracellular calcium mobilization stimulated by MIP-1alpha, several compounds were found to show modest but selective CCR5 antagonistic activity. After the rapid evaluation of these hit compounds, several single-digit nanomolar, low-molecular-weight CCR5 antagonists that can potently block the infectivity and replication of laboratory and clinical strains of HIV as well as those of highly drug-resistant HIV variants with minimal cytotoxicity have been identified. PMID:16821774

  14. Combinatorial peptide ligand libraries for the analysis of low-expression proteins: Validation for normal urine and definition of a first protein MAP.

    PubMed

    Santucci, Laura; Candiano, Giovanni; Bruschi, Maurizio; D'Ambrosio, Chiara; Petretto, Andrea; Scaloni, Andrea; Urbani, Andrea; Righetti, Pier G; Ghiggeri, Gian M

    2012-02-01

    In this review, we report the evolution on experimental conditions for the analysis of normal urine based on combinatorial peptide ligand library (CPLL) treatment and successive 2-DE and 2-DE/MS analysis. The main topics are (i) definition of the urine sample requirements, (ii) optimization of the urine/ligand ratio, (iii) essay conditions, (iv) en bloc elution. Overall, normal urine protein composition as studied by 2-DE includes over 2600 spots. Relevant data on inter and intraessay reproducibility obtained by the analysis of different normal urines repeated several times are also here presented. We found a 73% reproducibility upon analysis of the same sample and 68% correspondence of protein composition among different normal urine samples. Based on the above results, we are completing the characterization with LC-MS of 249 spots. The composition of normal urine proteins after CPLLs is finally shown with the indication of those spots which are currently under identification. This map will be completed in a near future; in the meantime this would represent the basic reference sample for newly developed studies on human diseases. PMID:22246922

  15. Combinatorial approach to flavor analysis. 2. Olfactory investigation of a library of S-methyl thioesters and sensory evaluation of selected components.

    PubMed

    Berger, C; Martin, N; Collin, S; Gijs, L; Khan, J A; Piraprez, G; Spinnler, H E; Vulfson, E N

    1999-08-01

    The odor characteristics of individual components present in a library comprised of S-methyl thioesters were determined independently by two laboratories using similar but not identical techniques. The odor potency was assessed by values of best estimate-GC-lower amount detected by sniffing (BE-GC-LOADS). For small and medium chain S-methyl thioesters, these values were found to increase from 6 ng for S-methyl thiobutanoate to 90 ng for S-methyl thiostearate. All assessors detected a "green", "floral", or "pineapple" odor for S-methyl thiohexanoate and described thioesters containing a 2-6 carbon chain length as "cheesy". The results of this preliminary analysis were confirmed by a more extensive study of selected compounds, namely S-methyl thioacetate, S-methyl thiopropionate, S-methyl thiobutanoate, and S-methyl thiohexanoate, using a trained panel of 18 subjects. The subjects confirmed the presence of the "green" and "fruity" notes in the odor of S-methyl thiohexanoate. The analysis also revealed a significant difference in the odor of S-methyl thiopropionate relative to that of S-methyl thioacetate and S-methyl thiobutanoate. When "cheesy" characteristics were mentioned, the majority of panelists clearly associated the flavor of S-methyl thiopropionate with Camembert with almost 20% of all the descriptors given referring specifically to this cheese variety as compared to about 2 and 5% in the case of S-methyl thioacetate and thiobutanoate, respectively. Prompted by this observation, two samples of Camembert prepared from unpasteurized and pasteurized milk were analyzed and relatively large amounts of S-methyl thiopropionate were found in the former but not in the latter cheese. The results obtained in the course of this work suggest that the sensory analysis of combinatorial libraries is a useful new approach in the search for new commercial flavors and/or identification of characteristic flavors in foods. PMID:10552644

  16. Discovery of small peptide antagonists of PED/PEA15-D4α interaction from simplified combinatorial libraries.

    PubMed

    Scognamiglio, Pasqualina Liana; Doti, Nunzianna; Grieco, Paolo; Pedone, Carlo; Ruvo, Menotti; Marasco, Daniela

    2011-05-01

    Most biological processes involve permanent and temporary interactions between different proteins: protein complexes often play key roles in human diseases and, as a consequence, molecules that prevent protein-protein interactions can be potential new therapeutic agents to treat diseases. Here, we describe a simplified approach by which small synthetic peptide libraries were screened to identify the inhibitors of the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes15 (PED/PEA15) and D4α, a functional domain of the phospholipase D1, that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. By using an enzyme-linked immunosorbent assay (ELISA)-based screening, performed on a fully automated platform, we analyzed two simplified peptide libraries in a positional scanning format. This screening led to the identification of small peptides able to inhibit PED/PEA15-D4α interaction. The selection of inhibitors was carried out employing combined competitive and direct experiments, through ELISA and surface plasmon resonance techniques, providing peptides with IC(50) values in the micromolar range. Our results showed that the protein complex PED/PEA15-D4α is susceptible to peptides having H-donor groups and aromatic rings on specific positions. These small sequences can be considered as promising scaffolds that could be converted into higher-affinity inhibitor compounds. PMID:21294846

  17. Probing a 2-Aminobenzimidazole Library for Binding to RNA Internal Loops via Two-Dimensional Combinatorial Screening

    PubMed Central

    Velegapudi, Sai Pradeep; Pushechnikov, Alexei; Labuda, Lucas P.; French, Jonathan M.; Disney, Matthew D.

    2012-01-01

    There are many potential RNA drug targets in bacterial, viral, and the human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition. PMID:22958065

  18. Probing a 2-aminobenzimidazole library for binding to RNA internal loops via two-dimensional combinatorial screening.

    PubMed

    Velagapudi, Sai Pradeep; Pushechnikov, Alexei; Labuda, Lucas P; French, Jonathan M; Disney, Matthew D

    2012-11-16

    There are many potential RNA drug targets in bacterial, viral, and human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition. PMID:22958065

  19. Bacterial expression and purification of recombinant bovine Fab fragments.

    PubMed

    O'Brien, Philippa M; Maxwell, Gavin; Campo, M Saveria

    2002-02-01

    We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli. PMID:11812221

  20. Two-Dimensional Combinatorial Screening (2DCS) of a Bacterial rRNA A-site-like Motif Library: Defining Privileged Asymmetric Internal Loops that Bind Aminoglycosides

    PubMed Central

    Tran, Tuan; Disney, Matthew D.

    2010-01-01

    RNAs have diverse structures that are important for biological function. These structures include bulges and internal loops that can form tertiary contacts or serve as ligand binding sites. The most commonly exploited RNA drug target for small molecule intervention is the bacterial ribosome, more specifically the ribosomal RNA aminoacyl-tRNA site (rRNA A-site) which is a major target for the aminoglycoside class of antibiotics. The bacterial A-site is composed of a 1×1 nucleotide all-U internal loop and a 2×1 nucleotide all-A internal loop separated by a single GC base pair. Therefore, we probed the molecular recognition of a small library of four aminoglycosides for binding a 16384-member bacterial rRNA A-site-like internal loop library using Two-Dimensional Combinatorial Screening (2DCS). 2DCS is a microarray-based method that probes RNA and chemical spaces simultaneously. These studies sought to determine if aminoglycosides select their therapeutic target if given a choice of binding all possible internal loops derived from an A-site-like library. Results show that the bacterial rRNA A-site was not selected by any aminoglycoside. Analyses of selected sequences using the RNA Privileged Space Predictor (RNA-PSP) program show that each aminoglycoside preferentially binds different types of internal loops. For three of the aminoglycosides, 6″-azido-kanamycin A, 5-O-(2-azidoethyl) neamine, and 6″-azido-tobramycin, the selected internal loops bind with ~10-fold higher affinity than the bacterial rRNA A-site. The internal loops selected to bind 5″-azido-neomycin B bind with similar affinity as the therapeutic target. Selected internal loops that are unique for each aminoglycoside have dissociation constants ranging from 25 to 270 nM and are specific for the aminoglycoside they were selected to bind compared to the other arrayed aminoglycosides. These studies further establish a database of RNA motifs that are recognized by small molecules that could be used to

  1. A human anti-c-Met Fab fragment conjugated with doxorubicin as targeted chemotherapy for hepatocellular carcinoma.

    PubMed

    Chen, Ximin; Ding, Guipeng; Gao, Qihe; Sun, Jian; Zhang, Qianqian; Du, Lijian; Qiu, Zhenning; Wang, Changjun; Zheng, Feng; Sun, Bowang; Ni, Jian; Feng, Zhenqing; Zhu, Jin

    2013-01-01

    c-Met is over-expressed in hepatocellular carcinoma(HCC) but is absent or expressed at low levels in normal tissues. Therefore we generated a novel conjugate of a human anti-c-Met Fab fragment (MetFab) with doxorubicin (DOX) and assessed whether it had targeted antitumor activity against HCC and reduced the side-effects of DOX. The MetFab was screened from human phage library, conjugated with DOX via chemical synthesis, and the conjugation MetFab-DOX was confirmed by HPLC. The drug release patterns, the binding efficacy, and cellular distribution of MetFab-DOX were assessed. MetFab-DOX was stable at pH7.2 PBS while release doxorubicin quickly at pH4.0, the binding efficacy of MetFab-DOX was similarly as MetFab, and the cellular distribution of the MetFab-DOX is distinct from free DOX. The cytotoxicity of MetFab-DOX was analyzed by the MTT method and the nude mouse HCC model. The MetFab-DOX demonstrated cytotoxic effects on c-Met expressing-tumor cells, but not on the cells without c-Met expression. MetFab-DOX exerted anti-tumor effect and significantly reduced the side effect of free DOX in mice model. Furthermore, the localization of conjugate was confirmed by immunofluorescence staining of tumor tissue sections and optical tumor imaging, respectively, and the tissue-distribution of drug was compared between free DOX and MetFab-DOX treatment by spectrofluorometer. MetFab-DOX can localize to the tumor tissue, and the concentration of doxorubicin in the tumor was higher after MetFab-DOX administration than after DOX administration. In summary, MetFab-DOX can target c-Met expressing HCC cells effectively and have obvious antitumor activity with decreased side-effects in preclinical models of HCC. PMID:23675455

  2. High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

    PubMed

    Tsotsou, Georgia Eleni; Cass, Anthony Edward George; Gilardi, Gianfranco

    2002-01-01

    A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts. PMID:11742743

  3. Towards an animal model of ovarian cancer: cataloging chicken blood proteins using combinatorial peptide ligand libraries coupled with shotgun proteomic analysis for translational research.

    PubMed

    Ma, Yingying; Sun, Zeyu; de Matos, Ricardo; Zhang, Jing; Odunsi, Kunle; Lin, Biaoyang

    2014-05-01

    Epithelial ovarian cancer is the most deadly gynecological cancer around the world, with high morbidity in industrialized countries. Early diagnosis is key in reducing its morbidity rate. Yet, robust biomarkers, diagnostics, and animal models are still limited for ovarian cancer. This calls for broader omics and systems science oriented diagnostics strategies. In this vein, the domestic chicken has been used as an ovarian cancer animal model, owing to its high rate of developing spontaneous epithelial ovarian tumors. Chicken blood has thus been considered a surrogate reservoir from which cancer biomarkers can be identified. However, the presence of highly abundant proteins in chicken blood has compromised the applicability of proteomics tools to study chicken blood owing to a lack of immunodepletion methods. Here, we demonstrate that a combinatorial peptide ligand library (CPLL) can efficiently remove highly abundant proteins from chicken blood samples, consequently doubling the number of identified proteins. Using an integrated CPLL-1DGE-LC-MSMS workflow, we identified a catalog of 264 unique proteins. Functional analyses further suggested that most proteins were coagulation and complement factors, blood transport and binding proteins, immune- and defense-related proteins, proteases, protease inhibitors, cellular enzymes, or cell structure and adhesion proteins. Semiquantitative spectral counting analysis identified 10 potential biomarkers from the present chicken ovarian cancer model. Additionally, many human homologs of chicken blood proteins we have identified have been independently suggested as diagnostic biomarkers for ovarian cancer, further triangulating our novel observations reported here. In conclusion, the CPLL-assisted proteomic workflow using the chicken ovarian cancer model provides a feasible platform for translational research to identify ovarian cancer biomarkers and understand ovarian cancer biology. To the best of our knowledge, we report here

  4. Combinatorial synthesis of ceramic materials

    DOEpatents

    Lauf, Robert J [Oak Ridge, TN; Walls, Claudia A [Oak Ridge, TN; Boatner, Lynn A [Oak Ridge, TN

    2010-02-23

    A combinatorial library includes a gelcast substrate defining a plurality of cavities in at least one surface thereof; and a plurality of gelcast test materials in the cavities, at least two of the test materials differing from the substrate in at least one compositional characteristic, the two test materials differing from each other in at least one compositional characteristic.

  5. Combinatorial synthesis of ceramic materials

    DOEpatents

    Lauf, Robert J.; Walls, Claudia A.; Boatner, Lynn A.

    2006-11-14

    A combinatorial library includes a gelcast substrate defining a plurality of cavities in at least one surface thereof; and a plurality of gelcast test materials in the cavities, at least two of the test materials differing from the substrate in at least one compositional characteristic, the two test materials differing from each other in at least one compositional characteristic.

  6. Combinatorial Chemistry for Optical Sensing Applications

    NASA Astrophysics Data System (ADS)

    Díaz-García, M. E.; Luis, G. Pina; Rivero-Espejel, I. A.

    The recent interest in combinatorial chemistry for the synthesis of selective recognition materials for optical sensing applications is presented. The preparation, screening, and applications of libraries of ligands and chemosensors against molecular species and metal ions are first considered. Included in this chapter are also the developments involving applications of combinatorial approaches to the discovery of sol-gel and acrylic-based imprinted materials for optical sensing of antibiotics and pesticides, as well as libraries of doped sol-gels for high-throughput optical sensing of oxygen. The potential of combinatorial chemistry applied to the discovery of new sensing materials is highlighted.

  7. Developing New Tools for the in vivo Generation/Screening of Cyclic Peptide Libraries. A New Combinatorial Approach for the Detection of Bacterial Toxin Inhibitors

    SciTech Connect

    Camarero, J A

    2006-11-28

    A new combinatorial approach for the biosynthesis and screening of small drug-like toxin inhibitors inside living cells is presented. This approach has been initially used as proof of principle for finding inhibitors against the LF factor from Bacillus anthracis. Key to our ''living combinatorial'' approach is the use of a living cell as a micro-chemical factory for both synthesis and screening of potential inhibitors for a given molecular recognition event (see Scheme 1). This powerful technique posses the advantage that both processes synthesis and screening happen inside the cell thus accelerating the whole screening/selection process.

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

    PubMed

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

    2015-04-01

    Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX(8)K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria. PMID:25370725

  9. Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library.

    PubMed

    Houimel, Mehdi; Dellagi, Koussay

    2009-11-01

    A human immune Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from rabies virus hyperimmune volunteers on phagemid vector. The size of the constructed Fab library was 2 x 10(7) Escherichia coli transformants. After four rounds of panning on whole inactivated rabies virus (PV-11), phage clones displaying rabies virus-specific human Fab were selected. The specificity of soluble Fab antibody fragments, derived from positive phage clones was verified by ELISA. Among 20 specific Fab clones, the genetic sequence of 6 of them (FabRV01, FabRV02, FabRV03, FabRV04, FabRV05, and FabRV06) was analyzed. The variable heavy (VH) and variable light (VL) domains were found to share 90% and 93% homology with sequences encoded by the corresponding human germline genes, respectively. The soluble Fab fragments, expressed in Escherichia coli were purified by a single step Nickel-NTA affinity chromatography via a hexa-histidine tag and their binding specificities to rabies virus were confirmed. Three of the Fab antibodies, FabRV01, FabRV02 and FabRV03, showed binding characteristics to rabies virus glycoprotein antigenic site III with affinities in the K(D) range 7 x 10(-9) to 5 x 10(-8)M. The Fab fragments showed dose-dependent neutralization properties for the challenge virus standard (CVS-11). PMID:19559727

  10. Library+

    ERIC Educational Resources Information Center

    Merrill, Alex

    2011-01-01

    This article discusses possible future directions for academic libraries in the post Web/Library 2.0 world. These possible directions include areas such as data literacy, linked data sets, and opportunities for libraries in support of digital humanities. The author provides a brief sketch of the background information regarding the topics and…

  11. Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication

    PubMed Central

    Lund, Anders H.; Duch, Mogens; Pedersen, Finn Skou

    2000-01-01

    Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNAPro molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retroviruses have revealed evidence of molecular adaptation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3′-end of tRNAPro, we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNAArg(CCU), tRNAPhe(GAA) and a hitherto unknown human tRNASer(CGA). PMID:10637332

  12. An affinity selection-mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries: Discovery of a novel antagonist of E. coli dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Annis, D. Allen; Athanasopoulos, John; Curran, Patrick J.; Felsch, Jason S.; Kalghatgi, Krishna; Lee, William H.; Nash, Huw M.; Orminati, Jean-Paul A.; Rosner, Kristin E.; Shipps, Gerald W., Jr.; Thaddupathy, G. R. A.; Tyler, Andrew N.; Vilenchik, Lev; Wagner, Carston R.; Wintner, Edward A.

    2004-11-01

    The NeoGenesis Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry (AS-MS) process consisting of a rapid size-exclusion chromatography stage integrated with reverse-phase chromatography, electrospray mass spectrometry, and novel data searching algorithms, was used to screen mass-encoded, 2500-member combinatorial libraries, leading to the discovery of a novel, bioactive ligand for the anti-infective target Escherichia coli dihydrofolate reductase (DHFR). Synthesis of the mass-encoded, ligand-containing library, discussion of the deconvolution process for verifying the structure of the ligand through independent synthesis and screening in a small mixture (sub-library) format, and ALIS-MS/MS techniques to assign its regioisomeric connectivity are presented. ALIS-based competition experiments between the newly discovered ligand and other, known DHFR ligands, and biological activity assessments with stereo- and regioisomers of the hit compound confirm its DHFR-specific biological activity. The method described requires no foreknowledge of the structure or biochemistry of the protein target, consumes less than 1 [mu]g protein to screen >2500 compounds in a single experiment, and enables screening of >250,000 compounds per system per day. These advantages highlight the potential of the ALIS method for drug discovery against genomic targets with unknown biological function, as well as validated targets for which traditional discovery efforts have failed.

  13. Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

    PubMed Central

    Hughes, Marcus D.; Zhang, Zhan-Ren; Sutherland, Andrew J.; Santos, Albert F.; Hine, Anna V.

    2005-01-01

    We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using ‘MAX’ randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40 000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants. PMID:15722478

  14. Jeffamine Derivatized TentaGel Beads and PDMS Microbead Cassettes for Ultra-high Throughput in situ Releasable Solution-Phase Cell-based Screening of OBOC Combinatorial Small Molecule Libraries

    PubMed Central

    Townsend, Jared B.; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S.

    2011-01-01

    A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated polydimethylsiloxane (PDMS) cassette for high-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting tri-functional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry, resulting in beads with increased loading capacity, hydrophilicity and porosity at the outer layer. We have found that such bead configuration can facilitate ultra high-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 minutes) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel® were then layered over the microbead cassette to immobilize the compound-beads. After 24 hours of incubation at 37°C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These

  15. New Electrocatalysts by Combinatorial Methods

    NASA Astrophysics Data System (ADS)

    Smotkin, Eugene S.; Diaz-Morales, Robert R.

    2003-08-01

    Combinatorial methods provide a means for accelerating the discovery of fuel cell catalysts. The first example of parallel fuel cell catalysts screening was an indirect method that used fluorescent chemosensors to detect changes in pH in proximity to electrocatalyst spots. Serial direct electrochemical methods have been developed that use voltammetry, chronoamperometry, and scanning electrochemical microscopy. An array fuel cell screens catalysts simultaneously, using high-performance fuel cell components. Heuristic models based on mechanistic and spectroscopic studies provide guidance for library development, and detailed studies of discovered catalysts can help to refine these models. The remaining challenges are the development of high throughput synthetic methods that can enable the use of discovery level and focus level screening. Until these synthetic methods are developed, a greater emphasis should be placed on smaller libraries with design of experiment strategies leveraged with informatics and data mining.

  16. Molecular insights of protein contour recognition with ligand pharmacophoric sites through combinatorial library design and MD simulation in validating HTLV-1 PR inhibitors.

    PubMed

    Selvaraj, Chandrabose; Omer, Ankur; Singh, Poonam; Singh, Sanjeev Kumar

    2015-01-01

    Retroviruses HIV-1 and HTLV-1 are chiefly considered to be the most dangerous pathogens in Homo sapiens. These two viruses have structurally unique protease (PR) enzymes, which are having common function of its replication mechanism. Though HIV PR drugs failed to inhibit HTLV-1 infections, they emphatically emphasise the need for designing new lead compounds against HTLV-1 PR. Therefore, we tried to understand the binding level interactions through the charge environment present in both ligand and protein active sites. The domino effect illustrates that libraries of purvalanol-A are attuned to fill allosteric binding site of HTLV-1 PR through molecular recognition and shows proper binding of ligand pharmacophoric features in receptor contours. Our screening evaluates seven compounds from purvalanol-A libraries, and these compounds' pharmacophore searches for an appropriate place in the binding site and it places well according to respective receptor contour surfaces. Thus our result provides a platform for the progress of more effective compounds, which are better in free energy calculation, molecular docking, ADME and molecular dynamics studies. Finally, this research provided novel chemical scaffolds for HTLV-1 drug discovery. PMID:25335799

  17. SECM characterization of Pt-Ru-WC and Pt-Ru-Co ternary thin film combinatorial libraries as anode electrocatalysts for PEMFC

    NASA Astrophysics Data System (ADS)

    Lu, Guojin; Cooper, James S.; McGinn, Paul J.

    Rapid screening of electrocatalytic activity of ternary Pt-Ru-WC and Pt-Ru-Co thin film gradient material libraries towards hydrogen oxidation in the presence or absence of CO adsorption was performed by scanning electrochemical microscopy. It was observed that the addition of WC or Co to Pt or Pt-Ru catalysts can improve their hydrogen oxidation reaction activity and CO tolerance, making them suitable as potential electrocatalysts for polymer electrolyte membrane fuel cells. The stability of WC and Co in the acidic electrolyte were enhanced by alloying with Pt. SECM offers the capability for both qualitative and quantitative characterization of electrocatalytic activity of thin films of potential fuel cell electrode material candidates. However, promising electrode compositions identified by this technique need to be verified by traditional electrode preparation and characterization techniques.

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

    PubMed

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

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. PMID:26271437

  19. Liquid-phase combinatorial synthesis.

    PubMed Central

    Han, H; Wolfe, M M; Brenner, S; Janda, K D

    1995-01-01

    A concept termed liquid-phase combinatorial synthesis (LPCS) is described. The central feature of this methodology is that it combines the advantages that classic organic synthesis in solution offers with those that solid-phase synthesis can provide, through the application of a linear homogeneous polymer. To validate this concept two libraries were prepared, one of peptide and the second of nonpeptide origin. The peptide-based library was synthesized by a recursive deconvolution strategy [Erb, E., Janda, K. D. & Brenner, S. (1994) Proc. Natl. Acad. Sci. USA 91, 11422-11426] and several ligands were found within this library to bind a monoclonal antibody elicited against beta-endorphin. The non-peptide molecules synthesized were arylsulfonamides, a class of compounds of known clinical bactericidal efficacy. The results indicate that the reaction scope of LPCS should be general, and its value to multiple, high-throughput screening assays could be of particular merit, since multimilligram quantities of each library member can readily be attained. PMID:7541541

  20. Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display.

    PubMed

    Patel, Seema; Mathonet, Pascale; Jaulent, Agnes M; Ullman, Christopher G

    2013-04-01

    WW domains are small β-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications. PMID:23378640

  1. Available pathways database (APD): an essential resource for combinatorial biology.

    PubMed

    Pirrung, M C; Silva, C M; Jaeger, J

    2000-10-01

    A relational database, the Available Pathways Database (APD), has been constructed of microbial natural products, their producing strains, and their biosynthetic pathways. The database allows the ready selection of donor strains for combinatorial biology experiments. It provides the same type of resource for combinatorial biology as the Available Chemicals Directory (ACD) does for combinatorial chemical library generation. Its cataloging ability can also provide insight into novel aspects of biosynthetic routes. In particular, no 10-unit Type I polyketides were found in the compilation of this edition of the APD (Version I). PMID:11076562

  2. Combinatorial protein design strategies using computational methods.

    PubMed

    Kono, Hidetoshi; Wang, Wei; Saven, Jeffery G

    2007-01-01

    Computational methods continue to facilitate efforts in protein design. Most of this work has focused on searching sequence space to identify one or a few sequences compatible with a given structure and functionality. Probabilistic computational methods provide information regarding the range of amino acid variability permitted by desired functional and structural constraints. Such methods may be used to guide the construction of both individual sequences and combinatorial libraries of proteins. PMID:17041256

  3. Combinatorial Geometry Printer Plotting.

    Energy Science and Technology Software Center (ESTSC)

    1987-01-05

    Picture generates plots of two-dimensional slices through the three-dimensional geometry described by the combinatorial geometry (CG) package used in such codes as MORSE and QAD-CG. These plots are printed on a standard line printer.

  4. Combinatorial Interdependence in Lottery

    ERIC Educational Resources Information Center

    Helman, Danny

    2005-01-01

    This paper examines a real life question of gamble facing lottery players. Combinatorial dependence plays a central role in shaping the game probabilistic structure, but might not carry the merited weight in punters' considerations.

  5. A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4.

    PubMed Central

    Bachelder, R E; Bilancieri, J; Lin, W; Letvin, N L

    1995-01-01

    To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection. PMID:7637018

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

    PubMed

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

    2016-01-16

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50=5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials. PMID:26562505

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

    PubMed Central

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

    2016-01-01

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials. PMID:26562505

  8. Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.

    PubMed

    Zeitoun, Ramsey I; Garst, Andrew D; Degen, George D; Pines, Gur; Mansell, Thomas J; Glebes, Tirzah Y; Boyle, Nanette R; Gill, Ryan T

    2015-06-01

    Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies. We used TRACE to map growth selection dynamics for Escherichia coli combinatorial libraries created by recursive multiplex recombineering at a depth 10(4)-fold greater than before. TRACE was used to identify genotype-to-phenotype correlations and to map the evolutionary trajectory of two individual combinatorial mutants in E. coli. Combinatorial mutations in the human ES2 ovarian carcinoma cell line were also assessed with TRACE. TRACE completes the combinatorial engineering cycle and enables more sophisticated approaches to genome engineering in both bacteria and eukaryotic cells than are currently possible. PMID:25798935

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

    ERIC Educational Resources Information Center

    Pagano, John

    2015-01-01

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

  10. Application of computer assisted combinatorial chemistry in antivirial, antimalarial and anticancer agents design

    NASA Astrophysics Data System (ADS)

    Burello, E.; Bologa, C.; Frecer, V.; Miertus, S.

    Combinatorial chemistry and technologies have been developed to a stage where synthetic schemes are available for generation of a large variety of organic molecules. The innovative concept of combinatorial design assumes that screening of a large and diverse library of compounds will increase the probability of finding an active analogue among the compounds tested. Since the rate at which libraries are screened for activity currently constitutes a limitation to the use of combinatorial technologies, it is important to be selective about the number of compounds to be synthesized. Early experience with combinatorial chemistry indicated that chemical diversity alone did not result in a significant increase in the number of generated lead compounds. Emphasis has therefore been increasingly put on the use of computer assisted combinatorial chemical techniques. Computational methods are valuable in the design of virtual libraries of molecular models. Selection strategies based on computed physicochemical properties of the models or of a target compound are introduced to reduce the time and costs of library synthesis and screening. In addition, computational structure-based library focusing methods can be used to perform in silico screening of the activity of compounds against a target receptor by docking the ligands into the receptor model. Three case studies are discussed dealing with the design of targeted combinatorial libraries of inhibitors of HIV-1 protease, P. falciparum plasmepsin and human urokinase as potential antivirial, antimalarial and anticancer drugs. These illustrate library focusing strategies.

  11. Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial

    PubMed Central

    Ruha, Anne-Michelle; Seifert, Steven A.; Morgan, David L.; Lewis, Brandon J.; Arnold, Thomas C.; Clark, Richard F.; Meggs, William J.; Toschlog, Eric A.; Borron, Stephen W.; Figge, Gary R.; Sollee, Dawn R.; Shirazi, Farshad M.; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D.; Boyer, Leslie V.

    2015-01-01

    Background. Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. Methods. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm3, fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. Results. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. Conclusions. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation

  12. Manipulating Combinatorial Structures.

    ERIC Educational Resources Information Center

    Labelle, Gilbert

    This set of transparencies shows how the manipulation of combinatorial structures in the context of modern combinatorics can easily lead to interesting teaching and learning activities at every level of education from elementary school to university. The transparencies describe: (1) the importance and relations of combinatorics to science and…

  13. Massively parallel high-order combinatorial genetics in human cells

    PubMed Central

    Wong, Alan S L; Choi, Gigi C G; Cheng, Allen A; Purcell, Oliver; Lu, Timothy K

    2016-01-01

    The systematic functional analysis of combinatorial genetics has been limited by the throughput that can be achieved and the order of complexity that can be studied. To enable massively parallel characterization of genetic combinations in human cells, we developed a technology for rapid, scalable assembly of high-order barcoded combinatorial genetic libraries that can be quantified with high-throughput sequencing. We applied this technology, combinatorial genetics en masse (CombiGEM), to create high-coverage libraries of 1,521 two-wise and 51,770 three-wise barcoded combinations of 39 human microRNA (miRNA) precursors. We identified miRNA combinations that synergistically sensitize drug-resistant cancer cells to chemotherapy and/or inhibit cancer cell proliferation, providing insights into complex miRNA networks. More broadly, our method will enable high-throughput profiling of multifactorial genetic combinations that regulate phenotypes of relevance to biomedicine, biotechnology and basic science. PMID:26280411

  14. Research on universal combinatorial coding.

    PubMed

    Lu, Jun; Zhang, Zhuo; Mo, Juan

    2014-01-01

    The conception of universal combinatorial coding is proposed. Relations exist more or less in many coding methods. It means that a kind of universal coding method is objectively existent. It can be a bridge connecting many coding methods. Universal combinatorial coding is lossless and it is based on the combinatorics theory. The combinational and exhaustive property make it closely related with the existing code methods. Universal combinatorial coding does not depend on the probability statistic characteristic of information source, and it has the characteristics across three coding branches. It has analyzed the relationship between the universal combinatorial coding and the variety of coding method and has researched many applications technologies of this coding method. In addition, the efficiency of universal combinatorial coding is analyzed theoretically. The multicharacteristic and multiapplication of universal combinatorial coding are unique in the existing coding methods. Universal combinatorial coding has theoretical research and practical application value. PMID:24772019

  15. Research on Universal Combinatorial Coding

    PubMed Central

    Lu, Jun; Zhang, Zhuo; Mo, Juan

    2014-01-01

    The conception of universal combinatorial coding is proposed. Relations exist more or less in many coding methods. It means that a kind of universal coding method is objectively existent. It can be a bridge connecting many coding methods. Universal combinatorial coding is lossless and it is based on the combinatorics theory. The combinational and exhaustive property make it closely related with the existing code methods. Universal combinatorial coding does not depend on the probability statistic characteristic of information source, and it has the characteristics across three coding branches. It has analyzed the relationship between the universal combinatorial coding and the variety of coding method and has researched many applications technologies of this coding method. In addition, the efficiency of universal combinatorial coding is analyzed theoretically. The multicharacteristic and multiapplication of universal combinatorial coding are unique in the existing coding methods. Universal combinatorial coding has theoretical research and practical application value. PMID:24772019

  16. Automated Combinatorial Chemistry in the Organic Chemistry Majors Laboratory

    ERIC Educational Resources Information Center

    Nichols, Christopher J.; Hanne, Larry F.

    2010-01-01

    A multidisciplinary experiment has been developed in which students each synthesize a combinatorial library of 48 hydrazones with the aid of a liquid-handling robot. Each product is then subjected to a Kirby-Bauer disk diffusion assay to assess its antibacterial activity. Students gain experience working with automation and at the…

  17. Introducing Dynamic Combinatorial Chemistry: Probing the Substrate Selectivity of Acetylcholinesterase

    ERIC Educational Resources Information Center

    Angelin, Marcus; Larsson, Rikard; Vongvilai, Pornrapee; Ramstrom, Olof

    2010-01-01

    In this laboratory experiment, college students are introduced to dynamic combinatorial chemistry (DCC) and apply it to determine the substrate selectivity of acetylcholinesterase (AChE). Initially, the students construct a chemical library of dynamically interchanging thioesters and thiols. Then, AChE is added and allowed to select and hydrolyze…

  18. A combinatorial approach to the discovery of advanced materials

    NASA Astrophysics Data System (ADS)

    Sun, Xiao-Dong

    This thesis discusses the application of combinatorial methods to the search of advanced materials. The goal of this research is to develop a "parallel" or "fast sequential" methodology for both the synthesis and characterization of materials with novel electronic, magnetic and optical properties. Our hope is to dramatically accelerate the rate at which materials are generated and studied. We have developed two major combinatorial methodologies to this end. One involves generating thin film materials libraries using a combination of various thin film deposition and masking strategies with multi-layer thin film precursors. The second approach is to generate powder materials libraries with solution precursors delivered with a multi-nozzle inkjet system. The first step in this multistep combinatorial process involves the design and synthesis of high density libraries of diverse materials aimed at exploring a large segment of the compositional space of interest based on our understanding of the physical and structural properties of a particular class of materials. Rapid, sensitive measurements of one or more relevant physical properties of each library member result in the identification of a family of "lead" compositions with a desired property. These compositions are then optimized by continuously varying the stoichiometries of a more focused set of precursors. Materials with the optimal composition are then synthesized in quantities sufficient for detailed characterization of their structural and physical properties. Finally, the information obtained from this process should enhance our predictive ability in subsequent experiments. Combinatorial methods have been successfully used in the synthesis and discovery of materials with novel properties. For example, a class of cobaltite based giant magnetoresistance (GMR) ceramics was discovered; Application of this method to luminescence materials has resulted in the discovery of a few highly efficient tricolor

  19. Immunoscintigraphy with 111In antimyosin Fab.

    PubMed

    Morguet, A J; Munz, D L; Kreuzer, H; Emrich, D

    1990-11-01

    Monoclonal 111In antimyosin Fab is a marker for myocytes which have lost their membrane integrity. Because of the slow blood pool clearance of the radiopharmaceutical, imaging is usually started 24-48 h after intravenous injection of 74 MBq of the tracer. This long postinjection interval restricts its utilization in the primary diagnosis of acute myocardial infarction. However, antimyosin may help to differentiate between necrotic and viable myocardium in the subacute stage of incomplete myocardial infarction. Serial endomyocardial biopsy for early detection of transplant rejection after heart transplantation may be partially replaced or supplemented by antimyosin scintigraphy. The compound may facilitate the diagnosis of myocarditis. Other potential indications may be prognostic assessment of dilated cardiomyopathy, monitoring cardiotoxic side-effects of chemotherapeutics, recognition of cardiac contusion as well as diagnosis of rhabdo- and leiomyosarcoma. In specific clinical situations 111In antimyosin Fab immunoscintigraphy may provide valuable diagnostic information. PMID:2277688

  20. Mask qualification strategies in a wafer fab

    NASA Astrophysics Data System (ADS)

    Jaehnert, Carmen; Kunowski, Angela

    2007-02-01

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

  1. Combinatorial Engineering of Dextransucrase Specificity

    PubMed Central

    Irague, Romain; Tarquis, Laurence; André, Isabelle; Moulis, Claire; Morel, Sandrine; Monsan, Pierre; Potocki-Véronèse, Gabrielle; Remaud-Siméon, Magali

    2013-01-01

    We used combinatorial engineering to investigate the relationships between structure and linkage specificity of the dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, and to generate variants with altered specificity. Sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to eight amino acids (D306, F353, N404, W440, D460, H463, T464 and S512) being targeted, randomized by saturation mutagenesis and simultaneously recombined. Screening of two libraries totaling 3.6.104 clones allowed the isolation of a toolbox comprising 81 variants which synthesize high molecular weight α-glucans with different proportions of α(1→3) linkages ranging from 3 to 20 %. Mutant sequence analysis, biochemical characterization and molecular modelling studies revealed the previously unknown role of peptide 460DYVHT464 in DSR-S linkage specificity. This peptide sequence together with residue S512 contribute to defining +2 subsite topology, which may be critical for the enzyme regiospecificity. PMID:24204991

  2. Combinatorial measurements of Hall effect and resistivity in oxide films.

    PubMed

    Clayhold, J A; Kerns, B M; Schroer, M D; Rench, D W; Logvenov, G; Bollinger, A T; Bozovic, I

    2008-03-01

    A system for the simultaneous measurement of the Hall effect in 31 different locations as well as the measurement of the resistivity in 30 different locations on a single oxide thin film grown with a composition gradient is described. Considerations for designing and operating a high-throughput system for characterizing highly conductive oxides with Hall coefficients as small as 10(-10) m3/C are discussed. Results from measurements on films grown using combinatorial molecular beam epitaxy show the usefulness of characterizing combinatorial libraries via both the resistivity and the Hall effect. PMID:18377026

  3. Characterization of FabG and FabI of the Streptomyces coelicolor dissociated fatty acid synthase.

    PubMed

    Singh, Renu; Reynolds, Kevin A

    2015-03-01

    Streptomyces coelicolor produces fatty acids for both primary metabolism and for biosynthesis of the secondary metabolite undecylprodiginine. The first and last reductive steps during the chain elongation cycle of fatty acid biosynthesis are catalyzed by FabG and FabI. The S. coelicolor genome sequence has one fabI gene (SCO1814) and three likely fabG genes (SCO1815, SCO1345, and SCO1846). We report the expression, purification, and characterization of the corresponding gene products. Kinetic analyses revealed that all three FabGs and FabI are capable of utilizing both straight and branched-chain β-ketoacyl-NAC and enoyl-NAC substrates, respectively. Furthermore, only SCO1345 differentiates between ACPs from both biosynthetic pathways. The data presented provide the first experimental evidence that SCO1815, SCO1346, and SCO1814 have the catalytic capability to process intermediates in both fatty acid and undecylprodiginine biosynthesis. PMID:25662938

  4. Combinatorial Mechanical Metamaterials

    NASA Astrophysics Data System (ADS)

    van Hecke, Martin

    The structure of most mechanical metamaterials is periodic so that their design space is that of the unit cell. Here we introduce a combinatorial strategy to create a vast number of distinct mechanical metamaterials, each with a unique spatial texture and response. These are aperiodic stackings of anisotropic building blocks, and their functionality rests on both the block design and their stacking configuration which is governed by a tiling problem. We realize such metamaterials by 3D printing, and show that they act as soft machines, capable of pattern recognition and pattern analysis.

  5. Development of a combinatorial atmospheric pressure cold plasma processor

    NASA Astrophysics Data System (ADS)

    Terajima, Takeshi; Koinuma, Hideomi

    2004-02-01

    Low-temperature plasma can be generated under atmospheric pressure by applying an RF (13.56 MHz) voltage between parallel electrodes, the surfaces of which are preferably covered with an insulator. Applications of this atmospheric pressure cold plasma include thin film deposition, chemical synthesis, etching, resist-ashing, surface treatment, and sterilization. For seeking further improvement of the system and more applications, we have developed a combinatorial atmospheric pressure cold plasma generator to fabricate composition spread thin films by synchronizing the variation of feeding gas ratio with the substrate stage motion. This system can be extended to fabricating a variety of combinatorial libraries by controlling other parameters in the operation such as the gas flow rate, the RF power, substrate temperature, and the treatment time. The utility of this combinatorial plasma process has been demonstrated with the plasma copolymerization of CO 2 with ethylene to fix CO 2 into the plasma polymerized film in the form of ester linkage.

  6. Cryptographic Combinatorial Securities Exchanges

    NASA Astrophysics Data System (ADS)

    Thorpe, Christopher; Parkes, David C.

    We present a useful new mechanism that facilitates the atomic exchange of many large baskets of securities in a combinatorial exchange. Cryptography prevents information about the securities in the baskets from being exploited, enhancing trust. Our exchange offers institutions who wish to trade large positions a new alternative to existing methods of block trading: they can reduce transaction costs by taking advantage of other institutions’ available liquidity, while third party liquidity providers guarantee execution—preserving their desired portfolio composition at all times. In our exchange, institutions submit encrypted orders which are crossed, leaving a “remainder”. The exchange proves facts about the portfolio risk of this remainder to third party liquidity providers without revealing the securities in the remainder, the knowledge of which could also be exploited. The third parties learn either (depending on the setting) the portfolio risk parameters of the remainder itself, or how their own portfolio risk would change if they were to incorporate the remainder into a portfolio they submit. In one setting, these third parties submit bids on the commission, and the winner supplies necessary liquidity for the entire exchange to clear. This guaranteed clearing, coupled with external price discovery from the primary markets for the securities, sidesteps difficult combinatorial optimization problems. This latter method of proving how taking on the remainder would change risk parameters of one’s own portfolio, without revealing the remainder’s contents or its own risk parameters, is a useful protocol of independent interest.

  7. Combinatorial investigation of ferromagnetic shape memory materials

    NASA Astrophysics Data System (ADS)

    Famodu, Olugbenga O.

    2005-07-01

    Combinatorial synthesis is research methodology which allows one to systemically study a large number of compositionally varying samples simultaneously. We apply this technique to the investigation of multifunctional materials. Different designs of combinatorial libraries and various characterization tools are implemented in order to rapidly map composition-structure-property relationships in a variety of materials systems. In this thesis, I will discuss combinatorial investigation of various shape memory alloys. We have utilized the combinatorial magnetron co-sputtering deposition technique for fabricating composition spreads of ternary alloy systems containing ferromagnetic shape memory alloys (FSMAs) and thermoelastic shape memory alloys (SMAs). Magnetic properties of the composition spreads were rapidly characterized using a room temperature scanning semiconducting quantum interference device (SQUID) microscope which provides mapping of the magnetic field emanating from different parts of the composition spreads. By applying the inversion technique to the mapping of the magnetic field distribution, we have mapped the magnetic phase diagram of the Ni-Mn-Ga and Ni-Mn-Al systems whose Heusler compositions Ni2MnGa and Ni2MnAl are well known ferromagnetic shape memory alloys (FSMAs). In addition, a rapid visual inspection technique was developed for detection of reversible martensites using arrays of micromachined cantilevers. A large, previously unexplored compositional region of FSMAs outside the Heusler composition was found. In search of novel FSMAs, we have also investigated a number of other ternary alloys systems. These systems included Ni-Mn-In, Gd-Ge-Si, Co-Mn-Ga, Ni-Fe-Al, and Co-Ni-Ga. A summary of the results from the investigation of these systems is presented. We have used the combinatorial technique to search for "ideal" SMAs with minimal hysteresis. For pursuing this, we had first set out to verify the geometric non-linear theory of martensites which

  8. The cosmology of the Fab-Four

    NASA Astrophysics Data System (ADS)

    Copeland, Edmund J.; Padilla, Antonio; Saffin, Paul M.

    2012-12-01

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

  9. The cosmology of the Fab-Four

    SciTech Connect

    Copeland, Edmund J.; Padilla, Antonio; Saffin, Paul M. E-mail: antonio.padilla@nottingham.ac.uk

    2012-12-01

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

  10. Combinatorial thin film composition mapping using three dimensional deposition profiles

    NASA Astrophysics Data System (ADS)

    Suram, Santosh K.; Zhou, Lan; Becerra-Stasiewicz, Natalie; Kan, Kevin; Jones, Ryan J. R.; Kendrick, Brian M.; Gregoire, John M.

    2015-03-01

    Many next-generation technologies are limited by material performance, leading to increased interest in the discovery of advanced materials using combinatorial synthesis, characterization, and screening. Several combinatorial synthesis techniques, such as solution based methods, advanced manufacturing, and physical vapor deposition, are currently being employed for various applications. In particular, combinatorial magnetron sputtering is a versatile technique that provides synthesis of high-quality thin film composition libraries. Spatially addressing the composition of these thin films generally requires elemental quantification measurements using techniques such as energy-dispersive X-ray spectroscopy or X-ray fluorescence spectroscopy. Since these measurements are performed ex-situ and post-deposition, they are unable to provide real-time design of experiments, a capability that is required for rapid synthesis of a specific composition library. By using three quartz crystal monitors attached to a stage with translational and rotational degrees of freedom, we measure three-dimensional deposition profiles of deposition sources whose tilt with respect to the substrate is robotically controlled. We exhibit the utility of deposition profiles and tilt control to optimize the deposition geometry for specific combinatorial synthesis experiments.

  11. Combinatorial thin film composition mapping using three dimensional deposition profiles.

    PubMed

    Suram, Santosh K; Zhou, Lan; Becerra-Stasiewicz, Natalie; Kan, Kevin; Jones, Ryan J R; Kendrick, Brian M; Gregoire, John M

    2015-03-01

    Many next-generation technologies are limited by material performance, leading to increased interest in the discovery of advanced materials using combinatorial synthesis, characterization, and screening. Several combinatorial synthesis techniques, such as solution based methods, advanced manufacturing, and physical vapor deposition, are currently being employed for various applications. In particular, combinatorial magnetron sputtering is a versatile technique that provides synthesis of high-quality thin film composition libraries. Spatially addressing the composition of these thin films generally requires elemental quantification measurements using techniques such as energy-dispersive X-ray spectroscopy or X-ray fluorescence spectroscopy. Since these measurements are performed ex-situ and post-deposition, they are unable to provide real-time design of experiments, a capability that is required for rapid synthesis of a specific composition library. By using three quartz crystal monitors attached to a stage with translational and rotational degrees of freedom, we measure three-dimensional deposition profiles of deposition sources whose tilt with respect to the substrate is robotically controlled. We exhibit the utility of deposition profiles and tilt control to optimize the deposition geometry for specific combinatorial synthesis experiments. PMID:25832242

  12. Combinatorial optimization games

    SciTech Connect

    Deng, X.; Ibaraki, Toshihide; Nagamochi, Hiroshi

    1997-06-01

    We introduce a general integer programming formulation for a class of combinatorial optimization games, which immediately allows us to improve the algorithmic result for finding amputations in the core (an important solution concept in cooperative game theory) of the network flow game on simple networks by Kalai and Zemel. An interesting result is a general theorem that the core for this class of games is nonempty if and only if a related linear program has an integer optimal solution. We study the properties for this mathematical condition to hold for several interesting problems, and apply them to resolve algorithmic and complexity issues for their cores along the line as put forward in: decide whether the core is empty; if the core is empty, find an imputation in the core; given an imputation x, test whether x is in the core. We also explore the properties of totally balanced games in this succinct formulation of cooperative games.

  13. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  14. Protein-Directed Dynamic Combinatorial Chemistry: A Guide to Protein Ligand and Inhibitor Discovery.

    PubMed

    Huang, Renjie; Leung, Ivanhoe K H

    2016-01-01

    Protein-directed dynamic combinatorial chemistry is an emerging technique for efficient discovery of novel chemical structures for binding to a target protein. Typically, this method relies on a library of small molecules that react reversibly with each other to generate a combinatorial library. The components in the combinatorial library are at equilibrium with each other under thermodynamic control. When a protein is added to the equilibrium mixture, and if the protein interacts with any components of the combinatorial library, the position of the equilibrium will shift and those components that interact with the protein will be amplified, which can then be identified by a suitable biophysical technique. Such information is useful as a starting point to guide further organic synthesis of novel protein ligands and enzyme inhibitors. This review uses literature examples to discuss the practicalities of applying this method to inhibitor discovery, in particular, the set-up of the combinatorial library, the reversible reactions that may be employed, and the choice of detection methods to screen protein ligands from a mixture of reversibly forming molecules. PMID:27438816

  15. Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

    PubMed Central

    2009-01-01

    Background The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci). Results We report that the FabF homologue located within the fatty acid biosynthetic gene cluster of C. acetobutylicium functions in synthesis of both unsaturated fatty acids and saturated fatty acids. Expression of this protein in E. coli functionally replaced both the FabB and FabF proteins of the host in vivo and replaced E. coli FabB in a defined in vitro fatty acid synthesis system. In contrast the single C. acetobutylicium FabZ homologue, although able to functionally replace E. coli FabZ in vivo and in vitro, was unable to replace FabA, the key dehydratase-isomerase of E. coli unsaturated fatty acid biosynthesis in vivo and lacked isomerase activity in vitro. Conclusion Thus, C. acetobutylicium introduces the double of unsaturated fatty acids by use of a novel and unknown enzyme. PMID:19493359

  16. Combinatorial synthesis of deuterium-enriched (S)-oxybutynin.

    PubMed

    Li, Feng; Jiang, Wenfeng; Czarnik, Anthony W; Li, Wenbao

    2016-08-01

    The concept of deuterium enrichment has gained more attention due to its advantages in the studies of clinical pharmacokinetics and metabolic profiles. In addition, it is cost and time efficient to develop deuterium-enriched drugs. Herein we built a combinatorial library of deuterated (S)-oxybutynins which all 8 D-compounds were characterized by MS, [Formula: see text] NMR and [Formula: see text]C NMR. PMID:26852022

  17. How fabulous is Fab 5 cosmology?

    SciTech Connect

    Linder, Eric V.

    2013-12-01

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

  18. Development of Combinatorial Methods for Alloy Design and Optimization

    SciTech Connect

    Pharr, George M.; George, Easo P.; Santella, Michael L

    2005-07-01

    The primary goal of this research was to develop a comprehensive methodology for designing and optimizing metallic alloys by combinatorial principles. Because conventional techniques for alloy preparation are unavoidably restrictive in the range of alloy composition that can be examined, combinatorial methods promise to significantly reduce the time, energy, and expense needed for alloy design. Combinatorial methods can be developed not only to optimize existing alloys, but to explore and develop new ones as well. The scientific approach involved fabricating an alloy specimen with a continuous distribution of binary and ternary alloy compositions across its surface--an ''alloy library''--and then using spatially resolved probing techniques to characterize its structure, composition, and relevant properties. The three specific objectives of the project were: (1) to devise means by which simple test specimens with a library of alloy compositions spanning the range interest can be produced; (2) to assess how well the properties of the combinatorial specimen reproduce those of the conventionally processed alloys; and (3) to devise screening tools which can be used to rapidly assess the important properties of the alloys. As proof of principle, the methodology was applied to the Fe-Ni-Cr ternary alloy system that constitutes many commercially important materials such as stainless steels and the H-series and C-series heat and corrosion resistant casting alloys. Three different techniques were developed for making alloy libraries: (1) vapor deposition of discrete thin films on an appropriate substrate and then alloying them together by solid-state diffusion; (2) co-deposition of the alloying elements from three separate magnetron sputtering sources onto an inert substrate; and (3) localized melting of thin films with a focused electron-beam welding system. Each of the techniques was found to have its own advantages and disadvantages. A new and very powerful technique for

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

    PubMed Central

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

    2016-01-01

    The results of clinical and experimental studies suggest that endotoxin/toll-like receptor 4 (TLR4)-mediated proinflammatory and profibrotic signaling activation is critical in the development of hepatic fibrosis. However, studies examining the role of specific TLR4 inhibitor are still lacking. The present study was aimed to prepare a human anti-TLR4 Fab fragment, named hTLR4-Fab01, and to explore its immune activity. We screened the positive clone of anti-human TLR4 phagemid from a human phage-display antibody library using recombinant TLR4 protein, which was used as template cDNA for the amplification of variable regions of the heavy (VH) chain and light chain (VL), then coupled with highly conserved regions of the heavy chain domain 1 (CH1) and the light chain (CL), respectively. Thus, the prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was constructed and transformed into Escherichia coli (E. coli) BL21. The characteristic of hTLR4-Fab01 was examined by SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay. Further, our data demonstrate that hTLR4-Fab01 could specifically bind to TLR4, and its treatment obviously attenuated the proinflammatory effect, characterized by less LPS-induced TNF-α, IL-1, IL-6 and IL-8 production in human macrophages. In conclusion, we have successfully prepared the hTLR4-Fab01 with efficient activity for blocking LPS-induced proinflammatory cytokines production, suggesting that the hTLR4-Fab01 may be a potential candidate for the treatment of hepatic fibrosis. PMID:26785354

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

    PubMed

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

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

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

    PubMed Central

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

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  2. EDITORIAL: Combinatorial and High-Throughput Materials Research

    NASA Astrophysics Data System (ADS)

    Potyrailo, Radislav A.; Takeuchi, Ichiro

    2005-01-01

    The success of combinatorial and high-throughput methodologies relies greatly on the availability of various characterization tools with new and improved capabilities [1]. Indeed, how useful can a combinatorial library of 250, 400, 25 000 or 2 000 000 compounds be [2-5] if one is unable to characterize its properties of interest fairly quickly? How useful can a set of thousands of spectra or chromatograms be if one is unable to analyse them in a timely manner? For these reasons, the development of new approaches for materials characterization is one of the most active areas in combinatorial materials science. The importance of this aspect of research in the field has been discussed in numerous conferences including the Pittsburgh Conferences, the American Chemical Society Meetings, the American Physical Society Meetings, the Materials Research Society Symposia and various Gordon Research Conferences. Naturally, the development of new measurement instrumentation attracts the attention not only of practitioners of combinatorial materials science but also of those who design new software for data manipulation and mining. Experimental designs of combinatorial libraries are pursued with available and realistic synthetic and characterization capabilities in mind. It is becoming increasingly critical to link the design of new equipment for high-throughput parallel materials synthesis with integrated measurement tools in order to enhance the efficacy of the overall experimental strategy. We have received an overwhelming response to our proposal and call for papers for this Special Issue on Combinatorial Materials Science. The papers in this issue of Measurement Science and Technology are a very timely collection that captures the state of modern combinatorial materials science. They demonstrate the significant advances that are taking place in the field. In some cases, characterization tools are now being operated in the factory mode. At the same time, major challenges

  3. Combinatorial synthesis and screening of fuel cell catalysts

    NASA Astrophysics Data System (ADS)

    Jayaraman, Shrisudersan

    Polymer electrolyte membrane fuel cells (PEMFCs) are compact power sources that can operate with high efficiencies and low emission of environmentally harmful gases. One of the major barriers impeding the development of PEMFCs as a competitive energy source is the inability of existing anode catalysts to oxidize fuels other than hydrogen at sufficient levels due to catalyst deactivation by carbon monoxide (CO) and other partial oxidation products. The focus of this research is the development and application of combinatorial strategies to construct and interrogate electrooxidation (anode) catalysts pertaining to PEMFCs to discover catalysts with enhanced performance in catalyst deactivating environments. A novel method (known as the "gel-transfer" method) for synthesizing catalyst composition gradient libraries for combinatorial catalyst discovery was developed. This method involved transferring a spatial concentration gradient of precursor metal salts created within a polymer gel on to a solid conducting substrate by electrochemical reduction. Chemically sensitive surface-imaging techniques, namely, scanning electrochemical microscopy (SECM) and optical screening with a pH-dependent fluorescence probe were used to characterize the combinatorial catalyst samples. The utility of SECM as a screening tool to measure the activity of multicomponent catalyst libraries towards fuel cell electrooxidation reactions was established with simple catalyst libraries including a platinum coverage gradient and platinum-ruthenium and platinum-ruthenium-molybdenum arrays. A platinum-ruthenium surface composition gradient was constructed through the gel-transfer method and its reactivity towards hydrogen oxidation in the presence of a catalyst poison (CO) was mapped using the SECM. Ruthenium composition between 20 and 30% exhibited superior performance than the rest of the binary. The gel-transfer method was extended to construct a ternary platinum-ruthenium-rhodium catalyst library

  4. Combinatorial Synthesis and Discovery of an Antibiotic Compound. An Experiment Suitable for High School and Undergraduate Laboratories

    NASA Astrophysics Data System (ADS)

    Wolkenberg, Scott E.; Su, Andrew I.

    2001-06-01

    An exercise demonstrating solution-phase combinatorial chemistry and its application to drug discovery is described. The experiment involves the synthesis of six libraries of three hydrazones, screening the libraries for antibiotic activity, and deconvolution to determine the active individual compound. The laboratory was designed for a high school classroom, though it can easily be expanded to suit a college introductory organic laboratory course.

  5. Mesofluidic Devices for DNA-Programmed Combinatorial Chemistry

    PubMed Central

    Weisinger, Rebecca M.; Marinelli, Robert J.; Wrenn, S. Jarrett; Harbury, Pehr B.

    2012-01-01

    Hybrid combinatorial chemistry strategies that use DNA as an information-carrying medium are proving to be powerful tools for molecular discovery. In order to extend these efforts, we present a highly parallel format for DNA-programmed chemical library synthesis. The new format uses a standard microwell plate footprint and is compatible with commercially available automation technology. It can accommodate a wide variety of combinatorial synthetic schemes with up to 384 different building blocks per chemical step. We demonstrate that fluidic routing of DNA populations in the highly parallel format occurs with excellent specificity, and that chemistry on DNA arrayed into 384 well plates proceeds robustly, two requirements for the high-fidelity translation and efficient in vitro evolution of small molecules. PMID:22479318

  6. Logical Structures Underlying Combinatorial Reasoning.

    ERIC Educational Resources Information Center

    Glick, Rochelle; Martorano, Suzanne

    This study was designed to examine the processes underlying developmental changes in children's (1) use of combinatorial strategy, and (2) comprehension of conjunctive and disjunctive propositional relationships. A total of 108 children from third, sixth and eighth grades participated in this study. Each child was administered three tasks…

  7. Combinatorial Evolution of Enzymes and Synthetic Pathways Using One-Step PCR.

    PubMed

    Jin, Peng; Kang, Zhen; Zhang, Junli; Zhang, Linpei; Du, Guocheng; Chen, Jian

    2016-03-18

    DNA engineering is the fundamental motive driving the rapid development of modern biotechnology. Here, we present a versatile evolution method termed "rapidly efficient combinatorial oligonucleotides for directed evolution" (RECODE) for rapidly introducing multiple combinatorial mutations to the target DNA by combined action of a thermostable high-fidelity DNA polymerase and a thermostable DNA Ligase in one reaction system. By applying this method, we rapidly constructed a variant library of the rpoS promoters (with activity of 8-460%), generated a novel heparinase from the highly specific leech hyaluronidase (with more than 30 mutant residues) and optimized the heme biosynthetic pathway by combinatorial evolution of regulatory elements and pathway enzymes (2500 ± 120 mg L(-1) with 20-fold increase). The simple RECODE method enabled researchers the unparalleled ability to efficiently create diverse mutant libraries for rapid evolution and optimization of enzymes and synthetic pathways. PMID:26751617

  8. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  9. Deciphering the Structural Requirements of Nucleoside Bisubstrate Analogues for Inhibition of MbtA in Mycobacterium tuberculosis: A FB-QSAR Study and Combinatorial Library Generation for Identifying Potential Hits.

    PubMed

    Maganti, Lakshmi; Das, Sanjit Kumar; Mascarenhas, Nahren Manuel; Ghoshal, Nanda

    2011-10-01

    The re-emergence of tuberculosis infections, which are resistant to conventional drug therapy, has steadily risen in the last decade. Inhibitors of aryl acid adenylating enzyme known as MbtA, involved in siderophore biosynthesis in Mycobacterium tuberculosis, are being explored as potential antitubercular agents. The ability to identify fragments that interact with a biological target is a key step in fragment based drug design (FBDD). To expand the boundaries of quantitative structure activity relationship (QSAR) paradigm, we have proposed a Fragment Based QSAR methodology, referred here in as FB-QSAR, for deciphering the structural requirements of a series of nucleoside bisubstrate analogs for inhibition of MbtA, a key enzyme involved in siderophore biosynthetic pathway. For the development of FB-QSAR models, statistical techniques such as stepwise multiple linear regression (SMLR), genetic function approximation (GFA) and GFAspline were used. The predictive ability of the generated models was validated using different statistical metrics, and similarity-based coverage estimation was carried out to define applicability boundaries. To aid the creation of novel antituberculosis compounds, a bioisosteric database was enumerated using the combichem approach endorsed mining in a lead-like chemical space. The generated library was screened using an integrated in-silico approach and potential hits identified. PMID:27468106

  10. Combinatorial 3D Mechanical Metamaterials

    NASA Astrophysics Data System (ADS)

    Coulais, Corentin; Teomy, Eial; de Reus, Koen; Shokef, Yair; van Hecke, Martin

    2015-03-01

    We present a class of elastic structures which exhibit 3D-folding motion. Our structures consist of cubic lattices of anisotropic unit cells that can be tiled in a complex combinatorial fashion. We design and 3d-print this complex ordered mechanism, in which we combine elastic hinges and defects to tailor the mechanics of the material. Finally, we use this large design space to encode smart functionalities such as surface patterning and multistability.

  11. YCRD: Yeast Combinatorial Regulation Database

    PubMed Central

    Wu, Wei-Sheng; Hsieh, Yen-Chen; Lai, Fu-Jou

    2016-01-01

    In eukaryotes, the precise transcriptional control of gene expression is typically achieved through combinatorial regulation using cooperative transcription factors (TFs). Therefore, a database which provides regulatory associations between cooperative TFs and their target genes is helpful for biologists to study the molecular mechanisms of transcriptional regulation of gene expression. Because there is no such kind of databases in the public domain, this prompts us to construct a database, called Yeast Combinatorial Regulation Database (YCRD), which deposits 434,197 regulatory associations between 2535 cooperative TF pairs and 6243 genes. The comprehensive collection of more than 2500 cooperative TF pairs was retrieved from 17 existing algorithms in the literature. The target genes of a cooperative TF pair (e.g. TF1-TF2) are defined as the common target genes of TF1 and TF2, where a TF’s experimentally validated target genes were downloaded from YEASTRACT database. In YCRD, users can (i) search the target genes of a cooperative TF pair of interest, (ii) search the cooperative TF pairs which regulate a gene of interest and (iii) identify important cooperative TF pairs which regulate a given set of genes. We believe that YCRD will be a valuable resource for yeast biologists to study combinatorial regulation of gene expression. YCRD is available at http://cosbi.ee.ncku.edu.tw/YCRD/ or http://cosbi2.ee.ncku.edu.tw/YCRD/. PMID:27392072

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

    SciTech Connect

    Gerard, Morgan Evan

    2011-12-01

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

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

    PubMed

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

    2013-12-15

    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days. PMID:24047962

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

    PubMed

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

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. PMID:26654254

  15. Improved radioimaging and tumor localization with monoclonal F(ab')2

    SciTech Connect

    Wahl, R.L.; Parker, C.W.; Philpott, G.W.

    1983-04-01

    Monoclonal anti-tumor antibodies have great promise for radioimmunodetection and localization of tumors. Fab and F(ab')2 fragments, which lack the Fc fragment of antibody (Ab), are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact Ab. In radioimaging studies using a murine monoclonal antibody to carcinoembryonic antigen in a human colon carcinoma xenografted into hamsters, F(ab')2 fragments were shown superior to Fab fragments and intact antibody for scintiscanning. In double-label experiments with anti-CEA antibody and control monoclonal IgG, F(ab')2 fragments were found to give better and more rapid specific tumor localization than intact antibody or Fab fragments. F(ab')2 fragments offer significant promise for tumor imaging and possibly therapy.

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

    SciTech Connect

    Obmolova, Galina Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L.

    2014-07-23

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

  17. Characterization of FAB1 phosphatidylinositol kinases in Arabidopsis pollen tube growth and fertilization.

    PubMed

    Serrazina, Susana; Dias, Fernando Vaz; Malhó, Rui

    2014-08-01

    In yeast and animal cells, phosphatidylinositol-3-monophosphate 5-kinases produce phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) and have been implicated in endomembrane trafficking and pH control in the vacuole. In plants, PtdIns(3,5)P2 is synthesized by the Fab1 family, four orthologs of which exist in Arabidopsis: FAB1A and FAB1B, both from the PIKfyve/Fab1 family; FAB1C and FAB1D, both without a PIKfyve domain and of unclear role. Using a reverse genetics and cell biology approach, we investigated the function of the Arabidopsis genes encoding FAB1B and FAB1D, both highly expressed in pollen. Pollen viability, germination and tube morphology were not significantly affected in homozygous mutant plants. In vivo, mutant pollen fertilized ovules leading to normal seeds and siliques. The same result was obtained when mutant ovules were fertilized with wild-type pollen. Double mutant pollen for the two genes was able to fertilize and develop plants no different from the wild-type. At the cellular level, fab1b and fab1d pollen tubes were found to exhibit perturbations in membrane recycling, vacuolar acidification and decreased production of reactive oxygen species (ROS). Subcellular imaging of FAB1B-GFP revealed that the protein localized to the endomembrane compartment, whereas FAB1D-GFP localized mostly to the cytosol and sperm cells. These results were discussed considering possible complementary roles of FAB1B and FAB1D. PMID:24807078

  18. Combinatorial Strategies for the Development of Bulk Metallic Glasses

    NASA Astrophysics Data System (ADS)

    Ding, Shiyan

    The systematic identification of multi-component alloys out of the vast composition space is still a daunting task, especially in the development of bulk metallic glasses that are typically based on three or more elements. In order to address this challenge, combinatorial approaches have been proposed. However, previous attempts have not successfully coupled the synthesis of combinatorial libraries with high-throughput characterization methods. The goal of my dissertation is to develop efficient high-throughput characterization methods, optimized to identify glass formers systematically. Here, two innovative approaches have been invented. One is to measure the nucleation temperature in parallel for up-to 800 compositions. The composition with the lowest nucleation temperature has a reasonable agreement with the best-known glass forming composition. In addition, the thermoplastic formability of a metallic glass forming system is determined through blow molding a compositional library. Our results reveal that the composition with the largest thermoplastic deformation correlates well with the best-known formability composition. I have demonstrated both methods as powerful tools to develop new bulk metallic glasses.

  19. Analysis of Combinatorial Epigenomic States.

    PubMed

    Soloway, Paul D

    2016-03-18

    Hundreds of distinct chemical modifications to DNA and histone amino acids have been described. Regulation exerted by these so-called epigenetic marks is vital to normal development, stability of cell identity through mitosis, and nongenetic transmission of traits between generations through meiosis. Loss of this regulation contributes to many diseases. Evidence indicates epigenetic marks function in combinations, whereby a given modification has distinct effects on local genome control, depending on which additional modifications are locally present. This review summarizes emerging methods for assessing combinatorial epigenomic states, as well as challenges and opportunities for their refinement. PMID:26555135

  20. Multispecies TASEP and combinatorial R

    NASA Astrophysics Data System (ADS)

    Kuniba, Atsuo; Maruyama, Shouya; Okado, Masato

    2015-08-01

    We identify the algorithm for constructing steady states of the n-species totally asymmetric simple exclusion process (TASEP) on an L site periodic chain by Ferrari and Martin with a composition of combinatorial R for the quantum affine algebra {U}q({\\widehat{{sl}}}L) in crystal base theory. Based on this connection and the factorized form of the R matrix derived recently from the tetrahedron equation, we establish a new matrix product formula for the steady state of the TASEP, which is expressed in terms of corner transfer matrices of the q-oscillator valued five-vertex model at q = 0. Dedicated to the memory of Professor Ryogo Hirota.

  1. Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching.

    PubMed

    Hevener, Kirk E; Mehboob, Shahila; Su, Pin-Chih; Truong, Kent; Boci, Teuta; Deng, Jiangping; Ghassemi, Mahmood; Cook, James L; Johnson, Michael E

    2012-01-12

    Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with submicromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented. PMID:22098466

  2. DNA Assembly Techniques for Next Generation Combinatorial Biosynthesis of Natural Products

    PubMed Central

    Cobb, Ryan E.; Ning, Jonathan C.; Zhao, Huimin

    2013-01-01

    Natural product scaffolds remain important leads for pharmaceutical development. However, transforming a natural product into a drug entity often requires derivatization to enhance the compound’s therapeutic properties. A powerful method by which to perform this derivatization is combinatorial biosynthesis, the manipulation of the genes in the corresponding pathway to divert synthesis towards novel derivatives. While these manipulations have traditionally been carried out via restriction digestion/ligation-based cloning, the shortcomings of such techniques limit their throughput and thus the scope of corresponding combinatorial biosynthesis experiments. In the burgeoning field of synthetic biology, the demand for facile DNA assembly techniques has promoted the development of a host of novel DNA assembly strategies. Here we describe the advantages of these recently-developed tools for rapid, efficient synthesis of large DNA constructs. We also discuss their potential to facilitate the simultaneous assembly of complete libraries of natural product biosynthetic pathways, ushering in the next generation of combinatorial biosynthesis. PMID:24127070

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

    PubMed

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

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy. PMID:27112931

  4. FAB overlapping: a strategy for sequencing homologous proteins

    NASA Astrophysics Data System (ADS)

    Ferranti, P.; Malorni, A.; Marino, G.; Pucci, P.; di Luccia, A.; Ferrara, L.

    1991-12-01

    Extensive similarity has been shown to exist between the primary structures of closely related proteins from different species, the only differences being restricted to a few amino acid variations. A new mass spectrometric procedure, which has been called FAB-overlapping, has been developed for sequencing highly homologous proteins based on the detection of these small differences as compared with a known protein used as a reference. Several complementary peptide maps are constructed using fast atom bombardment mass spectrometry (FAB-MS) analysis of different proteolytic digests of the unknown protein and the mass values are related to those expected on the basis of the sequence of the reference protein. The mass signals exhibiting unusual mass values identify those regions where variations have taken place; fine location of the mutations can be obtained by coupling simple protein chemistry methodologies with FAB-MS. Using the FAB-overlapping procedure, it was possible to determine the sequence of [alpha]1, [alpha]3 and [beta] globins from water buffalo (Bubalus bubalis hemoglobins (phenotype AA). Two amino acid substitutions were detected in the buffalo [beta] chain (Lys16 --> His and Asn118 --> His) whereas the [alpha]1 chains were found the [alpha]1 and [alpha]3 chains were found to contain four amino acid replacements, three of which were identical (Glu23 --> Asp, Glu71 --> Gly, Phe117 --> Cys), and the insertion of an alanine residue in position 124. The only differences between [alpha]1 and [alpha]3 globins were identified in the C -terminal region; [alpha]1 contains a Phe residue at position 130 whereas [alpha]3 shows serine at position 132.

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

    SciTech Connect

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

    1989-02-01

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

  6. Statistical approach to linewidth control in a logic fab

    NASA Astrophysics Data System (ADS)

    Pitter, Michael; Doleschel, Bernhard; Eibl, Ludwig; Steinkirchner, Erwin; Grassmann, Andreas

    1999-04-01

    We designed an adaptive line width controller specially tailored to the needs of a highly diversified logic fab. Simulations of different controller types fed with historic CD data show advantages of an SPC based controller over a Run by Run controller. This result confirms the SPC assumption that as long as a process is in statistical control, changing the process parameters will only increase the variability of the output.

  7. Crystallization and preliminary X-ray analysis of the complexes between a Fab and two forms of human insulin-like growth factor II

    PubMed Central

    Newman, Janet; Cohen, Edward H.; Cosgrove, Leah; Kopacz, Kris; Dransfield, Daniel T.; Adams, Timothy E.; Peat, Thomas S.

    2009-01-01

    Elevated expression of insulin-like growth factor II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon and liver cancer. As IGF-II can deliver a mitogenic signal through both the type 1 insulin-like growth factor receptor (IGF-IR) and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is an attractive therapeutic option. One method of doing this would be to find antibodies that bind tightly and specifically to the peptide, which could be used as protein therapeutics to lower the peptide levels in vivo and/or to block the peptide from binding to the IGF-IR or IR-A. To address this, Fabs were selected from a phage-display library using a biotinylated precursor form of the growth factor known as IGF-IIE as a target. Fabs were isolated that were specific for the E-­domain C-terminal extension and for mature IGF-II. Four Fabs selected from the library were produced, complexed with IGF-II and set up in crystallization trials. One of the Fab–IGF-II complexes (M64-F02–IGF-II) crystallized readily, yielding crystals that diffracted to 2.2 Å resolution and belonged to space group P212121, with unit-cell parameters a = 50.7, b = 106.9, c = 110.7 Å. There was one molecule of the complete complex in the asymmetric unit. The same Fab was also crystallized with a longer form of the growth factor, IGF-IIE. This complex crystallized in space group P212121, with unit-cell parameters a = 50.7, b = 107, c = 111.5 Å, and also diffracted X-rays to 2.2 Å resolution. PMID:19724140

  8. Strategy optimization for mask rule check in wafer fab

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  9. Losing Libraries, Saving Libraries

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2010-01-01

    This summer, as public libraries continued to get budget hit after budget hit across the country, several readers asked for a comprehensive picture of the ravages of the recession on library service. In partnership with 2010 Movers & Shakers Laura Solomon and Mandy Knapp, Ohio librarians who bought the Losing Libraries domain name, "LJ" launched…

  10. From combinatorial chemistry to cancer targeting nanotherapeutics

    NASA Astrophysics Data System (ADS)

    Xiao, Kai; Luo, Juntao; Li, Yuanpei; Xiao, Wenwu; Lee, Joyce S.; Gonik, Abby M.; Lam, Kit S.

    2010-04-01

    We have developed a number of amphiphilic polymers, comprised of a cluster of cholic acids (4 to 10) linked by a series of lysines and attached to one end of a linear polyethylene glycol chain (PEG, 2000-5000 Dalton). Under aqueous condition, such telodendrimers can self-assemble together with hydrophobic payloads to form highly stable micelles (15-150 nm diameter, size tunable). We used near infrared fluorescence (NIRF) optical imaging technique to study the in vivo passive accumulation of our nanocarriers (via EPR effect) in different types and stages of tumors. The results demonstrated that the micelle could preferentially accumulate in many types of tumor xenografts or synografts implanted in mice. Nanoparticle uptake in solid tumors was found to be much higher than that of lymphoma, which could be attributed to the relatively low microvascular density in the latter. We have also demonstrated that micelles smaller than 64 nm preferentially targeted xenografts with high efficiency and with low liver and lung uptake, whereas those micelles at 154 nm targeted the tumor poorly but with very high liver and lung uptake. Telodendrimers decorated with oligolysine or oligoaspartic acid resulted in high uptake of the nanoparticles into the liver. When decorated with cancer targeting ligands identified from the one-bead-one-compound (OBOC) combinatorial library methods, the drug-loaded nanoparticles were rapidly taken up by the target cultured tumor cells causing cell death. In vivo near infra-red optical imaging studies with hydrophobic fluorescent dye demonstrated that xenograft uptake of the micelles was greatly enhanced by the cancer targeting peptide.

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

    PubMed Central

    Obmolova, Galina; Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L.

    2014-01-01

    The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization. PMID:25084393

  12. Combinatorial Optimization of Heterogeneous Catalysts Used in the Growth of Carbon Nanotubes

    NASA Technical Reports Server (NTRS)

    Cassell, Alan M.; Verma, Sunita; Delzeit, Lance; Meyyappan, M.; Han, Jie

    2000-01-01

    Libraries of liquid-phase catalyst precursor solutions were printed onto iridium-coated silicon substrates and evaluated for their effectiveness in catalyzing the growth of multi-walled carbon nanotubes (MWNTs) by chemical vapor deposition (CVD). The catalyst precursor solutions were composed of inorganic salts and a removable tri-block copolymer (EO)20(PO)70(EO)20 (EO = ethylene oxide, PO = propylene oxide) structure-directing agent (SDA), dissolved in ethanol/methanol mixtures. Sample libraries were quickly assayed using scanning electron microscopy after CVD growth to identify active catalysts and CVD conditions. Composition libraries and focus libraries were then constructed around the active spots identified in the discovery libraries to understand how catalyst precursor composition affects the yield, density, and quality of the nanotubes. Successful implementation of combinatorial optimization methods in the development of highly active, carbon nanotube catalysts is demonstrated, as well as the identification of catalyst formulations that lead to varying densities and shapes of aligned nanotube towers.

  13. Accelerated luminophore discovery through combinatorial synthesis.

    PubMed

    Lowry, Michael S; Hudson, William R; Pascal, Robert A; Bernhard, Stefan

    2004-11-01

    A method for accelerating the discovery of ionic luminophores using combinatorial techniques is reported. The photophysical properties of the resulting transition-metal-based chromophores were compared against a series of analogous, traditionally prepared species. The strong overlap between these two sets confirms the identity of the parallel synthesis products and supports the truthfulness of the combinatorial results. Further support for the combinatorial method comes from the adherence of these complexes to the energy gap law. The relationship between the structure of a complex and its photophysical properties was also considered, and static DFT calculations were used to assess whether it is feasible to predict the luminescent behavior of novel materials. PMID:15506778

  14. Dynamic combinatorial synthesis of a catenane based on donor–acceptor interactions in water

    PubMed Central

    Au-Yeung, Ho Yu; Pantoş, G. Dan; Sanders, Jeremy K. M.

    2009-01-01

    A new type of neutral donor–acceptor [2]-catenane, containing both complementary units in the same ring was synthesized from a dynamic combinatorial library in water. The yield of the water soluble [2]-catenane is enhanced by increasing either building-block concentrations or ionic strength, or by the addition of an electron-rich template. NMR spectroscopy demonstrates that the template is intercalated between the 2 electron-deficient naphthalenediimide units of the catenane. PMID:19171892

  15. High Divergence of the Precursor Peptides in Combinatorial Lanthipeptide Biosynthesis

    PubMed Central

    2015-01-01

    Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and post-translationally modified peptides (RiPPs). These compounds are widely distributed in taxonomically distant species, and their biosynthetic systems and biological activities are diverse. A unique example of lanthipeptide biosynthesis is the prochlorosin synthetase ProcM from the marine cyanobacterium Prochlorococcus MIT9313, which transforms up to 29 different precursor peptides (ProcAs) into a library of lanthipeptides called prochlorosins (Pcns) with highly diverse sequences and ring topologies. Here, we show that many ProcM-like enzymes from a variety of bacteria have the capacity to carry out post-translational modifications on highly diverse precursor peptides, providing new examples of natural combinatorial biosynthesis. We also demonstrate that the leader peptides come from different evolutionary origins, suggesting that the combinatorial biosynthesis is tied to the enzyme and not a specific type of leader peptide. For some precursor peptides encoded in the genomes, the leader peptides apparently have been truncated at the N-termini, and we show that these N-terminally truncated peptides are still substrates of the enzymes. Consistent with this hypothesis, we demonstrate that about two-thirds of the ProcA N-terminal sequence is not essential for ProcM activity. Our results also highlight the potential of exploring this class of natural products by genome mining and bioengineering. PMID:25244001

  16. Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9.

    PubMed

    EauClaire, Steve F; Zhang, Jianzhong; Rivera, Corban Gregory; Huang, Lixuan L

    2016-07-01

    The yeast Saccharomyces cerevisiae is an important industrial platform for the production of grain and cellulosic ethanol, isobutanol, butanediol, isoprenoids, and other chemicals. The construction of a successful production strain usually involves multiple gene knockouts and chromosomal integration of expression cassettes to redirect the metabolic fluxes for the conversion of sugars and other feed stocks into the desired product. RNA-guided Cas9 based genome editing has been demonstrated in many prokaryotic and eukaryotic hosts including S. cerevisiae, in which it has been additionally exploited as a tool for metabolic engineering. To extend the utilization of RNA-guided Cas9 as a metabolic pathway building tool, we demonstrated the direct assembly and chromosomal integration of up to 17 overlapping DNA fragments encoding the beta-carotene biosynthetic pathway. Furthermore, we generated a combinatorial strain library for the beta-carotene biosynthetic pathway, directly integrated into the yeast genome to create a diverse library of strains. This enabled the screening of combinatorial libraries in stable chromosomally integrated strains for rapid improvements of product titers. This combinatorial approach for pathway assembly will significantly accelerate the current speed of metabolic engineering for S. cerevisiae as an industrial platform, and increase the number of strains that can be simultaneously evaluated for enzyme screening, expression optimization and protein engineering to achieve the titer, rate and yield necessary for the commercialization of new industrial fermentation products. PMID:27138038

  17. Early membrane damage during coronary reperfusion in dogs. Detection by radiolabeled anticardiac myosin (Fab')2.

    PubMed Central

    Frame, L H; Lopez, J A; Khaw, B A; Fallon, J T; Haber, E; Powell, W J

    1983-01-01

    There is currently great interest in acute coronary reperfusion as a therapeutic modality for severe myocardial ischemia. While some studies have demonstrated a reduction in the overall extent of necrosis by early reperfusion, other studies have identified potentially deleterious effects produced by reflow. Because membrane disruption may be an important mechanism of irreversible cell injury, we measured changes in cell membrane integrity early during reperfusion using radiolabeled anticardiac myosin (Fab')2 antibody fragments in dogs. Our method involved brief periods of exposure to the (Fab')2 so that the levels of (Fab')2 binding indicated the degree of membrane disruption at discrete times during the progression of cell injury. In the first protocol (Fab')2 fragments labeled with either 125I and 131I were injected into the left circumflex coronary artery at the onset of reflow and at 45 min of reflow after a 1-h circumflex artery occlusion. Coronary sinus flow was diverted for 5 min following each injection to prevent recirculation. The (Fab')2 binding ratio (ischemic/control) increased during the first 45 min of reflow in each of eight experiments (mean increase 170%, P less than 0.01). No significant increase in (Fab')2 binding was observed in five additional experiments in which nonspecific (Fab')2 was injected. This indicates that the increase in binding seen with antimyosin-specific (Fab')2 was due to changes in specific binding rather than to alterations in (Fab')2 delivery produced by changes in blood flow distribution. The increase in membrane damage during reflow was confirmed by a second protocol in which each animal received only a single left atrial injection of (Fab')2 followed by rapid excision of the heart. The (Fab')2 binding ratio was 1.7 +/- 0.3 (SEM) in the group that received (Fab')2 at the onset of reflow and 3.7 +/- 0.6 (SEM) (P less than 0.05) in the group that received (Fab')2 after 45 min of reflow. In a third set of experiments in

  18. Extending green technology innovations to enable greener fabs

    NASA Astrophysics Data System (ADS)

    Takahisa, Kenji; Yoo, Young Sun; Fukuda, Hitomi; Minegishi, Yuji; Enami, Tatsuo

    2015-03-01

    Semiconductor manufacturing industry has growing concerns over future environmental impacts as fabs expand and new generations of equipment become more powerful. Especially rare gases supply and price are one of prime concerns for operation of high volume manufacturing (HVM) fabs. Over the past year it has come to our attention that Helium and Neon gas supplies could be unstable and become a threat to HVM fabs. To address these concerns, Gigaphoton has implemented various green technologies under its EcoPhoton program. One of the initiatives is GigaTwin deep ultraviolet (DUV) lithography laser design which enables highly efficient and stable operation. Under this design laser systems run with 50% less electric energy and gas consumption compared to conventional laser designs. In 2014 we have developed two technologies to further reduce electric energy and gas efficiency. The electric energy reduction technology is called eGRYCOS (enhanced Gigaphoton Recycled Chamber Operation System), and it reduces electric energy by 15% without compromising any of laser performances. eGRYCOS system has a sophisticated gas flow design so that we can reduce cross-flow-fan rotation speed. The gas reduction technology is called eTGM (enhanced Total gas Manager) and it improves gas management system optimizing the gas injection and exhaust amount based on laser performances, resulting in 50% gas savings. The next steps in our roadmap technologies are indicated and we call for potential partners to work with us based on OPEN INNOVATION concept to successfully develop faster and better solutions in all possible areas where green innovation may exist.

  19. Applications of high throughput (combinatorial) methodologies to electronic, magnetic, optical, and energy-related materials

    NASA Astrophysics Data System (ADS)

    Green, Martin L.; Takeuchi, Ichiro; Hattrick-Simpers, Jason R.

    2013-06-01

    High throughput (combinatorial) materials science methodology is a relatively new research paradigm that offers the promise of rapid and efficient materials screening, optimization, and discovery. The paradigm started in the pharmaceutical industry but was rapidly adopted to accelerate materials research in a wide variety of areas. High throughput experiments are characterized by synthesis of a "library" sample that contains the materials variation of interest (typically composition), and rapid and localized measurement schemes that result in massive data sets. Because the data are collected at the same time on the same "library" sample, they can be highly uniform with respect to fixed processing parameters. This article critically reviews the literature pertaining to applications of combinatorial materials science for electronic, magnetic, optical, and energy-related materials. It is expected that high throughput methodologies will facilitate commercialization of novel materials for these critically important applications. Despite the overwhelming evidence presented in this paper that high throughput studies can effectively inform commercial practice, in our perception, it remains an underutilized research and development tool. Part of this perception may be due to the inaccessibility of proprietary industrial research and development practices, but clearly the initial cost and availability of high throughput laboratory equipment plays a role. Combinatorial materials science has traditionally been focused on materials discovery, screening, and optimization to combat the extremely high cost and long development times for new materials and their introduction into commerce. Going forward, combinatorial materials science will also be driven by other needs such as materials substitution and experimental verification of materials properties predicted by modeling and simulation, which have recently received much attention with the advent of the Materials Genome

  20. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

    PubMed Central

    Guo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, Lihui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, Junbiao

    2015-01-01

    It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts. PMID:25956650

  1. High-pressure combinatorial process integrating hot isostatic pressing.

    PubMed

    Fujimoto, Kenjiro; Morita, Hiroki; Goshima, Yuji; Ito, Shigeru

    2013-12-01

    A high-pressure combinatorial process integrating hot isostatic pressing (HIP) was developed by providing a reaction vessel with a high-pressure tightness based on a commercial flange. The reaction vessel can be used up to 200 MPa and 500 °C under HIP processing condition. Preparation of spinel-type MgAl2O4 from Mg(OH)2, Al(OH)3 and AlOOH was performed using the reaction vessel under 200 MPa and 500 °C as demonstration. The entire powder library was characterized using powder X-ray diffraction patterns, and the single phase of spinel-type MgAl2O4 was obtained from Mg(OH)2+Al(OH)3. These assessments corresponded with previously published data. PMID:24168067

  2. Combinatorial discovery of two-photon photoremovable protecting groups.

    PubMed

    Pirrung, Michael C; Pieper, Wolfgang H; Kaliappan, Krishna P; Dhananjeyan, Mugunthu R

    2003-10-28

    A design principle for a two-photon photochemically removable protecting group based on sequential one-photon processes has been established. The expected performance of such groups in spatially directed photoactivation/photodeprotection has been shown by a kinetic analysis. One particular molecular class fitting into this design, the nitrobenzyl ethers of o-hydroxycinnamates, has been presented. An initial demonstration of two-photon deprotection of one such group prompted further optimization with respect to photochemical deprotection rate. This was accomplished by the preparation and screening of a 135-member indexed combinatorial library. Optimum performance for lambda >350 nm deprotection in organic solvent was found with 4,5-dialkoxy and -cyano substitution in the nitrobenzyl group and 4-methoxy substitution in the cinnamate. PMID:14557545

  3. Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

    PubMed Central

    Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

    2013-01-01

    For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

  4. Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.

    PubMed

    Martínez-Ceron, María C; Marani, Mariela M; Taulés, Marta; Etcheverrigaray, Marina; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

    2011-05-01

    Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. PMID:21495625

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

    ERIC Educational Resources Information Center

    Pagano, John

    2005-01-01

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

  6. Library Cooperation.

    ERIC Educational Resources Information Center

    Lund, Patricia; And Others

    1993-01-01

    Includes nine articles that discuss cooperative library networking in Illinois. Highlights include library systems as cooperative agencies; PALI (Private Academic Libraries of Illinois); rural school and public library development; systemwide users; regional medical libraries; virtual libraries and the Coalition for Networked Information; a…

  7. Conformational diversity of bacterial FabH: implications for molecular recognition specificity.

    PubMed

    Mittal, Anuradha; Johnson, Michael E

    2015-02-01

    The molecular basis of variable substrate and inhibitor specificity of the highly conserved bacterial fatty acid synthase enzyme, FabH, across different bacterial species remains poorly understood. In the current work, we explored the conformational diversity of FabH enzymes to understand the determinants of diverse interaction specificity across Gram-positive and Gram-negative bacteria. Atomistic molecular dynamics simulations reveal that FabH from E. coli and E. faecalis exhibit distinct native state conformational ensembles and dynamic behaviors. Despite strikingly similar substrate binding pockets, hot spot assessment using computational solvent mapping identified quite different favorable binding interactions between the two homologs. Our data suggest that FabH utilizes protein dynamics and seemingly minor sequence and structural differences to modulate its molecular recognition and substrate specificity across bacterial species. These insights will potentially facilitate the rational design and development of antibacterial inhibitors against FabH enzymes. PMID:25437098

  8. Nebulized anti-IL-13 monoclonal antibody Fab' fragment reduces allergen-induced asthma.

    PubMed

    Hacha, Jonathan; Tomlinson, Kate; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noel, Agnès; Palframan, Roger; Gueders, Maud; Cataldo, Didier D

    2012-11-01

    IL-13 is a prototypic T helper type 2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis, and eosinophil infiltration. We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness, and remodeling in an experimental model of allergic asthma. Anti-IL-13 Fab' was administered to mice as a liquid aerosol generated by inExpose inhalation system in a tower allowing a nose-only exposure. BALB/c mice were treated by PBS, anti-IL-13 Fab', or A33 Fab' fragment and subjected to ovalbumin exposure for 1 and 5 weeks (short-term and long-term protocols). Our data demonstrate a significant antiasthma effect after nebulization of anti-IL-13 Fab' in a model of asthma driven by allergen exposure as compared with saline and nonimmune Fab fragments. In short- and long-term protocols, administration of the anti-IL-13 Fab' by inhalation significantly decreased bronchial responsiveness to methacholine, bronchoalveolar lavage fluid eosinophilia, inflammatory cell infiltration in lung tissue, and many features of airway remodeling. Levels of proinflammatory mediators and matrix metalloprotease were significantly lower in lung parenchyma of mice treated with anti-IL-13 Fab'. These data demonstrate that an inhaled anti-IL-13 Fab' significantly reduces airway inflammation, hyperresponsiveness, and remodeling. Specific neutralization of IL-13 in the lungs using an inhaled anti-IL-13 Fab' could represent a novel and effective therapy for the treatment of asthma. PMID:22904197

  9. A 3-D microfluidic combinatorial cell array.

    PubMed

    Liu, Mike C; Tai, Yu-Chong

    2011-02-01

    We present the development of a three-dimensional (3-D) combinatorial cell culture array device featured with integrated three-input, eight-output combinatorial mixer and cell culture chambers. The device is designed for cell-based screening of multiple compounds simultaneously on a microfluidic platform. The final assembled device is composed of a porous membrane integrated in between a Parylene 3-D microfluidic chip and a PDMS microfluidic chip. The membrane turned the cell culture chambers into two-level configuration to facilitate cell loading and to maintain cells in a diffusion dominated space during device operation. Experimentally, we first characterized the combined compound concentration profile at each chamber using a fluorescence method. We then successfully demonstrated the functionality of the quantitative cell-based assay by culturing B35 rat neuronal cells on this device and screening the ability of three compounds (1,5-dihydroxyisoquinoline, deferoxamine, and 3-aminobenzoic acid) to attenuate cell death caused by cytotoxic hydrogen peroxide. In another experiment, we assayed for the combinatorial effects of three chemotherapeutic compound exposures (vinorelbine, paclitaxel, and γ-linolenic acid) on human breast cancer cells, MDA-MB-231. The same technology will enable the construction of inexpensive lab-on-a-chip devices with high-input combinatorial mixer for performing high-throughput cell-based assay and highly parallel and combinatorial chemical or biochemical reactions. PMID:21063783

  10. Combinatorial stresses kill pathogenic Candida species.

    PubMed

    Kaloriti, Despoina; Tillmann, Anna; Cook, Emily; Jacobsen, Mette; You, Tao; Lenardon, Megan; Ames, Lauren; Barahona, Mauricio; Chandrasekaran, Komelapriya; Coghill, George; Goodman, Daniel; Gow, Neil A R; Grebogi, Celso; Ho, Hsueh-Lui; Ingram, Piers; McDonagh, Andrew; de Moura, Alessandro P S; Pang, Wei; Puttnam, Melanie; Radmaneshfar, Elahe; Romano, Maria Carmen; Silk, Daniel; Stark, Jaroslav; Stumpf, Michael; Thiel, Marco; Thorne, Thomas; Usher, Jane; Yin, Zhikang; Haynes, Ken; Brown, Alistair J P

    2012-10-01

    Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g., dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts. PMID:22463109

  11. Bulk Combinatorial Synthesis and High Throughput Characterization for Rapid Assessment of Magnetic Materials: Application of Laser Engineered Net Shaping (LENS™)

    NASA Astrophysics Data System (ADS)

    Geng, J.; Nlebedim, I. C.; Besser, M. F.; Simsek, E.; Ott, R. T.

    2016-07-01

    A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS™; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS™ system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. The Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

  12. Bulk Combinatorial Synthesis and High Throughput Characterization for Rapid Assessment of Magnetic Materials: Application of Laser Engineered Net Shaping (LENS™)

    NASA Astrophysics Data System (ADS)

    Geng, J.; Nlebedim, I. C.; Besser, M. F.; Simsek, E.; Ott, R. T.

    2016-04-01

    A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS™; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS™ system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. The Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

  13. Synthesis and biological evaluation of a beauveriolide analogue library.

    PubMed

    Nagai, Kenichiro; Doi, Takayuki; Sekiguchi, Takafumi; Namatame, Ichiji; Sunazuka, Toshiaki; Tomoda, Hiroshi; Omura, Satoshi; Takahashi, Takashi

    2006-01-01

    Synthesis of beauveriolide III (1b), which is an inhibitor of lipid droplet accumulation in macrophages, was achieved by solid-phase assembly of linear depsipeptide using a 2-chlorotrityl linker followed by solution-phase cyclization. On the basis of this strategy, a combinatorial library of beauveriolide analogues was carried out by radio frequency-encoded combinatorial chemistry. After automated purification using preparative reversed-phase HPLC, the library was tested for inhibitory activity of CE synthesis in macrophages to determine structure-activity relationships of beauveriolides. Among them, we found that diphenyl derivative 7{9,1} is 10 times more potent than 1b. PMID:16398560

  14. Exploration of polymethacrylate structure-property correlations: Advances towards combinatorial and high-throughput methods for biomaterials discovery

    PubMed Central

    Holmes, Paul F.; Bohrer, Mike; Kohn, Joachim

    2008-01-01

    In this review, we discuss the synthesis, characterization, physical properties, and applications of polymethacrylates and describe physical and biological structure-property correlations relevant to many high performance applications. We also track the advancement of material-property space from the ‘traditional’ mode of materials design to the emerging, state-of-the-art combinatorial and in silico methods. Particularly, this article places emphasis on recent advances in the automated combinatorial synthesis and development of high-throughput characterization methods. As a future perspective, we believe that the realization of combinatorial, high-throughput, and computational methods will allow for the rapid exploration of a vast polymethacrylate library property space. PMID:19649142

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

    PubMed

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

    2014-01-15

    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate. PMID:24246428

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

    PubMed

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

    1986-01-01

    Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils. PMID:2433859

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

    SciTech Connect

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

    1986-03-01

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

  18. Chromatographic purification of equine immunoglobulin G F(ab)2 from plasma.

    PubMed

    Fernandes, Aldon; Kaundinya, John Oswald; Daftary, Gautam; Saxena, Lalit; Banerjee, Subhasis; Pattnaik, Priyabrata

    2008-12-01

    The antibody fragments generated from hyperimmune equine IgG is widely used as anti-snake venom, anti-scorpion venom, anti-diphtheria, anti-tetanus, anti-gangrene and anti-rabies agents. Antibody fragments, F(ab)(2), because of their specificity and absence of undesired reactivity are preferred over complete IgG. This paper discusses a novel purification technique for chromatographic purification of anti-rabies immunoglobulin G (IgG) fragment F(ab)(2) from horse serum. F(ab)(2) was purified by two successive chromatography steps using Cellufine A-200 and ProSep-vA Ultra media. The purified F(ab)(2) was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified F(ab)(2) was reactive to rabies antigen in immuno-electrophoresis and diffusion tests. The purified F(ab)(2) was biologically functional and was found to show a potency of 1500 IU ml(-1). Comparative analysis of the purity with commercially available F(ab)(2) by HPLC analysis and SDS-PAGE indicated that the present product is better in purity. To our knowledge, this is the first report providing evidence on purification of equine antibody fragment using controlled pore glass based protein A chromatography media. PMID:19008160

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

    PubMed

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

    2016-04-01

    A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. PMID:26902097

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

    PubMed

    Stephenson, Makeda K; Dow, Douglas E

    2014-01-01

    Skill development in science, technology, engineering and math (STEM) education present one of the most formidable challenges of modern society. The Community FabLab platform presents a viable solution. Each FabLab contains a suite of modern computer numerical control (CNC) equipment, electronics and computing hardware and design, programming, computer aided design (CAD) and computer aided machining (CAM) software. FabLabs are community and educational resources and open to the public. Development of STEM based workforce skills such as digital fabrication and advanced manufacturing can be enhanced using this platform. Particularly notable is the potential of the FabLab platform in STEM education. The active learning environment engages and supports a diversity of learners, while the iterative learning that is supported by the FabLab rapid prototyping platform facilitates depth of understanding, creativity, innovation and mastery. The product and project based learning that occurs in FabLabs develops in the student a personal sense of accomplishment, self-awareness, command of the material and technology. This helps build the interest and confidence necessary to excel in STEM and throughout life. Finally the introduction and use of relevant technologies at every stage of the education process ensures technical familiarity and a broad knowledge base needed for work in STEM based fields. Biomedical engineering education strives to cultivate broad technical adeptness, creativity, interdisciplinary thought, and an ability to form deep conceptual understanding of complex systems. The FabLab platform is well designed to enhance biomedical engineering education. PMID:25570331

  1. Combinatorial pathway engineering for optimized production of the anti-malarial FR900098.

    PubMed

    Freestone, Todd S; Zhao, Huimin

    2016-02-01

    As resistance to current anti-malarial therapeutics spreads, new compounds to treat malaria are increasingly needed. One promising compound is FR900098, a naturally occurring phosphonate. Due to limitations in both chemical synthesis and biosynthetic methods for FR900098 production, this potential therapeutic has yet to see widespread implementation. Here we applied a combinatorial pathway engineering strategy to improve the production of FR900098 in Escherichia coli by modulating each of the pathway's nine genes with four promoters of different strengths. Due to the large size of the library and the low screening throughput, it was necessary to develop a novel screening strategy that significantly reduced the sample size needed to find an optimal strain. This was done by using biased libraries that localize searching around top hits and home in on high-producing strains. By incorporating this strategy, a significantly improved strain was found after screening less than 3% of the entire library. When coupled with culturing optimization, a strain was found to produce 96 mg/L, a 16-fold improvement over the original strain. We believe the enriched library method developed here can be used on other large pathways that may be difficult to engineer by combinatorial methods due to low screening throughput. PMID:26245694

  2. A Review on Platensimycin: A Selective FabF Inhibitor

    PubMed Central

    Sakha Ghosh, Partha; Manna, Kuntal

    2016-01-01

    Emerging resistance to existing antibiotics is an inevitable matter of concern in the treatment of bacterial infection. Naturally occurring unique class of natural antibiotic, platensimycin, a secondary metabolite from Streptomyces platensis, is an excellent breakthrough in recent antibiotic research with unique structural pattern and significant antibacterial activity. β-Ketoacyl-(acyl-carrier-protein (ACP)) synthase (FabF) whose Gram-positive bacteria need to biosynthesize cell membranes is the target of inhibition of platensimycin. So, isolation, retrosynthetic analysis, synthesis of platensimycin, and analogues of platensimycin synthesized till today are the objectives of this review which may be helpful to further investigate and to reveal untouched area on this molecule and to obtain a potential antibacterial lead with enhanced significant antibacterial activity. PMID:26942008

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

    PubMed

    Zheng, Chang Ji; Sohn, Mi-Jin; Lee, Sangku; Kim, Won-Gon

    2013-01-01

    Bacterial enoyl-acyl carrier protein reductase (FabI) is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria. PMID:24312171

  4. Meleagrin, a New FabI Inhibitor from Penicillium chryosogenum with at Least One Additional Mode of Action

    PubMed Central

    Zheng, Chang Ji; Sohn, Mi-Jin; Lee, Sangku; Kim, Won-Gon

    2013-01-01

    Bacterial enoyl-acyl carrier protein reductase (FabI) is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria. PMID:24312171

  5. Automated reticle inspection data analysis for wafer fabs

    NASA Astrophysics Data System (ADS)

    Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

    2009-04-01

    To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity Defect(R) data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

  6. Automated reticle inspection data analysis for wafer fabs

    NASA Astrophysics Data System (ADS)

    Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

    2008-10-01

    To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity DefecTM data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

  7. Automated reticle inspection data analysis for wafer fabs

    NASA Astrophysics Data System (ADS)

    Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

    2009-03-01

    To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity DefectTM data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

  8. Hypergraph-Based Combinatorial Optimization of Matrix-Vector Multiplication

    ERIC Educational Resources Information Center

    Wolf, Michael Maclean

    2009-01-01

    Combinatorial scientific computing plays an important enabling role in computational science, particularly in high performance scientific computing. In this thesis, we will describe our work on optimizing matrix-vector multiplication using combinatorial techniques. Our research has focused on two different problems in combinatorial scientific…

  9. A New Approach for Proving or Generating Combinatorial Identities

    ERIC Educational Resources Information Center

    Gonzalez, Luis

    2010-01-01

    A new method for proving, in an immediate way, many combinatorial identities is presented. The method is based on a simple recursive combinatorial formula involving n + 1 arbitrary real parameters. Moreover, this formula enables one not only to prove, but also generate many different combinatorial identities (not being required to know them "a…

  10. Combinatorial Development of Fe-Co-Nb Thin Film Magnetic Nanocomposites.

    PubMed

    Alexandrakis, Vasileios; Wallisch, Wolfgang; Hamann, Sven; Varvaro, Gaspare; Fidler, Josef; Ludwig, Alfred

    2015-11-01

    A Fe-Co-Nb thin film materials library was deposited by combinatorial magnetron sputtering and investigated by high-throughput methods to identify new noncubic ferromagnetic phases, indicating that combinatorial experimentation is an efficient method to discover new ferromagnetic phases adequate for permanent magnet applications. Structural analysis indicated the formation of a new magnetic ternary compound (Fe,Co)3Nb with a hexagonal crystal structure (C36) embedded in an FeCo-based matrix. This nanocomposite exhibits characteristics of a two-phase ferromagnetic system, the so-called hard-soft nanocomposites, indicating that the new phase (Fe,Co)3Nb is ferromagnetic. Magnetic hysteresis loops at various angles revealed that the magnetization reversal process is governed by a domain wall pinning mechanism. PMID:26401900