Sort Of Library Research Articles

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

The Walking Library: Mobilizing books, places, readers and reading

The Walking Library, inaugurated in 2012, has functioned as a mobile laboratory and art project for the ongoing exploration of the relationships between environments, books, reading and writing. In this essay, our focus turns to The Walking Library’s function as a library, asking: ‘What sort of library is a walking library? What does a walking library do—for its books and its borrowers and the places through which it moves? And what can it reveal or teach us about libraries, books, reading and environment?’ In a context in which data has become ‘mobile’, we explore the mobility of physical books through the Walking Library’s social and architextural designs and structures. The book on the move is recognised as the material of social bonding. The Walking Library depends upon and promotes the mobility of books through social networks by gifting, lending, borrowing and sharing; it is the social capacity—the social capital—of The Walking Library, and of walking and reading together, which concerns us most here. The Walking Library has offered temporary spaces for sociality, for shared contemplation, poetic spatiality and kinaesthetic comprehension. In doing so, it has generated a heightened sense of books’ sociability, spatiality and mobility through a stronger understanding of the inter-dependencies of reading, walking, time and place.

Optimizing sorting algorithms by using sorting networks

Abstract In this paper, we show how the theory of sorting networks can be applied to synthesize optimized general-purpose sorting libraries. Standard sorting libraries are often based on combinations of the classic Quicksort algorithm, with insertion sort applied as base case for small, fixed, numbers of inputs. Unrolling the code for the base case by ignoring loop conditions eliminates branching, resulting in code equivalent to a sorting network. By replacing it with faster sorting networks, we can improve the performance of these algorithms. We show that by considering the number of comparisons and swaps alone we are not able to predict any real advantage of this approach. However, significant speed-ups are obtained when taking advantage of instruction level parallelism and non-branching conditional assignment instructions, both of which are common in modern CPU architectures. Furthermore, a close control of how often registers have to be spilled to memory gives us a complete explanation of the performance of different sorting networks, allowing us to choose an optimal one for each particular architecture. Our experimental results show that using code synthesized from these efficient sorting networks as the base case for Quicksort libraries results in significant real-world speed-ups.

Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor.

Protein ligand charge can impact physiological delivery with charge reduction often benefiting performance. Yet neutralizing mutations can be detrimental to protein function. Herein, three approaches are evaluated to introduce charged-to-neutral mutations of three cations and three anions within an affibody engineered to bind epidermal growth factor receptor. These approaches-combinatorial library sorting or consensus design, based on natural homologs or library-sorted mutants-are used to identify mutations with favorable affinity, stability, and recombinant yield. Consensus design, based on 942 affibody homologs, yielded a mutant of modest function (Kd = 11 ±4 nM, Tm = 62°C, and yield = 4.0 ± 0.8 mg/L as compared to 5.3 ± 1.7 nM, 71°C, and 3.5 ± 0.3 mg/L for the parental affibody). Extension of consensus design to 10 additional mutants exhibited varied performance including a substantially improved mutant (Kd = 6.9 ± 1.4 nM, Tm = 71°C, and 12.7 ± 0.9 mg/L yield). Sorting a homolog-based combinatorial library of 7 × 10(5) mutants generated a distribution of mutants with lower stability and yield, but did identify one strongly binding variant (Kd = 1.2 ± 0.3 nM, Tm = 69°C, and 6.0 ± 0.4 mg/L yield). Synthetic consensus design, based on the amino acid distribution in functional library mutants, yielded higher affinities (P = 0.05) with comparable stabilities and yields. The best of four analyzed clones had Kd = 1.7 ± 0.5 nM, Tm = 68°C, and 7.0 ± 0.5 mg/L yield. While all three approaches were effective in creating targeted affibodies with six charged-to-neutral mutations, synthetic consensus design proved to be the most robust. Synthetic consensus design provides a valuable tool for ligand engineering, particularly in the context of charge manipulation. Biotechnol. Bioeng. 2016;113: 1628-1638. © 2016 Wiley Periodicals, Inc.

Screening and characterization of anti-SEB peptides using a bacterial display library and microfluidic magnetic sorting.

Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15-mer random library on the outer surface of Escherichia coli (E.coli), high-affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On-cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off-cell, using peptide-ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB-binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The Kd measured by peptide-ELISA off-cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.

The Medical Library Is History

Medical libraries are dying. Or at least some specific sorts of medical libraries—independent institutional libraries, owned by historic organizations, in historic buildings, with large historic collections—are under serious threat of themselves becoming part of the past. To mitigate this threat, there is a need to rethink the nature of the “historic” medical library. This involves reconsidering the library’s relationship to medicine and the history of medicine as disciplines, defining what is important about the nature of the library as a physical space and of its collections as material things, and reevaluating its audiences. Digitization has a role to . . .

Highly efficient selection of epitope specific antibody through competitive yeast display library sorting

Combinatory antibody library display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a yeast display antibody library using dengue virus envelope protein domain III as a model target. A domain III mutant protein with a key mutation inside a cross-reactive neutralizing epitope was designed, expressed, and used in the competitive panning of a yeast display naïve antibody library. All the yeast display antibodies that bound to the wild type domain III but not to the mutant were selectively sorted and characterized. Two unique clones were identified and showed cross-reactive binding to envelope protein domain IIIs from different serotypes. Epitope mapping of one of the antibodies confirmed that its epitope overlapped with the intended neutralizing epitope. This novel approach has implications for many areas of research where the isolation of epitope-specific antibodies is desired, such as selecting antibodies against conserved epitope(s) of viral envelope proteins from a library containing high titer, high affinity non-neutralizing antibodies, and targeting unique epitopes on cancer-related proteins.

You Say Goodbye, I Say Hello

Saying goodbye has never been a favorite part of my life. For the past eight years, beginning with RUSQ 44:3, I have been privileged to edit the readers' advisory column. What a wonderful time it has been. I have had the chance to talk with and work with some of the best writers and practitioners in the readers' advisory world. I will be forever grateful that they took the time out of their busy work schedules to put together articles for the journal, sharing their expertise and building a stronger readers' advisory community through these pieces. As I move on to take up the role of editor of RUSQ, I want to use this last column, or at least the first part of it, to both reflect and to look forward. I would be remiss in leaving not to thank several people, without whom I would not have been able to make the column as good as it has been. First would be the editors of RUSQ. I have had the good fortune to work with three superb editors. In 2004, Danny Wallace and Connie Van Fleet were generous enough to offer me the position of RA column editor for RUSQ, and to get me started in the business of finding authors and crafting articles. I learned a great deal from them about working with word counts, manuscripts, writers, and deadlines, and I am grateful for their guidance. When Danny and Connie finished their terms, I was fortunate to have the opportunity to work for Diane Zabel. For the past six years, Diane has encouraged and enthusiastically supported the readers' advisory column. The articles that have appeared in the RA column are in part a tribute to Danny, Connie, and Diane's patience and attention to detail. Any column editor would be lucky to have worked with and for three such fine readers and editors. I would also like to thank all of the authors who have written for the column. To me, their pieces reflect some of the best thinking about the intersection of reading, readers, and librarians. It is not always a simple thing to write a column to someone else's schedule and to accept editorial suggestions. Each of the authors I have worked with has done both of these things with grace and style. In developing the readers' advisory column, I was building on the work of Mary K. Chelton, who had previously edited the column. Under Mary K.'s guidance, the RA column offered the RUSQ community articles that presented practical advice about working with readers as well as examinations of the recent history and potential future of readers' advisory services in the United States. Her work was indeed a firm foundation on which to build. In the past eight years, the readers' advisory column has sought to blend theory and practice, looking not only at how to work with readers but also at why readers' advisory is essential in all sorts of libraries. Articles have covered a wide range of topics: book discussion groups (in both public and academic libraries as well as online), form-based readers' advisory, RA in library school education, theories of genre separation (one of my favorite topics), fiction and the brain, and readers' advisory and the library catalog among others. In each of these cases, and in the other articles too numerous to mention, the authors looked for new ways to approach readers and reading and began a conversation that often carried over into the broader RA world. That really is the goal, I believe, for any column, that it serve not as an ending point but as a beginning for reflection, discussion, and then application of the ideas it promotes. A real strength of RUSA is its varied and passionate membership. The opportunity to work with librarians outside your type of library is one that should not be under-valued. During my time in RUSA, and in particular as a column editor for RUSQ, I have learned a great deal from working closely with academic librarians and LIS faculty as well as with librarians in non-traditional roles. I hope that they have had the same experience in working with public librarians. …

Cell type–specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP

This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type-specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type-specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.

A high-performance sorting algorithm for multicore single-instruction multiple-data processors

Many sorting algorithms have been studied in the past, but there are only a few algorithms that can effectively exploit both single-instruction multiple-data (SIMD) instructions and thread-level parallelism. In this paper, we propose a new high-performance sorting algorithm, called aligned-access sort (AA-sort), that exploits both the SIMD instructions and thread-level parallelism available on today's multicore processors. Our algorithm consists of two phases, an in-core sorting phase and an out-of-core merging phase. The in-core sorting phase uses our new sorting algorithm that extends combsort to exploit SIMD instructions. The out-of-core algorithm is based on mergesort with our novel vectorized merging algorithm. Both phases can take advantage of SIMD instructions. The key to high performance is eliminating unaligned memory accesses that would reduce the effectiveness of SIMD instructions in both phases. We implemented and evaluated the AA-sort on PowerPC 970MP and Cell Broadband Engine platforms. In summary, a sequential version of the AA-sort using SIMD instructions outperformed IBM's optimized sequential sorting library by 1.8 times and bitonic mergesort using SIMD instructions by 3.3 times on PowerPC 970MP when sorting 32 million random 32-bit integers. Also, a parallel version of AA-sort demonstrated better scalability with increasing numbers of cores than a parallel version of bitonic mergesort on both platforms. Copyright © 2011 John Wiley & Sons, Ltd.

Modular model building for vehicular traffic systems with macroscopic dynamics

AbstractVehicular traffic modeling, analysis and supervision has became in the last year very attractive both for engineers and mathematicians with the aim of understanding, controlling and forecasting traffic phenomena. In this framework, the use of simulation tools has spread rapidly both to adequately perform the design of a road infrastructure, and as a support to simulate and verify the road infrastructure. Currently available simulators are in general closed systems where designers have no access to the implemented models, do not permit the integration of macroscopic and microscopic models together and are usually separated from design and verification tools. This paper presents a preliminary study on how to fill these gaps by using a modular model building approach. The developed elementary traffic models have been implemented into Matlab/Simulink environment and collected into a sort of library. The approach has been tested and validated using a real scenario and the available traffic measurements. Copyright ©2011 IFAC.

Epitope Mapping Using Gram‐Positive Surface Display

Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibody's binding characteristics will still be the main features determining the assay's reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.

Generation of a De Novo Actin Pointed-End Binding Protein

In order to create disease specific drugs, molecules that are designed to bind to any given target with high affinity and high specificity have been the motivation of the biotechnology field for some time. The natural immune system can produce antibodies that essentially recognize any antigenic target with high affinity and specificity. Therefore, employing this specificity through antibody related technologies should yield bioengineered proteins that are very useful to biomedical research. We performed a high-throughput phage display screen, which used restricted amino acid diversity phage display libraries to generate antibody fragments to F-actin. We generated synthetic actin-binding antibody fragments (sABs) that were further selected to bind to specific actin conformations. These sABs resemble the Fab fragment of an antibody, with a 55kDa heterodimer of heavy and light chain variable fragments, forming an antigen-binding site that was engineered to bind to a specific actin conformation by specific library sorting and subsequent phage ELISA characterization. A series of these generated sABs were characterized in vitro and in vivo. We identified three interesting candidates for study. The first construct severs F-actin in vitro and in vivo. The second sAB bundles F-actin and inhibits G-actin from polymerizing. And the most interesting sAB is a pointed-end actin binding molecule. This construct shows clear affinity for the pointed-end of actin by in vitro TIRF analysis. Furthermore, this construct also inhibits in vitro polymerization of actin over time. The application of this technology to generate molecules to specific conformations of proteins for analysis will be pivotal in the bioengineering field.

Genome‐scale analysis of library sorting (GALibSo): Isolation of secretion enhancing factors for recombinant protein production in Pichia pastoris

A method combining fluorescence activated cell sorting (FACS) and DNA microarray assisted clone identification was developed and termed Genome-Scale Analysis of Library Sorting (GALibSo). Genes enhancing the production of secreted heterologous proteins in Pichia pastoris were identified out of a cDNA library by cell surface display and FACS. The trends of gene enrichment during consecutive FACS rounds were monitored by DNA microarrays. In a case study a P. pastoris cDNA library was co-expressed in a strain secreting the Fab fragment of a monoclonal antibody against human immunodeficiency virus type 1 as a model protein. Three genes were identified, increasing the relative expression level of the surface-displayed model protein up to 45%. While one of these genes had a positive effect on three out of four tested proteins, the product specific effect of the other two suggested that the effects of the co-expressed secretion enhancing factors are partly dependent on the protein to be produced. The microarray based monitoring of the enrichment of genes causing enhanced protein secretory capacity led to novel insights into the limitation of protein secretion.

Accelerating phage-display library selection by reversible and site-specific biotinylation

Immobilization of a target molecule to a solid support is an indispensable step in phage display library sorting. Here we describe an immobilization method that addresses shortcomings of existing strategies. Our method is based on the use of a polyhistidine-tagged (His-tagged) target molecule and (BT)tris-NTA, a high-affinity capture reagent for His-tags that also contains a biotin moiety. (BT)tris-NTA provides a stable and reversible linkage between a His-tag and a streptavidin-coated solid support. Because His-tags are the de facto standard for recombinant protein purification, this method dramatically simplifies target preparation for phage display library sorting. Here, we demonstrate the utility of this method by selecting high-affinity binding proteins based on the fibronectin type III (FN3) scaffold to two His-tagged protein targets, yeast small ubiquitin-like modifier and maltose-binding protein. Notably, a significant number of FN3 clones binding either targets selected using the new immobilization method exhibited only very weak binding when the same target was immobilized by coating on a polystyrene surface. This suggests that the His-tag-mediated immobilization exposes epitopes that are masked by commonly used passive adsorption methods. Together, these results establish a method with the potential to streamline and enhance many binding-protein engineering experiments.

An automatically‐tuned sorting library

AbstractWe present ATSL, an automatically‐tuned sorting library. ATSL generates a sorting routine optimized to the target machine for a specific data type. ATSL finds a high‐performance sorting routine by searching an algorithmic space that we have defined. The search space includes basic sorting algorithms and automatically‐generated compositions of sorting algorithms. Performance measurements are used both for ranking candidate algorithms and for characterizing the behavior of candidates in specific settings (e.g. ranges of input sizes). These characterizations allow ATSL to generate hybrid algorithms that intelligently exploit the strengths of particular algorithms, such as high speed at specific input‐size ranges. Many sorting algorithms can be tuned using numeric parameters and ATSL searches these parameter spaces to find values that yield high performance on the target machine. The building blocks from which ATSL synthesizes sorting algorithms include adaptations of many of the most effective hand‐tuned sorting routines, including several that are tuned for cache efficiency. An extensive experimental evaluation shows that ATSL generates high‐performance codes that are well tuned for the target machine and data type. The experiments were conducted on six different machines, of several architectures, and with three different compilers. The algorithms that are generated are fast; in particular, they beat the hand‐tuned building blocks and the compiler's C++ built‐in sorting routine. The algorithms that ATSL generates on different machines and using different compilers are different from each other. Copyright © 2007 John Wiley & Sons, Ltd.

Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichia coli

The complex and integrated nature of both genetic and protein level factors influencing recombinant protein production in Escherichia coli makes it difficult to predict the optimal expression strategy for a given protein. Here, two combinatorial library strategies were evaluated for their capability of tuning recombinant protein production in the cytoplasm of E. coli. Large expression vector libraries were constructed through either conservative (ExLib1) or free (ExLib2) randomization of a seven-amino-acid window strategically located between a degenerated start codon and a sequence encoding a fluorescently tagged target protein. Flow cytometric sorting and analyses of libraries, subpopulations or individual clones were followed by SDS-PAGE, western blotting, mass spectrometry and DNA sequencing analyses. For ExLib1, intracellular accumulation of soluble protein was shown to be affected by codon specific effects at some positions of the common N-terminal extension. Interestingly, for ExLib2 where the same sequence window was randomized via seven consecutive NN(G/T) tri-nucleotide repeats, high product levels (up to 24-fold higher than a reference clone) were associated with a preferential appearance of novel SD-like sequences. Possible mechanisms behind the observed effects are discussed.

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.

Directed Evolution of Biocatalysts

Biocatalysis is the activity to synthesize chemicals using biological entities, in particular enzymes. Natural enzymes often need to be optimized before being used in industrial processes, and directed evolution is an approach that is well suited for the creation of these improved industrial biocatalysts. Directed evolution involves creating a library of mutant genes and then sorting them based on the associated phenotypes. Main library creation and sorting protocols are compared here, with an emphasis on semirational approaches to library creation and fast screening or pool selection techniques. Representative reports of improved catalytic activity, enzyme stability, and specificity are discussed. Finally, an insight is given on recent technological developments to make enzymes more suitable for biocatalysis. These new technologies could favor the switch of numerous processes from chemistry to biocatalysis soon.

Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display

We have investigated a staphylococcal surface display system for its potential future use as a protein library display system in combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1:1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections.