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Results for “Library Automation”

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PubMed1997

Combinatorial protein design: strategies for screening protein libraries.

Powerful strategies for screening protein libraries further strengthen the arguments for applying 'irrational' approaches to understanding and designing new proteins. Developments during the past year include the application of functional complementation and automation to reduce screening loads, as well as the use of computerized data acquisition to characterize whole protein libraries rather than just selected individuals.

Automation↗

PubMed2005

Solid-phase total synthesis of (-)-Phenylhistine and (-)-Aurantiamine. Synthesis of a diverse dehydro-2,5-diketopiperazine library. Part II.

The preparation of solid supported glycine phosphonate and its utilization for the total synthesis of two natural products is presented. The proposed protocol combines diversity with accessibility and speed, which makes this scaffold suitable for automated parallel synthesis and combinatorial chemistry. The preparation of a small library of dehydro-2,5-diketopiperazines, combining several natural amino acids with diverse heterocycles (including thiazoles, pyridines, indoles and imidazoles), is also demonstrated.

Indicators and Reagents↗

PubMed2004

Identification of a novel binding partners for tumor suppressor PTEN by a yeast two-hybrid approach.

AIM: To identify novel PTEN-binding partners. METHODS: The technique of yeast two-hybrid screening was used in this study. A panel of bait constructs was created, containing the C-terminal domain of PTEN, full length PTEN, activated and phosphatase-dead mutants. The expression of LexA-fused baits, their nuclear localization and autoactivation potential were tested according to the standard protocol of Duplex A system. CDNA libraries from Colon Cancer, HeLa and Mouse Embryo were screened with two selected bait constructs. Isolated positive clones were further analysed by mating assay and identified by automated DNA sequencing and database searching. RESULTS: Extensive screening of cDNA libraries with the full length and the C-terminal domain of PTEN led to the identification of 43 positive clones, which were confirmed in mating assay. Sequence analysis indicated that two clones encode AEBP1 (Adipocyte Enhancer Binding Protein 1). CONCLUSION: Our data indicate that the interaction between PTEN and AEBP1 is mediated by their C-terminal and N-terminal domains, respectively. The functional importance of PTEN-AEBP1 interaction is currently under investigation.

DNA, Complementary↗

PubMed2006

Novel two-stage screening procedure leads to the identification of a new class of transfection enhancers.

BACKGROUND: Non-viral gene transfer efficiency is low as compared to viral vector systems. Here we describe the discovery of new drugs that are capable of enhancing non-viral gene transfer into mammalian cells using a novel two-stage screening procedure. METHODS: First, potential candidates are preselected from a molecular library at various concentrations by a semi-automated yeast transfection screen (YTS). The maximal transfection efficiency of every positive drug is subsequently determined in independent experiments at the optimal concentration and compared to the inhibitory effect of the drug on cell growth (IC50). In a subsequent mammalian cell transfection screen (MTS), the maximal transfection efficiency and the IC50 are determined for all preselected drugs using a human cell line and a luciferase reporter gene construct. RESULTS: Employing our novel system we have been able to identify a new class of transfection enhancers, the tricyclic antidepressants (i.e. doxepin, maprotiline, desipramine and amoxapine). All positive drugs enhanced gene transfer in both yeast and human cell lines, but lower concentrations were sufficient for mammalian cells. With a triple combination of doxepin, amoxapine and chloroquine we obtained a transfection efficiency that exceeded that of chloroquine, one of the best-known transfection enhancers of mammalian cells, by nearly one order of magnitude. CONCLUSIONS: Non-viral gene transfer efficiency can be increased significantly using new transfection enhancers that are identified by a novel, semi-automated two-stage screening system employing yeast cells in the first and specific human target cells in the second round.

Animals↗

PubMed2002

Prediction of 'drug-likeness'.

Recent developments in combinatorial chemistry and high-throughput screening have dramatically increased the scale on which drug discovery programs are carried out. Along with these advances has come a need for automated methods of determining which compounds from a library should be synthesized and screened. These methods range from simple counting schemes to sophisticated machine learning techniques such as neural networks. While many of these methods have performed well in validation studies, the field is still in its formative stage. This paper reviews a number of computational techniques for identifying drug-like molecules and examines challenges facing the field.

Artificial Intelligence↗

PubMed1999

A systematic comparison of protein structure classifications: SCOP, CATH and FSSP.

BACKGROUND: Several methods of structural classification have been developed to introduce some order to the large amount of data present in the Protein Data Bank. Such methods facilitate structural comparisons and provide a greater understanding of structure and function. The most widely used and comprehensive databases are SCOP, CATH and FSSP, which represent three unique methods of classifying protein structures: purely manual, a combination of manual and automated, and purely automated, respectively. In order to develop reliable template libraries and benchmarks for protein-fold recognition, a systematic comparison of these databases has been carried out to determine their overall agreement in classifying protein structures. RESULTS: Approximately two-thirds of the protein chains in each database are common to all three databases. Despite employing different methods, and basing their systems on different rules of protein structure and taxonomy, SCOP, CATH and FSSP agree on the majority of their classifications. Discrepancies and inconsistencies are accounted for by a small number of explanations. Other interesting features have been identified, and various differences between manual and automatic classification methods are presented. CONCLUSIONS: Using these databases requires an understanding of the rules upon which they are based; each method offers certain advantages depending on the biological requirements and knowledge of the user. The degree of discrepancy between the systems also has an impact on reliability of prediction methods that employ these schemes as benchmarks. To generate accurate fold templates for threading, we extract information from a consensus database, encompassing agreements between SCOP, CATH and FSSP.

Databases, Factual↗

PubMed2002

High-throughput protein arrays: prospects for molecular diagnostics.

High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations.

Animals↗

PubMed2003

Optically active iridium imidazol-2-ylidene-oxazoline complexes: preparation and use in asymmetric hydrogenation of arylalkenes.

This work explores the potential of iridium complexes of the N-heterocyclic carbene oxazoline ligands 1 in asymmetric hydrogenations of arylalkenes. The accessible carbene precursors, imidazolium salts 2, and robust iridium complexes 5 facilitated a discovery/optimization approach that featured preparation of a small library of iridium complexes, parallel hydrogenation reactions, and automated analysis. Three of the complexes (5ab, 5ad, and 5dp) and a similar rhodium complex (6ap) were studied by single-crystal X-ray diffraction techniques. This revealed molecular features of 6ap, and presumably the corresponding iridium complex 5ap, that the others do not have. In enantioselective hydrogenations of arylalkenes complex 5ap was the best for many, but not all, substrates. The enantioselectivities and conversions observed were sensitive to minor changes to the catalyst and substrate structure. Ligands with aliphatic N-heterocyclic carbene substituents gave complexes that are inactive, and do not lose the 1,5-cyclooctadiene ligands under the hydrogenation conditions. Experiments to investigate this unexpected observation imply that it is of a steric, rather than an electronic, origin. Temperature and pressure effects on the conversions and enantioselectivities of these reactions had minimal effects for some alkenes, but profound effects for others. In one case, the enantioselectivities obtained at high-pressure/low-temperature conditions were opposite to those obtained under high-temperature/low-pressure conditions (-64% enantiomeric excess versus +89% enantiomeric excess); a transformation from one prevalent mechanism to another is inferred from this. The studies of pressure dependence revealed that many reactions proceeded with high conversions, and optimal enantioselectivities in approximately 2 h when only 1 bar of hydrogen was used. Deuterium-labeling experiments provide evidence for other types of competing mechanisms that lead to D-incorporation at positions that do not correspond to direct addition to the double bond.

Journal Article↗

PubMed2001

Solid-phase synthesis of peptidomimetic inhibitors for the hepatitis C virus NS3 protease.

The NS3 serine protease enzyme of the hepatitis C virus (HCV) is essential for viral replication. Short peptides mimicking the N-terminal substrate cleavage products of the NS3 protease are known to act as weak inhibitors of the enzyme and have been used as templates for the design of peptidomimetic inhibitors. Automated solid-phase synthesis of a small library of compounds based on such a peptidomimetic scaffold has led to the identification of potent and highly selective inhibitors of the NS3 protease enzyme.

Combinatorial Chemistry Techniques↗

PubMed1999

Organic-Fluorous Phase Switches: A Fluorous Amine Scavenger for Purification in Solution Phase Parallel Synthesis.

The synthesis of the fluorous amine scavenger [(C(6)F(13)CH(2)CH(2))(3)SiCH(2)CH(2)CH(2)](2)NH and its successful application in the automated solution phase parallel synthesis of a urea library are described. Ureas were made by robotic synthesis from organic amines and excess isocyanates. The amine scavenger reacts with excess isocyanate, and the fluorous tag serves to solubilize the resulting adduct in the fluorous phase so it can be removed by fluorous-organic extraction. Organic urea products are isolated in high yields and purities after liquid-liquid extraction. Preliminary biological evaluation shows that several of the ureas have ion channel modulation abilities. In contrast to polymer and ionic quenching methods, the fluorous quench works whether the product is soluble or insoluble in the reaction medium, and ionizable functional groups are tolerated in the products.

Journal Article↗

PubMed2001

The preparation of a series of nitrostilbene ester compounds using micro reactor technology.

The synthesis of stilbene esters using Wittig chemistry has been used to illustrate the generic diversity micro reactors offer in terms of chemical control and rapid method development. The micro reactor consisted of a 'T' design based on channel geometries 200 microns wide and 100 microns deep, etched into borosilicate glass and sealed with a borosilicate top plate using a thermal bonding technique. The movement of the reagent and products was achieved using electroosmotic flow (EOF), assisted by the incorporation of micro porous silica frits within the micro-channels to allow accurate solution control. To optimise the operating conditions methyl 4-formylbenzoate, premixed with sodium methoxide, was reacted with 2-nitrobenzyl-triphenylphosphonium bromide in dry degassed MeOH using flow conditions for both reagents of 0.40 microL min-1 for 20 min. A product yield of 70% (2:1 reaction stoichiometry with the aldehyde in excess) was obtained representing a 10% increase compared with the traditional batch synthesis. To demonstrate the capability of micro reactors to perform atom efficient synthesis a series of experiments based on an injection methodology (optimised to 30 s) were performed in the micro reactor at 1:1 stoichiometry resulting in a yield of 59%. Finally, the capability of micro reactors to perform a series of analogue reactions was investigated. The yields for a further three aldehydes indicated that the technology will be suitable for the development of automated device to support the generation of combinatorial libraries and rapid high throughput synthetic methods.

Journal Article↗

PubMed2005

Profiling of alopecia areata autoantigens based on protein microarray technology.

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Adult↗

PubMed2001

Construct validity of selected Automated Neuropsychological Assessment Metrics (ANAM) battery measures.

The Automated Neuropsychological Assessment Metrics (ANAM) is a computerized library of tests designed to assess neurocognitive functioning across administrations (Kane & Reeves, 1997). This study was designed to examine neuropsychological constructs measured by selected ANAM measures and to compare them with traditional measures putatively assessing similar domains. The sample consisted of 191 outpatients with suspected neurocognitive dysfunction. Correlations and regressions indicated significant relationships between traditional and computerized tests measuring similar constructs. PCA results yielded a three-factor solution: Factor I (Processing Speed/Efficiency), Factor II (Retention/Memory), and Factor III (Working Memory).

Adult↗

PubMed1994

Navigating the Brookhaven Protein Data Bank.

The Protein Data Bank maintained at Brookhaven National Laboratories has expanded to the point where even experienced users have difficulty understanding, exploring, and exploiting it. This paper describes a text file, an annotation of the Protein Data Bank, which helps users find information on related files and structures. The most recent version of this file includes information on homologous structures, including both sequence homology and structural homology. This file is in ASCII format and is available electronically. It is easy to search locally on any type of computer, using an editor or a pattern-matching program, such as grep.

Base Sequence↗

PubMed1991

Automatic evaluation of protein sequence functional patterns.

A procedure that automatically provides an evaluation of the diagnostic ability of a protein sequence functional pattern is described. The procedure relies on the identification of the closest definable set in terms of a (protein sequence) database functional annotation to the set of database instances containing a given pattern. Assuming annotation correctness and completeness in the protein sequence database, the degree of statistical association between these sets provides an appropriate measure of the diagnostic ability of the pattern. An experimental implementation of the procedure, using the NBRF/PIR protein database, has been applied to a diverse collection of published sequence patterns. Results obtained reveal that frequently it is not possible to define (in NBRF/PIR database terminology) the set of database instances containing a given pattern, suggesting either lack of pattern diagnostic ability or protein database annotation incompleteness and/or inconsistencies.

Algorithms↗

PubMed2001

On approximate string matching of unique oligonucleotides.

The current research considers the approximate string matching search for important subsequences from DNA sequences, which is essential for numerous bioinformatics computation tasks. We tested several approximate string matching algorithms and furthermore developed one for DNA data. Run times of the algorithms are important, since the amount of data is very large.

Algorithms↗

PubMed2005

From targets to leads: the importance of advanced data analysis for decision support in drug discovery.

Lead discovery is a complex process that is intimately linked to chemistry, but which is also increasingly driven by biological sciences. In an industrial pharmaceutical research environment the process is defined by highly automated technologies for target identification and validation, compound library screening, and compound efficacy assessment. The huge volumes and complex dependencies of data produced by such large-scale experiments have led to a reassessment of data analysis processes, resulting in the development of novel data analysis strategies tailored to drug discovery. In this review, recent progress in data-driven research applications is reported, focusing on the use and processing of transcriptomics, proteomics and high-throughput screening data. The successful application of specialized data analysis procedures in many companies is discussed, which has resulted in significant improvements in decision-making processes for progressing therapeutic targets to promising leads.

Animals↗

PubMed1993

Meeting the urgency for document delivery in clinical medicine.

A Document Delivery library project was designed to improve delivery of information to health professionals in the Washington DC/Baltimore area. The project goals were to enhance delivery of full text documents and accelerate interlibrary loan services. The aim was to provide direct library services in the clinical arena by facilitating access to the articles needed by practitioners and clinical investigators. The objectives were to (1) design, develop and implement a comprehensive Document Delivery System (DDS) for the Library Information System (LIS) which included interlibrary loan, photocopy services and facsimile transmission capabilities; (2) establish a multi-university Library Knowledge Network for resource sharing; and (3) evaluate the project. The DDS and facsimile service are described and project data and outcomes are reported. Today, the participating libraries can use electronic means to share interlibrary loans. Georgetown users have responded favorably to the DDS and Fax services.

Clinical Medicine↗