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At least 235 records · Page 13Linked to original sources

PubMed2004

Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries.

Early determinations of pharmaceutical properties can serve as predictors of a compound's likely development success. Our laboratory has implemented high throughput in vitro absorption, distribution, metabolism and excretion (ADME) assays which address absorption, metabolism, and physico-chemical properties in an effort to identify potential development liabilities early, thereby minimizing discovery to market attrition. In response to the throughput demands of parallel synthesis, we have incorporated a SAGIAN core robotics system for the determination of both metabolic stability in human liver microsomes (HLMs) and cytochrome P450 (CYP450) inhibition. This automated solution has led to an increase in capacity, throughput and reliability for both in vitro assays. The SAGIAN core robotics system integrates devices such as liquid handlers, plate hotels and incubators through the use of an ORCA robotic arm. The HLM stability assay utilizes a Multimek 96-channel pipettor for liquid handling. The incubation plates are transferred off-line for final semi-quantitative analysis using high throughput parallel LC/MS. The CYP inhibition method combines both liquid handlers and an integrated fluorescence plate reader to perform single concentration percent inhibition assays for 88 compounds. Cytochrome P450 inhibition is measured for both CYP3A4 and CYP2D6 isozymes. This system represents a fully integrated approach to high throughput ADME evaluation in support of drug discovery. The core system concept creates a plug-and-play approach, which combines a series of modular stations to build a robotic platform, which is flexible, upgradable, and easily reconfigured when assays change or are newly developed. The application of these strategies as a means of assessing metabolic stability and CYP inhibition of synthetic libraries is discussed.

Combinatorial Chemistry Techniques↗

PubMed1996

A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries.

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites--trinucleotide phosphotriesters--have been prepared using the phosphotriester approach without protection of the 3'-hydroxyl function. The structures of trinucleotide phosphotriesters and intermediates were confirmed by 1H- and 31P-NMR spectra, mass-spectra and by analysis of SPDE-hydrolysates of deprotected preparations. Purity of the target products has been confirmed by test reactions. The synthons have been used for automated synthesis of oligonucleotides and corresponding libraries by a phosphite-triester approach. A 54mer, containing 12 randomized internal bases, and a 72mer with 24 internal randomized bases have been synthesized.

Bacteriophages↗

PubMed2001

Functionalized Polymers-Emerging Versatile Tools for Solution-Phase Chemistry and Automated Parallel Synthesis.

As part of the dramatic changes associated with the need for preparing compound libraries in pharmaceutical and agrochemical research laboratories, industry searches for new technologies that allow for the automation of synthetic processes. Since the pioneering work by Merrifield polymeric supports have been identified to play a key role in this field however, polymer-assisted solution-phase synthesis which utilizes immobilized reagents and catalysts has only recently begun to flourish. Polymer-assisted solution-phase synthesis has various advantages over conventional solution-phase chemistry, such as the ease of separation of the supported species from a reaction mixture by filtration and washing, the opportunity to use an excess of the reagent to force the reaction to completion without causing workup problems, and the adaptability to continuous-flow processes. Various strategies for employing functionalized polymers stoichiometrically have been developed. Apart from reagents that are covalently or ionically attached to the polymeric backbone and which are released into solution in the presence of a suitable substrate, scavenger reagents play an increasingly important role in purifying reaction mixtures. Employing functionalized polymers in solution-phase synthesis has been shown to be extremely useful in automated parallel synthesis and multistep sequences. So far, compound libraries containing as many as 88 members have been generated by using several polymer-bound reagents one after another. Furthermore, it has been demonstrated that complex natural products like the alkaloids (+/-)-oxomaritidine and (+/-)-epimaritidine can be prepared by a sequence of five and six consecutive polymer-assisted steps, respectively, and the potent analgesic compound (+/-)-epibatidine in twelve linear steps ten of which are based on functionalized polymers. These developments reveal the great future prospects of polymer-assisted solution-phase synthesis.

Journal Article↗

PubMed2003

Prediction of deleterious functional effects of amino acid mutations using a library of structure-based function descriptors.

An automated, active site-focused, computational method is described for use in predicting the effects of engineered amino acid mutations on enzyme catalytic activity. The method uses structure-based function descriptors (Fuzzy Functional Forms trade mark or FFFs trade mark ) to automatically identify enzyme functional sites in proteins. Three-dimensional sequence profiles are created from the surrounding active site structure. The computationally derived active site profile is used to analyze the effect of each amino acid change by defining three key features: proximity of the change to the active site, degree of amino acid conservation at the position in related proteins, and compatibility of the change with residues observed at that position in similar proteins. The features were analyzed using a data set of individual amino acid mutations occurring at 128 residue positions in 14 different enzymes. The results show that changes at key active site residues and at highly conserved positions are likely to have deleterious effects on the catalytic activity, and that non-conservative mutations at highly conserved residues are even more likely to be deleterious. Interestingly, the study revealed that amino acid substitutions at residues in close contact with the key active site residues are not more likely to have deleterious effects than mutations more distant from the active site. Utilization of the FFF-derived structural information yields a prediction method that is accurate in 79-83% of the test cases. The success of this method across all six EC classes suggests that it can be used generally to predict the effects of mutations and nsSNPs for enzymes. Future applications of the approach include automated, large-scale identification of deleterious nsSNPs in clinical populations and in large sets of disease-associated nsSNPs, and identification of deleterious nsSNPs in drug targets and drug metabolizing enzymes.

Algorithms↗

PubMedDate not supplied

Synthesis and biological evaluation of a beauveriolide analogue library.

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.

Animals↗

PubMed1991

The establishment of an academic health sciences library in a developing country: a case study.

The development of a Faculty of Medical Sciences (FMS) and an academic health sciences library for the University of the West Indies (UWI) has proven to be a polemical and political issue due to the depressed economy of the country. Although FMS is still shrouded in politics and controversy after its inaugural year, the Medical Sciences Library (MSL) has expanded its dimensions and is actively developing a biomedical information network within the country. This will result in better dissemination and control of biomedical information. The library now participates in joint projects with other health sciences libraries in the country with the goal of joint automated listings of holdings and shared cataloging projects. This paper examines the development of the library and explains the difficulties experienced in its developmental stages due to politics, the delay in appointment of a medical sciences librarian, and the financial decline in the local economy.

Developing Countries↗

PubMed2004

A method for comparing multiple bacterial community structures from 16S rDNA clone library sequences.

Culture-independent approaches, based on 16S rDNA sequences, are extensively used in modern microbial ecology. Sequencing of the clone library generated from environmental DNA has advantages over fingerprint-based methods, such as denaturing gradient gel electrophoresis, as it provides precise identification and quantification of the phylotypes present in samples. However, to date, no method exists for comparing multiple bacterial community structures using clone library sequences. In this study, an automated method to achieve this has been developed, by applying pair wise alignment, hierarchical clustering and principle component analysis. The method has been demonstrated to be successful in comparing samples from various environments. The program, named CommCluster, was written in JAVA, and is now freely available, at http://chunlab.snu.ac.kr/commcluster/.

Algorithms↗

PubMed1977

Centralized automated cataloging of health science materials in the MLC/SUNY/OCLC shared cataloging service.

Since February 1976, The Medical Library Center of New York, with the assistance of the SUNY/OCLC Network, has offered, on a subscription basis, a centralized automated cataloging service to health science libraries in the greater metropolitan New York area. By using workforms and prints of OCLC record (amended by the subscribing participants), technical services personnel at the center have fed cataloging data, via a CRT terminal, into the OCLC system, which provides (1) catalog cards, received in computer filing order; (2) book card, spine, and pocket labels; (3) accessions lists; and (4) data for eventual production of book catalogs and union catalogs. The experience of the center in the development, implementation, operation, and budgeting of its shared cataloging service is discussed.

Book Classification↗

PubMed2000

An automated prediction of MHC class I-binding peptides based on positional scanning with peptide libraries.

Specificities of three mouse major histocompatibility complex (MHC) class I molecules, Kb, Db, and Ld, were analyzed by positional scanning using combinatorial peptide libraries. The result of the analysis was used to create a scoring program to predict MHC-binding peptides in proteins. The capacity of the scoring was then challenged with a number of peptides by comparing the prediction with the experimental binding. The score and the experimental binding exhibited a linear correlation but with substantial deviations of data points. Statistically, for approximately 80% of randomly chosen peptides, MHC-binding capacity could be predicted within one log concentration of peptides for a half-maximal binding. Known cytotoxic T-lymphocyte epitope peptides could be predicted, with a few exceptions. In addition, frequent findings of MHC-binding peptides with incomplete or no anchor amino acid(s) suggested a substantial bias introduced by natural antigen processing in peptide selection by MHC class I molecules.

Animals↗

PubMed2005

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer-Villiger monooxygenase catalysed lactone synthesis.

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L(-1) glycerol). Following induction of CHMO activity biomass concentrations of up to 6 gDCW L(-1) were obtained. Cells from each fermentation were subsequently used for the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L(-1)) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 microL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.

Acinetobacter calcoaceticus↗

PubMed2004

Solid-phase synthesis of an N-(phenylalkyl)cinnamide library via Horner-Wadsworth-Emmons reaction.

A readily automated solid-phase approach to the synthesis of diverse N-(phenylalkyl)cinnamides, analogues of the NR2B antagonist 2, is described. The procedure utilizes polymer supported N-(phenylalkyl)amines, (diethylphosphono)acetic acid and a wide range of commercially available hydroxybenzaldehydes. The key step, a Horner-Wadsworth-Emmons reaction is achieved under mild conditions and was found to be general for a large number of benzaldehydes. A 225-member focused library was synthesized using a Tecan Combitec synthesizer.

Cinnamates↗

PubMed2004

Combinatorial codon-based amino acid substitutions.

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Amino Acid Substitution↗

PubMed2002

Automated screening procedure for high-throughput generation of antibody fragments.

In the emerging field of proteomics, there is an urgent need for catcher molecules such as antibodies for detecting the proteome or parts of the proteome in a microarray format. A suitable source for providing a large diversity of binders is obtained by combinatorial libraries, such as phage display libraries of single chain antibody fragments (scFv) or Fab fragments. To find novel binders from the n-CoDeR libraries with a high throughput, we have automated the screening process with robotics. The automated system is configured to screen tens of thousands of clones per day to target antigens in various formats, including peptides and soluble proteins, as well as cell-bound targets; thus, it is well designed to meet demands from the proteomics area.

Antigens↗

PubMed2002

High-throughput analysis of natural product compound libraries by parallel LC-MS evaporative light scattering detection.

The application of an 8-way fully automated parallel LC-MS-ELSD system to the analysis of a library of 96 structurally diverse natural products is described. A 10-min separation incorporating a universal gradient allowed elution of 86 of these 96 compounds, all of which were detected by positive or negative mode electrospray ionization in conjunction with ELSD. This method is demonstrated to be one of the most universal means of detection for polar, nonvolatile, thermally labile natural products. It also allows an 8-fold increase in throughput. The analysis and profiling of constituents present in a library derived from plant material of Sarcostemma hirtellum, in terms of their retention times and mass spectra, are shown. This rapid characterization of plant constituents in terms of compound libraries is important in searching for new biologically active compounds. As a high-throughput tool to support our natural product discovery program, this method has been successfully used to analyze a library of 36,000 partially purified fractions derived from plant materials.

Biological Factors↗

PubMed2002

Triage protein fold prediction.

We have constructed, in a completely automated fashion, a new structure template library for threading that represents 358 distinct SCOP folds where each model is mathematically represented as a Hidden Markov model (HMM). Because the large number of models in the library can potentially dilute the prediction measure, a new triage method for fold prediction is employed. In the first step of the triage method, the most probable structural class is predicted using a set of manually constructed, high-level, generalized structural HMMs that represent seven general protein structural classes: all-alpha, all-beta, alpha/beta, alpha+beta, irregular small metal-binding, transmembrane beta-barrel, and transmembrane alpha-helical. In the second step, only those fold models belonging to the determined structural class are selected for the final fold prediction. This triage method gave more predictions as well as more correct predictions compared with a simple prediction method that lacks the initial classification step. Two different schemes of assigning Bayesian model priors are presented and discussed.

Animals↗

PubMed1994

The Modified Barium Swallow Database.

Every center that carries out the modified barium swallow (MBS) has a potential storehouse of useful clinical and radiological information filed away in the patient's files and in the center's video library. The modified barium swallow database automates the recording, storage, and analysis of this information. This project utilizes a standardized clinical and radiological data form which provides a consistent approach to the reporting of the MBS results. The information on the data form is entered into the MBS database software program for decoding and permanent storage. This database program, designed for the computer novice, is menu driven to simplify operation. Once the patient information is verified on the computer screen, the program can generate different types of reports for clinical and research purposes. The program has routines to search for patients with any clinical or radiological findings of interest. The ability to group and recall patients with specific findings has been an aid in teaching, peer review, and research. The program has been modified based upon feedback and experience at three centers in Toronto that have used it for 9 months.

Barium Sulfate↗

PubMed1997

Application of molecular biology in veterinary parasitology.

The number of applications of molecular biology in veterinary parasitology is increasing rapidly. The techniques used with eukaryotic cells are generally applicable to the study of parasites and their hosts. The polymerase chain reaction is particularly important for identification and diagnosis of parasites, as well as for many other applications. With species and type specific probes or primers, sensitivities and specificities unheard of with conventional techniques can be achieved. The accumulation of more information on the DNA sequences of parasites will reveal many more unique sequences which can be used for identification, diagnosis, molecular epidemiology, vaccine development and for studying the evolutionary biology and the physiology of parasites and the host-parasite relationship. Similarly, the completion of genome projects on host organisms will greatly assist efforts to select for hosts that are genetically resistant to parasite infection. The study of the molecular biology of antiparasitic drug receptors, potential targets for chemotherapy, and the molecular genetics of drug resistance will allow molecular screens to be used with combinatorial chemistry in the search for new antiparasitic drugs, improvements to existing chemotherapeutic families and better diagnosis and monitoring of drug resistance. While there is a proliferation of molecular biology techniques, the availability of simple kits and of automated techniques and services for sequencing, library construction and oligonucleotide synthesis and other procedures is making it easier for non-specialists to apply many of the common techniques of molecular biology. Molecular biology and the benefits from its application are relevant for veterinary parasitologists in developing countries as well as developed countries and we should introduce aspects of molecular biology to the teaching and training of veterinary parasitologists.

Animals↗

PubMed2003

Ribotyping characterisation of campylobacter isolates randomly collected from different sources in Italy.

In this study the potential for using the automated PstI ribotyping as a primary library typing method to survey Campylobacter and for identification of two thermophilic Campylobacter jejuni and Campylobacter coli species was evaluated. A total of 158 isolates randomly collected in Italy from different sources were analyzed. A large percentage of chicken (28%), turkey (27%) and turkey meat (25%) isolates shared their ribotyping profiles (ribotypes) with those of humans, whereas the swine isolates had unique profiles. The identification results obtained by ribotyping corresponded to those collected by using a multiplex PCR protocol specifically designed for C. jejuni and C. coli detection. The comparison of the PstI ribotyping profiles obtained in this research with those of the isolates collected over time will facilitate determining the ribotypes that are more frequently transmitted to humans in comparison to those that are normally harboured only in animals, foods and in the environment.

Abattoirs↗