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At least 1,045 records · Page 58Linked to original sources
PubMed2000

A high-throughput system for genome-wide measurement of genetic recombination in Arabidopsis thaliana based on transgenic markers.

A high-throughput system for the measurement of recombination frequencies in the genetic model plant, Arabidopsis thaliana, is described. It is based on 21 mono-transgenic isogenic lines harboring antibiotic resistance genes on all five chromosomes. Recombination between pairs of gene insertions in repulsion phase that confer resistance against kanamycin (kan) and hygromycin (hyg) is determined by a phenotypic assay of progeny (DART: Double Antibiotic Resistance Technique). DART allows testing for the influence of numerous environmental and genetic factors, including candidate genes, on recombination frequencies in specific genomic regions as well as the entire genome. Its usefulness is demonstrated by investigating the effects of UV treatment, different temperature and phosphorus supply regimes, and sex on recombination frequencies for all five chromosomes of A. thaliana.

Anti-Bacterial Agents↗
PubMed2005

Research perspectives for pre-screening alternatives to animal experimentation: on the relevance of cytotoxicity measurements, barrier passage determinations and high throughput screening in vitro to select potentially hazardous compounds in large sets of chemicals.

The MEIC study revealed a high predictivity of in vitro cytotoxicity data for human acute systemic toxicity. The idea, put forward by several authors, that compounds that show high cytotoxicity should not need further testing for confirmation but could be assumed toxic also in vivo provides a convenient concept for the selection of the most relevant compounds for further studies in large sets of chemicals, as in the REACH program. The automated techniques applied in high throughput screening (HTS) by the pharmaceutical and biotech industries to select hits in extensive compound collections represent an opportunity to significantly increase the capacity of cytotoxicity testing. Furthermore, it has been suggested that a combination of cytotoxicity data and some basic biokinetic information would greatly improve the accuracy in the extrapolation from in vitro to in vivo and thus make it possible to identify additional toxic compounds that might have escaped in the initial screen. Such information, which can be obtained in a medium throughput screening mode (MTS), includes biotransformation, absorption and some aspects of distribution. The measurement of the net flux of a compound over a cellular barrier, as the one formed in culture by human Caco-2 cells, gives useful, but limited, information on both gut absorption and blood-brain barrier penetration. The test procedures discussed here, as well as other supplementary in vitro tests, cannot always easily be described in terms of animal-based test replacements. In those instances, the necessary test validation cannot be carried out using animal reference data, and prediction models may have to be adapted to new ideas. Consequently, concepts of prospective validation to supplement the now well-established retrospective validation have to be developed.

Animal Testing Alternatives↗
PubMed2006

Nonmevalonate terpene biosynthesis enzymes as antiinfective drug targets: substrate synthesis and high-throughput screening methods.

The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.

Aldose-Ketose Isomerases↗
PubMed2003

Nucleic acid technology screening of Australian blood donors for hepatitis C and human immunodeficiency virus-1 RNA: comparison of two high-throughput testing strategies.

BACKGROUND AND OBJECTIVES: The aim of this study was to compare the performance of two high-throughput strategies for hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) RNA nucleic acid technology (NAT) screening in a volunteer blood-donor population. MATERIALS AND METHODS: The semiautomated Chiron Procleix HIV-1/HCV transcription mediated amplification (TMA) assay was used to screen 1 439 765 donations in two different testing configurations. Three sites (termed PDT sites) performed a mixture of individual donation (ID) and minipool (MP) testing, where 1 113 288 donations were screened as pools of 24 and an additional 32 003 donations were screened in ID format. A further two sites (termed SDT sites) screened 294 474 donations exclusively in ID format. RESULTS: A significantly higher proportion of initial NAT reactives that failed to react on follow-up testing [termed non-repeatably reactive (NRR)] was observed for ID testing at SDT sites than at PDT sites (0.082 vs. 0.047%: P < 0.01). Within the PDT sites, however, there was no significant difference between the NRR rate for MP or ID samples (0.037 vs. 0.047%; not significant). There was a significant difference in failed run rates between PDT and SDT sites (P < 0.01), with PDT sites having a higher run failure rate owing to non-amplification of the internal control. The PDT sites also had a significantly higher overall invalid sample rate. However, the invalid sample rate, specifically caused by known equipment failure, was significantly higher in the SDT sites, possibly attributable to greater usage (P < 0.0001). Four HCV NAT-positive/antibody-negative samples were identified in the course of the study. CONCLUSIONS: The comparison of the performance of PDT with SDT sites identified only minor differences that did not adversely impact on the timely release of blood products. Although both ID and MP strategies showed excellent specificity, irrespective of site configuration, the significantly increased NRR rate, observed exclusively for ID testing performed at SDT sites, indicates a potential for contamination that may limit the number of samples that can optimally be processed using ID testing. The performance data for ID testing in particular should serve as a useful benchmark for evaluating candidate NAT systems that are fully automated.

Australia↗
PubMed2000

Automated high throughput human CYP isoform activity assay using SPE-LC/MS method: application in CYP inhibition evaluation.

1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well format, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termination and metabolite extraction with solid phase extraction (SPE) plates. Metabolites from the seven reactions were pooled and quantified using a generic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on Km values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazole, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYPs 3A4, 2D6, 2C9, 2A6 land 2C19), 1A2 and 2E1, respectively, were obtained by this HTS method. Graphically obtained IC50 values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity improvement over traditional methods, and can be used for CYP inhibition assay and can be extended for liver activity profiling.

Automation↗
PubMed2004

High-throughput methods for measuring autoantibodies in systemic lupus erythematosus and other autoimmune diseases.

Numerous groups have now validated high-throughput approaches to autoantibody profiling in a variety of systems. Recently, we have used autoantigen microarray technology to identify distinct autoantibody profiles in H-2 congenic MRL/lpr mice (Sekine et al., manuscript in preparation), and we are expanding this platform to study human and mouse models of IDDM and RA. We are also developing protein arrays for multiplex analysis of serum antibody isotypes. Multiplexed methods for autoantibody profiling will undoubtedly continue to uncover novel aspects of autoimmunity and B cell biology. It is now time to move these technologies beyond the proof-of-concept phase, and start addressing the next series of important questions. These include, but certainly are not limited to: identifying "autoantibody signatures" associated with disease state or outcome; profiling autoantibodies during the natural course of murine and human disease; and monitoring changes in autoantibody profiles of patients in response to therapeutic intervention. However, the next set of challenges is just right around the corner. As data and statistical analysis tools become more robust, it will be possible to generate and approach new hypotheses at an unprecedented pace.

Autoantibodies↗
PubMed2001

TissueInfo: high-throughput identification of tissue expression profiles and specificity.

We describe TissueInfo, a knowledge-based method for the high-throughput identification of tissue expression profiles and tissue specificity. TissueInfo defines a set of tissue information calculations that can be computed for large numbers of genes, expressed sequence tags (ESTs) or proteins. Tissue information records that result from the TissueInfo calculations are used to generate tables suitable for data mining and for the selection of genes according to a given expression profile or specificity. When benchmarked against a test set of 116 proteins and literature information, TissueInfo was found to be accurate for 69% of identified tissue specificities and for 80% of expression profiles. The accuracy of the identifications can be increased if query sequences for which little information is available from dbEST are ignored. Thus, with 80% coverage, TissueInfo achieves an accuracy of 76% for specificity and 89% for expression. For the same set of proteins, the curated tissue specificity offered in SWISS-PROT was accurate in 78% of cases. TissueInfo can be useful for the selection of clones for custom microarrays, selection of training sets for ab initio identification of tissue information, gene discovery and genome-wide predictions. Further information about the program can be found at http://icb.mssm.edu/tissueinfo.

Computational Biology↗
PubMed2006

High-throughput method for the prediction of low-resolution, three-dimensional RNA structures.

Our aim is the high-throughput prediction of three-dimensional RNA structures at low resolution to answer questions posed by the RNA molecular biology. The strategy presented here is based on a computer program, called 3D-RNApredict, which implements and converts various structural data (RNA secondary structure, RNA motifs coordinates, experimental data) to create the input to the CYANA program (torsion-angle dynamics algorithm, TAD) which provides a fast engine for the 3D RNA structure calculation. The resultant RNA structure is refined using the X-PLOR program. The approach is amenable to automatization. Current predictions involve HIV-2 leader RNA domains and the sets of bacterial ncRNA.

Nucleic Acid Conformation↗
PubMed2003

Development and use of a high-throughput bacterial DNA gyrase assay to identify mammalian topoisomerase II inhibitors with whole-cell anticancer activity.

A high-throughput screen (HTS) was developed and used to identify inhibitors of bacterial DNA gyrase. Among the validated hits were 53 compounds that also inhibited mammalian topoisomerase II with IC(50) values of <12.5 micro g/mL for 51 of them. Using computational methods, these compounds were subjected to cluster analysis to categorize them according to their chemical and structural properties. Nine compounds from different clusters were tested for their whole-cell inhibitory activity against 3 cancer cell lines-NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS)-at a concentration of 100 micro M. Five compounds inhibited cell growth by >50% for all 3 cell lines tested. These compounds were tested further against a panel of 53 to 57 cell lines representing leukemia, melanoma, colon, CNS, ovarian, renal, prostate, breast, and non-small cell lung cancers. In this assay, PGE-7143417 was found to be the most potent compound, which inhibited the growth of all the cell lines by 50% at a concentration range of 0.31 to 2.58 micro M, with an average of 1.21 micro M. An additional 17 compounds were also tested separately against a panel of 10 cell lines representing melanoma, colon, lung, mammary, ovarian, prostate, and renal cancers. In this assay, 4 compounds-PGE-3782569, PGE-7411516, PGE-2908955, and PGE-3521917-were found to have activity with concentrations for 50% cell growth inhibition in the 0.59 to 3.33, 22.5 to 59.1, 7.1 to >100, and 24.7 to >100 micro M range.

Animals↗
PubMed2002

Developing soluble polymers for high-throughput synthetic chemistry.

Soluble polymers have emerged as viable alternatives to resin supports across the broad spectrum of high-throughput organic chemistry. As the application of these supports become more widespread, issues such as broad-spectrum solubility and loading are becoming limiting factors and therefore new polymers are required to overcome such limitations. This article details the approach made within our group to new soluble polymer supports and specifically focuses on parallel libraries of block copolymers, de novo poly(styrene-co-chloromethylstyrene), PEG- stealth stars, and substituted poly(norbornylene)s.

Combinatorial Chemistry Techniques↗
PubMed1999

A contiguous 3-Mb sequence-ready map in the S3-MX region on 21q22.2 based on high- throughput nonisotopic library screenings.

Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21-LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis.

Chromosome Mapping↗
PubMed2003

High-throughput screening for isolation of enhanced green fluorescent protein expressing transformants of filamentous fungus using an image analyzer.

High-throughput screening of enhanced green fluorescent protein (EGFP) gene-trap transformants of filamentous fungus was achieved for the first time using an image analyzer to measure their fluorescent intensity. For quantitative analysis of EGFP fluorescent intensity per unit cell mass, we developed a method for measurement of cell mass using the fluorescent dye SP-Dil. This method offers an effective and convenient tool for screening transformants expressing fluorescent protein as a reporter, and for quantitative analysis of fungal cell mass at jig levels.

Journal Article↗
PubMed2001

Development of a high throughput 96-well plate sample preparation method for the determination of trileptal (oxcarbazepine) and its metabolites in human plasma.

A high throughput preparation method for the determination of trileptal (oxcarbazepine, OXC) and its mono (MHD) and dihydroxy (DHD) metabolites in human plasma, using 96-well plate technology, has been developed and validated according to international regulatory requirements. Preparation of plasma samples (50 microl) containing the compounds to be analysed involved solid-phase extraction (SPE) on Empore C18 96-well SPE plates. Eluates from the plate were injected onto a reversed-phase column (Hypersil C18,3 microm) with UV detection at 210 nm. Detector response was linear over the ranges 0.2-10, 0.1-200 and 0.1-20 micromol/l, for OXC, MHD and DHD, respectively, with relative standard deviations from 1 to 10% and mean accuracies within 4% of the nominal values (number of standard curves=3 in duplicate). The limits of quantitation were 0.2, 0.1 and 0.1 micromol/l, respectively. The overall mean accuracies ranged from 96 to 106% and precision was in the range 4 to 11%. Cross validation indicated no significant difference between plasma concentrations obtained using the 96-well method and the previous method using a traditional SPE method with a 50 mg C18 cartridge. About a threefold increase in sample throughput and a twofold decrease of plasma volume required for the assays, were the main advantages obtained from the previous method. The method was applied for the determination of 3000 plasma samples from clinical studies.

Anticonvulsants↗
PubMed2004

Electrospray ionization mass spectrometric analysis of nucleic acids using high-throughput on-line desalting.

A rapid on-line desalting method utilizing ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was employed in tandem with negative electrospray ionization mass spectrometry (ESI-MS) for the routine analysis of nucleic acids. Desalting was performed on a short 10 x 2.1 mm guard column packed with 3.5 microm C(18) sorbent. The HPLC system was connected in-line to an orthogonal ESI-TOF mass spectrometer via a six-port, two-position switching valve, allowing desalting followed by mass analysis of nucleic acids. Duty cycle times for the method were as low as 1.5 min per sample. This allowed for the analysis of approximately 950 samples per 24-h time period, which is suitable for medium- to high-throughput applications. Average mass accuracy was determined to be 80 ppm for oligonucleotides up to 110 mer in length with external calibration. The method was utilized for synthetic oligonucleotide quality control and analysis of DNA genotyping fragments.

Animals↗
PubMed2006

High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.

Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.

Algorithms↗
PubMedDate not supplied

High throughput cloning and expression strategies for protein production.

Traditionally, the production of a recombinant protein requires a preliminary cloning step of the target gene into an expression vector before evaluating its cellular expression. Among current methods, site-specific recombination cloning techniques, which eliminate the use of restriction endonucleases and ligase, offer several advantages in the context of high throughput (HT) procedures. Rapid and highly efficient, the recombinational cloning technology is largely used for structural genomics and functional proteomics. However, the correct expression of some genes requires further optimization steps that are time-consuming and carried out at relatively late stages in the cloning-expression process. An alternative strategy is described where expression is tested in vitro before cloning the target gene. This technology, amenable to automation for HT studies, makes the expression of several hundreds of genes possible from PCR products in cell-free transcription-translation systems. Once this preliminary step is achieved, the PCR product, which gives satisfying expression levels, is selected, and then cloned in a plasmid for its cellular expression and perpetuation.

Animals↗
PubMed2003

Renaissance of immobilized catalysts. New types of polymer-supported catalysts, 'microencapsulated catalysts', which enable environmentally benign and powerful high-throughput organic synthesis.

Immobilized catalysts have been reinvestigated from two aspects; as keys to environmentally benign chemical processes and high-throughput organic synthesis for combinatorial chemistry. While most known polymer-supported catalysts are less active than the corresponding original catalysts, new types of polymer-supported catalysts, microencapsulated catalysts, have been developed. The catalysts were immobilized on to polymers using physical envelopment by polymer backbones and interaction between pi electrons of benzene rings of the polystyrenes used as polymer backbones and vacant orbitals of the catalysts. Microencapsulated Sc, Os, Pd and Ru catalysts have been successfully prepared and high activities have been attained. In all cases, no leaching of the catalysts occurred, and the immobilized catalysts were recovered quantitatively by simple filtration and reused without loss of activity. It is noted that this method enables direct immobilization of metals onto polymers, and that normally unstable species such as Pd(0)(PPh3) can be kept stable by this immobilization technique. It is expected that other metal catalysts can be immobilized using this microencapsulation technique.

Journal Article↗
PubMedDate not supplied

Novel statistical approach for primary high-throughput screening hit selection.

The standard activity threshold-based method (the "top X" approach), currently widely used in the high-throughput screening (HTS) data analysis, is ineffective at identifying good-quality hits. We have proposed a novel knowledge-based statistical approach, driven by the hidden structure-activity relationship (SAR) within a screening library, for primary hit selection. Application to an in-house ultrahigh-throughput screening (uHTS) campaign has demonstrated it can directly identify active scaffolds containing valuable SAR information with a greatly improved confirmation rate compared to the standard "top X" method (from 55% to 85%). This approach may help produce high-quality leads and expedite the hit-to-lead process in drug discovery.

Algorithms↗