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At least 1,567 records · Page 87Linked to original sources
PubMed2003

A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (EC 3.5.1.4.) is the enzyme responsible for the rapid degradation of lipid-derived chemical messengers such as anandamide, oleamide, and 2-arachidonoylglycerol. The pharmacological characterization of this enzyme in vivo has been hampered by the lack of selective and bioavailable inhibitors. We have developed a simple, radioactive, high-throughput-compatible assay for this enzyme based on the differential absorption of the substrate and its products to activated charcoal. The assay was validated using known inhibitors. It may be applied for the identification of new inhibitors from a compound library.

Absorption↗
PubMed2003

High throughput protein production for functional proteomics.

A major impact of genome projects on human health will be their contribution to the understanding of protein function. Proteins are the engines of biological systems, nearly all pharmaceuticals act on proteins and increasingly proteins themselves are used therapeutically. As biology enters the post-genomic era, researchers have begun to embrace the exciting opportunity of investigating proteins in high throughput (HT) experiments. The study of proteins includes a vast array of techniques ranging from enzyme catalysis assays to interaction and structural studies. Many of these methods depend on purified proteins. The discovery of thousands of novel protein-coding sequences and the increased availability of large cDNA collections provide the opportunity to investigate protein function in a systematic manner and at an unprecedented scale. This opportunity highlights the need for development of HT methods for protein isolation. This article describes the challenges faced and the approaches taken to develop proteome-scale protein expression systems.

Cell-Free System↗
PubMedDate not supplied

High-throughput identification of phage-derived imaging agents.

The use of phage-displayed peptide libraries is a powerful method for selecting peptides with desired binding properties. However, the validation and prioritization of "hits" obtained from this screening approach remains challenging. Here, we describe the development and testing of a new analysis method to identify and display hits from phage-display experiments and high-throughput enzyme-linked immunosorbent assay screens. We test the method using a phage screen against activated macrophages to develop imaging agents with higher specificity for active disease processes. The new methodology should be useful in identifying phage hits and is extendable to other library screening methods such as small-molecule and nanoparticle libraries.

Affinity Labels↗
PubMed2003

High-resolution, high-throughput magnetic paragraph sign resonance imaging of mouse embryonic paragraph sign anatomy using a fast gradient-echo sequence.

Embryonic development in normal and genetically modified mice is commonly analysed by histological sectioning. This procedure is time-consuming, prone to artefact, and results in the loss of three-dimensional (3D) information. Magnetic resonance imaging (MRI) of embryos has the potential of noninvasively acquiring a complete 3D data set. Published methods have used spin-echo techniques with inherently high signal-to-noise ratio (SNR); however, they required either perfusion of the embryo with a contrast agent, or prolonged acquisition times to improve contrast and resolution. Here, we show that a standard preparation (i.e. paraformaldehyde fixation) of 15.5 days post-coitum embryos followed by MRI using a fast gradient-echo sequence with T(1)-weighting achieves high resolution and high throughput for investigating mouse embryonic anatomy. 3D data sets were acquired in overnight experiments (<9 h) with an experimental resolution of approximately 25 microm(3). This spatial resolution is twofold higher than the values reported previously for comparable paraformaldehyde-fixed embryos, and it was obtained in less than a quarter of the time with sufficient SNR. Our approach combines speed, high resolution and contrast with a simple preparation technique and minimal operator time (<1 h). It allows rapid routine 3D characterisation of normal and abnormal mouse embryonic anatomy.

Anatomy, Cross-Sectional↗
PubMed2004

Fluorescence detection-based functional assay for high-throughput screening for MraY.

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N(epsilon)-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

Anti-Bacterial Agents↗
PubMed2004

High throughput screening of genetic polymorphisms by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

In the post genomic era, the screening of many different genetic polymorphisms in large populations represents a major goal that will facilitate the understanding of individual genetic variability in the development of multi factor diseases and in drug response and toxicities. The increasing interest in these pathogenetic and pharmacogenomic studies by both academic and pharmaceutical industry researchers has increased the demand for broad genome association studies. This demand has produced a boom in the development of new and robust high throughput screening methods for genotype analysis. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents an emerging and powerful technique for DNA analysis because of its high speed, accuracy, no label requirement, and cost-effectiveness. So far, many MALDI-TOF MS approaches have been developed for rapid screening of single nucleotide polymorphisms (SNPs), variable sequences repeat, epigenotype analysis, quantitative allele studies, and for the discovery of new genetic polymorphisms. The more established methods are based on single base primer extension and minisequencing implemented with new chemical features to overcome the limitations associated with DNA analysis using MALDI-TOF MS. These new promising methods of genotyping include both photochemical and other different chemical and enzyme cleavage strategies that facilitate sample automation and MS analysis for both real-time genotyping and resequencing screening. In this review, we analyze and discuss in depth the advantages and the limitations of the more recent developments in MALDI-TOF MS analysis for large-scale genomic studies applications.

Automation↗
PubMed1998

Automated high-throughput mass spectrometric analysis of synthetic oligonucleotides.

The use of automated sample preparation and data collection coupled with delayed ion extraction matrix-assisted laser desorption ionization with time-of-flight detection mass spectrometry (DE MALDI-TOF MS) enables the rapid mass analysis of oligonucleotide samples. We have incorporated a matrix-diluting and liquid-spotting automated work station and a DE MALDI mass spectrometer to provide high-throughput analysis of synthetic oligonucleotides. Automated sample preparation greatly speeds processing while the spotting function allows for precise sample placement on a 100-well sample plate. The DE MALDI MS typically determines the mass of each sample to within 0.13 +/- 0.10% of the theoretically expected mass. This mass accuracy allows confirmation of identity and detection of low-level impurities present in synthetic oligonucleotide samples with an unprecedented degree of confidence. Using this automation suite, sample preparation, spotting, and analysis of 100 samples ranging from 12 to 70 bases in length can be routinely completed in less than 90 min.

Calibration↗
PubMed2000

Anchor polymerase chain reaction display: a high-throughput method to resolve, score, and isolate dimorphic genetic markers based on interspersed repetitive DNA elements.

Genes which confer a disease when mutated, or for which population variability contributes to a quantitative trait such as longevity or disease susceptibility, can be localized in the genetic map by use of an appropriately dense set of polymorphic DNA markers. Here we describe an anchor PCR method for high-throughput genotyping, which can be used to amplify the DNA segments flanking an interspersed repetitive sequence such as a transposon, and to limit the number of product bands per reaction to facilitate marker resolution. We used this method to amplify and display DNA fragments flanking the Tc1 transposable elements from different strains of the nematode Caenorhabditis elegans, varying widely in insert number, and to analyze marker segregation in recombinant inbred lines generated from an interstrain cross. Since essentially all eukaryotic genomes contain abundant interspersed repeat families, many of which are dimorphic (for presence or absence of specific elements) among populations, this method can be used for rapid genotyping and fine-scale chromosomal mapping in many species, including those for which extensive mapping and sequencing data do not yet exist.

Animals↗
PubMed2007

High-throughput five minute microwave accelerated glycosylation approach to the synthesis of nucleoside libraries.

The Vorbrüggen glycosylation reaction was adapted into a one-step 5 min/130 degrees C microwave assisted reaction. Triethanolamine in acetontrile containing 2% water was determined to be optimal for the neutralization of trimethylsilyl triflate allowing for direct MPLC purification of the reaction mixture. When coupled with a NH3/methanol deprotection reaction, a high-throughput method of nucleoside library synthesis was enabled. The method was demonstrated by examining the ribosylation of 48 nitrogen containing heteroaromatic bases that included 25 purines, four pyrazolopyrimidines, two 8-azapurines, one 2-azapurine, two imidazopyridines, two benzimidazoles, three imidazoles, three 1,2,4-triazoles, two pyrimidines, two 3-deazapyrimidines, one quinazolinedione, and one alloxazine. Of these, 32 yielded single regioisomer products, and six resulted in separable mixtures. Seven examples provided inseparable regioisomer mixtures of -two to three compounds (16 nucleosides), and three examples failed to yield isolable products. For the 45 single isomers isolated, the average two-step overall yield +/- SD was 26 +/- 16%, and the average purity +/- SD was 95 +/- 6%. A total of 58 different nucleosides were prepared of which 15 had not previously been accessed directly from glycosylation/deprotection of a readily available base.

Databases, Genetic↗
PubMed2001

High-throughput genotyping of thiopurine S-methyltransferase by denaturing HPLC.

BACKGROUND: The thiopurine S:-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC). METHODS: To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR). RESULTS: The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals. CONCLUSIONS: DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.

Antimetabolites, Antineoplastic↗
PubMed1997

Monoclonal antibodies as surrogate receptors in a high throughput screen for compounds that enhance insulin sensitivity.

Monoclonal antibodies (MoAbs) were made to a known insulin sensitivity enhancer (ISE) compound, CS-045. The MoAbs were characterized with respect to binding other known thiazolidinedione ISE compounds using a CS-045 labeled with b-phycoerythrin in a competitive particle concentration fluorescence immunoassay (PCFIA). By comparing the rank order of IC50 values for each compound to its respective potency as an ISE, one MoAb (13E3) was selected for further characterization. This MoAb was also used as a surrogate receptor in a high throughput screen to identify novel compounds that compete for binding to CS-045. Some of the hits were found to have efficacy in reducing blood glucose. Subsequently, another group reported that several compounds with the core thiazolidinedione structure of the ISE compounds bound with high affinity to peroxisome proliferator-activating receptors (PPAR). Therefore, we used the MoAb assay to test these and other compounds that are known to bind to PPARgamma and noted crossreactivity with some of the compounds.

Animals↗
PubMed2006

High-throughput, library-based selection of a murine leukemia virus variant to infect nondividing cells.

Gammaretroviruses, such as murine leukemia virus (MLV), are functionally distinguished from lentiviruses, such as human immunodeficiency virus, by their inability to infect nondividing cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We utilized a transposon-based method to generate saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. High-throughput selection of the libraries via iterative retroviral infection of nondividing cells led to the identification of a novel variant that successfully transduced growth-arrested cells. Vector packaging by cotransfection of the gag-pol.NLS variant with wild-type gag-pol produced high-titer virions capable of infecting neurons in vitro and in vivo. The capacity of mutant virions to transduce nondividing cells could help to elucidate incompletely understood mechanisms of the viral life cycle and greatly broaden the gene therapy applications of retroviral vectors. Furthermore, the ability to engineer key intracellular viral infection steps has potential implications for the understanding, design, and control of other post-entry events. Finally, this method of library generation and selection for a desired phenotype directly in a mammalian system can be readily expanded to address other challenges in protein engineering.

Active Transport, Cell Nucleus↗
PubMed2007

Critical evaluation of methods to incorporate entropy loss upon binding in high-throughput docking.

Proper accounting of the positional/orientational/conformational entropy loss associated with protein-ligand binding is important to obtain reliable predictions of binding affinity. Herein, we critically examine two simplified statistical mechanics-based approaches, namely a constant penalty per rotor method, and a more rigorous method, referred to here as the partition function-based scoring (PFS) method, to account for such entropy losses in high-throughput docking calculations. Our results on the estrogen receptor beta and dihydrofolate reductase proteins demonstrate that, while the constant penalty method over-penalizes molecules for their conformational flexibility, the PFS method behaves in a more "DeltaG-like" manner by penalizing different rotors differently depending on their residual entropy in the bound state. Furthermore, in contrast to no entropic penalty or the constant penalty approximation, the PFS method does not exhibit any bias towards either rigid or flexible molecules in the hit list. Preliminary enrichment studies using a lead-like random molecular database suggest that an accurate representation of the "true" energy landscape of the protein-ligand complex is critical for reliable predictions of relative binding affinities by the PFS method.

Algorithms↗
PubMed2002

Software tools for high-throughput analysis and archiving of immunohistochemistry staining data obtained with tissue microarrays.

The creation of tissue microarrays (TMAs) allows for the rapid immunohistochemical analysis of thousands of tissue samples, with numerous different antibodies per sample. This technical development has created a need for tools to aid in the analysis and archival storage of the large amounts of data generated. We have developed a comprehensive system for high-throughput analysis and storage of TMA immunostaining data, using a combination of commercially available systems and novel software applications developed in our laboratory specifically for this purpose. Staining results are recorded directly into an Excel worksheet and are reformatted by a novel program (TMA-Deconvoluter) into a format suitable for hierarchical clustering analysis or other statistical analysis. Hierarchical clustering analysis is a powerful means of assessing relatedness within groups of tumors, based on their immunostaining with a panel of antibodies. Other analyses, such as generation of survival curves, construction of Cox regression models, or assessment of intra- or interobserver variation, can also be done readily on the reformatted data. Finally, the immunoprofile of a specific case can be rapidly retrieved from the archives and reviewed through the use of Stainfinder, a novel web-based program that creates a direct link between the clustered data and a digital image database. An on-line demonstration of this system is available at http://genome-www.stanford.edu/TMA/explore.shtml.

Cluster Analysis↗
PubMed2006

High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol.

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of approximately 14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.

Amino Acid Sequence↗
PubMed2005

Gene expression profiling of cerebellar development with high-throughput functional analysis.

We measured the expression levels of 450 genes during mouse postnatal cerebellar development by quantitative PCR using RNA purified from layers of the cerebellar cortex. Principal component analysis of the data matrix demonstrated that the first and second components corresponded to general levels of gene expression and gene expression patterns, respectively. We introduced 288 of the 450 genes into PC12 cells using a high-throughput transfection assay based on atelocollagen and determined the ability of each gene to promote neurite outgrowth or cell proliferation. Five genes induced neurite outgrowth, and seven genes enhanced proliferation. Evaluation of the functional data and gene expression patterns showed that none of these genes exhibited elevated expression at maturation, suggesting that genes characteristic of mature neurons are not likely to participate in neuronal development. These results demonstrate that functional data can facilitate interpretation of expression profiles and identification of new molecules that participate in biological processes.

Animals↗
PubMed2002

Extensive profiling of a complex microbial community by high-throughput sequencing.

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.

Animals↗
PubMed1999

High-throughput nonradioisotopic detection of picomole levels of phosphothreonine and phosphoserine containing peptides via biotinylation and enzyme-linked immunosorbent assay.

Determination of phosphorylation sites in proteins is usually accomplished using [gamma-32P]ATP. For low-abundance phosphoproteins, in vivo and intact cell studies usually require millicurie levels of 32P for a single experiment, making multiple experiments prohibitive. Here we describe a low picomole sensitivity, nonradioisotopic, high-throughput method for tracing phosphorylation sites in proteins and peptides. The method is based on in situ enzyme-linked immunosorbent assay (ELISA) plate biotinylation of nonphospho- and phosphopeptides, streptavidin capture, and ELISA detection using recently available anti-phospho-Thr and anti-phospho-Ser antibodies.

Amino Acid Sequence↗