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Results for “High Throughput Screening”

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At least 1,189 records · Page 66Linked to original sources
PubMed2001

A high-capacity LC/MS system for the bioanalysis of samples generated from plate-based metabolic screening.

HPLC/MS is a linear technique characterized by serial injection and analysis of individual samples. Parallel-format high-throughput screens for druglike properties present a significant analytical challenge. Analysis speed and system ruggedness are key requirements for bioanalysis of thousands of samples per day. The tasks involved in LC/MS analysis are readily divided into three areas, sample preparation/liquid handling, LC/MS method building/sample analysis, and data processing. Several automation and multitasking strategies were developed and implemented to minimize plating and liquid handling errors, reduce dead times within the analysis cycle, and allow for comprehensive review of data. Delivering multiple samples to multiple injectors allows the autosampler time to complete its wash cycles and aspirate the next set of samples while the previous set is being analyzed. A dual-column chromatography system provides column cycling and peak stacking and allows rapid throughput using conventional LC equipment. Collecting all data for a compound into a single file greatly reduces the number of data files collected, increases the speed of data collection, allows rugged and complete review of all data, and provides facile data management. The described systems have analyzed over 40 000 samples per month for two years and have the capacity for over 2000 samples per instrument per day.

Automation↗
PubMed2004

A rapid microtitre plate screening method for in vitro assessment of fibrinolysis: a preliminary report.

A novel and precise assay that facilitates high-throughput screening of fibrinolytic agents was developed based on the automated assessment of the euglobulin clot lysis time in microtitre plates. Euglobulin fractions from fresh plasma samples were assessed over 28 days to determine the inter-assay and intra-assay precision. The intra-assay (coefficient of variation range, 0.7-2.6%) and inter-assay precision (coefficient of variation range, 6.8-12.1%) was found to be well within limits required by the Food and Drug Administration. On day 1 and day 28, the results of the microtitre plate euglobulin clot lysis time method were compared with tissue plasminogen activator activity, plasminogen activator inhibitor activity and results produced on fibrin plates. All comparisons were found to correlate significantly. The validity of this method for assaying fibrinolytic agents was assessed by comparing dose-response curves for streptokinase produced using fibrin plates and this method. The critical influence of ambient temperature on the inter-assay reproducibility of this method was established by testing samples over a range of temperatures between 20 degrees C and 40 degrees C.

Adult↗
PubMed2003

[Screening C282Y mutation with double-stranded probes using synchronous fluorometry].

We described in the paper a new high-throughput screening method for Cys282Tyr mutation in hereditary haemochromatosis with double-stranded probe using synchronous fluorometry. The probe for wild type was labeled with Fam, the probe for mutant type was labeled with Joe. After PCR, reaction tubes were transferred to a spectrofluorometer, where synchronous spectra were scanned in a constant-wavelength mode. The genotype could be obtained through the appearance of the fluorescence peaks corresponding to each probe. The results were totally in agreement with restriction endonuclease analysis. Considering the simplicity,low cost and specificity, this approach could be generally applied to detect varieties of gene mutations.

English Abstract↗
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↗
PubMed1999

Screening for oligonucleotide binding affinity by a convenient fluorescence competition assay.

A competitive homogeneous quenched fluorescence assay system is described for the high throughput screening of DNA conjugates that bind to single-stranded DNA. Fluorescence signal is generated by competitive binding of the sample molecule to a target strand labelled with a quencher probe, which is otherwise hybridised to a complementary strand containing a fluorescent probe. Thus fluorescence generated is related to the affinity of the sample. Competitive analysis of a number of peptide-oligonucleotide conjugates gave data that correlated well with the corresponding UV melting data. The assay will be useful for screening of large numbers of potential single-stranded binding molecules.

Chromatography, High Pressure Liquid↗
PubMed2001

Phage display as a tool for rapid cloning of allergenic proteins.

Allergic diseases represent an immune disorder associated with the production of immunoglobulin E (IgE) against normally innocuous antigens (allergens). Almost 20% of the population in industrialized countries suffer from type I allergic symptoms such as allergic rhinitis, conjunctivitis, urticaria or asthma. Although the mechanisms responsible for these allergic reactions are quite well understood, knowledge about the repertoire of molecules able to elicit type I symptoms is still limited. To clone and characterize entire allergen repertoires from complex allergenic sources in a fast and efficient way, new technologies are required. The phage surface display of cDNA libraries described here has proven to be a versatile cloning system to selectively isolate allergens physically linked to their genetic information. The screening of cDNA libraries displayed on phage surfaces with immobilised serum IgE from allergic patients reduces the time required for the selection of candidate clones to a few weeks. Robot-assisted high-throughput screening of the enriched library provides a fast and cost-effective way to isolate complete allergen repertoires. The biotechnological production of recombinant allergens derived from these sequences bears a high potential for the improvement of the diagnosis of allergic diseases.

Allergens↗
PubMed2005

Transforming science: cancer gene identification.

Methods for cancer gene discovery include identification of viral oncogenes, identification of genes associated with recurrent chromosomal aberrations, and screens for genes capable of the transformation of cells in culture. In recent years, the completed genome sequence of human and model organisms has markedly enhanced cancer gene identification. Whole genome, high-throughput screens have been facilitated by the advent of new technologies such as murine leukemia virus-based mutagenesis, Sleeping Beauty-based mutagenesis, RNA interference, exon re-sequencing, and high-resolution methods for detecting chromosomal amplifications and deletions; these, in turn, have led to the identification of novel tumor suppressors and oncogenes. The identification of genes that are altered by mutation or expression and which are directly involved in tumor initiation and maintenance will be instrumental for understanding cancer phenotypic variation and for identifying crucial therapeutic targets.

Animals↗
PubMed1997

Random mutagenesis screen for dominant behavioral mutations in mice.

Large-scale mutagenesis and screening for altered phenotypes have been used effectively in many (lower) model organisms to identify mutations in genes that control biological processes. In the mouse, the cost of maintaining the large breeding colonies necessary to screen for recessive mutations makes it important to consider alternate approaches such as region-specific saturation mutagenesis or screening for mutations with a dominant mode of inheritance. In this article, a pilot screen for (semi)dominant visible and behavioral mutations in the mouse induced by a potent chemical mutagen, N-ethyl-N-nitrosourea (ENU), is described. An efficient protocol for ENU mutagenesis and strain-specific differences in the effect of mutagen on the sterility period and long-term survival are reported. In addition to a description of the screen for abnormal circadian wheel running activity that was used previously, the suitability of a high-throughput screen of mutagenized progeny in the Porsolt swim test, used to test the efficacy of antidepressant agents, and in the prepulse inhibition of the acoustic startle response, used to detect anomalies in sensorimotor gating, is tested. By demonstrating strain specific differences and prescreening 100 G1 progeny of mutagenized males, the feasibility of using these behavioral assays for a large-scale screen is illustrated. In this review, details of a mutagenesis screen for behavioral abnormalities are described and issues important in the initial characterization of novel ENU-induced mutations are considered.

Animals↗
PubMed2005

3,4-Dihydro-2H-benzoxazinones are 5-HT(1A) receptor antagonists with potent 5-HT reuptake inhibitory activity.

Starting from a high throughput screening hit, a series of 3,4-dihydro-2H-benzoxazinones has been identified with both high affinity for the 5-HT(1A) receptor and potent 5-HT reuptake inhibitory activity. The 5-(2-methyl)quinolinyloxy derivative combined high 5-HT(1A/1B/1D) receptor affinities with low intrinsic activity and potent inhibition of the 5-HT reuptake site (pK(i)8.2). This compound also had good oral bioavailability and brain penetration in the rat.

Animals↗
PubMed2005

Screening of active compounds as neuromedin U2 receptor agonist from natural products.

Reporter cell lines are often used for high-throughput screening of natural product libraries or chemical libraries to identify new receptor ligands. Screening of neuromedin U2 receptor (NMU2R) ligands may be very useful to treat obesity for the reason that centrally administered neuromedin U affects feeding behavior, energy expenditure, and pituitary. Here, we have developed a stable cell line of neuromedin U2 receptor (NMU2R) to screen for its agonist. The experimental results demonstrate that icariin, isolated from Herba epimedii, was a strong agonist for NMU2R, which could selectively activate NMU2R, but not M1R, MC4R, and negative cell lines.

Base Sequence↗
PubMed2006

Using small molecules to overcome drug resistance induced by a viral oncogene.

We used small molecule screening to discover compounds and mechanisms for overcoming E6 oncogene-mediated drug resistance. Using high-throughput screening in isogenic cell lines, we identified compounds that potentiate doxorubicin's lethality in E6-expressing colon cancer cells. Such compounds included quaternary ammonium salts, protein synthesis inhibitors, 11-deoxyprostaglandins, and two additional classes of compounds-analogs of 1,3-bis(4-morpholinylmethyl)-2-imidazolidinethione (a thiourea) and acylated secondary amines that we named indoxins. Indoxins upregulated topoisomerase IIalpha, the target of doxorubicin, thereby increasing doxorubicin lethality. We developed a photolabeling strategy to identify targets of indoxin and discovered a nuclear actin-related protein complex as a candidate indoxin target.

Alprostadil↗
PubMed2003

Homogeneous assays for adenosine 5'-monophosphate-activated protein kinase.

Allosteric activation of 5(')-AMP-activated protein kinase (AMPK) is currently of interest as an approach for the treatment of metabolic disorders because AMPK plays multiple roles in glucose and lipid metabolism. The availability of ultrafast, ultrasensitive, and robust assays suited to high-throughput screening (HTS) is key to obtaining small-molecule AMPK activators. In the absence of high-affinity and selective antiphospho Ser/Thr antibodies for AMPK substrates, we have developed two homogeneous AMPK assays with the commercially available antibody Anti-pS(133)-CREB and an engineered peptide ACC-CREBp. Anti-pS(133)-CREB antibody was raised against the phospho-CREB peptide derived from cAMP response element binding protein (CREB). ACC-CREBp was a variant (Arg to Pro) of ACC-CREB, a hybrid peptide consisting of a 9-amino-acid peptide from rat acetyl-CoA carboxylase (ACC), CREB peptide, and the addition of two hydrophobic Leu residues. ACC-CREBp showed increased suitability as a substrate for AMPK, eliminated phosphorylation by PKA, and preserved antibody binding. The homogeneous time-resolved fluorescence and AlphaScreen AMPK assays were developed using both Anti-pS(133)-CREB antibody and ACC-CREBp that are either labeled with a fluorescent probe or linked to a photoactivated bead, respectively. Thus, ACC-CREBp phosphorylation can be measured as a signal change resulting from the formation of antibody-antigen complex. This approach of adapting known antibody and antigenic peptide pairs to monitor alternate Ser/Thr kinases may be of general use.

AMP-Activated Protein Kinases↗
PubMed1996

Improved green fluorescent protein by molecular evolution using DNA shuffling.

Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

Animals↗
PubMed2005

Generation of cell lines for drug discovery through random activation of gene expression: application to the human histamine H3 receptor.

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

Binding, Competitive↗
PubMed2006

A morphology- and kinetics-based cascade for human neural cell high content screening.

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits represents a novel mechanism for treating a variety of central nervous system disorders. Using human neural precursor cultures and a variety of assays for studying stem cell behavior we have screened two libraries of commercially available compounds using an endpoint high content screening assay. We then performed detailed follow-up mechanistic studies on confirmed hits using endpoint and kinetics assays to characterize and differentiate the mechanisms of action of these compounds. The screening cascade employed successfully identified a number of active compounds with differing mechanisms of action. This approach shows how hits from a phenotypic screen can be prioritized and characterized by high content screening to identify potentially novel mechanisms and druggable targets to take forward into more conventional high-throughput screening approaches.

Animals↗
PubMed2004

Screening for presenilin inhibitors using the free-living nematode, Caenorhabditis elegans.

Caenorhabditis elegans contains 3 homologs of presenilin genes that are associated with Alzheimer s disease. Loss-of-function mutations in C. elegans genes cause a defect in egg laying. In humans, loss of presenilin-1 (PS1) function reduces amyloid-beta peptide processing from the amyloid protein precursor. Worms were screened for compounds that block egg laying, phenocopying presenilin loss of function. To accommodate even relatively high throughput screening, a semi-automated method to quantify egg laying was devised by measuring the chitinase released into the culture medium. Chitinase is released by hatching eggs, but little is shed into the medium from the body cavity of a hermaphrodite with an egg laying deficient (egl) phenotype. Assay validation involved measuring chitinase release from wild-type C. elegans (N2 strain), sel-12 presenilin loss-of-function mutants, and 2 strains of C. elegans with mutations in the egl-36 K(+) channel gene. Failure to find specific presenilin inhibitors in this collection likely reflects the small number of compounds tested, rather than a flaw in screening strategy. Absent defined biochemical pathways for presenilin, this screening method, which takes advantage of the genetic system available in C. elegans and its historical use for anthelminthic screening, permits an entry into mechanism-based discovery of drugs for Alzheimer's disease.

Animals↗
PubMed2005

Medical nanotechnology: shortening clinical trials and regulatory pathways?

Nanotechnology, the science of creating structures, devices, and systems with a length scale of approximately 1-100 nanometers, is poised to have a revolutionary effect on biomedical research and clinical science. By operating at the same scale as most biomacromolecules, nanoscale devices can afford a detailed view of the molecules and events that drive cellular systems and that lie at the heart of disease, and thus, nanotechnology can impact the drug discovery, development, and clinical testing of novel pharmaceuticals. Already, nanoscale drug delivery vehicles are in clinical use, but those successes represent just one way in which nanotechnology will prove useful. One promising nanoscale technology under development may provide real-time, in vivo measurements of apoptosis, and thus may afford an early signal of therapeutic efficacy, both in human clinical trials and in preclinical screening. Microfluidic systems, built of nanoscale components, can enable a host of rapid, massively parallel, high-throughput screening systems, while nanoscale sensors in a wide variety of formats are ready to provide multiplexed biochemical and genetic measurements in living systems. These advances could be utilized to shave time and expense from multiple stages of the drug discovery and development effort.

Biomedical Research↗