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Results for “High Throughput Screening”

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At least 793 records · Page 44Linked to original sources
PubMed2004

High-throughput differential screening of mRNAs by serial analysis of gene expression: decreased expression of trefoil factor 3 mRNA in thyroid follicular carcinomas.

To find mRNAs whose expression differs between thyroid follicular adenomas and carcinomas, a high-throughput analysis of mRNAs in these two tumours was performed. This method, named high-throughput differential screening by serial analysis of gene expression (HDSS), combines a modified method of serial analysis of gene expression (SAGE) and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). A total of 40 candidate tag sequences that showed extremely different expression levels between a follicular carcinoma and a follicular adenoma in the SAGE analysis were analysed by real-time quantitative RT-PCR, using RNAs from an additional four typical follicular carcinomas and adenomas. One sequence tag that represents trefoil factor 3 (TFF3) mRNA showed a clear difference in its expression level between adenomas and carcinomas. The expression levels of TFF3 mRNA in 48 follicular adenomas and 29 follicular carcinomas were measured by real-time quantitative RT-PCR using a specific probe for TFF3. They were significantly decreased in follicular carcinomas, especially in widely invasive types and those with evident metastases. These results indicate that the decreased expression of TFF3 mRNA is a marker of follicular carcinomas, especially those with a high risk of invasion or metastasis.

Adenocarcinoma, Follicular↗
PubMed1991

Endothelin-1 receptor binding assay for high throughput chemical screening.

An endothelin-1 (ET-1) receptor-binding assay, in microtiter format, has been developed for use in high throughput chemical or natural product screening. A rat smooth muscle, clonal cell line derived from embryonic thoracic aorta and designated A10 has been shown to consistently express high-affinity ET-1 receptors. A 96-well microtiter filtration plate, with individual PVDF membranes attached to the bottom of each well, was used for separation. In saturation binding assays, using [125I]Tyr13-ET-1, Scatchard analysis was monophasic, indicating a single high-affinity population of receptors with Kd = 0.12 nM with approximately 40,000 receptors per cell. The Ki values for peptides, known to bind at the ET receptor, were as follows: ET-1, 0.14 nM; ET-2, 0.16 nM; sarafotoxin S6b, 0.6 nM; VIC, 0.2 nM; ET-3, 16 nM; and big human ET, greater than 1 microM. ET receptors on A10 cells were stable for at least 2 months when stored at -20 degrees C. The assay is suitable for automation, because it is stable and reproducible. This method gave a 90% reduction in radioactive waste compared to tissue homogenate assays that use glass fiber filtration and cell harvesters.

Animals↗
PubMed2025

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Reverse Genetics↗
PubMed2005

Novel approaches to plant drug discovery based on high throughput pharmacological screening and genetic manipulation.

Plants are potentially important for novel therapeutic drug leads, but the slowness of conventional methods for investigation of plants limits enthusiasm in the pharmaceutical industry. To overcome some of the drawbacks, we have applied high throughput pharmacological screening (HTPS) to crude plant extracts. Using a "differential smart screen", (DSS) the spectrum of activity contained in a crude extract is measured at several closely related receptor subtypes. This spectrum is then compared to that of known compounds. A unique spectrum suggests that the extract merits further investigation. Evaluation of species and environmental libraries of whole plants has demonstrated the value of this approach for rapid prioritization of plants for investigation. In addition, genomic and genetic manipulation of plants and plant cell cultures can increase the value of DSS. For example, the whole genomic potential of a plant species for biodiversity can be accessed by using gain of function mutations to generate a "functional genomics library" of mutant clonal cultures, and the bioactivity of these cultures tested by DSS. Clones that overproduce activity differing from the wild-type plant can be identified in this way. This "Natural Products Genomics" (NPG) strategy is limited by the massive numbers of clonal cultures that are required to cover all possible gain-of-function mutations. The rapidity and efficiency of this process can be improved by using transgenic plants expressing appropriate mammalian proteins. These may be designed to make the plant cell resemble a human cell for a specific form of toxicity. Now, "unnatural selection" of resistant mutant clones can be used to provide a sub-population potentially enriched in useful compounds. Alternatively, transgenic plant cells can be used for "in situ screening" in which a mammalian receptor protein, linked to a reporter construct, such as green fluorescent protein, is expressed. Clonal cultures that produce ligands for this receptor can now be rapidly identified visually in an ultra-HTPS. Overall, our aim is to use pharmacological screening, together with functional genomic approaches, to make plant drug discovery competitive with combinatorial chemistry.

Drug Evaluation, Preclinical↗
PubMed2005

Cell microarray for screening feeder cells for differentiation of embryonic stem cells.

Microarrays are currently recognized as one of major tools in the assessment of gene expression via cDNA or RNA analysis and are now accepted as a powerful experimental tool for high-throughput screening of a large number of samples, such as cDNA and siRNAs. In this study, we examined the potential of the microarray methodology for high-throughput screening of candidate cells as feeder cells which effectively differentiate embryonic stem (ES) cells to the specific lineage. Cell arrays were prepared by applying three kinds of cells, PA6, human umbilical vein endothelial, and COS-1 cells, to circular spots, 2 mm in diameter, on a glass plate, followed by the application of mouse ES cells to the cell microarray. After 8 d in culture, TuJ1 (neuron-specific class III beta-tubulin) immunocytochemical staining clearly demonstrated that only PA6 cell spots had the capability to induce ES cells to neuronal differentiation. Although this is a model experiment, these findings clearly indicate that the cell microarray will become a powerful tool for high-throughput screening large numbers of candidate feeder cells for specific differentiation.

Animals↗
PubMed2000

High throughput mutation screening by automated capillary electrophoresis.

Molecular diagnosis of complex inherited disorders, population screening of genetic diseases, studies of the genetic basis of variable drug response (pharmacogenetics) as well as discovery and investigation of new drug targets (pharmacogenomics) involve screening for mutations in multiple DNA samples. Furthermore, the development of a third generation of the human genome map, based on single nucleotide polymorphisms (SNPs), requires screening for allelic variants through all of the three billion basepairs in the human genome. Thus, the need for high throughput mutation screening methods is great and is rapidly increasing. Traditional methods for mutation screening often involve slab-gel electrophoresis analyses which are laborious and difficult to automate. However, recent developments in capillary electrophoresis systems for DNA fragment analysis have made fully automated mutation screening possible and have dramatically increased the possible sample throughput. This review describes the recent advances in capillary electrophoresis of DNA and summarize the various methods for mutation screening based on this technique.

Automation↗
PubMed2003

Bioluminescence and chemiluminescence in drug screening.

Drug screening, that is, the evaluation of the biological activity of candidate drug molecules, is a key step in the drug discovery and development process. In recent years, high-throughput screening assays have become indispensable for early stage drug discovery because of the developments in synthesis technologies, such as combinatorial chemistry and automated synthesis, and the discovery of an increasing number of new pharmacological targets. Bioluminescence and chemiluminescence represent suitable detection techniques for high-throughput screening because they allow rapid and sensitive detection of the analytes and can be applied to small-volume samples. In this paper we report on recent applications of bioluminescence and chemiluminescence in drug screening, both for in vitro and in vivo assays. Particular attention is devoted to the latest and most innovative bioluminescence and chemiluminescence-based technologies for drug screening, such as assays based on genetically modified cells, bioluminescence resonance energy transfer (BRET)-based assays, and in vivo imaging assays using transgenic animals or bioluminescent markers. The possible relevance of bioluminescence and chemiluminescence techniques in the future developments of high-throughput screening technologies is also discussed.

Amoxicillin↗
PubMed2000

High throughput production, screening and analysis of adeno-associated viral vectors.

Recombinant adeno-associated viruses (rAAV) are promising candidates as gene vectors, as they transduce non-dividing cells and permit lasting transgene expression in a wide spectrum of tissues. In this paper, we describe a robust procedure for the high throughput production, screening and characterization of rAAV vectors. The technology includes the production of rAAV from rapid small scale plasmid preparations and the analysis of virus productivity (physical and infectious particles) and activity (transgene expression, replication). rAAV are produced by triple transfection (rAAV plasmid and AAV- and adenovirus (Ad)-helper plasmids) on 293 human embryo kidney (HEK) cells. The titers of physical and infectious particles are obtained by dot blot hybridization and by a serial dilution assay, followed by either dot blot hybridization or real-time PCR, respectively. rAAV can be produced and characterized from plasmid mixtures containing as little as 1/100 productive molecules. Experiments on rAAV replication kinetics and Ad helper functions are discussed. All steps are performed in 96-well microtiter plates. The process is reproducible, high throughput, linear and ready for automation.

Cell Line↗
PubMed2001

The use of a high-volume screening procedure to assess the effects of dietary flavonoids on human cyp1a1 expression.

We examined the effects of several agents, including dietary flavonoids, on CYP1A1 expression utilizing a recently developed high-throughput screening system for assessing human cytochrome P450 (CYP) induction. HepG2 cells, stably integrated with regulatory regions of human CYP1A1, were treated with resveratrol, apigenin, curcumin, kaempferol, green tea extract (GTE), (-)-epigallocatechin gallate (EGCG), quercetin, and naringenin. Of these flavonoids, resveratrol produced the greatest increase in CYP1A1-mediated luciferase activity (10-fold), whereas GTE, apigenin, curcumin, and kaempferol produced 2- to 3-fold increases in activity. Compared with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole, or benzanthracene, where increases in luciferase activity ranged from 12- to 35-fold, these flavonoids exhibited weak agonist activity. The remaining compounds, EGCG, quercetin, and naringenin, produced negligible effects. Cotreatment of cells with TCDD and GTE, naringenin, and apigenin resulted in 58, 77, and 74% reductions, respectively, in TCDD-mediated CYP1A1 induction, indicating that these flavonoids exhibit potential antagonist activity toward the aryl hydrocarbon (Ah) receptor. Furthermore, results also suggest that GTE and apigenin possess Ah receptor antagonist and weak agonist activities. Thus, we have shown that a 96-well plate assay allowing high-throughput screening for P450 induction in less than 24 h was efficient in determining the effects of flavonoids on human CYP1A expression. Signal-to-noise ratios were low, and well-to-well and replicate variability was below 10%, allowing induction to be easily detected in this system. These features illustrate the reliability and feasibility of this high-volume screening system for identifying CYP inducers. Furthermore, results produced with the stable cell line were corroborated in HepG2 cells and primary cultures of human hepatocytes, suggesting that stably integrated cell lines harboring enhancer elements of P450 genes may be highly conducive to high-throughput screening.

Benz(a)Anthracenes↗
PubMed2006

High-throughput, functional screening of the anti-HIV-1 humoral response by an enzymatic nanosensor.

The impact of antibodies on the target's epitope conformation is a major determinant of HIV-1 neutralization and a potential contributor to disease progression. We explore here a conformation-sensitive enzymatic nanosensor for the high-throughput functional screening of human anti-HIV-1 antibodies in sera. When displaying a model epitope from a gp41 immunodominant region (Env residues from 579 to 613), the sensing signal quantitatively distinguishes between adaptive and non-adaptive antibody binding. By using this tool, we have identified IgG4 as the immunoglobulin subpopulation most efficient in the structural modification of the target epitope.

Antibodies, Viral↗
PubMed2006

Development of high-throughput functional screening of therapeutic genes, using a hemagglutinating virus of Japan envelope vector.

Isolation of effective therapeutic genes is critical for the advancement of gene therapy for various diseases, including vascular diseases and cancers. The goal of the present study was to screen a human cDNA library, using a hemagglutinating virus of Japan envelope (HVJ-E) vector, to isolate candidate genes with potent therapeutic potential. The advantages of a high-throughput functional screening system based on the HVJ-E vector include (1) rapid preparation of the vector containing the DNA library, (2) effective fusion-mediated transfer of the plasmids to various cells with minimal toxicity, and (3) easy cloning of candidate genes by transformation of Escherichia coli. These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes. Screening of a human heart library for candidate genes to regulate endothelial cell growth identified three growth-stimulating genes, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and c-fos promoter activity, the products of which were more potent than vascular endothelial growth factor. Similarly, two growth-inhibiting genes were identified, the effects of which were similar to angiostatin. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.

Cell Line↗
PubMed2001

Combinatorial and Evolution-Based Methods in the Creation of Enantioselective Catalysts.

Combinatorial methods in the development of enantioselective homogeneous catalysts constitute a new branch of catalysis research. The goal is to prepare libraries of potential asymmetric catalysts, rather than choosing the traditional one-catalyst-at-a-time approach. Several conceptional advancements have been reported in the parallel preparation of chiral ligands. Currently the most meaningful systems constitute modularly constructed ligands on solid supports, which allow high degrees of structural diversity and thus the maximum probability of finding enantioselective catalysts or even new types of ligands for asymmetric catalysis. Search strategies have been developed which amongst other things, lead to catalysts not likely to have been discovered by traditional methods. Genuine application of such strategies involve thousands of catalysts and require high-throughput screening systems capable of assaying enantioselectivity. The first high-throughput ee-screening systems were in fact developed for use in the directed evolution of enantioselective enzymes, a process based on "evolution in the test tube" in which the appropriate methods of random mutagenesis, gene expression, and ee assays are combined. Since no screening system is likely to be universal, different approaches are necessary. Thus far these include assays based on UV/Vis, fluorescence, circular dichroism, mass spectrometry, and even modified gas chromatography as well as special forms of capillary electrophoresis. One of the most efficient systems involves the concept of the mass-spectrometric detection of deuterium-labeled pseudo-enantiomers and pseudo-prochiral compounds with which about 1000 exact ee determinations can be achieved per day, although the assay is restricted to kinetic resolution and/or reactions of prochiral compounds bearing enantiotopic groups. Super-high-throughput screening for enantioselectivity is possible in many cases by making use of chirally modified capillary array electrophoresis in a parallel step. Accordingly, 7000 to 30 000 ee determinations can be carried out per day. These and other analytical developments are expected to stimulate further research in the combinatorial search for asymmetric homogeneous catalysts and in the directed evolution of enantioselective enzymes for use in organic chemistry.

Journal Article↗
PubMed2006

Automated MEMS-based Drosophila embryo injection system for high-throughput RNAi screens.

We have developed an automated system based on microelectromechanical systems (MEMS) injectors for reliable mass-injection of Drosophila embryos. Targeted applications are high-throughput RNA interference (RNAi) screens. Our injection needles are made of silicon nitride. The liquid to be injected is stored in an integrated 500 nl reservoir, and an externally applied air pressure pulse precisely controls the injected volume. A steady-state water flow rate per applied pressure of 1.2 nl s(-1) bar(-1) was measured for a needle with channel width, height and length of 6.1 microm, 2.3 microm and 350 microm, respectively. A typical volume of 60 pl per embryo can be reliably and rapidly delivered within tens of milliseconds. Theoretical predictions of flow rates match measured values within +/-10%. Embryos are attached to a glass slide surface and covered with oil. Packages with the injector chip and the embryo slide are mounted on motorized xyz-stages. Two cameras allow the user to quickly align the needle tip to alignment marks on the glass slide. Our system then automatically screens the glass slide for embryos and reliably detects and injects more than 98% of all embryos. Survival rates after deionized (DI) water injection of 80% and higher were achieved. A first RNAi experiment was successfully performed with double-stranded RNA (dsRNA) corresponding to the segment polarity gene armadillo at a concentration of 0.01 microM. Almost 80% of the injected embryos expressed an expected strong loss-of-function phenotype. Our system can replace current manual injection technologies and will support systematic identification of Drosophila gene functions.

Animals↗
PubMed2006

High-throughput RNAi screening by time-lapse imaging of live human cells.

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

Automation↗
PubMed2006

A High-throughput two-phase partition assay to measure the activity of lipid-metabolizing enzymes.

A new method to measure the activity of lipid-metabolizing enzymes is described. Subsequent to an enzymatic reaction, a two-phase system (organic/aqueous) is established by the addition of a phase partition scintillation fluid (PPSF). The PPSF serves as a scintillation fluid, a phase partition agent, and a carrier/separator of an organic-soluble radiolabeled reaction substrate or product. Applying an empirically derived set of conditions typically enhances the separation of substrate from product whereby one species is effectively solubilized in the PPSF. In situ partitioning of the radionuclide-containing organic/lipid phase from the aqueous phase occurs within individual wells of 96-well or 384-well density PPSF-resistant microtiter plates without the requirement for multiple organic solvent extractions and aspirations, making this method applicable to high-throughput screening. The utility of this method for both kinetic characterization and high-throughput screening is demonstrated with acetyl-CoA carboxylase and fatty acid synthase.

Acetate-CoA Ligase↗
PubMed2006

A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.

Alcohol Dehydrogenase↗
PubMedDate not supplied

Use of fluorescence polarization detection for the measurement of fluopeptidetm binding to G protein-coupled receptors.

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 microL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.

Binding, Competitive↗
PubMed2003

Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch hydrolysis.

High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of reducing agents, particularly the glucose used in yeast growth media. The present investigation employed colorimetric and chemiluminescent detection systems that enable direct and rapid screening of activities on raw starch substrate. Active clones could be separated into two groups, based on high total activity or high specific activity.

Base Sequence↗