scholarly journals Development and validation of a high-throughput transcriptomic biomarker to address 21st century genetic toxicology needs

2017 ◽  
Vol 114 (51) ◽  
pp. E10881-E10889 ◽  
Author(s):  
Heng-Hong Li ◽  
Renxiang Chen ◽  
Daniel R. Hyduke ◽  
Andrew Williams ◽  
Roland Frötschl ◽  
...  
Keyword(s):  
High Throughput ◽  
Human Cancer ◽  
High Sensitivity ◽  
Chromosome Damage ◽  
Genetic Toxicology ◽  
Chemical Screening ◽  
Cell Assays ◽  
Development And Validation ◽  
Positive Results

Interpretation of positive genotoxicity findings using the current in vitro testing battery is a major challenge to industry and regulatory agencies. These tests, especially mammalian cell assays, have high sensitivity but suffer from low specificity, leading to high rates of irrelevant positive findings (i.e., positive results in vitro that are not relevant to human cancer hazard). We developed an in vitro transcriptomic biomarker-based approach that provides biological relevance to positive genotoxicity assay data, particularly for in vitro chromosome damage assays, and propose its application for assessing the relevance of the in vitro positive results to carcinogenic hazard. The transcriptomic biomarker TGx-DDI (previously known as TGx-28.65) readily distinguishes DNA damage-inducing (DDI) agents from non-DDI agents. In this study, we demonstrated the ability of the biomarker to classify 45 test agents across a broad set of chemical classes as DDI or non-DDI. Furthermore, we assessed the biomarker’s utility in derisking known irrelevant positive agents and evaluated its performance across analytical platforms. We correctly classified 90% (9 of 10) of chemicals with irrelevant positive findings in in vitro chromosome damage assays as negative. We developed a standardized experimental and analytical protocol for our transcriptomics biomarker, as well as an enhanced application of TGx-DDI for high-throughput cell-based genotoxicity testing using nCounter technology. This biomarker can be integrated in genetic hazard assessment as a follow-up to positive chromosome damage findings. In addition, we propose how it might be used in chemical screening and assessment. This approach offers an opportunity to significantly improve risk assessment and reduce cost.

How To Improve the Dosing of Chemicals in High-Throughput in Vitro Mammalian Cell Assays

2019 ◽  
Vol 32 (8) ◽  
pp. 1462-1468 ◽  
Author(s):  
Fabian C. Fischer ◽  
Luise Henneberger ◽  
Rita Schlichting ◽  
Beate I. Escher
Keyword(s):  
Mammalian Cell ◽  
High Throughput ◽  
Cell Assays

Genetic toxicology studies in SALATRIM structured triacylglycerols. 2. Lack of genetic damage in in vitro mammalian cell assays and the in vivo micronucleus assay

1994 ◽  
Vol 42 (2) ◽  
pp. 521-527 ◽  
Author(s):  
Johnnie R. Hayes ◽  
Colette J. Rudd ◽  
Jon C. Mirsalis ◽  
James P. Bakke ◽  
Richard A. Winegar ◽  
...  
Keyword(s):  
Mammalian Cell ◽  
Micronucleus Assay ◽  
Genetic Damage ◽  
Genetic Toxicology ◽  
Cell Assays ◽  
Structured Triacylglycerols

scholarly journals Development and validation of a sensitive, high throughput bioassay of radiolabeled E. coli adhesion to uroepithelial cell in vitro

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Bridget D Mathison ◽  
Lindsey L Kimble ◽  
Kerrie L Kaspar ◽  
Christina Khoo ◽  
Boon P Chew
Keyword(s):  
High Throughput ◽  
E Coli ◽  
Uroepithelial Cell ◽  
Development And Validation

scholarly journals Development and Validation of a High-Throughput Screen for Inhibitors of SARS CoV and Its Application in Screening of a 100,000-Compound Library

2006 ◽  
Vol 12 (1) ◽  
pp. 33-40 ◽  
Author(s):  
William E. Severson ◽  
Nice Shindo ◽  
Mindy Sosa ◽  
Thomas Fletcher ◽  
E. Lucile White ◽  
...  
Keyword(s):  
High Throughput ◽  
Cytopathic Effect ◽  
High Throughput Screen ◽  
Compound Library ◽  
Hit Rate ◽  
Compound Libraries ◽  
Potential Inhibitors ◽  
Development And Validation ◽  
Large Compound

The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV–induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The “hit” rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro—compounds which will serve as excellent lead candidates for further evaluation. At a 10-μM concentration, 3 compounds with selective indexes (SI50) of > 53 were discovered.

scholarly journals A Strategy for Discovery of Novel Broad-Spectrum Antibacterials Using a High-Throughput Streptococcus pneumoniae Transcription/Translation Screen

2004 ◽  
Vol 9 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Steven D. Pratt ◽  
Caroline A. David ◽  
Candace Black-Schaefer ◽  
Peter J. Dandliker ◽  
Xiaoling Xuei ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
High Throughput ◽  
Broad Spectrum ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
In Vitro Toxicity ◽  
Bacterial Cells ◽  
Biochemical Activity ◽  
Cell Assays

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays. ( Journal of Biomolecular Screening 2004:3-11)

scholarly journals High-throughput chemical screening identifies AG-490 as a stimulator of aquaporin 2 membrane expression and urine concentration

2014 ◽  
Vol 307 (7) ◽  
pp. C597-C605 ◽  
Author(s):  
Naohiro Nomura ◽  
Paula Nunes ◽  
Richard Bouley ◽  
Anil V. Nair ◽  
Stanley Shaw ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Throughput ◽  
Membrane Expression ◽  
Chemical Screening ◽  
Principal Cells ◽  
Kidney Slices ◽  
Aquaporin 2 ◽  
Urine Production

A reduction or loss of plasma membrane aquaporin 2 (AQP2) in kidney principal cells due to defective vasopressin (VP) signaling through the VP receptor causes excessive urine production, i.e., diabetes insipidus. The amount of AQP2 on the plasma membrane is regulated by a balance of exocytosis and endocytosis and is the rate limiting step for water reabsorption in the collecting duct. We describe here a systematic approach using high-throughput screening (HTS) followed by in vitro and in vivo assays to discover novel compounds that enhance vasopressin-independent AQP2 membrane expression. We performed initial chemical library screening with a high-throughput exocytosis fluorescence assay using LLC-PK1 cells expressing soluble secreted yellow fluorescent protein and AQP2. Thirty-six candidate exocytosis enhancers were identified. These compounds were then rescreened in AQP2-expressing cells to determine their ability to increase AQP2 membrane accumulation. Effective drugs were then applied to kidney slices in vitro. Three compounds, AG-490, β-lapachone, and HA14-1 increased AQP2 membrane accumulation in LLC-PK1 cells, and both AG-490 and β-lapachone were also effective in MDCK cells and principal cells in rat kidney slices. Finally, one compound, AG-490 (an EGF receptor and JAK-2 kinase inhibitor), decreased urine volume and increased urine osmolality significantly in the first 2–4 h after a single injection into VP-deficient Brattleboro rats. In conclusion, we have developed a systematic procedure for identifying new compounds that modulate AQP2 trafficking using initial HTS followed by in vitro assays in cells and kidney slices, and concluding with in vivo testing in an animal model.

scholarly journals Risk-Based High-Throughput Chemical Screening and Prioritization using Exposure Models and in Vitro Bioactivity Assays

2015 ◽  
Vol 49 (11) ◽  
pp. 6760-6771 ◽  
Author(s):  
Hyeong-Moo Shin ◽  
Alexi Ernstoff ◽  
Jon A. Arnot ◽  
Barbara A. Wetmore ◽  
Susan A. Csiszar ◽  
...  
Keyword(s):  
High Throughput ◽  
In Vitro Bioactivity ◽  
Chemical Screening ◽  
Exposure Models

High-throughput and ultra-sensitive single-cell profiling of multiple microRNAs and identification of human cancer

2019 ◽  
Vol 55 (70) ◽  
pp. 10404-10407 ◽  
Author(s):  
Lu Li ◽  
Meng Lu ◽  
Yuanyuan Fan ◽  
Lingling Shui ◽  
Shuting Xie ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Efficient Method ◽  
Human Cancer ◽  
High Sensitivity ◽  
Droplet Microfluidics ◽  
Single Cell Profiling

We established an efficient method for single-cell multiple miRNA analysis by droplet microfluidics with high sensitivity and high throughput.

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