Development and Application of a High Throughput Assay System for the Detection of Rieske Dioxygenase Activity

2021 ◽  
Author(s):  
Cristina Preston-Herrera ◽  
Aaron S Jackson ◽  
Brian O Bachmann ◽  
Jordan Thomas Froese
Keyword(s):  
High Throughput ◽  
Assay System ◽  
Fluorescent Detection ◽  
System P ◽  
Rieske Dioxygenase ◽  
High Throughput Assay ◽  
Dioxygenase Activity

Herein we report the development of a new periodate-based reactive assay system for the fluorescent detection of the cis-diol metabolites produced by Rieske dioxygenases. This sensitive and diastereoselective assay system...

A Cell-based High-throughput Assay System Reveals Modulation of Oxidative and Nonoxidative Glucose Metabolism due to Commonly Used Organic Solvents

2008 ◽  
Vol 40 (1) ◽  
pp. 29-37 ◽  
Author(s):  
S. Zimmermann ◽  
K. Zarse ◽  
T. Schulz ◽  
K. Siems ◽  
L. Müller-Kuhrt ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Organic Solvents ◽  
High Throughput ◽  
Assay System ◽  
A Cell ◽  
High Throughput Assay

scholarly journals GigaAssay – a high-throughput assay system for molecular functions and cell processes

2021 ◽  
Author(s):  
Martin Schiller ◽  
Ronald Benjamin ◽  
Christopher Giacoletto ◽  
Zachary FitzHugh ◽  
Daniel Eames ◽  
...  
Keyword(s):  
Structure Function ◽  
High Throughput ◽  
Assay System ◽  
Molecular Function ◽  
Typical Structure ◽  
Holistic View ◽  
Human Pathology ◽  
Cell Processes ◽  
Molecular Functions ◽  
High Throughput Assay

Abstract High-throughput assay systems have had a disproportionally large impact on uncovering how cells function, as well as how misregulation can lead to disease. However, no high-throughput assay systems have been developed to systematically address how mutations impact molecular functions or cell processes in human cells. This is arguably one of the most critical assays because human pathology and treatment are largely based upon molecular functions. To address this challenge, herein we engineered, developed, and tested the first modular high-throughput molecular function assay system. Note that this is not a selection lethality screen! This “GigaAssay” single cell / one-pot assay system was adapted to study how variants impact HIV Tat-driven transactivation of a green fluorescent protein (GFP) reporter. We assayed all 1,615 Tat single and 3,429 double amino acid substitutions with no single mutant dropout. Each mutant was assayed with replicate observations in LentiX293T and Jurkat cells with an average of 100s of separately barcoded cDNA molecules and cell groups for each mutant. Each mutant had ~2,000X-90,000X sequencing coverage to measure its transcriptional activity and had p value ranging as low as 10-271. Five independent assay performance assessments with benchmark data, individually tested clones, and replicate comparisons all indicate exceptional reproducibility, accuracy, and robustness. The shortcomings of alanine scanning mutagenesis and protein truncation studies are revealed by including exhaustive substitution tolerance and intragenic epistasis in the typical structure/function analysis(structure/function/tolerance/epistasis). This flexible and extensible technology enables a far more comprehensive holistic view of protein molecular function and yet with a highly simplified single-pot assay.

scholarly journals Development of a cell-based high throughput assay system and IN-house image analysis software for screening of active compounds against dengue virus

2011 ◽  
Vol 5 (S1) ◽  
Author(s):  
Deu John M Cruz ◽  
Andrea C Koishi ◽  
Ana Luiza M Pamplona ◽  
Auguste Genovesio ◽  
Claudia N Duarte dos Santos ◽  
...  
Keyword(s):  
Image Analysis ◽  
Dengue Virus ◽  
High Throughput ◽  
Assay System ◽  
Analysis Software ◽  
Active Compounds ◽  
Image Analysis Software ◽  
A Cell ◽  
High Throughput Assay

Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

2010 ◽  
Vol 130 (10) ◽  
pp. 471-475 ◽  
Author(s):  
Kanako Sugiura ◽  
Noritada Kaji ◽  
Yukihiro Okamoto ◽  
Manabu Tokeshi ◽  
Yoshinobu Baba
Keyword(s):  
High Throughput ◽  
Assay System ◽  
Cell Assay

A Functional High-Throughput Assay of Myelination in Vitro

2013 ◽  
Author(s):  
Michael J. Moore
Keyword(s):  
High Throughput ◽  
High Throughput Assay

Revealing the changes of IgG subclass‐specific N ‐glycosylation in colorectal cancer progression by high‐throughput assay

2021 ◽  
pp. 2000022
Author(s):  
Si Liu ◽  
Yuting Yu ◽  
Yuanyuan Liu ◽  
Jiajing Lin ◽  
Yang Fu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Progression ◽  
High Throughput ◽  
Igg Subclass ◽  
Colorectal Cancer Progression ◽  
High Throughput Assay

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

Sign in / Sign up

Export Citation Format

Share Document