Kinetic Assay for High-Throughput Screening of In Vitro Transthyretin Amyloid Fibrillogenesis Inhibitors

2005 ◽  
Vol 7 (2) ◽  
pp. 246-252 ◽  
Author(s):  
Ignacio Dolado ◽  
Joan Nieto ◽  
Maria João M. Saraiva ◽  
Gemma Arsequell ◽  
Gregori Valencia ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinetic Assay

scholarly journals Fluorescence-Based High-Throughput Screening of Dicer Cleavage Activity

2013 ◽  
Vol 19 (3) ◽  
pp. 417-426 ◽  
Author(s):  
Katerina Podolska ◽  
David Sedlak ◽  
Petr Bartunek ◽  
Petr Svoboda
Keyword(s):  
Rna Interference ◽  
High Throughput ◽  
Small Rnas ◽  
High Throughput Screening ◽  
Kinetic Assay ◽  
Ribonuclease Iii ◽  
Single Turnover ◽  
Small Molecule Modulators ◽  
Dicer Cleavage

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

scholarly journals Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

2021 ◽  
Vol 18 (7) ◽  
pp. 3332
Author(s):  
Olga V. Naidenko ◽  
David Q. Andrews ◽  
Alexis M. Temkin ◽  
Tasha Stoiber ◽  
Uloma Igara Uche ◽  
...  
Keyword(s):  
Immune System ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Specific Activity ◽  
Laboratory Animals ◽  
In Vitro Assays ◽  
Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals High content and high throughput screening to assess the angiogenic and neurogenic actions of mesenchymal stem cells in vitro

2015 ◽  
Vol 333 (1) ◽  
pp. 93-104 ◽  
Author(s):  
Jennifer J. Bara ◽  
Sarah Turner ◽  
Sally Roberts ◽  
Gareth Griffiths ◽  
Rod Benson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals DDIS-12. A FACILE, ROBUST AND PREDICTIVE IN VITRO BLOOD-BRAIN-BARRIER MODEL FOR HIGH-THROUGHPUT SCREENING AND DISCOVERY OF BRAIN-PENETRATING AGENTS

2016 ◽  
Vol 18 (suppl_6) ◽  
pp. vi49-vi50
Author(s):  
Choi-Fong Cho ◽  
Justin Wolfe ◽  
Colin Fazden ◽  
Kalvis Hornburg ◽  
E. Antonio Chiocca ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
High Throughput ◽  
High Throughput Screening ◽  
Brain Barrier ◽  
Blood Brain ◽  
Blood Brain Barrier Model ◽  
Barrier Model

scholarly journals New Approach for High-Throughput Screening of Drug Activity on Plasmodium Liver Stages

2006 ◽  
Vol 50 (4) ◽  
pp. 1586-1589 ◽  
Author(s):  
Audrey Gego ◽  
Olivier Silvie ◽  
Jean-François Franetich ◽  
Khemaïs Farhati ◽  
Laurent Hannoun ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Scanning System ◽  
New Approach ◽  
Drug Activity ◽  
Hepatic Cells ◽  
High Throughput Drug Screening ◽  
Prophylactic Drugs

ABSTRACT Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrared fluorescence scanning system. This method allowed us to count automatically and rapidly Plasmodium-infected hepatocytes, using different hepatic cells and different Plasmodium species, including Plasmodium falciparum. This new technique is well adapted for high-throughput drug screening and should facilitate the identification of new antimalarial compounds active on Plasmodium liver stages.

scholarly journals Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics

Metabolites ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 52
Author(s):  
Julia M. Malinowska ◽  
Taina Palosaari ◽  
Jukka Sund ◽  
Donatella Carpi ◽  
Gavin R. Lloyd ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
In Vitro Screening ◽  
Chemical Safety ◽  
Experimental Conditions ◽  
Well Location ◽  
Relative Standard ◽  
Automated Sample Preparation

Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated sample preparation of the low biomass samples typically used for in vitro screening. Building on previous work, we have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics of HepaRG cells in 96-well microplates using a Biomek i7 Hybrid Workstation (Beckman Coulter) and Orbitrap Elite (Thermo Scientific) high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS), across polar metabolites and lipids. The experimental conditions evaluated included the day of metabolite extraction, order of extraction of samples in 96-well microplates, position of the 96-well microplate on the instrument’s deck and well location within a microplate. By using the median relative standard deviation (mRSD (%)) of spectral features, we have demonstrated good repeatability of the workflow (final mRSD < 30%) with a low percentage of features outside the threshold applied for statistical analysis. To improve the quality of the automated workflow further, small method modifications were made and then applied to a large cohort study (4860 sample infusions across three nESI-DIMS assays), which confirmed very high repeatability of the whole workflow from cell culturing to metabolite measurements, whilst providing a significant improvement in sample throughput. It is envisioned that the automated in vitro metabolomics workflow will help to advance the application of metabolomics (as a part of NAMs) in chemical safety, primarily as an approach for high throughput screening and prioritisation.

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