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.

scholarly journals Integrating in vitro metabolomics with a 96-well high-throughput screening platform

Metabolomics ◽  
2022 ◽  
Vol 18 (1) ◽  
Author(s):  
Julia M. Malinowska ◽  
Taina Palosaari ◽  
Jukka Sund ◽  
Donatella Carpi ◽  
Mounir Bouhifd ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Toxicity Testing ◽  
Exposure Period ◽  
Solvent System ◽  
Extraction Methods ◽  
Metabolic Phenotype ◽  
Chemical Safety ◽  
Relative Standard

Abstract Introduction High-throughput screening (HTS) is emerging as an approach to support decision-making in chemical safety assessments. In parallel, in vitro metabolomics is a promising approach that can help accelerate the transition from animal models to high-throughput cell-based models in toxicity testing. Objective In this study we establish and evaluate a high-throughput metabolomics workflow that is compatible with a 96-well HTS platform employing 50,000 hepatocytes of HepaRG per well. Methods Low biomass cell samples were extracted for metabolomics analyses using a newly established semi-automated protocol, and the intracellular metabolites were analysed using a high-resolution spectral-stitching nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) method that was modified for low sample biomass. Results The method was assessed with respect to sensitivity and repeatability of the entire workflow from cell culturing and sampling to measurement of the metabolic phenotype, demonstrating sufficient sensitivity (> 3000 features in hepatocyte extracts) and intra- and inter-plate repeatability for polar nESI-DIMS assays (median relative standard deviation < 30%). The assays were employed for a proof-of-principle toxicological study with a model toxicant, cadmium chloride, revealing changes in the metabolome across five sampling times in the 48-h exposure period. To allow the option for lipidomics analyses, the solvent system was extended by establishing separate extraction methods for polar metabolites and lipids. Conclusions Experimental, analytical and informatics workflows reported here met pre-defined criteria in terms of sensitivity, repeatability and ability to detect metabolome changes induced by a toxicant and are ready for application in metabolomics-driven toxicity testing to complement HTS assays.

scholarly journals High-Throughput Screening for Human Galactokinase Inhibitors

2008 ◽  
Vol 13 (5) ◽  
pp. 415-423 ◽  
Author(s):  
Klaas J. Wierenga ◽  
Kent Lai ◽  
Peter Buchwald ◽  
Manshu Tang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Expression System ◽  
Chemical Compounds ◽  
Average Concentration ◽  
Effective Therapy ◽  
Experimental Conditions ◽  
Classic Galactosemia ◽  
Neonatal Lethality

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z′ factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 µM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia. ( Journal of Biomolecular Screening 2008:415-423)

Recent Advances in High-throughput Platforms with Engineered Biomaterial Microarrays for Screening of Cell and Tissue Behavior

2019 ◽  
Vol 24 (45) ◽  
pp. 5458-5470 ◽  
Author(s):  
Kijun Park ◽  
Yeontaek Lee ◽  
Jungmok Seo
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Continuous Monitoring ◽  
In Vitro Screening ◽  
Cellular Behavior ◽  
Current Trends ◽  
Recent Advances ◽  
Screening Platform ◽  
Review Current

In the last decades, bioengineers have developed myriad biomaterials for regenerative medicine. Development of screening techniques is essential for understanding complex behavior of cells in the biological microenvironments. Conventional approaches to the screening of cellular behavior in vitro have limitations in terms of accuracy, reusability, labor-intensive screening, and versatility. Thus, drug screening and toxicology test through in vitro screening platforms have been underwhelming. Recent advances in the high-throughput screening platforms somewhat overcome the limitations of in vitro screening platforms via repopulating human tissues’ biophysical and biomchemical microenvironments with the ability to continuous monitoring of miniaturized human tissue behavior. Herein, we review current trends in the screening platform in which a high-throughput system composed of engineered microarray devices is developed to investigate cell-biomaterial interaction. Furthermore, diverse methods to achieve continuous monitoring of cell behavior via developments of biosensor integrated high-throughput platforms, and future perspectives on high-throughput screening will be provided.

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

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 Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1019
Author(s):  
Teresa von Linde ◽  
Gzona Bajraktari-Sylejmani ◽  
Walter E. Haefeli ◽  
Jürgen Burhenne ◽  
Johanna Weiss ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Peptide Bond ◽  
Peptide Transporter ◽  
Systemic Absorption ◽  
Screening Assay ◽  
Limit Of Quantification

The peptide transporter PEPT-1 (SLC15A1) plays a major role in nutritional supply with amino acids by mediating the intestinal influx of dipeptides and tripeptides generated during food digestion. Its role in the uptake of small bioactive peptides and various therapeutics makes it an important target for the investigation of the systemic absorption of small peptide-like active compounds and prodrug strategies of poorly absorbed therapeutics. The dipeptide glycyl-sarcosine (Gly-Sar), which comprises an N-methylated peptide bond that increases stability against enzymatic degradation, is widely utilized for studying PEPT-1-mediated transport. To support experiments on PEPT-1 inhibitor screening to identify potential substrates, we developed a highly sensitive Gly-Sar quantification assay for Caco-2 cell lysates with a dynamic range of 0.1 to 1000 ng/mL (lower limit of quantification 0.68 nM) in 50 µL of cell lysate. The assay was validated following the applicable recommendations for bioanalytic method validation of the FDA and EMA. Sample preparation and quantification were established in 96-well cell culture plates that were also used for the cellular uptake studies, resulting in a rapid and robust screening assay for PEPT-1 inhibitors. This sample preparation principle, combined with the high sensitivity of the UPLC-MS/MS quantification, is suitable for screening assays for PEPT-1 inhibitors and substrates in high-throughput formats and holds the potential for automation. Applicability was demonstrated by IC50 determinations of the known PEPT-1 inhibitor losartan, the known substrates glycyl-proline (Gly-Pro), and valaciclovir, the prodrug of aciclovir, which itself is no substrate of PEPT-1 and consequently showed no inhibition in our assay.

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