Current In Vitro High Throughput Screening Approaches to Assess Nuclear Receptor Activation

Identifying novel marine-derived neuroactive chemicals with therapeutic potential is difficult due to inherent complexities of the central nervous system (CNS), our limited understanding of the molecular foundations of neuro-psychiatric conditions, as well as the limited applications of effective high-throughput screening models that recapitulate functionalities of the intact CNS. Furthermore, nearly all neuro-modulating chemicals exhibit poorly characterized pleiotropic activities often referred to as polypharmacology. The latter renders conventional target-based in vitro screening approaches very difficult to accomplish. In this context, chemobehavioural phenotyping using innovative small organism models such as planarians and zebrafish represent powerful and highly integrative approaches to study the impact of new chemicals on central and peripheral nervous systems. In contrast to in vitro bioassays aimed predominantly at identification of chemicals acting on single targets, phenotypic chemobehavioural analysis allows for complex multi-target interactions to occur in combination with studies of polypharmacological effects of chemicals in a context of functional and intact milieu of the whole organism. In this review, we will outline recent advances in high-throughput chemobehavioural phenotyping and provide a future outlook on how those innovative methods can be utilized for rapidly screening and characterizing marine-derived compounds with prospective applications in neuropharmacology and psychosomatic medicine.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa1213 ◽

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.

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

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18073332 ◽

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.

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

Nature Communications ◽

10.1038/s41467-021-23954-8 ◽

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.

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