Investigating the application of a nitroreductase-expressing transgenic zebrafish line for high-throughput toxicity testing

We validated a novel microfluidic permeation array for high-precision and high-throughput skin penetration and toxicity testing or screening of chemicals.

Download Full-text

A calibrated optogenetic toolbox of stable zebrafish opsin lines

eLife ◽

10.7554/elife.54937 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Paride Antinucci ◽

Adna Dumitrescu ◽

Charlotte Deleuze ◽

Holly J Morley ◽

Kristie Leung ◽

...

Keyword(s):

High Throughput ◽

Neural Activity ◽

Ion Selectivity ◽

Transgenic Zebrafish ◽

Substantial Variation ◽

Opsin Expression ◽

Electrophysiological Recordings ◽

Subcellular Resolution ◽

Kinetics Of

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

Download Full-text

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

Metabolomics ◽

10.1007/s11306-021-01867-3 ◽

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.

Download Full-text

Co-culture of human alveolar epithelial (A549) and macrophage (THP-1) cells to study the potential toxicity of ambient PM2.5: a comparison of growth under ALI and submerged conditions

Toxicology Research ◽

10.1093/toxres/tfaa072 ◽

2020 ◽

Vol 9 (5) ◽

pp. 636-651

Author(s):

Guanghe Wang ◽

Xiaofeng Zhang ◽

Xinyan Liu ◽

Jing Zheng

Keyword(s):

High Throughput ◽

Animal Studies ◽

Fine Particulate Matter ◽

Toxicity Testing ◽

Culture Conditions ◽

Ambient Atmosphere ◽

Submerged Condition ◽

Culture Methods ◽

Submerged Conditions

Abstract Fine particulate matter (PM2.5) in the ambient atmosphere is strongly associated with detrimental health effects. However, these particles from various sources and regions are unlikely equally toxic. While animal studies are impractical for high-throughput toxicity testing, appropriate in vitro models are urgently needed. Co-culture of A549 and THP-1 macrophages grown at air–liquid interface (ALI) or under submerged conditions was exposed to same concentrations of ambient PM2.5 to provide accurate comparisons between culture methods. Following 24-h incubation with PM2.5 collected in Harbin in China, biological endpoints being investigated include cytotoxicity, reactive oxygen species (ROS) levels and pro-inflammatory mediators. The co-culture grown under submerged condition demonstrated a significant increase in ROS levels and all tested pro-inflammatory indicators [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] in mRNA expression and released protein levels. Similar but a declining response trend was observed using the same PM2.5 incubation after grown at ALI. We further observed a significant increase of PM2.5-induced phosphorylation of p38 MAPK and activation of NF-κB p65 in a dose-dependent trend for co-cultures grown under submerged condition. These results provide important implications that culture conditions (ALI versus submerged) can induce different extents of biological responses to ambient PM2.5; the co-culture grown at ALI is less likely to produce false-positive results than submerged culture. Hence, culture conditions should be discussed when comparing in vitro methods used for high-throughput PM2.5 toxicity assessment in future.

Download Full-text