Comparison of base-line and chemical-induced transcriptomic responses in HepaRG and RPTEC/TERT1 cells using TempO-Seq.

The utilisation of genome-wide transcriptomics has played a pivotal role in advancing the field of toxicology, allowing the mapping of transcriptional signatures to chemical exposures. These activities have uncovered several transcriptionally regulated pathways that can be utilised for assessing the perturbation impact of a chemical and also the identification of toxic mode of action. However, current transcriptomic platforms are not very amenable to high-throughput workflows due to, high cost, complexities in sample preparation and relatively complex bioinformatic analysis. Thus, transcriptomic investigations are usually limited in dose and time dimensions and are, therefore, not optimal for implementation in risk assessment workflows. In this study, we investigated a new cost-effective, transcriptomic assay, TempO-Seq, which alleviates the aforementioned limitations. This technique was evaluated in a 6-compound screen, utilising differentiated kidney (RPTEC/TERT1) and liver (HepaRG) cells and compared to non-transcriptomic label-free sensitive endpoints of chemical-induced disturbances, namely phase contrast morphology, xCELLigence and glycolysis. Non-proliferating cell monolayers were exposed to six sub-lethal concentrations of each compound for 24 h. The results show that utilising a 2839 gene panel, it is possible to discriminate basal tissue-specific signatures, generate dose-response relationships and to discriminate compound-specific and cell type-specific responses. This study also reiterates previous findings that chemical-induced transcriptomic alterations occur prior to cytotoxicity and that transcriptomics provides in depth mechanistic information of the effects of chemicals on cellular transcriptional responses. TempO-Seq is a robust transcriptomic platform that is well suited for in vitro toxicity experiments.

In vitro model for assessing arrhythmogenic properties of drugs based on high-resolution impedance measurements.

BACKGROUND/AIMS: Cardiac dysfunction is one of the main cause of drug candidate failures in the preclinical and/or clinical studies and responsible for the retraction of large number of drugs from the market. The prediction of arrhythmic risk based on preclinical trials during drug development remains limited despite intensive and costly investigation. Moreover, methods for analyzing beating behavior of cardiomyocytes (CMs) in culture to diagnose arrhythmias are not well developed. METHODS: In this study, we combined two emerging technologies, induced pluripotent stem (iPS) cell-derived CMs and impedance-based real-time (xCELLigence RTCA Cardio Instrument) monitoring of CM electrical activity, to assess the effect of drugs known affect cardiac activity such as isoproterenol, carbachol, terfenadine, sotalol and doxorubicin. Cells were exposed to a drug in a single dose or repeated dose scenarios and data were analyzed using RTCA Cardio software, Poincaré plot and detrended fluctuation analysis. RESULTS: The results revealed significant changes in beating parameters of iPS-CMs induced by reference compounds. Heptanol, gap junction blocker, completely disrupted the synchronous beating pattern of iPS-CMs. Decrease of beating rate, amplitude and beat-to-beat signal variations of iPS-CMs monolayer observed in the presence of doxorubicin revealed severe abnormality detected by the system. Additionally, the irregular beating rhythms recorded in the presence of Terfenadine and Sotalol at high concentration, reflect abnormalities in cell contraction and/or relaxation which may lead to arrhythmia. CONCLUSIONS: All these results indicated that xCELLigence RTCA Cardio system combined with iPS cells, has the potential to be an attractive high-throughput tool for studying CMs during prolonged culture times and to screen potential drugs for cardiotoxic side effects.

Dynamic monitoring of beating periodicity of stem cell-derived cardiomyocytes as a predictive tool for preclinical safety assessment.

BACKGROUND AND PURPOSE: Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process. EXPERIMENTAL APPROACH: Beating of cardiomyocytes was measured by a recently developed microelectronic-based system using impedance readouts. We used mouse stem cell-derived cardiomyocytes to obtain dose-response profiles for over 60 compounds, including ion channel modulators, chronotropic/ionotropic agents, hERG trafficking inhibitors and drugs known to induce Torsades de Pointes arrhythmias. KEY RESULTS: This system sensitively and quantitatively detected effects of modulators of cardiac function, including some compounds missed by electrophysiology. Pro-arrhythmic compounds produced characteristic profiles reflecting arrhythmia, which can be used for identification of other pro-arrhythmic compounds. The time series data can be used to identify compounds that induce arrhythmia by complex mechanisms such as inhibition of hERG channels trafficking. Furthermore, the time resolution allows for assessment of compounds that simultaneously affect both beating and viability of cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: Microelectronic monitoring of stem cell-derived cardiomyocyte beating provides a high throughput, quantitative and predictive assay system that can be used for assessment of cardiac liability earlier in the drug discovery process. The convergence of stem cell technology with microelectronic monitoring should facilitate cardiac safety assessment.

Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system.

The dependence of efficiency of translation initiation on mRNA sequence parameters was investigated in an Escherichia coli in vitro expression system. We designed a large-scale expression experiment focussing on the influence of sequence variations in the translated region (TR) of the mRNA without changing the 5'-untranslated region (5'-UTR). The level of translated protein from 756 expression constructs was measured and the influence of a large number of possible effector attributes was statistically analyzed. Base exchanges immediately adjacent to the start codon up to nucleotide (+)25 had a profound effect on translational efficiency. Correlation analysis revealed a significant dependence on base pair probability and G+C content on the expression level, indicating that mRNA secondary structure in this region hampers translation. Using our training data, we developed a methodology to predict and improve the translation efficiency of open reading frames (ORFs).

RNA stem-loop enhanced expression of previously non-expressible genes.

The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem-loop (stem length, 7 bp; DeltaG(0) = -9.9 kcal/mol) in front of various gene sequences. In each case, the stem-loop was inserted 15 nt downstream from the start codon. Insertion of the stem-loop allowed in vitro expression of five previously non-expressible genes and enhanced the expression of all other genes investigated. Analysis of the RNA structure proved that the stem-loop was formed in vitro, and demonstrated that stabilization of the ribosome binding site is due to stem-loop introduction. By theoretical RNA structure analysis we showed that the inserted RNA stem-loop suppresses long-range interactions between the translation initiation domain and gene-specific mRNA sequences. Thus the inserted RNA stem-loop supports the formation of a separate translational initiation domain, which is more accessible to ribosome binding.