Toxicity assays in nanodrops combining bioassay and morphometric endpoints.
BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl2), Sodium Arsenate (NaAsO2) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl2 and NaAsO2, in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. CONCLUSIONS: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework. (+info)
The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.
BACKGROUND: The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources. METHODOLOGY: We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis. CONCLUSIONS: We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial analyses, population genetics, and phylogenetics. (+info)
High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes.
In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks. (+info)
A new classification system for the actions of IRS chemicals traditionally used for malaria control.
Knowledge of how mosquitoes respond to insecticides is of paramount importance in understanding how an insecticide functions to prevent disease transmission. A suite of laboratory assays was used to quantitatively characterize mosquito responses to toxic, contact irritant, and non-contact spatial repellent actions of standard insecticides. Highly replicated tests of these compounds over a range of concentrations proved that all were toxic, some were contact irritants, and even fewer were non-contact repellents. Of many chemicals tested, three were selected for testing in experimental huts to confirm that chemical actions documented in laboratory tests are also expressed in the field. The laboratory tests showed the primary action of DDT is repellent, alphacypermethrin is irritant, and dieldrin is only toxic. These tests were followed with hut studies in Thailand against marked-released populations. DDT exhibited a highly protective level of repellency that kept mosquitoes outside of huts. Alphacypermethrin did not keep mosquitoes out, but its strong irritant action caused them to prematurely exit the treated house. Dieldrin was highly toxic but showed no irritant or repellent action. Based on the combination of laboratory and confirmatory field data, we propose a new paradigm for classifying chemicals used for vector control according to how the chemicals actually function to prevent disease transmission inside houses. The new classification scheme will characterize chemicals on the basis of spatial repellent, contact irritant and toxic actions. (+info)
Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.
Technologies for transporter drug discovery.
Transporters represent attractive targets for drug discovery and are implicated in the pathophysiology of disorders across several therapeutic areas including asthma, cardiovascular disease, diabetes and neuroscience. However, the intrinsic mechanistic properties of transporters present significant challenges to the development of high-throughput screening methodologies. This review provides an update on potential transporter targets and evaluates the impact of available technologies to enable transporter screening, lead optimization and assessment of pharmacokinetics. (+info)
Medium- and high-throughput screening of neurotoxicants using C. elegans.