The rapid development of nanotechnology allowed the fabrication of a wide range of different nanomaterials, raising many questions about their safety and potential risks for the human health and environment. Most of the current nanotoxicology research is not standardized, hampering any comparison or reproducibility of the obtained results. Drosophotoxicology encompasses the plethora of methodological approaches addressing the use of Drosophila melanogaster as a choice organism in toxicology studies. Drosophila melanogaster model offers several important advantages, such as a relatively simple genome structure, short lifespan, low maintenance cost, readiness of experimental manipulation comparative to vertebrate models from both ethical and technical points of view, relevant gene homology with higher organisms, and ease of obtaining mutant phenotypes. The molecular pathways, as well as multiple behavioral and developmental parameters, can be evaluated using this model in lower, medium or high throughput type assays, allowing a systematic classification of the toxicity levels of different nanomaterials. The purpose of this paper is to review the current research on the applications of Drosophila melanogaster model for the in vivo assessment of nanoparticles toxicity and to reveal the huge potential of this model system to provide results that could enable a proper selection of different nanostructures for a certain biomedical application.
BackgroundA critical topic of insertional mutagenesis experiments performed on model organisms is mapping the hits of artificial transposons (ATs) at nucleotide level accuracy. Mapping errors may occur when sequencing artifacts or mutations as single nucleotide polymorphisms (SNPs) and small indels are present very close to the junction between a genomic sequence and a transposon inverted repeat (TIR). Another particular item of insertional mutagenesis is mapping of the transposon self-insertions and, to our best knowledge, there is no publicly available mapping tool designed to analyze such molecular events.ResultsWe developed Genome ARTIST, a pairwise gapped aligner tool which works out both issues by means of an original, robust mapping strategy. Genome ARTIST is not designed to use next-generation sequencing (NGS) data but to analyze ATs insertions obtained in small to medium-scale mutagenesis experiments. Genome ARTIST employs a heuristic approach to find DNA sequence similarities and harnesses a multi-step implementation of a Smith-Waterman adapted algorithm to compute the mapping alignments. The experience is enhanced by easily customizable parameters and a user-friendly interface that describes the genomic landscape surrounding the insertion. Genome ARTIST is functional with many genomes of bacteria and eukaryotes available in Ensembl and GenBank repositories. Our tool specifically harnesses the sequence annotation data provided by FlyBase for Drosophila melanogaster (the fruit fly), which enables mapping of insertions relative to various genomic features such as natural transposons. Genome ARTIST was tested against other alignment tools using relevant query sequences derived from the D. melanogaster and Mus musculus (mouse) genomes. Real and simulated query sequences were also comparatively inquired, revealing that Genome ARTIST is a very robust solution for mapping transposon insertions.ConclusionsGenome ARTIST is a stand-alone user-friendly application, designed for high-accuracy mapping of transposon insertions and self-insertions. The tool is also useful for routine aligning assessments like detection of SNPs or checking the specificity of primers and probes. Genome ARTIST is an open source software and is available for download at www.genomeartist.ro and at GitHub (https://github.com/genomeartist/genomeartist ).Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0061-0) contains supplementary material, which is available to authorized users.
The annotation of transposable elements (transposons) is a very dynamic field of genomics and various tools assigned to support this bioinformatics endeavor have been developed and described. Genome ARTIST v1.19 (GA_v1.19) software was conceived for mapping artificial transposons mobilized during insertional mutagenesis projects, but the new functions of GA_v2 qualify it as a tool for the mapping and annotation of natural transposons (NTs) in long reads, contigs and assembled genomes. The tabular export of mapping and annotation data for high-throughput data analysis, the generation of a list of flanking sequences around the coordinates of insertion or around the target site duplications and the computing of a consensus sequence for the flanking sequences are all key assets of GA_v2. Additionally, we developed a set of scripts that enable the user to annotate NTs, to harness annotations offered by FlyBase for Drosophila melanogaster genome, to convert sequence files from .fasta to .raw, and to extract junction query sequences essential for NTs mapping. Herein, we present the applicability of GA_v2 for a preliminary annotation of P-element and hobo class II NTs and copia retrotransposon in the genome of D. melanogaster strain Horezu_LaPeri (Horezu), Romania, which was sequenced with Nanopore technology in our laboratory. We used contigs assembled with Flye tool and a Q10 quality filter of the reads. Our results suggest that GA_v2 is a reliable autonomous tool able to perform mapping and annotation of NTs in genomes sequenced by long sequencing technology. GA_v2 is open-source software compatible with Linux, Mac OS and Windows and is available at GitHub repository and dedicated website.
Hypertrophic cardiomyopathy (HCM) has a special place among genetic cardiomyopathies, being one of the main causes of sudden death in young patients, mainly in performance athletes. Herein we report a deletion in the myosin binding protein C (MYBPC3) gene identified in a female patient affected by HCM. The mutation was initially pinpointed in an NGS screening, then it was confirmed by Sanger sequencing with original primers. Bioinformatics analysis revealed a deletion previously reported as c.2441_2443delAGA, but the precise breakpoints mapping appears to be difficult to conclude. Since alternative three nucleotides deletions unambiguously result in a net Lysine missing from a specific poly-Lysine protein domain, the absolute mapping of the mutation is yet elusive, an aspect which should be considered when reporting the genomic coordinates of this deletion.
We report an unusual overexpression pattern of the hypomorphic alleles γCop 11a , γCop 14a and γCop 16b of gammaCop gene from Drosophila melanogaster. Each allele contains a distinct short remnant of the P{lacW} transposon located in 5'UTR. Repeated crosses between γCop 14a /γCop 14a females and γCop 11a /γCop 11a or γCop 16b /γCop 16b males proved to be sterile, but the reciprocal ones are fertile.The most severe allele is γCop 14a since a γCop 14a /γCop 14a line cannot be obtained. Remarkably, qRT-PCR performed on mutant eggs, testes and embryos reveals that γCop 11a , γCop 14a and γCop 16b are overexpressed, which is counterintuitive for hypomorphic alleles. Moreover, the expression scale overlaps the hierarchy of the allelic phenotype severity.We found that the folding ΔG values of the mutant 5'UTRs are very low as compared to the folding ΔG value of the wild-type 5'UTR. This thermodynamic context may account for the uncommon expression pattern of the hypomorphic alleles described herein.
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