Automated phenotype recognition for zebrafish embryo based in vivo high throughput toxicity screening of engineered nano-materials.
ABSTRACT A phenotype recognition model was developed for high throughput screening (HTS) of engineered Nano-Materials (eNMs) toxicity using zebrafish embryo developmental response classified, from automatically captured images and without manual manipulation of zebrafish positioning, by three basic phenotypes (i.e., hatched, unhatched, and dead). The recognition model was built with a set of vectorial descriptors providing image color and texture information. The best performing model was attained with three image descriptors (color histogram, representative color, and color layout) identified as most suitable from an initial pool of six descriptors. This model had an average recognition accuracy of 97.40±0.95% in a 10-fold cross-validation and 93.75% in a stress test of low quality zebrafish images. The present work has shown that a phenotyping model can be developed with accurate recognition ability suitable for zebrafish-based HTS assays. Although the present methodology was successfully demonstrated for only three basic zebrafish embryonic phenotypes, it can be readily adapted to incorporate more subtle phenotypes.
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ABSTRACT: Group IV Nanowires have strong potential for several biomedical applications. However, to date their use remains limited because many are synthesised using heavy metal seeds and functionalised using organic ligands to make the materials water dispersible. This can result in unpredicted toxic side effects for mammalian cells cultured on the wires. Here, we describe an approach to make seedless and ligand free Germanium nanowires water dispersible using glutamic acid, a natural occurring amino acid that alleviates the environmental and health hazards associated with traditional functionalisation materials. We analysed the treated material extensively using Transmission electron microscopy (TEM), High resolution-TEM, and scanning electron microscope (SEM). Using a series of state of the art biochemical and morphological assays, together with a series of complimentary and synergistic cellular and molecular approaches, we show that the water dispersible germanium nanowires are non-toxic and are biocompatible. We monitored the behaviour of the cells growing on the treated germanium nanowires using a real time impedance based platform (xCELLigence) which revealed that the treated germanium nanowires promote cell adhesion and cell proliferation which we believe is as a result of the presence of an etched surface giving rise to a collagen like structure and an oxide layer. Furthermore this study is the first to evaluate the associated effect of Germanium nanowires on mammalian cells. Our studies highlight the potential use of water dispersible Germanium Nanowires in biological platforms that encourage anchorage-dependent cell growth.PLoS ONE 09/2014; 9(9):e108006. DOI:10.1371/journal.pone.0108006 · 3.53 Impact Factor
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ABSTRACT: Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100% agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification.PLoS ONE 01/2015; 10(1):e0116989. DOI:10.1371/journal.pone.0116989 · 3.53 Impact Factor
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ABSTRACT: Nanotechnology has been one of the major success stories of the early 21st century. The foundation for this success rests on the discovery that a small size confers completely new properties on materials. Nowadays, engineered nanomaterials (ENMs) are used in a plethora of applications such as paints, cosmetics, food products and electronics. These new properties, however, potentially make ENMs more reactive in biological systems than their large-scale counterparts. Already, asbestos-like effects have been described in mice after exposure to certain forms of carbon nanotubes (CNTs), while nano-sized titanium dioxide (nTiO2) has been shown to evoke inflammation in mouse lung. Therefore, extensive nanosafety studies have to be performed to ensure that no adverse effects are suffered by either workers or end-users of ENM products. This thesis has investigated health effects of ENMs by proteomic methods, first by evaluating the uptake and interactions of ENMs with plasma and cellular proteins followed by an analysis of the effects of ENM exposure on the intracellular proteome and secretome of human primary macrophages. The results revealed that ENM interactions with cellular proteins were governed by the surface reactivity of ENMs, whereas interactions with plasma proteins seemed to depend on the combination of both surface reactivity and active recognition, namely tagging of ENMs by opsonin proteins. The binding of cellular proteins to ENMs and subsequent interference with cellular processes might represent a novel cause of ENM toxicity, especially since transmission electron microscopy (TEM) micrographs indicated that several ENM species could be visualized free in the cytoplasm. The cytoplasmic protein expression changes after exposure to silica coated and uncoated nTiO2 revealed that silica coated TiO2 induced stronger protein expression changes in the macrophages. Most of the proteins with altered expression were related to phagocytosis, oxidative stress and inflammation. These proteome changes indicate that macrophages are actively engulfing ENMs and processing them. Moreover, the up-regulation of oxidative stress related proteins might be an indication of oxidative burst. Finally, nTiO2 treatment evoked acetylation of cytoplasmic proteins, a previously uncharacterized phenomenon in cells exposed to ENMs. The results from the macrophage secretome analysis showed that asbestos and long rigid carbon nanotubes (R CNTs) produced a similar response, while protein secretion profile of macrophages exposed to long tangled carbon nanotubes (T CNTs) exhibited a distinct profile. Bioinformatic analysis revealed that R CNTs evoked secretion of inflammation and apoptosis related proteins, possibly because of lysosomal damage. Functional assay confirmed that R CNT exposure triggered apoptosis in macrophages, while T CNTs and asbestos did not. This thesis offers new knowledge concerning the biological effects of engineered nanomaterials. Proteomic methods proved to be useful in the ENM protein interaction studies revealing that it would be beneficial to include the ENM-protein interaction experiments as part of the routine ENM characterization when assessing the health effects of ENMs. By employing quantitative proteomics, we obtained a global view of both cytoplasmic and secreted proteome changes of macrophages exposed to different ENMs.04/2014; Unigrafia., ISBN: 978-952-10-9809-3