Presently, nanotechnology is a multi-trillion dollar business sector that covers a wide range of industries, such as medicine, electronics and chemistry. In the current era, the commercial transition of nanotechnology from research level to industrial level is stimulating the world’s total economic growth. However, commercialization of nanoparticles might offer possible risks once they are liberated in the environment. In recent years, the use of zebrafish (Danio rerio) as an established animal model system for nanoparticle toxicity assay is growing exponentially. In the current in-depth review, we discuss the recent research approaches employing adult zebrafish and their embryos for nanoparticle toxicity assessment. Different types of parameters are being discussed here which are used to evaluate nanoparticle toxicity such as hatching achievement rate, developmental malformation of organs, damage in gill and skin, abnormal behavior (movement impairment), immunotoxicity, genotoxicity or gene expression, neurotoxicity, endocrine system disruption, reproduction toxicity and finally mortality. Furthermore, we have also highlighted the toxic effect of different nanoparticles such as silver nanoparticle, gold nanoparticle, and metal oxide nanoparticles (TiO2, Al2O3, CuO, NiO and ZnO). At the end, future directions of zebrafish model and relevant assays to study nanoparticle toxicity have also been argued.

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Zebrafish: A complete animal model to enumerate the nanoparticle toxicity.

Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.

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Rapid adaptive optical recovery of optimal resolution over large volumes

As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic.

Zebrafish in Toxicology and Environmental Health.

Advancements in zebrafish applications for 21st century toxicology.

Gene therapy in a global context involves correction of a genetic defect by introducing a normal version of a defective or missing gene thereby correcting an underlying disorder (Friedmann, 1992) Milestones on the path of developing gene therapies are presented in Figure 1 This concept was first advanced in the 1960s after observing that viruses could cause malignant transformation in cells by integrating their genetic information into the genomes of infected cells In 1966, Edward Tatum proposed the use of viruses in the genetic manipulation of somatic cells and its possible therapeutic applications (Tatum, 1966) A few years later, initial proof-of-concept for gene therapy was demonstrated using tobacco mosaic virus as a vector to introduce a polyadenylate stretch to viral RNA (Rogers and Pfuderer, 1968) Encouraged by these results, gene therapy was attempted in the 1970s to correct a urea cycle disorder by administering wild type Shope papilloma virus, encoding the arginase gene, to two severely handicapped young girls suffering from hyperarginemia (Rogers et al, 1973; Terheggen et al, 1975) Unfortunately, the desired outcome was not achieved The first successful therapeutic application of this gene therapy approach was evident in 1990 when a retrovirus-vector was used to mediate transfer of the gene encoding adenosine deaminase (ADA) into the T-cells of two children suffering from severe combined immunodeficiency (SCID) (Rosenberg et al, 1990) The response was positive for only one of the patients; however, debate arose since the patient simultaneously received enzyme replacement therapy alongside gene therapy Another study was conducted in an 18-year-old patient suffering from ornithine transcarbamylase deficiency, a relatively mild form of nitrogen metabolism disorder However, the application of gene therapy in patients was temporarily halted following the death of a patient due to vector-associated toxicity (Stolberg, 1999) In spite of the public backlash from this incident, in the last decades a growing body of evidences supported by positive human efficacy data confirmed that gene therapy could be used to correct certain debilitating conditions including Leber’s congenital amaurosis (Maguire et al, 2009), β-thalassemia (Cavazzana-Calvo et al, 2010), X-linked severe combined immunodeficiency (SCID-X1) (Hacein-Bey-Abina et al, 2010) and cancer (Wirth et al, 2013) Additionally, an increased understanding of the genetic basis of many diseases, improvement of vectors to minimize unwanted toxicity, advances in approaches for manipulating DNA expression and delivery in a target-specific manner has raised expectations that a clinical breakthrough may be imminent As a consequence, there have been over 1800 clinical trials already conducted or currently ongoing worldwide (Wirth et al, 2013)



Fig 1

The timelines and milestones in developing gene therapy approaches: from conception to clinical applications



Initially, gene therapy focused on rare (orphan) diseases mediated by detrimental monogenetic defects However, with advances in the field, various chronic and progressive diseases such as heart failure, neurodegeneration or metabolic disorders were also evaluated using gene therapy approaches These studies indicated that gene therapy techniques have broad potential applications, although cancer comprises over 60% of all ongoing clinical gene trials (Wirth et al, 2013) In this review, we focus on various gene therapy strategies that are currently employed, roadblocks and challenges in the field of cancer gene therapy, and a brief discussion of a few current successful applications of gene therapy for cancer

Gene Therapies for Cancer: Strategies, Challenges and Successes

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

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Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.

Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current ...

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Advances in zebrafish chemical screening technologies

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate ...

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations. We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing. Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.

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Jonathan R. Mathias

Papers

Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.

Advances in zebrafish chemical screening technologies

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner