Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated

Novel technologies for high-throughput and high-content studies on zebrafish larvae

The zebrafish larva is an ideal candidate for in vivo high-throughput screening: it is a small vertebrate, it is optically transparent, possesses complex organs, and is easy to culture. In addition, genetic mutants and models of human diseases are widely available. Despite these attractive features there are no tools capable of screening at sufficient throughput and resolution to fully exploit the zebrafish. Here, I present a collection of technologies that enable high-throughput studies on zebrafish larvae at cellular resolution.Engineering and Applied Science

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

Cytochrome P450 (CYP) enzymes for which there is no functional information are considered "orphan" CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to "deorphanization" CYP20A1 functions and its roles in health and disease

How microplastics and adsorbed pollutants affect zebrafish development?

A presença de microplásticos no ecossistema aquático é, nos dias de hoje, uma realidade extremamente preocupante que acarreta sérios riscos para o meio ambiente e para a saúde pública. A capacidade dos microplásticos de adsorverem poluentes orgânicos, como é o caso do benzo[α]pireno (BaP), levanta preocupações adicionais, pois cria uma nova rota de entrada de compostos tóxicos na cadeia alimentar. No entanto, o conhecimento atual sobre o impacto dos microplásticos, inalterados e/ou contaminados, nos organismos aquáticos é ainda insuficiente e requer estudos adicionais. O trabalho desenvolvido no âmbito desta tese pretende assim fornecer novos dados sobre os efeitos biológicos dos microplásticos utilizando como modelo o peixe-zebra (Danio rerio). Pequenos teleósteos, como é o caso do peixe-zebra, oferecem vantagens significativas relativamente aos modelos animais clássicos e são atualmente utilizados como organismos de primeira linha para avaliar os riscos ambientais associados aos compostos tóxicos presentes nos meios aquáticos. Estudos toxicológicos requerem o uso de materiais inertes e condições controladas, todavia, nenhum dos sistemas atualmente comercializados é adequado para avaliar o efeito tóxico dos microplásticos. Estes sistemas contêm componentes feitos de polímeros plásticos que podem libertar partículas plásticas micrométricas, lixiviar os constituintes químicos ou adsorver os compostos químicos em estudo. O sistema de aquários autônomo ZEB316 foi desenvolvido no âmbito deste trabalho com o objetivo de suprimir esta necessidade e de facultar uma solução económica e fácil de implementar que permita a realização de estudos toxicológicos de última geração. Este sistema é construído com materiais inertes e resistentes à corrosão e proporciona boas condições de alojamento através de um sistema eficiente de recirculação e filtragem da água. A avaliação dos parâmetros da água e do desempenho do crescimento dos peixes mostrou que o sistema ZEB316 oferece condições de alojamento comparáveis à dos sistemas comercialmente disponíveis. A maioria dos resultados obtidos no âmbito desta dissertação provêm da análise de imagens de microscopia de campo claro e/ou fluorescência. Embora o uso de imagens de microscopia seja comum na maioria dos laboratórios e permita obter uma quantidade considerável de informação, a análise destas imagens é geralmente um processo moroso, em parte devido à falta de ferramentas automáticas/semiautomáticas dedicadas que permitam a sua análise. Nesse sentido, desenvolvemos diversas macros para o software ImageJ, com o objetivo de reduzir o erro associado à análise pelo usuário, aumentando assim a reprodutibilidade dos dados e a padronização das metodologias experimentais. Uma vez que o foco deste trabalho está centrado no estudo da osteotoxicidade, é de salientar o desenvolvimento de um conjunto de macros específicas para esse fim e denominadas ZFBONE. Este conjunto de ferramentas permite aos usuários avaliar, a partir de imagens 2D, parâmetros morfométricos de várias estruturas ósseas (por exemplo, opérculo, raios e escamas da barbatana caudal), mas também a extensão e a intensidade das colorações específicas do osso. Além disso, outras macros foram desenvolvidas para outros fins, por exemplo, para analisar os vários parâmetros morfométricos relativos aos embriões de peixe ou para avaliar cortes histológicos. Uma vez desenvolvidos os meios e técnicas necessários para a execução dos ensaios toxicológicos, e subsequente análise dos resultados, procedeu-se à realização das várias experiências que visam compreender efeito biológico dos microplásticos e contaminates no peixe-zebra. Assim, larvas de peixe-zebra foram produzidas e mantidas no sistema previamente descrito, ZEB316, e expostas cronicamente, durante o seu desenvolvimento, a partículas de polietileno de 20- 27 μm, inalteradas (MP) ou contaminadas com benzo[α]pireno (MP-BaP). Estas partículas foram adicionadas à dieta dos peixes através de uma suplementação a 1% m/m. Apesar de não se ter registado qualquer alteração ao nível dos parâmetros morfológicos aos 30 dias pós-fertilização (dpf), a presença de MP e MP-BaP acabou por ter um efeito negativo no crescimento dos espécimes aos 90 e 360 dpf. A fecundidade relativa, a morfologia do ovo/embrião e a área do vitelo também sofreram um impacto negativo em peixes-zebra alimentados com MP-BaP. No que respeita ao estado geral do esqueleto, os peixes expostos a dietas experimentais contendo MP e MP-BaP sofreram um aumento significativo na incidência de deformações esqueléticas aos 30 dpf quando comparados com peixes alimentados com a dieta controlo, bem como um desenvolvimento anómalo da barbatana caudal e escamas, e uma diminuição da qualidade do osso aos 90 dpf. Um comprometimento da formação óssea intergeracional foi também observado na prole de espécimes expostos a MP ou MP-BaP, que se refletiu numa redução do osso opercular dos descendentes aos 6 dpf. Além de um claro efeito no desenvolvimento ósseo, a análise histológica do intestino revelou ainda um número reduzido de células caliciformes em peixes alimentados com dieta MP-BaP, um claro sinal de inflamação intestinal. Finalmente, a exposição das larvas a MP-BaP levou a um aumento da expressão de genes associados à via de resposta do BaP, ao mesmo tempo que impactou negativamente na expressão de genes envolvidos no estresse oxidativo. Os resultados obtidos indicaram um maior comprometimento do desenvolvimento ósseo no peixe-zebra quando sujeito a uma dieta contendo microplásticos contaminados com BaP (MP-BaP), por comparação com a dieta contendo microplásticos não contaminados (MP). Outros autores demostraram também que a presença de BaP afeta a formação das vértebras e o desenvolvimento generalizado do esqueleto, no entanto os mecanismos envolvidos nestes fenómenos permanecem pouco estudados. Desta forma, realizámos ensaios adicionais in vivo com o objetivo de avaliar os efeitos osteotóxicos do BaP durante o desenvolvimento e regeneração óssea em peixe-zebra. A exposição aguda de larvas de peixe-zebra entre os 3 e os 6 dpf ao BaP levou a uma redução do tamanho do osso opercular e uma diminuição quantitativa de células positivas para osteocalcina, indicando um efeito composto na maturação dos osteoblastos. Por sua vez, quando se trata de uma exposição crônica das larvas de peixe-zebra ao BaP, entre os 3 e os 30 dpf, verificou-se que o desenvolvimento do esqueleto axial é afetado, aumentando a incidência e gravidade das deformações esqueléticas. Em peixes jovens adultos, observou-se ainda que a exposição ao BaP afetou não só a mineralização dos raios da barbatana caudal e escamas recém-formados, como prejudicou também o seu padrão morfológico, fenómenos que têm por base uma remodelação óssea desequilibrada. Relativamente às análises de expressão genética de vários marcadores verificou-se que o BaP induziu a ativação das vias xenobióticas e metabólicas e impactou negativamente na formação e organização da matriz extracelular. Curiosamente, a exposição ao BaP regulou positivamente os marcadores de inflamação nas larvas e aumentou o recrutamento de neutrófilos. Uma interação direta entre neutrófilos e a matriz extracelular óssea, ou células responsáveis pela formação do osso, foi observada in vivo, o que sugere um papel dos neutrófilos nos mecanismos subjacentes à osteotoxicidade do BaP. Globalmente, os resultados obtidos no âmbito deste trabalho sugerem que a exposição crónica a microplásticos inalterados e/ou contaminados não prejudica apenas o crescimento dos peixes, mas também o seu desempenho a nível reprodutivo, saúde geral do seu esqueleto, e compromete a descendência por meio de efeitos intergeracionais. Além disso, este trabalho fornece novos dados sobre os principais mecanismos celulares e moleculares envolvidos na osteotoxicidade do BaP e aborda o possível papel dos neutrófilos na redução da resposta óssea inflamatória.The presence of microplastics in the aquatic ecosystem represents a major issue for the environment and human health. The capacity of organic pollutants such as benzo[α]pyrene (BaP) to adsorb onto microplastic particles raises additional concerns, as it creates a new route for toxic compounds to enter the food web. Current knowledge on the impact of pristine and/or contaminated microplastics on aquatic organisms remains insufficient, and this work aims at providing new insights by evaluating their biological effects in zebrafish (Danio rerio). The ZEB316, a standalone housing system built with inert materials, and a comprehensive set of ImageJ semi-automatic tools were first developed and optimized to perform state-of-the-art toxicological studies and obtain meaningful data from morphometric analysis of brightfield/ fluorescence images. Zebrafish larvae were exposed throughout their development to polyethylene microplastics, pristine or spiked with BaP, supplemented in the fish diet. While exposure up to 30 days post-fertilization only increased the incidence of skeletal deformities, long-term exposure to pristine/contaminated microplastics not only jeopardized fish growth, reproduction performance, and skeletal health, but it also caused an intergenerational effect. To further study the mechanisms underlying BaP osteotoxicity, several bone-related in vivo assays were used to evaluate the effects of waterborne exposure to BaP during bone development and regeneration. BaP inhibited osteoblast maturation and ECM mineralization and stimulated osteoclast activity, thus affecting bone remodeling. Transgenic and transcriptomic approaches suggested that besides the activation of xenobiotic and metabolic pathways, which may negatively impact extracellular matrix formation and organization, BaP activates inflammatory mechanisms that recruit neutrophils, which affect both osteoblast and osteoclast activity, possibly through a direct interaction of the neutrophils with the bone matrix. This work provides novel data on the effects of microplastics exposure during zebrafish development, in particular its osteotoxic effects, and gives new insights into the cellular and molecular players involved in BaP osteotoxicity.Marco Tarasco was supported by the Portuguese Foundation for Science and Technology (FCT) through the PhD grant SFRH/BD/128634/2017 and COVID/BD/151848/2021 and by NEUBIAS-COST STSM program as part of action CA15124. This work was funded by FCT through projects UID/Multi/04326/2019, UID/00350/2020, UIDB/04326/2020, UID/MAR/00350/2013 and from the operational programs MAR2020 and COMPETE 2020 through projects OSTEOMAR MAR-02.01.01-FEAMP-0057 and EMBRC.PT ALG-01-0145-FEDER-022121

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia. The Cancer Genome Atlas Research Network has demonstrated the increasing genomic complexity of acute myeloid leukemia (AML). In addition, the network has facilitated our understanding of the molecular events leading to this deadly form of malignancy for which the prognosis has not improved over past decades. AML is a highly heterogeneous disease, and cytogenetics and molecular analysis of the various chromosome aberrations including deletions, duplications, aneuploidy, balanced reciprocal translocations and fusion of transcription factor genes and tyrosine kinases has led to better understanding and identification of subgroups of AML with different prognoses. Furthermore, molecular classification based on mRNA expression profiling has facilitated identification of novel subclasses and defined high-, poor-risk AML based on specific molecular signatures. However, despite increased understanding of AML genetics, the outcome for AML patients whose number is likely to rise as the population ages, has not changed significantly. Until it does, further investigation of the genomic complexity of the disease and advances in drug development are needed. In this review, leading AML clinicians and research investigators provide an up-to-date understanding of the molecular biology of the disease addressing advances in diagnosis, classification, prognostication and therapeutic strategies that may have significant promise and impact on overall patient survival

Development of lab-on-a-chip devices for automated zebrafish embryo bioassay

Zebrafish embryos have become one of the most popular model systems in biomedical and environmental research. However, current testing protocols using conventional multiwell plates rely heavily on time-consuming and labour-intensive manual handing. Static culture environments and low-throughput data collection are outdated with regards to meet the requirements of modern compound library screening. Herein, this research presents steps towards the development of a miniaturised and automated system for manipulating zebrafish embryos by using both innovative microfluidic lab-on-a-chip technologies and three-dimensional printing technologies. Four steps were taken to achieve this goal: (i) 3D printing technologies were explored to fabricate the lab-on-a-chip device. While 3D printing provided rapid manufacture of devices with high definition and optical transparency, as evidenced by SEM and confocal microscopy results, it caused significant toxicity in fish embryos after long-term exposure. (ii) The toxicity profile of a selection of 3D printing polymers was then extensively investigated using standard biotests. A chemical analysis was performed to reveal the compounds contributing to the toxicity. (iii) To avoid the use of toxic materials, a chip-based embryo trapping array was fabricated using biocompatible material PMMA. The chip allowed for automatic embryo loading, continuous reagent perfusion, and convenient image acquisition. The device was validated using both CFD simulations and biological experiments using reference toxicants. In addition, the embryo chip device was further developed to enable real-time metabolic level detection. (iv) A miniaturised and automated imaging platform, together with the high-throughput embryo trapping array and customised fluidic actuators, were prototyped

Identifying the pathological changes caused by familial Alzheimer’s disease-like mutations in zebrafish psen2

This project aimed to investigate the pathological changes caused by familial Alzheimer's disease-related (fAD-related) mutations in the gene PSEN2 based on a zebrafish model system. The CRISPR/Cas9 system was used to generate fAD-like mutations in the zebrafish genome. Although this mutagenesis system failed to generate mutations directly equivalent to the fAD mutations Nl411 and Vl481, (Nl40I and Vl471 in zebrafishpsen2), a fAD-like mutation psen2T141_L142delinsMISLISV [T141_L142delinsMISLISV superscript], a truncation mutation psen2N140fs [N140fs superscript], and a putative null mutation psen2S4Ter [S4Ter superscript], were generated. Allele­ specific expression tests using digital quantitative PCR were applied to all three mutations, and nonsense mediated decay was found to occur for the psen2N140fs [N140fs superscript] transcript. RNA-seq-based transcriptomic analysis was performed on brains from psen2S4Ter [S4Ter superscript] heterozygous and wild type siblings from a single family and revealed a likely relationship between psen2 and mitochondrial formation and function, which are thought to be associated with AD pathology. A GFP-Lc3a-GFP construct was designed to assay changes in autophagic flux in zebrafish larvae. By applying this assay to psen2S4Ter [S4Ter superscript]-carrying mutant larvae, as well as larvae possessing mutations in psen1, the Psen2 protein was seen to play a role in autophagy, which is also thought to be a process important in the development of AD pathology. Thus, the results of this project may contribute to a better understanding of the pathological mechanisms underlying AD.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 201