As a matter of fact, concordance between studies ran on zebrafish and - "Zebrafish angiogenesis: A new model for drug screening" Figure 3. In fact, zebrafish … Error bars indicate SDs. S4). Keywords:Zebrafish, drug, toxicity screening, high throughput assay, neurotoxicity, hepatotoxicity, cardiotoxicity. Genotype and dystrophin expression of treated fish with seven candidate chemicals from the longer screen. However, there was no chemical exhibiting less than 7.5% of mutant fish with abnormal birefringence. Long-term treatment of dystrophin-null fish selected to have abnormal birefringence at 4 dpf with chemicals 1–7 from 4 to 30 dpf (n = 3). Muscular dystrophy is a disease in which the muscle forms normally at first but then degenerates faster than it can be repaired. This disorder results in a muscle pathology that can be detected by birefringence under polarizing light and usually results in death by 7–9 dpf. the zebrafish can serve as an early preclinical drug screening tool and can also help personalize cancer therapy by providing real-time data on the response of the human cells to treatment. Here we briefly review screening … Zebrafish are indeed excellent models for whole animal drug screening with excellent throughput. However, the homozygous dystrophin-null fish that showed no abnormality of birefringence showed no immunoreactivity with anti-dystrophin, and the same lack of staining was found in the nontreated dystrophin-null fish (Fig. S3). Biology, Husbandry, Diseases, and Research Applications, American College of Laboratory Animal Medicine, Zebrafish as a Platform for Drug Screening. (E) Assay of cAMP-dependent PK activity (n = 3). (D–J) Dystrophin immunostaining of fish with restored muscle structure with each of the seven chemicals (chemicals 1–7 in Table 2). These studies recognized the advantages of using transparent 48–72 hpf larvae to monitor the effects of compounds on cardiac rhythmicity using relatively simple light microscopy imaging. (B) Immunostaining of WT, nontreated, and chemical 4-treated fish with anti-myosin heavy chain and anti-laminin antibody. The remaining four chemicals groups resulted in death of all three strains (+/+, +/−, and −/− dystrophin) (Fig. 4B, arrows). Due to their small size and transparency, such testing requires a small mass of test article, very little lab space, and data can be collected noninvasively over time in vivo. In 20 embryos, five apparently affected fish are caused by a homozygous mutation in the zebrafish dystrophin gene, which is inherited in a recessive manner (25%; arrows). Nontreated dystrophin-null fish have broken and disturbed structure (arrows) of skeletal muscle fibers. 3), and indeed, some were toxic to all fish. Keywords:Zebrafish, drug discovery, drug testing, large-scale screen, chemical genetics, pre-clinical studies. To whom correspondence should be addressed at Drug Safety Evaluation, Research Laboratory for Development, Shionogi and Co., Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan. All sequencing was accomplished in the Intellectual and Developmental Disabilities Research Center Molecular Core Laboratory supported by National Institute of Child Health and Human Development Grant 2P30HD018655-26. Pairs of heterozygous sapje fish were mated, and fertilized eggs were cultured at 28.5 °C. (D) Ratio of phosphorylated PKA and PKA in 30 dpf surviving fish (n = 3). The one of the features that the zebrafish heart can be dissected rapidly and maintained ex vivo for several days makes zebrafish an excellent model for assessment of cardiovascular toxicity. These embryos also showed markedly reduced expression of dystrophin in their myosepta. Zebrafish were introduced as a drug screening platform in the context of LQTS almost 20 years ago (Langheinrich et al., 2003; Milan et al., 2003; see Table 1). Zebrafish can be used to assess the toxicity of drug candidates in early screening assays, sometimes in a high-throughput manner. Take cardiovascular toxicity test as an example, which is a key limiting factor in drug discovery. 18. The dystrophin-null fish with treated aminophylline have normal structure in skeletal muscle. We have already looked into other PDF inhibitors through our identification of aminophylline in this screen as targets for therapy. Zebrafish can be used to study vertebrate development and gene … For drug screening involving PTZ induction, 5 dpf zebrafish were treated with 10 mM PTZ for 10 min, followed by 20 μM (final concentration) of drug for 20 min under ambient light, and assayed for total locomotor activity, burst activity, distance moved and inactive duration, and tracking under 100% light in ZebraBox for 20 min. Zebrafish: A Model for Marine Peptide Based Drug Screening [electronic resource] / by Saravanan Ramachandran, Senthilkumar Rajagopal. Copyright © 2021 National Academy of Sciences. These results indicate that some of the dystrophin-null fish treated throughout the first 4 d of development failed to develop the abnormalities seen in some of their clutch mates. After washing three times, sections were incubated with secondary antibodies and examined as described in ref. In the study by Hong and colleagues, on-target and off-target effects could be … For H&E staining of frozen muscle sections, sections were stained with Mayer’s hematoxylin solution (Fluka) for 10 min and 1% eosin solution (Sigma) for 30 s after washing with water. Chemical compounds of relatively small molecular weight can bind to specific proteins and alter their function, resulting in nonheritable phenotype changes. Interestingly, sildenafil citrate, a PDE5 inhibitor (Fig. Thank you for your interest in spreading the word on PNAS. Overall, this study highlights the predictive value of the zebrafish larva as a drug screening model for SCI. Similarly, the zebrafish is also an attractive screening tool for cardiovascular risk assessment after treatment with atypical antipsychotic drugs, as it facilitates the evaluation of the … Zebrafish are increasingly swimming into the view of large-scale drug screening projects. The orthologs of many dystrophin–glycoprotein complex (DGC) components are expressed in zebrafish (19). The expression of PKA was detected in all samples. Among the 108 groups that survived treatment, some contained pools of chemicals that seemed to influence the ratio of affected fish relative to normal-seeming fish (Fig. Fax: +81 … Immunohistochemical staining of whole-mutant and morphant fish bodies was done as described in ref. All restored fish with chemicals 1–7 showed no expression of dystrophin, similar to those of untreated dystrophin-null fish (C). Recently, a number of chemical and drug screens have been published using zebrafish embryos (7–11). Zebrafish can also be integrated to validate the efficacy and toxicities of compounds that are identified as novel therapeutics by other approaches, such as computational drug … Interestingly, myosin heavy-chain staining of treated dystrophin-null fish, which survived until 30 dpf, showed normal myofiber structure, similar to that of the WT fish, whereas untreated dystrophin-null fish showed clear abnormalities of muscle (Fig. Scale bar = 100 lm. Although the evidence is still limited, a growing body of research suggests music may have beneficial effects for diseases such as Parkinson’s. The phenotypes of the disease model zebrafish can be used to identify novel compounds and/or new indications for old drugs (i.e., drug repositioning) that ameliorate the abnormal phenotypes of the zebrafish disease models. laboratory for their experimental advice and help in performing these experiments and drafting this manuscript. Light blue, WT fish (normal birefringence). These results show that activated phosphorylated PKA and the activity of PKA were increased in aminophylline-treated fish compared with WT and untreated dystrophin-null fish, which suggests indirectly that intracellular cAMP is increased with aminophylline treatment. 4B). As such, our laboratory has two available fish models of DMD with dystrophin deficiency, sapje (12) and sapje-like fish (15). 1E). (2003), tested the effects of … The zebrafish is introduced as a model organism, in particular for cardiovascular biology. Translating results to what we might see in humans requires an appropriate (vertebrate) model suited for large-scale studies. Chemical screening in a zebrafish model of doxorubicin-induced cardiotoxicity identified the small molecules visnagin and diphenylurea, which rescued doxorubicin-induced cardiotoxicity in zebrafish and mice; visnagin bound to mitochondrial malate dehydrogenase (MDH2), and several MDH2 inhibitors exhibited similar cardioprotective effects, supporting visnagin and modulators of MDH2 activity as potential options for treating doxorubicin-induced … The Molecular Genetics Core Facility at Children’s Hospital Boston sequenced PCR products after PCR purification using a standard kit (Qiagen). So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Zebrafish has proven to be an ideal preclinical model due to the high percentage of genes associated with human disorders. Many of these screens have been highly successful in disease modeling and drug screening (8–10), making the zebrafish ideal for high-throughput whole-organism screening of candidate compounds. (A) H&E staining of WT, nontreated, and chemical 4-treated fish. Our results indicate that the activity of PKA is clearly up-regulated in aminophylline-treated dystrophin-null fish. 5, closed and opened diamonds), exhibited no influence on the percentage of affected sapje fish. 2C). Creative Biostructure establishes and provides the MagHelix™ Zebrafish Screening Platforms for scientific and medical research. After excising the negatively charged phosphorylated and positively charged nonphosphorylated bands from the gel, the gels were melted at 95 °C. The results are plotted (Fig. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for large-scale drug screening. Zebrafish model for rapid drug discovery There are six major reasons for supporting zebrafish as a good vertebrate model for drug screening, specifically on visual problems: 1) zebrafish's eye, particularly the retina, is anatomically similar to many vertebrates including human10. Chemical compounds of relatively small molecular weight can bind to specific proteins and alter their function, resulting in nonheritable phenotype changes. wrote the paper. S1), 140 chemical pools from a total of 1,120 chemicals in the Prestwick library were tested using embryos from matings of sapje heterozygous pairs. However, PDE3 inhibitors, enoximone and milrinone (Fig. The morpholino sequences for dystrophin were MO: 5′-TTGAGTCCTTTAATCCTACAATTTT-3′; 6 ng morpholinos were injected into the yolk of one- to two-cell stage embryos as described in ref. 4 C and D, P < 0.05). Nontreated dystrophin-null fish have disorganized structure (arrow) in skeletal muscle. By continuing you agree to the use of cookies. 3). ZEBRAFISH DRUG SCREENS. The motility of surviving affected fish was not relatively altered compared with that of WT. At 1 dpf, 20 injected embryos were arrayed in a 24-well plate and were treated with individual chemicals (1–7). The use of zebrafish embryos in early stages of drug safety‐screening is discussed. These preclinical studies quickly assess toxicity and efficacy of new drugs, saving both time and money, necessities in this age of great pressure to do both. To assay the activity of PKA, the PepTag Assay for Non-Radioactive Detection of cAMP-Dependent Protein Kinase (Promega) was used following the manufacturer’s protocol. Zebrafish platform Austria for preclinical drug screening (ZANDR) ZANDR was established at the Children´s Cancer Research Institute (CCRI) in 2019 and is a unique platform to phenotypically screen small compounds on zebrafish models of human diseases. The SickKids Zebrafish Genetics and Disease Models Core aims to bridge human disease modeling to drug discovery using zebrafish as a model system. The effects of molecule antineoplastic drugs have often been detected by biochemical assays or in cell line models, but the outcomes were unsatisfactory. Treated fish with chemicals 1 (◇), 2 (X), 3 (+), 4 (■), 5 (), 6 (△), 7 (—), and WT (●) are shown. In fact, zebrafish is a diurnal animal The expression of dystrophin was examined by immunostaining individual fish bodies with anti-dystrophin antibodies. Abstract. (A) Genotyping of each of 40 fish treated with each of the seven potential candidate chemicals. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Analysis of skeletal muscle of dystrophin-null fish that survive 30 d after treatment with chemical 4, aminophylline. S1). Phenotype-based, cost-effective whole-organism chemical screening in zebrafish offers a variety of advantages including the identification of disease-modifying drugs without knowledge of a … To this end, the expression, phosphorylation, and activation of PKA in dystrophin-null fish treated with aminophylline for 25 d were examined. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for large-scale drug screening. 2 D–J). Zebrafish-based drug screens have identified novel compounds that modulate pathological phenotypes arising from a wide range of diseases, including cancer, bone marrow failure, cardiac and vascular abnormalities, metabolic disease, and behavioral disorders . Moreover, they are ideally suited for use in chemical screens to select drug candidates capable of correcting the muscle phenotype. Two known zebrafish dystrophin mutants, sapje and sapje-like (sapc/100), represent excellent small-animal models of human muscular dystrophy. Extracted samples were incubated with PepTag A1 peptide and buffer and separated by eletrophoresis on 0.8% agarose gels. Behavioral screens can be used as a first-line detection tool for new drug effects, and their popularity … The number of surviving fish was counted and recorded every other day, and at 30 dpf, their genotypes were determined. These preclinical studies quickly assess toxicity and efficacy of new drugs… performed research; G.K. contributed new reagents/analytic tools; G.K. and L.M.K. Abstract:Over the past decade, zebrafish are being increasingly used in assessing the … This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Using a two-tiered screening strategy, first, pooling compounds and then, screening individual compounds, resulted in identification of seven individual chemicals that decreased the percentage of phenotypically affected fish, which suggests that these seven chemicals might rescue the muscle phenotype found in the dystrophin-null fish. Zebrafish are ideal for this type of translational biology because of their large clutch size, rapid early development, and transparent embryos, making them perfect for both CRISPR-Cas genome editing and high-throughput drug screening. The muscle degeneration phenotype in these mutant fish is transmitted in a recessive manner, such that 25% of the offspring show dystrophic features of skeletal muscle after 3 d postfertilization (dpf). In light of recent results, they’re not so sure. 18. Co., Ltd., Toyonaka, Osaka 561-0825, Japan. Rapid and cost-effective in vivo assays to screen potential environmental neurodevelopmental toxicants are necessary to address the … Zebrafish model for rapid drug discovery There are six major reasons for supporting zebrafish as a good vertebrate model for drug screening, specifically on visual problems: 1) zebrafish's eye, particularly the retina, is anatomically similar to many vertebrates including human10. This analysis may indicate common and specific pathways of action of these candidate drugs. For long-term treatment of fish from 1 to 30 dpf, WT, heterozygous, and dystrophin-null fish from heterozygous fish matings were treated with candidate chemicals 1–7. Currently, prednisone is the only treatment option available for muscular dystrophy patients in the United States, although there are currently other options through approved clinical trials. This is a tall order, but one A screening approach that detects sedative-hypnotic drug activity in zebrafish larvae, based on inhibition of movements in response to brief bright light stimuli (photomotor responses), was established and used to screen several hundred organic compounds with drug … In the experimental groups tested with the chemical pools, the distribution of the affected fish ranged between 2.5% and 27.5%; 6 groups had less than 7.5% of mutant fish showing abnormal birefringence, and 16 groups had less than 10% (Fig. In this study, we evaluated the usefulness of adult zebrafish as a small in vivo system for detecting drug-induced kidney … Keywords:Zebrafish, drug discovery, drug testing, large-scale screen, chemical genetics, pre-clinical studies. These fish are also small enough to be permeable to small molecules and can be assayed in large numbers. High-Throughput Zebrafish Screening Core (HTZSC) The HTZSC is a unit to support high-throughput screening of drugs or toxins to assess safety, effectiveness, or toxicity for humans using zebrafish. At 5 dpf, they were cultured at room temperature in 100 mL fish water containing individual chemical (2.5 μg/mL) and monitored to 30 dpf. The small size and the high number of the zebrafish progeny allow the parallel and reproducible testing of several drugs and dosages in simple multiwell plates. Blue bars, untreated fish (n = 28; independent determination). The 20 embryos were arrayed in 24-well plates and cultured in 1 mL fish water containing individual chemicals at 1 dpf. Proteins were analyzed with Western blot as described in ref. We have also shown the usefulness of the dystrophin-null zebrafish in screens of compounds that might ameliorate symptoms. These results suggest that treatment with chemical 4 restored the muscle structure of these dystrophin-null fish, because they had abnormal structure when selected at 4 dpf before chemical treatment. Some chemical pools decreased the percentage of affected fish showing abnormal birefringence compared with untreated fish and might contain a compound able to restore normal muscle structure. The larval zebrafish xenograft assay has unique characteristics that make it unlike all other ex vivo assays and a top choice for in vivo drug screening. The number of surviving fish at every other day intervals was determined, and the genotypes of surviving fish were determined at 30 dpf. Drug screening in a zebrafish model of Duchenne muscular dystrophy Genri Kawahara, Jeremy A. Karpf, Jennifer A. Myers, Matthew S. Alexander, Jeffrey R. Guyon, and Louis M. Kunkel … Survival of zebrafish after treatment with pools or individual chemicals from the Prestwick library. (A and C) Bright image. Abstract:Over the past decade, zebrafish are being increasingly used in assessing the effects of chemical compounds.