2B). Previously, other groups have reported that a PDE5 inhibitor restores mdx mouse muscle to normal (20, 21, 26). 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. analyzed data; and G.K. and L.M.K. Translating results to what we might see in humans requires an appropriate (vertebrate) model suited for large-scale studies. - "Zebrafish angiogenesis: A new model for drug screening" Figure 3. In the study by Hong and colleagues, on-target and off-target effects could be … After excising the negatively charged phosphorylated and positively charged nonphosphorylated bands from the gel, the gels were melted at 95 °C. 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 … 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. Zebrafish embryos were collected and raised at 28.5 °C according to standard procedures and criteria. The orthologs of many dystrophin–glycoprotein complex (DGC) components are expressed in zebrafish (19). Here, we demonstrate that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation. Light blue, nontreated WT fish. Drug screening in zebrafish. 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. Red, dystrophin-null unaffected fish (normal birefringence). Astronomers thought they’d finally figured out where gold and other heavy elements in the universe came from. 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-like biophysical properties. Error bars indicate SDs. Zebrafish platform Austria for preclinical drug screening (ZANDR) Zebrafish and in particular zebrafish larvae is a well suited vertebrate model system to investigate various human diseases and to perform … In light of recent results, they’re not so sure. The other compounds identified in our screen target additional pathways and represent additional targets for therapy. 56,57 The zebrafish system is emerging as a valuable model organism for drug … Zebrafish xenograft assays are ideal for testing drug sensitivity directly from patient biopsies in a personalized medicine context. 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. Enoximone (PDE3 inhibitor; ◆), milrinone (PDE3 inhibitor; ◇), ibudilast (PDE4 inhibitor; ▲), rolipram (PDE4 inhibitor; △), sildenafil citrate salt (PDE5 inhibitor; ●), dipyridamole (PDE5 inhibitor; ○), aminophylline (nonselective PDE inhibitor; ■), and DMSO (vehicle; X). 3), and indeed, some were toxic to all fish. At 5 dpf, treated fish were cultured at room temperature in 100 mL fish water containing individual chemicals (2.5 μg/mL) until 30 dpf at room temperature. 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). 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). Red bars, chemically treated fish with 108 chemical pools (in duplicate). Nontreated dystrophin-null fish have disorganized structure (arrow) in skeletal muscle. Fish treated with chemical 4 that survived to 30 dpf were sectioned, and they were stained by H&E and immunostained with anti-dystrophin, laminin, and myosin heavy-chain antibodies. The number of surviving fish at every other day intervals was determined, and the genotypes of surviving fish were determined at 30 dpf. These candidate drugs will be tested in other strains of muscular dystrophy in laminin-deficient zebrafish. Extracted samples were incubated with PepTag A1 peptide and buffer and separated by eletrophoresis on 0.8% agarose gels. Co., Ltd., Toyonaka, Osaka 561-0825, Japan. Zebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high throughput, microscopy-based screens. Zebrafish can be used to study vertebrate development and gene … For the first screen (schematically outlined in Fig. Dear Editor, Here we report findings of zebrafish behavior and development during drug screening. On the other hand, mammalian models are excellent tools to study disease mechanisms and test therapies but are difficult to use for large scale drug screening. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. (B) WT. In the second screen, pools were separated into individual chemicals for a total 48 molecules and tested to identify which individual compound influenced the ratio of fish showing abnormal birefringence. Behavioral screens can be used as a first-line detection tool for new drug effects, and their popularity continues to grow. To test whether other PDE inhibitors might also ameliorate dystrophic symptoms in sapje fish, 20 embryos from matings of heterozygous sapje fish were treated with a series of PDE inhibitors from 1 to 4 dpf (in triplicate). The identification of compounds, especially PDE inhibitors, that moderate the muscle phenotype in these dystrophin-null zebrafish validates the screening protocol described here and may lead to candidate molecules to be used as therapeutic interventions in human muscular dystrophy. Recently, a number of chemical and drug screens have been published using zebrafish embryos (7–11). 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. Moreover, other PDE inhibitors also reduced the percentage of affected sapje fish. 4 A and B). Milan et al. Twenty pairs of heterozygous sapje fish or sapje-like mutants were mated, and fertilized eggs were cultured at 28.5 °C. In this secondary screen, seven chemicals influenced muscle structure by decreasing the percentage of affected fish using both sapje (used in the first screen) and sapje-like fish, which has an independent dystrophin mutation (Table 2). 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. 1E). It is generally known that PDE inhibitors cause an increase in intracellular cAMP and/or cGMP. More phosphorylated PKA was detected in aminophylline-treated fish compared with WT and untreated dystrophin-null fish (Fig. Proteins were analyzed with Western blot as described in ref. (E) Distribution of the percentage of affected fish treated with chemical pools and untreated fish. The number of surviving fish was counted and recorded every other day, and at 30 dpf, their genotypes were determined. S2). The role of ZEBRAFISH in drug discovery By Dr Paul Goldsmith and Roberto Solari The NIH has ranked the Zebrafish as the third most important experimental organism after man, the Sanger … Our results indicate that the activity of PKA is clearly up-regulated in aminophylline-treated dystrophin-null fish. However, treatment with each of the seven chemicals reduced the percentages of affected fish showing abnormal birefringence (Fig. ZEBRAFISH DRUG SCREENS. Similar results were obtained in sapje-like fish, where some of the homozygous dystrophin-null fish had apparently normal birefringence reduction (Fig. Scale bar = 100 lm. Screening assays, Zebrafish, Drug discovery; Get e-Alerts. This library contains 1,120 small molecules composed of 90% marketed drugs and 10% bioactive alkaloids or related substances. They are widely recognised as an ideal model for HTS chemical screening as they are closer to mammals from an evolutionary point of view than worms or flies. After the identification of pools that decreased the number of affected fish, each individual compound in these pools was screened as outlined in Fig. The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD), representing more than 90% of the diagnosed cases. Other treatments currently being tested or considered for treating muscular dystrophy include the small molecule PTC124, which promotes read-through of nonsense mutations (3), encouraging muscle development by myostatin down-regulation (4, 5), and the use of oligonucleotides to promote exon skipping to restore dystrophin expression (6). 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. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene, A cDNA clone from the Duchenne/Becker muscular dystrophy gene, PTC124 targets genetic disorders caused by nonsense mutations, Functional improvement of dystrophic muscle by myostatin blockade, Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse, Local dystrophin restoration with antisense oligonucleotide PRO051, Zebrafish: A model system for the study of human disease, Chemical discovery and global gene expression analysis in zebrafish, Small molecules that delay S phase suppress a zebrafish bmyb mutant, Small molecule developmental screens reveal the logic and timing of vertebrate development, The zebrafish as a model for muscular dystrophy and congenital myopathy, Delta-sarcoglycan is required for early zebrafish muscle organization, The dystrophin associated protein complex in zebrafish, Genetic isolation and characterization of a splicing mutant of zebrafish dystrophin, Genes required for functional glycosylation of dystroglycan are conserved in zebrafish, Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of fukutin-related protein (FKRP), Zebrafish models for human FKRP muscular dystrophies, Zebrafish orthologs of human muscular dystrophy genes, Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy, Sarcolemma-localized nNOS is required to maintain activity after mild exercise, Theophylline: New perspectives for an old drug, Effects of aminophylline on cytokines and pulmonary function in patients undergoing valve replacement, Anti-inflammatory effects of low-dose oral theophylline in atopic asthma, Anti-oxidative effects of theophylline on human neutrophils involve cyclic nucleotides and protein kinase A, Primary role of functional ischemia, quantitative evidence for the two-hit mechanism, and phosphodiesterase-5 inhibitor therapy in mouse muscular dystrophy, An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics, Protein kinase A signalling via CREB controls myogenesis induced by Wnt proteins, Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase, Stages of embryonic development of the zebrafish, Proceedings of the National Academy of Sciences, Earth, Atmospheric, and Planetary Sciences, www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102116108/-/DCSupplemental, Science & Culture: At the nexus of music and medicine, some see disease treatments, News Feature: Tracing gold's cosmic origins, Journal Club: Friends appear to share patterns of brain activity, Transplantation of sperm-producing stem cells. 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