scholarly journals Cardioluminescence in transgenic zebrafish embryos: a calcium imaging tool to study drug effects and pathological modeling

2021 ◽  
Author(s):  
Manuel Vicente ◽  
Jussep Salgado-Almario ◽  
Michelle M. Collins ◽  
Antonio Martinez-Sielva ◽  
Masafumi Minoshima ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Light Output ◽  
Drug Effects ◽  
Study Drug ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Cardiovascular Research ◽  
Imaging Tool ◽  
Adrenergic Blocker

The zebrafish embryo has emerged as an excellent model in cardiovascular research. The existing techniques to monitor Ca2+ in the heart based on fluorescent Ca2+ biosensors are limited due to phototoxicity and photobleaching. To overcome these issues, we have used bioluminescence. We generated a transgenic line expressing GFP-Aequorin in the heart, Tg(cmlc2:GA), and optimized an in vivo aequorin reconstitution protocol to improve the luminescence capacity. This allowed imaging Ca2+ in long duration recordings in embryos of 3 to 5 days post-fertilization. The analogs diacetyl h-coelenterazine and f-coelenterazine enhanced the light output and signal-to-noise ratio from the embryos. With this cardioluminescence model, we monitored the time-averaged Ca2+ levels and beat-to-beat Ca2+ oscillations. Changes in Ca2+ levels were observed by incubation with BayK8644, an L-type Ca2+ channel agonist, the channel blocker nifedipine, and β-adrenergic blocker propranolol. Treatment of zebrafish embryos with terfenadine for 24 hours has been proposed as a model of heart failure. Tg(cmlc2:GA) embryos treated with terfenadine showed a 2:1 atrioventricular block and a decrease in the ventricular Ca2+ levels.

scholarly journals Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1294
Author(s):  
Manuel Vicente ◽  
Jussep Salgado-Almario ◽  
Michelle M. Collins ◽  
Antonio Martínez-Sielva ◽  
Masafumi Minoshima ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Output ◽  
Transgenic Line ◽  
Light Output ◽  
Study Drug ◽  
Calcium Oscillations ◽  
Transgenic Zebrafish ◽  
Calcium Dynamics ◽  
Cardiovascular Research ◽  
Zebrafish Larvae

Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetyl h-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.

scholarly journals An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

2018 ◽  
Author(s):  
Inês Garcez Palha ◽  
Isabelle Anselme ◽  
Sylvie Schneider-Maunoury ◽  
François Giudicelli
Keyword(s):  
Protein Synthesis ◽  
Protease Inhibitor ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Reporter System ◽  
Control Of Gene Expression ◽  
A Cell

ABSTRACTControl of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive, and reliable procedures to visualise sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique (Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. Addition of a cell-permeable protease inhibitor instantly stabilizes newly produced, tagged protein allowing to distinguish recently synthesized protein from more ancient one. After a brief protease inhibitor treatment, the ratio of tagged vs non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged protein it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.

scholarly journals Overexpression of Lifeact-GFP Disrupts F-Actin Organization in Cardiomyocytes and Impairs Cardiac Function

2021 ◽  
Vol 9 ◽  
Author(s):  
Rui Xu ◽  
Shaojun Du
Keyword(s):  
Actin Filament ◽  
Heart Development ◽  
Molecular Probe ◽  
Actin Dynamics ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Gfp Expression ◽  
Actin Organization ◽  
Cardiac Muscles

Lifeact-GFP is a frequently used molecular probe to study F-actin structure and dynamic assembly in living cells. In this study, we generated transgenic zebrafish models expressing Lifeact-GFP specifically in cardiac muscles to investigate the effect of Lifeact-GFP on heart development and its application to study cardiomyopathy. The data showed that transgenic zebrafish with low to moderate levels of Lifeact-GFP expression could be used as a good model to study contractile dynamics of actin filaments in cardiac muscles in vivo. Using this model, we demonstrated that loss of Smyd1b, a lysine methyltransferase, disrupted F-actin filament organization in cardiomyocytes of zebrafish embryos. Our studies, however, also demonstrated that strong Lifeact-GFP expression in cardiomyocytes was detrimental to actin filament organization in cardiomyocytes that led to pericardial edema and early embryonic lethality of zebrafish embryos. Collectively, these data suggest that although Lifeact-GFP is a good probe for visualizing F-actin dynamics, transgenic models need to be carefully evaluated to avoid artifacts induced by Lifeact-GFP overexpression.

scholarly journals Development of High-Content Assays for Kidney Progenitor Cell Expansion in Transgenic Zebrafish

2013 ◽  
Vol 18 (10) ◽  
pp. 1193-1202 ◽  
Author(s):  
Subramaniam Sanker ◽  
Maria Cecilia Cirio ◽  
Laura L. Vollmer ◽  
Natasha D. Goldberg ◽  
Lee A. McDermott ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Image Analysis ◽  
Time Course ◽  
Kidney Development ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Kidney Regeneration ◽  
Kidney Organogenesis

Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence–based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence–based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

scholarly journals Visualizing Drug Efficacy In Vivo

2005 ◽  
Vol 4 (2) ◽  
pp. 153535002005051 ◽  
Author(s):  
Weisheng Zhang ◽  
Min Chen ◽  
David B. West ◽  
Anthony F. Purchio
Keyword(s):  
Enzyme Activity ◽  
Drug Metabolism ◽  
Drug Effects ◽  
Study Drug ◽  
Drug Efficacy ◽  
Biochemical Assays ◽  
Metabolism Enzymes ◽  
First Time ◽  
Cytochrome P 450

Many enzymes are therapeutic targets for drug discovery, whereas other enzymes are important for understanding drug metabolism and pharmacokinetics during compound testing in animals. Testing of drug efficacy and metabolism in an animal model requires the measurement of disease endpoints as well as assays of enzyme activity in specific tissues at selected time points during treatment. This requires the removal of tissue and biochemical assays. Techniques to noninvasively assess drug effects on enzyme activity using imaging technology would facilitate understanding of drug efficacy, pharmacokinetics, and drug metabolism. Using a commercially available cytochrome P−450 3A substrate whose oxidized product is a luciferase substrate, we show for the first time that cytochrome P−450 enzyme activity can be measured in vivo in real time by bioluminescent imaging. This imaging approach could be applicable to study drug effects on therapeutic target enzymes, as well as drug metabolism enzymes.

Fish in chips: an automated microfluidic device to study drug dynamics in vivo using zebrafish embryos

2014 ◽  
Vol 50 (8) ◽  
pp. 981-984 ◽  
Author(s):  
Chunhong Zheng ◽  
Hongwei Zhou ◽  
Xinxing Liu ◽  
Yuhong Pang ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Study Drug ◽  
Zebrafish Embryos

scholarly journals In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis

PLoS ONE ◽  
2016 ◽  
Vol 11 (2) ◽  
pp. e0149497 ◽  
Author(s):  
Ling Li ◽  
François Bonneton ◽  
Marie Tohme ◽  
Laure Bernard ◽  
Xiao Yong Chen ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
In Vivo Screening

scholarly journals Py 3 -FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes

Open Biology ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 200241
Author(s):  
Zhaotong Wang ◽  
Takamasa Mizoguchi ◽  
Takahito Kuribara ◽  
Masaya Nakajima ◽  
Mayuu Iwata ◽  
...  
Keyword(s):  
Normal Development ◽  
Early Stage ◽  
Human Cells ◽  
The Body ◽  
Zebrafish Embryos ◽  
Imaging Tool ◽  
Bulbus Arteriosus ◽  
Cultured Human Cells ◽  
Low Toxicity

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo . Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py 3 -FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py 3 -FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py 3 -FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py 3 -FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py 3 -FITC solution. Therefore, Py 3 -FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Acyclovir Loaded Solid Lipid Nanoparticle Based Cream: A Novel Drug Delivery System

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
EL- Assal I. A. ◽  
Retnowati .
Keyword(s):  
Drug Delivery ◽  
Drug Release ◽  
Antiviral Agent ◽  
Study Drug ◽  
Particle Size Analysis ◽  
Stability Study ◽  
Size Analysis ◽  
Solid Lipid ◽  
Dermal Delivery

Objective of the present investigation was enthused by the possibility to develop solid lipid nanoparticles (SLNs) of hydrophilic drug acyclovir. Also study vitro and vivo drug delivery. Methods: Drug loaded SLNs (ACV-SLNs) were prepared by high pressure homogenization of aqueous surfactant solutions containing the drug-loaded lipids in the melted or in the solid state with formula optimization study (Different lipid concentration, drug loaded, homogenization / stirring speed and compritol 888ATO: drug ratio). ACV - SLN incorporated in cream base. The pH was evaluated and rheological study. Drug release was evaluated and compared with simple cream- drug, ACV – SLN with compritol 888ATO and marketed cream. The potential of SLN as the carrier for dermal delivery was studied. Results: Particle size analysis of SLNs prove small, smooth, spherical shape particle ranged from 150 to 200 nm for unloaded and from 330 to 444 nm for ACV loaded particles. The EE% for optimal formula is 72% with suitable pH for skin application. Rheological behavior is shear thinning and thixotropic. Release study proved controlled drug release for SLNs especially in formula containing compritol88 ATO. Stability study emphasized an insignificant change in SLNs properties over 6 month. In-vivo study showed significantly higher accumulation of ACV in stratum corneum, dermal layer, and receptor compartment compared with blank skin. Conclusion: AVC-loaded SLNs might be beneficial in controlling drug release, stable and improving dermal delivery of antiviral agent(s).

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