Zebrafish Yolk Research Articles

Super-resolution imaging lysosome vesicles and establishing a gallbladder-visualizable zebrafish model via a fluorescence probe

Advanced probes for imaging viscous lipids microenvironment in vitro and in vivo are desirable for the study of membranous organelles and lipids traffic. Herein, a reaction-based dihydroquinoline probe (DCQ) was prepared via linking a diethylamino coumarin fluorophore with a N-methylquinoline moiety. DCQ is stable in low viscous aqueous mediums and exhibits green fluorescence, which undergoes fast autoxidation in high viscous mediums to form a fluorescent product with deep-red to near-infrared (NIR) emission, rendering the ability for dual-color imaging. Living cell imaging indicated that DCQ can effectively stain lysosomal membranes with deep-red fluorescence. Super-resolution imaging of lysosome vesicles has been achieved by DCQ and stimulated emission depletion (STED) microscopy. In addition, DCQ realizes multiple organs imaging in zebrafish, whose dual-color emission can perfectly discriminate zebrafish's yolk sac, digestive tract and gallbladder. Most importantly, DCQ has been successfully used to establish a gallbladder-visualizable zebrafish model for the evaluation of drug stress.

Hexyltrimethylammonium ion enhances potential copper-chelating properties of ammonium thiomolybdate in an in vivo zebrafish model

Ammonium and hexyltrimethylammonium thiomolybdates (ATM and ATM-C6) and thiotungstates (ATT and ATT-C6) were synthesized. Their toxicity was evaluated using both in vitro and in vivo approaches via the zebrafish embryo acute toxicity assay (ZFET), while the copper-thiometallate interaction was studied using cyclic voltammetry, as well as in an in vivo assay. Cyclic voltammetry suggests that all thiometallates form complexes with copper in a 2:1 Cu:thiometallate ratio. Both in vitro and in vivo assays demonstrated low toxicity in BALB/3T3 cells and in zebrafish embryos, with high IC50 and LC50 values. Furthermore, the hexyltrimethylammonium ion played a crucial role in enhancing viability and reducing toxicity during prolonged treatments for ATM and ATT. In particular, the ZEFT assay uncovered the accumulation of ATM in zebrafish yolk, averted by the incorporation of the hexyltrimethylammonium ion. Notably, the copper-thiometallate interaction assay highlighted the improved viability of embryos when cultured in CuCl2 and ATM-C6, even at high CuCl2 concentrations. The hatching assay further confirmed that copper-ATM-C6 interaction mitigates inhibitory effects induced by thiomolybdates and CuCl2 when administered individually. These results suggest that the incorporation of the hexyltrimethylammonium ion in ATM increase its ability to interact with copper and its potential application as a copper chelator.

In vitro and in vivo anti-inflammatory models demonstrate oligopeptides play a significant role in anti-inflammatory properties of white tea

White tea is a kind of tea abundant in oligopeptides. The study aims to reveal the potential association between white tea oligopeptides and anti-inflammatory ability. Crude oligopeptide extracts of two subtype white tea (named BaiMudan and ShouMei) were prepared by removing known active substances including pigments, polyphenols, caffeine, polysaccharides, and free amino acids. Four abundant oligopeptides (LCAY, LLLSKKL, EVFAG, and ECDSCG) were identified through high-resolution mass spectrometry sequencing, and synthesized for anti-inflammation evaluation. In vitro experiments showed, expect there was no effect of EVFAG on TNF-α, the rests oligopeptides were all good at inhibit NO, IL-6, and TNF-α. In vivo experiments indicated a notable decrease in neutrophils of zebrafish yolk sac. Specifically, performance of LCAY was particularly outstanding, with its anti-inflammatory effect at 250 μg·mL−1 could exceed 100 μM dexamethasone. The prominent significance of this study is that it confirms white tea oligopeptides have a good anti-inflammatory activity.

Protective effect of N-(E)-p-coumaroyltyrosine on LPS-induced acute inflammatory injury and signaling pathway analysis

N-trans-p-coumaroyltyrosine (N-(E)-p-coumaroyltyrosine, NPCT), extracted and purified from Abri Mollis Herba, is an amino acid amide. The defense mechanism of NPCT against inflammatory response is still unknown. In this study, lipopolysaccharide (LPS)-induced zebrafish acute inflammatory injury model was established to observe the inhibitory effect of NPCT on the aggregation of inflammatory cells in the yolk sac of zebrafish, as well as the inhibitory effect of NPCT on inflammatory and gas signaling factors. Results show that NPCT could inhibit inflammatory cell infiltration in zebrafish yolk sac, the migration and aggregation of macrophages and neutrophils to the site of inflammation, and the release of Nitric Oxide (NO) and Reactive Oxygen Species (ROS) in zebrafish, indicating that NPCT could substantially significantly prevent the development of LPS-induced acute systemic inflammation. In addition, the analysis results of RNA-seq showed that in the model group versus the administered group, the differentially expressed genes were mainly enriched to inflammatory signaling pathways, such as the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway, which were down-regulated in the administered group. The TLR4, MyD88, IRAK4, NF-κB, IκB, NLRP3, Caspase-1, ASC, IL-1β, and IL-6 genes were significantly different in the transcripts, and the overall trend of the qPCR results was consistent with the transcriptome sequencing results. Therefore, NPCT had a significant inhibitory effect on LPS-induced acute inflammatory injury in zebrafish, and its anti-inflammatory mechanism may be through the regulation of key genes on the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway, thereby affecting the release of relevant inflammatory cytokines.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Skin pigmentation improvement with resveratrol microemulsion gel using polyoxyethylene hydrogenated castor oil

Objective To investigate the safety and efficacy of resveratrol microemulsion gel in improving pigmentation. Methods Resveratrol microemulsion gel was prepared by the microemulsion solubilization method, and its quality was evaluated. The transdermal and drug retention rates of resveratrol in vivo were assessed using a transdermal test. The inhibitory effects of resveratrol suspension and microemulsion on tyrosinase activity and melanin production of A375 human melanocytes and zebrafish embryos were compared. A skin patch test was used to investigate the safety of the gel on 15 volunteers. Results The microemulsion gel was homogeneous and stable. Compared with suspension and microemulsion, the drug penetration rate and skin retention in the microemulsion gel group were significantly increased. Compared with the suspension group, the activity of melanocyte tyrosinase in A375 human melanocyte was significantly inhibited in the microemulsion group, and the melanin production rate of A375 human melanocyte and the melanin area of zebrafish yolk was decreased. All 15 volunteers tested negative for the human skin patch. Conclusions The microemulsion gel could significantly enhance the ability of resveratrol to inhibit the formation of melanin without causing side effects. These data provide the experimental basis for developing and applying the preparation for improving pigmentation.

Abstract B007: Patterns and quantitation of migration and metastasis in a zebrafish xenograft model of ewing sarcoma

Abstract Ewing sarcoma (ES) is one of the most common and lethal pediatric cancers with a 5-year survival rate of less than 30% for those with metastatic disease. Approximately 25% of ES patients have metastatic disease at time of diagnosis. Current treatment for ES rests on an intensive regimen of five chemotherapeutic agents with a high level of toxicity. Recurrent ES is incurable. Hence, there is a desperate need for more effective and less toxic treatments. A major obstacle in developing new, more effective therapies against ES is the lack of animal models. Zebrafish offers a robust vertebrate model for investigating cancer and evaluating the efficacy of anti-cancer drugs. Many genes, signaling pathways, and mechanisms of carcinogenesis and metastasis are highly conserved between humans and zebrafish. As zebrafish do not develop an adaptive immune response until approximately four weeks of age, they present an ideal system for xenograft tolerance in an immunocompetent organism. In addition, the transparency of zebrafish embryos permits high-resolution optical imaging in vivo and allows for easy tracking of tumor cell migration and metastasis. Our lab has created a ES xenograft model in zebrafish by injecting mCherry-labeled ES cell lines into the transgenic strain Tg(fli:GFP). This strain marks the zebrafish vasculature green, allowing for effective tracking of the injected red cancer cells. Our lab transduced the human ES cell lines A673 and MHH-ES-1, both of which contain the EWSR1:FLI fusion and TP53 mutations, with the mCherry construct. The top 10% brightest cells were selected to allow for easy visualization within the zebrafish. Approximately 200 labeled cells were injected into the yolk at two days post fertilization (2 dpf). Cell growth and migration were tracked until 5 dpf. Using fluorescent microscopy, images of each injected fish were collected at one day post injection (1 dpi), corresponding to 3 dpf, and at 3dpi, corresponding to 5 dpf. Using ImageJ software, these images were carefully analyzed for tumor growth, cell migration, and metastasis. To quantify tumor invasion and metastatic dissemination, we used flow cytometry to compare total ES cell numbers between 1 dpi and 3 dpi within the zebrafish. Our results show that metastasis within the zebrafish, which is indicated by ES cell growth within the tail region, occurs in approximately 15% of injected fish. Data from fluorescent microscopy and flow cytometry demonstrate primary tumor growth, within the zebrafish yolk, occurs in nearly all injected fish. Flow cytometry work is ongoing to quantify the percent increase in tumor growth within injected fish at 5dpf. Large–scale drug screens can be easily performed in the zebrafish due to their small size and ease of drug delivery, which consists simply of immersing fish into drug solutions. Ongoing work includes testing our xenograft model using two known chemotherapeutic agents against ES, doxorubicin and ifosfamide, with the goal of providing a benchmark for novel drugs’ responses on cell migration and metastases. Citation Format: Rebecca A. Anderson, Usua Oyarbide Cuervas-Mon, Seth Corey. Patterns and quantitation of migration and metastasis in a zebrafish xenograft model of ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B007.

Morphological characteristics of zebrafish's yolk sac for malformation based on orthogonal-polarization-gating optical coherence tomography.

In this study, an automatic algorithm combining an ellipsoid approximation and U-net has been presented for the characterization of a zebrafish's yolk sac. The polarization-difference-balanced-detection image of zebrafish was obtained based on orthogonal-polarization-gating optical coherence tomography and used to segment the yolk sac region. And ellipsoid can approximate the shape of the three-dimensional yolk sac, and the multiple parameters of volume and the three principal axes (k, l and m) can be used to quantify the yolk sac. In addition, the multiple parameters of two principal axes (l and m) and volume can distinguish the malformation from the normal controlled group. Finally, the volume malformation of the yolk sac calculated by the proposed algorithm ranges from 16.55% to 46.05%. Thus, the degree of malformation can be applied for toxicity analysis. And this method provides a potential application for an accurate judgment index for biotoxicological testing.

Light-sheet fluorescence microscopy for the in vivo study of microtubule dynamics in the zebrafish embryo.

During its first hours of development, the zebrafish embryo presents a large microtubule array in the yolk region, essential for its development. Despite of its size and dynamic behavior, this network has been studied only in limited field of views or in fixed samples. We designed and implemented different strategies in Light Sheet Fluorescence microscopy for imaging the entire yolk microtubule (MT) network in vivo. These have allowed us to develop a novel image analysis from which we clearly observe a cyclical re-arrangement of the entire MT network in synchrony with blastoderm mitotic waves. These dynamics also affect a previously unreported microtubule array deep within the yolk, here described. These findings provide a new vision of the zebrafish yolk microtubules arrangement, and offers novel insights in the interaction between mitotic events and microtubules reorganization.

The effect of an isoquinoline alkaloid on treatment of periodontitis by regulating the neutrophils chemotaxis.

Neutrophil plays a critical role in the progression of periodontitis. In general, its chemotaxis and activation are benefit for the host defense of bacterial infection and inflammation. However, previous studies have reported that the hyperactive and reactive neutrophils appear to be one of the reasons for tissue destruction in periodontitis tissues. In this study, we investigated an isoquinoline alkaloid Litcubanine A (LA), which from the Traditional Chinese medicinal plant, Litsea cubeba. We found LA showed significant activity in inhibiting neutrophils chemotaxis in the zebrafish yolk sac microinjection model in vivo and in mouse neutrophils in vitro. Further investigation proved that LA could inhibit the expression levels of neutrophil respiratory burst-related and inflammation-related genes CYBB and NCF2, as well as inhibit the activation of MAPK signaling pathway. Moreover, using LA, we successfully achieved the effect of reducing periodontitis bone loss by regulating neutrophil chemotaxis and related functions in a mouse ligature-induced periodontitis model.

Caffeine Inhibits Direct and Indirect Angiogenesis in Zebrafish Embryos.

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.

The Zebrafish Xenograft Platform-A Novel Tool for Modeling KSHV-Associated Diseases.

Kaposi’s sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic, but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally induced lymphoma in zebrafish, whereby KSHV-infected PEL tumor cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well vascularized, and by using fluorogenic assays, we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment, and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.

Gradient-Enhanced Electromagnetic Actuation System With a New Core Shape Design for Microrobot Manipulation

Electromagnetic-based actuation is receiving increasing attention for driving microparticles, particularly for in vivo biomedical applications. In this article, we present a core shape design for electromagnetic coils to develop a magnetic gradient field-based actuation system that can enable microrobotic manipulation. Based on the mathematical model using the finite-element method, the shape of the iron core and the size of the probe are optimized, aiming to increase the gradient at the focused area. A thin disc is also attached to the end of the iron core for reducing the waste of magnetic field energy. Through this design, the magnetic field generated by the electromagnetic actuation system can be considerably enhanced to propel microrobots in the in vivo environments. The designed system is tested in different in vitro environments, including pure water, artificial cerebrospinal fluid, mouse blood, and a special environment with an extremely high viscosity coefficient. The system is also tested for driving microrobots in an in vivo environment, namely, zebrafish yolk. Experimental results effectively demonstrate the capacity of the designed platform in manipulating microrobots for in vitro and in vivo applications.

Xenotransplantation of Human glioblastoma in Zebrafish larvae: in vivo imaging and proliferation assessment.

ABSTRACTGlioblastoma (GBM) is the most prevalent type of primary brain tumor. Treatment options include maximal surgical resection and drug-radiotherapy combination. However, patient prognosis remains very poor, prompting the search for new models for drug discovery and testing, especially those that allow assessment of in vivo responses to treatment. Zebrafish xenograft models have an enormous potential to study tumor behavior, proliferation and cellular interactions. Here, an in vivo imaging and proliferation assessment method of human GBM xenograft in zebrafish larvae is introduced. Zebrafish larvae microinjected with fluorescently labeled human GBM cells were screened daily using a stereomicroscope and imaged by light sheet fluorescence microscopy (LSFM); volumetric modeling and composite reconstructions were done in single individuals. Larvae containing tumors were enzymatically dissociated, and proliferation of cancer cells was measured using dye dilution by flow cytometry. GBM micro-tumors formed mainly in the zebrafish yolk sac and perivitelline space following injection in the yolk sac, with an engraftment rate of 73%. Daily image analysis suggested cellular division, as micro-tumors progressively grew with differentiated fluorescence intensity signals. Using dye dilution assay by flow cytometry, at least three GBM cells' division cycles were identified. The combination of LSFM and flow cytometry allows assessment of proliferation and tumor growth of human GBM inside zebrafish, making it a useful model to identify effective anti-proliferative agents in a preclinical setting.

Exploring the Antiangiogenic Potential of Solomonamide A Bioactive Precursors: In Vitro and in Vivo Evidences of the Inhibitory Activity of Solo F-OH During Angiogenesis.

Marine sponges are a prolific source of bioactive compounds. In this work, the putative antiangiogenic potential of a series of synthetic precursors of Solomonamide A, a cyclic peptide isolated from a marine sponge, was evaluated. By means of an in vitro screening, based on the inhibitory activity of endothelial tube formation, the compound Solo F–OH was selected for a deeper characterization of its antiangiogenic potential. Our results indicate that Solo F–OH is able to inhibit some key steps of the angiogenic process, including the proliferation, migration, and invasion of endothelial cells, as well as diminish their capability to degrade the extracellular matrix proteins. The antiangiogenic potential of Solo F–OH was confirmed by means of two different in vivo models: the chorioallantoic membrane (CAM) and the zebrafish yolk membrane (ZFYM) assays. The reduction in ERK1/2 and Akt phosphorylation in endothelial cells treated with Solo F–OH denotes that this compound could target the upstream components that are common to both pathways. Taken together, our results show a new and interesting biological activity of Solo F–OH as an inhibitor of the persistent and deregulated angiogenesis that characterizes cancer and other pathologies.

Zebrafish yolk sac microinjection of thalidomide for assessment of developmental toxicology.

Zebrafish offer advantages over traditional rodent models in developmental toxicology studies, including low maintenance costs, high fecundity, genetic diversity, and reduced animal welfare concerns (1,2).

Honokiol Eliminates Glioma/Glioblastoma Stem Cell-Like Cells Via JAK-STAT3 Signaling and Inhibits Tumor Progression by Targeting Epidermal Growth Factor Receptor.

Malignant gliomas are the most aggressive forms of brain tumors; whose metastasis and recurrence contribute to high rates of morbidity and mortality. Glioma stem cell-like cells are a subpopulation of tumor-initiating cells responsible for glioma tumorigenesis, metastasis, recurrence and resistance to therapy. Epidermal growth factor receptor (EGFR) has been reported to be dysregulated in most cancers, including gliomas and its functions are closely linked to initiating tumor metastasis and a very poor prognosis. In search for compounds that may reduce the tumorigenic potential of gliomas/glioblastomas honokiol attracted our attention. Honokiol, purified from the bark of traditional Chinese herbal medicine Magnolia species, is beneficial in vitro and in animal models via a variety of pharmacological effects, including anti-inflammatory, anti-angiogenetic, anti-arrhythmic and antioxidant activities, as well as anti-proliferative and proapoptotic effects in a wide range of human cancer cells. However, its effects on glioma cells are unknown. Here, we used different concentrations of honokiol in treating U251 and U-87 MG human glioma/glioblastoma cells in cell culture. Results showed that honokiol inhibited glioma cell viability and colony formation and promoted apoptosis. It also inhibited glioma cell migration/proliferation and invasion. In addition, honokiol promoted apoptosis and reduced Bcl-2 expression, accompanied by increase in Bax expression. Honokiol reduced expression of EGFR, CD133 and Nestin. Moreover, honokiol inhibited the activation of both AKT and ERK signaling pathways, increased active caspase-3 level and reduced phosphorylation of STAT3. U-87 MG xenografts in nude mice and in immunotolerant zebrafish yolk sac showed that honokiol inhibits tumor growth and metastasis. Altogether, results indicate that honokiol reduces tumorigenic potentials, suggesting hopes for honokiol to be useful in the clinical management of glioma/glioblastoma.

Zebrafish as a Model for Toxicological Perturbation of Yolk and Nutrition in the Early Embryo.

Developmental toxicity assessments often focus on structural outcomes and overlook subtle metabolic differences which occur during the early embryonic period. Deviant embryonic nutrition can result in later-life disease, including diabetes, obesity, and cardiovascular disease. Prior to placenta-mediated nutrient exchange, the human embryo requires maternally supplied nutritional substrates for growth, called yolk. Here, we compare the biology of the human and zebrafish yolk and review examples of toxicant-mediated perturbation of yolk defects, composition, and utilization. Zebrafish embryos, like human embryos, have a protruding yolk sac that serves as a nutritional cache. Aberrant yolk morphology is a common qualitative finding in fish embryotoxicity studies, but quantitative assessment and characterization provides an opportunity to uncover mechanistic targets of toxicant effects on embryonic nutrition. The zebrafish and the study of its yolk sac is an excellent model for uncovering toxicant disruptions to early embryonic nutrition and has potential to discover mechanistic insights into the developmental origins of health and disease.

Progesterone modulates microtubule dynamics and epiboly progression during zebrafish gastrulation

Control of microtubule dynamics is crucial for cell migration. We analyzed regulation of microtubule network dynamics in the zebrafish yolk cell during epiboly, the earliest coordinated gastrulation movement. We labeled microtubules with EMTB-3GFP and EB3-mCherry to visualize and measure microtubule dynamics by TIRF microscopy live imaging. Yolk cell microtubules dynamics is temporally modulated during epiboly progression. We used maternal zygotic Pou5f3 mutant (MZspg) embryos, which develop strong distortions of microtubule network organization and epiboly retardation, to investigate genetic control of microtubule dynamics. In MZspg embryos, microtubule plus-end growth tracks move slower and are less straight compared to wild-type. MZspg embryos have altered steroidogenic enzyme expression, resulting in increased pregnenolone and reduced progesterone levels. We show that progesterone positively affects microtubule plus-end growth and track straightness. Progesterone may thus act as a non-cell-autonomous regulator of microtubule dynamics across the large yolk cell, and may adjust differing demands on microtubule dynamics and stability during initiation and progression phases of epiboly.

Evaluation in zebrafish model of the toxicity of rhodamine B-conjugated crotamine, a peptide potentially useful for diagnostics and therapeutics.

Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4μM, which effectively kill zebrafish larvae in less than 10min. With non-lethal concentrations (<1μM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.