Extra-Intestinal Effects of C. difficile Toxin A and B: An In Vivo Study Using the Zebrafish Embryo Model

C.difficile infection (CDI) is not a merely “gut-confined” disease as toxemia could drive the development of CDI-related extra-intestinal effects. These effects could explain the high CDI-associated mortality, not just justified by diarrhea and dehydration. Here, the extra-intestinal effects of toxin A (TcdA) and B (TcdB) produced by C. difficile have been studied in vivo using the zebrafish embryo model. Noteworthy, protective properties of human serum albumin (HSA) towards toxins-induced extra-intestinal effects were also addressed. Zebrafish embryos were treated with TcdA, TcdB and/or HSA at 24 h post-fertilization. Embryos were analyzed for 48 h after treatment to check vital signs and morphological changes. Markers related to cardio-vascular damage and inflammation were evaluated by Real-Time quantitative PCR and/or western blotting. Both toxins induced cardiovascular damage in zebrafish embryos by different mechanisms: (i) direct toxicity (i.e., pericardial edema, cardiac chambers enlargement, endothelial alteration); (ii) increased hormonal production and release (i.e., atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), (iii) alteration of the vascular system through the increase of the vascular endothelial growth factor (VEGF-A) levels, as well as of its receptors, (iv) pro-inflammatory response through high cytokines production (i.e., CXCL8, IL1B, IL6 and TNFα) and (v) cell-mediated damage due to the increase in neutrophils number. In addition to cardiovascular damage, we observe skin alteration and inflammation. Finally, our data indicate a protective effect of HSA toward the toxins induced extra-intestinal effects. Together, our findings can serve as a starting point for humans’ studies to substantiate and understand the extra-intestinal effects observed in CDI patients.

Download Full-text

An Orthotopic Model of Uveal Melanoma in Zebrafish Embryo: A Novel Platform for Drug Evaluation

Biomedicines ◽

10.3390/biomedicines9121873 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1873

Author(s):

Chiara Tobia ◽

Daniela Coltrini ◽

Roberto Ronca ◽

Alessandra Loda ◽

Jessica Guerra ◽

...

Keyword(s):

Uveal Melanoma ◽

Zebrafish Embryo ◽

Drug Evaluation ◽

Metastatic Tumor ◽

Melanoma Cells ◽

Response To Therapy ◽

Neoplastic Disease ◽

Zebrafish Embryos ◽

Orthotopic Model

Uveal melanoma is a highly metastatic tumor, representing the most common primary intraocular malignancy in adults. Tumor cell xenografts in zebrafish embryos may provide the opportunity to study in vivo different aspects of the neoplastic disease and its response to therapy. Here, we established an orthotopic model of uveal melanoma in zebrafish by injecting highly metastatic murine B16-BL6 and B16-LS9 melanoma cells, human A375M melanoma cells, and human 92.1 uveal melanoma cells into the eye of zebrafish embryos in the proximity of the developing choroidal vasculature. Immunohistochemical and immunofluorescence analyses showed that melanoma cells proliferate during the first four days after injection and move towards the eye surface. Moreover, bioluminescence analysis of luciferase-expressing human 92.1 uveal melanoma cells allowed the quantitative assessment of the antitumor activity exerted by the canonical chemotherapeutic drugs paclitaxel, panobinostat, and everolimus after their injection into the grafted eye. Altogether, our data demonstrate that the zebrafish embryo eye is a permissive environment for the growth of invasive cutaneous and uveal melanoma cells. In addition, we have established a new luciferase-based in vivo orthotopic model that allows the quantification of human uveal melanoma cells engrafted in the zebrafish embryo eye, and which may represent a suitable tool for the screening of novel drug candidates for uveal melanoma therapy.

Download Full-text

Ce(SO4)2-catalysed the highly diastereoselective synthesis of tetrahydroquinolines via an imino Diels Alder ABB′ type reaction and their in vivo toxicity and imaging in zebrafish embryos

RSC Advances ◽

10.1039/c6ra04325a ◽

2016 ◽

Vol 6 (44) ◽

pp. 37478-37486 ◽

Cited By ~ 9

Author(s):

Carlos A. Martínez Bonilla ◽

Carlos E. Puerto Galvis ◽

Leonor Y. Vargas Méndez ◽

Vladimir V. Kouznetsov

Keyword(s):

Zebrafish Embryo ◽

Diels Alder ◽

Zebrafish Embryos ◽

Diastereoselective Synthesis ◽

In Vivo Toxicity ◽

Type Reaction ◽

Povarov Reaction

The synthesis of tetrahydroquinolines via the Povarov reaction has been developed using Ce(SO2)4 as a catalyst. This efficient protocol allowed the toxicity and phenotypic study of these products using a zebrafish embryo model.

Download Full-text

Quantifying endothelial cell proliferation in the zebrafish embryo

F1000Research ◽

10.12688/f1000research.73130.1 ◽

2021 ◽

Vol 10 ◽

pp. 1032

Author(s):

George Bowley ◽

Timothy JA Chico ◽

Jovana Serbanovic-Canic ◽

Paul C Evans

Keyword(s):

Cell Proliferation ◽

Endothelial Cell ◽

Zebrafish Embryo ◽

Time Lapse ◽

Endothelial Cell Proliferation ◽

Small Scale ◽

Zebrafish Embryos ◽

Time Lapse Imaging

Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research.

Download Full-text

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy

Nature Communications ◽

10.1038/s41467-020-19827-1 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Håkon Høgset ◽

Conor C. Horgan ◽

James P. K. Armstrong ◽

Mads S. Bergholt ◽

Vincenzo Torraca ◽

...

Keyword(s):

Zebrafish Embryo ◽

Spectroscopic Imaging ◽

Wound Response ◽

Three Dimensions ◽

Zebrafish Embryos ◽

Biological Processes ◽

Raman Spectroscopic ◽

Confocal Raman ◽

Biomolecular Imaging

AbstractZebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.

Download Full-text

Thermal proteome profiling in zebrafish reveals effects of napabucasin on retinoic acid metabolism

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002273 ◽

2020 ◽

pp. mcp.RA120.002273

Author(s):

Niels M Leijten ◽

Petra Bakker ◽

Herman P. Spaink ◽

Jeroen den Hertog ◽

Simone Lemeer

Keyword(s):

Retinoic Acid ◽

Zebrafish Embryo ◽

Acid Metabolism ◽

Zebrafish Embryos ◽

Developmental Defects ◽

Protein Targets ◽

Proteome Profiling ◽

Stat3 Signaling

Thermal proteome profiling (TPP) allows for the unbiased detection of drug – target protein engagements in vivo. Traditionally, one cell type is used for TPP studies, with the risk of missing important differentially expressed target proteins. The use of whole organisms would circumvent this problem. Zebrafish embryos are amenable to such an approach. Here, we used TPP on whole zebrafish embryo lysate to identify protein targets of napabucasin, a compound that may affect Signal transducer and activator of transcription 3 (Stat3) signaling through an ill-understood mechanism. In zebrafish embryos, napabucasin induced developmental defects consistent with inhibition of Stat3 signaling. TPP profiling showed no distinct shift in Stat3 upon napabucasin treatment, but effects were detected on the oxidoreductase, Pora, which might explain effects on Stat3 signaling. Interestingly, thermal stability of several aldehyde dehydrogenases (Aldhs) was affected. Moreover, napabucasin activated ALDH enzymatic activity in vitro. Aldhs have crucial roles in retinoic acid metabolism and functionally we validated napabucasin-mediated activation of the retinoic acid pathway in zebrafish in vivo. We conclude that TPP profiling in whole zebrafish embryo lysate is feasible and facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets.

Download Full-text

In vivo toxicity of bioreactor-grown biomass and exopolysaccharides from Malaysian tiger milk mushroom mycelium for potential future health applications

Scientific Reports ◽

10.1038/s41598-021-02486-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Siti Rokhiyah Ahmad Usuldin ◽

Wan Abd Al Qadr Imad Wan-Mohtar ◽

Zul Ilham ◽

Adi Ainurzaman Jamaludin ◽

Nur Raihan Abdullah ◽

...

Keyword(s):

Zebrafish Embryo ◽

Therapeutic Interventions ◽

Zebrafish Embryos ◽

Mycelial Biomass ◽

Future Health ◽

Potential Value ◽

Embryo Toxicity ◽

Delayed Hatching ◽

Health Applications

AbstractNatural mycelial biomass (MB) and exopolysaccharides (EPS) of Malaysian tiger milk mushroom Lignosus rhinocerus are considered high-end components due to their high commercial potential value in drug discovery. This study aims to evaluate the toxicity of the mushroom extracts’ generated in a bioreactor using the zebrafish embryo toxicity (ZFET) model assay as a new therapy for treating asthma. Both MB and EPS extracts, at concentrations 0.16–10 mg/mL, were tested for ZFET and early development effects on Zebrafish Embryos (ZE) during 24–120 h post-fertilisation (HPF). Findings revealed that MB was deemed safe with an LC50 of 0.77 mg/mL; the EPS were non-toxic (LC50 of 0.41 mg/mL). Neither MB nor EPS delayed hatching nor teratogenic defects in the treated ZE at a 2.5 mg/mL dose. There were no significant changes in the ZE heart rate after treatments with MB (130 beats/min) and EPS (140 beats/min), compared to that of normal ZE (120–180 beats/min). Mixing both natural compounds MB and EPS did not affect toxicity using ZFET testing; thus, intimating their safe future use as therapeutic interventions. This represents the first study to have used the ZFET assay on MB and EPS extracts of L. rhinocerus for future health applications.

Download Full-text

Effects of Five Substances with Different Modes of Action on Cathepsin H, C and L Activities in Zebrafish Embryos

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph16203956 ◽

2019 ◽

Vol 16 (20) ◽

pp. 3956

Author(s):

Eberhard Küster ◽

Stefan Kalkhof ◽

Silke Aulhorn ◽

Martin von Bergen ◽

Ulrike Gündel

Keyword(s):

Enzyme Activity ◽

Zebrafish Embryo ◽

Statistical Significance ◽

Cathepsin L ◽

Atp Synthesis ◽

Chemical Exposure ◽

Zebrafish Embryos ◽

Modes Of Action ◽

Cathepsin H

Cathepsins have been proposed as biomarkers of chemical exposure in the zebrafish embryo model but it is unclear whether they can also be used to detect sublethal stress. The present study evaluates three cathepsin types as candidate biomarkers in zebrafish embryos. In addition to other functions, cathepsins are also involved in yolk lysosomal processes for the internal nutrition of embryos of oviparous animals until external feeding starts. The baseline enzyme activity of cathepsin types H, C and L during the embryonic development of zebrafish in the first 96 h post fertilisation was studied. Secondly, the effect of leupeptin, a known cathepsin inhibitor, and four embryotoxic xenobiotic compounds with different modes of action (phenanthrene—baseline toxicity; rotenone—an inhibitor of electron transport chain in mitochondria; DNOC (Dinitro-ortho-cresol)—an inhibitor of ATP synthesis; and tebuconazole—a sterol biosynthesis inhibitor) on in vivo cathepsin H, C and L total activities have been tested. The positive control leupeptin showed effects on cathepsin L at a 20-fold lower concentration compared to the respective LC50 (0.4 mM) of the zebrafish embryo assay (FET). The observed effects on the enzyme activity of the four other xenobiotics were not or just slightly more sensitive (factor of 1.5 to 3), but the differences did not reach statistical significance. Results of this study indicate that the analysed cathepsins are not susceptible to toxins other than the known peptide-like inhibitors. However, specific cathepsin inhibitors might be identified using the zebrafish embryo.

Download Full-text

Morpholino knockdown of the ubiquitously expressed transmembrane serine protease TMPRSS4a in zebrafish embryos exhibits severe defects in organogenesis and cell adhesion

Biological Chemistry ◽

10.1515/bc.2011.070 ◽

2011 ◽

Vol 392 (7) ◽

Cited By ~ 7

Author(s):

Anke Ohler ◽

Christoph Becker-Pauly

Keyword(s):

Cell Differentiation ◽

Serine Protease ◽

Vascular System ◽

Human Cancer ◽

Zebrafish Embryos ◽

Tissue Development ◽

Rt Pcr ◽

Morpholino Knockdown ◽

Transmembrane Serine Protease

Abstract Over the past years the members of the type II transmembrane serine protease (TTSP) family have emerged as new players in mammalian biology. TMPRSS4 (transmembrane protease/serine) is overexpressed in several human cancer tissues, promoting invasion, migration, and metastasis. However, the physiological function has not yet been elucidated. Here, we present morpholino knockdown studies targeting TMPRSS4a, a homolog of human TMPRSS4 in zebrafish embryos. By RT-PCR, we could demonstrate an expression of this protease already 5 h post-fertilization, suggesting important functions in the early stages of embryonic development. Indeed, in vivo gene silencing caused severe defects in tissue development and cell differentiation including a disturbed skeletal muscle formation, a decelerated heartbeat, and a degenerated vascular system. Scanning electron microscopy revealed strong defects in epidermal skin organization, with clearly altered cell-cell contacts, resulting in the detachment of keratinocytes from the underneath tissue. The disturbed organogenesis in general is consistent with RT-PCR results which exhibited a ubiquitous expression of TMPRSS4a, predominantly in kidney, skin, heart, and gills. Our results demonstrate the importance of TMPRSS4a in tissue development and cell differentiation. Whether its proteolytic activity is directed towards adhesion molecules or leads to the activation of other proteases needs to be investigated further.

Download Full-text

The Anti-Tumoral Potential of Phosphonate Analog of Sulforaphane in Zebrafish Xenograft Model

Cells ◽

10.3390/cells10113219 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3219

Author(s):

Magdalena Rudzinska-Radecka ◽

Łukasz Janczewski ◽

Anna Gajda ◽

Marlena Godlewska ◽

Malgorzata Chmielewska-Krzesinska ◽

...

Keyword(s):

Morphological Changes ◽

Xenograft Model ◽

Living Organism ◽

Significant Inhibition ◽

Zebrafish Embryos ◽

Cancer Cell Growth ◽

Strong Activity ◽

Hatching Time ◽

Fluorescence Dye

Isothiocyanates (ITCs) show strong activity against numerous human tumors. Five structurally diverse ITCs were tested in vivo using the zebrafish embryos 6 and 48 h post-fertilization (hpf). The survival rate, hatching time, and gross morphological changes were assessed 24, 48, and 72 h after treatment with all compounds in various doses (1–10 µM). As a result, we selected a phosphonate analog of sulforaphane (P-ITC; 1–3 µM) as a non-toxic treatment for zebrafish embryos, both 6 and 48 hpf. Furthermore, the in vivo anti-cancerogenic studies with selected 3 µM P-ITC were performed using a set of cell lines derived from the brain (U87), cervical (HeLa), and breast (MDA-MB-231) tumors. For the experiment, cells were labeled using red fluorescence dye Dil (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine, 10 μg/mL) and injected into the hindbrain ventricle, yolk sac region and Cuvier duct of zebrafish embryos. The tumor size measurement after 48 h of treatment demonstrated the significant inhibition of cancer cell growth in all tested cases by P-ITC compared to the non-treated controls. Our studies provided evidence for P-ITC anti-cancerogenic properties with versatile activity against different cancer types. Additionally, P-ITC demonstrated the safety of use in the living organism at various stages of embryogenesis.

Download Full-text

Evaluation of Noxious Effect of Environmental Contaminants Using Zebrafish Model

Proceedings International ◽

10.33263/proceedings21.037037 ◽

2020 ◽

Vol 2 (1) ◽

pp. 37

Keyword(s):

Cell Death ◽

Developmental Stages ◽

Morphological Changes ◽

Cell Damage ◽

Developmental Toxicity ◽

Environmental Pollutant ◽

Toxicity Study ◽

Zebrafish Embryos ◽

Zebrafish Model

Effects of the inorganic chemicals Calcium Fluoride (CaF2) and Hexaflurosilicilic acid (H2SiF2) have been studied due to its excessive usage in drinking water plants, glass manufacturing etc. Toxicity studies on Zebrafish embryos have been carried out for CaF2 and H2SiF2 during the embryonic developmental stages to observe the changes taken place during the growth, development. These changes can be observed in cell differentiation, larval movements, delay in hatching, and by the changes in behavior. Due to the ease with the transparency of zebrafish embryos, it can be observed and manipulated. In the field of early developmental studies, these zebrafish embryos have been vital because they have faster development by which the whole organs get developed in 3 days. Thus it plays a significant role in the discovery and analysis of changes in the developmental aspects of their teratology study. Toxicity study in Adults Zebrafish can be studied through the histology analysis where the cell damage and cell death due to fluorides and acid ions which may also lead to morphological changes due to this environmental pollutant. This toxicity study can be studied based on behavioral effects, LC50 determination, and immunohistochemistry of the brain to observe the developmental neurotoxicity. This study describes the effect of the inorganic chemicals is leading to developmental toxicity, cell deformities, and cell death with the high mortality rate in the In vivo Zebrafish model.

Download Full-text