基于斑马鱼胚胎急性毒性测试预测食用油毒性可行性研究

探讨利用斑马鱼胚胎预测食用油毒性的可行性。用乙腈和正己烷联合提取采自我国台湾地区的7个不同品质的猪油样本，并用甲醇溶解，然后用斑马鱼胚胎测试提取物的毒性，最后用非靶标高分辨率复合四极杆质谱仪偶联超高液相色谱/质谱系统进一步分析低毒、中毒、高毒代表性样本中的化学组成，结合ChemSpider、CT-link和Vega软件分析预测有毒物质的结构和功能。结果表明，不同品质的猪油样本毒性差异大，高毒性猪油的特征性质荷比信号相对应的化学成分为脂肪酸的氧化物，是样本的潜在有毒成分。斑马鱼胚胎急性毒性测试可准确地预测猪油品质，该方法为评估食用油安全提供了一个新的思路。     

Expression of TRPV4 in the zebrafish retina during development

The transient receptor potential (TRP) channels are involved in sensing mechanical/physical stimuli such as temperature, light, pressure, as well as chemical stimuli. Some TRP channels are present in the vertebrate retina, and the occurrence of the multifunctional channel TRP vanilloid 4 (TRPV4) has been reported in adult zebrafish. Here, we investigate the expression and distribution of TRPV4 in the retina of zebrafish during development using polymerase chain reaction (PCR), Western blot, and immunohistochemistry from 3 days post fertilization (dpf) until 100 dpf. TRPV4 was detected at the mRNA and protein levels in the eye of zebrafish at all ages sampled. Immunohistochemistry revealed the presence of TRPV4 in a population of the retinal cells identified as amacrine cells on the basis of their morphology and localization within the retina, as well as the co-localization of TRPV4 with calretinin. TRPV4 was first (3 dpf) found in the soma of cells localized in the inner nuclear and ganglion cell layers, and thereafter (10 dpf) also in the inner plexiform layer. The adult pattern of TRPV4 expression was achieved by 40 dpf the expression being restricted to the soma of some cells in the inner nuclear layer and ganglion cell layers. These data demonstrate the occurrence and developmental changes in the expression and localization of TRPV4 in the retina of zebrafish, and suggest a role of TRPV4 in the visual processing.     

短链氯化石蜡C10 (50.2% Cl)对斑马鱼胚胎的发育毒性

为评价短链氯化石蜡对斑马鱼胚胎的发育毒性,以斑马鱼为模式生物,观察不同质量浓度的短链氯化石蜡C10(50.2%Cl)暴露24,48,72和96 h后斑马鱼胚胎/幼鱼的死亡率、孵化率、畸形率和体长.结果表明,C10(50.2%Cl)在斑马鱼的早期发育阶段可引起一系列非致死效应和致死效应.高质量浓度(1 000和10 000μg/L)短链氯化石蜡可导致96 h斑马鱼胚胎死亡率显著升高(达100%),48 h抑制胚胎的孵化,并诱发一系列的发育畸形.C10(50.2%Cl)在斑马鱼幼鱼中引起的畸形表型主要包括脊柱弯曲,卵黄畸形,心包囊肿,尾部畸形和鱼鳔发育缺陷.低质量浓度组和高质量浓度组C10(50.2%Cl)均能够抑制斑马鱼幼鱼的生长速度,目前的环境质量浓度已具有一定程度的水生生态风险.     

Effects of Nanotoxicity on Female Reproductivity and Fetal Development in Animal Models

The extensive application of nanomaterials in industry, medicine and consumer products has raised concerns about their potential toxicity. The female population is particularly vulnerable and deserves special attention because toxicity in this group may impact both female reproductivity and fetal development. Mouse and zebrafish models each have their own unique features and studies using these models to examine the potential toxicity of various nanoparticles are compared and summarized in this review. Several nanoparticles exhibit detrimental effects on female reproductivity as well as fetal development, and these adverse effects are related to nanoparticle composition, surface modification, dose, exposure route and animal species. Limited studies on the mechanisms of nanotoxicity are also documented and reviewed herein.     

Generation of a Novel Transgenic Zebrafish for Studying Adipocyte Development and Metabolic Control

Zebrafish have become a popular animal model for studying various biological processes and human diseases. The metabolic pathways and players conserved among zebrafish and mammals facilitate the use of zebrafish to understand the pathological mechanisms underlying various metabolic disorders in humans. Adipocytes play an important role in metabolic homeostasis, and zebrafish adipocytes have been characterized. However, a versatile and reliable zebrafish model for long-term monitoring of adipose tissues has not been reported. In this study, we generated stable transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) in adipocytes. The transgenic zebrafish harbored adipose tissues that could be detected using GFP fluorescence and the morphology of single adipocyte could be investigated in vivo. In addition, we demonstrated the applicability of this model to the long-term in vivo imaging of adipose tissue development and regulation based on nutrition. The transgenic zebrafish established in this study may serve as an excellent tool to advance the characterization of white adipose tissue in zebrafish, thereby aiding the development of therapeutic interventions to treat metabolic diseases in humans.     

Embryo zebrafish segmentation using an improved hybrid method

Zebrafish is an invaluable vertebrate model in life science research and has been widely used in biological pathway analysis, molecular screening and disease modelling, among others. As a result, microscopic imaging has become an essential step in zebrafish phenotype analysis, and image segmentation thus plays an important role in the zebrafish microscopy analysis. Due to the nonuniform distribution of intensity and weak boundary in zebrafish microscope images, the traditionally used segmentation methods may lead to unsatisfactory result. Here, a novel hybrid method that integrates region and boundary information into active contour model is proposed to segment zebrafish embryos from the background, which performs better than traditional segmentation models. Meanwhile, how to utilize the gradient information effectively in image segmentation is still an open problem. In this paper, we propose to improve the aforementioned hybrid method in two aspects. Firstly, the mean grey value of background is estimated by the expectation maximization (EM) algorithm to constrain the active curve evolution. Secondly, an edge stopping function sensitive to gradient information is designed to stop curve evolution when the active curve reaches the embryo boundary. Experimental results show that the proposed methods can provide superior segmentation results compared to existing algorithms.     

Inner Ear and Muscle Developmental Defects in Smpx-Deficient Zebrafish Embryos

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.     

Generation of GCaMP6s-Expressing Zebrafish to Monitor Spatiotemporal Dynamics of Calcium Signaling Elicited by Heat Stress

The ability of organisms to quickly sense and transduce signals of environmental stresses is critical for their survival. Ca²⁺ is a versatile intracellular messenger involved in sensing a wide variety of stresses and regulating the subsequent cellular responses. So far, our understanding for calcium signaling was mostly obtained from ex vivo tissues and cultured cell lines, and the in vivo spatiotemporal dynamics of stress-triggered calcium signaling in a vertebrate remains to be characterized. Here, we describe the generation and characterization of a transgenic zebrafish line with ubiquitous expression of GCaMP6s, a genetically encoded calcium indicator (GECI). We developed a method to investigate the spatiotemporal patterns of Ca²⁺ events induced by heat stress. Exposure to heat stress elicited immediate and transient calcium signaling in developing zebrafish. Cells extensively distributed in the integument of the head and body trunk were the first batch of responders and different cell populations demonstrated distinct response patterns upon heat stress. Activity of the heat stress-induced calcium signaling peaked at 30 s and swiftly decreased to near the basal level at 120 s after the beginning of exposure. Inhibition of the heat-induced calcium signaling by LaCl₃ and capsazepine and treatment with the inhibitors for CaMKII (Ca²²/calmodulin-dependent protein kinase II) and HSF1 (Heat shock factor 1) all significantly depressed the enhanced heat shock response (HSR). Together, we delineated the spatiotemporal dynamics of heat-induced calcium signaling and confirmed functions of the Ca²⁺-CaMKII-HSF1 pathway in regulating the HSR in zebrafish.     

Antimicrobial and Anti-Biofilm Peptide Octominin for Controlling Multidrug-Resistant Acinetobacter baumannii

Acinetobacter baumannii is a serious nosocomial pathogen with multiple drug resistance (MDR), the control of which has become challenging due to the currently used antibiotics. Our main objective in this study is to determine the antibacterial and antibiofilm activities of the antimicrobial peptide, Octominin, against MDR A. baumannii and derive its possible modes of actions. Octominin showed significant bactericidal effects at a low minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of 5 and 10 µg/mL, respectively. Time-kill kinetic analysis and bacterial viability tests revealed that Octominin showed a concentration-dependent antibacterial activity. Field-emission scanning electron microscopy (FE-SEM) analysis revealed that Octominin treatment altered the morphology and membrane structure of A. baumannii. Propidium iodide (PI) and reactive oxygen species (ROS) generation assays showed that Octominin increased the membrane permeability and ROS generation in A. baumannii, thereby causing bacterial cell death. Further, a lipopolysaccharides (LPS) binding assay showed an Octominin concentration-dependent LPS neutralization ability. Biofilm formation inhibition and eradication assays further revealed that Octominin inhibited biofilm formation and showed a high biofilm eradication activity against A. baumannii. Furthermore, up to a concentration of 100 µg/mL, Octominin caused no hemolysis and cell viability changes in mammalian cells. An in vivo study in zebrafish showed that the Octominin-treated group had a significantly higher relative percentage survival (54.1%) than the untreated group (16.6%). Additionally, a reduced bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octominin in vivo. Collectively, these data suggest that Octominin exhibits significant antibacterial and antibiofilm activities against the multidrug-resistant A. baumannii, and this AMP can be developed further as a potent AMP for the control of antibiotic resistance.     

Zebrafish as a Model for the Study of Lipid-Lowering Drug-Induced Myopathies

Drug-induced myopathies are classified as acquired myopathies caused by exogenous factors. These pathological conditions develop in patients without muscle disease and are triggered by a variety of medicaments, including lipid-lowering drugs (LLDs) such as statins, fibrates, and ezetimibe. Here we summarise the current knowledge gained via studies conducted using various models, such as cell lines and mammalian models, and compare them with the results obtained in zebrafish (Danio rerio) studies. Zebrafish have proven to be an excellent research tool for studying dyslipidaemias as a model of these pathological conditions. This system enables in-vivo characterization of drug and gene candidates to further the understanding of disease aetiology and develop new therapeutic strategies. Our review also considers important environmental issues arising from the indiscriminate use of LLDs worldwide. The widespread use and importance of drugs such as statins and fibrates justify the need for the meticulous study of their mechanism of action and the side effects they cause.