Po Kwok Chan

Acute and long-term effects after single loading of functionalized multi-walled carbon nanotubes into zebrafish (Danio rerio)

Carbon nanotubes (CNTs) are widely explored for biomedical applications, but there is very limited information regarding their in vivo biodistribution and biocompatibility. Here, we report the in vivo biodistribution and long-term effects of functionalized multi-walled carbon nanotubes (MWCNTs) in developing zebrafish. The fluorescent-labeled MWCNTs were introduced into zebrafish embryos at 1-cell stage and at 72 h post fertilization through microinjection. After single injection, both acute and long-term interactions between zebrafish and functionalized MWCNTs were studied. The injected FITC-BSA-MWCNTs (at 1-cell stage) were allocated to all blastoderm cells of the embryos through proliferation, and were distinctively excluded from the yolk cell. When introduced into the circulation system, FITC-BSA-MWCNTs moved easily in the compartments and finally were cleaned out by the body at 96 h after the loading. At early stages, the treated zebrafish embryos generated immune response by accumulating circulating white blood cells at the trunk region. Under transmission electron microscope, many lysosome-like vesicles were observed in the blastoderm cells of the treated embryos. The zebrafish loaded with MWCNTs had normal primordial germ cells at early stage and produced second generation later on. However, the larvae of the second generation had obviously lower survival rates as compared to the untreated groups, suggesting a negative effect on the reproduction potential. These results suggest that extensive purification and functionalization processes can help improve the biocompatibility of CNTs. This study also indicates that purified CNTs may have long-term toxicity effects when they were delivered into the body.

Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos

BackgroundZebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis.ResultsThe heart rate measured by this noninvasive method in zebrafish embryos at 52 hour post-fertilization was similar to that determined by direct visual counting of ventricle beating (p > 0.05). In addition, the method was validated by a known cardiotoxic drug, terfenadine, which affects heartbeat regularity in humans and induces bradycardia and atrioventricular blockage in zebrafish. A significant decrease in heart rate was found by our method in treated embryos (p < 0.01). Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (p < 0.01) of treated embryos as shown by Poincare plot.ConclusionThe data support and validate this rapid, simple, noninvasive method, which includes video image analysis and frequency analysis. This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos. With the advantages of rapid sample preparation procedures, automatic image analysis and data analysis, this method can potentially be applied to cardiotoxicity screening assay.

The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium

Embryonic vascular patterns in zebrafish (Danio rerio) could be visualised by confocal microscopy coupled with microinjected fluorescent microbeads. This microangiographic technique was adopted here, for the first time, to study the effects of cadmium on cardiovascular development in zebrafish embryos. Zebrafish embryos were incubated in culture medium containing 100 microM cadmium from 5 h post fertilisation (hpf) to 48 hpf. At 48 hpf, embryos were examined for viability and occurrence of malformations. The 100 microM cadmium caused 32.21 +/- 3.65% mortality and 20.33 +/- 4.04% visible malformations in surviving embryos. In the remaining embryos with no visible signs of malformations, further assessments for less obvious abnormalities were performed. Assessments on craniofacial development were made by digital measurements on areas of brains and eyes. Cardiac development was assessed by immunostaining the heart with the antibody MF20 specific for myosin heavy chain. Body lengths of the embryos were also measured. Embryonic development of brains, eyes, hearts and body lengths of visibly healthy embryos in the cadmium treatment group showed no significant difference from the controls. Embryonic vasculature of these visibly healthy embryos was then studied by microinjecting fluorescent microbeads of diameter 0.02 microm into the circulation. All the cadmium treated embryos showed localised vascular defects in the dorsal aortae, segmental and cranial vessels while none of the control embryos showed any aberrant patterns in the networking of the vasculature. Improved image analyses on the anterior regions revealed that cadmium treated embryos had markedly less complex networks of cranial vessels with fewer vessels perfusing the craniofacial regions. The number of branch points in the vascular network was counted. In untreated embryos, there were 135.6 +/- 51 branches in the vasculature in entire body. In the cadmium treated embryos, there were 64.5+/-31 branches. The difference was significant when assessed with Students t-test. It appeared that although cadmium did not cause any signs of external malformations in these visibly healthy embryos, nonetheless induced impaired branching and anastomsis of the cranial vessels. This study revealed, for the first time, that vital vascular structures in fish embryos could be affected by exposure to cadmium. This technique allowed visualisation of vascular anomalies in embryos showing no external signs of malformations. The impairment of anatomical features during embryonic development might serve as meaningful health endpoints in ecotoxicological studies and in risk assessment.

A novel zebrafish jak2aV581F model shared features of human JAK2V617F polycythemia vera

OBJECTIVE The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV. MATERIALS AND METHODS Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites. RESULTS Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019). CONCLUSION The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.

A rapid low-cost high-density DNA-based multi-detection test for routine inspection of meat species.

The increasing occurrence of food frauds suggests that species identification should be part of food authentication. Current molecular-based species identification methods have their own limitations or drawbacks, such as relatively time-consuming experimental steps, expensive equipment and, in particular, these methods cannot identify mixed species in a single experiment. This project proposes an improved method involving PCR amplification of the COI gene and detection of species-specific sequences by hybridisation. Major innovative breakthrough lies in the detection of multiple species, including pork, beef, lamb, horse, cat, dog and mouse, from a mixed sample within a single experiment. The probes used are species-specific either in sole or mixed species samples. As little as 5 pg of DNA template in the PCR is detectable in the proposed method. By designing species-specific probes and adopting reverse dot blot hybridisation and flow-through hybridisation, a low-cost high-density DNA-based multi-detection test suitable for routine inspection of meat species was developed.

Automated segmentation in confocal images using a density clustering method

Confocal microscopy provides a powerful tool for biologists to investigate gene expression in a 3D manner. However, due to the inherent properties of confocal images, it is difficult to accurately segregate foreground signals from the background using direct thresholding. Therefore, there is a need for a segmentation algorithm that can be used with fluorescent confocal images of gene expression. We present an automatic segmentation algorithm for thresholding confocal images of gene expression in biological samples. The algorithm, called density-based segmentation (DBS), is modified from a noise-tolerant data clustering algorithm (DENCLUE). We demonstrate the utility of this algorithm in different synthetic images as well as in confocal images of zebrafish embryos, with comparison to Otsus algorithm, which employs direct thresholding. The results of segmentation in synthetic images show that the DBS algorithm is noise-tolerant and is able to distinguish two objects located close to each other. In addition, the results of segmentation in confocal images show that the DBS algorithm can threshold objects while preserving morphological details of internal structures. Therefore, the proposed DBS algorithm is a better segmentation technique than direct thresholding in the segmentation of fluorescent confocal images.

Data mining for chinese materia medica and pharmacological research.

Chinese materia medica (CMM) is becoming increasingly important in modern health care, with the potential for new or improved clinical protocols and reduction in treatment costs. Conventional approaches to drug discovery are based on knowledge of biological systems and screen phenotypes in the context of a whole organism. It will be valuable to identify the CMM that would induce certain biological responses (such as angiogenesis). The authors have developed a database that they plan to commercialize that contains traditional knowledge of Chinese medicine and pharmacology along with their own experimental data from controlled scientific observations by using the zebrafish as a model of CMM-induced pathology. The database is visualized and functions via the World Wide Web by subscription or license. The authors have also written software for personal digital assistant (PDA) devices that supports multiple users performing screening experiments worldwide. This provides a platform for the study of CMM, and data mining of this resource will help evaluate CMM in the context of experimental observations of biological aberrations. (Journal of Biomolecular Screening 2008:390-395)