Trace Determination of Grouper Nervous Necrosis Virus in Contaminated Larvae and Pond Water Samples Using Label-Free Fiber Optic Nanoplasmonic Biosensor.

We developed a fast (<20 min), label-free fiber optic particle plasmon resonance (FOPPR) immunosensing method to detect nervous necrosis virus (NNV), which often infects high-value economic aquatic species, such as grouper. Using spiked NNV particles in a phosphate buffer as samples, the standard calibration curve obtained was linear (R2 = 0.99) and the limit of detection (LOD) achieved was 2.75 × 104 TCID50/mL, which is superior to that obtained using enzyme-linked immunosorbent assay (ELISA). By using an enhancement method called fiber optic nanogold-linked immunosorbent assay (FONLISA), the LOD can be further improved to <1 TCID50/mL, which is comparable to that found by the conventional qPCR method. Employing the larvae homogenate samples of NNV-infected grouper, the results obtained by the FOPPR biosensor agree with those obtained by the quantitative polymerase chain reaction (qPCR) method. We also examined pond water samples from an infected container in an indoor aquaculture facility. The lowest detectable level of NNV coat protein was found to be 0.17 µg/mL, which is one order lower than the LOD reported by ELISA. Therefore, we demonstrated the potential of the FOPPR biosensor as an outbreak surveillance tool, which is able to give warning indication even when the trend of larvae death toll increment is still not clear.

Progranulin A Promotes Compensatory Hepatocyte Proliferation via HGF/c-Met Signaling after Partial Hepatectomy in Zebrafish.

Compensatory hepatocyte proliferation and other liver regenerative processes are activated to sustain normal physiological function after liver injury. A major mitogen for liver regeneration is hepatocyte growth factor (HGF), and a previous study indicated that progranulin could modulate c-met, the receptor for HGF, to initiate hepatic outgrowth from hepatoblasts during embryonic development. However, a role for progranulin in compensatory hepatocyte proliferation has not been shown previously. Therefore, this study was undertaken to clarify whether progranulin plays a regulatory role during liver regeneration. To this end, we established a partial hepatectomy regeneration model in adult zebrafish that express a liver-specific fluorescent reporter. Using this model, we found that loss of progranulin A (GrnA) function by intraperitoneal-injection of a Vivo-Morpholino impaired and delayed liver regeneration after partial hepatectomy. Furthermore, transcriptome analysis and confirmatory quantitative real-time PCR suggested that cell cycle progression and cell proliferation was not as active in the morphants as controls, which may have been the result of comparative downregulation of the HGF/c-met axis by 36 h after partial hepatectomy. Finally, liver-specific overexpression of GrnA in transgenic zebrafish caused more abundant cell proliferation after partial hepatectomy compared to wild types. Thus, we conclude that GrnA positively regulates HGF/c-met signaling to promote hepatocyte proliferation during liver regeneration.

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

BACKGROUND: Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. RESULTS: Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1ß, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. CONCLUSIONS: Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

The activity of carp muscle-specific creatine kinase at low temperature is enhanced by decreased hydrophobicity of residue 268.

Abstract The muscle-specific forms of creatine kinase in rabbit (RM-CK) and carp (M1-CK) exhibit different temperature-dependent functional properties. Replacing the glycine at residue 268 of RM-CK with asparagine increases the enzyme's activity at 10°C. In this study, we investigated how hydrophobicity of residue 268 affects the biochemical properties of RM-CK and M1-CK at low temperature. We generated three mutants of both RM-CK and M1-CK: Asp268, Lys268, and Leu268. The secondary structures of these mutants were similar, as revealed by their circular dichroism spectra. Similar to the Asn268 mutants, the Asp268 and Lys268 mutants of RM-CK and M1-CK exhibited higher specific activities at 10°C and pH 8.0. However, no such effect was observed for the RM-CK and M1-CK Leu268 mutants. While in the presence of cryoprotectant (sucrose or trehalose), the activities of wild-type RM-CK and M1-CK mutant enzymes with a hydrophobic residue at 268 were higher, and the effect was more profound at pH 8.0. It may be inferred that water molecules affect protein conformation around residue 268, thereby influencing protein stability at low temperature.

Zebrafish HSC70 promoter to express carp muscle-specific creatine kinase for acclimation under cold condition.

Zebrafish (Danio rerio) is used as a model system for in vivo studies. To expand the research scope of physical, biochemical and physiological studies, a cold-tolerant model of zebrafish was developed. The common carp (Cyprinus carpio) muscle form of creatine kinase (CK, EC 2.7.3.2) can maintain enzymatic activity at a temperature of around 15°C. However, a cold-inducible promoter of zebrafish, hsc 70 (heat shock protein 70 cognate), is able to increase the expression of gene product by 9.8 fold at a temperature of 16°C. Therefore, the carp CK gene was promoted by hsc 70 and transfected into zebrafish embryos. Resulting transgenic zebrafish survived and could maintain its swimming behavior at 13°C, which was not possible with the wild-type zebrafish. The swimming distance of the transgenic fish was 42% greater than that of the wild type at 13°C. This new transgenic fish model is ideal for studies of ectothermal vertebrates in low-temperature environments.

Activity and function of rabbit muscle-specific creatine kinase at low temperature by mutation at gly268 to asn268.

Carp muscle-specific creatine kinase M1 isoenzyme (M1-CK) seems to have evolved to adapt to synchronized changes in body temperature and intracellular pH. When gly(268) in rabbit muscle-specific creatine kinase was substituted with asn(268) as found in carp M1-CK, the rabbit muscle-specific CK G286N mutant specific activity at pH 8.0 and 10°C was more than 2-fold higher than that in the wild-type rabbit enzyme. Kinetic studies showed that K(m) values of the rabbit CK G268N mutant were similar to those of the wild-type rabbit enzyme, yet circular dichroism spectra showed that the overall secondary structures of the mutant enzyme, at pH 8.0 and 5°C, were almost identical to the carp M1-CK enzyme. The X-ray diffraction pattern of the mutant enzyme crystal revealed that amino acid residues involved in substrate binding are closer to one another than in the rabbit enzyme, and the cysteine283 active site of the mutant enzyme points away from the ADP binding site. At pH 7.4-8.0 and 35-10°C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the wild-type rabbit enzyme and more similar to the carp M1-CK enzyme. Thus, the smaller active site of the RM-CK G268N mutant may be one of the reasons for its improved activity at low temperature.