Reprocessing seafood waste: challenge to develop aquatic clean meat from fish cells.

Fish consumption has been increasing worldwide as per capita consumption of fish rises along with population growth. At the same time, overfishing is increasing all over the world, causing enormous damage to the ecosystem. There is an urgent need to secure sustainable fishery resources to meet the expanding demand for fish. The present study focused on the cells obtained from fish fins, which were often discarded as food waste, and which had the potential to change their morphology with simple treatments, creating the possibility of processing fish fin cells into clean meat (i.e., meat produced in vitro; artificial, lab-cultured meat using tissue engineering techniques). The fin-derived fibroblast-like cells demonstrated an interesting characteristic; changing the sera or culture media supported differentiation of the fibroblast-like cells to various cell morphologies, such as neurofilaments and adipocytes, etc., without genetic manipulation. Furthermore, it was possible to culture the cells in multi-layered and three-dimensional forms that were suitable for processing and shaping. Taking advantage of the cells' characteristics, 'aquatic clean meat' was produced successfully at the prototype stage. Our results suggest that fish fins, which are often treated as waste material, thus, are easy to procure, simple to process, and could be used to create a sustainable food resource.

Ca2+ dynamics in zebrafish morphogenesis.

Intracellular calcium ion (Ca2+) signaling is heavily involved in development, as illustrated by the use of a number of Ca2+ indicators. However, continuous Ca2+ patterns during morphogenesis have not yet been studied using fluorescence resonance energy transfer to track the Ca2+ sensor. In the present study, we monitored Ca2+ levels during zebrafish morphogenesis and differentiation with yellow cameleon, YC2.12. Our results show not only clear changes in Ca2+ levels but also continuous Ca2+ patterns at 24 hpf and later periods for the first time. Serial Ca2+dynamics during early pharyngula period (Prim-5-20; 24-33 hpf) was successfully observed with cameleon, which have not reported anywhere yet. In fact, high Ca2+ level occurred concurrently with hindbrain development in segmentation and pharyngula periods. Ca2+ patterns in the late gastrula through segmentation periods which were obtained with cameleon, were similar to those obtained previously with other Ca2+sensor. Our results suggested that the use of various Ca2+ sensors may lead to novel findings in studies of Ca2+ dynamics. We hope that these results will prove valuable for further research in Ca2+ signaling.

A novel fluorescent protein from the deep-sea anemone Cribrinopsis japonica (Anthozoa: Actiniaria).

A fluorescent protein was identified and cloned from the deep-sea anemone Cribrinopsis japonica. Bioluminescence and fluorescence expression were examined by direct observations of live specimens and RNA-Seq analysis. Both approaches revealed a novel green fluorescent protein in the tentacles of the anemone, but bioluminescence was not observed. Behavioural observations revealed that a blue light excited the fluorescence in the tentacles, and initiated a behavioural response whereby the fluorescent tentacles became fully exposed to the blue light. The excitation and emission peaks of C. japonica's fluorescent protein were at 500 and 510 nm, respectively, which were greener than those reported in homologs. Furthermore, this protein was highly tolerant of increased temperatures and repeated freeze-thaw treatments. The current study presents an example of fluorescence in a deep-sea cnidarian, demonstrating that fluorescent proteins could have important roles, regardless of the presence or absence of strong sunlight. It also demonstrates that this deep-sea fluorescent protein has unique characteristics, including high stability, perhaps as an adaptation to the extreme environment.

Methyl viologen induces neural differentiation on murine P19 cells.

Methyl viologen is a highly effective contact herbicide, a neurotoxic compound and an inducer of reactive oxygen species, which generate oxidative stress in cells. Reactive oxygen species has been known to function as an important messenger in cell differentiation. In this study, we investigated the effect of methyl viologen on neural cell differentiation using pluripotent mouse embryonal carcinoma P19 cells, which reportedly differentiate into neural cells upon exposure to retinoic acid. Methyl viologen, an inducer of intracellular reactive oxygen species, induced the differentiation of P19 cells into neural cells in the presence of neurofilament. Reduced glutathione, an eliminator of reactive oxygen species, also induced neural differentiation in P19 cells. These results suggest that P19 cells differentiate into neural cells, conceivably independent of intracellular reactive oxygen species.

Loss of wwox expression in zebrafish embryos causes edema and alters Ca(2+) dynamics.

We investigated the role of the WW domain-containing oxidoreductase (wwox) gene in the embryonic development of zebrafish, with particular emphasis on intracellular Ca(2+) dynamics because Ca(2+) is an important intracellular messenger. Comparisons between zebrafish wwox and human WWOX sequences identified highly conserved domain structures. wwox was expressed in developing heart tissues in the zebrafish embryo. Moreover, wwox knockdown induced pericardial edema with similarities to conditions observed in human breast cancer. The wwox knockdown embryos with the edema died within a week. High Ca(2+) levels were observed at the boundary between the edema and yolk in wwox knockdown embryos.

Characteristics of established KSG cells derived from the scorpionfish Sebastiscus marmoratus: what happens under the hydrostatic pressure like the deep sea?

Advances in cell biology depend, partly, on the development of new cell lines and culture methods. Our research focused on a fibroblast-like cell line, "KSG," which is derived from scorpionfish fin tissue (Sebastiscus marmoratus). Cells were grown in Leibovitz's L-15 medium with 10% fetal bovine serum following standard procedures. The optimum growth temperatures for these lines ranged from 15°C to 25°C. All cells survived storage for at least 3 yr at -80°C. Subsequently, they were continuously cultured until the 78th generation without evident changes in their morphology. Moreover, we were able to culture KSG cells in the absence of fetal bovine serum in a culture medium containing the fish serum "SeaGrow." Optimum SeaGrow concentrations for these cells ranged from 5% to 20%. The growth rate of KSG cells decreased when the concentration of SeaGrow was reduced to 1%. However, this decrease could be partially reversed by adding 0.5% "Hy-Fish." In addition, the inclusion of Hy-Fish improved cell adhesion. KSG cells that were cultured in serum-free culture media containing 0.5% and 1% Hy-Fish had been added and were able to survive at low densities. Furthermore, we successfully transfected this cell line with a commercial plasmid vector coding a fluorescent protein using the cationic lipid. Finally, the analyses of cell behavior under hydrostatic pressure showed that some pressures (10 MPa) helped the cells to proliferate more.

Salinisphaera japonica sp. nov., a moderately halophilic bacterium isolated from the surface of a deep-sea fish, Malacocottus gibber, and emended description of the genus Salinisphaera.

A moderately halophilic, slightly acidophilic, aerobic bacterium, designated strain YTM-1(T), was isolated from the body surface of Malacocottus gibber. Cells were Gram-stain-negative, short rods or cocci, approximately 0.9-1.1 µm long and 1.0-1.8 µm wide. Strain YTM-1(T) was able to grow with 1-30% NaCl (optimum, 7.5-10%, w/v), at 4-30 °C (optimum, 20-25 °C) and at pH 3.8-9.5 (optimum, pH 5.0-5.5). Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain YTM-1(T) belonged to the genus Salinisphaera with low similarity values to the type strains of recognized species of this genus (<94.8-94.4%). The polar lipids of strain YTM-1(T) consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, three unknown phospholipids and one unknown lipid. The predominant isoprenoid quinone was Q-8. The major fatty acids were C19:0ω8c cyclo, C18:1ω7c, C16:1ω5c and C16:0. The DNA G+C content of strain YTM-1(T) was 67.3 mol%. These phylogenetic, physiological and chemotaxonomic data indicated that strain YTM-1(T) represents a novel species of the genus Salinisphaera, for which the name Salinisphaera japonica sp. nov. is proposed. The type strain is YTM-1(T) (=JCM 18087(T)=CECT 8012(T)). An emended description of the genus Salinisphaera is also proposed.

Real-time monitoring of dynamic intracellular Ca(2+) movement during early embryogenesis through expression of yellow cameleon.

Intracellular calcium ion (Ca2+) is important in a variety of developmental events in the vertebrate embryo through wide-ranging signal transduction pathways. The purpose of this study was to detect dynamic changes in Ca2+ during gastrulation using yellow cameleon 2.12 (YC2.12), a Ca2+ indicator protein based on cyan and yellow fluorescent proteins. mRNA for YC2.12 was synthesized in vitro, and injected into early embryos at the single-cell stage. Fluorescence images were obtained using a fluorescence microscope equipped with a cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) filter. Image acquisition and analysis were executed using the fluorescence resonance energy transfer (FRET) software. Embryos injected with YC2.12 mRNA became larvae normally, indicating that YC2.12 may not be toxic. FRET analysis enabled us to trace dynamic changes of Ca2+ up to 30 hours postfertilization (hpf). The highest concentration of Ca2+ was observed during gastrulation. Although the fertilized eggs were symmetrical by microscopic observation, FRET images clearly indicated asymmetrical distribution and dynamic movement of Ca2+. When Fluo-3, a chemical Ca2+ indicator, was used to monitor Ca2+, fluorescence images could be obtained only during gastrulation (<6 hpf), and the images were not clear compared to when YC2.12 was used. These results suggested that YC2.12 is useful for noninvasive and real-time detection of dynamic Ca2+ change throughout gastrulation.

Enhancing the tolerance of zebrafish (Danio rerio) to heavy metal toxicity by the expression of plant phytochelatin synthase.

Phytochelatin synthase (PC synthase) catalyzes a biosynthesis of phytochelatins (PCs), which are small molecules and glutathione (GSH)-derived metal-binding peptides that are essential for the detoxification of heavy metal ions in plants, fungi and worms. In order to enhance tolerance to heavy metal cytotoxicity, mRNA coding for PC synthase from Arabidopsis thaliana (AtPCS1) was introduced into the early embryos of zebrafish. As a result, the heterogeneous expression of PC synthase and the synthesis of PCs from GSH in embryos could be detected. The developing embryos expressing PC synthase (PC-embryos) became more tolerant to Cd toxicity (500 microM exposure). PC-embryos had significantly longer apparent lethal times for 50% of the population (LT50) of 8.17+/-1.08 days, although control embryos had apparent LT50 of 5.43+/-0.66 days. These data suggest that PC synthase can function in developmental zebrafish, and that PCs are highly effective in detoxifying Cd toxicity even in the whole body of a vertebrate species.