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For the in situ and sensitive detection of benthic dinoflagellates, we have established an integrated loop-mediated isothermal amplification (LAMP) assay based on Ostreopsis cf. ovata and Amphidinium massartii. To detect the two species, a set of species-specific primers was constructed between the ITS gene and D1–D6 LSU gene, and the reaction temperature, time, and buffer composition were optimized to establish this method. In addition, the specificity of the LAMP primers was verified both in strains established in the laboratory and in field samples collected from the Jeju coastal waters, Korea. With the LAMP assay, the analysing time was within 45 to 60 min, which may be shorter than that with the conventional PCR. The detection sensitivity of the LAMP assay for O. cf. ovata or A. massartii was comparable to other molecular assays (PCR and quantitative PCR (qPCR)) and microscopy examination. The detection limit of LAMP was 0.1 cell of O. cf. ovata and 1 cell of A. massartii. The optimized LAMP assay was successfully applied to detect O. cf. ovata and A. massartii in field samples. Thus, this study provides an effective method for detecting target benthic dinoflagellate species, and could be further implemented to monitor phytoplankton in field surveys as an altenative.

The temporal and spatial distribution of the toxic epiphytic dinoflagellate Ostreopsis cf. ovata was investigated off the Jeju coastal waters, Korea, from July 2016 to January 2019. The results showed that the presence of Ostreopsis cf. ovata in 184 macroalgae was 79.3%, and it was more frequently attached to red algae and brown algae than to green algae. The abundance of Ostreopsis cf. ovata as determined by quantitative real-time polymerase chain reactions (qPCR) and microscopic analysis was 4&ndash, 3204 cells g&minus, 1, and the maximum abundance observed in September 2018, when the water temperature was 24.4 &deg, C. The abundance was higher in summer and autumn than in spring and winter. Spatially, high abundance was observed in autumn on the northern coast of Jeju Island and, in summer, in the southern and eastern coastal waters. The water temperature of Jeju coastal waters in winter remained higher than 15 &deg, C, and this species could be overwintering in the Jeju waters. Therefore, further monitoring and research are needed to evaluate the proliferation of Ostreopsis cf. ovata, which contains a novel toxin with unidentified effects on humans.

Bioactive peptides have positive effects on human health, including antioxidant, antihypertensive, and antimicrobial. The aim of this study is to produce anti-adipogenic bioactive peptides from ark shell protein and to evaluate the mechanisms that inhibit adipogenesis in mesenchymal stem cells (MSCs). Exposure of ark shell protein hydrolysates (ASPH) produced at a pepsin/substrate ratio of 1 : 500 to 240 min hydrolysis led to a decrease in the intracellular lipid accumulation and increase in lipolysis. Molecular weight fractionation revealed that ASPH III exerts the highest inhibitory effect on the intracellular lipid accumulation and increases lipolysis. At the molecular level, ASPH III significantly inhibits adipogenesis by down-regulating the adipocyte-specific protein expression including peroxisome proliferator-activated receptor γ (PPAR γ), CCAAT/enhancer-binding protein α (C/EBP α), and sterol regulatory element-binding protein 1c (SREBP 1c), as well as downstream lipoprotein lipase (LPL) and fatty acid synthase (FAS) expression. Moreover, treatment with ASPH III increases hormone-sensitive lipase (HSL) and leptin expression in the differentiated adipocytes from MSCs.

Chitosan and its derivatives have been reported to have anti-inflammatory effects in vitro and in vivo. It is also suggested that chitosan and its derivatives could be up-regulating heme oxygenase-1 (HO-1) in different models. However, the up-regulation of HO-1 by chitosan oligosaccharides (COS) remains unexplored in regard to anti-inflammatory action in lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells). Treatment with COS induced HO-1 expression in LPS-stimulated RAW264.7 cells, whereas the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased. Pretreatment with ZnPP, a HO-1 inhibitor, reduced the COS-mediated anti-inflammatory action. HO-1 induction is mediated by activating the nuclear translocation of NF-E2-related factor 2 (Nrf2) using COS. Moreover, COS increased the phosphorylation of extracellular signal regulated kinase (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), and p38 MAPK. However, specific inhibitors of ERK, JNK, and p38 reduced COS-mediated nuclear translocation of Nrf2. Therefore, HO-1 induction also decreased in RAW264.7 cells. Collectively, COS exert an anti-inflammatory effect through Nrf2/MAPK-mediated HO-1 induction.

Osteoporosis is a common bone disease resulting from imbalance between bone formation and bone resorption. Currently, anti-resorptive agents that inhibit bone resorption are the most available drugs on the market. Biosphosphonates, anti-resorptive drugs most commonly used to treat osteoporosis, are limited by their side effects for long-term continuous treatment. It is important to develop appropriate therapeutic stragegies capable of promoting bone formation to counteract osteoporotic bone loss. Thus, anabolic agents that stimulate bone formation are undoubtedly of interest. Here, we purified and identified two novel osteogenic peptides AWLNH and PHDL from ark shell protein hydrolysates. AWLNH and PHDL stimulated osteoblast differentiation via mitogen-activated protein kinase (MAPK) and bone morphogenetic protein-2 (BMP-2) pathways. The activation of BMP-2 pathway stimulated by AWLNH and PHDL was abolished by treating noggin, BMP antagonist, in bone marrow-derived mesenchymal stem cells (BMMSCs), but not the phosphorylation of JNK1/2, ERK1/2, and p38 MAPK. However, treatment with MAPK inhibitors in BMMSCs downregulated the expression of BMP-2 and p-Smad1/5 and inhibited alkaline phosphatase activity. The dominant inhibitory effects by JNK inhibitor and ERK inhibitor are observed. In ovariectomized (OVX) mice, a reduction of femoral bone mineral density (BMD) was significantly observed, however, AWLNH and PHDL (0.2 mg/kg/per day) injection restored BMD as well as the osteoporotic conditions in OVX mice. Moreover, the increased serum osteocalcin and alkaline phosphatase activity in OVX mice were significantly reduced in AWLNH and PHDL injected-OVX mice. These results suggest that two novel osteogenic peptides AWLNH and PHDL could be attractive therapeutic agents for osteoporosis treatment.

Aging of human bone is characterized by decreased bone formation and bone mass. In this study, ark shell protein hydrolysates (ASPHs) were prepared by peptic hydrolysis with optimal conditions and were fractionated into 3–10 kDa, 1–3 kDa and

Seafood provides a range of health benefits due to its high-protein level. In this study, the osteogenic effect of blue mussel (Mytilus edulis) protein hydrolysates (BMPH) on osteoblast differentiation were examined using mouse mesenchymal stem cells (MSCs). A preparation we called BMPH