Presence of Microplastics in Four Types of Shellfish Purchased at Fish Markets in Okayama City, Japan

The worldwide microplastic pollution in our environment is a matter of great concern. Harmful effects of plastics have been reported in various types of organisms including murine animals. We examined the presence of microplastics in four types of shellfish purchased from fish markets in Okayama, Japan and served to the public: short-neck clam (Ruditapes philippinarum, asari in Japanese), hard-shell clam (Meretrix lusoria, hamaguri), brackishwater clam (Cyrenidae, shijimi), and oyster (Crassostrea gigas, kaki). Our analyses demonstrated that approx. 3 pieces of microplastics were present per single shellfish, based on the division of the total number of pieces of microplastic obtained from all 4 types of shellfish by the total number of shellfish examined. Since health problems in humans due to microplastics have not yet been confirmed, further examinations of the effects of ingested microplastics are needed

Comparison of Various Methods of Assaying the Cytotoxic Effects of Ethanol on Human Hepatoblastomaells (HUH-6 Line)

The sensitivity of five kinds of cytotoxicity assays using ethanol on human hepatoblastoma cells (HUH-6 line), which were cultured as monolayers or spheroids, was compared. Ethanol was chosen as a test because it acts on cell membranes directly without being metabolized and exerts its cytotoxicity. The assay methods used were as follows: 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), colony formation, cell growth and DNA assays. The sensitivity of the assays was: LDH &#60; DNA &#60; cell growth &#60; MTT &#60; colony formation. LDH assay had the advantage that the same culture could be used for multiple assays, but when a small number of cells were assayed, no significant increase in the release of LDH was detected in the assay cultures compared with the control cultures. Although the DNA and cell growth assays were more sensitive than the LDH assay, the extent of cell damage may be underestimated because the damaged cells and DNA present in the cultures are included in the assay samples. On the other hand, both MTT and colony formation assays showed a high sensitivity. The MTT assay was done within 24 h after ethanol was added to the cultures and was applicable to both monolayer and spheroid cultures, while the colony formation assay required 1-2 weeks and it was applicable only to monolayer cultures. Taken together, the MTT assay was the most suitable method to evaluate the cytotoxic effects of ethanol on HUH-6 cells cultured as either monolayers or spheroids.</p

Spheroid Cultures of Human Hepatoblastoma Cells (HuH-6 Line) and Their Application for Cytotoxicity Assay of Alcohols

&#60;P&#62;Spheroid cultures of human hepatoblastoma cells (HuH-6 line) were established by rotating 3 x 10(6) cells/3 ml culture medium in 25-ml Erlenmeyer flasks on a gyratory shaker. The size of the spheroids rapidly increased until 4 days of culture, and thereafter their size gradually increased until 8 days of culture. A considerable amount of lactate dehydrogenase (LDH) was detected in the culture medium at 24h after seeding because of cell damage by subculturing, but thereafter the amount released was small, indicating that the spheroids were in healthy condition. Albumin production, one of the differentiated functions of hepatocytes, was higher in spheroid cultures than in monolayer cultures. Using this spheroid culture model, the cytotoxic effects of alcohols on HuH-6 cells were studied by measuring the activity of LDH released in the medium from damaged cells. The results indicate that the increasing order of toxicity of the alcohols was as follows: methanol &#60; ethanol &#60; propanol.&#60;/P&#62;</p

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10–35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression

Total Synthesis of (+)-Clavilactone A and (−)-Clavilactone B by Ring-Opening/Ring-Closing Metathesis

The enantioselective total synthesis of natural enantiomers of clavilactones A and B has been achieved. A key feature of the synthesis is the use of a ring-opening/ring-closing metathesis, which allows the one-pot transformation of a strained cyclobutenecarboxylate into a γ-butenolide

Novel polychrome staining distinguishing osteochondral tissue and bone cells in decalcified paraffin sections

The bone is a dynamic and metabolically active organ in which growth and resorption of the osteochondral matrix is orchestrated by osteoblasts and osteoclasts. For decalcified paraffin-embedded specimens, decalcifying agents alter the staining intensity, and excess decalcification interferes with bone staining. Robust bone staining methods independent of the decalcification conditions and animal species are lacking. In this study, we have developed a novel polychrome staining method, named JFRL staining, which stains the components of osteochondral tissue in different colors. With this staining we could visualize the hyaline cartilage as blue by alcian blue, osteoid as red by picrosirius red, and mineralized bone as green by picro-light green SF or picro-naphthol green B and easily distinguished osteoblasts, osteocytes, and osteoclasts. In mineralized bone, this staining revealed the obvious lamellar structures and woven bone. Notably, this staining was independent of the decalcification conditions and experimental animal species examined. To verify the usefulness of JFRL staining, we observed cotton rat tail which has shorter length and shows a false autotomy. The caudal vertebrae were normally developed via endochondral ossification without a fracture plane. At 6 months of age, the number of chondrocytes declined and the hypertrophic zone was absent at the epiphyseal plate, which might reflect the shorter tail. In conclusion, JFRL staining is the first method to simultaneously distinguish osteochondral matrix and bone cells in one section regardless of decalcifying conditions. This robust staining will provide new information for a wide number of biomedical fields, including bone development, physiology, and pathology