Endolithic microbial habitats hosted in carbonate nodules currently forming within sediment at a high methane flux site in the sea of Japan

Concretionary carbonates in deep-sea methane seep fields are formed as a result of microbial methane degradation, called anaerobic oxidation of methane (AOM). Recently, active microorganisms, including anaerobic methanotrophic archaea, were discovered from methane seep-associated carbonate outcroppings on the seafloor. However sedimentary buried carbonate nodules are a hitherto unknown microbial habitat. In this study, we investigated the microbial community structures in two carbonate nodules collected from a high methane flux site in a gas hydrate field off the Oki islands in the Sea of Japan. The nodules were formed around sulfate-methane interfaces (SMI) corresponding to 0.7 and 2.2 m below the seafloor. Based on a geochemical analysis, light carbon isotopic values ranging from −54.91‰ to −37.32‰ were found from the nodules collected at the shallow SMI depth, which were attributed to the high contributions of AOM-induced carbonate precipitation. Signatures of methanotrophic archaeal populations within the sedimentary buried nodule were detected based on microbial community composition analyses and quantitative real-time PCR targeted 16S rRNA, and functional genes for AOM. These results suggest that the buried carbonate nodule currently develops AOM-related microbial communities, and grows depending on the continued AOM under high methane flux conditions

Enhancement of Chemosensitivity to Fluoropyrimidines by Retroviral Transduction of Thymidine Phosphorylase cDNA: an in vitro and in vivo Study

Thymidine phospholyrase (TP) is an essential enzyme in activating 5’-deoxy-5-fluorocytidine (5’- DFUR) into 5-fluorouracil (5-FU) and for the conversion of 5-FU into 5-fluoro-2’-deoxyuridine. The purpose of this study was to examine the therapeutic efficacy of the retroviral vector-mediated TP gene in the sensitivity to fluoropyrimidines, 5-FU and its prodrugs, 5’-DFUR and capecitabine, in MC38 murine colon adenocarcinoma cells in vitro and in vivo. After retroviral infection with or without human TP cDNA, we obtained MC38 cells having the stable expression of TP (MC38-TP) and control-vector transfected cells (MC38-Neo). There was no significant difference in the doubling time in vitro and tumor growth rate in vivo among parental MC38 cells (MC38-P), MC38- Neo and MC38-TP, demonstrating that the TP gene was not directly toxic. The in vitro study showed significant increases in sensitivities to 5-FU, 5’-DFUR and capecitabine in MC38-TP cells. The 50% growth inhibitory concentration (IC50) of MC38-TP cells to 5-FU, 5’-DFUR and capecitabine, respectively, was about 10-fold, 800-fold and 40-fold higher than that of MC38-P cells and MC38-Neo cells. The in vivo study showed significant increases in sensitivities to 5-FU, 5’- DFUR and capecitabine in MC38-TP tumors. There was no significant difference in the sensitivities to 5-FU, 5’-DFUR and capecitabine between MC38 and MC38-Neo tumors. The tumor-cure rate in MC38-TP tumors treated with capecitabine was 100% and that treated with 5’-DFUR was 63%. In conclusion, our results demonstrate that the stable expression of TP gene by using recombinant retroviral vector could dramatically increase the anticancer effect of fluoropyrimidines

PDGF inhibits BMP2-induced bone healing

Abstract Bone regeneration depends on a pool of bone/cartilage stem/progenitor cells and signaling mechanisms regulating their differentiation. Using in vitro approach, we have shown that PDGF signaling through PDGFRβ inhibits BMP2-induced osteogenesis, and significantly attenuates expression of BMP2 target genes. We evaluated outcomes of treatment with two anabolic agents, PDGF and BMP2 using different bone healing models. Targeted deletion of PDGFRβ in αSMA osteoprogenitors, led to increased callus bone mass, resulting in improved biomechanical properties of fractures. In critical size bone defects BMP2 treatment increased proportion of osteoprogenitors, while the combined treatment of PDGF BB with BMP2 decreased progenitor number at the injury site. BMP2 treatment induced significant bone formation and increased number of osteoblasts, while in contrast combined treatment with PDGF BB decreased osteoblast numbers. This is in vivo study showing that PDGF inhibits BMP2-induced osteogenesis, but inhibiting PDGF signaling early in healing process does not improve BMP2-induced bone healing