Reactive oxygen species (ROS) participate in various physiological and pathological functions following generation from different types of cells. Here we explore ROS functions on spontaneous tail regeneration using gecko model. ROS were mainly produced in the skeletal muscle after tail amputation, showing a temporal increase as the regeneration proceeded. Inhibition of the ROS production influenced the formation of autophagy in the skeletal muscles, and as a consequence, the length of the regenerating tail. Transcriptome analysis has shown that NADPH oxidase (NOX2) and the subunits (p40phox and p47phox) are involved in the ROS production. ROS promoted the formation of autophagy through regulation of both ULK and MAPK activities. Our results suggest that ROS produced by skeletal muscles are required for the successful gecko tail regeneration.

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The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi. The presence of aprN, a gene-encoding nattokinase, was verified with PCR method. The predicted amino acid sequence was aligned with homologous sequences, and a phylogenetic tree was constructed. Nattokinase was sublimated with ammonium sulfate, using a DEAE-Sepharose Fast Flow column, a CM-Sepharose Fast Flow column and a Sephadex G-75 gel filtration column. SDS-PAGE analysis indicated that the molecular weight of the purified nattokinase from Bacillus subtilis MX-6 was about 28 kDa. Fermentation of Bacillus subtilis MX-6 nattokinase showed that nattokinase production was maximized after 72 h; the diameter of clear zone reached 21.60 mm on the plasminogen-free fibrin plate. Nattokinase production by Bacillus subtilis MX-6 increased significantly after supplementation with supernatant I, supernatant II and soy peptone but decreased substantially after the addition of amino acids. This result indicated that the nattokinase production by B. subtilis MX-6 might be induced by soybean polypeptides. The addition of MgSO and CaCl increased B. subtilis MX-6 nattokinase production.

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