Micturition behavior follows regular day/night fluctuations, and unwanted increase in micturition could occur during night in jet lag condition. To clarify the effect of jet lag on micturition behavior, we simultaneously detected circadian micturition patterns and locomotor activity rhythms of mice under experimental jet lag conditions, by applying the improved automated Voided Stain on Paper (aVSOP) method. When wild-type (WT) mice were phase-advanced for 8 hours, day-night variation of micturition was disrupted suddenly, and this irregular daily micturition continued until 8 days, although their activity rhythms entrained gradually day by day until 8 days. We also examined how jet lag induced changes of micturition in Per-null mice lacking Per1, Per2 and Per3 genes, whose endogenous clock is completely disrupted. We found both micturition and locomotor activity of Per-null mice promptly entrained to the new LD cycle. These findings suggest that the irregular micturition during jet lag is caused along with the gradual shift of the endogenous clock, and paradoxically, jet lag-associated abnormality was absent when endogenous circadian oscillations were genetically disrupted.

The epithelial basal lamina of the small intestine forms a felt-like sheet at the base of the epithelium. Previous studies have shown that the basal lamina has numerous fenestrations, which are produced by leukocytes penetrating through the basal lamina. In this study, we aimed to directly visualize fenestrations of the rat basal lamina in intestinal villi by scanning electron microscopy (SEM) after removal of the villous epithelium by osmium maceration and ultrasonic treatment. Structural changes in fenestrations were then investigated in relation to dietary conditions. SEM of these tissues revealed the presence of fenestrations in the villous epithelial basal lamina in all segments of the small intestine, although the number was the highest in the jejunum. The present study also showed that the number and size of fenestrations increased after feeding in the jejunum, whereas changes were unclear in the ileum. These findings suggested that the basal lamina fenestrations were changed through the dynamics of migrating leukocytes in dietary conditions and may also be related to the regulation of nutrient absorption, particularly as lipids are transported from the intercellular space of the epithelium to the lamina propria.

Separation of sister chromatids is a drastic and irreversible step in the cell cycle. The key biochemistry behind this event is the proteolysis mediated by the ubiquitin ligase called the anaphase promoting complex, or APC/C. Securin and cyclin B1 are the two established substrates for APC/C whose degradation releases separase and inactivates cyclin B1-dependent kinase 1 (cdk1), respectively, at the metaphase-to-anaphase transition. In this study, we have combined biochemical quantifications with mathematical simulations to characterize the kinetic regulation of securin and cyclin B1, in the cytoplasmic and chromosomal compartments, and found that they are differentially distributed and degraded with different rates. Modeling their interaction with separase predicted that activation timing of separase well coincides with the decline of securin-separase concentration in the cytoplasm. Notably, it also coincides with the peak of cyclin B1-separase level on chromosomes, which appeared crucial to coordinate the timing for separase activation and cdk1 inhibition. We have also conducted phosphoproteomic analysis and identified Ki67 as a chromosomal cdk1 substrate whose dephosphorylation is facilitated by cyclin B1-separase interaction in anaphase.

Chloroform is a common contaminant in the drinking water. Exposure of human to chloroform leads to severe hepatotoxicity. In the present study, chloroform-induced acute liver injury was investigated in mice using 3-methyadenine (3-MA), a common autophagy inhibitor. At 24 h after intraperitoneal injection of 0.5 mL/kg chloroform, serum alanine aminotransferase (ALT) levels were increased significantly; extensive necrosis and inflammation occurred as identified by histological examinations. Moreover, chloroform induced an increase in lipid peroxidation as demonstrated by increased formation of malondialdehyde (MDA) in the liver tissues. Hepatic antioxidants including glutathione (GSH) and superoxide dismutase (SOD) were decreased by chloroform treatment. All these changes were significantly inhibited by 3-MA treatment. Further mechanistic insights demonstrated that chloroform up-regulated pro-inflammatory cytokine, IL-1β, in the livers and blood, which was suppressed by 3-MA. Surprisingly, Western blots results showed that after 24-hours of chloroform treatment 3-MA activated autophagy as indicated by decreased levels of LC3B II and p62 protein. Co-treatment of chloroquine with 3-MA to inhibit autophagy would abrogate the hepatoprotection of 3-MA in chloroform hepatotoxicity. Taken together, findings in the present study suggested that a widely-used autophagy inhibitor, 3-MA, significantly reduced chloroform hepatotoxicity in mice via autophagy activation. Findings in this study also suggested that caution should be exercised when using 3-MA to modulate autophagy in vivo.

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco’s modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.