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Objective To investigate the role and possible mechanism of the core rhythm molecule BMAL1 in the prognosis of osteosarcoma (OS). Methods After BMAL1 was overexpressed in U2OS cells by Lipofectamine3000, the expression of BMAL1 was detected with qRT-PCR, and the clone formation and invasion ability of the overexpressed cells were observed with clonogenic assay and Transwell assay. Then, the BMAL1 overexpressed U2OS cells or the cells with control vector transfection were cocultured with peripheral blood mononuclear cells (PBMCs), the direct killing effects of PBMCs on U2OS cells was observed with Calcein AM staining, and the CD107a expression by CD8+ T and NK cells was measured with flow cytometry. Results The OS patients with higher BMAL1 mRNA level had significantly longer survival time when compared with those with lower expression (P < 0.001). BMAL1 overexpression had no obvious effects on the invasion and clone formation of U2OS cells. However, cell death was significantly increased in BMAL1 overexpressed U2OS cells than the control vector transfected U2OS cells when co-cultured with PBMCs in vitro [PBMCs:U2OS cells=5:1, (81.00±0.58)% vs (73.33±0.67)%, P < 0.01; PBMCs:U2OS cells=10:1, (82.67±1.86)% vs (76.67±0.33)%, P < 0.01]. There was no significant difference in the expression level of CD107a between T cells and NK cells in the BMAL1 overexpressed U2OS cells than the control vector transfected U2OS cells when co-cultured with PBMCs (E:T=5:1, CD3+ CD56- T cells, P=0.46, CD3-CD56+ NK cells, P=0.96; E:T=10:1, CD3+ CD56- T cells, P=0.93; CD3-CD56+ NK cells, P=0.21). Conclusion The expression level of BMAL1 is positively correlated with the prognosis of OS patients, which might be mainly due to the enhanced immunological surveillance, but not by affecting the ability of invasion and clone formation of OS cells.

Melatonin affects physiological processes that have circadian and seasonal cycles and causes behavioural changes in nearly all organisms, ranging from bacteria to mammals. However, no studies have focused on the presence, biosynthetic pathway, and rhythms of melatonin in bivalves, which are considered as the class of molluscs with the highest economic value. Here, the metabolic pathway and tissue distribution of melatonin, tissue transcription profiles of melatonin-related genes [key synthases arylalkylamine N-acetyltransferase type 2 and 5 (aanat2 , aanat5), N-acetylserotonin methyltransferase (asmt), and melatonin receptor type 1 A1 and C3 (MTNR1A1 , MTNR1C3)], daily changes in melatonin, transcripts of genes, and effects of four lights (blue, cyan, orange, and red) during the night on melatonin secretion were studied in a bivalve species, razor clam Sinonovacula constricta. Our results showed the presence of complete metabolic and circadian entrainment pathways of melatonin. The melatonin content in the lymphocytes and labial palp was higher than that in the other studied tissues, and aanat2 , aanat5 , asmt , MTNR1A1 , and MTNR1C3 mRNA levels were the highest in the labial palp. Melatonin fluctuated rhythmically in lymphocytes and labial palps and increased at night. Similarly, aanat2 , aanat5 , asmt , MTNR1A1 , and MTNR1C3 mRNA levels were rhythmic in the labial palp, peaking at night. In addition, blue light effectively inhibited the nocturnal secretion of melatonin in the clam. In summary, our results suggest that melatonin plays a role in light signal transduction and the circadian system in S. constricta. • Melatonin could be biosynthesized by the clam Sinonovacula constricta. • Labial palp probably is an important tissue where melatonin exerted its function. • The biosynthesis and secretion of melatonin in the clam are enhanced under the dark phase of the photoperiod. • Blue light inhibits melatonin production in the clam. [ABSTRACT FROM AUTHOR]

Sinonovacula constricta is an important bivalve with strong photosensitivity and a robust circadian rhythm. To elucidate the light entrainment of the molecular circadian clock in this species, this study identified the clock genes, characterized their tissue distribution, and investigated their transcriptional variations under different light regimes: LD (12 h light/12 h dark cycle), LL (continuous light), and DD (continuous dark). The results showed that the S. constricta clock system consists of seven genes [ Circadian locomotor output cycles kaput (ScClock), Brain and muscle ARNT-like protein (ScBmal), Cryptochrome (ScCry1 and 2), Period (ScPer), and Timeless (ScTimeless1 and 2)] that are closely related to their orthologs in marine mollusks and contained corresponding conserved functional regions, indicating the conserved evolution of the circadian clock among organisms. These genes were expressed in all tissues, with high expression in the labial palps and hepatopancreas, suggesting a complex peripheral biological clock in S. constricta and the critical role of these two tissues in circadian functions. When exposed to different light regimes in labial palps, genes from the positive loop (ScClock and ScBmal) displayed a daily/circadian rhythm under both LD and DD and their acrophases were shifted by 5–7 h, whereas genes from the negative loop displayed daily rhythms (ScCry1 and ScPer) under LD and circadian rhythms (ScCry2 and ScTimeless2) under DD. However, only one gene (ScTimeless2) displayed a circadian rhythm under LL conditions. These results suggest that the S. constricta circadian system may be regulated by different clock genes to adapt to various light environments. Although similar phenomena were observed in the hepatopancreas, markedly different clock gene expression patterns occurred between the two tissues, indicating a tissue-specific regulated circadian system. Collectively, this study reveals how the circadian oscillators of S. constricta respond to light and may promote light environment optimization in the S. constricta farming industry. • The molecular circadian clock was conserved in S. constricta. • A peripheral and tissue-specific clock might exist in S. constricta. • The S. constricta circadian clock was synchronized by photoperiod. • A functional clock exists in S. constricta. [ABSTRACT FROM AUTHOR]