The circadian clock coordinates physiology, metabolism, and behavior with the 24-h cycles of environmental light. Fundamental mechanisms of how the circadian clock regulates organ physiology and metabolism have been elucidated at a rapid speed in the past two decades. Here we review circadian networks in more than six organ systems associated with complex disease, which cluster around metabolic disorders, and seek to propose critical regulatory molecules controlled by the circadian clock (named clock-controlled checkpoints) in the pathogenesis of complex disease. These include clock-controlled checkpoints such as circadian nuclear receptors in liver and muscle tissues, chemokines and adhesion molecules in the vasculature. Although the progress is encouraging, many gaps in the mechanisms remain unaddressed. Future studies should focus on devising time-dependent strategies for drug delivery and engagement in well-characterized organs such as the liver, and elucidating fundamental circadian biology in so far less characterized organ systems, including the heart, blood, peripheral neurons, and reproductive systems.

Canities is an obvious sign of aging in mouse and human, shown as hair graying. Melanocytes in the hair follicle show cyclic activity with hair cycling, which transitions from anagen, catagen to telogen. How the hairs turn gray during aging is not completely uncovered. Here, by using immunostaining and LacZ staining in Dct-LacZ mice, we show that β-catenin is expressed in melanocytes during hair cycling. RT-PCR, western blot and immunostaining show that β-catenin expression is significantly increased in both anagen and telogen skin of aged mice, when compared to the anagen and telogen skin of young mice, respectively. Overexpression of Wnt10b not only accelerates hair follicle to enter anagen phase, but also promotes melanocytes differentiation in young adult mice (2-month old), with increased β-catenin expression in melanocytes at the secondary hair germ and matrix region of regenerated hair follicles. Overexpression of Wnt10b also promotes melanocyte progenitor cells differentiation in vitro. Our data suggest that increased Wnt signaling promotes excessive differentiation of melanocytes, leading to exhaustion of melanocyte stem cells and eventually canities in aged mice.

Reperfusion through thrombolytic therapy or primary percutaneous coronary intervention is commonly used to deal with acute myocardial infarction. However, the reperfusion procedure is accompanied by myocardial ischemia-reperfusion injury (MIRI). Currently, there is no therapeutics that can effectively deal with MIRI in clinical practice. Herein, the potential of ceria nanoparticles (CNPs) coated by different ligands in the treatment of rat MIRI is evaluated. The results demonstrate that CNPs can effectively modulate the oxidative stress in the heart tissue through the elimination of reactive oxygen species (ROS) and stimulation of endogenous antioxidant system. The inhibition of oxidative stress results in the reduction of p-Drp1 (Ser 616) which is critical in driving the fission and fragmentation of mitochondria. The improved mitochondrial dynamics saves the cardiomyocytes from apoptosis and reduces the acute injury of left ventricular wall during the MIRI. The ejection function of the left ventricle for both the short-term and long-term MIRI rats is well preserved. We therefore believe based on these results that the administration of CNPs is beneficial in the attenuation of MIRI during the acute stage. These findings provide useful information for the future fabrication of inorganic antioxidant nanomedicine for the treatment of MIRI.

Inverted feeding is a paradigm to study synchronization of circadian clocks by feeding rhythm in tissues more directly. Here, we provide a protocol for performing inverted feeding in mice and analyzing circadian rhythmicity in mouse tissues. We describe setting up inverted feeding and performing tissue dissection, followed by RNA extraction and gene expression analysis, and lastly R software-based analysis of circadian rhythmicity. This protocol can be combined with the use of CircaMetDB database for mechanistic studies of inverted feeding.

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

Hypobaric hypoxia (HH)-induced acute lung injury (ALI) is a growing concern. Tryptophan metabolite 5-Methoxytryptophan (5-MTP) is a recently identified endothelial protective factor. However, the role of 5-MTP in HH-induced ALI remains unclear. Forty healthy male human participants were enrolled in Chongqing (200 m) to travel to Golmud (4260 m). We found the decreased plasma 5-MTP level was correlated with HH-induced blood oxygen desaturation and acute mountain sickness. ELISA showed 5-MTP levels decreased in the plasma and lungs of ALI mice (male 6-8 weeks C57BL/6J), and also in the supernatant of mouse pulmonary microvascular endothelial cells (PMVECs) after hypoxia. Immunoblotting showed the synthetase Asmt was downregulated in mice lungs and PMVECs after hypoxia, which could be restored by si-Hif1α or NF-κB inhibitor. ChIP assays confirmed that hypoxia enhanced NF-κB p50 binding in Asmt promotor. Application of 5-MTP protected the hyperpermeability in ALI. The transmission electron microscope of mice lungs showed 5-MTP alleviated the endothelial barrier disruption and lipoperoxidation injury. Using limited proteolysis combined with mass spectrometry, we revealed Prdx6-Ser32 as the direct target of 5-MTP, consistent with molecular docking. Molecular dynamics simulation, cellular thermal shift assays and microscale thermophoresis confirmed the interaction. 5-MTP prevented the hypoxia-induced decline of Prdx6 and lipoperoxidation. By MG132 and BafA1, we found hypoxia promoted the lysosomal degradation of Prdx6. Immunoblotting and immunofluorescence showed 5-MTP could reduce hypoxia-induced increased lysosomal localization of Prdx6. Prdx6-S32A impaired the protective effects of 5-MTP on PMVECs, and also the lysosomal localization of Prdx6. Adeno-associated viruses were injected intratracheally to confirm Prdx6-S32A also impaired the therapeutic effects of 5-MTP on ALI mice. 5-MTP is a predictive biomarker of hypoxic maladaptation. The restoration of 5-MTP attenuated ALI by inhibiting lipoperoxidation and protecting endothelial barrier via directly interaction with Ser32 site of Prdx6 to prevent its lysosomal degradation.