Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PE-RIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER proteindependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.keratinocyte | p54nrb | RNA-binding protein | paraspeckle protein T he circadian clock adapts organisms to their daily surroundings both behaviorally and physiologically. In animals, not only are complex behaviors such as sleep and mood governed by this oscillator, but also different body functions such as digestion, circulation, and respiration (1). The basic mechanism of this clock is cell-autonomous in all studied species possessing a circadian clock. In mammals, individual clocks in most cells are synchronized by a brain "master clock" in the suprachiasmatic nucleus of the hypothalamus to orchestrate all rhythmic physiology (2). On a cellular level, circadian physiology extends even to processes such as proliferation (3-7), apoptosis (8), and DNA damage repair (6, 9), which are thought to play important roles in cancer control (8,10).In individual cells, the circadian clock mechanism consists of oscillating feedback loops of transcription of "core" oscillator genes and posttranslational modifications of their protein products that regulate protein stability, activity, and/or localization. For example, in mammals the transcription of periods (Per) and cryptochomes (Cry) are activated by BMAL1:CLOCK heterodimers at cisacting elements called E-boxes, and their protein products form complexes that repress their own transcription (11). We originally identified the RNA-binding protein NONO (also called p54nrb) biochemically as a new member of this circadian transcriptional repressor complex in mice, and mutation of its ortholog NonA in flies resulted in severe attenuation of circadian rhythmicity (12). However, apart from its interaction with this circadian repressor complex, NONO's mechanism of action within the clock remains unknown.The mechanism of the cell cycle has been reviewed extensively elsewhere (13,14). Rathe...

More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus 1 . Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection [1][2][3][4] . We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8 + T cell-dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8 + T cell-dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8 + T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.Infection of mice with the noncytopathic lymphocytic choriomeningitis virus (LCMV) caused hepatocyte damage, as revealed by elevated serum alanin-aminotransferase (ALT) activities and serum bilirubin concentrations 5 (Fig. 1a). Virus-specific T cells are a major determinant of the outcome of hepatitis-these cells contribute to the early control of chronic hepatitis viruses and mediate immunopathology during persistent virus infection 1-4 . In a model of LCMV-induced hepatitis, complete depletion of CD8 + T cells enhanced virus replication, but strongly reduced hepatocyte damage (Fig. 1a), showing that the activation of virus-specific CD8 + T cells by viral antigen and not LCMV replication alone is responsible for liver cell damage, confirming earlier results 5,6 . To induce a delayed appearance of CD8 + T cells within the liver, we treated C57BL/6 mice with a 1:10 dilution of a CD8 cell-depleting antibody, which leads to a transient absence of CD8 + T cells. After 8-12 days, CD8 + T cells re-emerged (Fig. 1b). The delayed CD8 + T cell response within the liver was associated with prolonged viral replication and enhanced ALT levels in the serum when CD8 + T cells re-emerged (Fig. 1b).Therefore, similarly to the situation in human hepatitis 7-11 , delayed control of LCMV in the liver enhanced the overall immunopathology in the liver.Infection and virus-induced hepatitis have been linked to platelet activation [12][13][14] . To study the role of platelet activation in LCMVtriggered CD8 + T cell-dependent hepatiti...

Macrovesicular hepatic steatosis has a lower tolerance to reperfusion injury than microvesicular steatosis with an abnormally high ratio of omega-6 (n-6): omega-3 (n-3) polyunsaturated fatty acids (PUFAs). We investigated the influence of PUFAs on microcirculation in steatotic livers and the potential to minimize reperfusion injury in the macrosteatotic liver by normalization of PUFAs. Ob/ob mice were used as a model of macrovesicular hepatic steatosis and C57/Bl6 mice fed a choline-deficient diet for microvesicular steatosis. Steatotic and lean livers were subjected to 45 minutes of ischemia and 3 hours of reperfusion. Hepatic content of omega-3 and omega-6 PUFAs was determined. Microcirculation was investigated using intravital fluorescence microscopy. A second group of ob/ob mice was supplemented with dietary omega-3 PUFAs and compared with the control diet-fed group. Microcirculation, AST, and Kupffer cell activity were assessed. Macrosteatotic livers had significant microcirculatory dysfunction correlating with high omega-6: omega-3 PUFA ratio. Dietary omega-3 PUFA resulted in normalization of this ratio, reduction of intrahepatic lipids, and decrease in the extent of macrosteatosis. Defective microcirculation was dramatically ameliorated with significant reduction in Kupffer cell activity and protection against hepatocellular injury both before ischemia and after reperfusion. Conclusion: Macrosteatotic livers disclosed an abnormal omega-6: omega-3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury. Thus, protective strategies applied during or after ischemia are unlikely to be useful. Preoperative dietary omega-3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool. (

No abstract

This new model allows for repetitive analysis of the microcirculation during skin graft healing. It provides ideal in vivo conditions to further delineate the exact mechanisms of blood vessel interconnection during the complex process of angiogenesis, and may also allow study of the vascularization of tissue-engineered skin substitutes.