The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks

BACKGROUND: Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure. RESULTS: We developed the in vitro real-time oscillation monitoring system (IV-ROMS) by measuring the activity of luciferase coupled to the oscillatory gene promoter using photomultiplier tubes and applied this system to screen and identify factors able to influence circadian rhythmicity. Using this IV-ROMS as the primary screening of entrainment factors for circadian clocks, we identified 12 candidates as the potential entrainment factor in a total of 299 peptides and bioactive lipids. Among them, four candidates (endothelin-1, all-trans retinoic acid, 9-cis retinoic acid, and 13-cis retinoic acid) have already been reported as the entrainment factors in vivo and in vitro. We demonstrated that one of the novel candidates, 15-deoxy-Δ(12,14)-prostaglandin J(2 )(15d-PGJ(2)), a natural ligand of the peroxisome proliferator-activated receptor-γ (PPAR-γ), triggers the rhythmic expression of endogenous clock genes in NIH3T3 cells. Furthermore, we showed that 15d-PGJ(2 )transiently induces Cry1, Cry2, and Rorα mRNA expressions and that 15d-PGJ(2)-induced entrainment signaling pathway is PPAR-γ – and MAPKs (ERK, JNK, p38MAPK)-independent. CONCLUSION: Here, we identified 15d-PGJ(2 )as an entrainment factor in vitro. Using our developed IV-ROMS to screen 299 compounds, we found eight novel and four known molecules to be potential entrainment factors for circadian clocks, indicating that this assay system is a powerful and useful tool in initial screenings

Human Placental Extract Delays In Vitro Cellular Senescence through the Activation of NRF2-Mediated Antioxidant Pathway

Senescent cells accumulate in the organs of aged animals and exacerbate organ dysfunction, resulting in age-related diseases. Oxidative stress accelerates cellular senescence. Placental extract, used in the alleviation of menopausal symptoms and promotion of wound healing and liver regeneration, reportedly protects against oxidative stress. In this study, we investigated the effects of human placental extract (HPE) on cellular senescence in normal human dermal fibroblasts (NHDFs) under oxidative stress conditions. We demonstrated that HPE delays the onset of cellular senescence. Next-generation sequencing analysis revealed that under oxidative stress conditions, HPE treatment enhanced the expression of the antioxidant genes CYGB, APOE, NQO1, and PTGS1. Further, HPE treatment under oxidative stress conditions increased the protein level of nuclear factor-erythroid factor 2-related factor 2 (NRF2)—a vital molecule in the antioxidant pathway—via post-transcriptional and/or post-translational regulations. These findings indicate that HPE treatment in NHDFs, under chronic oxidative stress, delays cellular senescence by mitigating oxidative stress via upregulation of the NRF2-mediated antioxidant pathway, and HPE treatment could potentially ameliorate skin-aging-associated damage, in vivo

Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock

Cellular Senescence Triggers Altered Circadian Clocks With a Prolonged Period and Delayed Phases

Senescent cells, which show the permanent growth arrest in response to various forms of stress, accumulate in the body with the progression of age, and are associated with aging and age-associated diseases. Although the senescent cells are growth arrested, they still demonstrate high metabolic rate and altered gene expressions, indicating that senescent cells are still active. We recently showed that the circadian clock properties, namely phase and period of the cells, are altered with the establishment of replicative senescence. However, whether cellular senescence triggers the alteration of circadian clock properties in the cells is still unknown. In this study we show that the oxidative stress-induced premature senescence induces the alterations of the circadian clock, similar to the phenotypes of the replicative senescent cells. We found that the oxidative stress-induced premature senescent cells display the prolonged period and delayed phases. In addition, the magnitude of these changes intensified over time, indicating that cellular senescence changes the circadian clock properties. Our current results corroborate with our previous findings and further confirm that cellular senescence induces altered circadian clock properties, irrespective of the replicative senescence or the stress-induced premature senescence

Human Placental Extract Delays In Vitro Cellular Senescence through the Activation of NRF2-Mediated Antioxidant Pathway

Senescent cells accumulate in the organs of aged animals and exacerbate organ dysfunction, resulting in age-related diseases. Oxidative stress accelerates cellular senescence. Placental extract, used in the alleviation of menopausal symptoms and promotion of wound healing and liver regeneration, reportedly protects against oxidative stress. In this study, we investigated the effects of human placental extract (HPE) on cellular senescence in normal human dermal fibroblasts (NHDFs) under oxidative stress conditions. We demonstrated that HPE delays the onset of cellular senescence. Next-generation sequencing analysis revealed that under oxidative stress conditions, HPE treatment enhanced the expression of the antioxidant genes CYGB, APOE, NQO1, and PTGS1. Further, HPE treatment under oxidative stress conditions increased the protein level of nuclear factor-erythroid factor 2-related factor 2 (NRF2)—a vital molecule in the antioxidant pathway—via post-transcriptional and/or post-translational regulations. These findings indicate that HPE treatment in NHDFs, under chronic oxidative stress, delays cellular senescence by mitigating oxidative stress via upregulation of the NRF2-mediated antioxidant pathway, and HPE treatment could potentially ameliorate skin-aging-associated damage, in vivo

Getting traceability right, from fish to advanced bio-technological products: a review of legislation

Accepted manuscript version. Published version at <a href=http://doi.org/10.1016/j.jclepro.2015.05.003>http://doi.org/10.1016/j.jclepro.2015.05.003</a>. License in accordance with the journal's policy - <a href=http://creativecommons.org/licenses/by-nc-nd/4.0/>CC-BY-NC-ND</a>.Traceability is a tool used by regulators to manage risk in multiple supply chains, including supply chains of goods derived from genetically modified organisms, human blood, seafood products, toys and hazardous waste. This tool may help support a variety of claims that range from concern for nature to consumer satisfaction and health. This paper examines the consistency of European Union legislation with the declared objective of the law, i.e., to implement a traceability system through the entire supply chain. This analysis is undertaken by benchmarking 30 European Union laws that introduced traceability in the supply chain of 16 groups of products. The conclusion is that one-half of these norms lack basic effective principles of traceability. The approaches implemented were strongly correlated with the original driver for risk management (for example, concern for environmental sustainability), moderately correlated with the type of goods involved and uncorrelated with their trade value. The paper forecasts traceability approaches for new products, and indicates how traceability systems can become operative, regardless of product and driver. In addition, the importance that the legal provisions are consistent with the declared objective of the law is discussed. This integrated view is useful for regulators, industry and consumers in general and provides legislators and businesses with guidelines for consistent application of traceability, which facilitates other processes, such as life cycle analysis. Concurrently, it provides the public with an understanding of what lies behind the (often) inaccessible wording of legal norms