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Abstract Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light and feeding). The presence of tissue niche-dependent physiological time cues has been proposed, allowing tissues the ability of circadian phase adjustment based on local signals. However, to date, such stimuli have remained elusive. Here we show that daily patterns of mechanical loading and associated osmotic challenge within physiological ranges reset circadian clock phase and amplitude in cartilage and intervertebral disc tissues in vivo and in tissue explant cultures. Hyperosmolarity (but not hypo-osmolarity) resets clocks in young and ageing skeletal tissues and induce genome-wide expression of rhythmic genes in cells. Mechanistically, RNAseq and biochemical analysis revealed the PLD2-mTORC2-AKT-GSK3β axis as a convergent pathway for both in vivo loading and hyperosmolarity-induced clock changes. These results reveal diurnal patterns of mechanical loading and consequent daily oscillations in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync.

Abstract The circadian clock participates in maintaining homeostasis in peripheral tissues, including intervertebral discs (IVDs). Abnormal mechanical loading is a known risk factor for intervertebral disc degeneration (IDD). Based on the rhythmic daily loading pattern of rest and activity, we hypothesized that abnormal mechanical loading could dampen the IVD clock, contributing to IDD. Here, we investigated the effects of abnormal loading on the IVD clock and aimed to inhibit compression-induced IDD by targeting the core clock molecule brain and muscle Arnt-like protein-1 (BMAL1). In this study, we showed that BMAL1 KO mice exhibit radiographic features similar to those of human IDD and that BMAL1 expression was negatively correlated with IDD severity by systematic analysis based on 149 human IVD samples. The intrinsic circadian clock in the IVD was dampened by excessive loading, and BMAL1 overexpression by lentivirus attenuated compression-induced IDD. Inhibition of the RhoA/ROCK pathway by Y-27632 or melatonin attenuated the compression-induced decrease in BMAL1 expression. Finally, the two drugs partially restored BMAL1 expression and alleviated IDD in a diurnal compression model. Our results first show that excessive loading dampens the circadian clock of nucleus pulposus tissues via the RhoA/ROCK pathway, the inhibition of which potentially protects against compression-induced IDD by preserving BMAL1 expression. These findings underline the importance of the circadian clock for IVD homeostasis and provide a potentially effective therapeutic strategy for IDD.

Developmentally, the articular joints are derived from lateral plate (LP) mesoderm. However, no study has produced both LP derived prechondrocytes and preosteoblasts from human pluripotent stem cells (hPSC) through a common progenitor in a chemically defined manner. Differentiation of hPSCs through the authentic route, via an LP-osteochondral progenitor (OCP), may aid understanding of human cartilage development and the generation of effective cell therapies for osteoarthritis. We refined our existing chondrogenic protocol, incorporating knowledge from development and other studies to produce a LP-OCP from which prechondrocyte- and preosteoblast-like cells can be generated. Results show the formation of an OCP, which can be further driven to prechondrocytes and preosteoblasts. Prechondrocytes cultured in pellets produced cartilage like matrix with lacunae and superficial flattened cells expressing lubricin. Additionally, preosteoblasts were able to generate a mineralised structure. This protocol can therefore be used to investigate further cartilage development and in the development of joint cartilage for potential treatments.

Circadian rhythm governs many aspects of liver physiology and its disruption exacerbates chronic disease. CLOCKΔ19 mice disrupted circadian rhythm and spontaneously developed obesity and metabolic syndrome, a phenotype that parallels the progression of non-alcoholic fatty liver disease (NAFLD). NAFLD represents an increasing health burden with an estimated incidence of around 25% and is associated with an increased risk of progression towards inflammation, fibrosis and carcinomas. Excessive extracellular matrix deposition (fibrosis) is the key driver of chronic disease progression. However, little attention was paid to the impact of disrupted circadian rhythm in hepatic stellate cells (HSCs) which are the primary mediator of fibrotic ECM deposition. Here, we showed in vitro and in vivo that liver fibrosis is significantly increased when circadian rhythm is disrupted by CLOCK mutation. Quiescent HSCs from CLOCKΔ19 mice showed higher expression of RhoGDI pathway components and accelerated activation. Genes altered in this primed CLOCKΔ19 qHSC state may provide biomarkers for early liver disease detection, and include AOC3, which correlated with disease severity in patient serum samples. Integration of CLOCKΔ19 microarray data with ATAC-seq data from WT qHSCs suggested a potential CLOCK regulome promoting a quiescent state and downregulating genes involved in cell projection assembly. CLOCKΔ19 mice showed higher baseline COL1 deposition and significantly worse fibrotic injury after CCl4 treatment. Our data demonstrate that disruption to circadian rhythm primes HSCs towards an accelerated fibrotic response which worsens liver disease.

The mammalian circadian clock exerts control of daily gene expression through cycles of DNA binding. Here, we develop a quantitative model of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We used quantitative imaging to track dynamic changes in endogenous labelled proteins across peripheral tissues and the SCN. We determine the contribution of multiple rhythmic processes coordinating BMAL1 DNA binding, including cycling molecular abundance, binding affinities, and repression. We find nuclear BMAL1 concentration determines corresponding CLOCK through heterodimerisation and define a DNA residence time of this complex. Repression of CLOCK:BMAL1 is achieved through rhythmic changes to BMAL1:CRY1 association and high-affinity interactions between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.

Abstract Background Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. Methods We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. Results Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. Conclusion Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.

Circadian clocks regulate physiological and behavioural rhythms. Here, the authors show that the stiffness of the extracellular environment regulates circadian clocks in murine breast epithelium via Rho signalling, and explain how extracellular matrix stiffening in aging affects circadian rhythm, with implication in disease.

Lithium salt has been widely used in treatment of Bipolar Disorder, a mental disturbance associated with circadian rhythm disruptions. Lithium mildly but consistently lengthens circadian period of behavioural rhythms in multiple organisms. To systematically address the impacts of lithium on circadian pacemaking and the underlying mechanisms, we measured locomotor activity in mice in vivo following chronic lithium treatment, and also tracked clock protein dynamics (PER2::Luciferase) in vitro in lithium-treated tissue slices/cells. Lithium lengthens period of both the locomotor activity rhythms, as well as the molecular oscillations in the suprachiasmatic nucleus, lung tissues and fibroblast cells. In addition, we also identified significantly elevated PER2::LUC expression and oscillation amplitude in both central and peripheral pacemakers. Elevation of PER2::LUC by lithium was not associated with changes in protein stabilities of PER2, but instead with increased transcription of Per2 gene. Although lithium and GSK3 inhibition showed opposing effects on clock period, they acted in a similar fashion to up-regulate PER2 expression and oscillation amplitude. Collectively, our data have identified a novel amplitude-enhancing effect of lithium on the PER2 protein rhythms in the central and peripheral circadian clockwork, which may involve a GSK3-mediated signalling pathway. These findings may advance our understanding of the therapeutic actions of lithium in Bipolar Disorder or other psychiatric diseases that involve circadian rhythm disruptions.

GPR50 is an orphan G-protein coupled receptor most closely related to the melatonin receptors. The physiological function of GPR50 remains unclear, although our previous studies implicate the receptor in energy homeostasis. Here, we reveal a role for GPR50 as a signalling partner and modulator of the transcriptional co-activator TIP60. This interaction was identified in a yeast-two-hybrid screen, and confirmed by co-immunoprecipitation and co-localisation of TIP60 and GPR50 in HEK293 cells. Co-expression with TIP60 increased perinuclear localisation of full length GPR50, and resulted in nuclear translocation of the cytoplasmic tail of the receptor, suggesting a functional interaction of the two proteins. We further demonstrate that GPR50 can enhance TIP60-coactiavtion of glucocorticoid receptor (GR) signalling. In line with in vitro results, repression of pituitary Pomc expression, and induction of gluconeogenic genes in liver in response to the GR agonist, dexamethasone was attenuated in Gpr50(-/-) mice. These results identify a novel role for GPR50 in glucocorticoid receptor signalling through interaction with TIP60.

Studies in shift workers and model organisms link circadian disruption to breast cancer. However, molecular circadian rhythms in noncancerous and cancerous human breast tissues and their clinical relevance are largely unknown. We reconstructed rhythms informatically, integrating locally collected, time-stamped biopsies with public datasets. For noncancerous breast tissue, inflammatory, epithelial-mesenchymal transition (EMT), and estrogen responsiveness pathways show circadian modulation. Among tumors, clock correlation analysis demonstrates subtype-specific changes in circadian organization. Luminal A organoids and informatic ordering of luminal A samples exhibit continued, albeit dampened and reprogrammed rhythms. However, CYCLOPS magnitude, a measure of global rhythm strength, varied widely among luminal A samples. Cycling of EMT pathway genes was markedly increased in high-magnitude luminal A tumors. Surprisingly, patients with high-magnitude tumors had reduced 5-y survival. Correspondingly, 3D luminal A cultures show reduced invasion following molecular clock disruption. This study links subtype-specific circadian disruption in breast cancer to EMT, metastatic potential, and prognosis. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) is the noncellular scaffolding component present within all tissues and organs. It provides crucial biochemical and biomechanical cues to instruct cellular behavior and has been shown to be under circadian clock regulation, a highly conserved cell-intrinsic timekeeping mechanism that has evolved with the 24-hour rhythmic environment. Aging is a major risk factor for many diseases, including cancer, fibrosis, and neurodegenerative disorders. Both aging and our modern 24/7 society disrupt circadian rhythms, which could contribute to altered ECM homeostasis. Understanding the daily dynamics of ECM and how this mechanism changes with age will have a profound impact on tissue health, disease prevention, and improving treatments. Maintaining rhythmic oscillations has been proposed as a hallmark of health. On the other hand, many hallmarks of aging turn out to be key regulators of circadian timekeeping mechanisms. In this review, we summarize new work linking the ECM with circadian clocks and tissue aging. We discuss how the changes in the biomechanical and biochemical properties of ECM during aging may contribute to circadian clock dysregulation. We also consider how the dampening of clocks with age could compromise the daily dynamic regulation of ECM homeostasis in matrix-rich tissues. This review aims to encourage new concepts and testable hypotheses about the two-way interactions between circadian clocks and ECM in the context of aging. [ABSTRACT FROM AUTHOR]

Circadian clocks in the brain and peripheral tissues temporally coordinate local physiology to align with the 24 hours rhythmic environment through light/darkness, rest/activity and feeding/fasting cycles. Circadian disruptions (during ageing, shift work and jet-lag) have been proposed as a risk factor for degeneration and disease of tissues, including the musculoskeletal system. The intervertebral disc (IVD) in the spine separates the bony vertebrae and permits movement of the spinal column. IVD degeneration is highly prevalent among the ageing population and is a leading cause of lower back pain. The IVD is known to experience diurnal changes in loading patterns driven by the circadian rhythm in rest/activity cycles. In recent years, emerging evidence indicates the existence of molecular circadian clocks within the IVD, disruption to which accelerates tissue ageing and predispose animals to IVD degeneration. The cell-intrinsic circadian clocks in the IVD control key aspects of physiology and pathophysiology by rhythmically regulating the expression of ~3.5% of the IVD transcriptome, allowing cells to cope with the drastic biomechanical and chemical changes that occur throughout the day. Indeed, epidemiological studies on long-term shift workers have shown an increased incidence of lower back pain. In this review, we summarise recent findings of circadian rhythms in health and disease, with the IVD as an exemplar tissue system. We focus on rhythmic IVD functions and discuss implications of utilising biological timing mechanisms to improve tissue health and mitigate degeneration. These findings may have broader implications in chronic rheumatic conditions, given the recent findings of musculoskeletal circadian clocks. [ABSTRACT FROM AUTHOR]

The circadian system in mammals is responsible for the temporal coordination of multiple physiological and behavioural processes that are necessary for homeostasis. In the skeleton, it has long been known that metabolic functions of chondrocytes, osteoblasts and osteoclasts exhibit intrinsic circadian rhythms. In addition, results from animal models reveal a close connection between the disruption of circadian rhythms and skeletal disorders such as rheumatoid arthritis, osteoarthritis and osteoporosis. In this review, we summarise the latest insights into the genetic and biochemical mechanisms linking cartilage and bone physiology to the circadian clock system. We also discuss how this knowledge can be utilised to improve human health. [ABSTRACT FROM AUTHOR]

Cell-autonomous circadian clocks coordinate tissue homeostasis with a 24-hourly rhythm. The molecular circadian clock machinery controls tissue- and cell type-specific sets of rhythmic genes. Disruptions of clock mechanisms are linked to an increased risk of acquiring diseases, especially those associated with aging, metabolic dysfunction and cancer. Despite rapid advances in understanding the cyclic outputs of different tissue clocks, less is known about how the clocks adapt to their local niche within tissues. We have discovered that tissue stiffness regulates circadian clocks, and that this occurs in a cell-type-dependent manner. In this Review, we summarise new work linking the extracellular matrix with differential control of circadian clocks. We discuss how the changes in tissue structure and cellular microenvironment that occur throughout life may impact on the molecular control of circadian cycles. We also consider how altered clocks may have downstream impacts on the acquisition of diseases. [ABSTRACT FROM AUTHOR]

Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of Clock subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10-12 hearts (63, no core circadian genes) than in E17-19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of Clock mRNA, which we showed was due to Dicer/Dgcr8-dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of Clock plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development. [ABSTRACT FROM AUTHOR]

Objectives: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration.Methods: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1.Results: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs.Conclusions: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a prevalent and debilitating joint disease for which ageing, obesity and chronic inflammation are known risk factors. The central, peripheral and autonomic nervous systems are essential in all metabolic systems, and emerging evidence suggests a role for these systems in OA. In the past few years, metabolic diseases, such as obesity or diabetes, have been linked to disruption of circadian rhythms that are tightly regulated by the nervous system, whereas inflammatory and autoimmune diseases are known to be linked to disruption of the cholinergic vagus nerve reflex. Interestingly, metabolism, inflammation and circadian rhythms have all been linked to the development and progression of OA. This article reviews current knowledge of the direct and indirect roles of the nervous system and circadian system in the initiation and/or progression of OA, and highlights the directions for future research in this emerging field. [ABSTRACT FROM AUTHOR]

The night and day cycle governs the circadian (24 hourly) rhythm of activity and rest in animals and humans. This is reflected in daily changes of the global gene expression pattern and metabolism, but also in the local physiology of various tissues. A central clock in the brain co-ordinates the rhythmic locomotion behaviour, as well as synchronizing various local oscillators, such as those found in the musculoskeletal system. It has become increasingly recognized that the internal molecular clocks in cells allow a tissue to anticipate the rhythmic changes in their local environment and the specific demands of that tissue. Consequently, the majority of the rhythmic clock controlled genes and pathways are tissue specific. The concept of the tissuespecific function of circadian clocks is further supported by the diverse musculoskeletal phenotypes in mice with deletions or mutations of various core clock components, ranging from increased bone mass, dwarfism, arthropathy, reduced muscle strength and tendon calcification. The present review summarizes the current understanding of the circadian clocks in muscle, bone, cartilage and tendon tissues, with particular focus on the evidence of circadian rhythms in tissue physiology, their entrainment mechanisms and disease links, and the tissue-specific clock target genes/pathways. Research in this area holds strong potential to advance our understanding of how circadian rhythms control the health and disease of the musculoskeletal tissues,which hasmajor implications in diseases associated with advancing age. It could also have potential implications in sports performance and sports medicine. [ABSTRACT FROM AUTHOR]

Tendons are prominent members of the family of fibrous connective tissues (FCTs), which collectively are the most abundant tissues in vertebrates and have crucial roles in transmitting mechanical force and linking organs. Tendon diseases are among the most common arthropathy disorders; thus knowledge of tendon gene regulation is essential for a complete understanding of FCT biology. Here we show autonomous circadian rhythms in mouse tendon and primary human tenocytes, controlled by an intrinsic molecular circadian clock. Time-series microarrays identified the first circadian transcriptome of murine tendon, revealing that 4.6% of the transcripts (745 genes) are expressed in a circadian manner. One of these genes was Grem2, which oscillated in antiphase to BMP signaling. Moreover, recombinant human Gremlin-2 blocked BMP2-induced phosphorylation of Smad1/5 and osteogenic differentiation of human tenocytes in vitro.We observed dampened Grem2 expression, deregulated BMP signaling, and spontaneously calcifying tendons in young CLOCKΔ19 arrhythmic mice and aged wild-type mice. Thus, disruption of circadian control, through mutations or aging, of Grem2/BMP signaling becomes a new focus for the study of calcific tendinopathy, which affects 1-in-5 people over the age of 50 years. [ABSTRACT FROM AUTHOR]

Circadian pacemaking in the suprachiasmatic nucleus (SCN) revolves around a transcriptional/posttranslational feedback loop in which period (Per) and cryptochrome (Cry) genes are negatively regulated by their protein products. Genetically specified differences in this oscillator underlie sleep and metabolic disorders, and dictate diurnal/nocturnal preference. A critical goal, therefore, is to identify mechanisms that generate circadian phenotypic diversity, through both single gene effects and gene interactions. The individual stabilities of PER or CRY proteins determine pacemaker period, and PER/CRY complexes have been proposed to afford mutual stabilization, although how PER and CRY proteins with contrasting stabilities interact is unknown. We therefore examined interactions between two mutations in male mice: Fbxl3Afh, which lengthens period by stabilizing CRY, and Csnk1ϵtm1Asil (CK1ϵTau), which destabilizes PER, thereby accelerating the clock. By intercrossing these mutants, we show that the stabilities of CRY and PER are independently regulated, contrary to the expectation of mutual stabilization. Segregation of wild-type and mutant alleles generated a spectrum of periods for rest-activity behavior and SCN bioluminescence rhythms. The mutations exerted independent, additive effects on circadian period, biased toward shorter periods determined by CK1ϵTau. Notably, Fbxl3Afh extended the duration of the nadir of the PER2-driven bioluminescence rhythm but CK1ϵTau reversed this, indicating that despite maintained CRY expression, CK1ϵTau truncated the interval of negative feedback. These results argue for independent, additive biochemical actions of PER and CRY in circadian control, and complement genome-wide epistatic analyses, seeking to decipher the multigenic control of circadian pacemaking. [ABSTRACT FROM AUTHOR]

Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) e orfi can alterthe circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1δ and CKTh using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to, reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1δ is the principal regulator of the clock period: pharmacological inhibition of CK1δ, but not CK1δ. significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1ö inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CKTh inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2-/- mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2:Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2-/- mutation or by constant light, daily treatment with PF670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1δ offers an avenue for therapeutic modulation of perturbed circadian behavior. [ABSTRACT FROM AUTHOR]

Abstract: Coal mining subsidence forms anthropogenic wetland. Nutrients in cropland around the wetland will be transferred into catchment along the slope of the coal mining wetland especially by the water washing in raining season. On the other hand, the washed nutrients from the cropland will be accumulated in the sediments of the catchment and eutrophicate the water body of catchment potentially. Nitrogen and phosphorus were selected as the biogeochemical indicators to address the environment effect of coal mining subsidence wetland in Panbei Coal Mine, Huainan, China. Nine water samples, nine sediment samples in the catchment, and twenty-seven topsoil samples around the catchment were collected. Contents of total nitrogen (TN), NO3-N, NH4-N, total phosphorus (TP) and available phosphorus of water, sediment and topsoil in coal mining subsidence wetland were measured. Distribution characteristics of nitrogen and phosphorus contents in water, sediments and topsoil in coal mining subsidence wetland indicated that their contents in water were relative lower, and highest contents in sediments were found, which reflected the nutrient absorption and accumulation in the sediment. In Panbei coal mining, the characteristics of distributions of nitrogen and phosphorus contents in topsoil of coal mining wetland generally decreased along the slope from flooded area to top of the basin. The phenomena indicated that the gradient formed by coal underground mining led to the nutrient losses in cropland and nutrient accumulaiton in sediments of catchment. [Copyright &y& Elsevier]

Circadian clocks respond to environmental time cues to coordinate 24-hour oscillations in almost every tissue of the body. In the breast, circadian clocks regulate the rhythmic expression of numerous genes. Disrupted expression of circadian genes can alter breast biology and may promote cancer. Here we overview circadian mechanisms, and the connection between the molecular clock and breast biology. We describe how disruption of circadian genes contributes to cancer via multiple mechanisms, and link this to increased tumour risk in women who work irregular shift patterns. Understanding the influence of circadian rhythms on breast cancer could lead to more efficacious therapies, reformed public health policy and improved patient outcome. [ABSTRACT FROM AUTHOR]

The nuclear receptor REV-ERBα is a key negative-feedback regulator of the biological clock. REV-ERBα binds to ROR elements of the Bmal1 (Arntl) promoter and represses Bmal1 transcription. This stabilizing negative loop is important for precise control of the circadian pacemaker. In the present study, we identified a novel synthetic REV-ERBα ligand, which enhances the recruitment of nuclear receptor co-repressor (NCoR) to REV-ERBα. In order to explore REV-ERBα action on resetting responses of the molecular clock, we first established the rhythmic transcription profile and expression level of REV-ERBα in Rat-1 fibroblasts. When applied at different phases of the circadian oscillation to cell models containing stably transfected Bmal1::Luc or Per2::Luc, the REV-ERBα ligand induced phase-dependent bi-directional phase shifts. When the phase changes were plotted against time, a clear phase response curve was revealed, with a significant peak-to-trough amplitude of ca. 5 hours. The phase-resetting effect was also observed when the compound was applied to primary lung fibroblasts and ectopic lung slices from transgenic PER2::Luc mice. Therefore, similar regulation of REV-ERBα function by endogenous ligands, such as heme, is likely to be an important mechanism for clock resetting. In addition, we identify a new means to generate phasic shifts in the clock. [ABSTRACT FROM AUTHOR]