Circadian Clock Mechanism Research Articles

The Genome of Winter Moth (Operophtera brumata) Provides a Genomic Perspective on Sexual Dimorphism and Phenology.

The winter moth (Operophtera brumata) belongs to one of the most species-rich families in Lepidoptera, the Geometridae (approximately 23,000 species). This family is of great economic importance as most species are herbivorous and capable of defoliating trees. Genome assembly of the winter moth allows the study of genes and gene families, such as the cytochrome P450 gene family, which is known to be vital in plant secondary metabolite detoxification and host-plant selection. It also enables exploration of the genomic basis for female brachyptery (wing reduction), a feature of sexual dimorphism in winter moth, and for seasonal timing, a trait extensively studied in this species. Here we present a reference genome for the winter moth, the first geometrid and largest sequenced Lepidopteran genome to date (638 Mb) including a set of 16,912 predicted protein-coding genes. This allowed us to assess the dynamics of evolution on a genome-wide scale using the P450 gene family. We also identified an expanded gene family potentially linked to female brachyptery, and annotated the genes involved in the circadian clock mechanism as main candidates for involvement in seasonal timing. The genome will contribute to Lepidopteran genomic resources and comparative genomics. In addition, the genome enhances our ability to understand the genetic and molecular basis of insect seasonal timing and thereby provides a reference for future evolutionary and population studies on the winter moth.

Nondestructive and intuitive determination of circadian chlorophyll rhythms in soybean leaves using multispectral imaging.

The circadian clock, synchronized by daily cyclic environmental cues, regulates diverse aspects of plant growth and development and increases plant fitness. Even though much is known regarding the molecular mechanism of circadian clock, it remains challenging to quantify the temporal variation of major photosynthesis products as well as their metabolic output in higher plants in a real-time, nondestructive and intuitive manner. In order to reveal the spatial-temporal scenarios of photosynthesis and yield formation regulated by circadian clock, multispectral imaging technique has been employed for nondestructive determination of circadian chlorophyll rhythms in soybean leaves. By utilizing partial least square regression analysis, the determination coefficients R2, 0.9483 for chlorophyll a and 0.8906 for chlorophyll b, were reached, respectively. The predicted chlorophyll contents extracted from multispectral data showed an approximately 24-h rhythm which could be entrained by external light conditions, consistent with the chlorophyll contents measured by chemical analyses. Visualization of chlorophyll map in each pixel offers an effective way to analyse spatial-temporal distribution of chlorophyll. Our results revealed the potentiality of multispectral imaging as a feasible nondestructive universal assay for examining clock function and robustness, as well as monitoring chlorophyll a and b and other biochemical components in plants.

PER2 Differentially Regulates Clock Phosphorylation versus Transcription by Reciprocal Switching of CK1ε Activity

Casein kinase 1ε (CK1ε) performs key phosphorylation reactions in the circadian clock mechanism that determine period. We show that the central clock protein PERIOD2 (PER2) not only acts as a transcriptional repressor but also inhibits the autoinactivation of CK1ε, thereby promoting CK1ε activity. Moreover, PER2 reciprocally regulates CK1ε's ability to phosphorylate other substrates. On output pathway substrates (e.g., P53), PER2 inhibits the activity of CK1ε. However, in the case of central clock proteins (e.g., CRYPTOCHROME2), PER2 stimulates the CK1ε-mediated phosphorylation of CRY2. CK1ε activity is temperature compensated on the core clock substrate CRY2 but not on output substrates, for example, the physiological output protein substrate P53 and its nonphysiological correlate, bovine serum albumin (BSA). These results indicate heretofore unrecognized pivotal roles of PER2; it not only regulates the central transcription/translation feedback loop but also differentially controls kinase activity CK1ε in its phosphorylation of central clock (e.g., CRY2) versus output (e.g., P53) substrates.

A computational approach to persistence, permanence, and endotacticity of biochemical reaction systems.

We introduce a mixed-integer linear programming (MILP) framework capable of determining whether a chemical reaction network possesses the property of being endotactic or strongly endotactic. The network property of being strongly endotactic is known to lead to persistence and permanence of chemical species under genetic kinetic assumptions, while the same result is conjectured but as yet unproved for general endotactic networks. The algorithms we present are the first capable of verifying endotacticity of chemical reaction networks for systems with greater than two constituent species. We implement the algorithms in the open-source online package CoNtRol and apply them to a large sample of networks from the European Bioinformatics Institute's BioModels Database. We use strong endotacticity to establish for the first time the permanence of a well-studied circadian clock mechanism.

Waking up to the alarm: sleep, clocks, and making memory (s)tick.

Waking up to the alarm: sleep, clocks, and making memory (s)tick.

Therapeutic applications of circadian rhythms for the cardiovascular system.

The cardiovascular system exhibits dramatic time-of-day dependent rhythms, for example the diurnal variation of heart rate, blood pressure, and timing of onset of adverse cardiovascular events such as heart attack and sudden cardiac death. Over the past decade, the circadian clock mechanism has emerged as a crucial factor regulating these daily fluctuations. Most recently, these studies have led to a growing clinical appreciation that targeting circadian biology offers a novel therapeutic approach toward cardiovascular (and other) diseases. Here we describe leading-edge therapeutic applications of circadian biology including (1) timing of therapy to maximize efficacy in treating heart disease (chronotherapy); (2) novel biomarkers discovered by testing for genomic, proteomic, metabolomic, or other factors at different times of day and night (chronobiomarkers); and (3) novel pharmacologic compounds that target the circadian mechanism with potential clinical applications (new chronobiology drugs). Cardiovascular disease remains a leading cause of death worldwide and new approaches in the management and treatment of heart disease are clearly warranted and can benefit patients clinically.

Development of circadian oscillators in neurosphere cultures during adult neurogenesis.

Circadian rhythms are common in many cell types but are reported to be lacking in embryonic stem cells. Recent studies have described possible interactions between the molecular mechanism of circadian clocks and the signaling pathways that regulate stem cell differentiation. Circadian rhythms have not been examined well in neural stem cells and progenitor cells that produce new neurons and glial cells during adult neurogenesis. To evaluate circadian timing abilities of cells undergoing neural differentiation, neurospheres were prepared from the mouse subventricular zone (SVZ), a rich source of adult neural stem cells. Circadian rhythms in mPer1 gene expression were recorded in individual spheres, and cell types were characterized by confocal immunofluorescence microscopy at early and late developmental stages in vitro. Circadian rhythms were observed in neurospheres induced to differentiate into neurons or glia, and rhythms emerged within 3–4 days as differentiation proceeded, suggesting that the neural stem cell state suppresses the functioning of the circadian clock. Evidence was also provided that neural stem progenitor cells derived from the SVZ of adult mice are self-sufficient clock cells capable of producing a circadian rhythm without input from known circadian pacemakers of the organism. Expression of mPer1 occurred in high frequency oscillations before circadian rhythms were detected, which may represent a role for this circadian clock gene in the fast cycling of gene expression responsible for early cell differentiation.

The cardiomyocyte molecular clock regulates the circadian expression of Kcnh2 and contributes to ventricular repolarization.

Sudden cardiac death (SCD) follows a diurnal variation. Data suggest the timing of SCD is influenced by circadian (~24-hour) changes in neurohumoral and cardiomyocyte-specific regulation of the heart's electrical properties. The basic helix-loop-helix transcription factors brain muscle arnt-like1 (BMAL1) and circadian locomotor output control kaput (CLOCK) coordinate the circadian expression of select genes. We sought to test whether Bmal1 expression in cardiomyocytes contributes to K(+) channel expression and diurnal changes in ventricular repolarization. We used transgenic mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1(-/-)). We used quantitative polymerase chain reaction, voltage clamping, promoter-reporter bioluminescence assays, and electrocardiographic telemetry. Although several K(+) channel gene transcripts were downregulated in iCSΔBmal1(-/-)mouse hearts, only Kcnh2 exhibited a robust circadian pattern of expression that was disrupted in iCSΔBmal1(-/-) hearts. Kcnh2 underlies the rapidly activating delayed-rectifier K(+) current, and the rapidly activating delayed-rectifier K(+) current recorded from iCSΔBmal1(-/-) ventricular cardiomyocytes was ~50% smaller than control ventricular myocytes. Promoter-reporter assays demonstrated that the human Kcnh2 promoter is transactivated by the coexpression of BMAL1 and CLOCK. Electrocardiographic analysis showed that iCSΔBmal1(-/-) mice developed a prolongation in the heart rate-corrected QT interval during the light (resting) phase. This was secondary to an augmented circadian rhythm in the uncorrected QT interval without a corresponding change in the RR interval. The molecular clock in the heart regulates the circadian expression of Kcnh2, modifies K(+) channel gene expression, and is important for normal ventricular repolarization. Disruption of the cardiomyocyte circadian clock mechanism likely unmasks diurnal changes in ventricular repolarization that could contribute to an increased risk of cardiac arrhythmias/SCD.

Ube3a Imprinting Impairs Circadian Robustness in Angelman Syndrome Models

The paternal allele of Ube3a is silenced by imprinting in neurons, and Angelman syndrome (AS) is a disorder arising from a deletion or mutation of the maternal Ube3a allele, which thereby eliminates Ube3a neuronal expression. Sleep disorders such as short sleep duration and increased sleep onset latency are very common in AS. We found a unique link between neuronal imprinting of Ube3a and circadian rhythms in two mouse models of AS, including enfeebled circadian activity behavior and slowed molecular rhythms in exvivo brain tissues. As a consequence of compromised circadian behavior, metabolic homeostasis is also disrupted in AS mice. Unsilencing the paternal Ube3a allele restores functional circadian periodicity in neurons deficient in maternal Ube3a but does not affect periodicity inperipheral tissues that are not imprinted for uniparental Ube3a expression. The ubiquitin ligase encoded by Ube3a interacts with the central clock components BMAL1 and BMAL2. Moreover, inactivation of Ube3a expression elevates BMAL1 levels in brain regions that control circadian behavior of AS-model mice, indicating an important role for Ube3a in modulating BMAL1 turnover. Ube3a expression constitutes a direct mechanistic connection between symptoms of a human neurological disorder and the central circadian clock mechanism. The lengthened circadian period leads to delayed phase, which could explain the short sleep duration and increased sleep onset latency of AS subjects. Moreover, we report the pharmacological rescue of an AS phenotype, in this case, altered circadian period. These findings reveal potential treatments for sleep disorders in AS patients.

The role of casein kinase I in the Drosophila circadian clock.

The circadian clock mechanism in organisms as diverse as cyanobacteria and humans involves both transcriptional and posttranslational regulation of key clock components. One of the roles for the posttranslational regulation is to time the degradation of the targeted clock proteins, so that their oscillation profiles are out of phase with respect to those of the mRNAs from which they are translated. In Drosophila, the circadian transcriptional regulator PERIOD (PER) is targeted for degradation by a kinase (DOUBLETIME or DBT) orthologous to mammalian kinases (CKIɛ and CKIδ) that also target mammalian PER. Since these kinases are not regulated by second messengers, the mechanism (if any) for their regulation is not known. We are investigating the possibility that regulation of DBT is conferred by other proteins that associate with DBT and PER. In this chapter, the methods we are employing to identify and analyze these factors are discussed. These methods include expression of wild type and mutant proteins with the GAL4/UAS binary expression approach, analysis of DBT in Drosophila S2 cells, in vitro kinase assays with DBT isolated from S2 cells, and proteomic analysis of DBT-containing complexes and of DBT phosphorylation with mass spectrometry. The work has led to the discovery of a previously unrecognized circadian rhythm component (Bride of DBT, a noncanonical FK506-binding protein) and the mapping of autophosphorylation sites within the DBT C-terminal domain with potential regulatory roles.

Identification of small-molecule modulators of the circadian clock.

Chemical biology or chemical genetics has emerged as an interdisciplinary research area applying chemistry to understand biological systems. The development of combinatorial chemistry and high-throughput screening technologies has enabled large-scale investigation of the biological activities of diverse small molecules to discover useful chemical probes. This approach is applicable to the analysis of the circadian clock mechanisms through cell-based assays to monitor circadian rhythms using luciferase reporter genes. We and others have established cell-based high-throughput circadian assays and have identified a variety of novel small-molecule modulators of the circadian clock by phenotype-based screening of hundreds of thousands of compounds. The results demonstrated the effectiveness of chemical biology approaches in clock research field. This technique will become more and more common with propagation of high-throughput screening facilities. This chapter describes assay development, screening setups, and their optimization for successful screening campaigns.

Melatonin feedback on clock genes: a theory involving the proteasome.

The expression of 'clock' genes occurs in all tissues, but especially in the suprachiasmatic nuclei (SCN) of the hypothalamus, groups of neurons in the brain that regulate circadian rhythms. Melatonin is secreted by the pineal gland in a circadian manner as influenced by the SCN. There is also considerable evidence that melatonin, in turn, acts on the SCN directly influencing the circadian 'clock' mechanisms. The most direct route by which melatonin could reach the SCN would be via the cerebrospinal fluid of the third ventricle. Melatonin could also reach the pars tuberalis (PT) of the pituitary, another melatonin-sensitive tissue, via this route. The major 'clock' genes include the period genes, Per1 and Per2, the cryptochrome genes, Cry1 and Cry2, the clock (circadian locomotor output cycles kaput) gene, and the Bmal1 (aryl hydrocarbon receptor nuclear translocator-like) gene. Clock and Bmal1 heterodimers act on E-box components of the promoters of the Per and Cry genes to stimulate transcription. A negative feedback loop between the cryptochrome proteins and the nucleus allows the Cry and Per proteins to regulate their own transcription. A cycle of ubiquitination and deubiquitination controls the levels of CRY protein degraded by the proteasome and, hence, the amount of protein available for feedback. Thus, it provides a post-translational component to the circadian clock mechanism. BMAL1 also stimulates transcription of REV-ERBα and, in turn, is also partially regulated by negative feedback by REV-ERBα. In the 'black widow' model of transcription, proteasomes destroy transcription factors that are needed only for a particular period of time. In the model proposed herein, the interaction of melatonin and the proteasome is required to adjust the SCN clock to changes in the environmental photoperiod. In particular, we predict that melatonin inhibition of the proteasome interferes with negative feedback loops (CRY/PER and REV-ERBα) on Bmal1 transcription genes in both the SCN and PT. Melatonin inhibition of the proteasome would also tend to stabilize BMAL1 protein itself in the SCN, particularly at night when melatonin is naturally elevated. Melatonin inhibition of the proteasome could account for the effects of melatonin on circadian rhythms associated with molecular timing genes. The interaction of melatonin with the proteasome in the hypothalamus also provides a model for explaining the dramatic 'time of day' effect of melatonin injections on reproductive status of seasonal breeders. Finally, the model predicts that a proteasome inhibitor such as bortezomib would modify circadian rhythms in a manner similar to melatonin.

Circadian rhythms, Wnt/beta-catenin pathway and PPAR alpha/gamma profiles in diseases with primary or secondary cardiac dysfunction.

Circadian clock mechanisms are far-from-equilibrium dissipative structures. Peroxisome proliferator-activated receptors (PPAR alpha, beta/delta, and gamma) play a key role in metabolic regulatory processes, particularly in heart muscle. Links between circadian rhythms (CRs) and PPARs have been established. Mammalian CRs involve at least two critical transcription factors, CLOCK and BMAL1 (Gekakis et al., 1998; Hogenesch et al., 1998). PPAR gamma plays a major role in both glucose and lipid metabolisms and presents circadian properties which coordinate the interplay between metabolism and CRs. PPAR gamma is a major component of the vascular clock. Vascular PPAR gamma is a peripheral regulator of cardiovascular rhythms controlling circadian variations in blood pressure and heart rate through BMAL1. We focused our review on diseases with abnormalities of CRs and with primary or secondary cardiac dysfunction. Moreover, these diseases presented changes in the Wnt/beta-catenin pathway and PPARs, according to two opposed profiles. Profile 1 was defined as follows: inactivation of the Wnt/beta-catenin pathway with increased expression of PPAR gamma. Profile 2 was defined as follows: activation of the Wnt/beta-catenin pathway with decreased expression of PPAR gamma. A typical profile 1 disease is arrhythmogenic right ventricular cardiomyopathy, a genetic cardiac disease which presents mutations of the desmosomal proteins and is mainly characterized by fatty acid accumulation in adult cardiomyocytes mainly in the right ventricle. The link between PPAR gamma dysfunction and desmosomal genetic mutations occurs via inactivation of the Wnt/beta-catenin pathway presenting oscillatory properties. A typical profile 2 disease is type 2 diabetes, with activation of the Wnt/beta-catenin pathway and decreased expression of PPAR gamma. CRs abnormalities are present in numerous pathologies such as cardiovascular diseases, sympathetic/parasympathetic dysfunction, hypertension, diabetes, neurodegenerative diseases, cancer which are often closely inter-related.

A homeostatic sleep-stabilizing pathway in Drosophila composed of the sex peptide receptor and its ligand, the myoinhibitory peptide.

Author SummarySleep is a common trait in animals, from insects to mammals, and it needs to be coordinated with other critical activities such as feeding and reproduction. However, the mechanisms by which this is achieved are not fully understood. The fruit fly Drosophila melanogaster has become a key model organism for sleep research and it has been shown that reproduction is one of the factors that can modulate sleep in these animals. Researchers have observed that mating reduces the daytime sleep of female flies and shown that the seminal fluid protein Sex Peptide (SP), a ligand of the Sex Peptide Receptor (SPR) that is transferred to females during copulation, is responsible for this reduction of siesta sleep. Here, we investigated further the role of SPR in sleep regulation in Drosophila. We show that SPR is required for sleep stabilization in both sexes and that in mutant flies lacking SPR or its ligand myoinhibitory peptide (MIP) sleep is fragmented independently of reproduction. Unlike SP, MIP is expressed in the brain of both sexes and acts on SPR to silence specific neurons that keep flies awake, stabilizing sleep. Hence, our results reveal that SPR interacts with two distinct ligands to control different behaviors: SP for reproduction and MIP for sleep.

Translational regulation of the Drosophila post-translational circadian mechanism.

Translational regulation of the Drosophila post-translational circadian mechanism.

Circadian regulation of myocardial sarcomeric Titin-cap (Tcap, telethonin): identification of cardiac clock-controlled genes using open access bioinformatics data.

Circadian rhythms are important for healthy cardiovascular physiology and are regulated at the molecular level by a circadian clock mechanism. We and others previously demonstrated that 9–13% of the cardiac transcriptome is rhythmic over 24 h daily cycles; the heart is genetically a different organ day versus night. However, which rhythmic mRNAs are regulated by the circadian mechanism is not known. Here, we used open access bioinformatics databases to identify 94 transcripts with expression profiles characteristic of CLOCK and BMAL1 targeted genes, using the CircaDB website and JTK_Cycle. Moreover, 22 were highly expressed in the heart as determined by the BioGPS website. Furthermore, 5 heart-enriched genes had human/mouse conserved CLOCK:BMAL1 promoter binding sites (E-boxes), as determined by UCSC table browser, circadian mammalian promoter/enhancer database PEDB, and the European Bioinformatics Institute alignment tool (EMBOSS). Lastly, we validated findings by demonstrating that Titin cap (Tcap, telethonin) was targeted by transcriptional activators CLOCK and BMAL1 by showing 1) Tcap mRNA and TCAP protein had a diurnal rhythm in murine heart; 2) cardiac Tcap mRNA was rhythmic in animals kept in constant darkness; 3) Tcap and control Per2 mRNA expression and cyclic amplitude were blunted in ClockΔ19/Δ19 hearts; 4) BMAL1 bound to the Tcap promoter by ChIP assay; 5) BMAL1 bound to Tcap promoter E-boxes by biotinylated oligonucleotide assay; and 6) CLOCK and BMAL1 induced tcap expression by luciferase reporter assay. Thus this study identifies circadian regulated genes in silico, with validation of Tcap, a critical regulator of cardiac Z-disc sarcomeric structure and function.

The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells.

Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanism that is responsible for generating the negative feedback loop that sustains the clock. Its relevance to human disease is underlined by alterations in its function that affect numerous biochemical and physiological processes. When absent, it results in the development of various cancers and an increase in the cell's susceptibility to genotoxic stress. Thus we sought to define a yet-uncharacterized checkpoint node in which circadian components integrate environmental stress signals to the DNA-damage response. We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53's negative regulator, Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from being ubiquitinated and targeting hp53 by the proteasome. Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes. Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

The day/night proteome in the murine heart.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

CREB signalling in bipolar disease (commentary on Gaspar et al.): commentary on Gaspar et al. 2014.

Approximately one in 30 individuals across the world suffers from bipolar disorder (BD). With this prevalence, the pathophysiology of BD represents one of the most important unsolved challenges in neuroscience. Although there appears to be a strong hereditary component to the disorder, genome-wide association studies have failed to identify any obvious underlying developmental or signalling pathways. In this issue, Gaspar et al. (2014) report two significant developments in this area. The first is the report of alterations in the cAMP–CREB signalling pathway in BD, and the second is the possibility of a biomarker and assay specific for the condition. The authors derived fibroblasts from skin biopsy specimens, and used viral luciferase-based reporter systems to investigate the extent to which eight major signalling pathways were activated by specific pharmacological agents. Surprisingly, they found large inter-individual differences in signal pathway transduction in response to the same drug, and these were mirrored by smaller, harder to detect, differences at the transcriptional level. Furthermore, they were able to correlate the degree to which melatonin suppression in response to light occurs in each individual with the strength of the cAMP–CREB signal induced by forskolin in their fibroblasts. They extended these studies to patients with BD, and found a three-fold increase in the cAMP–CREB signalling response in BD patients as compared with age-matched matched controls. This is a particularly interesting finding, given the emerging links between cAMP–CREB signalling and circadian rhythms and their disruption in BD. Several groups have reported disruption of normal sleep and circadian rhythms in BD, and recently, mechanistic links between the two have emerged. For example, lithium, which is the mainstay pharmacotherapy, has well-documented effects on the circadian clock, and at least two clock mutant mice show a mania-like phenotype. cAMP–CREB signalling plays a fundamental role in regulating the circadian clock, both at the level of clock gene rhythmicity, and by setting the phase of the clock in response to environmental time signals such as light. An upregulated CREB-mediated input pathway in bipolar patients may provide one route by which these disruptions of the circadian clock and other CREB-regulated mechanisms, such as synaptic transmission, are produced. Perhaps most exciting is that this study, alongside other recent reports, suggests that CREB-mediated pathways may provide novel pharmacological targets for the treatment of BD. In addition to a lack of validated targets, drug discovery for BD has been slow, because of the scarcity of both biomarkers and relevant research models that can be used for high-throughput analysis. The authors provide one such assay, the strength of cAMP–CREB signalling in patient-derived cells, which may serve as a valuable tool with which to fight this widespread and debilitating condition. This study raises many questions and avenues for future work. Do these changes in cAMP–CREB signalling underlie altered neuronal signalling in BD? Do they underlie circadian disruption? Is cAMP–CREB signalling altered in high-risk populations, and can this be used as a predictive tool for the risk of developing disease? Further studies on the fibroblasts from bipolar patients should yield valuable information for addressing such questions.

Characterisation of circadian rhythms of various duckweeds.

The plant circadian clock controls various physiological phenomena that are important for adaptation to natural day-night cycles. Many components of the circadian clock have been identified in Arabidopsis thaliana, the model plant for molecular genetic studies. Recent studies revealed evolutionary conservation of clock components in green plants. Homologues of clock-related genes have been isolated from Lemna gibba and Lemna aequinoctialis, and it has been demonstrated that these homologues function in the clock system in a manner similar to their functioning in Arabidopsis. While clock components are widely conserved, circadian phenomena display diversity even within the Lemna genus. In order to survey the full extent of diversity in circadian rhythms among duckweed plants, we characterised the circadian rhythms of duckweed by employing a semi-transient bioluminescent reporter system. Using a particle bombardment method, circadian bioluminescent reporters were introduced into nine strains representing five duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna gibba, L. aequinoctialis and Wolffia columbiana. We then monitored luciferase (luc+) reporter activities driven by AtCCA1, ZmUBQ1 or CaMV35S promoters under entrainment and free-running conditions. Under entrainment, AtCCA1::luc+ showed similar diurnal rhythms in all strains. This suggests that the mechanism of biological timing under day-night cycles is conserved throughout the evolution of duckweeds. Under free-running conditions, we observed circadian rhythms of AtCCA1::luc+, ZmUBQ1::luc+ and CaMV35S::luc+. These circadian rhythms showed diversity in period length and sustainability, suggesting that circadian clock mechanisms are somewhat diversified among duckweeds.