A Systematic Review of Utilisation of Diurnal Timing Information in Clinical Trial Design for Long QT Syndrome.

Diurnal oscillations in human cardiac electrophysiology are thought to be under the control of the endogenous circadian clock. The incidence of arrhythmic events in patients with Long QT syndrome (LQTS) varies diurnally. The diurnal variation in QT interval has previously been identified as a potential for error in clinical trials which utilise ECG measurement. We performed a systematic review of clinical trials for LQTS to identify practice around specification of timing information for point electrocardiogram (ECG) measurements, analysis of continual ECG recordings ≥24 h, and drug delivery. Despite guidelines having been issued around the analysis of 24-h ECG recordings, we identify a lack of usage of detailed time of day information in trial design for LQTS studies, which has the potential to affect the interpretation of the results of drug trials. We identify that, in contrast, clinical trials for QT prolonging drugs demonstrate increased incorporation of time of day information of both QT analysis and drug dosing. We provide a visual portal to allow trial designers and clinicians to better understand timing of common cardiac-targeting drugs, and to bear this concept in mind in the design of future clinical trials.

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Recent advances in understanding regulation of the Arabidopsis circadian clock by local cellular environment.

Circadian clocks have evolved to synchronise an organism's physiology with the environmental rhythms driven by the Earth's rotation on its axis. Over the past two decades, many of the genetic components of the Arabidopsis thaliana circadian oscillator have been identified. The interactions between these components have been formulized into mathematical models that describe the transcriptional translational feedback loops of the oscillator. More recently, focus has turned to the regulation and functions of the circadian clock. These studies have shown that the system dynamically responds to environmental signals and small molecules. We describe advances that have been made in discovering the cellular mechanisms by which signals regulate the circadian oscillator of Arabidopsis in the context of tissue-specific regulation.

TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation.

The Arabidopsis genome contains three genes encoding proteins of the TRANSPARENT TESTA GLABRA 1 (TTG1) WD-repeat (WDR) subfamily. TTG1 is a known regulator of epidermal cell differentiation and pigment production, while LIGHT-REGULATED WD1 and LIGHT-REGULATED WD2 are known regulators of the circadian clock. Here, we discovered a new central role for TTG1 WDR proteins as regulators of the circadian system, as evidenced by the lack of detectable circadian rhythms in a triple lwd1 lwd2 ttg1 mutant. This shows that there has been subfunctionalization via protein changes within the angiosperms, with some TTG1 WDR proteins developing a stronger role in circadian clock regulation while losing the protein characteristics essential for pigment production and epidermal cell specification, and others weakening their ability to drive circadian clock regulation. Our work shows that even where proteins are very conserved, small changes can drive big functional differences.

Dynamical differential expression (DyDE) reveals the period control mechanisms of the Arabidopsis circadian oscillator.

The circadian oscillator, an internal time-keeping device found in most organisms, enables timely regulation of daily biological activities by maintaining synchrony with the external environment. The mechanistic basis underlying the adjustment of circadian rhythms to changing external conditions, however, has yet to be clearly elucidated. We explored the mechanism of action of nicotinamide in Arabidopsis thaliana, a metabolite that lengthens the period of circadian rhythms, to understand the regulation of circadian period. To identify the key mechanisms involved in the circadian response to nicotinamide, we developed a systematic and practical modeling framework based on the identification and comparison of gene regulatory dynamics. Our mathematical predictions, confirmed by experimentation, identified key transcriptional regulatory mechanisms of circadian period and uncovered the role of blue light in the response of the circadian oscillator to nicotinamide. We suggest that our methodology could be adapted to predict mechanisms of drug action in complex biological systems.

Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock.

In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).

Gene regulatory network models in response to sugars in the plant circadian system.

Circadian entrainment is the process by which internal circadian oscillators staying in synchronization with the local environmental rhythms. Circadian clocks are entrained by adjusting phase and period in response to environmental and metabolic signals. In Arabidopsis thaliana, light and sugar signals differentially affect the circadian phase; the former advances the phase in the late of the subjective night and delays around dusk, while the latter advances the phase mainly in the morning, which is optimal to maintain sucrose homeostasis. We have proposed that the phase adjustment of the A. thaliana circadian oscillator by sugar signals contributes to the realization of carbon homeostasis and the increase of plant growth under fluctuating day-night cycles. However, which genes in the circadian oscillator are targets of sucrose signals and how the potential target genes should be regulated by sucrose to realize sucrose homeostasis has not been studied from the theoretical perspective. Here we investigate the effect of sugar on the phase response property of the plant circadian oscillator using clock gene-regulatory network models. We simulated phase response curves (PRCs) to sucrose pulses, which were compared with an experimental PRC. Our analyses of the gene-regulatory network model demonstrated that target genes of the sugar signal could be members of the PSEUDO-RESPONSE REGULATOR gene family and the evening complex components. We also examined the phase response property using a single feedback-loop model and elucidated how phase advance is induced in the subjective morning under certain conditions of a target clock gene of sucrose and its regulatory property.

Circadian Entrainment in Arabidopsis by the Sugar-Responsive Transcription Factor bZIP63.

Synchronization of circadian clocks to the day-night cycle ensures the correct timing of biological events. This entrainment process is essential to ensure that the phase of the circadian oscillator is synchronized with daily events within the environment [1], to permit accurate anticipation of environmental changes [2, 3]. Entrainment in plants requires phase changes in the circadian oscillator, through unidentified pathways, which alter circadian oscillator gene expression in response to light, temperature, and sugars [4-6]. To determine how circadian clocks respond to metabolic rhythms, we investigated the mechanisms by which sugars adjust the circadian phase in Arabidopsis [5]. We focused upon metabolic regulation because interactions occur between circadian oscillators and metabolism in several experimental systems [5, 7-9], but the molecular mechanisms are unidentified. Here, we demonstrate that the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) regulates the circadian oscillator gene PSEUDO RESPONSE REGULATOR7 (PRR7) to change the circadian phase in response to sugars. We find that SnRK1, a sugar-sensing kinase that regulates bZIP63 activity and circadian period [10-14] is required for sucrose-induced changes in circadian phase. Furthermore, TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1), which synthesizes the signaling sugar trehalose-6-phosphate, is required for circadian phase adjustment in response to sucrose. We demonstrate that daily rhythms of energy availability can entrain the circadian oscillator through the function of bZIP63, TPS1, and the KIN10 subunit of the SnRK1 energy sensor. This identifies a molecular mechanism that adjusts the circadian phase in response to sugars.

BIG Regulates Dynamic Adjustment of Circadian Period in Arabidopsis thaliana.

Circadian clocks drive rhythms with a period near 24 h, but the molecular basis of the regulation of the period of the circadian clockis poorly understood. We previously demonstrated that metabolites affect the free-running period of the circadian oscillator of Arabidopsis (Arabidopsis thaliana), with endogenous sugars acting as an accelerator and exogenous nicotinamide acting as a brake. Changes in circadian oscillator period are thought to adjust the timing of biological activities through the process of entrainment, in which the circadian oscillator becomes synchronized to rhythmic signals such as light and dark cycles as well as changes in internal metabolism. To identify the molecular components associated with the dynamic adjustment of circadian period, we performed a forward genetic screen. We identified Arabidopsis mutants that were either period insensitive to nicotinamide (sin) or period oversensitive to nicotinamide (son). We mapped son1 to BIG, a gene of unknown molecular function that was shown previously to play a role in light signaling. We found that son1 has an early entrained phase, suggesting that the dynamic alteration of circadian period contributes to the correct timing of biological events. Our data provide insight into how the dynamic period adjustment of circadian oscillators contributes to establishing a correct phase relationship with the environment and show that BIG is involved in this process.

Adjustment of the Arabidopsis circadian oscillator by sugar signalling dictates the regulation of starch metabolism.

Arabidopsis plants store part of the carbon fixed by photosynthesis as starch to sustain growth at night. Two competing hypotheses have been proposed to explain this diel starch turnover based on either the measurement of starch abundance with respect to circadian time, or the sensing of sugars to feedback to the circadian oscillator to dynamically adjust the timing of starch turnover. We report a phase oscillator model that permitted derivation of the ideal responses of the circadian regulation of starch breakdown to maintain sucrose homeostasis. Testing the model predictions using a sugar-unresponsive mutant of Arabidopsis demonstrated that the dynamics of starch turnover arise from the circadian clock measuring and responding to the rate of change of cellular sucrose. Our theory and experiments suggest that starch turnover is controlled by the circadian clock acting as a dynamic homeostat responding to sucrose signals to maintain carbon homeostasis.

ELF3 controls thermoresponsive growth in Arabidopsis.

Plant development is highly responsive to ambient temperature, and this trait has been linked to the ability of plants to adapt to climate change. The mechanisms by which natural populations modulate their thermoresponsiveness are not known. To address this, we surveyed Arabidopsis accessions for variation in thermal responsiveness of elongation growth and mapped the corresponding loci. We find that the transcriptional regulator EARLY FLOWERING3 (ELF3) controls elongation growth in response to temperature. Through a combination of modeling and experiments, we show that high temperature relieves the gating of growth at night, highlighting the importance of temperature-dependent repressors of growth. ELF3 gating of transcriptional targets responds rapidly and reversibly to changes in temperature. We show that the binding of ELF3 to target promoters is temperature dependent, suggesting a mechanism where temperature directly controls ELF3 activity.

Measuring circadian oscillations of cytosolic-free calcium in Arabidopsis thaliana.

Circadian oscillations of cytosolic-free calcium can be measured in plants by observing luminescence of the bioluminescent calcium binding protein aequorin. Here we describe the use of intensified photon-counting CCD cameras to measure circadian oscillations in aequorin bioluminescence from Arabidopsis thaliana.

Metabolic regulation of circadian clocks.

Circadian clocks are 24-h timekeeping mechanisms, which have evolved in plants, animals, fungi and bacteria to anticipate changes in light and temperature associated with the rotation of the Earth. The current paradigm to explain how biological clocks provide timing information is based on multiple interlocking transcription-translation negative feedback loops (TTFL), which drive rhythmic gene expression and circadian behaviour of growth and physiology. Metabolism is an important circadian output, which in plants includes photosynthesis, starch metabolism, nutrient assimilation and redox homeostasis. There is increasing evidence in a range of organisms that these metabolic outputs can also contribute to circadian timing and might also comprise independent circadian oscillators. In this review, we summarise the mechanisms of circadian regulation of metabolism by TTFL and consider increasing evidence that rhythmic metabolism contributes to the circadian network. We highlight how this might be relevant to plant circadian clock function.

Function and evolution of 'green' GSK3/Shaggy-like kinases.

Glycogen synthase kinase 3 (GSK3) proteins, also known as SHAGGY-like kinases, have many important cell signalling roles in animals, fungi and amoebae. In particular, GSK3s participate in key developmental signalling pathways and also regulate the cytoskeleton. GSK3-encoding genes are also present in all land plants and in algae and protists, raising questions about possible ancestral functions in eukaryotes. Recent studies have revealed that plant GSK3 proteins are actively implicated in hormonal signalling networks during development as well as in biotic and abiotic stress responses. In this review, we outline the mechanisms of Arabidopsis GSK3 action, summarize GSK3 functions in dicot and monocot flowering plants, and speculate on the possible functions of GSK3s in the earliest-evolving land plants.