Clock Mechanism Research Articles

No Evidence That Homologs of Key Circadian Clock Genes Direct Circadian Programs of Development or mRNA Abundance in Verticillium dahliae

Many organisms harbor circadian clocks that promote their adaptation to the rhythmic environment. While a broad knowledge of the molecular mechanism of circadian clocks has been gained through the fungal model Neurospora crassa, little is known about circadian clocks in other fungi. N. crassa belongs to the same class as many important plant pathogens including the vascular wilt fungus Verticillium dahliae. We identified homologs of N. crassa clock proteins in V. dahliae, which showed high conservation in key protein domains. However, no evidence for an endogenous, free-running and entrainable rhythm was observed in the daily formation of conidia and microsclerotia. In N. crassa the frequency (frq) gene encodes a central clock protein expressed rhythmically and in response to light. In contrast, expression of Vdfrq is not light-regulated. Temporal gene expression profiling over 48 h in constant darkness and temperature revealed no circadian expression of key clock genes. Furthermore, RNA-seq over a 24 h time-course revealed no robust oscillations of clock-associated transcripts in constant darkness. Comparison of gene expression between wild-type V. dahliae and a ΔVdfrq mutant showed that genes involved in metabolism, transport and redox processes are mis-regulated in the absence of Vdfrq. In addition, VdΔfrq mutants display growth defects and reduced pathogenicity in a strain dependent manner. Our data indicate that if a circadian clock exists in Verticillium, it is based on alternative mechanisms such as post-transcriptional interactions of VdFRQ and the WC proteins or the components of a FRQ-less oscillator. Alternatively, it could be that whilst the original functions of the clock proteins have been maintained, in this species the interactions that generate robust rhythmicity have been lost or are only triggered when specific environmental conditions are met. The presence of conserved clock genes in genomes should not be taken as definitive evidence of circadian function.

Circadian clock mechanism driving mammalian photoperiodism

The annual photoperiod cycle provides the critical environmental cue synchronizing rhythms of life in seasonal habitats. In 1936, Bünning proposed a circadian-based coincidence timer for photoperiodic synchronization in plants. Formal studies support the universality of this so-called coincidence timer, but we lack understanding of the mechanisms involved. Here we show in mammals that long photoperiods induce the circadian transcription factor BMAL2, in the pars tuberalis of the pituitary, and triggers summer biology through the eyes absent/thyrotrophin (EYA3/TSH) pathway. Conversely, long-duration melatonin signals on short photoperiods induce circadian repressors including DEC1, suppressing BMAL2 and the EYA3/TSH pathway, triggering winter biology. These actions are associated with progressive genome-wide changes in chromatin state, elaborating the effect of the circadian coincidence timer. Hence, circadian clock-pituitary epigenetic pathway interactions form the basis of the mammalian coincidence timer mechanism. Our results constitute a blueprint for circadian-based seasonal timekeeping in vertebrates.

Blockchain at the Edge: Performance of Resource-Constrained IoT Networks

The proliferation of IoT in various technological realms has resulted in the massive spurt of unsecured data. The use of complex security mechanisms for securing these data is highly restricted owing to the low-power and low-resource nature of most of the IoT devices, especially at the Edge. In this article, we propose to use blockchains for extending security to such IoT implementations. We deploy a Ethereum blockchain consisting of both regular and constrained devices connecting to the blockchain through wired and wireless heterogeneous networks. We additionally implement a secure and encrypted networked clock mechanism to synchronize the non-real-time IoT Edge nodes within the blockchain. Further, we experimentally study the feasibility of such a deployment and the bottlenecks associated with it by running necessary cryptographic operations for blockchains in IoT devices. We study the effects of network latency, increase in constrained blockchain nodes, data size, Ether, and blockchain node mobility during transaction and mining of data within our deployed blockchain. This study serves as a guideline for designing secured solutions for IoT implementations under various operating conditions such as those encountered for static IoT nodes and mobile IoT devices.

Involvement of Bmal1 and circadian clock signaling in chondrogenic differentiation of ATDC5 cells by fluoride

Skeletal fluorosis causes growth plate impairment and growth retardation during bone development. However, the mechanism of how fluoride impairs chondrocyte is unclear. To explore the effect of fluoride on chondrocyte differentiation and the regulation of circadian clock signaling pathway during chondrogenesis, we treated ATDC5 cells with fluoride and carried out a series of experiments. 10−3 M fluoride inhibited cell viability and significantly decreased the expression of Sox9 and Col2a1 (P < 0.05). Fluoride inhibited proteoglycan synthesis and decreased significantly the expression of Aggrecan, Ihh and Col10a1 (P < 0.05). Meanwhile, fluoride significantly inhibited the expression of Bmal1 and disrupted circadian clock signaling pathway (P < 0.05). Furthermore, fluoride disrupted the time-dependent expression of circadian clock molecules and stage-specific differentiation markers. Overexpression of Bmal1 by lentivirus reversed the adverse effects of fluoride on chondrogenesis. These results suggested that fluoride inhibited chondrocyte viability and delayed chondrocyte differentiation. Fluoride delayed chondrogenesis partly via interfering with Bmal1 and circadian clock signaling pathway. Nevertheless, the specific mechanism of circadian clock in fluoride-induced cartilage damage needs to be further studied.

MCD diet-induced steatohepatitis generates a diurnal rhythm of associated biomarkers and worsens liver injury in Klf10 deficient mice

A large number of hepatic functions are regulated by the circadian clock and recent evidence suggests that clock disruption could be a risk factor for liver complications. The circadian transcription factor Krüppel like factor 10 (KLF10) has been involved in liver metabolism as well as cellular inflammatory and death pathways. Here, we show that hepatic steatosis and inflammation display diurnal rhythmicity in mice developing steatohepatitis upon feeding with a methionine and choline deficient diet (MCDD). Core clock gene mRNA oscillations remained mostly unaffected but rhythmic Klf10 expression was abolished in this model. We further show that Klf10 deficient mice display enhanced liver injury and fibrosis priming upon MCDD challenge. Silencing Klf10 also sensitized primary hepatocytes to apoptosis along with increased caspase 3 activation in response to TNFα. This data suggests that MCDD induced steatohepatitis barely affects the core clock mechanism but leads to a reprogramming of circadian gene expression in the liver in analogy to what is observed in other experimental disease paradigms. We further identify KLF10 as a component of this transcriptional reprogramming and a novel hepato-protective factor.

Entrainment mechanism of the cyanobacterial circadian clock induced by oxidized quinone**Project supported by the National Natural Science Foundation of China (Grant No. 11672177).

The circadian clock is a self-sustained biological oscillator which can be entrained by environmental signals. The cyanobacteria circadian clock is the simplest one, which is composed of the proteins KaiA, KaiB and KaiC. The phosphorylation/dephosphorylation state of KaiC exhibits a circadian oscillator. KaiA and KaiB activate KaiC phosphorylation and dephosphorylation respectively. CikA competing with KaiA for the same binding site on KaiB affects the phosphorylation state of KaiC. Quinone is a signaling molecule for entraining the cyanobacterial circadian clock which is oxidized at the onset of darkness and reduced at the onset of light, reflecting the environmental light–dark cycle. KaiA and CikA can sense external signals by detecting the oxidation state of quinone. However, the entrainment mechanism is far from clear. We develop an enhanced mathematical model including oxidized quinone sensed by KaiA and CikA, with which we present a detailed study on the entrainment of the cyanobacteria circadian clock induced by quinone signals. We find that KaiA and CikA sensing oxidized quinone pulse are related to phase advance and delay, respectively. The time of oxidized quinone pulse addition plays a key role in the phase shifts. The combination of KaiA and CikA is beneficial to the generation of entrainment, and the increase of signal intensity reduces the entrainment phase. This study provides a theoretical reference for biological research and helps us understand the dynamical mechanisms of cyanobacteria circadian clock.

Time-of-day dependent effects of contractile activity on the phase of the skeletal muscle clock.

Key points Disruptions in circadian rhythms across an organism are associated with negative health outcomes, such as cardiometabolic and neurodegenerative diseases.Exercise has been proposed as a time cue for the circadian clock in rodents and humans.In this study, we assessed the effect of a single bout of endurance exercise on the skeletal muscle clock in vivo and a bout of muscle contractions in vitro.Timing of exercise or contractions influences the directional response of the muscle clock phase in vivo and in vitro.Our findings demonstrate that muscle contractions, as a component of exercise, can directly modulate the expression of muscle clock components in a time‐of‐day dependent manner. Exercise has been proposed to be a zeitgeber for the muscle circadian clock mechanism. However, this is not well defined and it is unknown if exercise timing induces directional shifts of the muscle clock. Our purpose herein was to assess the effect of one bout of treadmill exercise on skeletal muscle clock phase changes. We subjected PERIOD2::LUCIFERASE mice (n = 30F) to one 60 min treadmill exercise bout at three times of day. Exercise at ZT5, 5 h after lights on, induced a phase advance (100.2 ± 25.8 min; P = 0.0002), whereas exercise at ZT11, 1 h before lights off, induced a phase delay (62.1 ± 21.1 min; P = 0.0003). Exercise at ZT17, middle of the dark phase, did not alter the muscle clock phase. Exercise induces diverse systemic changes so we developed an in vitro model system to examine the effects of contractile activity on muscle clock phase. Contractions applied at peak or trough Bmal1 expression induced significant phase delays (applied at peak: 27.2 ± 10.2 min; P = 0.0017; applied at trough: 64.6 ± 6.5 min, P < 0.0001). Contractions applied during the transition from peak to trough Bmal1 expression induced a phase advance (49.8 ± 23.1 min; P = 0.0051). Lastly, contractions at different times of day resulted in differential changes of core clock gene expression, demonstrating an exercise and clock interaction, providing insight into potential mechanisms of exercise‐induced phase shifts. These data demonstrate that muscle contractions, as part of exercise, are sufficient to shift the muscle circadian clock phase, likely through changes in core clock gene expression. Additionally, our findings that exercise induces directional muscle clock phase changes confirms that exercise is a bona fide environmental time cue for skeletal muscle.

The Molecular Genetic Interaction Between Circadian Rhythms and Susceptibility to Seizures and Epilepsy

Seizure patterns observed in patients with epilepsy suggest that circadian rhythms and sleep/wake mechanisms play some role in the disease. This review addresses key topics in the relationship between circadian rhythms and seizures in epilepsy. We present basic information on circadian biology, but focus on research studying the influence of both the time of day and the sleep/wake cycle as independent but related factors on the expression of seizures in epilepsy. We review studies investigating how seizures and epilepsy disrupt expression of core clock genes, and how disruption of clock mechanisms impacts seizures and the development of epilepsy. We focus on the overlap between mechanisms of circadian-associated changes in SCN neuronal excitability and mechanisms of epileptogenesis as a means of identifying key pathways and molecules that could represent new targets or strategies for epilepsy therapy. Finally, we review the concept of chronotherapy and provide a perspective regarding its application to patients with epilepsy based on their individual characteristics (i.e., being a “morning person” or a “night owl”). We conclude that better understanding of the relationship between circadian rhythms, neuronal excitability, and seizures will allow both the identification of new therapeutic targets for treating epilepsy as well as more effective treatment regimens using currently available pharmacological and non-pharmacological strategies.

219-LB: The Hepatocyte Clock and Feeding Interdependently Control Chrono-Homeostasis of Multiple Liver Cell Types

Most cells in the body contain a cell autonomous molecular clock, but the requirement of peripheral clocks for circadian rhythmicity, and their effects on physiology, are not well understood. Here we show that deletion of core clock components REV-ERB alpha and β in adult mouse hepatocytes disrupted circadian rhythmicity of many liver genes, as expected, but also led to maintained and even gained rhythmicity of other genes without altering feeding behavior. The loss of REV-ERBs from hepatocytes led to an exaggerated circadian rhythm of de novo lipogenesis. Liver function is also influenced by non-hepatocytic cells, and remarkably the loss of REV-ERBs in hepatocytes remodeled the circadian transcriptomes and metabolomes of multiple cell types within the liver, indicating that hepatocytes communicated time signals to non-hepatocytic cells. Finally, alteration of food availability demonstrated strong interdependence of the cell-autonomous hepatocyte clock mechanism and non-cell-autonomous environmental change. Together these studies reveal previously unsuspected roles of the hepatocyte clock in the physiological coordination of nutritional signals and cell-cell communication controlling rhythmic metabolism. Disclosure D. Guan: None. M.A. Lazar: Board Member; Self; Pfizer Inc. Consultant; Self; Calico, Madrigal Pharmaceuticals, Inc., Novartis Pharmaceuticals Corporation. Funding National Institute of Diabetes and Digestive and Kidney Diseases (F32DK116519)

STUDY OF EFFECTS OF CIRCADIAN RHYTHMS ON SOLID TUMOR CELLS PROLIFERATION AND CHEMORESISTANCE

Circadian clock is a complex mechanism regulating many different physiological processes. Preclinical, epidemiological and clinical studies demonstrate association between circadian rhythms disruption and tumor initiation. Study of modulation of solid tumor cells biological properties through enhancement of clock mechanisms could attribute to the development of more effective chemo- and hormone therapy approaches. Aim: Evaluate the effects of ovarian and lung tumor cells synchronization with dexamethasone in vitro on cells sensitivity to cisplatin. Materials and methods: Metastatic ovarian cancer (n=3) and lung cancer (n=3) cell lines were obtained from patients tumors. Tumor cell cultivation was performed in accordance with the protocol. Artificial synchronization was performed with dexamethasone 200 nM introduction to the cell cultures. Doses of cisplatin used were 1.5 and 3.0 mg/ml. xCELLigence Real-Time Cell Analysis and Cell-IQ was used to measure proliferation and chemoresistance of tumor cells. Results: Each cell-line had individual morphological characteristics and proliferation parameters. Preliminary incubation with dexamethasone (2 h) had a stimulating effect on proliferation of all tumor cell lines (Slope min -4.3(0.3)хЕ ‘х10-3 - max 36.8(0.6)хЫх10'3, min 2.2(0.2)хЕ1х10'3- max 50.4(0.8)хЕ1х10'3), and increased their sensitivity to cisplatin (min -43(2.6)хЕ1х10-3 - max 57.5(0.6)хЕ1х10-3 и min -217,3(2,2) -1,9(0,1)хч-1х10-3 - max -1,9(0,1)хч'1х10'3, respectively. Conclusion: These results should be the platform for future studies of the interaction of clock mechanisms, cell cycle regulation and viability of tumor cells.

Friction and Dynamics of Verge and Foliot: How the Invention of the Pendulum Made Clocks Much More Accurate

The tower clocks designed and built in Europe starting from the end of the 13th century employed the “verge and foliot escapement” mechanism. This mechanism provided a relatively low accuracy of time measurement. The introduction of the pendulum into the clock mechanism by Christiaan Huygens in 1658–1673 improved the accuracy by about 30 times. The improvement is attributed to the isochronicity of small linear vibrations of a mathematical pendulum. We develop a mathematical model of both mechanisms. Using scaling arguments, we show that the introduction of the pendulum resulted in accuracy improvement by approximately π/μ ≈ 30 times, where μ ≈ 0.1 is the coefficient of friction. Several historic clocks are discussed, as well as the implications of both mechanisms to the history of science and technology.

Circadian Rhythms and Epilepsy: A Suitable Case for Absence Epilepsy.

Many physiological processes such as sleep, hormonal secretion, or thermoregulation, are expressed as daily rhythms orchestrated by the circadian timing system. A powerful internal clock mechanism ensures proper synchronization of vital functions within an organism on the one hand, and between the organism and the external environment on the other. Some of the pathological processes developing in the brain and body are subjected to circadian modulation as well. Epilepsy is one of the conditions which symptoms often worsen at a very specific time of a day. Variation in peak occurrence depends on the syndrome and localization of the epileptic focus. Moreover, the timing of some types of seizures is closely related to the sleep-wake cycle, one of the most prominent circadian rhythms. This review focuses on childhood absence epilepsy (CAE), a genetic generalized epilepsy syndrome, in which both, the circadian and sleep influences play a significant role in manifestation of symptoms. Human and animal studies report rhythmical occurrence of spike-wave discharges (SWDs), an EEG hallmark of CAE. The endogenous nature of the SWDs rhythm has been confirmed experimentally in a genetic animal model of the disease, rats of the WAG/Rij strain. Well-known detrimental effects of circadian misalignment were demonstrated to impact the severity of ongoing epileptic activity. SWDs are vigilance-dependent in both humans and animal models, occurring most frequently during passive behavioral states and light slow-wave sleep. The relationship with the sleep-wake cycle seems to be bidirectional, while sleep shapes the rhythm of seizures, epileptic phenotype changes sleep architecture. Circadian factors and the sleep-wake states dependency have a potential as add-ons in seizures' forecasting. Stability of the rhythm of recurrent seizures in individual patients has been already used as a variable which refines existing algorithms for seizures' prediction. On the other hand, apart from successful pharmacological approach, circadian hygiene including sufficient sleep and avoidance of internal desynchronization or sleep loss, may be beneficial for patients with epilepsy in everyday management of seizures.

Elevated serum melatonin under constant darkness enhances neural repair in spinal cord injury through regulation of circadian clock proteins expression

We investigated the effects of environmental lighting conditions regulating endogenous melatonin production on neural repair, following experimental spinal cord injury (SCI). Rats were divided into three groups randomly: the SCI + L/D (12/12‐h light/dark), SCI + LL (24‐h constant light), and SCI + DD (24‐h constant dark) groups. Controlled light/dark cycle was pre‐applied 2 weeks before induction of spinal cord injury. There was a significant increase in motor recovery as well as body weight from postoperative day (POD) 7 under constant darkness. However, spontaneous elevation of endogenous melatonin in cerebrospinal fluid was seen at POD 3 in all of the SCI rats, which was enhanced in SCI + DD group. Augmented melatonin concentration under constant dark condition resulted in facilitation of neuronal differentiation as well as inhibition of primary cell death. In the rostrocaudal region, elevated endogenous melatonin concentration promoted neural remodeling in acute phase including oligodendrogenesis, excitatory synaptic formation, and axonal outgrowth. The changes were mediated via NAS‐TrkB‐AKT/ERK signal transduction co‐regulated by the circadian clock mechanism, leading to rapid motor recovery. In contrast, exposure to constant light exacerbated the inflammatory responses and neuroglial loss. These results suggest that light/dark control in the acute phase might be a considerable environmental factor for a favorable prognosis after SCI.Support or Funding InformationNational Research Foundation (NRF‐2013R1A2A2A01067169, NRF‐2015R1C1A2A01055691, NRF‐2017R1D1A1B03029565), Korea.

Programming the Sequential Release of DNA.

This study presents a mechanism for releasing a series of different short DNA sequences from sequestered complexes, one after another, using coupled biochemical reactions. The process uses stages of coupled DNA strand-displacement reactions that first release an output molecule and then trigger the initiation of the next release stage. We demonstrate the sequential release of 25 nM of four different sequences of DNA over a day, both with and without a centralized "clock" mechanism to regulate release timing. We then demonstrate how the presence of a target input molecule can determine which of several different release pathways are activated, analogous to branching conditional statements in computer programming. This sequential release circuit offers a means to schedule downstream chemical events, such as steps in the assembly of a nanostructure, or stages in a material's response to a stimulus.

Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae

Organisms possess an endogenous molecular clock which enables them to adapt to environmental rhythms and to synchronize their metabolism and behavior accordingly. Circadian rhythms govern daily oscillations in numerous physiological processes, and the underlying molecular components have been extensively described from fruit flies to mammals. Drosophila larvae have relatively simple nervous system compared to their adult counterparts, yet they both share a homologous molecular clock with mammals, governed by interlocking transcriptional feedback loops with highly conserved constituents. Larvae exhibit a robust light avoidance behavior, presumably enabling them to avoid predators and desiccation, and DNA-damage by exposure to ultraviolet light, hence are crucial for survival. Circadian rhythm has been shown to alter light-dark preference, however it remains unclear how distinct behavioral strategies are modulated by circadian time. To address this question, we investigate the larval visual navigation at different time-points of the day employing a computer-based tracking system, which allows detailed evaluation of distinct navigation strategies. Our results show that due to circadian modulation specific to light information processing, larvae avoid light most efficiently at dawn, and a functioning clock mechanism at both molecular and neuro-signaling level is necessary to conduct this modulation.

Role of Prolyl Isomerization in Metamorphosis of the Clock Protein, KaiB

Metamorphic proteins are an emerging class of proteins that defy the classic 'one structure-one fold' paradigm by alternating between two conformations despite having the same primary structure (i.e. sequence). KaiB is the only metamorphic protein known to exist in any circadian clock. It is a component of the cyanobacterial clock, adopting one fold during the day and a distinctly different one at night. Our recent findings suggest that three out of seven prolyl residues in KaiB- P63, P70 and P72- adopt the trans configuration during the day and cis at night. Also, there is a fourth prolyl residue, P51, which even though is trans in both folds is critical to clock mechanism. Binding assays of prolyl-to-alanyl KaiB mutants suggest hierarchical roles of the prolyl residues and raise the possibility that they isomerize sequentially: P63->P70 ->P72.

Disrupting the Circadian Clock Mechanism in Cardiomyocytes Exacerbates the LQT3-related Phenotype in Scn5aΔKPQ/+Mice

Disrupting the Circadian Clock Mechanism in Cardiomyocytes Exacerbates the LQT3-related Phenotype in Scn5aΔKPQ/+Mice

Design and Implementation of 802.15.4 Transceiver for Wireless Personal Area Networks (WPANs) on FPGA

The IEEE 802.15.4 standard provides mainly accessing, monitoring, and controlling capability of the Wireless devices. This standard supports short-range wireless communications and Low Rate (LR) - Wireless Personal Area Networks (WPANs). This manuscript presents the fully integrated digital 802.15.4 Transceiver, which suitable to ZigBee Device Standard at 2.4GHz range. The 802.15.4 Transceiver design includes an 8-bit input data sequence mapped to lower and Upper Symbols followed by Chip-sequence conversion as per the IEEE standard. The chip sequences are mapped separately as even and odd sequences used for the Offset-QPSK Modulation. The chip synchronization achieved by using a proper clocking mechanism on the receiver side. The 802.15.4 Transceiver design is implemented on Artix-7 FPGA using Xilinx Environment. The hardware constraints like Area (Slices), Frequency, and Power are analyzed. The proposed work also compared with existing similar approaches with more significant improvements in chip area and Power.

The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock

Circadian rhythms are the endogenous oscillation of biological reactions and behaviors in most organisms on Earth. Circadian clocks are the pacemakers regulating circadian rhythms, and the transcription-translation dependent feedback loop (TTFL) model was supposed to be the sole model of circadian clocks. However, recent years have witnessed rapid progresses in the study of non-TTFL circadian clocks. The cyanobacterial circadian clock consists of three proteins (KaiA, KaiB, and KaiC), and is extensively studied as a non-TTFL circadian clock model. Although containing only three proteins, the molecular mechanism of the KaiABC circadian clock remains elusive. We recently noticed that KaiA has an auto-inhibition conformation during the oscillation, but how this auto-inhibition is regulated is unclear. Here, we started from the design of light modulated KaiAs to investigate this mechanism. We designed different KaiA constructs fused with the light modulable LOV2 protein, and used light-modulated KaiAs to regulate the phosphorylation and dephosphorylation of KaiC. Our data indicated that the N-terminal domain of KaiA is important for KaiA’s reversible off/on switching during the unidirectional oscillation of the KaiABC system. This work provides an updated model to explain the molecular mechanism of the KaiABC circadian clock.

CFE: a convenient, flexible, and efficient clocking scheme for quantum‐dot cellular automata

Quantum-dot cellular automata (QCA) is a highly attractive alternative to CMOS for future digital circuit designs. The circuits in QCA have been extensively studied, which suffer from negative impacts due to the limits of the manual design process, even for a modest complexity. In the process of QCA circuit design, the problems caused by clock distribution and long transmission lines have to be considered. In order to solve such problems, some researchers have proposed several clock schemes and expected to realise electronic design automation. These clock schemes, however, have drawbacks; the most important one is the limitation of flexibility on designing complex circuits. In this regard, this study proposes a convenient, flexible, and efficient (CFE) clocking scheme by using diamond structures for clock zones, each of which has the same neighbouring clock zone and four information transmission directions. It has main advantages of existing clocking schemes and can commendably increase the flexibility in circuit design process. In order to show the suitability of the proposed CFE scheme using standard clock mechanism in QCA, several types of circuits proposed in this study reveal its advantages in comparison with an existing state-of-the-art clocking scheme.