Main Clock Research Articles

The new CLBv4 for the KM3NeT Neutrino Telescope

The KM3NeT collaboration aims at the construction of a deepsea neutrino observatory in the Mediterranean Sea equipped with thousands of glass spheres, the so-called Digital Optical Modules, each of which contains 31 photomultipliers of small photocathode area. These devices will be used for the detection of the Cherenkov light induced by charged particles produced by the interaction of neutrinos with matter inside or in the vicinity of the KM3NeT detector. The signal acquired by each photomultiplier is sent to a Time to Digital Converter which is part of a Central Logic Board. The Time to Digital Converter resolution is 1 ns and the White Rabbit technology is used to guarantee time synchronization between the optical modules. Due to the large volume to be instrumented by KM3NeT, a cost reduction of the different systems is important so different versions of the Central Logic Board have been designed. The newest version was designed also to also reduce the phase noise in the main clock, to improve the stability of the synchronization.

Entrainment range affected by the heterogeneity in the amplitude relaxation rate of suprachiasmatic nucleus neurons**Project supported by the National Natural Science Foundation of China (Grant Nos. 11875042, 11505114, and 10975099) and the Program for Professor of Special Appointment (Orientational Scholar) at Shanghai Institutions of Higher Learning, China (Grant Nos. QD2015016 and D-USST02).

Adaption of circadian rhythms in behavioral and physiological activities to the external light–dark cycle is achieved through the main clock, i.e., the suprachiasmatic nucleus (SCN) of the brain in mammals. It has been found that the SCN neurons differ in the amplitude relaxation rate, which represents the rigidity of the neurons to the external amplitude disturbance. Thus far, the appearance of that difference has not been explained. In the present study, an alternative explanation based on the Poincaré model is given which takes into account the effect of the difference in the entrainment range of the SCN. Both our simulation results and theoretical analyses show that the largest entrainment range is obtained with suitable difference in the case that only a part of SCN neurons are sensitive to the light information. Our findings may give an alternative explanation for the appearance of that difference (heterogeneity) and shed light on the effects of the heterogeneity in the neuronal properties on the collective behaviors of the SCN neurons.

Phase Noise and Frequency Stability of the Red-Pitaya Internal PLL.

In field-programmable gate array platforms, the main clock is generally a low-cost quartz oscillator whose stability is of the order of 10-9 to 10-10 in the short term and 10-7 to 10-8 in the medium term, with the uncertainty of tens of ppm. Better stability is achieved by feeding an external reference into the internal phase-locked loop (PLL). We report the noise characterization of the internal PLL of Red-Pitaya platform, an open-source embedded system architected around the Zynq 7010 System on Chip, with analog-to-digital and digital-to-analog converters. Our experiments show that, providing an external 10-MHz reference, the PLL exhibits a residual frequency stability of 1.2×10-12 at 1 s and 1.3×10-15 at 4000 s, Allan deviation in 5-Hz bandwidth. These results help to predict the PLL stability as a function of frequency and power of the external reference, and provide guidelines for the design of precision instrumentation, chiefly intended for time and frequency metrology.

Implementation of Clock Gating for Power Optimizing in Synchronous Design

Huffman coding is very important technique in information theory. Compression technique is the technology for reducing the amount of data used to denote any content without decreasing the quality. Furthermore, Clock gating is an effective method for decreasing power consumption in a sequence design. It saves more power by dividing the main clock and distributing the clock to the logic blocks only when there is a need for those blocks to be activated. This paper aim to design Huffman coding and decoding process with proposing a novel method of clock gating to achieve low power consumption. Huffman design is executed by expending ASIC design procedures. With the purpose of executing the encoder and decoder structures, 130 nm typical cell technology libraries are utilized for ASIC implementation. The simulations are completed by utilizing Modelsim tool. The design of coding and decoding process has been made using Verilog HDL language. Moreover, it carried out using Quartus II 14.1 Web Edition (64-Bit).

Effects of Light and Temperature on Daily Activity and Clock Gene Expression in Two Mosquito Disease Vectors.

Most organisms feature an endogenous circadian clock capable of synchronization with their environment. The most well-known synchronizing agents are light and temperature. The circadian clock of mosquitoes, vectors of many pathogens, drives important behaviors related to vectoral capacity, including oviposition, host seeking, and hematophagy. Main clock gene expression, as well as locomotor activity patterns, has been identified in Aedes aegypti and Culex quinquefasciatus under artificial light-dark cycles. Given that these mosquito species thrive in tropical areas, it is reasonable to speculate that temperature plays an important role in the circadian clock. Here, we provide data supporting a different hierarchy of light and temperature as zeitgebers of two mosquito species. We recorded their locomotor activity and quantified mRNA expression of the main clock genes in several combinations of light and temperature cycles. We observed that A. aegypti is more sensitive to temperature, while C. quinquefasciatus is more responsive to light. These variations in clock gene expression and locomotor activity may have affected the mosquito species' metabolism, energy expenditure, fitness cost, and pathogen transmission efficiency. Our findings are relevant to chronobiology studies and also have epidemiological implications.

A robust high-efficiency cross-coupled charge pump circuit without blocking transistors

A fully integrated cross-coupled charge pump circuit with a new clock scheme has been presented in this paper. The new clock scheme ensures that all NMOS pre-charge transistors are turned off when the voltages of main clock signals are high. Notably, all PMOS transfer transistors will be turned off when the voltages of the main clock signals are low. As a result, the charge pump eliminates all of the reversion power loss and reduces the ripple voltage. The proposed charge pump has a better performance even in scenarios where the main clock signals are mismatched. The proposed charge pump circuit was simulated using spectre in the TSMC 0.18 µm CMOS process. The simulation results show that the proposed charge pump circuit has a high voltage conversion efficiency and low ripple voltage.

Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria.

The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC-binding EnvZ-like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer ) and occasionally as a hexamer (SasA6mer ) in vitro. Previously, the molecular mass of the SasA-KaiCDD complex, where KaiCDD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel-filtration chromatography to be larger than 670kDa. This value disagrees with the theoretical estimation of 480kDa for a SasA3mer -KaiC hexamer (KaiC6mer ) complex with a 1:1 molecular ratio. To clarify the structure of the SasA-KaiC complex, we analyzed KaiCDD with 0.1mmol/L ATP and 5mmol/L MgCl2 (Mg-ATP), SasA and a mixture containing SasA and KaiCDD6mer with Mg-ATP by atomic force microscopy (AFM). KaiCDD images were classified into two types with height distribution corresponding to KaiCDD monomer (KaiCDD1mer ) and KaiCDD6mer , respectively. SasA images were classified into two types with height corresponding to SasA3mer and SasA6mer , respectively. The AFM images of the SasA-KaiCDD mixture indicated not only KaiCDD1mer , KaiCDD6mer , SasA3mer and SasA6mer , but also wider area "islands," suggesting the presence of a polymerized form of the SasA-KaiCDD complex.

Functional characterization of the circadian clock in the Antarctic krill, Euphausia superba

Antarctic krill (Euphausia superba) is a key species in Southern Ocean ecosystem where it plays a central role in the Antarctic food web. Available information supports the existence of an endogenous timing system in krill enabling it to synchronize metabolism and behavior with an environment characterized by extreme seasonal changes in terms of day length, food availability, and surface ice extent. A screening of our transcriptome database “KrillDB” allowed us to identify the putative orthologues of 20 circadian clock components. Mapping of conserved domains and phylogenetic analyses strongly supported annotations of the identified sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to define the role of the main clock components. Our findings provide an overall picture of the molecular mechanisms underlying the functioning of the endogenous circadian clock in the Antarctic krill and shed light on their evolution throughout crustaceans speciation. Interestingly, the core clock machinery shows both mammalian and insect features that presumably contribute to an evolutionary strategy to cope with polar environment’s challenges. Moreover, despite the extreme variability characterizing the Antarctic seasonal day length, the conserved light mediated degradation of the photoreceptor EsCRY1 suggests a persisting pivotal role of light as a Zeitgeber.

Dispersion of the intrinsic neuronal periods affects the relationship of the entrainment range to the coupling strength in the suprachiasmatic nucleus.

Living beings on the Earth are subjected to and entrained (synchronized) to the natural 24-h light-dark cycle. Interestingly, they can also be entrained to an external artificial cycle of non-24-h periods. The range of these periods is called the entrainment range and it differs among species. In mammals, the entrainment range is regulated by a main clock located in the suprachiasmatic nucleus (SCN) which is composed of 10000 neurons in the brain. Previous works have found that the entrainment range depends on the cellular coupling strength in the SCN. In particular, the entrainment range decreases with the increase of the cellular coupling strength, provided that all the neuronal oscillators are identical. However, the SCN neurons differ in the intrinsic periods that follow a normal distribution in a range from 22 to 28 h. In the present study, taking the dispersion of the intrinsic neuronal periods into account, we examined the relationship between the entrainment range and the coupling strength. Results from numerical simulations and theoretical analyses both show that the relationship is altered to be paraboliclike if the intrinsic neuronal periods are nonidentical, and the maximal entrainment range is obtained with a suitable coupling strength. Our results shed light on the role of the cellular coupling in the entrainment ability of the SCN network.

Coordination between Differentially Regulated Circadian Clocks Generates Rhythmic Behavior.

Specialized groups of neurons in the brain are key mediators of circadian rhythms, receiving daily environmental cues and communicating those signals to other tissues in the organism for entrainment and to organize circadian physiology. In Drosophila, the "circadian clock" is housed in seven neuronal clusters, which are defined by their expression of the main circadian proteins, Period, Timeless, Clock, and Cycle. These clusters are distributed across the fly brain and are thereby subject to the respective environments associated with their anatomical locations. While these core components are universally expressed in all neurons of the circadian network, additional regulatory proteins that act on these components are differentially expressed, giving rise to "local clocks" within the network that nonetheless converge to regulate coherent behavioral rhythms. In this review, we describe the communication between the neurons of the circadian network and the molecular differences within neurons of this network. We focus on differences in protein-expression patterns and discuss how such variation can impart functional differences in each local clock. Finally, we summarize our current understanding of how communication within the circadian network intersects with intracellular biochemical mechanisms to ultimately specify behavioral rhythms. We propose that additional efforts are required to identify regulatory mechanisms within each neuronal cluster to understand the molecular basis of circadian behavior.

Differences in intrinsic amplitudes of neuronal oscillators improve synchronization in the suprachiasmatic nucleus.

In mammals, a main clock located in the suprachiasmatic nucleus (SCN) regulates the ∼24 h rhythms of behavioral and physiological activities exposed to a natural 24 light-dark cycle or even under constant darkness. The rhythms originate from self-sustained oscillations of the SCN neurons, which differ in both intrinsic periods and intrinsic amplitudes. The intrinsic periods and the intrinsic amplitudes were found to be bound to specific regions in the previous experiments. In particular, neurons of smaller amplitudes and larger periods are located in a ventrolateral part, and neurons of larger amplitudes and smaller periods are in a dorsomedial part. In the present study, we examined the effects of the differences in the intrinsic frequencies and the differences in the intrinsic amplitudes of neuronal oscillators on the synchronization, respectively. We found that the differences in the intrinsic frequencies weaken the synchronization, whereas the differences in the intrinsic amplitudes strengthen the synchronization. Our finding may shed light on the effects of the heterogenous properties of individual neurons on the collective behaviors of the SCN network and provide a way to enhance the synchronization.

Melatonin in regulation of inflammatory pathways in rheumatoid arthritis and osteoarthritis: involvement of circadian clock genes.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the two most prevalent joint diseases. A such, they are important causes of pain and disability in a substantial proportion of the human population. A common characteristic of these diseases is the erosion of articular cartilage and consequently joint dysfunction. Melatonin has been proposed as a link between circadian rhythms and joint diseases including RA and OA. This hormone exerts a diversity of regulatory actions through binding to specific receptors and intracellular targets as well as having receptor-independent actions as a free radical scavenger. Cytoprotective effects of melatonin involve a myriad of prominent receptor-mediated pathways/molecules associated with inflammation, of which the role of omnipresent NF-κB signalling is crucial. Likewise, disturbance of circadian timekeeping is closely involved in the aetiology of inflammatory arthritis. Melatonin is shown to stimulate cartilage destruction/regeneration through direct/indirect modulation of the expression of the main circadian clock genes, such as BMAL, CRY and/or DEC2. In the current article, we review the effects of melatonin on RA and OA, focusing on its ability to regulate inflammatory pathways and circadian rhythms. We also review the possible protective effects of melatonin on RA and OA pathogenesis. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

The asymmetry of the entrainment range induced by the difference in intrinsic frequencies between two subgroups within the suprachiasmatic nucleus.

The rhythms of physiological and behavioral activities in mammals, which are regulated by the main clock suprachiasmatic nucleus (SCN) in the brain, can not be only synchronized to the natural 24 h light-dark cycle, but also to cycles with artificial periods. The range of the artificial periods that the animal can be synchronized to is called entrainment range. In the absence of the light-dark cycle, the animal can also maintain the circadian rhythm with an endogenous period close to 24 h. Experiments found that the entrainment range is not symmetrical with respect to the endogenous period. In the present study, an explanation is given for the asymmetry based on a Kuramoto model which describes the neuronal network of the SCN. Our numerical simulations and theoretical analysis show that the asymmetry results from the difference in the intrinsic frequencies between two subgroups of the SCN, as well as the entrainment range is affected by the difference.

The role of PDF neurons in setting the preferred temperature before dawn in Drosophila.

Animals have sophisticated homeostatic controls. While mammalian body temperature fluctuates throughout the day, small ectotherms, such as Drosophila achieve a body temperature rhythm (BTR) through their preference of environmental temperature. Here, we demonstrate that pigment dispersing factor (PDF) neurons play an important role in setting preferred temperature before dawn. We show that small lateral ventral neurons (sLNvs), a subset of PDF neurons, activate the dorsal neurons 2 (DN2s), the main circadian clock cells that regulate temperature preference rhythm (TPR). The number of temporal contacts between sLNvs and DN2s peak before dawn. Our data suggest that the thermosensory anterior cells (ACs) likely contact sLNvs via serotonin signaling. Together, the ACs-sLNs-DN2s neural circuit regulates the proper setting of temperature preference before dawn. Given that sLNvs are important for sleep and that BTR and sleep have a close temporal relationship, our data highlight a possible neuronal interaction between body temperature and sleep regulation.

Entrainment range of the suprachiasmatic nucleus affected by the difference in the neuronal amplitudes between the light-sensitive and light-insensitive regions.

Mammals not only can be synchronized to the natural 24-h light-dark cycle, but also to a cycle with a non-24-h period. The range of the period of the external cycle, for which the animals can be entrained to, is called the entrainment range, which differs among species. The entrainment range as a characteristic of the animal is determined by the main circadian clock, i.e., the suprachiasmatic nucleus (SCN) in the brain. The SCN is composed of ∼10000 heterogeneous neurons, which can be divided into two subgroups, i.e., the ventrolateral subgroup (VL) directly receiving the light information from the retina and relaying the information to the dorsomedial subgroup (DM). Among the SCN neurons, the amplitudes are different; however, it is unclear that the amplitude is related to the location of the neurons in experiments. In the present study, we examined the effect of the difference in the neuronal amplitude between the VL and the DM on the entrainment range of the SCN, based on a mathematical model, i.e., the Poincaré model, which is used to describe the circadian clock. We find that the maximal entrainment range is obtained when the difference is equal to a critical point. If the difference of the amplitudes of the VL neurons to the amplitudes of the DM neurons is smaller than a critical point, with the increase of the difference, the entrainment range of the SCN increases, while if the difference is larger than the critical point, the entrainment range decreases with the increase of the difference. Our finding may give a potential explanation for the diversity of the entrainment range among species.

Circadian rhythms of hedonic drinking behavior in mice

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the main circadian clock, synchronized by the light–dark cycle, which generates behavioral rhythms like feeding, drinking and activity. Notwithstanding, the main role of the SCN clock on the control of all circadian rhythms has been questioned due to the presence of clock activity in many brain areas, including those implicated in the regulation of feeding and reward. Moreover, whether circadian rhythms of particular motivated behaviors exist is unknown. Here, we evaluated the spontaneous daily and circadian behavior of consumption of a sweet caloric solution (5–10% sucrose), and the effects of sucrose intake on the expression of clock genes in the mouse brain. Mice showed a daily (in a light–dark cycle) and a circadian (in constant darkness conditions) rhythm in the intake and sucrose preference with a rise for both parameters at night (or subjective night). In addition, we observed changes in the circadian day-night expression of the clock gene Per2 in the SCN, cortex and striatum of animals ingesting sucrose compared to control mice on pure water. Finally, daily rhythms of sucrose intake and preference were abolished in Per2Brdm1- and double Per1−/−Per2Brdm1-mutant animals. These data indicate that the expression of circadian rhythms of hedonic feeding behaviors may be controlled by brain circadian clocks and Per gene expression.

Genetic basis of allochronic differentiation in the fall armyworm

BackgroundVery little is known on how changes in circadian rhythms evolve. The noctuid moth Spodoptera frugiperda (Lepidoptera: Noctuidae) consists of two strains that exhibit allochronic differentiation in their mating time, which acts as a premating isolation barrier between the strains. We investigated the genetic basis of the strain-specific timing differences to identify the molecular mechanisms of differentiation in circadian rhythms.ResultsThrough QTL analyses we identified one major Quantitative trait chromosome (QTC) underlying differentiation in circadian timing of mating activity. Using RADtags, we identified this QTC to be homologous to Bombyx mori C27, on which the clock gene vrille is located, which thus became the major candidate gene. In S. frugiperda, vrille showed strain-specific polymorphisms. Also, vrille expression differed significantly between the strains, with the rice-strain showing higher expression levels than the corn-strain. In addition, RT-qPCR experiments with the other main clock genes showed that pdp1, antagonist of vrille in the modulatory feedback loop of the circadian clock, showed higher expression levels in the rice-strain than in the corn-strain.ConclusionsTogether, our results indicate that the allochronic differentiation in the two strains of S. frugiperda is associated with differential transcription of vrille or a cis-acting gene close to vrille, which contributes to the evolution of prezygotic isolation in S. frugiperda.

Dissociation between two subgroups of the suprachiasmatic nucleus affected by the number of damped oscillated neurons

In mammals, the main clock located in the suprachiasmatic nucleus (SCN) of the brain synchronizes the body rhythms to the environmental light-dark cycle. The SCN is composed of about 2×10^{4} neurons which can be classified into three oscillatory phenotypes: self-sustained oscillators, damped oscillators, and arrhythmic neurons. Exposed to an artificial external light-dark cycle with a period of 22h instead of 24h, two subgroups of the SCN can become desynchronized (dissociated). The ventrolateral (VL) subgroup receives photic input and is entrained to the external cycle and a dorsomedial (DM) subgroup oscillates with its endogenous (i.e., free running) period and is synchronized to the external light-dark cycle through coupling from the VL. In the present study, we examined the effects of damped oscillatory neurons on the dissociation between VL and DM under an external 22h cycle. We found that, with increasing numbers of damped oscillatory neurons located in the VL, the dissociation between the VL and DM emerges, but if these neurons are increasingly present in the DM the dissociation disappears. Hence, the damped oscillatory neurons in different subregions of the SCN play distinct roles in the dissociation between the two subregions of the SCN. This shows that synchrony between SCN subregions is affected by the number of damped oscillatory neurons and the location of these cells. We suggest that more knowledge on the number and the location of these cells may explain why some species do show a dissociation between the subregions and others do not, as the distribution of oscillatory types of neurons offers a plausible and novel candidate mechanism to explain heterogeneity.

Diet-Induced Obesity and Circadian Disruption of Feeding Behavior.

Feeding behavior shows a rhythmic daily pattern, which in nocturnal rodents is observed mainly during the dark period. This rhythmicity is under the influence of the hypothalamic suprachiasmatic nucleus (SCN), the main biological clock. Nevertheless, various studies have shown that in rodent models of obesity, using high-energy diets, the general locomotor activity and feeding rhythms can be disrupted. Here, we review the data on the effects of diet-induced obesity (DIO) on locomotor activity and feeding patterns, as well as the effect on the brain sites within the neural circuitry involved in metabolic and rewarding feeding behavior. In general, DIO may alter locomotor activity by decreasing total activity. On the other hand, DIO largely alters eating patterns, producing increased overall ingestion and number of eating bouts that can extend to the resting period. Furthermore, within the hypothalamic areas, little effect has been reported on the molecular circadian mechanism in DIO animals with ad libitum hypercaloric diets and little or no data exist so far on its effects on the reward system areas. We further discuss the possibility of an uncoupling of metabolic and reward systems in DIO and highlight a gap of circadian and metabolic research that may help to better understand the implications of obesity.

Octagonal CMOS Image Sensor for Endoscopic Applications

An octagonal shaped CMOS image sensor (CIS) has been developed for endoscopic applications. The octagonal shape of the outlined die is also applied to the pixel matrix which has suppressed corners, resulting in an octagonal pixel matrix of 422'640 effective pixels. The sensor die fits on a circumference of 3.2 mm diameter being this the best die cut ratio between the cut corners and the pixel placement. The missing pixels on the corners are internally compensated by the readout electronics to maintain a rectangular image output format of 680x680 pixels. This paper presents the solutions found to overcome the layout challenges of fitting both row and column logic on the same side of the diagonally cut corners. Furthermore, the sensor features an area efficient column parallel ADC converter, having on the column readout side, only 402 μm overhead total space between the utmost pixel row until the edge of the silicon die. This space is used to place CDS, ADC, column addressing, readout, additional electronics and power routing. The sensor also has a fully autonomous operation, requiring only an external clock supply to drive the internal state machine. The interface requires just 6 connections: 2 for power supply, 2 for bidirectional data and 2 for main clock supply. The frame rate and the exposure are configurable by register. The maximum frame rate is 50 frames per second (FPS) and data is delivered over LVDS serial data link. The integration time can be configured from 147 μs to 20.1 ms. The power supply is 3.3 V with internal Power-On-Reset (POR) and has a power consumption of 91 mW manufactured in 180 nm CIS process.