The timing of eukaryotic evolution: Does a relaxed molecular clock reconcile proteins and fossils?

The use of nucleotide and amino acid sequences allows improved understanding of the timing of evolutionary events of life on earth. Molecular estimates of divergence times are, however, controversial and are generally much more ancient than suggested by the fossil record. The limited number of genes and species explored and pervasive variations in evolutionary rates are the most likely sources of such discrepancies. Here we compared concatenated amino acid sequences of 129 proteins from 36 eukaryotes to determine the divergence times of several major clades, including animals, fungi, plants, and various protists. Due to significant variations in their evolutionary rates, and to handle the uncertainty of the fossil record, we used a Bayesian relaxed molecular clock simultaneously calibrated by six paleontological constraints. We show that, according to 95% credibility intervals, the eukaryotic kingdoms diversified 950-1,259 million years ago (Mya), animals diverged from choanoflagellates 761-957 Mya, and the debated age of the split between protostomes and deuterostomes occurred 642-761 Mya. The divergence times appeared to be robust with respect to prior assumptions and paleontological calibrations. Interestingly, these relaxed clock time estimates are much more recent than those obtained under the assumption of a global molecular clock, yet bilaterian diversification appears to be approximate to100 million years more ancient than the Cambrian boundary.

Characterisation of an amphioxus Fringe gene and the evolution of the vertebrate segmentation clock

In mouse and chick embryos, cyclic expression of lunatic fringe has an important role in the regulation of mesoderm segmentation. We have isolated a Fringe gene from the protochordate amphioxus. Amphioxus is the closest living relative of the vertebrates, and has mesoderm that is definitively segmented in a manner that is similar to, and probably homologous with, that of vertebrates. AmphiFringe is placed basal to vertebrate Fringe genes in molecular phylogenetic analyses, indicating that the duplications that formed radical-, manic- and lunatic fringe are specific to the vertebrate lineage. AmphiFringe expression was detected in the anterior neural plate of early neurulae, where it resolved into a series of segmental patches by the mid-neurulae stage. No AmphiFringe transcripts were detected in the mesoderm. Based on these observations, we propose a model depicting a successive recruitment of Fringe in the maintenance then regulation of segmentation during vertebrate evolution.

A low clock frequency FFT core implementation for multiband full-rate ultra-wideband (UWB) receivers

This paper discusses the design, implementation and synthesis of an FFT module that has been specifically optimized for use in the OFDM based Multiband UWB system, although the work is generally applicable to many other OFDM based receiver systems. Previous work has detailed the requirements for the receiver FFT module within the Multiband UWB ODFM based system and this paper draws on those requirements coupled with modern digital architecture principles and low power design criteria to converge on our optimized solution particularly aimed at a low-clock rate implementation. The FFT design obtained in this paper is also applicable for implementation of the transmitter IFFT module therefore only needing one FFT module in the device for half-duplex operation. The results from this paper enable the baseband designers of the 200Mbit/sec variant of Multiband UWB systems (and indeed other OFDM based receivers) using System-on-Chip (SoC), FPGA and ASIC technology to create cost effective and low power consumer electronics product solutions biased toward the very competitive market.

Modeling the observed proton aurora and ionospheric convection responses to changes in the IMF clock angle: 2. Persistence of ionospheric convection

We apply a numerical model of time-dependent ionospheric convection to two directly driven reconnection pulses during a 15-min interval of southward IMF on 26 November 2000. The model requires an input magnetopause reconnection rate variation, which is here derived from the observed variation in the upstream IMF clock angle, q. The reconnection rate is mapped to an ionospheric merging gap, the MLT extent of which is inferred from the Doppler-shifted Lyman-a emission on newly opened field lines, as observed by the FUV instrument on the IMAGE spacecraft. The model is used to reproduce a variety of features observed during this event: SuperDARN observations of the ionospheric convection pattern and transpolar voltage; FUV observations of the growth of patches of newly opened flux; FUVand in situ observations of the location of the Open-Closed field line Boundary (OCB) and a cusp ion step. We adopt a clock angle dependence of the magnetopause reconnection electric field, mapped to the ionosphere, of the form Enosin4(q/2) and estimate the peak value, Eno, by matching observed and modeled variations of both the latitude, LOCB, of the dayside OCB (as inferred from the equatorward edge of cusp proton emissions seen by FUV) and the transpolar voltage FPC (as derived using the mapped potential technique from SuperDARN HF radar data). This analysis also yields the time constant tOCB with which the open-closed boundary relaxes back toward its equilibrium configuration. For the case studied here, we find tOCB = 9.7 ± 1.3 min, consistent with previous inferences from the observed response of ionospheric flow to southward turnings of the IMF. The analysis confirms quantitatively the concepts of ionospheric flow excitation on which the model is based and explains some otherwise anomalous features of the cusp precipitation morphology.

Modeling the observed proton aurora and ionospheric convection responses to changes in the IMF clock angle: 1. Persistence of cusp proton aurora

We employ a numerical model of cusp ion precipitation and proton aurora emission to fit variations of the peak Doppler-shifted Lyman-a intensity observed on 26 November 2000 by the SI-12 channel of the FUV instrument on the IMAGE satellite. The major features of this event appeared in response to two brief swings of the interplanetary magnetic field (IMF) toward a southward orientation. We reproduce the observed spatial distributions of this emission on newly opened field lines by combining the proton emission model with a model of the response of ionospheric convection. The simulations are based on the observed variations of the solar wind proton temperature and concentration and the interplanetary magnetic field clock angle. They also allow for the efficiency, sampling rate, integration time and spatial resolution of the FUV instrument. The good match (correlation coefficient 0.91, significant at the 98% level) between observed and modeled variations confirms the time constant (about 4 min) for the rise and decay of the proton emissions predicted by the model for southward IMF conditions. The implications for the detection of pulsed magnetopause reconnection using proton aurora are discussed for a range of interplanetary conditions.

Clock Genes, Melanopsins, Melatonin, and Dopamine Key Enzymes and Their Modulation by Light and Glutamate in Chicken Embryonic Retinal Cells

The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 mu M glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 mu M glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. (Author correspondence: amdlcast@ib.usp.br).

Melanopsin and Clock Genes: Regulation by Light and Endothelin in the Zebrafish ZEM-2S Cell Line

It is well known that clocks are present in brain regions other than the suprachiasmatic nucleus and in many peripheral tissues. In the teleost, Danio rerio, peripheral oscillators can be directly synchronized by light. Danio rerio ZEM-2S embryonic cells respond to light with differential growth: cells kept in constant light exhibited a strong inhibition of proliferation, whereas in cells kept in light:dark (LD) cycles (14L:10D and 10L:14D) or in constant darkness (DD), the doubling times were not statistically different. We demonstrated by RT-PCR followed by PCR that ZEM-2S cells express two melanopsins, Opn4x and Opn4m, and the six Cry genes. The presence of the protein OPN4x was demonstrated by immunocytochemistry. The pattern of temporal expression of the genes Opn4x, Per1, Cry1b, and Clock was studied in ZEM-2S cells kept for five days in 12L:12D or DD. In 12L:12D, the clock genes Per 1 and Cry1b exhibited robust circadian expression, while Opn4x and Clock expression seemed to vary in an ultradian pattern. Both Per1 and Cry1b genes had higher expression during the L phase; Clock gene had an increase in expression coincident with the D phase, and during the subjective night. In DD, the temporal variation of Per1 and Cry1b genes was greatly attenuated but not extinguished, and the higher expressions were shifted to the transition times between subjective day and night, demonstrating that Per and Cry1b were synchronized by the LD cycle. Clock and Opn4x kept the ultradian oscillation, but the rhythm was not statistically significant. As endothelins (ET) have been reported to be a potent stimulator of Per genes in rodents, we investigated the effect of endothelin on ZEM-2S cells, which express ETA receptors. Cells were kept in 12D:12L for five days, and then treated with 10-11 to 10-8M ET-1 for 24h. ET-1 exhibited a biphasic effect on Opn4x expression. At 10-11M, the hormone exerted a highly significant stimulation of Opn4x expression during the L phase and introduced a circadian oscillatory pattern. At 10-10M, a significant increase was seen at ZT21 and ZT0 (i.e., at the end of the D phase and beginning of the L phase), whereas 10-9 and 10-8M ET-1 inhibited the expression of Opn4x at most ZTs. Clock expression was unaffected by 10-8M ET-1; however, in the presence of lower concentrations, the expression was enhanced at some ZTs, strengthening the ultradian oscillation. ET-1 at 10-11 and 10-10M had no effect on Per1 circadian expression; however, 10-9 and 10-8M ET-1 reduced the amplitude of Per1 expression in the beginning of the L phase. ET-1 effects were less evident on Cry 1b. For both genes, the reduction in expression was not sufficient to abolish the circadian oscillatory pattern. Based on these results and data in the literature, a link between ET-1 stimulation of ETA receptors may be established by E4BP4 binding to the promoters and consequent inhibition of gene expression.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Disruption of the circadian clock within the cardiomyocyte influences myocardial contractile function, metabolism, and gene expression

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology. We, therefore, generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse to test this hypothesis. At 12 wk of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography. Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities). Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype. Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80 mmHg plus 1 mu M epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase vs. the light (inactive) phase. Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, whereas cardiac efficiency was decreased, in CCM hearts. These observations were associated with no alterations in mitochondrial content or structure and modest mitochondrial dysfunction in CCM hearts. Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice. These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Laser locking to the Hg-199 S-1(0)-P-3(0) clock transition with 5.4x10(-15)/root tau fractional frequency instability

With Hg-199 atoms confined in an optical lattice trap in the Lamb-Dicke regime, we obtain a spectral line at 265.6 nm for which the FWHM is similar to 15 Hz. Here we lock an ultrastable laser to this ultranarrow S-1(0) - P-3(0) clock transition and achieve a fractional frequency instability of 5.4 x 10(-15) / root tau for tau <= 400 s. The highly stable laser light used for the atom probing is derived from a 1062.6 nm fiber laser locked to an ultrastable optical cavity that exhibits a mean drift rate of -6.0 x 10(-17) s-(1) (-16.9 mHzs(-1) at 282 THz) over a six month period. A comparison between two such lasers locked to independent optical cavities shows a flicker noise limited fractional frequency instability of 4 x 10(-16) per cavity. (c) 2012 Optical Society of America

Architectures, stability and optimization for clock distribution networks

Synchronous telecommunication networks, distributed control systems and integrated circuits have its accuracy of operation dependent on the existence of a reliable time basis signal extracted from the line data stream and acquirable to each node. In this sense, the existence of a sub-network (inside the main network) dedicated to the distribution of the clock signals is crucially important. There are different solutions for the architecture of the time distribution sub-network and choosing one of them depends on cost, precision, reliability and operational security. In this work we expose: (i) the possible time distribution networks and their usual topologies and arrangements. (ii) How parameters of the network nodes can affect the reachability and stability of the synchronous state of a network. (iii) Optimizations methods for synchronous networks which can provide low cost architectures with operational precision, reliability and security. (C) 2011 Elsevier B. V. All rights reserved.

Modeling the Emergence of Circadian Rhythms in a Clock Neuron Network

Circadian rhythms in pacemaker cells persist for weeks in constant darkness, while in other types of cells the molecular oscillations that underlie circadian rhythms damp rapidly under the same conditions. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms leading to damped or self-sustained oscillations remain largely unknown. There exist many mathematical models that reproduce the circadian rhythms in the case of a single cell of the Drosophila fly. However, not much is known about the mechanisms leading to coherent circadian oscillation in clock neuron networks. In this work we have implemented a model for a network of interacting clock neurons to describe the emergence (or damping) of circadian rhythms in Drosophila fly, in the absence of zeitgebers. Our model consists of an array of pacemakers that interact through the modulation of some parameters by a network feedback. The individual pacemakers are described by a well-known biochemical model for circadian oscillation, to which we have added degradation of PER protein by light and multiplicative noise. The network feedback is the PER protein level averaged over the whole network. In particular, we have investigated the effect of modulation of the parameters associated with (i) the control of net entrance of PER into the nucleus and (ii) the non-photic degradation of PER. Our results indicate that the modulation of PER entrance into the nucleus allows the synchronization of clock neurons, leading to coherent circadian oscillations under constant dark condition. On the other hand, the modulation of non-photic degradation cannot reset the phases of individual clocks subjected to intrinsic biochemical noise.