Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse liver.

Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known about the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological light-dark conditions using stable isotope labeling by amino acids quantitative MS. Our analysis identified over 5,000 proteins, of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-life data. Moreover, a group comprising about one-half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or posttranslational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock-deficient animals subjected to rhythmic feeding, suggesting that food-related entrainment signals influence rhythms in circulating plasma factors.

Role of CLOCK and circadian rhythms in calcium homeostasis

Le maintien d'une concentration sanguine constante de calcium est d'une importance cruciale et trois organes participent à la balance calcique normale : les reins, les intestins et les os. La concentration plasmatique de calcium est strictement régulée par l'hormone parathyroïdienne (PTH) et par la vitamine D. Des variations circadiennes de la PTH, de la vitamine D ainsi que du calcium plasmatique ont été décrites précédemment chez l'humain ainsi que chez le rat. Ces rythmes de PTH dans le sérum sont importants pour la régulation du remodelage de l'os. En effet, il a été montré chez les souris C57BL/6J que des injections de PTH une fois par jour mènent à une augmentation de la densité minérale de l'os alors que l'infusion en continu de PTH est associée à une diminution de cette densité. La vitamine D joue également un rôle fondamental dans la physiologie osseuse, car un déficit en vitamine D peut conduire à une ostéomalacie. Cependant la fonction des oscillations de vitamine D au niveau de l'homéostasie osseuse reste inconnue. L'horloge circadienne est un système interne de contrôle biologique du temps générant des rythmes de 24 heures dans l'expression des gènes, ainsi que dans la physiologie et le comportement. Ce contrôle s'opère par des boucles rétroactives positives et négatives de l'expression de gènes circadiens tels que CLOCK, BMAL1, CRY1 et 2 ou PERI et 2. Dans ce travail, nous avons émis l'hypothèse que l'homéostasie calcique est sous le contrôle de l'horloge circadienne. Dans un premier temps, nous avons montré chez les souris C57BL/6J des variations journalières des concentrations de calcium, de PTH et de vitamine D dans le sang, ainsi que de calcium dans les urines. Nous avons également démontré des changements au niveau de l'expression rénale des gènes importants dans l'homéostasie du calcium, tant au niveau de l'ARN messager que des protéines. Ensuite, pour analyser le rôle du système de l'horloge circadienne dans l'homéostasie du calcium, nous avons étudié des souris dans lesquelles a été supprimé le gène CLOCK crucial pour la fonction de l'horloge et nous avons comparé ces souris à des souris de type sauvage de même portée. Les souris CLOCK-I- étaient hypercalciuriques à chaque moment de la journée. Cependant le rythme circadien de l'excrétion de calcium était préservé. Le taux de calcium plasmatique ne différait pas entre les génotypes, mais les souris CLOCK -/- ne montraient pas de variations journalières de ce paramètre. Une perte du rythme journalier était également observée pour les niveaux de vitamine D, perte qui pourrait être une cause de l'altération de la micro-architecture osseuse révélée chez les souris CLOCK-/-. En effet, ces souris montrent une diminution du nombre de trabécules, de leur volume ainsi que de leur surface, ce qui suggère la présence d'ostéoporose. Nous avons également trouvé que le rythme de l'expression de l'ARN messager de CYP27B1 était aboli dans les reins des souris CLOCK -/-, ce qui peut expliquer l'altération du rythme de la vitamine D. Les taux sanguins de PTH étaient comparables entre les souris CLOCK -/- et de type sauvage. Dans les reins, une augmentation de l'expression de l'ARN messager de TRPV5 et NCX1 a été constatée, ce qui suggérerait une augmentation de la réabsorption de calcium dans le tubule convoluté distal et dans le tubule connecteur. Dans les intestins, la réabsorption calcique était diminuée, chez les souris CLOCK-I-, fait confirmé par une diminution des niveaux d'ARN messager de TRPV6 et PMCAL. En résumé, la suppression du gène CLOCK chez les souris a conduit à une hypercalciurie, une altération du rythme des taux plasmatiques de calcium et de vitamine D et à une détérioration de l'architecture osseuse. Pour conclure, ces résultats montrent que l'horloge circadienne est essentielle à l'homéostasie calcique ainsi qu'à la physiologie des os. - L'ostéoporose affecte environ 22 millions de femmes et 5.5 millions d'hommes en Europe, réduisant significativement leur qualité de vie et a causé 3.5 millions de nouvelles fractures en 2010. Les dépenses totales liées à ces fractures ont atteint 37 milliards d'euro et ce coût devrait augmenter de 25% d'ici à 2025. Le nombre de nouvelles fractures dues à l'ostéoporose à travers le monde est estimé à environ 1000 par heure. Parmi les causes de l'ostéoporose, le déficit én calcium et/ou en vitamine D joue un rôle important, mais il existe également des causes génétiques ou liées à des facteurs comme les hormones sexuelles (estrogènes, testostérone), l'âge, le tabac, le poids corporel, certains médicaments,... La vie est rythmique : ceci est dû à l'alternance naturelle du jour et de la nuit et de ses effets sur le corps. La prise alimentaire, par exemple, est un processus qui a lieu pendant la phase active, qui est prévisible (il se produit toujours au même moment) et qui peut être anticipé par le corps. Pour cela, une horloge interne est présente dans chaque cellule du corps et est synchronisée par la lumière du jour, entre autres stimuli. Cette horloge indique la phase du jour et régule l'expression de gènes impliqués dans les différents processus qui nécessitent une anticipation. Pendant mon travail de thèse, je me suis demandé si des îythmes circadiens (c'est-à-dire d'une durée d'environ 24 heures et indépendants des stimuli externes) étaient observables'pour les gènes régulant les flux de calcium dans le corps et si l'interruption de ces rythmes pouvait mener à des altérations de la qualité de l'os. J'ai d'abord travaillé avec des souris normales et j'ai pu montrer la présence de rythmes au niveau du calcium sanguin et urinaire, mais également au niveau des hormones et gènes qui contrôlent le métabolisme du calcium dans le corps, comme la vitamine D et l'hormone parathyroidienne. De manière intéressante, j'ai observé que la plupart de ces gènes ont un rythme synchronisé. J'ai ensuite utilisé un modèle de souris dans lequel l'horloge interne a été génétiquement invalidée et j'ai montré que ces souris présentent une augmentation de leur excrétion urinaire de calcium et un rythme circadien altéré de la vitamine D dans le sang. Ces souris absorbent aussi moins bien le calcium intestinal et présentent une ostéoporose marquée. Ce travail montre donc que l'horloge interne est nécessaire pour établir un rythme circadiens de certains facteurs influant les flux de calcium dans l'organisme, comme la vitamine D, et que la perturbation de ces rythmes mène à une dérégulation du métabolisme osseux. Ainsi, la perturbation de l'horloge interne peut causer une ostéoporose et une hypercalciurie qui pourraient aboutir à la formation de fractures et de calculs rénaux. L'extrapolation de ces observations chez l'homme ou à des changements plus subtiles des rythmes circadiens, comme le décalage horaire, restent à montrer. Cette recherche a démontré que les rythmes circadiens des mécanismes de régulation des flux de calcium dans l'organisme sont essentiels au maintien d'un squelette normal et suggère que les perturbations des rythmes circadiens pourraient être une nouvelle cause de l'ostéoporose. - Maintaining constant calcium concentration in the plasma is of a crucial importance and three organs participate in normal calcium balance - kidney, gut and bone. Plasma calcium concentration is strictly regulated by parathyroid hormone (PTH) and vitamin D. Circadian variations of PTH, vitamin D and plasma calcium were previously described in humans, as well as in rats. Rhythms in serum PTH are important for balanced bone remodelling. Indeed in C57BL/6J mice, PTH injection once per day leads to an increase in bone mineral density (BMD), whilst continuous infusion is associated with decreased BMD. Vitamin D also plays a crucial role in bone physiology, since the deficiency in vitamin D can lead to rickets/osteomalacia. However, the role of vitamin D rhythms in bone homeostasis remains unknown. The circadian clock is an. internal time-keeping system generating rhythms in gene expression with 24h periodicity, in physiology and in behaviour. It is operated by positive- and negative-feedback loops of circadian genes, such as CLOCK, BMAL1, CRY1 and 2 or PERI and 2. In this work, we hypothesized, that calcium homeostasis is under the control of the circadian clock. First, we showed daily variations in urinary calcium and serum calcium, PTH and l,25(OH)2 vitamin D, together with renal mRNA and protein levels of genes involved in calcium homeostasis in C57BL/6J mice. Second, and to investigate the role of the circadian clock system in calcium handling, we studied mice lacking the gene CLOCK crucial for fonction of the clock system and compared them to the WT littermates. CLOCK-/- mice were hypercalciuric at all timepoints of the day. However, the circadian rhythm of calcium excretion was preserved. Serum calcium levels did not differ between the genotypes, but CLOCK-/- mice did not exhibit daily variation for this parameter. Loss of rhythm was observed also for serum l,25(OH)2 vitamin D levels, which may be one of the causes of altered bone microarchitecture that was revealed in CLOCK-/- mice. They displayed increased trabecular separation and decreased trabecular number, trabecular bone volume and trabecular bone surface, suggestive of osteoporosis. We found that the rhythm of the mRNA expression of CYP27B1 was abolished in the kidney of CLOCK-/- mice, which could induce the altered rhythm of l,25(OH)2 vitamin. Serum PTH levels were comparable between CLOCK-/- and WT mice. In the kidney, increased mRNA expression of TRPV5 and NCX1 suggests increased calcium reabsorption in the distal convoluted and connecting tubule. In the gut, intestinal calcium absorption was decreased in CLOCK¬/- mice, confirmed by decreased mRNA levels of TRPV6 and PMCA1. In summary, deletion of the CLOCK gene in mice conducts to hypercalciuria, alteration of the rhythm in serum calcium and l,25(OH)2D levels, and impainnent of their bone microarchitecture. In conclusion, these data show that the circadian clock system is essential in calcium homeostasis and bone physiology.

Circadian clock orchestration of signaling pathways influences mouse metabolism

Circadian clocks, present in organisms leaving in a rhythmic environment, constitute the mechanisms allowing anticipation and adaptation of behavior and physiology in response to these environmental variations. As a consequence, most aspects of metabolism and behavior are under the control of this circadian clock. At a molecular level, in all the studied species, the rhythmic expression of the genes involved are generated by interconnected transcriptional and translational feedback loops. In mammals, the heterodimer composed of BMAL1 and its partners CLOCK or NPAS2 constitutes a transcriptional activator regulating transcription of Per and Cry genes. These genes encode for repressors of the activity of BMAL1:CLOCK or BMAL1: NPAS2 heterodimers, thus closing a negative feedback loop that generates rhythms of approximately 24 hours. The aim of my doctoral work consisted in the investigation of the role of circadian clock in the regulation of different aspects of mouse metabolism through the rhythmic activation of signaling pathways. First, we showed that one way how the circadian clock exerts its function as an oscillator is through the regulation of mRNA translation. Indeed, we present evidence showing that circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. In the second part, we showed the involvement of the circadian clock in lipid metabolism. Indeed, the three PAR bZip transcription factors DBP, TEF and HLF, are regulated by the molecular clock and play key roles in the control of lipid metabolism. Here we present evidence concerning the circadian expression and activity of PPARα via the circadian transcription of genes involved in the release of fatty acids, natural ligands of PPARα. It leads to the rhythmic activation of PPARα itself which could then play its role in the transcription of genes encoding proteins involved in lipid, cholesterol and glucose metabolism. In addition, we considered the possible role of lipid transporters, here SCP2, in the modulation of circadian activation of signaling pathways such as TORC1, PPARα and SREBP, linked to metabolism, and its feedback on the circadian clock. In the last part of this work, we studied the effects of these circadian clock-orchestrated pathways in physiology, as clock disruptions have been shown to be linked to metabolic disorders. We performed in vivo experiments on genetically and high-fat induced obese mice devoid of functional circadian clock. The results obtained showed that clock disruption leads to impaired triglycerides and glucose homeostasis in addition to insulin secretion and sensitivity. -- Les rythmes circadiens, présents chez tout organisme vivant dans un environnement rythmique, constituent l'ensemble de mécanismes permettant des réponses comportementales et physiologiques anticipées et adaptées aux variations environnementales. De ce fait, la plupart des aspects liés au métabolisme et au comportement de ces organismes apparaissent être sous le contrôle de l'horloge circadienne contrôlant ces rythmes. Au niveau moléculaire, dans toutes les espèces étudiées, l'expression rythmique de gènes impliqués sont générés par l'interconnexion de boucles de contrôle transcriptionnelles et traductionnelles. Chez les mammifères, l'hétérodimère composé de BMAL1 et de ses partenaires CLOCK ou NPAS2 constitue un activateur transcriptionnel régulant la transcription des gènes Per et Cry. Ces gènes codent pour des répresseurs de l'activité des hétérodimères BMAL1:CLOCK ou BMAL1:NPAS2. Cela a pour effet de fermer la boucle négative, générant ainsi des rythmes d'environ 24 heures. Le but de mon travail de thèse a consisté en l'investigation du rôle de l'horloge circadienne dans la régulation de certains aspects du métabolisme chez la souris via la régulation de l'activation rythmique des voies de signalisation. Nous avons tout d'abord montré que l'horloge circadienne exerce sa fonction d'oscillateur notamment au niveau de la régulation de la traduction des ARNm. En effet, nous présentons des preuves montrant que l'horloge circadienne influence la traduction temporelle d'un groupe d'ARNm impliqués dans la biogénèse des ribosomes en contrôlant la transcription de facteurs d'initiation de la traduction ainsi que l'activation rythmique des voies de signalisation qui sont impliquées dans leur régulation. De plus, l'oscillateur circadien régule la transcription d'ARNm codant pour les protéines ribosomales et d'ARN ribosomaux. De cette façon, l'horloge circadienne exerce un rôle majeur dans la coordination des étapes de transcription et traduction permettant la biogénèse des ribosomes. Dans la deuxième partie, nous montrons les implications de l'horloge circadienne dans le métabolisme des lipides. En effet, DBP, TEF et HLF, trois facteurs de transcription de la famille des PAR bZip qui sont régulés par l'horloge circadienne, jouent un rôle clé dans le contrôle du métabolisme des lipides par l'horloge circadienne. Nous apportons ici des preuves concernant l'expression et l'activité rythmiques de PPARα via la transcription circadienne de gènes impliqués dans le relargage d'acides gras, ligands naturels de PPARα, conduisant à l'activation circadienne de PPARα lui-même, pouvant ainsi jouer son rôle de facteur de transcription de gènes codant pour des protéines impliquées dans le métabolisme des lipides, du cholestérol et du glucose. De plus, nous nous sommes penchés sur le rôle possible de transporteurs de lipides, ici SCP2, dans la modulation de l'activation circadienne de voies de signalisation, telles que TORC1, PPARα et SREBP, qui sont liées au métabolisme, ainsi que son impact sur l'horloge elle-même. Dans la dernière partie de ce travail, nous avons étudié les effets de l'activation de ces voies de signalisation régulées par l'horloge circadienne dans le contexte physiologique puisqu'il a été montré que la perturbation de l'horloge pouvait être associée à des désordres métaboliques. Pour ce faire, nous avons fait des expériences in vivo sur des souris déficientes pour l'horloge moléculaire pour lesquelles l'obésité est induite génétiquement ou induite par la nourriture riche en lipides. Les résultats que nous obtenons montrent des dérèglements au niveau de l'homéostasie des triglycérides et du glucose ainsi que sur l'expression et la réponse à l'insuline.

Impact of round-the-clock CSF Gram stain on empirical therapy for suspected central nervous system infections.

The impact of round-the-clock cerebrospinal fluid (CSF) Gram stain on overnight empirical therapy for suspected central nervous system (CNS) infections was investigated. All consecutive overnight CSF Gram stains between 2006 and 2011 were included. The impact of a positive or a negative test on empirical therapy was evaluated and compared to other clinical and biological indications based on institutional guidelines. Bacterial CNS infection was documented in 51/241 suspected cases. Overnight CSF Gram stain was positive in 24/51. Upon validation, there were two false-positive and one false-negative results. The sensitivity and specificity were 41 and 99 %, respectively. All patients but one had other indications for empirical therapy than Gram stain alone. Upon obtaining the Gram result, empirical therapy was modified in 7/24, including the addition of an appropriate agent (1), addition of unnecessary agents (3) and simplification of unnecessary combination therapy (3/11). Among 74 cases with a negative CSF Gram stain and without formal indication for empirical therapy, antibiotics were withheld in only 29. Round-the-clock CSF Gram stain had a low impact on overnight empirical therapy for suspected CNS infections and was associated with several misinterpretation errors. Clinicians showed little confidence in CSF direct examination for simplifying or withholding therapy before definite microbiological results.

Twist1 Is a TNF-Inducible Inhibitor of Clock Mediated Activation of Period Genes.

BACKGROUND: Activation of the immune system affects the circadian clock. Tumor necrosis factor (TNF) and Interleukin (IL)-1β inhibit the expression of clock genes including Period (Per) genes and the PAR-bZip clock-controlled gene D-site albumin promoter-binding protein (Dbp). These effects are due to cytokine-induced interference of E-box mediated transcription of clock genes. In the present study we have assessed the two E-box binding transcriptional regulators Twist1 and Twist2 for their role in cytokine induced inhibition of clock genes. METHODS: The expression of the clock genes Per1, Per2, Per3 and of Dbp was assessed in NIH-3T3 mouse fibroblasts and the mouse hippocampal neuronal cell line HT22. Cells were treated for 4h with TNF and IL-1β. The functional role of Twist1 and Twist2 was assessed by siRNAs against the Twist genes and by overexpression of TWIST proteins. In luciferase (luc) assays NIH-3T3 cells were transfected with reporter gene constructs, which contain a 3xPer1 E-box or a Dbp E-box. Quantitative chromatin immunoprecipitation (ChIP) was performed using antibodies to TWIST1 and CLOCK, and the E-box consensus sequences of Dbp (CATGTG) and Per1 E-box (CACGTG). RESULTS: We report here that siRNA against Twist1 protects NIH-3T3 cells and HT22 cells from down-regulation of Period and Dbp by TNF and IL-1β. Overexpression of Twist1, but not of Twist2, mimics the effect of the cytokines. TNF down-regulates the activation of Per1-3xE-box-luc, the effect being prevented by siRNA against Twist1. Overexpression of Twist1, but not of Twist2, inhibits Per1-3xE-box-luc or Dbp-E-Box-luc activity. ChIP experiments show TWIST1 induction by TNF to compete with CLOCK binding to the E-box of Period genes and Dbp. CONCLUSION: Twist1 plays a pivotal role in the TNF mediated suppression of E-box dependent transactivation of Period genes and Dbp. Thereby Twist1 may provide a link between the immune system and the circadian timing system.

Ribosome profiling reveals the rhythmic liver translatome and circadian clock regulation by upstream open reading frames.

Mammalian gene expression displays widespread circadian oscillations. Rhythmic transcription underlies the core clock mechanism, but it cannot explain numerous observations made at the level of protein rhythmicity. We have used ribosome profiling in mouse liver to measure the translation of mRNAs into protein around the clock and at high temporal and nucleotide resolution. We discovered, transcriptome-wide, extensive rhythms in ribosome occupancy and identified a core set of approximately 150 mRNAs subject to particularly robust daily changes in translation efficiency. Cycling proteins produced from nonoscillating transcripts revealed thus-far-unknown rhythmic regulation associated with specific pathways (notably in iron metabolism, through the rhythmic translation of transcripts containing iron responsive elements), and indicated feedback to the rhythmic transcriptome through novel rhythmic transcription factors. Moreover, estimates of relative levels of core clock protein biosynthesis that we deduced from the data explained known features of the circadian clock better than did mRNA expression alone. Finally, we identified uORF translation as a novel regulatory mechanism within the clock circuitry. Consistent with the occurrence of translated uORFs in several core clock transcripts, loss-of-function of Denr, a known regulator of reinitiation after uORF usage and of ribosome recycling, led to circadian period shortening in cells. In summary, our data offer a framework for understanding the dynamics of translational regulation, circadian gene expression, and metabolic control in a solid mammalian organ.

Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature.

Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. Synchronized circadian clocks improve fitness and are crucial for our physical and mental well-being. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light, but clock-resetting is also achieved by alternating day and night temperatures with only 2-4 °C difference. This temperature sensitivity is remarkable considering that the circadian clock period (~24 h) is largely independent of surrounding ambient temperatures. Here we show that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. We propose that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna, revealing the existence of novel periphery-to-brain temperature signalling channels.

Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals.

The liver is a key organ of metabolic homeostasis with functions that oscillate in response to food intake. Although liver and gut microbiome crosstalk has been reported, microbiome-mediated effects on peripheral circadian clocks and their output genes are less well known. Here, we report that germ-free (GF) mice display altered daily oscillation of clock gene expression with a concomitant change in the expression of clock output regulators. Mice exposed to microbes typically exhibit characterized activities of nuclear receptors, some of which (PPARα, LXRβ) regulate specific liver gene expression networks, but these activities are profoundly changed in GF mice. These alterations in microbiome-sensitive gene expression patterns are associated with daily alterations in lipid, glucose, and xenobiotic metabolism, protein turnover, and redox balance, as revealed by hepatic metabolome analyses. Moreover, at the systemic level, daily changes in the abundance of biomarkers such as HDL cholesterol, free fatty acids, FGF21, bilirubin, and lactate depend on the microbiome. Altogether, our results indicate that the microbiome is required for integration of liver clock oscillations that tune output activators and their effectors, thereby regulating metabolic gene expression for optimal liver function.

Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals.

The liver is a key organ of metabolic homeostasis with functions that oscillate in response to food intake. Although liver and gut microbiome crosstalk has been reported, microbiome-mediated effects on peripheral circadian clocks and their output genes are less well known. Here, we report that germ-free (GF) mice display altered daily oscillation of clock gene expression with a concomitant change in the expression of clock output regulators. Mice exposed to microbes typically exhibit characterized activities of nuclear receptors, some of which (PPARα, LXRβ) regulate specific liver gene expression networks, but these activities are profoundly changed in GF mice. These alterations in microbiome-sensitive gene expression patterns are associated with daily alterations in lipid, glucose, and xenobiotic metabolism, protein turnover, and redox balance, as revealed by hepatic metabolome analyses. Moreover, at the systemic level, daily changes in the abundance of biomarkers such as HDL cholesterol, free fatty acids, FGF21, bilirubin, and lactate depend on the microbiome. Altogether, our results indicate that the microbiome is required for integration of liver clock oscillations that tune output activators and their effectors, thereby regulating metabolic gene expression for optimal liver function.

Physiological and Genetic Bases of the Circadian Clock in Plants and Their Relationship with Herbicides Efficacy

In order to adapt to daily environmental changes, especially in relation to light availability, many organisms, such as plants, developed a vital mechanism that controls time-dependent biological events: the circadian clock. The circadian clock is responsible for predicting the changes that occur in the period of approximately 24 hours, preparing the plants for the following phases of the cycle. Some of these adaptations can influence the response of weeds to the herbicide application. Thus, the objectives of this review are to describe the physiological and genetic mechanisms of the circadian clock in plants, as well as to demonstrate the relationship of this phenomenon with the effectiveness of herbicides for weed control. Relationships are described between the circadian clock and the time of application of herbicides, leaf angle and herbicide interception, as well as photosynthetic activity in response to the circadian clock and herbicide efficiency. Further, it is discussed the role of phytochrome B (phyB) in the sensitivity of plants to glyphosate herbicide. The greater understanding of the circadian clock in plants is essential to achieve greater efficiency of herbicides and hence greater control of weeds and higher crop yields.

The search for circadian clock components in humans: new perspectives for association studies

Individual circadian clocks entrain differently to environmental cycles (zeitgebers, e.g., light and darkness), earlier or later within the day, leading to different chronotypes. In human populations, the distribution of chronotypes forms a bell-shaped curve, with the extreme early and late types _ larks and owls, respectively _ at its ends. Human chronotype, which can be assessed by the timing of an individual's sleep-wake cycle, is partly influenced by genetic factors - known from animal experimentation. Here, we review population genetic studies which have used a questionnaire probing individual daily timing preference for associations with polymorphisms in clock genes. We discuss their inherent limitations and suggest an alternative approach combining a short questionnaire (Munich ChronoType Questionnaire, MCTQ), which assesses chronotype in a quantitative manner, with a genome-wide analysis (GWA). The advantages of these methods in comparison to assessing time-of-day preferences and single nucleotide polymorphism genotyping are discussed. In the future, global studies of chronotype using the MCTQ and GWA may also contribute to understanding the influence of seasons, latitude (e.g., different photoperiods), and climate on allele frequencies and chronotype distribution in different populations.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Important state papers : declaration of war, Washington, June 18, 4 o'clock, P.M.

Message to the Senate and House of Representatives of the United States / James Madison -- Report : the Committee on Foreign relations, to whom was referred the message of the President of the United States of the 1st of June, 1812 -- An Act, declaring war between the United Kingdom of Great Britain and Ireland and the dependencies thereof, and the United States of America and their territories -- Address of the Senate to the people of the Commonwealth of Massachusetts.

Important state papers : declaration of war, Washington, June 18, 4 o'clock, P.M

Message to the Senate and House of Representatives of the United States / James Madison -- Report : the Committee on Foreign relations, to whom was referred the message of the President of the United States of the 1st of June, 1812 -- An Act, declaring war between the United Kingdom of Great Britain and Ireland and the dependencies thereof, and the United States of America and their territories -- Address of the Senate to the people of the Commonwealth of Massachusetts.