Associations of postoperative mortality with the time of day, week and year.

Studies that have investigated circadian, weekday and seasonal variation in postoperative mortality have been relatively small or have been for scheduled surgery. We retrospectively tested a large mixed surgical cohort from a German tertiary care university hospital for the presence of cyclical variation in all-cause in-hospital mortality after operations performed between 2006 and 2013. We analysed mortality rates after 247,475 operations, adjusted for age, sex, comorbidities, location, urgency and duration of the surgery, and intra-operative blood transfusions. The mortality odds ratio (95%CI) after operations started in the morning (08:00-11:00) were lowest, 0.73 (0.66-0.80), p < 0.001 and highest for operations started in the afternoon (13:00-17:00), 1.29 (1.18-1.40), p < 0.001. Mortality at the weekend was the same as during the week. There was no seasonal variation in mortality, p = 0.12. However, the interference of four-yearly and ten-monthly cycle amplitudes resulted in higher mortality odds ratio (95%CI) in winter 2008-2009, 1.41 (1.18-1.69), p < 0.001, and lower mortality in spring 2011 and 2012, 0.70 (0.56-0.85) and 0.67 (0.53-0.85), p < 0.001 and p = 0.001, respectively. The ability to predict cyclical phenomena would facilitate the design of interventional studies, aimed at reducing mortality following surgery in the afternoon and when cycles interfere constructively.

A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.

Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism.

Circadian clocks: Omnes viae Romam ducunt.

The circadian clock in all organisms is so intimately linked to light reception that it appears as if evolution has simply wired a timer into the mechanism that processes photic information. Several recent studies have provided new insights into the role of light input pathways in the circadian system of Arabidopsis.

The circadian cycle: is the whole greater than the sum of its parts?

The term 'circadian rhythm' describes an oscillatory behavior in the absence of exogenous environmental cues, with a period of about a day. As yet, we don't fully understand which biological mechanisms join together to supply a stable and self-sustained oscillation with such a long period. By chipping away at the molecular mechanism with genetic approaches, some common features are emerging. In combining molecular analyses and physiological experiments, those features that are crucial for structuring a circadian day could be uncovered.

Light-induced phase responses in Gonyaulax are drastically altered by creatine.

The mammalian phosphagen creatine has been shown to shorten the circadian period (tau) of the bioluminescence rhythm in the marine alga Gonyaulax polyedra from 23 to 18 hr. The studies described here concern the interactive effects of creatine and light, mainly on the bioluminescence rhythm. We have found that the tau-shortening effect of creatine is greater in blue light, suggesting that it acts on a blue-sensitive light input pathway. In addition, creatine affects the phase response mechanism in Gonyaulax, which is also known to be mainly blue-sensitive. The responses to 4-hr light pulses are dramatically increased under the influence of creatine. The unusual phase response curve (PRC) of the Gonyaulax circadian system, which has no phase delays in the early night, is changed in the presence of creatine to a more typical type 0 PRC, with delays of up to 12 hr. Creatine also amplifies the cells' phototactic response, suggesting that the blue-sensitive light input pathway is shared by the phase-shifting mechanism of the bioluminescence oscillator and the mechanism responsible for phototaxis.

Molecular circadian oscillators: an alternative hypothesis.

Results from experiments in different organisms have shown that elements of input pathways can themselves be under circadian control and that outputs might feed back into the oscillator. In addition, it has become clear that there might be redundancies in the generation of circadian rhythmicity, even within single cells. In view of these results, it is worth reevaluating our current working hypotheses about the pacemaker's molecular mechanisms and the involvement of single autoregulatory genes. On one hand, redundancies in the generation of circadian rhythmicity might make the approach of defining a discrete circadian oscillator with the help of single gene mutations extremely difficult. On the other hand, many examples show that components of signal transduction pathways can indeed be encoded by single genes. The authors have constructed a model placing an autoregulatory gene and its products on an input pathway feeding into a separate oscillator. The behavior of this model can explain the majority of results of molecular circadian biology published to date. In addition, it shows that different qualities of the circadian system might be associated with different cellular functions that can exist independently and, only if put together, will lead to the known circadian phenotype.

Circadian systems and metabolism.

Circadian systems direct many metabolic parameters and, at the same time, they appear to be exquisitely shielded from metabolic variations. Although the recent decade of circadian research has brought insights into how circadian periodicity may be generated at the molecular level, little is known about the relationship between this molecular feedback loop and metabolism both at the cellular and at the organismic level. In this theoretical paper, we conjecture about the interdependence between circadian rhythmicity and metabolism. A mathematical model based on the chemical reactions of photosynthesis demonstrates that metabolism as such may generate rhythmicity in the circadian range. Two additional models look at the possible function of feedback loops outside of the circadian oscillator. These feedback loops contribute to the robustness and sustainability of circadian oscillations and to compensation for long- and short-term metabolic variations. The specific circadian property of temperature compensation is put into the context of metabolism. As such, it represents a general compensatory mechanism that shields the clock from metabolic variations.

Seasonality and photoperiodism in fungi.

This review gives a retrospective of what is known about photoperiodism in fungi, which is largely based on reports about seasonal spore concentrations. Relatively few species have been investigated under laboratory conditions, so that our knowledge whether seasonal reproduction in fungi is mainly a direct response to environmental conditions or whether it involves a photoperiodic machinery with memory capacities and a relationship to the circadian system is extremely limited. To form a basis for further experimental endeavors into fungal photoperiodism, we review the reports about endogenous rhythms and photobiology. Finally, we will look at the possibilities of using the fungal circadian model system of Neurospora crassa for future work on photoperiodism.

Annual rhythm of human reproduction: I. Biology, sociology, or both?

The annual rhythm of human reproduction was analyzed on the basis of more than 3000 years of monthly birth rates covering 166 regions of the globe. The following variables were used to characterize the annual rhythm of human conception (birth minus 9 months): amplitude, phase of maximum and minimum, phase and length of the time span when rates are above the annual mean (alpha), and the steepest upward slope (delta max) of the curve fitted to the rates. The waveform of the annual rhythm is characteristic for geographical regions (e.g., unimodal or bimodal) and persists as such for many years. In most countries, the onset of alpha coincides with delta max and lies close to the spring equinox. This phase of the rhythm is the most stable over time. In many populations, the rhythm has changed in recent years, specifically in amplitude and phase. The phase of the rhythm depends on latitude, with a 6-month difference between the Northern and Southern Hemispheres. The latitudinal distribution of amplitudes is less systematic. In spite of the many social influences on timing of conceptions, we conclude that the seasonal component in human reproduction is based on biological factors.

Annual rhythm of human reproduction: II. Environmental correlations.

Annual rhythms of human conception rates (based on a worldwide selection of statistics) were correlated with photoperiod, monthly averages of daily hours of sunshine, minimum and maximum temperature, and humidity. Our results show for the first time on a global scale that photoperiod, as shown for many animals, may also influence the physiology of human reproduction. At higher latitudes, where changes in daylength are pronounced, a steep increase in human conceptions coincides with the vernal equinox. Temperature also appears to be a major influencing factor. Conception rates are above the annual mean at temperatures between 5 degrees and 20 degrees C, and temperature extremes decrease the probability of conceptions. In regions with cold winters and moderate summers, the number of conceptions correlates positively with temperature; close to the equator, where winters are more moderate than the hot summers, this correlation is negative. Regions with both hot summers and cold winters tend to have a bimodal conception rhythm. The influence of photoperiod is dominant before 1930, whereas the impact of temperature dominates in later years. With industrialization, people are increasingly shielded from both photoperiod (by indoor work) and temperature (by heating and air conditioning), which may explain the deseasonalization of the human conception rhythm. Photoperiod and temperature and the specific changes in their impact can account for several features of the annual human conception rhythm: latitude dependence, waveform, phase and amplitude, and their specific changes over time.

Phased protein synthesis at several circadian times does not change protein levels in Gonyaulax.

The synthesis rates of 13 individual proteins in Gonyaulax polyedra, resolved by two-dimensional gel electrophoresis, were estimated from the amount of 35S methionine incorporated during in vivo pulse labeling at 2-h intervals over one circadian period. The synthesis rates of three proteins, taken as controls, varied 2-3 fold, and no systematic pattern to these variations was apparent. In contrast, the synthesis rates of 10 other proteins varied at least tenfold and in a smooth and systematic pattern. The patterns of protein synthesis were placed into three different groups, the first occurring during the late day/early night phase, the second during the middle of the night phase, and the third during the late night/early day phase. The length of time that individual proteins within each group could incorporate radiolabel was variable, raising the possibility that additional groups might be present. However, both a replicate experiment in continuous light and a light:dark experiment confirmed the presence of at least three different groups of protein synthesis patterns. Unlike the circadian changes in the synthesis rate of the luciferin binding protein, which produces variations in protein levels that correlate with the bioluminescence rhythm, no substantial changes were found in the levels of any other rhythmically synthesized proteins examined.

Different phase responses of the two circadian oscillators in Gonyaulax.

Two circadian oscillators have been previously shown to exist in the unicellular alga Gonyaulax polyedra by virtue of different periods for two different rhythms, which occur under certain experimental conditions. Here we show that phase shifts in response to light pulses also differ for these two rhythms. The phase response curves (PRCs) for white or blue light pulses are similar in shape and amplitude, but are somewhat displaced in time and have a slightly larger delay portion for the aggregation rhythm, corresponding to the A oscillator. In contrast, the aggregation rhythm PRC for dark pulses in a red light background has a much larger amplitude than that for the bioluminescence rhythm (B oscillator). These results suggest that the light input mechanism of the B oscillator is mainly blue-sensitive, whereas that of the A oscillator is sensitive to both red and blue light. This is supported by entrainment experiments: Dark pulses given as a skeleton 24-hr scotoperiod in a blue light background act as a strong zeitgeber for both oscillators. But if red light is used as background, there is bistability in the entrainment of the B oscillator, whereas the A oscillator is entrained to a definitive phase angle, regardless of the cells' prior phase of entrainment. Finally, the two oscillators can be differentially entrained with 22-hr T cycles using 3-hr dark pulses interrupting red light (19 hr), whereas both oscillators are entrained when the dark pulses interrupt blue light.

A circadian rhythm of population behavior in Gonyaulax polyedra.

Like other flagellates, Gonyaulax polyedra exhibits diurnal vertical migration and pattern formation. Shape and size of the aggregations depend on container type, light intensity, and cell density. In Petri dishes, cells form oval "swarms"; within these, cells move downward in the highly dense center and rise up at the periphery. We have investigated the daily rhythm of this swarming activity in Petri dishes illuminated from the side, using time-lapse video recordings. At night, a "lawn" of cells forms at the bottom of the dish toward the light source (independent of light intensity). Before dawn, cells rise toward the surface and aggregate in swarms. The daily vertical migration occurs independent of light direction and intensity. The diurnal swarms, however, form every day at the same location within the dish, at a distance from the light that depends on light intensity, indicating a self-selection of light intensity. In constant light and temperature and with negligible vertical nutrient differences, all aspects of the rhythm continue to oscillate for up to 3 weeks, when the rhythm of the population becomes desynchronized. Under cycles of bright white-dim red light (WR), cell entrain to WR 10:10 but free run in WR 8:8 and shorter cycles, showing relative coordination (von Holst, 1939) to the driving light cycle. They also entrain to the 24-hr multiple of WR 6:6. Under nonentrained conditions, swarming activity is still influenced by light changes, and in spite of the apparent free run, the phasing of the averaged activity varies systematically with different T-cycle frequencies.

Assembling a clock for all seasons: are there M and E oscillators in the genes?

The hypothesis is advanced that the circadian pacemaker in the mammalian suprachiasmatic nucleus (SCN) is composed at the molecular level of a nonredundant double complex of circadian genes (per1, cry1, and per2, cry2). Each one of these sets would be sufficient for the maintenance of endogenous rhythmicity and thus constitute an oscillator. Each would have slightly different temporal dynamics and light responses. The per1/cry1 oscillator is accelerated by light and decelerated by darkness and thereby tracks dawn when day length changes. The per2 /cry2 oscillator is decelerated by light and accelerated by darkness and thereby tracks dusk. These M (morning) and E (evening) oscillators would give rise to the SCN's neuronal activity in an M and an E component. Suppression of behavioral activity by SCN activity in nocturnal mammals would give rise to adaptive tuning of the endogenous behavioral program to day length. The proposition-which is a specification of Pittendrigh and Daan's E-M oscillator model-yields specific nonintuitive predictions amenable to experimental testing in animals with mutations of circadian genes.

Daily rhythm of temporal resolution in the auditory system.

Over a period of 24 hours, fusion thresholds (click durations 100 micros) were assessed in 7 subjects. Over the same period, order thresholds (click duration of 1 ms) were measured in 10 subjects (12 independent sessions). Auditory fusion thresholds showed a diurnal rhythm with a maximum performance (shortest intervals) around midnight. In contrast, order thresholds appear to be independent on the time of day. Sex specific differences in threshold levels were only observed in order thresholds but not in fusion thresholds.

Are the effects of light on phase and period of the Gonyaulax clock mediated by different pathways?

The effects of different wavelengths of light on period, phase shifting, entrainment and after-effects of the circadian clock of the motile marine dinoflagellate Gonyaulax polyedra are described. Phase shifting and entrainment by light can be explained by the action of a single blue sensitive light input pathway. However, tonic effects of light on the period under free-running conditions, and also after-effects on period resulting from single 4 h light exposures, appear to involve two input pathways with different absorption and temperature characteristics. These results suggest different mechanisms for the control of phase and period of the circadian clock.

Daily rhythm of vigilance assessed by temporal resolution of the visual system.

To assess the daily distribution of temporal resolution in visual detection, binocular double-pulse resolution (DPR) was measured over a 24 h period in six healthy subjects. DPR showed a significant daily variation with an amplitude for the foveal stimulus of up to 60%. Like in other vigilance-dependent daily rhythms, optimal performance occurred around midday. The DPR measurements described here are an excellent method for assessment of vigilance and mental alertness (e.g. in pharmacological studies). They show strong time-of-day differences, are highly reliable across successive measurements, and can be fully automated.

Several clocks may simplify the circadian system of Gonyaulax.

The circadian system of Gonyaulax involves at least two pacemaking oscillators. These oscillators have been shown to be distinct at the physiological level by an examination of their rhythmic periods under different conditions, their phase response to pulses of light and darkness and their ability to compensate the rhythms for changes in temperature. It is also known that Gonyaulax can restrict synthesis of different proteins to at least three different times of day. We suggest that the two known oscillators may each control synthesis of a differently timed class of proteins.

Sleep timing is more important than sleep length or quality for medical school performance.

Overwhelming evidence supports the importance of sleep for memory consolidation. Medical students are often deprived of sufficient sleep due to large amounts of clinical duties and university load, we therefore investigated how study and sleep habits influence university performance. We performed a questionnaire-based study with 31 medical students of the University of Munich (second and third clinical semesters; surgery and internal medicine). The students kept a diary (in 30-min bins) on their daily schedules (times when they studied by themselves, attended classes, slept, worked on their thesis, or worked to earn money). The project design involved three 2-wk periods (A: during the semester; B: directly before the exam period--pre-exam; C: during the subsequent semester break). Besides the diaries, students completed once questionnaires about their sleep quality (Pittsburgh Sleep Quality Index [PSQI]), their chronotype (Munich Chronotype Questionnaire [MCTQ]), and their academic history (previous grades, including the previously achieved preclinical board exam [PBE]). Analysis revealed significant correlations between the actual sleep behavior during the semester (MS(diary); mid-sleep point averaged from the sleep diaries) during the pre-exam period and the achieved grade (p = 0.002) as well as between the grades of the currently taken exam and the PBE (p = 0.002). A regression analysis with MS(diary) pre-exam and PBE as predictors in a model explained 42.7% of the variance of the exam grade (effect size 0.745). Interestingly, MS(diary)--especially during the pre-exam period-was the strongest predictor for the currently achieved grade, along with the preclinical board exam as a covariate, whereas the chronotype did not significantly influence the exam grade.