The period length of fibroblast circadian gene expression varies widely among human individuals

Mammalian circadian behavior is governed by a central clock in the suprachiasmatic nucleus of the brain hypothalamus, and its intrinsic period length is believed to affect the phase of daily activities. Measurement of this period length, normally accomplished by prolonged subject observation, is difficult and costly in humans. Because a circadian clock similar to that of the suprachiasmatic nucleus is present in most cell types, we were able to engineer a lentiviral circadian reporter that permits characterization of circadian rhythms in single skin biopsies. Using it, we have determined the period lengths of 19 human individuals. The average value from all subjects, 24.5 h, closely matches average values for human circadian physiology obtained in studies in which circadian period was assessed in the absence of the confounding effects of light input and sleep–wake cycle feedback. Nevertheless, the distribution of period lengths measured from biopsies from different individuals was wider than those reported for circadian physiology. A similar trend was observed when comparing wheel-running behavior with fibroblast period length in mouse strains containing circadian gene disruptions. In mice, inter-individual differences in fibroblast period length correlated with the period of running-wheel activity; in humans, fibroblasts from different individuals showed widely variant circadian periods. Given its robustness, the presented procedure should permit quantitative trait mapping of human period length.     

A three-oscillator model of the human circadian system controlling the core temperature rhythm and the sleep-wake cycle

Even during “free-running” experiments, in which subjects lived in caves or cellars without any time cues, various circadian rhythms such as core body temperature and the sleep-wake cycle remained for a long time mutually synchronized in one group of subjects. In another group of subjects, or later in the same subjects, a number of unusually long sleep-wake cycles occurred while body temperature persisted in a near-24 hr rhythm. This has been termed “internal desynchronization” by Aschoff & Wever (1962) to emphasize the uncoupling of rhythms. Zulley (1980) and Czeisler et al. (1980) found that the duration of sleep depends regularly on the phase of the sleep onset in the body temperature rhythm, even in the apparently “random and irregular” sleep-wake pattern. The graph which plots, the sleep duration against the sleep onset phase is called sleep duration in this paper. We develop a quantitative, multi-oscillator model of human circadian system following Wever (1979) and Kronauer et al. (1982). Because the simplest model, which describes the state of each component oscillator by only one variable (ptlase) was adopted for each component oscillator, we can determine the intFraction between oscillators using sleep duration. It is found that a three-oscillator model can simulate several qualitative features of human circadian rhythms, such as an irregular free-running pattern and sleep duration. Moreover we find that the model reproduces the mysterious phenomenon of “forbidden wake up”, although we do not incorporate a priori any mechanism to explain it.     

The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts

Background

Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior.

Methodology/Principal Findings

In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer.

Conclusions/Significance

We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.     

So close and yet so different: The importance of considering temporal dynamics to understand habitat selection

Habitat selection of animals is influenced by spatial heterogeneity as well as temporal environmental dynamics. In addition, human activities potentially have severe influences on the habitat selection of animals, often resulting in more nocturnal behavior. We investigated seasonal and circadian habitat selection patterns of red deer (Cervus elaphus) on a military training area in Bavaria (Germany). Individual deer ranged on two neighboring, but non-overlapping sites differing in landscape composition and human activity. Using GPS telemetry data, we visually investigated selection patterns and then fitted step-selection functions to assess multiple approaches to account for temporal (i.e., diel and seasonal) effects and for the impact of habitat conditions on selection. We first showed that the way in which time of day is considered in step-selection functions is essential for obtaining ecologically meaningful results, and that the usual ways of including time (i.e., either based on clock-time or based on night vs. day categories) can lead to incomplete or misleading conclusions. Furthermore, we found that individuals followed either circadian or seasonal habitat selection patterns, depending on the site they inhabited. This can be explained by differences in disturbance between the two sites. Except in winter, individuals selected for open habitats during the night irrespective of human activity level, but only individuals from the more disturbed site selected for covered habitats during the day. Our results highlight the importance of carefully considering the appropriate temporal scale for habitat selection analyses. Our findings also indicate that red deer are not a crepuscular or nocturnal species per se, as is often observed in human-dominated landscapes. Instead, our results imply that nocturnality in red deer is an effect of adaption to human activities.     

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ERA) gene also oscillates in a circadian fashion. In contrast, ERA-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ERA-positive breast cancer cells do not display circadian oscillation of ERA expression. Our findings suggest that estrogen signaling could be affected not only in ERA-negative breast cancer, but also in ERA-positive breast cancer due to lack of circadian availability of ERA. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.Key words: circadian rhythm, clock genes, estrogen receptor alpha (ERA), breast cancer cells, entrainment, serum shock     

A useful relationship between epidemiology and queueing theory: The distribution of the number of infectives at the moment of the first detection

In this paper we establish a relation between the spread of infectious diseases and the dynamics of so called M/G/1 queues with processor sharing. The relation between the spread of epidemics and branching processes, which is well known in epidemiology, and the relation between M/G/1 queues and birth death processes, which is well known in queueing theory, will be combined to provide a framework in which results from queueing theory can be used in epidemiology and vice versa.In particular, we consider the number of infectious individuals in a standard SIR epidemic model at the moment of the first detection of the epidemic, where infectious individuals are detected at a constant per capita rate. We use a result from the literature on queueing processes to show that this number of infectious individuals is geometrically distributed.     

Ultradian Components of the Sleep-Wake Cycle in Babies

Behavioral states may be analyzed as expressions of underlying cyclic activity involving several physiological systems. The human sleep-wake cycle in the first year of life shows, in addition to the establishment of circadian rhythmic-ity around the second month, the dynamics of its ultradian components, as can be seen in the more or less gradual decline of the polyphasic pattern. To detect these changes, we have analyzed the sleep-wake cycle of five babies of different ages (3, 4, 9, 11, and 13 months) observed for 5 consecutive days (Monday through Friday), 10 h (08:00-18:00 h) per day at a kindergarten by the first author, and during the night (18:00-08:00 h) by the parents. Behavioral observations were designed for minimizing interference with the babies' habits. Sleep/wake data were arranged in 60-min intervals, and the relative amount of time spent asleep per interval constituted the time series submitted for statistical analysis. The five resulting time series were submitted to spectral analysis for detecting the composition of frequencies contributing to the observed sleep/wake cycle. Several frequencies were thus obtained for each baby in the ultradian and circadian domain, ranging from one cycle in 2.0 h to one cycle in 24 h. The circadian component was the strongest rhythmic influence for all individuals except for the youngest (3-month-old) baby, who showed a semicircadian component as the main frequency in the power spectrum. Three individuals showed ultradian frequencies in the domain of 3-4 h. Differences in the spectra derive from three possible, and probably not exclusive. causes: 1) ontogenetic changes, 2) different masking effects, and 3) individual differences.     

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ER A) gene also oscillates in a circadian fashion. In contrast, ER A-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ER A-positive breast cancer cells do not display circadian oscillation of ER A expression. Our findings suggest that estrogen signaling could be affected not only in ER A-negative breast cancer, but also in ER A-positive breast cancer due to lack of circadian availability of ER A. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.     

A randomized,double-blind and placebo-controlled crossover trial on the effect of l-ornithine ingestion on the human circadian clock

ABSTRACT

In mammals, daily physiological events are regulated by the circadian rhythm, which comprises two types of internal clocks: the central clock and peripheral clocks. Circadian rhythm plays an important role in maintaining physiological functions including the sleep-wake cycle, body temperature, metabolism and organ functions. Circadian rhythm disorder, which is caused, for example, by an irregular lifestyle or long-haul travel, increases the risk of developing disease; therefore, it is important to properly maintain the rhythm of the circadian clock. Food and the circadian clock system are known to be closely linked. Studies on rodents suggest that ingesting specific food ingredients, such as the flavonoid nobiletin, fish oil, the polyphenol resveratrol and the amino acid L-ornithine affects the circadian clock. However, there are few reports on the foods that affect these circadian clocks in humans. In this study, therefore, we examined whether L-ornithine affects the human central clock in a crossover design placebo-controlled human trial. In total, 28 healthy adults (i.e. ≥20 years) were randomly divided into two groups and completed the study protocol. In the 1st intake period, participants were asked to take either L-ornithine (400 mg) capsules or placebo capsules for 7 days. After 7 days’ interval, they then took the alternative test capsules for 7 days in the 2nd intake period. On the final day of each intake period, saliva was sampled at various time points in the dim light condition, and the concentration of melatonin was quantified to evaluate the phase of the central clock. The results revealed that dim light melatonin onset, a recognized marker of central circadian phase, was delayed by 15 min after ingestion of L-ornithine. Not only is this finding an indication that L-ornithine affects the human central clock, but it also demonstrates that the human central clock can be regulated by food ingredients.     

Reproductive phase dependent circadian variation in hypothalamic concentration of serotonin, dopamine and peripheral thyroxine levels in Japanese Quail following 5-HTP and L-DOPA administration at specific time intervals

Temporal phase relations of circadian hypothalamic neurotransmitters are reported to regulate seasonal reproduction in some avian species. Present experiments were designed to study circadian variation in the hypothalamic concentration of neurotransmitters (serotonin and dopamine) and the plasma thyroxine level in sexually active (long day) and inactive (short day) Japanese Quail. A significant circadian cycle was noted in the hypothalamic content of both serotonin and dopamine, but with different patterns. In breeding Quail, peak activity of serotonin and dopamine was noted at 10.00 A.M. and 10.00 P.M. respectively i.e. at the interval of 12 hours. However, during sexually quiescent condition, peaks of both neurotransmitters occurred at 2.00 P.M. i.e. having a 0-hour temporal relationship. During the breeding phase, the plasma thyroxine level showed a biphasic pattern with two circadian peaks at 10.00 A.M. and 10.00 P.M. whereas in the non-breeding condition a single peak was observed at 10.00 A.M. In the second experiment, to study the effect of temporal synergism of neurotransmitter precursor drugs on circadian cycles, two groups of Quail were administered daily with serotonin precursor 5-HTP (5-hydroxytryptophan) and dopamine precursor L-DOPA (L-dihydroxyphenylalanine) (5 mg/100 g body weight) 12 hour (12-hr) and 8 hour (8-hr) apart over a period of 11 days under continuous conditions of light and then transferred to long day length for 15 days when the experiment was terminated. When compared to controls, the 12-hr condition induced breeding while the 8-hr condition led to a non-breeding condition. The circadian pattern of serotonin levels of control and 12-hr Quail was similar to that of a normal sexually active bird, while that of the 8-hr Quail showed the pattern of a sexually inactive bird. The plasma thyroxine level exhibited a biphasic pattern in 12-hr Quail, which was similar to a normally breeding bird, whereas unlike sexually inactive birds, the thyroxine concentration in 8-hr Quail was relatively low and did not show significant cyclicity. Interestingly, the plasma testosterone level of 12-hr Quail followed a more or less similar pattern with peak activities coinciding with that of thyroxine i.e. biphasic in the sexually active condition (12-hr and control) but a single peak in the quiescent (8-hr) condition. These findings suggest that the temporal phase relation of circadian serotonergic and dopaminergic oscillator varies as a function of reproductive status of the bird, and breeding/non-breeding conditions may be induced experimentally by changing the phase relation of these oscillations.     

Plasticity of the intrinsic period of the human circadian timing system

Human expeditions to Mars will require adaptation to the 24.65-h Martian solar day-night cycle (sol), which is outside the range of entrainment of the human circadian pacemaker under lighting intensities to which astronauts are typically exposed. Failure to entrain the circadian time-keeping system to the desired rest-activity cycle disturbs sleep and impairs cognitive function. Furthermore, differences between the intrinsic circadian period and Earth's 24-h light-dark cycle underlie human circadian rhythm sleep disorders, such as advanced sleep phase disorder and non-24-hour sleep-wake disorders. Therefore, first, we tested whether exposure to a model-based lighting regimen would entrain the human circadian pacemaker at a normal phase angle to the 24.65-h Martian sol and to the 23.5-h day length often required of astronauts during short duration space exploration. Second, we tested here whether such prior entrainment to non-24-h light-dark cycles would lead to subsequent modification of the intrinsic period of the human circadian timing system. Here we show that exposure to moderately bright light ( approximately 450 lux; approximately 1.2 W/m(2)) for the second or first half of the scheduled wake episode is effective for entraining individuals to the 24.65-h Martian sol and a 23.5-h day length, respectively. Estimations of the circadian periods of plasma melatonin, plasma cortisol, and core body temperature rhythms collected under forced desynchrony protocols revealed that the intrinsic circadian period of the human circadian pacemaker was significantly longer following entrainment to the Martian sol as compared to following entrainment to the 23.5-h day. The latter finding of after-effects of entrainment reveals for the first time plasticity of the period of the human circadian timing system. Both findings have important implications for the treatment of circadian rhythm sleep disorders and human space exploration.     

A minimal model of light-induced circadian rhythms of nitrate reductase activity in leaves of barley

Observed circadian rhythms of nitrate reductase (NR) (EC 1.6.6.1) activity in leaves of barley ( Hordeum vulgare L. cv. Herta) under continuous light conditions are described by a simple kinetic model. The oscillatory mechanism has been decomposed into the negative and positive feedback loops which are necessary according to present theories of chemical oscillating systems. Our results indicate that the decrease of NR activity in darkness can be considered as a reversible unimolecular conversion of the active form of NR into an inactive form, forming a negative stabilizing feedback loop. The light-induced increase of NR activity is related to a positive destabilizing feedback loop. In our treatment this process is represented as an autocatalytical reaction.     

Long ultradian rhythms in red blood cells and ghost suspensions: Possible involvement of cell membrane

Summary Oscillations in glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD) activities were recorded in suspensions of intact human red blood cells (RBCs) exposed to various light regimens. The periods of these oscillations, defined as “long ultradian,” ranged between 13 and 18 h regardless of light regimen. The patterns of enzymatic activities were the same when assayed at each time point, in full hypotonic hemolysates, and membrane-free hemolysates. However, if hemolysates were prepared by sonication the activity pattern did not exhibit significant oscillations and the activity was higher than that recorded in hypotonic hemolysates. The observed rhythms may reflect a time-dependent attachment and detachment of enzyme molecules from cell membrane, suggesting that at the bound state the enzyme molecules are (temporarily) inactive. Oscillations with similar long ultradian periods were also observed in Ca⁺⁺ concentration of suspended RBCs and in the binding of Ca⁺⁺⁴⁵ to human RBC ghosts. Treatment of the RBCs with A₂C or Diamide before the preparation of the ghosts changed or distorted the rhythmic pattern of Ca⁺⁺⁴⁵ binding. These results point to the role of the membrane in processing the long ultradian oscillations. The relation between this type of oscillations to circadian rhythm is discussed.     

Chronobiological background to cathemerality: circadian rhythms in Eulemur fulvus albifrons (Prosimii) and Aotus azarai boliviensis (Anthropoidea)

Cathemeral activity, in which the animals' motor activity is almost evenly distributed throughout the dark and the light portion of the day, has been described in various lemur genera (Eulemur, Hapalemur) and in the owl monkey Aotus azarai of the Argentinean Chaco. Proximate and ultimate factors responsible for this behaviour are still being debated. However, the chronobiological background of the behaviour has largely been ignored. We studied E. fulvus albifrons and A. a. boliviensis under controlled laboratory conditions to assess whether their activity rhythm is endogenously regulated by a circadian timing system that obeys general rules found in other mammals, or whether there are characteristic differences. To this end, we carried out long-term activity recordings on individuals of both subspecies kept under constant light and various light-dark cycles (LDs) using a PC-controlled electro-acoustic device in combination with telemetric body temperature measurements. Both subspecies developed free-running circadian activity and body temperature rhythms with periods deviating from 24 h in constant light, and LDs turned out to be the most efficient Zeitgeber synchronizing this endogenous rhythmicity to the external 24-hour day. The luminosity prevailing during the dark time of the LD had a decisive effect on levels of activity in the lemurs and induced strong masking effects on their circadian activity pattern. The results indicate that, from a chronobiological viewpoint, both species should be considered as dark active primates. Their diel activity rhythm is regulated by a normally responding circadian timing system and strong activity inhibiting or enhancing direct effects of light intensity. Thus, hypotheses on proximate and/or ultimate factors of cathemerality in primates must also consider its circadian background.     

Task Partitioning in Insect Societies. I. Effect of Colony Size on Queueing Delay and Colony Ergonomic Efficiency

The collection and handling of colony resources such as food, water, and nest construction material is often divided into subtasks in which the material is passed from one worker to another. This is known as task partitioning. When material is transferred directly from one individual to another, queueing delays frequently occur because individuals must sometimes wait for a transfer partner. A stochastic simulation model was written to study the effect of colony size on these delays. Queueing delay decreases roughly exponentially with colony size because stochastic fluctuations in the arrival of individuals are lower in larger colonies. These results support empirical studies of Polybia occidentalis and other theoretical studies of honeybees. The effect of the relative number of individuals in the two subtask groups was also studied. There is a unique optimal ratio of the number of workers associated with each of the subtasks that simultaneously minimizes mean queueing delay and maximizes colony nectar-processing rate. Deviations from this optimal ratio, for example, as a result of forager mortality or changes in nectar productivity that affect foraging trip duration, increase mean queueing delays greatly, especially in smaller colonies.     

Rhythmic changes in the activities of NAD kinase and NADP phosphatase in the achlorophyllous ZC mutant of Euglena gracilis Klebs (strain Z)

NAD kinase and NADP phosphatase activities were detected in the supernatant and the pellet fractions prepared by sonication and centrifugation of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis. A detailed study of substrate concentration-velocity curves enabled us to define the saturating substrate concentrations that were used in the enzyme assays. An analysis of the reproducibility of the entire assay procedure indicated that the pooled standard error was about 14%. We report circadian variations in the activities of NAD kinase and NADP phosphatase in the soluble and membrane-bound fractions of both synchronously dividing and nondividing cultures maintained in constant darkness. Bimodal circadian rhythms in total NADP phosphatase activity were found in dividing cells (peaks at circadian times [CT] 00 and 12). The peak observed at CT 00-03 disappeared when the cells had ceased dividing, a result that suggests that it might be regulated by the cell division cycle. NAD kinase activity displayed unimodal circadian rhythms (peak at CT 12) in dividing cells, which persisted with the same phase after the culture entered the stationary phase of growth. Results are discussed with reference to a model (K. Goto, D. L. Laval-Martin, and L. N. Edmunds, Jr., 1985, Science 228, 1284-1288) in which we have proposed that the Ca2(+)-transport system, Ca2+, calmodulin, NAD kinase, and NADP phosphatase could represent clock "gears" that might constitute a self-sustained circadian oscillating loop.