Personalising circadian hygiene educational initiatives aimed at university students-"He who has ears to hear, let him hear".

The aim of the present study was to characterise "early drop-outs" (n = 3185) out of a group of university students (n = 7766) engaged in an ongoing circadian education initiative, to evaluate its efficacy and direct its developments. The initiative is aimed at improving sleep timing/quality through one of two sets of circadian hygiene advice covering the timing of sleep, meals, exercise and light exposure, and it has already been shown to have a positive effect on sleep timing. This second, interim analysis confirmed the high prevalence of disturbed night sleep and social jetlag amongst students at Padova University. Three-thousand, one-hundred and eighty-five (41.0%) students were early drop-outs. These were more commonly males (46.4 versus 37.6%; χ2 = 58, p < 0.0001), had later sleep-wake habits, more daytime sleepiness and worse night sleep quality. Chronotype distribution was also different, with a slight but significantly higher proportion of extremely evening/evening types amongst early drop-outs (χ2 = 10, p < 0.05). These results suggest that the more evening the student, the lower their likelihood of choosing/being able to follow circadian advice.

Circadian rhythms and the liver.

This narrative review briefly describes the mammalian circadian timing system, the specific features of the liver clock, also by comparison with other peripheral clocks, the role of the liver clock in the preparation of food intake, and its relationship with energy metabolism. It then goes on to provide a chronobiological perspective of the pathophysiology and management of several types of liver disease, with a particular focus on metabolic-associated fatty liver disease (MAFLD), decompensated cirrhosis and liver transplantation. Finally, it provides some insight into the potential contribution of circadian principles and circadian hygiene practices in preventing MAFLD, improving the prognosis of advanced liver disease and modulating liver transplantation outcomes.

Hyperammonaemia disrupts daily rhythms reversibly by elevating glutamate in the central circadian pacemaker.

Patients with cirrhosis exhibit features of circadian disruption. Hyperammonaemia has been suggested to impair both homeostatic and circadian sleep regulation. Here, we tested if hyperammonaemia directly disrupts circadian rhythm generation in the central pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus. Wheel-running activity was recorded from mice fed with a hyperammonaemic or normal diet for ~35 days in a 12:12 light-dark (LD) cycle followed by ~15 days in constant darkness (DD). The expression of the clock protein PERIOD2 (PER2) was recorded from SCN explants before, during and after ammonia exposure, ±glutamate receptor antagonists. In LD, hyperammonaemic mice advanced their daily activity onset time by ~1 h (16.8 ± 0.3 vs. 18.1 ± 0.04 h, p = .009) and decreased their total activity, concentrating it during the first half of the night. In DD, hyperammonaemia reduced the amplitude of daily activity (551.5 ± 27.7 vs. 724.9 ± 59 counts, p = .007), with no changes in circadian period. Ammonia (≥0.01 mM) rapidly and significantly reduced PER2 amplitude, and slightly increased circadian period. The decrease in PER2 amplitude correlated with decreased synchrony among circadian cells in the SCN and increased extracellular glutamate, which was rescued by AMPA glutamate receptor antagonists. These data suggest that hyperammonaemia affects circadian regulation of rest-activity behaviour by increasing extracellular glutamate in the SCN.

The Alarm Clock Against the Sun: Trends in Google Trends Search Activity Across the Transitions to and from Daylight Saving Time.

The human circadian timing system depends on the light/dark cycle as its main cue to synchronize with the environment, and thus with solar time. However, human activities depend also on social time, i.e. the set of time conventions and restrictions dictated by society, including Daylight Saving Time (DST), which adds an hour to any degree of desynchrony between social and solar time. Here, we used Google Trends as a data source to analyze diurnal variation, if any, and the daily peak in the relative search volume of 26 Google search queries in relation to the transitions to/from DST in Italy from 2015 to 2020. Our search queries of interest fell into three categories: sleep/health-related, medication and random non sleep/health-related. After initial rhythm and phase analysis, 11 words were selected to compare the average phase of the 15 days before and after the transition to/from DST. We observed an average phase advance after the transition to DST, and a phase delay after the transition to civil time, ranging from 25 to 60 minutes. Advances or delays shorter than 60 minutes, which were primarily observed in the sleep/health-related category, may suggest that search timing for these queries is at least partially driven by the endogenous circadian rhythm. Finally, a significant trend in phase anticipation over the years was observed for virtually all words. This is most likely related to an increase in age, and thus in earlier chronotypes, amongst Google users.

Driving fatigue increases after the Spring transition to Daylight Saving Time in young male drivers: A pilot study.

The Spring transition to Daylight Saving Time (DST) has been associated with several health and road safety issues. Previous literature has focused primarily on the analysis of historical crash and hospitalization data, without investigating specific crash contributing factors, such as driving fatigue. The present study aims to uncover the effects of DST-related circadian desynchrony and sleep deprivation on driving fatigue, by means of a driving simulator experiment. Eighteen participants (all males, age range 21-30 years, mean = 24.2, SD = 2.9) completed two 50-minute trials (at one week distance, same time and same day of the week) on a monotonous highway environment, the second one taking place in the week after the Spring transition to DST. Driving fatigue was evaluated by analysing several different variables (including driving-based, physiological and subjective indices) and by comparison with a historical cohort of pertinent, matched controls who had also undergone two trials, but in the absence of any time change in between. Results showed a considerable rise in fatigue levels throughout the driving task in both trials, but with significantly poorer performance in the post-DST trial, documented by a worsening in vehicle lateral control and an increase in eyelid closure. However, participants seemed unable to perceive this decrease in their alertness, which most likely prevented them from implementing fatigue-coping strategies. These findings indicate that DST has a detrimental effect on driving fatigue in young male drivers in the week after the Spring transition, and provide valuable insights into the complex relationship between DST and road safety.

Peptidergic signaling from clock neurons regulates reproductive dormancy in Drosophila melanogaster.

With the approach of winter, many insects switch to an alternative protective developmental program called diapause. Drosophila melanogaster females overwinter as adults by inducing a reproductive arrest that is characterized by inhibition of ovarian development at previtellogenic stages. The insulin producing cells (IPCs) are key regulators of this process, since they produce and release insulin-like peptides that act as diapause-antagonizing hormones. Here we show that in D. melanogaster two neuropeptides, Pigment Dispersing Factor (PDF) and short Neuropeptide F (sNPF) inhibit reproductive arrest, likely through modulation of the IPCs. In particular, genetic manipulations of the PDF-expressing neurons, which include the sNPF-producing small ventral Lateral Neurons (s-LNvs), modulated the levels of reproductive dormancy, suggesting the involvement of both neuropeptides. We expressed a genetically encoded cAMP sensor in the IPCs and challenged brain explants with synthetic PDF and sNPF. Bath applications of both neuropeptides increased cAMP levels in the IPCs, even more so when they were applied together, suggesting a synergistic effect. Bath application of sNPF additionally increased Ca2+ levels in the IPCs. Our results indicate that PDF and sNPF inhibit reproductive dormancy by maintaining the IPCs in an active state.

The role of astrocytes in generating circadian rhythmicity in health and disease.

Evidence is accumulating that the mammalian circadian clock system is considerably more complex than previously believed, also in terms of the cell types that actually contribute to generating the oscillation within the master clock, in the suprachiasmatic nuclei of the hypothalamus. Here we review the evidence that has lead to the identification of a bona fide astrocytic circadian clock, and that of the potential contribution of such clock to the generation of circadian and seasonal rhythmicity in health and in neurodegenerative disorders. Finally, we speculate on the role of the astrocytic clock in determining some of the clinical features of hepatic encephalopathy, a reversible neuropsychiatric syndrome associated with advanced liver disease, which is characterized by transient, profound morphological and functional astrocytic abnormalities, in the absence of significant, structural neuronal changes.

Modulation of miR-210 alters phasing of circadian locomotor activity and impairs projections of PDF clock neurons in Drosophila melanogaster.

Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.

Sleep, Circadian Rhythmicity and Response to Chronotherapy in University Students: Tips from Chronobiology Practicals.

Chronobiology is not routinely taught to biology or medical students in most European countries. Here we present the results of the chronobiology practicals of a group of students of the University of Padova, with a view to highlight some interesting features of this group, and to share a potentially interesting cross-faculty teaching experience. Thirty-eight students (17 males; 22.9 ± 1.6 yrs) completed a set of self-administered electronic sleep quality [Pittsburgh Sleep Quality Index (PSQI)], chronotype and sleepiness [Epworth Sleepiness Scale (ESS)] questionnaires. They then went on to complete sleep diaries for two weeks. Sixteen also wore an actigraph, 8 wore wireless sensors for skin temperature, and 8 underwent a course of chronotherapy aimed at anticipating their sleep-wake timing. Analyses were performed as practicals, together with the students. Average PSQI score was 5.4 ± 1.9, with 15 (39%) students being poor sleepers. Average ESS score was 6.5 ± 3.3, with 3 (8%) students exhibiting excessive daytime sleepiness. Seven classified themselves as definitely/moderately morning, 25 as intermediates, 6 as moderately/definitely evening. Students went to bed/fell asleep significantly later on weekends, it took them less to fall asleep and they woke up/got up significantly later. Diary-reported sleep onset time coincided with the expected decrease in proximal skin temperature. Finally, during chronotherapy they took significantly less time to fall asleep. In conclusion, significant abnormalities in the sleep-wake patterns of a small group of university students were observed, and the students seemed to benefit from chronotherapy. We had a positive impression of our teaching experience, and the chronobiology courses obtained excellent student feedback.

Unexpected features of Drosophila circadian behavioural rhythms under natural conditions.

Circadian clocks have evolved to synchronize physiology, metabolism and behaviour to the 24-h geophysical cycles of the Earth. Drosophila melanogaster's rhythmic locomotor behaviour provides the main phenotype for the identification of higher eukaryotic clock genes. Under laboratory light-dark cycles, flies show enhanced activity before lights on and off signals, and these anticipatory responses have defined the neuronal sites of the corresponding morning (M) and evening (E) oscillators. However, the natural environment provides much richer cycling environmental stimuli than the laboratory, so we sought to examine fly locomotor rhythms in the wild. Here we show that several key laboratory-based assumptions about circadian behaviour are not supported by natural observations. These include the anticipation of light transitions, the midday 'siesta', the fly's crepuscular activity, its nocturnal behaviour under moonlight, and the dominance of light stimuli over temperature. We also observe a third major locomotor component in addition to M and E, which we term 'A' (afternoon). Furthermore, we show that these natural rhythm phenotypes can be observed in the laboratory by using realistic temperature and light cycle simulations. Our results suggest that a comprehensive re-examination of circadian behaviour and its molecular readouts under simulated natural conditions will provide a more authentic interpretation of the adaptive significance of this important rhythmic phenotype. Such studies should also help to clarify the underlying molecular and neuroanatomical substrates of the clock under natural protocols.

Drosophila circadian rhythms in seminatural environments: Summer afternoon component is not an artifact and requires TrpA1 channels.

Under standard laboratory conditions of rectangular light/dark cycles and constant warm temperature, Drosophila melanogaster show bursts of morning (M) and evening (E) locomotor activity and a "siesta" in the middle of the day. These M and E components have been critical for developing the neuronal dual oscillator model in which clock gene expression in key cells generates the circadian phenotype. However, under natural European summer conditions of cycling temperature and light intensity, an additional prominent afternoon (A) component that replaces the siesta is observed. This component has been described as an "artifact" of the TriKinetics locomotor monitoring system that is used by many circadian laboratories world wide. Using video recordings, we show that the A component is not an artifact, neither in the glass tubes used in TriKinetics monitors nor in open-field arenas. By studying various mutants in the visual and peripheral and internal thermo-sensitive pathways, we reveal that the M component is predominantly dependent on visual input, whereas the A component requires the internal thermo-sensitive channel transient receptor potential A1 (TrpA1). Knockdown of TrpA1 in different neuronal groups reveals that the reported expression of TrpA1 in clock neurons is unlikely to be involved in generating the summer locomotor profile, suggesting that other TrpA1 neurons are responsible for the A component. Studies of circadian rhythms under seminatural conditions therefore provide additional insights into the molecular basis of circadian entrainment that would otherwise be lost under the usual standard laboratory protocols.

Nucleotide excision repair efficiency in quiescent human fibroblasts is modulated by circadian clock.

The efficiency of Nucleotide Excision Repair (NER)process is crucial for maintaining genomic integrity because in many organisms, including humans, it represents the only system able to repair a wide range of DNA damage. The aim of the work was to investigate whether the efficiency of the repair of photoproducts induced by UV-light is affected by the circadian phase at which irradiation occurred. NER activity has been analyzed in human quiescent fibroblasts (in the absence of the cell cycle effect), in which circadian rhythmicity has been synchronized with a pulse of dexamethasone. Our results demonstrate that both DNA damage induction and repair efficiency are strictly dependent on the phase of the circadian rhythm at which the cells are UV-exposed. Furthermore, the differences observed between fibroblasts irradiated at different circadian times (CTs) are abolished when the clock is obliterated. In addition, we observe that chromatin structure is regulated by circadian rhythmicity. Maximal chromatin relaxation occurred at the same CT when photoproduct formation and removal were highest. Our data suggest that the circadian clock regulates both the DNA sensitivity to UV damage and the efficiency of NER by controlling chromatin condensation mainly through histone acetylation.

F-ATPase of Drosophila melanogaster forms 53-picosiemen (53-pS) channels responsible for mitochondrial Ca2+-induced Ca2+ release.

Mitochondria of Drosophila melanogaster undergo Ca(2+)-induced Ca(2+) release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca(2+) and H(+). We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg(2+)/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R(+) cells sensitizes the mCrC to Ca(2+) but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca(2+) and thiol oxidants and inhibited by Mg(2+)/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.

UCP4C mediates uncoupled respiration in larvae of Drosophila melanogaster.

Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F-ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva-to-adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.

Fly cryptochrome and the visual system.

Cryptochromes are flavoproteins, structurally and evolutionarily related to photolyases, that are involved in the development, magnetoreception, and temporal organization of a variety of organisms. Drosophila CRYPTOCHROME (dCRY) is involved in light synchronization of the master circadian clock, and its C terminus plays an important role in modulating light sensitivity and activity of the protein. The activation of dCRY by light requires a conformational change, but it has been suggested that activation could be mediated also by specific "regulators" that bind the C terminus of the protein. This C-terminal region harbors several protein-protein interaction motifs, likely relevant for signal transduction regulation. Here, we show that some functional linear motifs are evolutionarily conserved in the C terminus of cryptochromes and that class III PDZ-binding sites are selectively maintained in animals. A coimmunoprecipitation assay followed by mass spectrometry analysis revealed that dCRY interacts with Retinal Degeneration A (RDGA) and with Neither Inactivation Nor Afterpotential C (NINAC) proteins. Both proteins belong to a multiprotein complex (the Signalplex) that includes visual-signaling molecules. Using bioinformatic and molecular approaches, dCRY was found to interact with Neither Inactivation Nor Afterpotential C through Inactivation No Afterpotential D (INAD) in a light-dependent manner and that the CRY-Inactivation No Afterpotential D interaction is mediated by specific domains of the two proteins and involves the CRY C terminus. Moreover, an impairment of the visual behavior was observed in fly mutants for dCRY, indicative of a role, direct or indirect, for this photoreceptor in fly vision.

Leigh syndrome in Drosophila melanogaster: morphological and biochemical characterization of Surf1 post-transcriptional silencing.

Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LS(Surf1) patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R(+) cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS.

Functional characterization of drim2, the Drosophila melanogaster homolog of the yeast mitochondrial deoxynucleotide transporter.

The CG18317 gene (drim2) is the Drosophila melanogaster homolog of the Saccharomyces cerevisiae Rim2 gene, which encodes a pyrimidine (deoxy)nucleotide carrier. Here, we tested if the drim2 gene also encodes for a deoxynucleotide transporter in the fruit fly. The protein was localized to mitochondria. Drosophila S2R(+) cells, silenced for drim2 expression, contained markedly reduced pools of both purine and pyrimidine dNTPs in mitochondria, whereas cytosolic pools were unaffected. In vivo drim2 homozygous knock-out was lethal at the larval stage, preceded by the following: (i) impaired locomotor behavior; (ii) decreased rates of oxygen consumption, and (iii) depletion of mtDNA. We conclude that the Drosophila mitochondrial carrier dRIM2 transports all DNA precursors and is essential to maintain mitochondrial function.

Sleep-wake abnormalities in patients with cirrhosis.

A considerable proportion of patients with cirrhosis exhibit insomnia, delayed sleep habits, and excessive daytime sleepiness. These have been variously attributed to hepatic encephalopathy and impaired hepatic melatonin metabolism, but the understanding of their pathophysiology remains limited and their treatment problematic. Sleep is regulated by the interaction of a homeostatic and a circadian process. The homeostatic process determines sleep propensity in relation to sleep-wake history, thus the need to sleep increases with the duration of the waking period. The circadian process, which is marked by the 24-hour rhythm of the hormone melatonin, is responsible for the alternation of high/low sleep propensity in relation to dark/light cues. Circadian sleep regulation has been studied in some depth in patients with cirrhosis, who show delays in the 24-hour melatonin rhythm, most likely in relation to reduced sensitivity to light cues. However, while melatonin abnormalities are associated with delayed sleep habits, they do not seem to offer a comprehensive explanation to the insomnia exhibited by these patients. Fewer data are available on homeostatic sleep control: it has been recently hypothesized that patients with cirrhosis and hepatic encephalopathy might be unable, due to excessive daytime sleepiness, to accumulate the need/ability to produce restorative sleep. This review will describe in some detail the features of sleep-wake disturbances in patients with cirrhosis, their mutual relationships, and those, if any, with hepatic failure/hepatic encephalopathy. A separate section will cover the available information on their pathophysiology. Finally, etiological treatment will be briefly discussed.

Evaluation of the steric impact of flavin adenine dinucleotide in Drosophila melanogaster cryptochrome function.

Photoreceptors are crucial components for circadian rhythm entrainment in animals, plants, fungi and cyanobacteria. Cryptochromes (CRYs) are flavin adenine dinucleotide (FAD) containing photoreceptors, and FAD is responsible for signal transduction, in contrast to photolyases where it promotes DNA-damage repair. In this work, we investigated an alternative role for FAD in CRY. We analyzed the Drosophila melanogaster CRY crystal structure by means of molecular dynamics, elucidating how this large co-factor within the receptor could be crucial for CRY structural stability. The co-factor appears indeed to improve receptor motility, providing steric hindrance. Moreover, multiple sequence alignments revealed that conserved motifs in the C-terminal tail could be necessary for functional stability.

A PDZ scaffolding/CaM-mediated pathway in Cryptochrome signaling.

Cryptochromes are cardinal constituents of the circadian clock, which orchestrates daily physiological rhythms in living organisms. A growing body of evidence points to their participation in pathways that have not traditionally been associated with circadian clock regulation, implying that cryptochromes may be subject to modulation by multiple signaling mechanisms. In this study, we demonstrate that human CRY2 (hCRY2) forms a complex with the large, modular scaffolding protein known as Multi-PDZ Domain Protein 1 (MUPP1). This interaction is facilitated by the calcium-binding protein Calmodulin (CaM) in a calcium-dependent manner. Our findings suggest a novel cooperative mechanism for the regulation of mammalian cryptochromes, mediated by calcium ions (Ca2+ ) and CaM. We propose that this Ca2+ /CaM-mediated signaling pathway may be an evolutionarily conserved mechanism that has been maintained from Drosophila to mammals, most likely in relation to its potential role in the broader context of cryptochrome function and regulation. Further, the understanding of cryptochrome interactions with other proteins and signaling pathways could lead to a better definition of its role within the intricate network of molecular interactions that govern circadian rhythms.