Multiplex influences on vigilance and biochemical variables induced by sleep deprivation.

Introduction: Sleep loss and sleep deprivation (SD) cause deleterious influences on health, cognition, mood and behaviour. Nevertheless, insufficient sleep and SD are prevalent across many industries and occur in various emergencies. The deleterious consequences of SD have yet to be fully elucidated. This study aimed to assess the extensive influences of SD on physiology, vigilance, and plasma biochemical variables. Methods: Seventeen volunteers were recruited to participate in a 32.5-h SD experiment. Multiple physiological and cognitive variables, including tympanic temperature, blood oxygen saturation (SaO2), and vigilance were recorded. Urinal/salivary samples were collected and subjected to cortisol or cortisone analysis, and plasma samples were subjected to transcriptomic analysis of circular RNA (circRNA) expression using microarray. Plasma neurotransmitters were measured by targeted metabolic analysis, and the levels of inflammatory factors were assessed by antibody microarray. Results: The volunteers showed significantly increased sleepiness and decreased vigilance during SD, and the changes in circadian rhythm and plasma biochemistry were observed. The plasma calcium (p = 0.0007) was induced by SD, while ischaemia-modified albumin (IMA, p = 0.0030) and total bile acid (TBA, p = 0.0157) decreased. Differentially expressed circRNAs in plasma were identified, which are involved in multiple signaling pathways including neuronal regulation and immunity. Accordingly, SD induced a decrease in 3-hydroxybutyric acid (3OBH, p = 0.0002) and an increase in thyroxine (T4, p < 0.0001) in plasma. The plasma anti-inflammatory cytokine IL-10 was downregulated while other ten inflammatory factors were upregulated. Conclusion: This study demonstrates that SD influences biochemical, physiological, cognitive variables, and the significantly changed variables may serve as candidates of SD markers. These findings may further our understanding of the detrimental consequence of sleep disturbance at multiple levels.

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species.

Circadian clocks drive a large array of physiological and behavioral activities. At the molecular level, circadian clocks are composed of positive and negative elements that form core oscillators generating the basic circadian rhythms. Over the course of the circadian period, circadian negative proteins undergo progressive hyperphosphorylation and eventually degrade, and their stability is finely controlled by complex post-translational pathways, including protein modifications, genetic codon preference, protein-protein interactions, chaperon-dependent conformation maintenance, degradation, etc. The effects of phosphorylation on the stability of circadian clock proteins are crucial for precisely determining protein function and turnover, and it has been proposed that the phosphorylation of core circadian clock proteins is tightly correlated with the circadian period. Nonetheless, recent studies have challenged this view. In this review, we summarize the research progress regarding the function, regulation, and mechanism of protein stability in the circadian clock systems of multiple model organisms, with an emphasis on Neurospora crassa, in which circadian mechanisms have been extensively investigated. Elucidation of the highly complex and dynamic regulation of protein stability in circadian clock networks would greatly benefit the integrated understanding of the function, regulation, and mechanism of protein stability in a wide spectrum of other biological processes.

Differential regulation of phosphorylation, structure, and stability of circadian clock protein FRQ isoforms.

Neurospora crassa is an important model organism for circadian clock research. The Neurospora core circadian component FRQ protein has two isoforms, large FRQ (l-FRQ) and small FRQ (s-FRQ), of which l-FRQ bears an additional N-terminal 99-amino acid fragment. However, how the FRQ isoforms operate differentially in regulating the circadian clock remains elusive. Here, we show l-FRQ and s-FRQ play different roles in regulating the circadian negative feedback loop. Compared to s-FRQ, l-FRQ is less stable and undergoes hypophosphorylation and faster degradation. The phosphorylation of the C-terminal l-FRQ 794-aa fragment was markedly higher than that of s-FRQ, suggesting the l-FRQ N-terminal 99-aa region may regulate the phosphorylation of the entire FRQ protein. Quantitative label-free LC/MS analysis identified several peptides that were differentially phosphorylated between l-FRQ and s-FRQ, which were distributed in FRQ in an interlaced fashion. Furthermore, we identified two novel phosphorylation sites, S765 and T781; mutations S765A and T781A showed no significant effects on conidiation rhythmicity, although T781 conferred FRQ stability. These findings demonstrate that FRQ isoforms play differential roles in the circadian negative feedback loop and undergo different regulations of phosphorylation, structure, and stability. The l-FRQ N-terminal 99-aa region plays an important role in regulating the phosphorylation, stability, conformation, and function of the FRQ protein. As the FRQ circadian clock counterparts in other species also have isoforms or paralogues, these findings will also further our understanding of the underlying regulatory mechanisms of the circadian clock in other organisms based on the high conservation of circadian clocks in eukaryotes.