PM2.5 induces a senescent state in mouse AT2 cells.

PM2.5 is known to induce lung injury, but its toxic effects on lung regenerative machinery and the underlying mechanisms remain unknown. In this study, primary mouse alveolar type 2 (AT2) cells, considered stem cells in the gas-exchange barrier, were sorted using fluorescence-activated cell sorting. By developing microfluidic technology with constricted microchannels, we observed that both passage time and impedance opacities of mouse AT2 cells were reduced after PM2.5, indicating that PM2.5 induced a more deformable mechanical property and a higher membrane permeability. In vitro organoid cultures of primary mouse AT2 cells indicated that PM2.5 is able to impair the proliferative potential and self-renewal capacity of AT2 cells but does not affect AT1 differentiation. Furthermore, cell senescence biomarkers, p53 and γ-H2A.X at protein levels, P16ink4a and P21 at mRNA levels were increased in primary mouse AT2 cells after PM2.5 stimulations as shown by immunofluorescent staining and quantitative PCR analysis. Using several advanced single-cell technologies, this study sheds light on new mechanisms of the cytotoxic effects of atmospheric fine particulate matter on lung stem cell behavior.

Microbial changes and associated metabolic responses modify host plant adaptation in Stephanitis nashi.

Symbiotic microorganisms are essential for the physiological processes of herbivorous pests, including the pear lace bug Stephanitis nashi, which is known for causing extensive damage to garden plants and fruit trees due to its exceptional adaptability to diverse host plants. However, the specific functional effects of the microbiome on the adaptation of S. nashi to its host plants remains unclear. Here, we identified significant microbial changes in S. nashi on 2 different host plants, crabapple and cherry blossom, characterized by the differences in fungal diversity as well as bacterial and fungal community structures, with abundant correlations between bacteria or fungi. Consistent with the microbiome changes, S. nashi that fed on cherry blossom demonstrated decreased metabolites and downregulated key metabolic pathways, such as the arginine and mitogen-activated protein kinase signaling pathway, which were crucial for host plant adaptation. Furthermore, correlation analysis unveiled numerous correlations between differential microorganisms and differential metabolites, which were influenced by the interactions between bacteria or fungi. These differential bacteria, fungi, and associated metabolites may modify the key metabolic pathways in S. nashi, aiding its adaptation to different host plants. These results provide valuable insights into the alteration in microbiome and function of S. nashi adapted to different host plants, contributing to a better understanding of pest invasion and dispersal from a microbial perspective.

Visible-Light-Driven NO Release from Postmodified MOFs via Photoinduced Electron Transfer for Antibacterial Application.

Photoresponsive nitric oxide (NO)-releasing materials (NORMs) enable the spatiotemporal delivery of NO to facilitate their potential applications in physiological conditions. Here two novel metal-organic frameworks (MOFs)-based photoactive NORMs achieved by the incorporation of prefunctionalized NO donors into the photosensitive Fe-MOFs via a postmodification strategy is reported. The modified Fe-MOFs display superior photoactivity of NO release when exposed to visible light (up to 720 nm). Significantly, the visible-light-driven NO release properties are further corroborated by their efficient antibacterial performance.

Short-term PM2.5 exposure induces transient lung injury and repair.

Exposure to fine atmospheric particulate matter (PM) is known to induce lung inflammation and injury; however, the way in which sophisticated endogenous lung repair and regenerative programs respond to this exposure remains unknown. In this study, we established a whole-body mouse exposure model to mimic real scenarios. Exposure to fine PM (PM with an aerodynamic diameter ≤ 2.5 µm [PM2.5]; mean 1.05 mg/m3) for 1-month elicited inflammatory infiltration and epithelial alterations in the lung, which were resolved 6 months after cessation of exposure. Immune cells that responded to PM2.5 exposure mainly included macrophages and neutrophils. During PM2.5 exposure, alveolar epithelial type 2 cells initiated rapid repair of alveolar epithelial mucosa through proliferation. However, the reparative capacity of airway progenitor cells (club cells) was impaired, which may have been related to the oxidative production of neutrophils or macrophages, as suggested in organoid co-cultures. These data suggested that the pulmonary toxic effects of short-term exposure to fine atmospheric PM at a certain dosage could be overcome through tissue reparative mechanisms.

SIRT6 is an epigenetic repressor of thoracic aortic aneurysms via inhibiting inflammation and senescence.

Thoracic aortic aneurysms (TAAs) develop asymptomatically and are characterized by dilatation of the aorta. This is considered a life-threating vascular disease due to the risk of aortic rupture and without effective treatments. The current understanding of the pathogenesis of TAA is still limited, especially for sporadic TAAs without known genetic mutation. Sirtuin 6 (SIRT6) expression was significantly decreased in the tunica media of sporadic human TAA tissues. Genetic knockout of Sirt6 in mouse vascular smooth muscle cells accelerated TAA formation and rupture, reduced survival, and increased vascular inflammation and senescence after angiotensin II infusion. Transcriptome analysis identified interleukin (IL)-1ß as a pivotal target of SIRT6, and increased IL-1ß levels correlated with vascular inflammation and senescence in human and mouse TAA samples. Chromatin immunoprecipitation revealed that SIRT6 bound to the Il1b promoter to repress expression partly by reducing the H3K9 and H3K56 acetylation. Genetic knockout of Il1b or pharmacological inhibition of IL-1ß signaling with the receptor antagonist anakinra rescued Sirt6 deficiency mediated aggravation of vascular inflammation, senescence, TAA formation and survival in mice. The findings reveal that SIRT6 protects against TAA by epigenetically inhibiting vascular inflammation and senescence, providing insight into potential epigenetic strategies for TAA treatment.

Comparing the Performance of Hyphantria cunea (Lepidoptera: Arctiidae) on Artificial and Natural Diets: Feasibility of Mass-Rearing on Artificial Diets.

In China, Hyphantria cunea (Drury) is an invasive phytophagous pest; it attacks nearly all species of defoliated trees. To develop integrated pest management programs (IPM) for H. cunea, we need to ensure the availability of insects by mass-rearing them on artificial diets under laboratory conditions. This study compared the growth characteristics, nutritional indices, growth indices, and digestive enzyme activity of insects reared on Pterocarya stenoptera C.DC (Fagales: Juglandaceae), the Chinese wingnut, and an artificial diet. We also investigated the correlation between diet components and growth indices using principal components analysis and Pearson correlation analysis. We found that mass-rearing of H. cunea on an artificial diet was feasible. It led to a shorter developmental period, with heavier larvae and pupae than natural diets. The principal components analysis indicated that the growth indices and α-Amylase were significantly positively associated with PC1, which explained 82.45% of the total data variability. Pearson correlation analysis showed a significant correlation between digestion, absorption parameters, and growth. Developing a mass-rearing program to produce H. cunea on an artificial diet will be valuable for improving IPM strategies. Understanding the mechanism of the responses of phytophagous insect populations to anthropogenic diet regulation can provide new ideas and methods for pest control.

iTRAQ Proteomic Analysis of Interactions Between 20E and Phospholipase C in Apolygus lucorum (Meyer-Dür).

Polyphagous Apolygus lucorum has become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. Hormone 20-hydroxyecdysone (20E) regulates multiple insect development and physiology events. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes and related proteins that can be affected by PLC still remain unknown. Here, isobaric tag for relative and absolute quantitation (iTRAQ) and immunoblotting techniques were used to compare differentially expressed proteins (DEPs) in A. lucorum in response to the treatment of 20E and the PLC inhibitor U73122 as well as their combination. A total of 1,624 non-redundant proteins and 97, 248, 266 DEPs were identified in the 20E/control, U73122/control, and 20E + U73122/control groups, respectively. Only 8 DEPs, including pathogenesis-related protein 5-like, cuticle protein 19.8, trans-sialidase, larval cuticle protein A2B-like, cathepsin L1, hemolymph juvenile hormone-binding protein, ATP-dependent RNA helicase p62-like, and myosin-9 isoform X1, were detected in all three groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEPs were involved in diverse signaling pathways. The results were validated by immunoblotting, which highlighted the reliability of proteomics analysis. These findings provided novel insights into the function of PLC in 20E signaling pathway in A. lucorum.

The long non-coding RNA UPAT promotes gastric cancer cell progression via UHRF1.

BACKGROUND: LncRNA ubiquitin-like with PHD and RING finger domains 1 (UHRF1) protein associated transcript (UPAT) regulates the progression of many cancers. However, its role in gastric cancer (GC) is less frequently reported. OBJECTIVE: In the context of the promoting effect of lncRNA on modulating GC progression, detailed insights into the role and underlying mechanism of UPAT in GC are the foothold in this study. METHODS: Overall survival was calculated. The mRNA expressions of UPAT and UHRF1 were measured by qRT-PCR, and the protein expressions of UHRF1, Cyclin D1 and cleaved caspase-3 were determined by western blot. Cell viability, growth, migration and invasion were assessed by CCK-8, colony formation, wound healing and Transwell assays, respectively. Apoptosis rate and cell cycle were assayed by flow cytometry. RESULTS: UPAT was overexpressed in GC tissue and cell lines. Decreased UPAT level was associated with higher overall survival. Down-regulation of UPAT diminished cell proliferation, Cyclin D1 expression, and migration and invasion rates, increased apoptosis rate and cleaved caspase-3 expression, and blocked cell cycle in AGS and NCI-N87 cells. UPAT expression in GC was positively correlated with UHRF1 expression. UHRF1 overexpression offset the inhibitory effects of UPAT down-regulation on cell proliferation, migration, invasion and cell cycle, and partially reversed the positive effect of UPAT down-regulation on apoptosis. CONCLUSION: UPAT might positively regulate the progression of GC via interacting with UHRF1. The UHRF1/UPAT axis revealed in the present study may provide a promising approach to intervene in the progression of GC.

LKB1 deficiency upregulates RELM-α to drive airway goblet cell metaplasia.

Targeting airway goblet cell metaplasia is a novel strategy that can potentially reduce the chronic obstructive pulmonary disease (COPD) symptoms. Tumor suppressor liver kinase B1 (LKB1) is an important regulator of the proliferation and differentiation of stem/progenitor cells. In this study, we report that LKB1 expression was downregulated in the lungs of patients with COPD and in those of cigarette smoke-exposed mice. Nkx2.1Cre; Lkb1f/f mice with conditional loss of Lkb1 in mouse lung epithelium displayed airway mucus hypersecretion and pulmonary macrophage infiltration. Single-cell transcriptomic analysis of the lung tissues from Nkx2.1Cre; Lkb1f/f mice further revealed that airway goblet cell differentiation was altered in the absence of LKB1. An organoid culture study demonstrated that Lkb1 deficiency in mouse airway (club) progenitor cells promoted the expression of FIZZ1/RELM-α, which drove airway goblet cell differentiation and pulmonary macrophage recruitment. Additionally, monocyte-derived macrophages in the lungs of Nkx2.1Cre; Lkb1f/f mice exhibited an alternatively activated M2 phenotype, while expressing RELM-α, which subsequently aggravated airway goblet cell metaplasia. Our findings suggest that the LKB1-mediated crosstalk between airway progenitor cells and macrophages regulates airway goblet cell metaplasia. Moreover, our data suggest that LKB1 agonists might serve as a potential therapeutic option to treat respiratory disorders associated with goblet cell metaplasia.

Insights into chemosensory genes of Pagiophloeus tsushimanus adults using transcriptome and qRT-PCR analysis.

Pagiophloeus tsushimanus is a new, destructive, and monophagous weevil pest that thrives on Cinnamomum camphora, found in Shanghai. The functions of chemosensory genes involved in the host location and intraspecific communication of P. tsushimanus remain unknown. The male-female transcriptomes of P. tsushimanus adults were assembled using Illumina sequencing, and we focused on all chemosensory genes in transcriptomes. In general, 58,088 unigenes with a mean length of 1018.19 bp were obtained. In total, 39 odorant binding proteins (OBPs), 10 chemosensory proteins (CSPs), 22 olfactory receptors (ORs), 16 gustatory receptors (GRs), eight ionotropic receptors (IRs), and five sensory neuron membrane proteins (SNMPs) were identified. PtsuOBPs comprised four subfamilies (20 Minus-C, one Plus-C, two Dimer, and 15 Classic). Both PtsuOBPs and PtsuCSPs contained a highly conserved sequence motif of cysteine residues. PtsuORs including one olfactory receptor co-receptors (Ptsu/Orco) comprised seven predicted transmembrane domains. Phylogenetic analysis revealed that PtsuOBPs, PtsuCSPs, and PtsuORs in P. tsushimanus exhibited low homology compared to other insect species. The results of tissue- and sex-specific expression patterns indicated that PtsuOBPs and PtsuORs were highly abundant in the antennae; whereas, PtsuCSPs were not only highly abundant in antennae, but also abdominal apexes, wings, and legs. In conclusion, these results enrich the gene database of P. tsushimanus, which may serve as a basis for identifying novel targets to disrupt olfactory key genes and may provide a reverse validation method to identify attractants for formulating potential eco-friendly control strategies for this pest.

Phospholipase C gamma (PLCγ) regulates soluble trehalase in the 20E-induced fecundity of Apolygus lucorum.

Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis (Bt) cotton in China. Additionally, 20-hydroxyecdysone (20E) has important functions in many biological processes, including insect reproduction. Phospholipase C (PLC), which is an essential enzyme for phosphoinositide metabolism, is involved in 20E signal transduction, but its function in 20E-mediated reproduction in A. lucorum remains unclear. In this study, 20E increased AlPLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis. The 20E treatment also induced the considerable accumulation of two second messengers, inositol triphosphate and diacylglycerol. The expression levels of genes encoding vitellogenin (AlVg) and soluble trehalase (AlTre-1) were similar to those of AlPLCγ, and were upregulated in response to 20E. The silencing of AlPLCγ resulted in downregulated expression of AlTre-1 and AlVg. However, the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression. Moreover, the silencing of AlVg did not alter AlTre-1 expression. Furthermore, an examination of the insect specimens indicated that AlPLCγ is required for female adult reproduction, and that downregulated expression of this gene is associated with decreases in fecundity, adult longevity, and egg hatching rate as well as delayed oocyte maturation. We propose that 20E regulates AlTre-1 expression via AlPLCγ and affects Vg expression as well as ovary development to facilitate the reproductive activities of A. lucorum females.

Life history of aggression in Anastatus disparis (Hymenoptera: Eupelmidae) with extreme male-male combat.

Aggressive behaviour is common in animals and typically has lifetime consequences. As younger males have higher residual reproductive value than older males and lose more from injuries than older males do, the propensity for fighting tends to increase with age in many empirical reports and species. However, fighting patterns in those empirical reports cannot confirm the hypothesis that individuals cannot readily inflict injuries on their opponents. To address this shortcoming, a parasitoid wasp species, Anastatus disparis (Hymenoptera: Eupelmidae), was used as an experimental model to explore the characteristics of aggression from a life-history perspective; this wasp exhibits extreme fighting, resulting in contestants experiencing injury and death. Results showed that the energetic costs of fighting to injury significantly shortened life and caused the loss of most mating ability. Inconsistent with general predictions, the frequency and intensity of fighting in A. disparis significantly decreased with male age. Further study results showed significantly more young males were received by and successfully mated with virgin females, and most genes related to energy metabolism were downregulated in aged males. Our study provided supporting evidence that young A. disparis males show more aggression likely because of their resource holding potential and sexual attractiveness decline with age.

Bioinspired Hydrogel-Polymer Hybrids with a Tough and Antifatigue Interface via One-Step Polymerization.

Hydrogel hybrids are one of the key factors in life activities and biomimetic science; however, their development and utilization are critically impeded by their inadequate adhesive strength and intricate process. In nature, barnacles can stick to a variety of solid surfaces firmly (adhesive strength above 300 kPa) using a hydrophobic interface, which inspires us to firmly combine hydrogels and polymers through introducing an adhesive layer. By spreading a hydrophobic liquid membrane directly, tough combination of a hydrogel and a polymer substrate could be achieved after one-step polymerization. The fracture energy of the hydrogel attached to the surface of polyvinyl chloride was up to 1200 J m-2 and the tensile strength reached 1.21 MPa. Furthermore, the adhesion samples with this method exhibit an antifatigue performance, having withstood large bends and twists. It should be pointed out that this approach can also be applied to a variety of complicated surfaces. This work may expand the application range of hydrogels and provides an inspiration for hydrogel adhesion.

Experimental demonstration of a tunable photonic hook by a partially illuminated dielectric microcylinder.

In this Letter, we report the experimental observations of a tunable curved photonic nanojet (photonic hook) generated by a 5 µm polydimethylsiloxane microcylinder deposited on a silicon substrate and illuminated by 405 nm laser beam. A moveable opaque aluminum-mask is mounted in front of the microcylinder implementing partial illumination and imparting spatial curvature to the photonic nanojet. Experimental results of main parameters (tilt angle, width, and intensity) of emerging photonic hooks exhibit close agreement with numerical predictions of the near-field optical structures. The experimentally measured full widths at half-maximum of photonic hooks are 0.48λ, 0.56λ, and 0.76λ for tilt angles of θ=0∘, 5.7°, and 20.1°, respectively. Photonic hooks possess great potential in complex manipulation such as super-resolution imaging, surface fabrication, and optomechanical manipulation along curved trajectories.

Sexual dimorphism and sex-biased gene expression in an egg parasitoid species, Anastatus disparis.

BACKGROUND: Differences in the expression of genes present in both sexes are assumed to contribute to sex differences including behavioural, physiological and morphological dimorphisms. For enriching our knowledge of gender differences in an important egg parasitoid wasp, Anastatus disparis (Hymenoptera: Eupelmidae), sex-biased differences in gene expression were investigated using Illumina-based transcriptomic analysis. RESULTS: A total of 15,812 resulting unigenes were annotated, and a large set of genes accounting for 50.09% of the total showed sex-biased expression and included 630 sex-specific genes. Gene Ontology (GO) enrichment analyses showed that the functional categories associated with sex-biased genes were mainly related to reproduction. In addition, the transcriptome data provided evidence that sex pheromones in A. disparis are produced by the female, and activity of Δ12-desaturases appear to have been replaced by Δ9-desaturases playing roles in sex pheromone production. The large set of sex-biased genes identified in this study provide a molecular background for sexually dimorphic traits such as flyability, longevity, and aggression in this species and suggests candidate venom proteins expressed only in females that could be used for biological control. CONCLUSIONS: This study provides comprehensive insight into sexually dimorphic traits of a parasitoid wasp and can inform future research into the molecular mechanisms underlying such traits and the application of parasitoids to the biological control of pest species.

Defecation function and quality of life in patients with slow-transit constipation after colectomy.

BACKGROUND: Although total or subtotal colectomy for slow-transit constipation (STC) has been proven to be a definite treatment, the associated defecation function and quality of life (QOL) are rarely studied. AIM: To evaluate the effectiveness of surgery for STC regarding defecation function and QOL. METHODS: From March 2013 to September 2017, 30 patients undergoing surgery for STC in our department were analyzed. Preoperative, intra-operative, and postoperative 3-mo, 6-mo, 1-year, and 2-year follow-up details were recorded. Defecation function was assessed by bowel movements, abdominal pain, bloating, straining, laxative, enema use, diarrhea, and the Wexner constipation and incontinence scales. QOL was evaluated using the gastrointestinal QOL index and the 36-item short form survey. RESULTS: The majority of patients (93.1%, 27/29) stated that they benefited from the operation at the 2-year follow-up. At each time point of the follow-up, the number of bowel movements per week significantly increased compared with that of the preoperative conditions (P < 0.05). Similarly, compared with the preoperative values, a marked decline was observed in bloating, straining, laxative, and enema use at each time point of the follow-up (P < 0.05). Postoperative diarrhea could be controlled effectively and notably improved at the 2-year follow-up. The Wexner incontinence scores at 6-mo, 1-year, and 2-year were notably lower than those at the 3-mo follow-up (P < 0.05). Compared with those of the preoperative findings, the Wexner constipation scores significantly decreased following surgery (P < 0.05). Thus, it was reasonable to find that the gastrointestinal QOL index scores clearly increase (P < 0.05) and that the 36-item short form survey results displayed considerable improvements in six spheres (role physical, role emotional, physical pain, vitality, mental health, and general health) following surgery. CONCLUSION: Total or subtotal colectomy for STC is not only effective in alleviating constipation-related symptoms but also in enhancing patients' QOL.

Effect of Winning Experience on Aggression Involving Dangerous Fighting Behavior in Anastatus disparis (Hymenoptera: Eupelmidae).

Aggressive behavior is widely observed in animal species for acquiring important resources and usually includes both dangerous and nondangerous fighting patterns. Only a few species show dangerous fighting patterns that are defined by fights ending with contestants being severely injured or killed. Prior experience, an important factor in many species, has been demonstrated to affect a contestant's subsequent fighting behavior. Few studies have focused on the effect of experience on aggression involving dangerous fighting patterns. Here, an egg parasitoid wasp, Anastatus disparis, which shows extreme and dangerous fighting behavior to acquire mating opportunities, was used as an experimental model. Our results showed that the fighting intensity of the winning males significantly decreased subsequent fighting behavior, which was inconsistent with general predictions. Transcriptomic analyses showed that many genes related to energy metabolism were downregulated in winners, and winners increased their fighting intensity after dietary supplementation. Our study suggested that fighting in A. disparis is a tremendous drain on energy. Thus, although males won at combat, significant reductions in available energy constrained the intensity of subsequent fights and influenced strategic decisions. In addition, winners might improve their fighting skills and abilities from previous contests, and their fighting intensity after dietary supplementation was significantly higher than that of males without any fighting experience. Generally, in A. disparis, although winners increased their fighting ability with previous experience, the available energy in winners was likely to be a crucial factor affecting the intensity and strategic decisions in subsequent fights.

Vascular Transcriptome Profiling Reveals Aging-Related Genes in Angiotensin â ¡-Induced Hypertensive Mouse Aortas.

Objective Angiotensin Ⅱ (Ang Ⅱ)-induced vascular damage is a major risk of hypertension. However, the underlying molecular mechanism of AngⅡ-induced vascular damage is still unclear. In this study, we explored the novel mechanism associated with Ang II-induced hypertension. Methods We treated 8- to 12-week-old C57BL/6J male mice with saline and Ang Ⅱ(0.72 mg/kg·d) for 28 days, respectively. Then the RNA of the media from the collected mice aortas was extracted for transcriptome sequencing. Principal component analysis was applied to show a clear separation of different samples and the distribution of differentially expressed genes was manifested by Volcano plot. Functional annotations including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed to reveal the molecular mechanism of Ang Ⅱ-induced hypertension. Finally, the differentially expressed genes were validated by using quantitative real-time PCR. Results The result revealed that a total of 773 genes, including 599 up-regulated genes and 174 down-regulated genes, were differentially expressed in the aorta of Ang Ⅱ-induced hypertension mice model. Functional analysis of differentially expressed genes manifested that various cellular processes may be involved in the Ang Ⅱ-induced hypertension, including some pathways associated with hypertension such as extracellular matrix, inflammation and immune response. Interestingly, we also found that the differentially expressed genes were enriched in vascular aging pathway, and further validated that the expression levels of insulin-like growth factor 1 and adiponectin were significantly increased (P<0.05). Conclusion We identify that vascular aging is involved in Ang Ⅱ-induced hypertension, and insulin-like growth factor 1 and adiponectin may be important candidate genes leading to vascular aging.

Diurnal oscillations of endogenous H2O2 sustained by p66Shc regulate circadian clocks.

Redox balance, an essential feature of healthy physiological steady states, is regulated by circadian clocks, but whether or how endogenous redox signalling conversely regulates clockworks in mammals remains unknown. Here, we report circadian rhythms in the levels of endogenous H2O2 in mammalian cells and mouse livers. Using an unbiased method to screen for H2O2-sensitive transcription factors, we discovered that rhythmic redox control of CLOCK directly by endogenous H2O2 oscillations is required for proper intracellular clock function. Importantly, perturbations in the rhythm of H2O2 levels induced by the loss of p66Shc, which oscillates rhythmically in the liver and suprachiasmatic nucleus (SCN) of mice, disturb the rhythmic redox control of CLOCK function, reprogram hepatic transcriptome oscillations, lengthen the circadian period in mice and modulate light-induced clock resetting. Our findings suggest that redox signalling rhythms are intrinsically coupled to the circadian system through reversible oxidative modification of CLOCK and constitute essential mechanistic timekeeping components in mammals.