Journal of Biological Rhythms最新文献

{"title":"Chronotype, Social Jetlag, and Nicotine Use.","authors":"Neda Ghotbi,&nbsp;Andrea Rabenstein,&nbsp;Luísa K Pilz,&nbsp;Tobias Rüther,&nbsp;Till Roenneberg","doi":"10.1177/07487304231177197","DOIUrl":"https://doi.org/10.1177/07487304231177197","url":null,"abstract":"<p><p>Late chronotype, which often leads to higher social jetlag (SJL), is strongly associated with the prevalence of smoking. Any circadian disruption, strain, or misalignment, results in people not being able to live according to their biological time as is described by SJL, which we will therefore use as umbrella term. We hypothesized two scenarios potentially explaining the association between smoking and SJL: (A) If smoking delays the clock, circadian phase should advance upon quitting. (B) If people smoke more to compensate the consequences of SJL, circadian phase should not change upon quitting. To distinguish between these two hypotheses, we accompanied participants of a smoking cessation program (not involving nicotine replacement products) across the cessation intervention (3 weeks prior and 6 weeks after) by monitoring their circadian behavior, sleep quality, and daytime sleepiness via questionnaires and actimetry. Our results show no effects of cessation on SJL, chronotype, sleep quality, or daytime sleepiness, thereby favoring scenario (B). Thus, smoking may be a consequence of rather than a cause for SJL. Daytime sleepiness was a significant predictor for the outcome in our model but did not improve with cessation.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/27/f6/10.1177_07487304231177197.PMC10336714.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9799744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modern Language for Modern Circadian Biologists: The End of the \"Slave\" Oscillator.","authors":"Casey-Tyler Berezin","doi":"10.1177/07487304231152982","DOIUrl":"https://doi.org/10.1177/07487304231152982","url":null,"abstract":"323 As I sat at the computer in my living room, I was not entirely shocked at the words on the slide before me. In the spring of 2021, I was in a graduate-level course on neuronal circuits and behavior; I usually attended the virtual class from home before heading into the lab to continue my research on the role of intrinsically photosensitive retinal ganglion cells on circadian behavior. Naturally, I was thrilled to get to the circadian biology lectures. That day, we discussed the role of the “master” clock in regulating “slave” oscillators throughout the body. As circadian biologists, it’s not unusual to encounter these terms, but that doesn’t mean we should continue using them.1 In his early work, Colin S. Pittendrigh, often dubbed the founder of circadian biology, discussed the light-sensitive A-oscillator and autonomous B-oscillator (Pittendrigh et al., 1958). His co-author on this 1958 paper, Peter Kaus, was a physicist credited with providing mathematical expertise for Pittendrigh’s work. The emergence of the circadian “slave oscillator” appears to originate with Kaus in 1976 (Kaus, 1976), and was likely born from the electronics field where the term had been used since at least the 1940s (Alsberg and Leed, 1949). Master-slave terminology likely caught on because it was an “easy” metaphor, and at the time, “there were few Black engineers to object,” says ethno-mathematician Ron Eglash (All Together, Society of Women Engineers, 2020). Over the past 50 years, generations of circadian biologists have been taught these terms, and their use should not be a source of individual blame. However, we scientists have a duty to overcome the role science has played in the United States’s continued history of racism2 (Nobles et al., 2022). We are trained in the importance of precise language and leaving a rigorous path to follow. With a new generation of scientists comes new expectations for the way we communicate and conduct ourselves. Eliminating the “slave” oscillator won’t undo years of scientific racism, but perhaps it can be one less reason a prospective student might not come our way. It’s encouraging that it’s already more common to see the phrase “peripheral” oscillator than “slave” oscillator in research articles (Figure 1). Yet it would be remiss to attribute the growth in “peripheral” oscillators simply to changes in societal thinking. Rather, scientific advancements in the 1990s elucidated the autonomous nature of circadian oscillators proposed decades before (Pittendrigh et al., 1958). Isolated neurons were shown to retain circadian rhythms in culture (Michel et al., 1993; Welsh et al., 1995), and circadian oscillations (e.g., rhythmic clock gene expression) were found widespread throughout the periphery (Balsalobre et al., 1998; Plautz et al., 1997; Zylka et al., 1998). As such, a modern hierarchical view of circadian rhythms features a coordinating “pacemaker” rather than a vague, all-powerful “master,” and rightfully recognizes “slave” ","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9787477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular and Neural Mechanisms of Temperature Preference Rhythm in <i>Drosophila melanogaster</i>.","authors":"Tadahiro Goda, Yujiro Umezaki, Fumika N Hamada","doi":"10.1177/07487304231171624","DOIUrl":"10.1177/07487304231171624","url":null,"abstract":"<p><p>Temperature influences animal physiology and behavior. Animals must set an appropriate body temperature to maintain homeostasis and maximize survival. Mammals set their body temperatures using metabolic and behavioral strategies. The daily fluctuation in body temperature is called the body temperature rhythm (BTR). For example, human body temperature increases during wakefulness and decreases during sleep. BTR is controlled by the circadian clock, is closely linked with metabolism and sleep, and entrains peripheral clocks located in the liver and lungs. However, the underlying mechanisms of BTR are largely unclear. In contrast to mammals, small ectotherms, such as <i>Drosophila</i>, control their body temperatures by choosing appropriate environmental temperatures. The preferred temperature of <i>Drosophila</i> increases during the day and decreases at night; this pattern is referred to as the temperature preference rhythm (TPR). As flies are small ectotherms, their body temperature is close to that of the surrounding environment. Thus, <i>Drosophila</i> TPR produces BTR, which exhibits a pattern similar to that of human BTR. In this review, we summarize the regulatory mechanisms of TPR, including recent studies that describe neuronal circuits relaying ambient temperature information to dorsal neurons (DNs). The neuropeptide diuretic hormone 31 (DH31) and its receptor (DH31R) regulate TPR, and a mammalian homolog of DH31R, the calcitonin receptor (CALCR), also plays an important role in mouse BTR regulation. In addition, both fly TPR and mammalian BTR are separately regulated from another clock output, locomotor activity rhythms. These findings suggest that the fundamental mechanisms of BTR regulation may be conserved between mammals and flies. Furthermore, we discuss the relationships between TPR and other physiological functions, such as sleep. The dissection of the regulatory mechanisms of <i>Drosophila</i> TPR could facilitate an understanding of mammalian BTR and the interaction between BTR and sleep regulation.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/86/53/10.1177_07487304231171624.PMC10330063.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9795020","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"S-Cone Photoreceptors Regulate Daily Rhythms and Light-Induced Arousal/Wakefulness in Diurnal Grass Rats (<i>Arvicanthis niloticus</i>).","authors":"Antony B Kim, Emma M Beaver, Stephen G Collins, Lance J Kriegsfeld, Steven W Lockley, Kwoon Y Wong, Lily Yan","doi":"10.1177/07487304231170068","DOIUrl":"10.1177/07487304231170068","url":null,"abstract":"<p><p>Beyond visual perception, light has non-image-forming effects mediated by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). The present study first used multielectrode array recordings to show that in a diurnal rodent, Nile grass rats (<i>Arvicanthis niloticus</i>), ipRGCs generate rod/cone-driven and melanopsin-based photoresponses that stably encode irradiance. Subsequently, two ipRGC-mediated non-image-forming effects, namely entrainment of daily rhythms and light-induced arousal, were examined. Animals were first housed under a 12:12 h light/dark cycle (lights-on at 0600 h) with the light phase generated by a low-irradiance fluorescent light (F12), a daylight spectrum (D65) stimulating all photoreceptors, or a narrowband 480 nm spectrum (480) that maximized melanopsin stimulation and minimized S-cone stimulation (λ_max 360 nm) compared to D65. Daily rhythms of locomotor activities showed onset and offset closer to lights-on and lights-off, respectively, in D65 and 480 than in F12, and higher day/night activity ratio under D65 versus 480 and F12, suggesting the importance of S-cone stimulation. To assess light-induced arousal, 3-h light exposures using 4 spectra that stimulated melanopsin equally but S-cones differentially were superimposed on F12 background lighting: D65, 480, 480 + 365 (narrowband 365 nm), and D65 - 365. Compared to the F12-only condition, all four pulses increased in-cage activity and promoted wakefulness, with 480 + 365 having the greatest and longest-lasting wakefulness-promoting effects, again indicating the importance of stimulating S-cones as well as melanopsin. These findings provide insights into the temporal dynamics of photoreceptor contributions to non-image-forming photoresponses in a diurnal rodent that may help guide future studies of lighting environments and phototherapy protocols that promote human health and productivity.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10364626/pdf/nihms-1888368.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10292645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Persistence of Nocturnality in Decapitated and Bisected Flatworms.","authors":"Shauni E T Omond,&nbsp;John A Lesku","doi":"10.1177/07487304231158947","DOIUrl":"https://doi.org/10.1177/07487304231158947","url":null,"abstract":"<p><p>The ability of flatworms to regenerate entire brain structures, and indeed much of their body from mere fragments of the whole animal, presents the unique opportunity to observe the development of day-night rhythms in adult animals. In many animals, young are arrhythmic, and their species-specific timing of activity develops as the animal matures. In this study, we created two flatworm cohorts, housed in isolation, that were regenerating either (1) the brain in a decapitated animal, or (2) major body structures in a bisected, tailless animal. In this way, we observed how bisection influenced the level of activity and diel rhythmicity, and how these developed as each flatworm regenerated. Here, we demonstrate that intact flatworms were predominantly active at night, with peaks in activity seen in the hours after lights-off and before lights-on. While decapitated and tailless flatworms could still move, both were less active than the original animal, and both segments retained a nocturnal lifestyle. Furthermore, decapitated flatworms, once regenerated, again showed a U-shaped pattern of nocturnal activity reminiscent of the two night-time peaks seen in the original animal. These results could be used to further investigate how regeneration may affect motor control and motor output, or to further investigate the presence of a clock in the flatworm brain.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10278384/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10039687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Voluntary Wheel Running Exercise Does Not Attenuate Circadian and Cardiac Dysfunction Caused by Conditional Deletion of <i>Bmal1</i>.","authors":"Musharraf Yusifova,&nbsp;Aykhan Yusifov,&nbsp;Sydney M Polson,&nbsp;William D Todd,&nbsp;Emily E Schmitt,&nbsp;Danielle R Bruns","doi":"10.1177/07487304231152398","DOIUrl":"https://doi.org/10.1177/07487304231152398","url":null,"abstract":"<p><p>Circadian misalignment occurs with age, jet lag, and shift work, leading to maladaptive health outcomes including cardiovascular diseases. Despite the strong link between circadian disruption and heart disease, the cardiac circadian clock is poorly understood, prohibiting identification of therapies to restore the broken clock. Exercise is the most cardioprotective intervention identified to date and has been suggested to reset the circadian clock in other peripheral tissues. Here, we tested the hypothesis that conditional deletion of core circadian gene <i>Bmal1</i> would disrupt cardiac circadian rhythm and function and that this disruption would be ameliorated by exercise. To test this hypothesis, we generated a transgenic mouse with spatial and temporal deletion of <i>Bmal1</i> only in adult cardiac myocytes (Bmal1 cardiac knockout [cKO]). Bmal1 cKO mice demonstrated cardiac hypertrophy and fibrosis concomitant with impaired systolic function. This pathological cardiac remodeling was not rescued by wheel running. While the molecular mechanisms responsible for the profound cardiac remodeling are unclear, it does not appear to involve activation of the mammalian target of rapamycin (mTOR) signaling or changes in metabolic gene expression. Interestingly, cardiac deletion of <i>Bmal1</i> disrupted systemic rhythms as evidenced by changes in the onset and phasing of activity in relationship to the light/dark cycle and by decreased periodogram power as measured by core temperature, suggesting cardiac clocks can regulate systemic circadian output. Together, we suggest a critical role for cardiac <i>Bmal1</i> in regulating both cardiac and systemic circadian rhythm and function. Ongoing experiments will determine how disruption of the circadian clock causes cardiac remodeling in an effort to identify therapeutics to attenuate the maladaptive outcomes of a broken cardiac circadian clock.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10277226/pdf/nihms-1863524.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9657159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antisense Transcription of the <i>Neurospora</i> Frequency Gene Is Rhythmically Regulated by CSP-1 Repressor but Dispensable for Clock Function.","authors":"Ibrahim A Cemel,&nbsp;Axel C R Diernfellner,&nbsp;Michael Brunner","doi":"10.1177/07487304231153914","DOIUrl":"https://doi.org/10.1177/07487304231153914","url":null,"abstract":"<p><p>The circadian clock of <i>Neurospora crassa</i> is based on a negative transcriptional/translational feedback loops. The <i>frequency</i> (<i>frq</i>) gene controls the morning-specific rhythmic transcription of a sense RNA encoding FRQ, the negative element of the core circadian feedback loop. In addition, a long noncoding antisense RNA, <i>qrf</i>, is rhythmically transcribed in an evening-specific manner. It has been reported that the <i>qrf</i> rhythm relies on transcriptional interference with <i>frq</i> transcription and that complete suppression of <i>qrf</i> transcription impairs the circadian clock. We show here that <i>qrf</i> transcription is dispensable for circadian clock function. Rather, the evening-specific transcriptional rhythm of <i>qrf</i> is mediated by the morning-specific repressor CSP-1. Since CSP-1 expression is induced by light and glucose, this suggests a rhythmic coordination of <i>qrf</i> transcription with metabolism. However, a possible physiological significance for the circadian clock remains unclear, as suitable assays are not available.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10278383/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10021738","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Associations Between Multidimensional Sleep Health Parameters and Adolescents' Self-reported Light Exposure in the Free-living Environment.","authors":"Gabrielle Gauthier-Gagné,&nbsp;Sujata Saha,&nbsp;Jana Jensen,&nbsp;Gail Sommerville,&nbsp;Reut Gruber","doi":"10.1177/07487304231152987","DOIUrl":"https://doi.org/10.1177/07487304231152987","url":null,"abstract":"<p><p>The objective of this study was to characterize the associations between light exposure in the free-living environment and multiple dimensions of sleep health of typically developing adolescents. Fifty-six (29 girls, 27 boys) typically developing adolescents (mean age = 13.59, SD = 0.89, range = 12-17 years) participated. For six consecutive nights, sleep was assessed in the home environment using actigraphy. During the same period, participants were asked to fill out a daily sleep log and a daily light exposure log, and to complete questionnaires regarding their alertness and subjective sleep satisfaction. Longer self-reported exposure to daylight in the morning was associated with longer objectively measured sleep duration. Longer self-reported exposures to electronic devices in the evening were associated with later objectively measured sleep onset and offset times, shorter sleep duration, and greater day-to-day sleep variability. Longer morning exposure to outdoor light was associated with a longer sleep duration. Self-reported light exposure was not associated with sleep satisfaction, alertness/sleepiness, or sleep efficiency. Among the covariates, circadian preference accounted for the highest percentage of variance. Adolescents' sleep health is associated with the self-reported duration of exposure to daylight in the morning and to electronic devices in the evening.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9639211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sensitive Timing: A Reappraisal of Chronobiology's Foundational Texts.","authors":"Patrick Emery,&nbsp;André Klarsfeld,&nbsp;Ralf Stanewsky,&nbsp;Orie T Shafer","doi":"10.1177/07487304231169080","DOIUrl":"https://doi.org/10.1177/07487304231169080","url":null,"abstract":"<p><p>The origin of experimental chronobiology can be traced to observations made in the 18^th and 19^th centuries on the sensitive plant <i>Mimosa</i>, which were described in two seminal reports: Jean-Jacques d'Ortous de Mairan's \"<i>Observation Botanique</i>\" (A <i>Botanical Observation</i>) and Augustin Pyramus de Candolle's \"<i>Du sommeil des feuilles</i>\" (<i>On the sleep of leaves</i>). Both report observations of the striking daily closing and opening of <i>Mimosa</i> leaves in controlled environments. This review presents translations of both texts with the aim of staying as faithful as possible to the original French texts. We also present the historical context in which these texts were written and link them to subsequent experiments that aimed at testing the veracity of their central conclusions. In particular, we definitely establish that Mairan himself presented his work to the French Royal Academy of Sciences, while the published report of his observation was authored by Fontenelle, the Secretary of the Academy. In addition, we offer a translation of Mairan's own presentation, based on the hand-written minutes of the academy. Finally, we discuss the decades of work on plant rhythms that laid the foundation for modern experimental chronobiology, including translations and discussion of the insightful and prescient reports by Charles François de Cisternay Dufay, Henri Louis Duhamel du Monceau, Johann Gottfried Zinn, and Wilhelm Pfeffer, which describe their efforts to reproduce and extend Mairan's pioneering observations.</p>","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9655761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pittendrigh Remembered.","authors":"Gene D Block,&nbsp;Fred C Davis,&nbsp;Carl Hirschie Johnson,&nbsp;Colin Sandy Pittendrigh,&nbsp;William J Schwartz,&nbsp;Fred W Turek,&nbsp;Russell N Van Gelder","doi":"10.1177/07487304221148590","DOIUrl":"https://doi.org/10.1177/07487304221148590","url":null,"abstract":"","PeriodicalId":15056,"journal":{"name":"Journal of Biological Rhythms","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9639178","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}