Journal of Biological Rhythms

Circadian clocks govern daily physiological and behavioral processes and are crucial for health, yet disruptions can lead to various diseases. Chronotype, the state of circadian timing, varies between individuals and is reflected in behaviors such as sleep-wake patterns, cognitive performance, and physical activity. This interindividual variability is influenced by both genetic factors and environmental cues, but the relative contributions of each remain unclear, particularly in terms of plasticity - how much chronotype can shift in response to lifestyle and environmental factors. The gold standard for chronotype assessment, dim-light melatonin onset (DLMO), is invasive and impractical for large-scale studies, while blood-based molecular biomarker tests, which estimate internal time, show promise but are limited by practicality. Here, we introduce HairTime, a novel assay that estimates chronotype from a single hair sample collected at one point during the day. HairTime was developed and evaluated in two studies: a training study and a validation study, where it demonstrated a strong ability to predict chronotype, with DLMO as the comparison. This non-invasive method is suitable for large-scale, longitudinal studies and clinical practice. We assessed HairTime using over 4,000 samples, observing a normal distribution of chronotype across the population, with its estimation associating with age, sex, and notably, work schedules. The association with work schedules reveals the plasticity of chronotype, as workdays circadian timing earlier, highlighting that societal factors can influence and modify an individuals internal rhythm. Additionally, we explore the concept of circadian amplitude, finding that lower amplitude rhythms in hair follicle cells are linked to reduced chronotype prediction accuracy. Our results highlight that both intrinsic circadian mechanisms and external factors, such as lifestyle and work schedules, shape chronotype. HairTime offers an innovative tool for understanding circadian rhythms, facilitating personalized chronotherapy to improve health outcomes by aligning treatments with an individuals biological rhythms.

Proper entrainment of the bodys circadian rhythms to the environment is critical to human health. Light is one of the strongest cues driving circadian photoentrainment of the central circadian pacemaker, the suprachiasmatic nucleus (SCN), via projections from the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Circadian research has historically centered males, and recent work has revealed multiple sex-differences in circadian circuitry and function, indicating that our understanding of this system in females is severely limited. Moreover, while recent studies have investigated the role of hormonal modulation of light responses, the additional possibility that ipRGC inputs may also be sex-dependent has not been directly tested. Here, we report that not only do ipRGCs in female mice show higher levels of melanopsin expression, but that melanopsin also plays a larger role in shaping circadian photic responses in females compared to males. Collectively, these results define a new retinal source for sex-dependent differences in circadian behavior.

Transcriptomic studies of human circadian rhythm are limited in efficacy by the invasiveness of sampling. Studies of circadian dynamics should ideally use multiple blood samples drawn at regular intervals over the course of a day. This is difficult to achieve, particularly at night, without disrupting the very circadian rhythm that is being studied. We propose a method by which blood is drawn at a single initial timepoint, then cultured and repeatedly sampled over the course of a day. This method is minimally invasive to the subject. Our results demonstrate that the expression levels of circadian genes are more closely correlated between the cultured (ex vivo) cells and live (in vivo) samples than non-circadian genes, suggesting that this method can be used for effective circadian analysis.

Reproducibility is a persistent challenge in preclinical research. We used multi-week rodent machine vision home cage monitoring at three different pharmaceutical companies to examine factors governing replication of genotype differences in activity. Interlaboratory replication of genotype effects was surprisingly high. Longer study durations reduced noise, improving replication and reducing replicable sample sizes. These findings demonstrate the potential of long-term home cage digital monitoring as a method to enhance reproducibility.

A ubiquitous feature of circadian clocks across life forms is its organization as a network of coupled cellular oscillators. Individual cellular oscillators of the network often exhibit a considerable degree of heterogeneity in their intrinsic periods. While the interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clocks entrainability, our knowledge of mechanisms governing network heterogeneity remains elusive. In this study, we aimed to explore the principles that underlie inter-cellular period variation in circadian clock networks (clonal period-heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 h to 28 h. We report that while a single parent clone can produce progeny clones with a wide distribution of circadian periods, heterogeneity is not entirely stochastically driven but has a strong heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our panel, we found that inheritance of different expression patterns in at least three clock genes might govern clonal period-heterogeneity in circadian clock networks. Furthermore, we provide preliminary evidence suggesting that epigenetic variation might underlie such gene expression variation.

Both active movement profiles and robust circadian rhythms are linked to improved health outcomes, yet the underlying mechanisms remain partially understood. We investigated this relationship in young adults (n = 169, aged 18-30 years) under real-world conditions using actigraphy data. We performed k-means clustering on 12 accelerometer-based features capturing magnitude, duration, frequency, and intensity distribution to derive movement behavior profiles. As a proxy of circadian rhythms integrity we computed the Circadian Function Index (CFI), which combines intradaily variability, interdaily stability, and relative amplitude. We also assessed circadian phase and sleep quality parameters. Additionally, we quantified light exposure and physical activity over 3-hour daily intervals. The unsupervised algorithm identified two non-overlapping profiles among participants, the More Active (MA) and the Less Active (LA) profiles. MA exhibited a higher CFI (0.81 {+/-} 0.06 vs. 0.69 {+/-} 0.06, p <0.001), which was also positively associated with early-evening physical activity, but not with light exposure. MA also showed an earlier circadian phase, estimated as the midpoint of the five least active hours (L5c, 04:30 {+/-} 01:03 vs. 04:59 {+/-} 01:15, p adj. = 0.04), which was inversely associated with early-morning physical activity and late-morning light exposure. We found no differences in sleep quality between MA and LA. Our results underscore the association between movement behavior and overall circadian rhythms integrity. Importantly, these findings reinforce actigraphy as a multidimensional tool for both health research and clinical applications.

The circadian system evolved under natural light-dark cycles, while modern humans spend much of their time indoors under electric lighting that differs substantially from daylight in intensity, spectral composition, and temporal structure. How such lighting environments influence circadian system has not been systematically examined in a diurnal animal model under ecologically relevant conditions. In this study, we used the diurnal Nile grass rat (Arvicanthis niloticus) to assess daily locomotor rhythms across four lighting conditions designed to approximate common human exposure scenarios: rectangular daylight (D65-R; [~]5,600 lux), semi-sigmoidal daylight mimicking natural intensity dynamics (D65-S; matched peak intensity with [~]50% lower cumulative energy), fluorescent indoor light (F12; [~]150 lux), and fluorescent light supplemented with a one-hour midday daylight pulse (F12+D65-P). Using a within-subject design (n = 8), male grass rats were housed under each condition for two weeks. D65-R produced the highest daytime activity levels and the strongest day/night activity ratio, consistent with robust circadian entrainment. Despite matching peak intensity, D65-S did not yield comparable circadian outcomes, indicating that cumulative photon exposure, rather than peak intensity alone, contributes to entrainment strength. Notably, the addition of a one-hour midday daylight pulse (D65-P) partially increased circadian amplitude under otherwise fluorescent conditions, with higher periodogram amplitude relative to F12 alone. A separate cohort of males was exposed to D65-R or F12 for six weeks (n = 10/condition) to assess physiological outcomes, including metabolic and reproductive measures. Compared with the D65-R group, F12 group showed higher diabetic rate (10% vs. 40%) and reduced sperm mobility (45{+/-}6 vs. 19{+/-}1 %), consistent with potential downstream correlates of circadian rhythm disruption. Together, these findings demonstrate that lighting conditions characteristic of indoor environments produce weaker circadian organization than daylight-equivalent lighting in a diurnal rodent, which underscore the importance of light quality in shaping circadian rhythms and downstream physiological processes.

Arrhythmia is considered the most disrupted state of the biological circadian clock, and usually occurs when circadian regulatory genes are rendered non-functional, or the master clock (Suprachiasmatic Nucleus) is ablated. Since clock gene expression is aligned by the external solar day-night cycle to exhibit a 24-hour rhythm, we hypothesized that ill-timed light and dark exposure could negatively impact endogenous circadian clock function in mice. In this study, we present an environmentally driven approach to induce arrhythmia in mice that is also reversible. Using the previously characterized fragmented day-night cycle (FDN) where the 8-hour night is split into four 2-hour fragments and equally distributed across the 24-hour day, we show that mice gradually exposed to the FDN for 1 month lose their circadian rhythmicity. Furthermore, subsequent exposure to constant light or constant dark conditions does not yield typical circadian rhythms, but instead, reveals circadian arrhythmia. Finally, we show that the arrhythmic locomotion phenotype is reversible with one week of reintroduction to a 12 hr day-12 hr night cycle. This is the first study to show how the light-dark environment induces arrhythmia of an intact circadian clock and how it can be reversed.

Historically, the primary method for measuring murine circadian activity in vivo has been monitoring voluntary wheel running. Recently, passive infrared (PIR) motion sensors have emerged as an alternative that is not reliant on voluntary behaviour. While research has examined the differences between the two methods for measuring circadian parameters, little focus has been placed on how these techniques may confound the assessment of therapeutic interventions. Here, we show that wheel running activity is disproportionately affected by daily oral gavage of saline compared to sham gavage treatment. In contrast, PIR-monitored activity indicates little difference between the two treatments. Both PIR and running-wheel-measured activity show a reduction in circadian amplitude and an increase in intradaily variability during both types of gavage, likely reflecting the stress of daily gavage, though the mice showed no weight loss. This finding indicates that pre- and post-intervention comparisons will misattribute gavage effects to the intervention unless appropriate sham and vehicle controls are included. More broadly, the choice of circadian measurement technique fundamentally shapes the interpretation of pharmacological interventions and must be considered in experimental design.

The suprachiasmatic nucleus (SCN) of the hypothalamus is a principal light-responsive circadian clock that adjusts circadian rhythms in mammalian physiology and behavior to changes in external light signals. Although mechanisms underlying how light acutely resets the timing of circadian rhythms have been characterized, it remains elusive how light signals induce lasting changes in circadian period, so-called period after-effects. Here we have found that the period after-effects on circadian behavior of changing photoperiods are blocked by application of DNA methyltransferase inhibitors directed to the SCN. At the level of single light pulses that act as clock-resetting stimulations, pharmacologically inhibiting DNA methylation in the SCN significantly attenuates period after-effects following acute phase shifts in behavioral rhythms in vivo, and blocks period after-effects on clock gene rhythms in the isolated ex vivo SCN. Acute clock resetting shifts themselves, however, do not appear to require DNA methylation at the SCN and behavioral levels, in contrast to subsequent period plasticity. Our results indicate that DNA methylation in the SCN mediates light-induced period after-effects in response to photoperiods, and single light pulses, and together with previous studies showing that DNA methylation in the SCN is essential for period after-effects of non-24hr light cycles (T-cycles), suggest that DNA methylation in the SCN is a widespread mechanism of light-induced circadian period plasticity.

Circadian rhythms play a crucial role in regulating behavior, physiology, and health. Sexual dimorphism, a widespread phenomenon across species, influences circadian behaviors. Additionally, post-mating physiological changes in females are known to modulate various behaviors, yet their effects on circadian rhythms remain underexplored. Here, using Drosophila melanogaster, a powerful model for studying circadian mechanisms, we systematically assessed the impact of sex and mating status on circadian behavior. We measured circadian period length and rhythm strength in virgin and mated males and females, including females mated to males lacking Sex Peptide (SP), a key mediator of post-mating changes. Across four wild-type and control strains, we found that males consistently exhibited shorter circadian periods than females, regardless of mating status, suggesting that circadian period length is a robust sexually dimorphic trait. In contrast, rhythm strength was influenced by the interaction between sex and mating status, with female mating generally reducing rhythm strength in the presence of SP signaling. Notably, genetic background significantly modulated these effects on rhythm strength. Our findings demonstrate that while circadian period length is a stable sex-specific trait, rhythm strength is shaped by a complex interplay between sex, mating status, and genetic background. This study advances our understanding of how sex and mating influence circadian rhythms in Drosophila and provides a foundation for future research into sexually dimorphic mechanisms underlying human diseases associated with circadian disruptions.

Circadian clocks coordinate physiological and behavioral rhythms by synchronizing biological processes with environmental cues. These rhythms emerge during development, but it remains unclear whether their component genes are activated by a common program or assembled through distinct regulatory pathways. To address this, we used longitudinal luciferase reporters to monitor per3 and per2 expression across zebrafish embryonic and larval development. Although both genes are canonical components of the circadian clock, they showed strikingly different developmental regulation. Two temporal frames of circadian gene expression were identified: an embryonic stage and a larval stage, each evident under different entrainment conditions. Per3 displayed early rhythmic expression in light/dark conditions, which was independent of per2 and cry1a light-entrainment regulation, but required bmal activity. Meanwhile, per2 displayed light-responsive transcription and remained largely bmal-independent. At the same time, both genes exhibited an endogenous embryonic expression that could not be explained solely by light-driven regulation, indicating that developmental inputs contribute to clock gene activation before mature larval rhythms are established. These findings demonstrate that the zebrafish circadian system is not assembled through a single synchronized onset of clock gene expression, but through gene-specific regulatory programs that shift across development.

Food anticipatory activity (FAA) is a robust behavioral output of food-entrained circadian rhythms, characterized by increased locomotor activity prior to scheduled feeding. Despite the social nature of rodents, FAA is almost exclusively studied in singly housed animals, leaving the influence of social context largely unexplored. Here, we used implanted wireless devices to measure individual locomotor activity and subcutaneous body temperature in group-housed mice and compared these measures to singly housed controls. Social housing significantly suppressed FAA in both male and female mice. In parallel, preprandial increases in body temperature were markedly attenuated in group-housed animals. These findings demonstrate that FAA is a flexible, state-dependent behavior that reflects both circadian timing and energetic demand. Together, these results identify social context as an important and underappreciated determinant of food-entrained circadian biology.

2)BackgroundCircadian rhythm desynchrony (CD) occurs when there is a mismatch between the circadian clock and local time, such as shift work. Mouse models are commonly employed to study CD, but may have significant shortcomings such as environmental masking, a focus only on sleep physiology, and significant variability between study designs. ObjectiveThis study used in vivo telemetry for simultaneous, real-time monitoring of locomotor activity (LA), core body temperature (CBT), and brain activity (EEG) in freely moving C57BL/6J mice to assess CD effects. MethodsFour-month-old C57BL/6J mice (n=11) were surgically implanted with telemeters enabling simultaneous real-time recording of LA, CBT, EEG.: Mice were sequentially exposed to a control condition standard 12:12h light-dark cycle (T24) then 4, 8-day CD paradigms: 10:10 h short day (T20), social jet lag (SJL), repeated 6h phase advances (6A2), and a 3:3 h ultradian cycle (T6)For each paradigm, the final 48h of data (250 Hz) were analyzed. ResultsWe found clear differences in the severity of the effects of each CD paradigm on sleep and circadian fitness, where T20[~]T6>SJL>6A2. CBT revealed broader disruption, but EEG outputs proved the most sensitive indicators of internal desynchrony. ConclusionsEach CD paradigm produced a unique profile across behavioral, physiological, and neural domains. We have also identified Gamma CV as a novel, sensitive metric of CD. These results highlight the necessity of multimodal monitoring to accurately characterize the impact of ecologically relevant stressors on circadian and sleep physiology. Statement of SignificanceCircadian rhythm desynchrony (CD), driven by shift work, jet lag, and modern irregular light exposure, is a major health burden linked to metabolic, neurodegenerative, and neuropsychiatric diseases. However, standard methods for measuring CD in laboratory models often rely on simple locomotor activity, which can "mask" the true extent of internal circadian stress. In this study, we simultaneously monitored brain EEG activity, core body temperature, and motion across four distinct models of circadian stress. We discovered that locomotor activity is a deceptive indicator of health; while mice appeared to show no alterations under several stress paradigms, their brain waves and body temperatures revealed the underlying impact of CD. Specifically, we identified "Gamma CV" as a highly sensitive new brain-wave marker that detects early circuit instability even when behavior appears normal and sleep quantity is preserved. These findings provide a marker for identifying early neurological vulnerability to irregular light schedules, offering a potential bridge to understanding similar gamma brain-wave alterations seen in addiction, early-stage Alzheimers disease, and other disorders.

The effects of real-world stimuli and how they vary across individuals remains largely unknown for lack of a continuous real-world circadian marker. Here, we show how an underlying circadian rhythm in heart rate (CRHR) can be extracted from several widely used wearables. Analysis of over 130,000 days of data from medical interns on rotating shifts shows how CRHR dynamics are distinct from those of sleep-wake timing and vary greatly among individuals. Analysis of circadian timekeeping in travelers in 5 continents shows how the circadian timekeeping more carefully controls wake time rather than sleep time. We determine a personalized phase response curve (PRC) of CRHR to activity for each individual, representing the first passive and personalized determination of how human circadian timekeeping continuously changes due to real-world stimuli. These results collectively establish CRHR as a practical method to study circadian rhythms in the real world. eTOC BlurbWe show how the circadian rhythm in heart rate can be extracted from real world data collected by wearables. Studying data from a large cohort of medical interns working on shifts, we find very interesting dynamics of this circadian rhythm that are independent of the acute effects of activity or sleep-wake timing. These techniques can also determine personalized parameters of circadian timekeeping.

BackgroundThe suprachiasmatic nucleus (SCN) is the central circadian clock in mammals, regulating many daily physiological and behavioral rhythms. Dysregulation of the SCN is associated with various circadian disorders, highlighting the potential therapeutic benefits of targeting its neurons and output pathways. Vasopressin signaling is one of the main regulators of the synchronicity and the functional output of the SCN. MethodsWe investigated the effect of a single dose (30mg/kg) of the vasopressin V1a receptor (V1aR) antagonist, balovaptan, on resynchronization of locomotor activity rhythms in mice after a 6-hour phase advance of the light-dark cycle. To mechanistically model the effect of V1aR antagonism, we developed a mathematical framework simulating the SCN, its control of circadian biomarkers (melatonin, core body temperature), and the impact of V1aR antagonism. ResultsA single administration of the V1a antagonist balovaptan significantly accelerated resynchronization of locomotor activity rhythms to new light-dark cycles. To mechanistically understand this effect, we devised a mathematical model of the SCN that successfully captures this accelerated synchronization of circadian rhythms under V1aR antagonism. Additionally, the model replicates well-established SCN behaviors in both humans and rodents, including the phase response curve triggered by a light pulse at various circadian phases, and the desynchronization of the SCN observed in forced desynchronization experiments. Mechanistically, our model suggests that weakening vasopressin signaling via V1aR antagonism strengthens the SCNs resistance to internal desynchronization. Additionally, our model suggests a strong link between the endogenous period (tau) and the phase of circadian biomarkers, with longer tau values resulting in delayed biomarker rhythms. Importantly, the model predicts that V1aR antagonism induces a phase advance proportional to tau. The model predicts that individuals with longer endogenous periods, who consequently exhibit greater phase delays in their circadian rhythms, could experience more substantial phase advances in response to V1aR antagonism. DiscussionWe show that targeting V1aR is enough to cause a faster resynchronization to a new light-dark cycle in the jet lag paradigm and establish a computational framework for investigating its therapeutic potential in circadian rhythm disorders. This framework, adaptable to incorporate pharmacodynamic data, can be used to design clinical trials evaluating V1aR antagonism for treating circadian disorders.

BackgroundThe human gut microbiota undergoes daily fluctuations, yet its interaction with sleep-wake patterns during infancy remains largely uncharted. This study aims to elucidate the relationship between gut microbiota rhythmicity and the development of sleep patterns in infants over the first year of life. We continuously monitored 162 healthy infants across multiple days at 3, 6, and 12 months of age using ankle actigraphy and 24-hour diaries. The Circadian Function Index (CFI) was computed as a proxy for sleep-wake rhythm maturation. Stool samples were collected to profile gut microbiota taxa composition via 16S rRNA gene amplicon sequencing, and microbial oscillations were assessed through sine and cosine fitting to detect 24-hour patterns. ResultsOur findings revealed that the relative abundance of bacterial taxa exhibited rhythmic patterns, with 26 zOTUs (1.74%) following a sine pattern and 100 zOTUs (6.69%) displaying cosine rhythmicity. Cosine rhythmicity became more pronounced with age, showing strong maturation: 7 zOTUs at 3 months, 2 zOTUs at 6 months, and 86 zOTUs at 12 months. Notably, 105 zOTUs (7.02%) were associated with CFI, demonstrating a significant relationship between gut microbiota rhythms and sleep development. Among these, 27 zOTUs with sine dynamics and 96 zOTUs with cosine dynamics were linked to CFI, with this association strengthening as infants aged. ConclusionsThese results highlight the increasing synchronization between gut microbiota rhythmicity and sleep-wake cycles during infancy, pointing to a critical window for potential health interventions. This novel observation, previously reported in rodents and adults, underscores the role of gut microbiota in early human development, offering new avenues for enhancing developmental outcomes through targeted interventions.

Seasonal daylength, or circadian photoperiod, is a pervasive environmental signal that profoundly influences physiology and behavior. In mammals, the central circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus where it receives retinal input and synchronizes, or entrains, organismal physiology and behavior to the prevailing light cycle. The process of entrainment induces sustained plasticity in the SCN, but the molecular mechanisms underlying SCN plasticity are incompletely understood. Entrainment to different photoperiods persistently alters the timing, waveform, period, and light resetting properties of the SCN clock and its driven rhythms. To elucidate novel molecular mechanisms of photoperiod plasticity, we performed RNAseq on whole SCN dissected from mice raised in Long (LD 16:8) and Short (LD 8:16) photoperiods. Fewer rhythmic genes were detected in Long photoperiod and in general the timing of gene expression rhythms was advanced 4-6 hours. However, a few genes showed significant delays, including Gem. There were significant changes in the expression clock-associated gene Timeless and in SCN genes related to light responses, neuropeptides, GABA, ion channels, and serotonin. Particularly striking were differences in the expression of the neuropeptide signaling genes Prokr2 and Cck, as well as convergent regulation of the expression of three SCN light response genes, Dusp4, Rasd1, and Gem. Transcriptional modulation of Dusp4 and Rasd1, and phase regulation of Gem, are compelling candidate molecular mechanisms for plasticity in the SCN light response through their modulation of the critical NMDAR-MAPK/ERK-CREB/CRE light signaling pathway in SCN neurons. Modulation of Prokr2 and Cck may critically support SCN neural network reconfiguration during photoperiodic entrainment. Our findings identify the SCN light response and neuropeptide signaling gene sets as rich substrates for elucidating novel mechanisms of photoperiod plasticity.

Many genes exhibit circadian rhythms in expression. The amplitude of oscillation, both in core clock and circadian output genes, may differ from person to person. Mutations in core clock genes are known to alter global rhythmic properties, and researchers often informally discuss "circadian amplitude." Yet, it remains unclear whether, in the general population, differences in transcriptional amplitude are largely gene-specific, or if they reflect a global, transcriptome-wide pattern -- whether some individuals have globally higher or lower amplitude across the set of all rhythmic genes. We used Cosinor regression to reanalyze four human skin time-series transcriptomic datasets (paired epidermis/dermis samples, N=11, N=19) and found that, using either absolute or relative amplitude measures, distributions of gene amplitudes tended to cluster by subject. Using a non-parametric, permutation-based statistical test, we found that in many subjects this global amplitude trend was statistically significant (p [&le;] 0.01). Furthermore, we found that when rhythmic genes were divided into two sets based on peak time (genes peaking before-noon and after-noon), the subjects global amplitude in one set predicted global amplitude in the other set (p [&le;] 0.05). We also found that in the paired epidermis/dermis datasets, subjects global amplitude in epidermis predicted their global amplitude in the dermis (p [&le;] 0.05). After identifying these trends in the skin datasets, we then found that evidence for subject-specific transcriptional rhythm strength replicated across 6 additional human time-course datasets from adipose, muscle, and blood. Perhaps surprisingly, across datasets, we found that neither established metrics of core clock transcriptional organization nor the amplitude of core clock transcription were strongly correlated with subject-specific global amplitude.

Transcriptomic studies of human circadian rhythm are limited in efficacy by the invasiveness of sampling. Studies of circadian dynamics should ideally use multiple blood samples drawn at regular intervals over the course of a day. This is difficult to achieve, particularly at night, without disrupting the very circadian rhythm that is being studied. We propose a method by which blood is drawn at a single initial timepoint, then cultured and repeatedly sampled over the course of a day. This method is minimally invasive to the subject. Our results demonstrate that the expression levels of circadian genes are more closely correlated between the cultured (ex vivo) cells and live (in vivo) samples than non-circadian genes, suggesting that this method can be used for effective circadian analysis.