Diet Diurnally Regulates Small Intestinal Microbiome-Epithelial-Immune Homeostasis and Enteritis.

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10+ lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.

The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.

Minute-scale oscillatory sequences in medial entorhinal cortex.

The medial entorhinal cortex (MEC) hosts many of the brain's circuit elements for spatial navigation and episodic memory, operations that require neural activity to be organized across long durations of experience1. Whereas location is known to be encoded by spatially tuned cell types in this brain region2,3, little is known about how the activity of entorhinal cells is tied together over time at behaviourally relevant time scales, in the second-to-minute regime. Here we show that MEC neuronal activity has the capacity to be organized into ultraslow oscillations, with periods ranging from tens of seconds to minutes. During these oscillations, the activity is further organized into periodic sequences. Oscillatory sequences manifested while mice ran at free pace on a rotating wheel in darkness, with no change in location or running direction and no scheduled rewards. The sequences involved nearly the entire cell population, and transcended epochs of immobility. Similar sequences were not observed in neighbouring parasubiculum or in visual cortex. Ultraslow oscillatory sequences in MEC may have the potential to couple neurons and circuits across extended time scales and serve as a template for new sequence formation during navigation and episodic memory formation.

Periodic inhibition of Erk activity drives sequential somite segmentation.

Sequential segmentation creates modular body plans of diverse metazoan embryos1-4. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation4. However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1-Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish5. Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species3,4,6-8. Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients.

The whisking oscillator circuit.

Central oscillators are primordial neural circuits that generate and control rhythmic movements1,2. Mechanistic understanding of these circuits requires genetic identification of the oscillator neurons and their synaptic connections to enable targeted electrophysiological recording and causal manipulation during behaviours. However, such targeting remains a challenge with mammalian systems. Here we delimit the oscillator circuit that drives rhythmic whisking-a motor action that is central to foraging and active sensing in rodents3,4. We found that the whisking oscillator consists of parvalbumin-expressing inhibitory neurons located in the vibrissa intermediate reticular nucleus (vIRtPV) in the brainstem. vIRtPV neurons receive descending excitatory inputs and form recurrent inhibitory connections among themselves. Silencing vIRtPV neurons eliminated rhythmic whisking and resulted in sustained vibrissae protraction. In vivo recording of opto-tagged vIRtPV neurons in awake mice showed that these cells spike tonically when animals are at rest, and transition to rhythmic bursting at the onset of whisking, suggesting that rhythm generation is probably the result of network dynamics, as opposed to intrinsic cellular properties. Notably, ablating inhibitory synaptic inputs to vIRtPV neurons quenched their rhythmic bursting, impaired the tonic-to-bursting transition and abolished regular whisking. Thus, the whisking oscillator is an all-inhibitory network and recurrent synaptic inhibition has a key role in its rhythmogenesis.

Sleep Spindles: Mechanisms and Functions.

Sleep spindles are burstlike signals in the electroencephalogram (EEG) of the sleeping mammalian brain and electrical surface correlates of neuronal oscillations in thalamus. As one of the most inheritable sleep EEG signatures, sleep spindles probably reflect the strength and malleability of thalamocortical circuits that underlie individual cognitive profiles. We review the characteristics, organization, regulation, and origins of sleep spindles and their implication in non-rapid-eye-movement sleep (NREMS) and its functions, focusing on human and rodent. Spatially, sleep spindle-related neuronal activity appears on scales ranging from small thalamic circuits to functional cortical areas, and generates a cortical state favoring intracortical plasticity while limiting cortical output. Temporally, sleep spindles are discrete events, part of a continuous power band, and elements grouped on an infraslow time scale over which NREMS alternates between continuity and fragility. We synthesize diverse and seemingly unlinked functions of sleep spindles for sleep architecture, sensory processing, synaptic plasticity, memory formation, and cognitive abilities into a unifying sleep spindle concept, according to which sleep spindles 1) generate neural conditions of large-scale functional connectivity and plasticity that outlast their appearance as discrete EEG events, 2) appear preferentially in thalamic circuits engaged in learning and attention-based experience during wakefulness, and 3) enable a selective reactivation and routing of wake-instated neuronal traces between brain areas such as hippocampus and cortex. Their fine spatiotemporal organization reflects NREMS as a physiological state coordinated over brain and body and may indicate, if not anticipate and ultimately differentiate, pathologies in sleep and neurodevelopmental, -degenerative, and -psychiatric conditions.

Time is of the essence: The importance of considering biological rhythms in an increasingly polluted world.

Biological rhythms have a crucial role in shaping the biology and ecology of organisms. Light pollution is known to disrupt these rhythms, and evidence is emerging that chemical pollutants can cause similar disruption. Conversely, biological rhythms can influence the effects and toxicity of chemicals. Thus, by drawing insights from the extensive study of biological rhythms in biomedical and light pollution research, we can greatly improve our understanding of chemical pollution. This Essay advocates for the integration of biological rhythmicity into chemical pollution research to gain a more comprehensive understanding of how chemical pollutants affect wildlife and ecosystems. Despite historical barriers, recent experimental and technological advancements now facilitate the integration of biological rhythms into ecotoxicology, offering unprecedented, high-resolution data across spatiotemporal scales. Recognizing the importance of biological rhythms will be essential for understanding, predicting, and mitigating the complex ecological repercussions of chemical pollution.

Cetaceans are the next frontier for vocal rhythm research.

While rhythm can facilitate and enhance many aspects of behavior, its evolutionary trajectory in vocal communication systems remains enigmatic. We can trace evolutionary processes by investigating rhythmic abilities in different species, but research to date has largely focused on songbirds and primates. We present evidence that cetaceans-whales, dolphins, and porpoises-are a missing piece of the puzzle for understanding why rhythm evolved in vocal communication systems. Cetaceans not only produce rhythmic vocalizations but also exhibit behaviors known or thought to play a role in the evolution of different features of rhythm. These behaviors include vocal learning abilities, advanced breathing control, sexually selected vocal displays, prolonged mother-infant bonds, and behavioral synchronization. The untapped comparative potential of cetaceans is further enhanced by high interspecific diversity, which generates natural ranges of vocal and social complexity for investigating various evolutionary hypotheses. We show that rhythm (particularly isochronous rhythm, when sounds are equally spaced in time) is prevalent in cetacean vocalizations but is used in different contexts by baleen and toothed whales. We also highlight key questions and research areas that will enhance understanding of vocal rhythms across taxa. By coupling an infraorder-level taxonomic assessment of vocal rhythm production with comparisons to other species, we illustrate how broadly comparative research can contribute to a more nuanced understanding of the prevalence, evolution, and possible functions of rhythm in animal communication.

The Dynamic Basis of Respiratory Rhythm Generation: One Breath at a Time.

Rhythmicity is a universal timing mechanism in the brain, and the rhythmogenic mechanisms are generally dynamic. This is illustrated for the neuronal control of breathing, a behavior that occurs as a one-, two-, or three-phase rhythm. Each breath is assembled stochastically, and increasing evidence suggests that each phase can be generated independently by a dedicated excitatory microcircuit. Within each microcircuit, rhythmicity emerges through three entangled mechanisms: ( a) glutamatergic transmission, which is amplified by ( b) intrinsic bursting and opposed by ( c) concurrent inhibition. This rhythmogenic triangle is dynamically tuned by neuromodulators and other network interactions. The ability of coupled oscillators to reconfigure and recombine may allow breathing to remain robust yet plastic enough to conform to nonventilatory behaviors such as vocalization, swallowing, and coughing. Lessons learned from the respiratory network may translate to other highly dynamic and integrated rhythmic systems, if approached one breath at a time.

Pattern dynamics and stochasticity of the brain rhythms.

Our current understanding of brain rhythms is based on quantifying their instantaneous or time-averaged characteristics. What remains unexplored is the actual structure of the waves-their shapes and patterns over finite timescales. Here, we study brain wave patterning in different physiological contexts using two independent approaches: The first is based on quantifying stochasticity relative to the underlying mean behavior, and the second assesses "orderliness" of the waves' features. The corresponding measures capture the waves' characteristics and abnormal behaviors, such as atypical periodicity or excessive clustering, and demonstrate coupling between the patterns' dynamics and the animal's location, speed, and acceleration. Specifically, we studied patterns of θ, γ, and ripple waves recorded in mice hippocampi and observed speed-modulated changes of the wave's cadence, an antiphase relationship between orderliness and acceleration, as well as spatial selectiveness of patterns. Taken together, our results offer a complementary-mesoscale-perspective on brain wave structure, dynamics, and functionality.

Inhibitory Subpopulations in preBötzinger Complex Play Distinct Roles in Modulating Inspiratory Rhythm and Pattern.

Inhibitory neurons embedded within mammalian neural circuits shape breathing, walking, and other rhythmic motor behaviors. At the core of the neural circuit controlling breathing is the preBötzinger Complex (preBötC), where GABAergic (GAD1/2+) and glycinergic (GlyT2+) neurons are functionally and anatomically intercalated among glutamatergic Dbx1-derived (Dbx1+) neurons that generate rhythmic inspiratory drive. The roles of these preBötC inhibitory neurons in breathing remain unclear. We first characterized the spatial distribution of molecularly defined preBötC inhibitory subpopulations in male and female neonatal double reporter mice expressing either tdTomato or EGFP in GlyT2+, GAD1+, or GAD2+ neurons. We found that the majority of preBötC inhibitory neurons expressed both GlyT2 and GAD2 while a much smaller subpopulation also expressed GAD1. To determine the functional role of these subpopulations, we used holographic photostimulation, a patterned illumination technique, in rhythmically active medullary slices from neonatal Dbx1tdTomato;GlyT2EGFP and Dbx1tdTomato;GAD1EGFP double reporter mice of either sex. Stimulation of 4 or 8 preBötC GlyT2+ neurons during endogenous rhythm prolonged the interburst interval in a phase-dependent manner and increased the latency to burst initiation when bursts were evoked by stimulation of Dbx1+ neurons. In contrast, stimulation of 4 or 8 preBötC GAD1+ neurons did not affect interburst interval or latency to burst initiation. Instead, photoactivation of GAD1+ neurons during the inspiratory burst prolonged endogenous and evoked burst duration and decreased evoked burst amplitude. We conclude that GlyT2+/GAD2+ neurons modulate breathing rhythm by delaying burst initiation while a smaller GAD1+ subpopulation shapes inspiratory patterning by altering burst duration and amplitude.

Speech rhythms and their neural foundations.

The recognition of spoken language has typically been studied by focusing on either words or their constituent elements (for example, low-level features or phonemes). More recently, the 'temporal mesoscale' of speech has been explored, specifically regularities in the envelope of the acoustic signal that correlate with syllabic information and that play a central role in production and perception processes. The temporal structure of speech at this scale is remarkably stable across languages, with a preferred range of rhythmicity of 2- 8 Hz. Importantly, this rhythmicity is required by the processes underlying the construction of intelligible speech. A lot of current work focuses on audio-motor interactions in speech, highlighting behavioural and neural evidence that demonstrates how properties of perceptual and motor systems, and their relation, can underlie the mesoscale speech rhythms. The data invite the hypothesis that the speech motor cortex is best modelled as a neural oscillator, a conjecture that aligns well with current proposals highlighting the fundamental role of neural oscillations in perception and cognition. The findings also show motor theories (of speech) in a different light, placing new mechanistic constraints on accounts of the action-perception interface.

Marmosets mutually compensate for differences in rhythms when coordinating vigilance.

Synchronization is widespread in animals, and studies have often emphasized how this seemingly complex phenomenon can emerge from very simple rules. However, the amount of flexibility and control that animals might have over synchronization properties, such as the strength of coupling, remains underexplored. Here, we studied how pairs of marmoset monkeys coordinated vigilance while feeding. By modeling them as coupled oscillators, we noted that (1) individual marmosets do not show perfect periodicity in vigilance behaviors, (2) nevertheless, marmoset pairs started to take turns being vigilant over time, a case of anti-phase synchrony, (3) marmosets could couple flexibly; the coupling strength varied with every new joint feeding bout, and (4) marmosets could control the coupling strength; dyads showed increased coupling if they began in a more desynchronized state. Such flexibility and control over synchronization require more than simple interaction rules. Minimally, animals must estimate the current degree of asynchrony and adjust their behavior accordingly. Moreover, the fact that each marmoset is inherently non-periodic adds to the cognitive demand. Overall, our study provides a mathematical framework to investigate the cognitive demands involved in coordinating behaviors in animals, regardless of whether individual behaviors are rhythmic or not.

Visual periodicity reveals distinct attentional signatures for face and non-face categories.

Observers can selectively deploy attention to regions of space, moments in time, specific visual features, individual objects, and even specific high-level categories-for example, when keeping an eye out for dogs while jogging. Here, we exploited visual periodicity to examine how category-based attention differentially modulates selective neural processing of face and non-face categories. We combined electroencephalography with a novel frequency-tagging paradigm capable of capturing selective neural responses for multiple visual categories contained within the same rapid image stream (faces/birds in Exp 1; houses/birds in Exp 2). We found that the pattern of attentional enhancement and suppression for face-selective processing is unique compared to other object categories: Where attending to non-face objects strongly enhances their selective neural signals during a later stage of processing (300-500 ms), attentional enhancement of face-selective processing is both earlier and comparatively more modest. Moreover, only the selective neural response for faces appears to be actively suppressed by attending towards an alternate visual category. These results underscore the special status that faces hold within the human visual system, and highlight the utility of visual periodicity as a powerful tool for indexing selective neural processing of multiple visual categories contained within the same image sequence.

Autonomous Metabolic Oscillations Robustly Gate the Early and Late Cell Cycle.

Eukaryotic cell division is known to be controlled by the cyclin/cyclin dependent kinase (CDK) machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic triggers for cell-cycle initiation and progression. Using microfluidics, cell-cycle reporters, and single-cell metabolite measurements, we found that metabolism of budding yeast is a CDK-independent oscillator that oscillates across different growth conditions, both in synchrony with and also in the absence of the cell cycle. Using environmental perturbations and dynamic single-protein depletion experiments, we found that the metabolic oscillator and the cell cycle form a system of coupled oscillators, with the metabolic oscillator separately gating and maintaining synchrony with the early and late cell cycle. Establishing metabolism as a dynamic component within the cell-cycle network opens new avenues for cell-cycle research and therapeutic interventions for proliferative disorders.

Fast and Slow Rhythms of Naturalistic Reading Revealed by Combined Eye-Tracking and Electroencephalography.

Neural oscillations are thought to support speech and language processing. They may not only inherit acoustic rhythms, but might also impose endogenous rhythms onto processing. In support of this, we here report that human (both male and female) eye movements during naturalistic reading exhibit rhythmic patterns that show frequency-selective coherence with the EEG, in the absence of any stimulation rhythm. Periodicity was observed in two distinct frequency bands: First, word-locked saccades at 4-5 Hz display coherence with whole-head theta-band activity. Second, fixation durations fluctuate rhythmically at ∼1 Hz, in coherence with occipital delta-band activity. This latter effect was additionally phase-locked to sentence endings, suggesting a relationship with the formation of multi-word chunks. Together, eye movements during reading contain rhythmic patterns that occur in synchrony with oscillatory brain activity. This suggests that linguistic processing imposes preferred processing time scales onto reading, largely independent of actual physical rhythms in the stimulus.SIGNIFICANCE STATEMENT The sampling, grouping, and transmission of information are supported by rhythmic brain activity, so-called neural oscillations. In addition to sampling external stimuli, such rhythms may also be endogenous, affecting processing from the inside out. In particular, endogenous rhythms may impose their pace onto language processing. Studying this is challenging because speech contains physical rhythms that mask endogenous activity. To overcome this challenge, we turned to naturalistic reading, where text does not require the reader to sample in a specific rhythm. We observed rhythmic patterns of eye movements that are synchronized to brain activity as recorded with EEG. This rhythmicity is not imposed by the external stimulus, which indicates that rhythmic brain activity may serve as a pacemaker for language processing.

Rhythmic Entrainment Echoes in Auditory Perception.

Rhythmic entrainment echoes-rhythmic brain responses that outlast rhythmic stimulation-can demonstrate endogenous neural oscillations entrained by the stimulus rhythm. Here, we tested for such echoes in auditory perception. Participants detected a pure tone target, presented at a variable delay after another pure tone that was rhythmically modulated in amplitude. In four experiments involving 154 human (female and male) participants, we tested (1) which stimulus rate produces the strongest entrainment echo and, inspired by the tonotopical organization of the auditory system and findings in nonhuman primates, (2) whether these are organized according to sound frequency. We found the strongest entrainment echoes after 6 and 8 Hz stimulation, respectively. The best moments for target detection (in phase or antiphase with the preceding rhythm) depended on whether sound frequencies of entraining and target stimuli matched, which is in line with a tonotopical organization. However, for the same experimental condition, best moments were not always consistent across experiments. We provide a speculative explanation for these differences that relies on the notion that neural entrainment and repetition-related adaptation might exercise competing opposite influences on perception. Together, we find rhythmic echoes in auditory perception that seem more complex than those predicted from initial theories of neural entrainment.SIGNIFICANCE STATEMENT Rhythmic entrainment echoes are rhythmic brain responses that are produced by a rhythmic stimulus and persist after its offset. These echoes play an important role for the identification of endogenous brain oscillations, entrained by rhythmic stimulation, and give us insights into whether and how participants predict the timing of events. In four independent experiments involving >150 participants, we examined entrainment echoes in auditory perception. We found that entrainment echoes have a preferred rate (between 6 and 8 Hz) and seem to follow the tonotopic organization of the auditory system. Although speculative, we also found evidence that several, potentially competing processes might interact to produce such echoes, a notion that might need to be considered for future experimental design.

Rhythm in the Premature Neonate Brain: Very Early Processing of Auditory Beat and Meter.

The ability to extract rhythmic structure is important for the development of language, music, and social communication. Although previous studies show infants' brains entrain to the periodicities of auditory rhythms and even different metrical interpretations (e.g., groups of two vs three beats) of ambiguous rhythms, whether the premature brain tracks beat and meter frequencies has not been explored previously. We used high-resolution electroencephalography while premature infants (n = 19, 5 male; mean age, 32 ± 2.59 weeks gestational age) heard two auditory rhythms in the incubators. We observed selective enhancement of the neural response at both beat- and meter-related frequencies. Further, neural oscillations at the beat and duple (groups of 2) meter were phase aligned with the envelope of the auditory rhythmic stimuli. Comparing the relative power at beat and meter frequencies across stimuli and frequency revealed evidence for selective enhancement of duple meter. This suggests that even at this early stage of development, neural mechanisms for processing auditory rhythms beyond simple sensory coding are present. Our results add to a few previous neuroimaging studies demonstrating discriminative auditory abilities of premature neural networks. Specifically, our results demonstrate the early capacities of the immature neural circuits and networks to code both simple beat and beat grouping (i.e., hierarchical meter) regularities of auditory sequences. Considering the importance of rhythm processing for acquiring language and music, our findings indicate that even before birth, the premature brain is already learning this important aspect of the auditory world in a sophisticated and abstract way.SIGNIFICANCE STATEMENT Processing auditory rhythm is of great neurodevelopmental importance. In an electroencephalography experiment in premature newborns, we found converging evidence that when presented with auditory rhythms, the premature brain encodes multiple periodicities corresponding to beat and beat grouping (meter) frequencies, and even selectively enhances the neural response to meter compared with beat, as in human adults. We also found that the phase of low-frequency neural oscillations aligns to the envelope of the auditory rhythms and that this phenomenon becomes less precise at lower frequencies. These findings demonstrate the initial capacities of the developing brain to code auditory rhythm and the importance of special care to the auditory environment of this vulnerable population during a highly dynamic period of neural development.

Switching neuron contributions to second network activity.

Network flexibility is important for adaptable behaviors. This includes neuronal switching, where neurons alter their network participation, including changing from single- to dual-network activity. Understanding the implications of neuronal switching requires determining how a switching neuron interacts with each of its networks. Here, we tested 1) whether "home" and second networks, operating via divergent rhythm generation mechanisms, regulate a switching neuron and 2) if a switching neuron, recruited via modulation of intrinsic properties, contributes to rhythm or pattern generation in a new network. Small, well-characterized feeding-related networks (pyloric, ∼1 Hz; gastric mill, ∼0.1 Hz) and identified modulatory inputs make the isolated crab (Cancer borealis) stomatogastric nervous system (STNS) a useful model to study neuronal switching. In particular, the neuropeptide Gly1-SIFamide switches the lateral posterior gastric (LPG) neuron (2 copies) from pyloric-only to dual-frequency pyloric/gastric mill (fast/slow) activity via modulation of LPG-intrinsic properties. Using current injections to manipulate neuronal activity, we found that gastric mill, but not pyloric, network neurons regulated the intrinsically generated LPG slow bursting. Conversely, selective elimination of LPG from both networks using photoinactivation revealed that LPG regulated gastric mill neuron-firing frequencies but was not necessary for gastric mill rhythm generation or coordination. However, LPG alone was sufficient to produce a distinct pattern of network coordination. Thus, modulated intrinsic properties underlying dual-network participation may constrain which networks can regulate switching neuron activity. Furthermore, recruitment via intrinsic properties may occur in modulatory states where it is important for the switching neuron to actively contribute to network output.NEW & NOTEWORTHY We used small, well-characterized networks to investigate interactions between rhythmic networks and neurons that switch their network participation. For a neuron switching into dual-network activity, only the second network regulated its activity in that network. In addition, the switching neuron was sufficient but not necessary to coordinate second network neurons and regulated their activity levels. Thus, regulation of switching neurons may be selective, and a switching neuron is not necessarily simply a follower in additional networks.

DiffCircaPipeline: a framework for multifaceted characterization of differential rhythmicity.

SUMMARY: Circadian oscillations of gene expression regulate daily physiological processes, and their disruption is linked to many diseases. Circadian rhythms can be disrupted in a variety of ways, including differential phase, amplitude and rhythm fitness. Although many differential circadian biomarker detection methods have been proposed, a workflow for systematic detection of multifaceted differential circadian characteristics with accurate false positive control is not currently available. We propose a comprehensive and interactive pipeline to capture the multifaceted characteristics of differentially rhythmic biomarkers. Analysis outputs are accompanied by informative visualization and interactive exploration. The workflow is demonstrated in multiple case studies and is extensible to general omics applications. AVAILABILITY AND IMPLEMENTATION: R package, Shiny app and source code are available in GitHub (https://github.com/DiffCircaPipeline) and Zenodo (https://doi.org/10.5281/zenodo.7507989). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.