Biology subjects

Ananthasubramaniam, B.

Publications and source records attributed to Ananthasubramaniam, B..

11 recordsLinked to original sources

bioRxiv2026

Different, same, or arrhythmic: an empirically informed benchmark of differential rhythmicity methods

Circadian rhythms represent a key biological dynamic across all kingdoms of life. These cell-autonomous rhythms generated by a gene-regulatory network regulate processes in a time-of-day-specific manner primarily via transcriptional rhythms in a significant fraction of genes. The controlled experiment paradigm can be used to study causal effects at the molecular level using transcriptomic RNA-seq data even in this dynamic scenario. Multiple tools for the analysis of such data, termed differential rhythmicity (DR) analysis, are available but guidance on method choice remains limited. Here, we present a systematic benchmarking study of 12 different methods from 5 packages using a synthetic model of RNA-seq count data that incorporates both empirically estimated mean-dispersion trends and concomitant differential expression changes. By linking method performance to experimental conditions and analytical goals, we provide practical and simple rules of thumb that will help biologists choose appropriate DR methods and extract reliable biological insight from time series RNA-seq data.

bioinformatics↗

bioRxiv2026

rhythmCI: an R package for rhythm detection using harmonic regression amplitude confidence intervals

Circadian clocks are evolutionarily conserved systems that orchestrate daily rhythms in human physiology, tissue behavior, and cellular function. In circadian research, the NanoString nCounter platform has emerged as a robust alternative to microarray and RNA sequencing. However, raw instrument output requires specialized preprocessing to handle technical noise. Furthermore, while harmonic regression is widely used to detect periodic signals in biological time-series data, evaluating rhythmicity remains highly challenging in clinical studies characterized by sparse sampling and small sample sizes. To address these limitations, we developed rhythmCI, an easy-to-use R package that integrates established normalization methods with a complete workflow for circadian data analysis. The package incorporates a bootstrapping procedure to estimate confidence intervals for harmonic regression amplitudes, offering a statistically transparent way to quantify rhythmicity in small-cohort studies. rhythmCI provides an end-to-end workflow for NanoString nCounter data, including automated preprocessing, normalization, and visualization. We evaluated the utility and accuracy of the package using synthetic time-series data generated with the Simphony framework, demonstrating that rhythmCI produces interpretable and reliable rhythmicity classifications under controlled simulation conditions. rhythmCI bridges a critical gap in circadian bioinformatics by offering a reliable method to characterize periodicity under challenging experimental designs typical of clinical research. By streamlining NanoString workflows and improving the statistical rigor of rhythm detection in small datasets, rhythmCI enhances the accessibility and reliability of circadian biology analysis.

bioinformatics↗

bioRxiv2026

Divergent molecular and circuit mechanisms underlie light entrainment of retinal and suprachiasmatic nucleus circadian clocks

The circadian system aligns behavior and physiology with the 24-hour environmental cycle through a distributed network of clocks including the master pacemaker in the suprachiasmatic nucleus (SCN) and an autonomous retinal clock critical for local retinal physiology and function. Although both clocks are entrained by light, they differ in their photoreceptor inputs and light sensitivity. The specific contributions and mechanisms by which distinct photoreceptor pathways drive their photoentrainment, however, remain incompletely understood. In this study, we conducted a comprehensive transcriptomic and integrative comparative analysis of retinal and SCN circadian light responses using mouse models lacking specific photoreceptors or key components of signaling pathways. Under photopic conditions, we found that each tissue displays distinct light-responsive transcriptional signatures across genotypes, yet both shared a conserved cluster of rod-driven immediate early-genes. Strikingly, the light-evoked transcriptional response was not sufficient to shift the phase of the SCN clock, in contrast to its robust phase-shifting effect on the retinal clock. Furthermore, by genetically disrupting rod/cone electrical coupling and pharmacologically isolating rod pathways, we identified the OFF-cone bipolar cell circuit as both necessary and sufficient to mediate light-induced phase resetting of the retinal clock. Together, these findings delineate the specialized retinal circuitry that underlies circadian entrainment and reveal a fundamental divergence between retinal and SCN mechanisms of photic timekeeping.

neuroscience↗

bioRxiv2024

Time series-free rhythm profiling using COFE reveals multi-omic circadian rhythms in in-vivo human cancers

The study of ubiquitous circadian rhythms in human physiology requires regular measurements across time. Repeated sampling of the different internal tissues that house circadian clocks is both practically and ethically infeasible. Here, we present a novel unsupervised machine learning approach (COFE) that can use single high-throughput omics samples (without time labels) from individuals to reconstruct circadian rhythms across cohorts. COFE can simultaneously assign time labels to samples and identify rhythmic data features used for temporal reconstruction, while also detecting invalid orderings. With COFE, we discovered widespread de novo circadian gene expression rhythms in 11 different human adenocarcinomas using data from the TCGA database. The arrangement of peak times of core clock gene expression was conserved across cancers and resembled a healthy functional clock except for the mistiming of a few key genes. Moreover, rhythms in the transcriptome were strongly associated with the cancer-relevant proteome. The rhythmic genes and proteins common to all cancers were involved in metabolism and the cell cycle. Although these rhythms were synchronized with the cell cycle in many cancers, they were uncoupled with clocks in healthy matched tissue. The targets of most of FDA-approved and potential anti-cancer drugs were rhythmic in tumor tissue with different amplitudes and peak times. These findings emphasize the utility of considering "time" in cancer therapy, and suggest a focus on clocks in healthy tissue rather than free-running clocks in cancer tissue. Our approach thus creates new opportunities to repurpose data without time labels to study circadian rhythms.

systems biology↗

bioRxiv2024

Dietary methionine/choline deficiency affects behavioural and molecular circadian rhythms

The feeding/fasting cycles controlled by our circadian clock impose great daily metabolic and physiological changes, and yet investigations into the consequences of metabolic deficiencies, either dietary or genetic, have often ignored the time of day or the circadian time of the animals or subjects. In addition, these deficiencies may themselves disrupt our circadian clock, causing secondary metabolic, physiological and behavioural disorders. Dietary methionine/choline deficiency in rodents is a common model for human non-alcoholic steatohepatitis, but methionine and choline are nutrients essential for many other processes beyond fatty acid synthesis in the liver, including biological methylations and 1-carbon metabolism, regulation of translation notably via the mTOR pathway, phospholipid synthesis, polyamine pathway and glutathione synthesis. We have previously shown that circadian rhythms in many organisms are highly sensitive to deficiency or excesses of 1-carbon metabolites. Using a methionine/choline deficient diet in mice, we illustrate the nutrigenomic crosstalk between circadian rhythms and 1-carbon metabolism. We show not only that circadian locomotor activity behaviour is profoundly, rapidly and reversibly affected by methionine/choline deficiency, but also that the effects of methionine/choline deficiency on gene expression and 1-carbon metabolites are dependent on circadian time, illustrating the importance of considering circadian rhythms in metabolic studies. This study also highlights the impact of what we eat, or dont, on our behaviour and biological rhythms.

physiology↗

bioRxiv2022

Gene expression signatures predict circadian rhythms in oncogenic pathways

Day-night environmental cycles together with our own adaptive rhythms in behavior and physiology lead to rhythmicity of various processes on the cellular level, including cell signaling. Despite many implications of such daily changes in signaling, the quantification of such rhythms and estimates of peak phases of pathway activities in various tissues are missing. Governed mainly by posttranslational modifications, a pathway activity might not be well quantified via the expression level of pathway components. Instead, a gene expression signatures approach can be used to score activity of various pathways. Here, we apply such gene expression signatures on circadian time series transcriptomics data to infer rhythmicity in cellular signaling. We show that, across multiple datasets, the gene expression signatures predict the presence of rhythmicity in EGFR, PI3K and p53 pathways in mouse liver. With the focus on EGFR pathway, we pinpoint the most influential signature genes for the overall rhythmicity in the activity scores for this pathway. These findings suggest that time of the day is an important factor to consider in studies on signaling. Simultaneously, this study provides a new paradigm to use circadian transcriptomics to get at temporal dynamics of pathway activation.

systems biology↗

bioRxiv2022

Inter-layer and inter-subject variability of circadian gene expression in human skin

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports circadian gene expression in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep schedules. 10% of the expressed genes showed rhythms at the population level, of which only a third differed between the two layers. Broadly, expression magnitudes of circadian genes were consistent across subjects in each layer. Amplitude and phases of circadian gene expression, however, varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in the their variability across subjects. We used this data to identify small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin to realize the potential of circadian medicine as well as a novel methodology to quantify sources of variability in human circadian rhythms.

bioinformatics↗

bioRxiv2020

Studies overestimate the extent of circadian rhythm reprogramming in response to dietary and genetic changes

The circadian clock modulates key physiological processes in many organisms. This widespread role of circadian rhythms is typically characterized at the molecular level by profiling the transcriptome at multiple time points. Subsequent analysis identifies transcripts with altered rhythms between control and perturbed conditions, i.e., are differentially rhythmic (DiffR). Commonly, Venn Diagram analysis (VDA) compares lists of rhythmic transcripts to catalog transcripts with rhythms in both conditions or have gained or lost rhythms. However, unavoidable errors in the rhythmicity detection propagate to the final DiffR classification resulting in overestimated DiffR. We show using artificial experiments constructed from biological data that VDA indeed produces excessive false DiffR hits both in the presence and absence of true DiffR transcripts. We present a hypothesis testing and a model selection approaches in an R-package compareRhythms that instead compare circadian amplitude and phase of transcripts between the two conditions. These methods identify transcripts with gain, loss, change or have the same rhythms; the third category is missed by VDA. We reanalyzed three studies on the interplay between metabolism and the clock in the mouse liver that used VDA. We found not only fewer DiffR transcripts than originally reported, but VDA overlooked many relevant DiffR transcripts. Our analyses confirmed some and contradicted other conclusions in the original studies and also generated novel hypotheses. Our insights also generalize easily to studies using other -omics technologies. We trust that avoiding Venn Diagrams and using our R-package will contribute to improved reproducibility in chronobiology.

bioinformatics↗

bioRxiv2019

Ultradian regulation of rest and activity bouts in mice

The suprachiasmatic nucleus (SCN), which serves as the central pacemaker in mammals, regulates the 24-hour rhythm in behavioral activity. However, it is currently unclear whether and how bouts of activity and rest are regulated within the 24-hour cycle (i.e., over ultradian time scales). Therefore, we used passive infrared sensors to measure behavior in mice housed under either a light-dark (LD) cycle or continuous darkness (DD). We found that a probabilistic Markov model captures the ultradian changes in the behavioral state over a 24-hour cycle. In this model, the animals behavioral state in the next time interval is determined solely by the animals current behavioral state and by the "toss" of a proverbial "biased coin". We found that the bias of this "coin" is regulated by light input and by the phase of the clock. Moreover, the bias of this "coin" for an animal is related to the average length of rest and activity bouts in that animal. In LD conditions, the average length of rest bouts was greater during the day compared to during the night, whereas the average length of activity bouts was greater during the night compared to during the day. Importantly, we also found that day-night changes in the rest bout lengths were significantly greater than day-night changes in the activity bout lengths. Finally, in DD conditions, the activity and rest bouts also differed between subjective night and subjective day, albeit to a lesser extent compared to LD conditions. The persistent differences in bout length over the circadian cycle following loss of the external LD cycle indicate that the central pacemaker plays a role in regulating rest and activity bouts on an ultradian time scale.

physiology↗

bioRxiv2019

Amplitude effects allow short jetlags and large seasonal phase shifts in minimal clock models

Mathematical models of varying complexity have helped shed light on different aspects of circadian clock function. In this work, we question whether minimal clock models (Goodwin models) are sufficient to reproduce essential phenotypes of the clock: a small phase response curve (PRC), fast jetlag and seasonal phase shifts. Instead of building a single best model, we take an approach where we study the properties of a set of models satisfying certain constraints; here a one-hour pulse PRC with a range of three hours and clock periods between 22h and 26h. Surprisingly, almost all these randomly parameterized models showed a four hour change in phase of entrainment between long and short days and jetlag durations of three to seven days in advance and delay. Moreover, intrinsic clock period influenced jetlag duration and entrainment amplitude and phase. Fast jetlag was realized in this model by means of an interesting amplitude effect: the association between clock amplitude and clock period termed twist. This twist allows amplitude changes to speed up and slow down clocks enabling faster shifts. These findings were robust to the addition of positive feedback to the model. In summary, the known design principles of rhythm generation - negative feedback, long delay and switch-like inhibition (we review these in detail) - are sufficient to reproduce the essential clock phenotypes. Furthermore, amplitudes play a role in determining clock properties and must be always considered, although they are difficult to measure.

systems biology↗

bioRxiv2019

Susceptibility rhythm to bacterialendotoxin in myeloid clock-knockout mice

Local circadian clocks are active in most cells of our body. However, their impact on circadian physiology is still under debate. Mortality by endotoxic (LPS) shock is highly time-of-day dependent and local circadian immune function such as the cytokine burst after LPS challenge has been assumed to be causal for the large differences in survival. Here, we investigate the roles of light and myeloid clocks on mortality by endotoxic shock. Strikingly, mice in constant darkness (DD) show a three-fold increased susceptibility to LPS as compared to mice in light-dark conditions. Mortality by endotoxic shock as a function of circadian time is independent of light-dark cycles as well as myeloid CLOCK or BMAL1 as demonstrated in conditional knockout mice. Unexpectedly, despite the lack of a myeloid clock these mice still show rhythmic patterns of pro- and anti-inflammatory cytokines such as TNF, MCP-1, IL-18 and IL-10 in peripheral blood as well as time-of-day and site dependent traffic of myeloid cells. We speculate that systemic time-cues are sufficient to orchestrate innate immune response to LPS by driving immune functions such as cell trafficking and cytokine expression.

immunology↗