Network switches and their role in circadian clocks.

Circadian rhythms are generated by complex interactions among genes and proteins. Self-sustained ∼24 h oscillations require negative feedback loops and sufficiently strong nonlinearities that are the product of molecular and network switches. Here, we review common mechanisms to obtain switch-like behavior, including cooperativity, antagonistic enzymes, multisite phosphorylation, positive feedback, and sequestration. We discuss how network switches play a crucial role as essential components in cellular circadian clocks, serving as integral parts of transcription-translation feedback loops that form the basis of circadian rhythm generation. The design principles of network switches and circadian clocks are illustrated by representative mathematical models that include bistable systems and negative feedback loops combined with Hill functions. This work underscores the importance of negative feedback loops and network switches as essential design principles for biological oscillations, emphasizing how an understanding of theoretical concepts can provide insights into the mechanisms generating biological rhythms.

Coupling allows robust mammalian redox circadian rhythms despite heterogeneity and noise.

Circadian clocks are endogenous oscillators present in almost all cells that drive daily rhythms in physiology and behavior. There are two mechanisms that have been proposed to explain how circadian rhythms are generated in mammalian cells: through a transcription-translation feedback loop (TTFL) and based on oxidation/reduction reactions, both of which are intrinsically stochastic and heterogeneous at the single cell level. In order to explore the emerging properties of stochastic and heterogeneous redox oscillators, we simplify a recently developed kinetic model of redox oscillations to an amplitude-phase oscillator with 'twist' (period-amplitude correlation) and subject to Gaussian noise. We show that noise and heterogeneity alone lead to fast desynchronization, and that coupling between noisy oscillators can establish robust and synchronized rhythms with amplitude expansions and tuning of the period due to twist. Coupling a network of redox oscillators to a simple model of the TTFL also contributes to synchronization, large amplitudes and fine-tuning of the period for appropriate interaction strengths. These results provide insights into how the circadian clock compensates randomness from intracellular sources and highlight the importance of noise, heterogeneity and coupling in the context of circadian oscillators.

Are circadian amplitudes and periods correlated? A new twist in the story.

Three parameters are important to characterize a circadian and in general any biological clock: period, phase and amplitude. While circadian periods have been shown to correlate with entrainment phases, and clock amplitude influences the phase response of an oscillator to pulse-like zeitgeber signals, the co-modulations of amplitude and periods, which we term twist, have not been studied in detail. In this paper we define two concepts: parametric twist refers to amplitude-period correlations arising in ensembles of self-sustained clocks in the absence of external inputs, and phase space twist refers to the co-modulation of an individual clock's amplitude and period in response to external zeitgebers. Our findings show that twist influences the interaction of oscillators with the environment, facilitating entrainment, fastening recovery to pulse-like perturbations or modifying the response of an individual clock to coupling. This theoretical framework might be applied to understand the emerging properties of other oscillating systems.

Feasibility of Telemedicine in the Management Strategy of Patients With Lymphoma Amid the COVID-19 Pandemic in Spain: Prospective Observational Study.

BACKGROUND: On March 14, 2020, a state of alarm was declared in Spain due to the spread of SARS-CoV-2. Beyond this date, COVID-19 in the country changed the practice of oncologic care. OBJECTIVE: Since recurrent hospital visits were a potential risk factor for contagion, the aim of this prospective observational study was to analyze the consequences of the COVID-19 pandemic in the health care of patients with lymphoma. METHODS: All data were obtained from the electronic medical record. Variables such as age, sex, reason of the visit, use of the patient portal, changes in management, enrollment in clinical trials, and COVID-19 infection were recorded. RESULTS: In all, 290 patients visited the lymphoma clinic, totaling 437 appointments. The median age was 66 (range 18-94) years, and 157 (54.1%) patients were male. Of them, 214 (73.8%) patients had only 1 visit to the clinic. Only 23 (7.9%) patients did not have access to the patient portal. Amid the COVID-19 pandemic, 78 (26.9%) patients remained in active treatment, 35 (12.1%) experienced delays in their treatments, and 6 (2.1%) experienced treatment discontinuation. During the follow-up, only 7 (2.4%) patients had a COVID-19 infection (6 cases with confirmed polymerase chain reaction test and 1 case with clinical suspicion). Despite the implementation of telemedicine strategies to avoid visits to the hospital, 66 (22.8%) patients had in-person visits at the lymphoma clinic. Patients who attended in-person consultations were younger than those who preferred telemedicine consultations (62 vs 66 years; P=.10) and had less use of the patient portal (17/224, 7.6% vs 6/66, 9%; P=.10), although these differences did not reach statistical significance. Patients who attended in-person visits were more likely to have had only 1 visit to the hospital (29/66, 43.9% vs 185/224, 82.6%; P<.001). Regarding the reason of in-person consultations, more patients were on active treatment in comparison to those using telemedicine resources (37/66, 56.1% vs 42/224, 18.3%; P<.001). Patients with a preference for telemedicine strategies had more surveillance visits (147/224, 65.6% vs 24/66, 36.4%; P<.001). Regarding treatment modifications, more treatment delays (29/224, 12.9% vs 6/66, 9.1%; P=.10) and more definite treatment discontinuations (6/224, 2.7% vs 0/66, 0%; P=.10) were seen in patients using telemedicine resources when compared to patients attending in-person visits, although these differences did not reach statistical significance. Regarding the type of therapy, patients attending in-person visits were more likely to receive an intravenous treatment rather than those using telemedicine (23/66, 62.2% vs 17/224, 40.5%; P<.001). CONCLUSIONS: Telemedicine such as patient portals are feasible strategies in the management of patients with lymphoma during the COVID-19 pandemic, with a reduction of in-person visits to the hospital and a very low contagion rate.

Inter-layer and inter-subject variability of diurnal 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 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression 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 their variability across subjects. We also identified 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 and presents a novel methodology to quantify sources of variability in human circadian rhythms.

Intercellular coupling between peripheral circadian oscillators by TGF-ß signaling.

Coupling between cell-autonomous circadian oscillators is crucial to prevent desynchronization of cellular networks and disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus, couple intercellularly, coupling among peripheral oscillators is controversial and the molecular mechanisms are unknown. Using two- and three-dimensional mammalian culture models in vitro (mainly human U-2 OS cells) and ex vivo, we show that peripheral oscillators couple via paracrine pathways. We identify transforming growth factor-ß (TGF-ß) as peripheral coupling factor that mediates paracrine phase adjustment of molecular clocks through transcriptional regulation of core-clock genes. Disruption of TGF-ß signaling causes desynchronization of oscillator networks resulting in reduced amplitude and increased sensitivity toward external zeitgebers. Our findings reveal an unknown mechanism for peripheral clock synchrony with implications for rhythmic organ functions and circadian health.

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Nonlinear phenomena in models of the circadian clock.

The mammalian circadian clock is well-known to be important for our sleep-wake cycles, as well as other daily rhythms such as temperature regulation, hormone release or feeding-fasting cycles. Under normal conditions, these daily cyclic events follow 24 h limit cycle oscillations, but under some circumstances, more complex nonlinear phenomena, such as the emergence of chaos, or the splitting of physiological dynamics into oscillations with two different periods, can be observed. These nonlinear events have been described at the organismic and tissue level, but whether they occur at the cellular level is still unknown. Our results show that period-doubling, chaos and splitting appear in different models of the mammalian circadian clock with interlocked feedback loops and in the absence of external forcing. We find that changes in the degradation of clock genes and proteins greatly alter the dynamics of the system and can induce complex nonlinear events. Our findings highlight the role of degradation rates in determining the oscillatory behaviour of clock components, and can contribute to the understanding of molecular mechanisms of circadian dysregulation.

A Robust Model for Circadian Redox Oscillations.

The circadian clock is an endogenous oscillator that controls daily rhythms in metabolism, physiology, and behavior. Although the timekeeping components differ among species, a common design principle is a transcription-translation negative feedback loop. However, it is becoming clear that other mechanisms can contribute to the generation of 24 h rhythms. Peroxiredoxins (Prxs) exhibit 24 h rhythms in their redox state in all kingdoms of life. In mammalian adrenal gland, heart and brown adipose tissue, such rhythms are generated as a result of an inactivating hyperoxidation reaction that is reduced by coordinated import of sulfiredoxin (Srx) into the mitochondria. However, a quantitative description of the Prx/Srx oscillating system is still missing. We investigate the basic principles that generate mitochondrial Prx/Srx rhythms using computational modeling. We observe that the previously described delay in mitochondrial Srx import, in combination with an appropriate separation of fast and slow reactions, is sufficient to generate robust self-sustained relaxation-like oscillations. We find that our conceptual model can be regarded as a series of three consecutive phases and two temporal switches, highlighting the importance of delayed negative feedback and switches in the generation of oscillations.

3-Iodothyronamine Decreases Expression of Genes Involved in Iodide Metabolism in Mouse Thyroids and Inhibits Iodide Uptake in PCCL3 Thyrocytes.

BACKGROUND: 3-Iodothyronamine (3-T1AM) is an endogenous decarboxylated thyroid hormone (TH) metabolite. Pharmacological doses of 3-T1AM decrease heart rate, body temperature, and metabolic rate in rodents-effects that are contrary to classic TH excess. Furthermore, a single dose of 3-T1AM was shown to suppress the hypothalamic-pituitary-thyroid (HPT) axis in rats. It was hypothesized that 3-T1AM might play a role in the fine-tuning of TH action and might have a direct regulatory effect on the thyroid gland. METHODS: This study tested whether repeated 3-T1AM treatment interfered with thyroid function and the HPT axis in mice. Therefore, male C57BL/6 mice were intraperitoneally injected with 5 mg/kg of 3-T1AM or vehicle daily for seven days. Additionally, the effects of 3-T1AM on the differentiated rat thyrocyte cell line PCCL3 were analyzed. RESULTS: Repeated administration of 3-T1AM decreased thyroidal mRNA content of the sodium iodide symporter (Nis), thyroglobulin, and pendrin in mice. No interference with the HPT axis was observed, as determined by unaltered pituitary mRNA levels of triiodothyronine-responsive genes, including thyrotropin subunit ß. Furthermore, 3-T1AM treatment did not change transcript levels of hepatic triiodothyronine-responsive genes, such as deiodinase 1. In line with this, serum TH concentrations were not changed after the treatment period of seven days. In concordance with the in vivo findings, 3-T1AM decreased the thyrotropin-dependent expression of Nis and functional iodide uptake in PCCL3 cells in vitro. Additionally, uptake and metabolism of 3-T1AM by PCCL3 cells was observed, as well as 3-T1AM-dependent changes in intracellular Ca2+ concentration that might be involved in mediating the reported effects. CONCLUSIONS: In conclusion, 3-T1AM application decreased expression of selected TH synthesis genes by acting directly on the thyroid gland, and it might therefore affect TH synthesis without involvement of the HPT axis.