Kalyanaraman, B. | Search bioRxiv

bioRxiv2026

ThermoClock: A Novel Automated Temperature Regulation Device that Can Model Circadian Entrainment and Disruption in 2D and 3D in Vitro Models

Circadian rhythms are critical for maintaining homeostasis and regulating physiological functions, and consequentially impact disease progression; yet, they remain largely overlooked in in vitro models used in preclinical research. One major barrier to rigorously testing the role of circadian rhythms in these models is the lack of accessible tools that seamlessly integrate into standard culture setups and are capable of sustainably delivering time cues to cells and tissues in long term experiments. Here, we present the ThermoClock, a low-cost, Arduino-based automated temperature control system capable of delivering independent temperature programs to multiple cultures simultaneously. Using circadian reporter U2OS cell lines (Bmal1:Luc and Per2:Luc), we demonstrated that ThermoClock-driven temperature cycles (36{degrees}C/38.5{degrees}C, 12h:12h) produced significantly higher amplitude entrainment than a programmable incubator delivering identical temperature trajectories, suggesting that the ramp time to setpoint is a critical determinant of entrainment strength. We further applied ThermoClock to skin explants from keratinocyte-specific Dbp:Luc reporter mice, showing that circadian temperature cycles (T24: 12h:12h and T25: 12.5h:12.5h) extended synchronized circadian rhythms ex vivo, while a shortened T-cycle (T20: 10h:10h) induced rhythm disruptions. We also observed reduced cell migration in T20 temperature-entrained explants wounded ex vivo, closely recapitulating attenuated wound healing observed in T20 light-cycle-disrupted mice in vivo. Finally, we show that wounding can act as a phase-resetting cue but its efficacy depends on pre-injury entrainment state, with circadian entrained tissues (T25) resisting reset, while disrupted (T20) and unentrained tissues showed resetting sensitivity. These findings establish ThermoClock as a versatile platform for incorporating circadian regulation and, for the first time, disruption into 2D and 3D in vitro systems and demonstrate that peripheral clock disruption and its functional consequences can be modeled ex vivo.

bioengineering↗

bioRxiv2026

c-MYC is Transcribed in a Circadian Manner and Acts as a Clock Disruptor whose Timing Minimizes its Impacts

The c-MYC proto-oncogene regulates cellular proliferation, and its aberrant expression drives a range of human cancers. It also has a bidirectional regulatory relationship with the mammalian core circadian clock, with emerging evidence suggesting that MYC overexpression leads to clock disruption and loss of rhythms. While prior studies have probed MYCs role in clock disruption by overexpressing or mutating the c-MYC gene, our understanding of the endogenous nature of c-MYC is limited. A major gap in knowledge is whether MYC itself is expressed rhythmically and if so, how its timing relates to that of core clock components. To address these shortcomings, we generated a c-MYC reporter and assessed its circadian nature, comparing it to BMAL1 and PER2, and developed a computational model based on these and previous findings to evaluate its role(s). We developed lentiviral constructs for and established a U2OS (common circadian model) reporter cell line expressing luciferase (luc) driven by a human-derived c-MYC promoter sequence. To facilitate comparisons, as part of this work, we also developed a human-sequence derived BMAL1 promoter reporter to more readily recapitulate its behaviors. Using luminometry studies and subsequent data analyses, we demonstrated that the c-MYC promoter oscillated rhythmically in U2OS cells, which possess inherently low levels of c-MYC. Furthermore, we found that c-MYC oscillates out-of-phase relative to BMAL1 and PER2. Using this information, we built a mathematical model to better understand how c-MYCs oscillations at both basal and over-expressed levels affect the clock and vice versa. The model reproduced expected alterations to the core clock resulting from c-MYC overexpression and showed that MYCs role is as a disruptor, although the timing of MYC regulation can minimize its negative impact(s) on circadian timekeeping. This work is the first to assess c-MYCs phase relationships relative to the core clock and to provide evidence for its circadian nature. Author summaryc-MYC is a transcription factor that is highly regulated and plays an important role in cellular proliferation. In cancers, deregulation of c-MYC causes its overexpression, resulting in tumorigenesis. There have been multiple connections demonstrated between MYC and the circadian clock, including the clocks role in MYC expression and that its overexpression can lead to disruptions to the core circadian clock. However, knowledge of the expression patterns of MYC are limited, including whether they occur in a circadian manner. To address this, we developed a c-MYC-luciferase reporter in a human circadian cell model (U2OS). For the first time, we were able to directly assess the rhythmic nature of c-MYC using this tool. Subsequently, we developed a mathematical model to gain insights into the disruptive role of MYC in clock regulation under disease-like conditions and, in turn, the effects of the circadian clock on MYC. We found that c-MYC oscillated in a circadian manner in U2OS cells and that the MYC proteins role is as a disruptor, but its timing can minimize its negative impact(s) on circadian rhythms.

molecular biology↗