The miR199 and miR214 genes occupy an intronic cluster located on the opposite strand of the Dynamin3 gene. These miRNAs play major roles in a broad variety of developmental processes and diseases, including skeletal development and several types of cancer. In the work reported here, we first deciphered the origin of the miR199 and miR214 families by following evolution of miR paralogs and their host Dynamin paralogs. We then examined the expression patterns of miR199 and miR214 in developing zebrafish embryos and demonstrated their regulation through a common primary transcript. Results suggest an evolutionarily conserved regulation across vertebrate lineages. Our expression study showed predominant expression patterns for both miR in tissues surrounding developing craniofacial skeletal elements consistent with expression data in mouse and human, thus indicating a conserved role of miR199 and miR214 in vertebrate skeletogenesis.
The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we show that cellular protein O-GlcNAcylation exhibits robust 24-hour rhythm and represents a key post-translational mechanism that regulates circadian physiology. We observe strong correlation between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms are severely dampened when we subject flies to time-restricted feeding at unnatural feeding time. This suggests the presence of clock-regulated buffering mechanisms that prevent excessive O-GlcNAcylation at non-optimal times of the day-night cycle. We show that this buffering mechanism is mediated by the expression and activity of GFAT, OGT, and OGA, which are regulated through integration of circadian and metabolic signals. Finally, we generate a mathematical model to describe the key factors that regulate daily O-GlcNAcylation rhythm.
The stress of water-borne hormone collection process was examined in sailfin mollies Poecilia latipinna. Baseline release rates of the stress hormone cortisol were measured and minimum confinement time for water sampling was evaluated for a standard 60 min v. a 30 min protocol. A 30 min hormone collection period reflects release rates over 60 min. Potential stress response to confinement in the beaker for the water-borne collection process was tested over 4 days. There was no evidence of stress due to the collection methods, as cortisol release rates did not differ significantly across four sequential days of handling for P. latipinna. Males and females did not differ significantly in baseline cortisol release rates. Baseline cortisol release rates from fish immediately after being collected in the field were also not significantly different than those in the 4 day confinement experiment. After exposure to a novel environment, however, P. latipinna mounted a stress response. Stress may also affect sex steroids and behaviour but cortisol release rates were not significantly correlated with sex steroids [11-ketotestosterone (KT), testosterone, or oestradiol], or mating attempts. The correlation between water-borne release rates and plasma steroid levels was validated for both cortisol and KT. Finally, normalizing cortisol release rates using standard length in lieu of mass is viable and accurate. Water-borne hormone assays are a valuable tool for investigating questions concerning the role of hormones in mediating stress responses and reproductive behaviours in P. latipinna and other livebearing fishes.
Intracellular signaling dynamics play fundamental roles in cell biology. Precise modulation of the amplitude, duration, and frequency of signaling activation will be a powerful approach to investigate molecular mechanisms as well as to engineer signaling to control cell behaviors. Here, we showed a practical approach to achieve precise amplitude modulation (AM), frequency modulation (FM), and duration modulation (DM) of MAP kinase activation. Alternating current (AC) electrical stimulation induced synchronized ERK activation. Amplitude and duration of ERK activation were controlled by varying stimulation strength and duration. ERK activation frequencies were arbitrarily modulated with trains of short AC applications with accurately defined intervals. Significantly, ERK dynamics coded by well-designed AC can rewire PC12 cell fate independent of growth factors. This technique can be used to synchronize and modulate ERK activation dynamics, thus would offer a practical way to control cell behaviors in vivo without the use of biochemical agents or genetic manipulation.
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