Convergent accelerated evolution of mammal-specific conserved non-coding elements in hibernators.

Mammals maintain their body temperature, yet hibernators can temporarily lower their metabolic rate as an energy-saving strategy. It has been proposed that hibernators evolved independently from homeotherms, and it is possible that the convergent evolution of hibernation involved common genomic changes among hibernator-lineages. Since hibernation is a seasonal trait, the evolution of gene regulatory regions in response to changes in season may have been important for the acquisition of hibernation traits. High-frequency accumulation of mutations in conserved non-coding elements (CNEs) could, in principle, alter the expression of neighboring genes and thereby contribute to the acquisition of new traits. To address this possibility, we performed a comparative genomic analysis of mammals to identify accelerated CNEs commonly associated with hibernation. We found that accelerated CNEs are common to hibernator-lineages and could be involved with hibernation. We also found that common factors of genes that located near accelerated CNEs and are differentially expressed between normal and hibernation periods related to gene regulation and cell-fate determination. It suggests that the molecular mechanisms controlling hibernation have undergone convergent evolution. These results help broaden our understanding of the genetic adaptations that facilitated hibernation in mammals and may offer insights pertaining to stress responses and energy conservation.

Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry for Automated Analysis of Four Sulfated Steroid Metabolites in Saliva Samples.

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17ß-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC-MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01-2 ng mL-1 for PREGS, DHEAS, and CRTS and of 0.05-10 ng mL-1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL-1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6-112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 µL saliva samples.