RESUMO
The orphan G protein-coupled receptor 35 (GPR35) is a potential target for the treatment of pain, inflammation, and metabolic diseases. Although many GPR35 agonists have been discovered, research on functional GPR35 ligands, such as fluorescent probes, is still limited. Herein, we developed a series of GPR35 fluorescent probes by conjugating a BODIPY fluorophore to DQDA, a known GPR35 agonist. All probes exhibited excellent GPR35 agonistic activity and desired spectroscopic properties, as determined by the DMR assay, bioluminescence resonance energy transfer (BRET)-based saturation, and kinetic binding experiments. Notably, compound 15 showed the highest binding potency and the weakest nonspecific BRET binding signal (K d = 3.9 nM). A BRET-based competition binding assay with 15 was also established and used to determine the binding constants and kinetics of unlabeled GPR35 ligands.
RESUMO
The anther is one of the most vulnerable organs to temperature stress. Many previous works focused on the genes regulating anthers development, but few results of miRNA in anther development were reported. In order to investigate the transcriptional regulation of temperature-sensitive anther development, RNA-Sequencing was used to study micRNA in anthers of Arabidopsis thaliana under 16 °C and 27 °C. A total of 46.26 million clean reads were generated and mapped to 715,748 small RNA sequences containing 281 miRNAs. Then 13 differentially expressed (DE) miRNAs, containing 3 novel miRNAs were found. Comprehensive analysis of miRNA expression showed 7 miRNAs were down-regulated and 6 miRNAs were up-regulated. Furthermore, 13 DE miRNAs putatively regulated 614 DE mRNAs. Among them, 20 important anther genes were predicted as target genes of MIR319A, MIR447A, MIR447B and MIR398B, respectively. Over-expression MIR319A and MIR447A could effectively inhibit the transcription of target genes and lead to male sterile. It suggested that DE miRNAs might mediate temperature signals and regulate anther and pollen development. Our work will provide a broader idea and valuable data information for further understanding the mechanism of thermo-sensitive male fertility in plants.
