Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.
Obesity results from numerous, interacting genetic, behavioral, and physiological factors. Adipogenesis is partially regulated by several adipocyte-selective microRNAs (miRNAs) and transcription factors that regulate proliferation and differentiation of human adipose-derived mesenchymal stem cells (hMSCs-Ad). In this study, we examined the roles of adipocyte-selective miRNAs in the differentiation of hMSCs-Ad to adipocytes. Results showed that the levels of miR-148a, miR-26b, miR-30, and miR-199a increased in differentiating hMSCs-Ad. Among these miRNAs, miR-148a exhibited significant effects on increasing PPRE luciferase activity (it represents PPAR-dependent transcription, a major factor in adipogenesis) than others. Furthermore, miR-148a expression levels increased in adipose tissues from obese people and mice fed high-fat diet. miR-148a acted by suppressing its target gene, Wnt1, an endogenous inhibitor of adipogenesis. Ectopic expression of miR-148a accelerated differentiation and partially rescued Wnt1-mediated inhibition of adipogenesis. Knockdown of miR-148a also inhibited adipogenesis. Analysis of the upstream region of miR-148a locus identified a 3 kb region containing a functional cAMP-response element-binding protein (CREB) required for miR-148a expression in hMSCs-Ad. The results suggest that miR-148a is a biomarker of obesity in human subjects and mouse model, which represents a CREB-modulated miRNA that acts to repress Wnt1, thereby promoting adipocyte differentiation.
The therapeutic promise of induced pluripotent stem cells (iPSCs) has spurred efforts to circumvent genome alteration when reprogramming somatic cells to pluripotency. Approaches based on episomal DNA, Sendai virus, and messenger RNA (mRNA) can generate “footprint-free” iPSCs with efficiencies equaling or surpassing those attained with integrating viral vectors. The mRNA method uniquely affords unprecedented control over reprogramming factor (RF) expression while obviating a cleanup phase to purge residual traces of vector. Currently, mRNA-based reprogramming is relatively laborious due to the need to transfect daily for ~2 weeks to induce pluripotency, and requires the use of feeder cells that add complexity and variability to the procedure while introducing a route for contamination with non-human-derived biological material. We accelerated the mRNA reprogramming process through stepwise optimization of the RF cocktail and leveraged these kinetic gains to establish a feeder-free, xeno-free protocol which slashes the time, cost and effort involved in iPSC derivation.
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