Enhanced small green fluorescent proteins as a multisensing platform for biosensor development.

Engineered light, oxygen, and voltage (LOV)-based proteins are able to fluoresce without oxygen requirement due to the autocatalytic incorporation of exogenous flavin as a chromophore thus allowing for live cell imaging under hypoxic and anaerobic conditions. They were also discovered to have high sensitivity to transition metal ions and physiological flavin derivatives. These properties make flavin-binding fluorescent proteins (FPs) a perspective platform for biosensor development. However, brightness of currently available flavin-binding FPs is limited compared to GFP-like FPs creating a need for their further enhancement and optimization. In this study, we applied a directed molecular evolution approach to develop a pair of flavin-binding FPs, named miniGFP1 and miniGFP2. The miniGFP proteins are characterized by cyan-green fluorescence with excitation/emission maxima at 450/499 nm and a molecular size of ∼13 kDa. We carried out systematic benchmarking of miniGFPs in Escherichia coli and cultured mammalian cells against spectrally similar FPs including GFP-like FP, bilirubin-binding FP, and bright flavin-binding FPs. The miniGFPs proteins exhibited improved photochemical properties compared to other flavin-binding FPs enabling long-term live cell imaging. We demonstrated the utility of miniGFPs for live cell imaging in bacterial culture under anaerobic conditions and in CHO cells under hypoxia. The miniGFPs' fluorescence was highly sensitive to Cu(II) ions in solution with Kd values of 67 and 68 nM for miniGFP1 and miniGFP2, respectively. We also observed fluorescence quenching of miniGFPs by the reduced form of Cu(I) suggesting its potential application as an optical indicator for Cu(I) and Cu(II). In addition, miniGFPs showed the ability to selectively bind exogenous flavin mononucleotide demonstrating a potential for utilization as a selective fluorescent flavin indicator. Altogether, miniGFPs can serve as a multisensing platform for fluorescence biosensor development for in vitro and in-cell applications.

Combined Shikonin-Loaded MPEG-PCL Micelles Inhibits Effective Transition of Endothelial-to-Mesenchymal Cells.

Introduction: Shikonin is well known for its anti-inflammatory activity in cardiovascular diseases. However, the application of shikonin is limited by its low water solubility and poor bioavailability. Methoxy poly (ethylene glycol)-b-poly (ε-caprolactone) (MPEG-PCL) is considered a promising delivery system for hydrophobic drugs. Therefore, in this study, we prepared shikonin-loaded MPEG-PCL micelles and investigated their effect on endothelial-to-mesenchymal transition (EndMT) induced by inflammatory cytokines. Methods: Shikonin was encapsulated in MPEG-PCL micelles using an anti-solvent method and the physiochemical characteristics of the micelles (particle size, zeta potential, morphology, critical micelle concentration (CMC), drug loading and encapsulation efficiency) were investigated. Cellular uptake of micelles in human umbilical vein endothelial cells (HUVECs) was evaluated using fluorescence microscopy. In vitro EndMT inhibition was explored in HUVECs by quantitative real-time PCR analysis. Results: The average particle size of shikonin-loaded MPEG-PCL micelles was 54.57±0.13 nm and 60 nm determined by dynamic light scattering and transmission electron microscopy, respectively. The zeta potential was -6.23±0.02 mV. The CMC of the micelles was 6.31×10-7mol/L. The drug loading and encapsulation efficiency were 0.88±0.08% and 43.08±3.77%, respectively. The MPEG-PCL micelles significantly improved the cellular uptake of cargo with low water solubility. Real-time PCR analysis showed that co-treatment with TNF-α and IL-1ß successfully induced EndMT in HUVECs, whereas this process was significantly inhibited by shikonin and shikonin-loaded MPEG-PCL micelles, with greater inhibition mediated by the shikonin-loaded MPEG-PCL micelles. Conclusion: Shikonin-loaded MPEG-PCL micelles significantly improved the EndMT-inhibiting effect of the free shikonin. MPEG-PCL is suitable for use more generally as a lipophilic drug carrier.

HDAC3 inhibitor suppresses endothelial-to-mesenchymal transition via modulating inflammatory response in atherosclerosis.

A total number of 18 different isoforms of histone deacetylases (HDACs) which were categorized into 4 classes have been identified in human. HDAC3 is categorized as class I HDACs and is closely related to the occurrence and development of atherosclerosis. Recent evidence has pointed to endothelial-to-mesenchymal transition (EndMT) as a key process in vascular inflammation in atherosclerosis. However, little is known about the effect of HDAC3 on EndMT in atherosclerosis. Therefore, we aimed to investigate the effect of HDAC3 specific inhibitor on EndMT in ApoE-/- mice fed a Western diet and human umbilical vein endothelial cells (HUVECs) induced by inflammatory cytokines. Firstly, we found that HDAC3 expression was up-regulated and EndMT occurred in the aortas of ApoE-/- mice compared with C57BL/6J mice. However, HDAC3 specific inhibitor RGFP966 alleviated atherosclerotic lesions and inhibited EndMT of the atherosclerotic plaque in ApoE-/- mice. Then, in vitro study showed that inflammatory cytokines TNF-α and IL-1ß co-treatment increased the expression of HDAC3 and induced EndMT in HUVECs. HDAC3 inhibition by siRNA or specific inhibitor RGFP966 suppressed EndMT in HUVECs stimulated with TNF-α and IL-1ß. By contrast, HDAC3 overexpression by adenovirus further promoted EndMT of HUVECs. In addition, we found that HDAC3 also regulated the inflammatory response of HUVECs by modulating the expression of inflammatory cytokines and the number of monocytes attached to HUVECs. These above results suggest that HDAC3 inhibitor suppresses EndMT via modulating inflammatory response in ApoE-/- mice and HUVECs.