Induced proximity labeling and editing for epigenetic research.

Epigenetic regulation plays a pivotal role in various biological and disease processes. Two key lines of investigation have been pursued that aim to unravel endogenous epigenetic events at particular genes (probing) and artificially manipulate the epigenetic landscape (editing). The concept of induced proximity has inspired the development of powerful tools for epigenetic research. Induced proximity strategies involve bringing molecular effectors into spatial proximity with specific genomic regions to achieve the probing or manipulation of local epigenetic environments with increased proximity. In this review, we detail the development of induced proximity methods and applications in shedding light on the intricacies of epigenetic regulation.

Quantification of bone quality and distribution of the proximal humerus with dual-energy computed tomography.

Background: The proximal humerus is a common site of osteoporotic fractures, and bone quality is a predictor of surgical reduction quality. Dual-energy computed tomography (DECT) is assuming an increasingly important role in the quantification of bone mineral density (BMD) due it is ability to perform three-material decomposition. We aimed to analyze the bone quality and distribution of the proximal humerus with DECT quantitatively. Methods: Sixty-five consecutive patients (average age 49.5±15.2 years; male: female ratio 32:33) without proximal humerus fractures who had undergone DECT were retrospectively selected. The humeral head was divided into 4 regions on a cross section in the medial plane between the greater tuberosity and the surgical neck. The quantitative parameters, including virtual noncalcium (VNCa) value, computed tomography value of calcium (CaCT), computed tomography value of mixed-energy images (regular CT value) (rCT), and relative calcium density (rCaD), were measured. The correlations between the quantitative parameters and age and body mass index (BMI) were analyzed, and the correlations of age, sex, BMI, region of the humeral head, and VNCa value on CaCT were evaluated. Results: The differences in CaCT, rCT, and rCaD between the 4 regions of proximal humerus were statistically significant (P<0.001), while the difference in VNCa values was not (P=0.688). The calcium concentration (CaCT and rCaD) was the densest in the posteromedial zone. The differences of CaCT, rCT, and rCaD between males and females in the 4 regions of proximal humerus were statistically significant (P<0.05), while those of the posterolateral zone were not (rCT; P>0.05). The differences in VNCa values between males and females were also not significant (P>0.05). Multivariable linear regression analysis indicated that sex, age, BMI, regions, and VNCa were significant (P<0.05) predictors of the CaCT value. Conclusions: The concentration of calcium was the densest in the posteromedial region of proximal humerus, and the VNCa value of DECT may be used for quantifying the BMD of the proximal humerus.

MFG-E8 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells through GSK3ß/ß-catenin signaling pathway.

Fracture nonunion and bone defects are challenging for orthopedic surgeons. Milk fat globule-epidermal growth factor 8 (MFG-E8), a glycoprotein possibly secreted by macrophages in a fracture hematoma, participates in bone development. However, the role of MFG-E8 in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. We investigated the osteogenic effect of MFG-E8 in vitro and in vivo. The CCK-8 assay was used to assess the effect of recombinant human MFG-E8 (rhMFG-E8) on the viability of hBMSCs. Osteogenesis was investigated using RT-PCR, Western blotting, and immunofluorescence. Alkaline phosphatase (ALP) and Alizarin red staining were used to evaluate ALP activity and mineralization, respectively. An enzyme-linked immunosorbent assay was conducted to evaluate the secretory MFG-E8 concentration. Knockdown and overexpression of MFG-E8 in hBMSCs were established via siRNA and lentivirus vector transfection, respectively. Exogenous rhMFG-E8 was used to verify the in vivo therapeutic effect in a tibia bone defect model based on radiographic analysis and histological evaluation. Endogenous and secretory MFG-E8 levels increased significantly during the early osteogenic differentiation of hBMSCs. Knockdown of MFG-E8 inhibited the osteogenic differentiation of hBMSCs. Overexpression of MFG-E8 and rhMFG-E8 protein increased the expression of osteogenesis-related genes and proteins and enhanced calcium deposition. The active ß-catenin to total ß-catenin ratio and the p-GSK3ß protein level were increased by MFG-E8. The MFG-E8-induced enhanced osteogenic differentiation of hBMSCs was partially attenuated by a GSK3ß/ß-catenin signaling inhibitor. Recombinant MFG-E8 accelerated bone healing in a rat tibial-defect model. In conclusion, MFG-E8 promotes the osteogenic differentiation of hBMSCs by regulating the GSK3ß/ß-catenin signaling pathway and so, is a potential therapeutic target.

A Split CRISPR/Cas13b System for Conditional RNA Regulation and Editing.

The CRISPR/Cas13b system has been demonstrated as a robust tool for versatile RNA studies and relevant applications. New strategies enabling precise control of Cas13b/dCas13b activities and minimal interference with native RNA activities will further facilitate the understanding and regulation of RNA functions. Here, we engineered a split Cas13b system that can be conditionally activated and deactivated under the induction of abscisic acid (ABA), which achieved the downregulation of endogenous RNAs in dosage- and time-dependent manners. Furthermore, an ABA inducible split dCas13b system was generated to achieve temporally controlled deposition of m6A at specific sites on cellular RNAs through conditional assembly and disassembly of split dCas13b fusion proteins. We also showed that the activities of split Cas13b/dCas13b systems can be modulated by light via using a photoactivatable ABA derivative. Overall, these split Cas13b/dCas13b platforms expand the existing repertoire of the CRISPR and RNA regulation toolkit to achieve targeted manipulation of RNAs in native cellular environments with minimal functional disruption to these endogenous RNAs.

Glycyrrhizic Acid Promotes Osteogenic Differentiation of Human Bone Marrow Stromal Cells by Activating the Wnt/ß-Catenin Signaling Pathway.

Glycyrrhizic acid (GA) is a major triterpene glycoside isolated from liquorice root that has been shown to inhibit osteoclastogenesis. However, there have been no reports regarding the effect of GA on osteogenic differentiation. Therefore, this study was performed to explore the effects and mechanism of action of GA on osteogenesis. A CCK-8 array was used to assess cell viability. The osteogenic capability was investigated by real-time quantitative PCR, western blotting and immunofluorescence analyses. ALP staining and ARS were used to evaluate ALP activity and mineralization, respectively. GA-GelMA hydrogels were designed to verify the therapeutic effects of GA in vivo by radiographic analysis and histological evaluation. Our results show that GA had no significant influence on the viability or proliferation of human bone marrow stromal cells (hBMSCs). GA promoted osteogenic differentiation and enhanced calcium deposition. Furthermore, ratio of active ß-catenin and total ß-catenin protein increased after treatment with GA. Wnt/catenin signaling inhibitor partially attenuated the effects of GA on osteogenic differentiation. In a mouse femoral fracture model, GA-GelMA hydrogels accelerated bone healing. Our results show that GA promotes the osteogenic differentiation of hBMSCs by modulating the Wnt/ß-catenin signaling pathway. GA-GelMA hydrogels promoted bone fracture healing. GA has potential as a cost-effective treatment of bone defects.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.