Stemness maintenance of stem cells derived from human exfoliated deciduous teeth (SHED) in 3D spheroid formation through the TGF-ß/Smad signaling pathway.

Mesenchymal stem cells (MSCs) have shown great potential as important therapeutic tools for dental pulp tissue engineering, with the maintenance and enhancement of their stemness being crucial for successful therapeutic application in vivo and three-dimensional (3D) spheroid formation considered a reliable technique for enhancing their pluripotency. Human exfoliated deciduous tooth stem cells (SHED) were cultured in a low attachment plate to form aggregates for five days. Then, the resulting spheroids were analyzed for pluripotent marker expression, paracrine secretory function, proliferation, signaling pathways involved, and distribution of key proteins within the spheroids. The results indicated that 3D spheroid formation significantly increased the activation of the transforming growth factor beta (TGF-ß)/Smad signaling pathway and upregulated the secretion and mRNA expression levels of TGF-ß, which in turn enhanced the expression of pluripotency markers in SHED spheroids. The activation of the TGF-ß/Smad signaling pathway through 3D spheroid formation was found to preserve the stemness properties of SHED. Thus, understanding the mechanisms behind pluripotency maintenance of SHED culture through 3D spheroid formation could have implications for the therapeutic application of MSCs in regenerative medicine and tissue engineering.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: The Novel Therapeutic Option for Regenerative Dentistry.

Dental mesenchymal stem cells (MSCs) are characterized by unlimited self-renewal ability and high multidirectional differentiation potential. Since dental MSCs can be easily isolated and exhibit a high capability to differentiate into odontogenic cells, they are considered as attractive therapeutic agents in regenerative dentistry. Recently, MSC-derived extracellular vesicles (MSC-EVs) have attracted widespread attention as carriers for cell-free therapy due to their potential functions. Many studies have shown that MSC-EVs can mediate microenvironment at tissue damage site, and coordinate the regeneration process. Additionally, MSC-EVs can mediate intercellular communication, thus affecting the phenotypes and functions of recipient cells. In this review, we mainly summarized the types of MSCs that could be potentially applied in regenerative dentistry, the possible molecular cargos of MSC-EVs, and the major effects of MSC-EVs on the therapeutic induction of osteogenic differentiation.

Molecular Engineering of Bacillus paralicheniformis Acid Urease To Degrade Urea and Ethyl Carbamate in Model Chinese Rice Wine.

Bacillus paralicheniformis urease (BpUrease) has been shown to be a promising biocatalyst for degrading the carcinogenic chemical ethyl carbamate (EC or urethane) in rice wine. However, low EC affinity and catalytic efficiency limit the practical application of BpUrease. In this study, we improved the EC degradation capability of BpUrease by site-saturation mutagenesis (SSM). The best variant L253P/L287N showed a 49% increase in EC affinity, 1027% increase in catalytic efficiency ( kcat/ Km), and 583% increase in half-life ( t1/2) at 70 °C. Homology modeling analysis suggest that mutation of Leu253 to Pro increased the BpUrease EC specificity by affecting the interaction between Arg339 with the catalytic residue His323, while Leu287Asn mutation benefits EC specificity and affinity by changing the interaction networks among the residues in the catalytic pocket. Our results show that the L253P/L287N variant efficiently degraded urea and EC in a model rice wine, making it a good candidate for practical application in the food industry.

High-yield secretory production of stable, active trypsin through engineering of the N-terminal peptide and self-degradation sites in Pichia pastoris.

Streptomyces griseus trypsin (SGT) possesses enzymatic properties similar to mammalian trypsins and has great potential applications in the leather processing, bioethanol, detergent and pharmaceutical industry. Here, a new strategy was reported for improving its stable, active secretory production through engineering of its propeptide and self-degradation sites. By rationally introducing hydrophobic mutations into the N-terminus of SGT Exmt (R145I), replacing the propeptide with FPVDDDDK and engineering the α-factor signal peptide, trypsin production (amidase activity) was improved to 177.85±2.83U·mL-1 in a 3-L fermenter (a 3.75-fold increase). Subsequently, all of the residues involved in autolysis that were identified by mass spectrometry were mutated and the resulting proteins were evaluated. In particular, the variant tbcf (K101A) demonstrated high stability and production (227.65±6.51U·mL-1 and 185.71±5.68mg·L-1, respectively). The recombinant strain constructed here has great potential for large-scale production of active trypsin.