Background Exosomes are extracellular vesicles of nano-structures and represent an emerging nano-scale acellular therapy in recent years. Tendon regeneration is a sophisticated process in the field of microsurgery due to its poor natural healing ability. To date, no successful long-term solution has been provided for the healing of tendon injuries. Functional recovery requires advanced treatment strategies. Human umbilical cord mesenchymal stem cell-derived exosomes (HUMSC-Exos) are considered as promising cell-free therapeutic agents. However, few studies reported their potential in the tendon repair previously. In this study, we explored the roles and underlying mechanisms of HUMSC-Exos in the tendon regeneration. Results Expression of tendon‐specific markers in, and collagen deposition by, tendon-derived stem cells (TDSCs) treated with HUMSC-Exos increased in vitro. In a rat Achilles tendon injury model, treatment with HUMSC-Exos improved the histological structure, enhanced tendon-specific matrix components, and optimized biomechanical properties of the Achilles tendon. Findings in miRNA sequencing indicated a significant increase in miR-29a-3p in HUMSC-Exo-treated Achilles tendons. Next, luciferase assay in combination with western blot identified phosphatase and tensin homolog (PTEN) as the specific target of miR-29a-3p. Furthermore, we applied a miR-29a-3p-specific agonist to engineer HUMSC-Exos. These HUMSC-Exos overexpressing miR-29a-3p amplified the gain effects of HUMSC-Exos on tendon healing in vivo. To explore the underlying mechanisms, a transforming growth factor-β1 (TGF-β1) inhibitor (SB-431542), mTOR inhibitor (rapamycin), and engineered HUMSC-Exos were employed. The results showed that TGF-β1 and mTOR signaling were involved in the beneficial effects of HUMSC-Exos on tendon regeneration. Conclusion The findings in our study suggest that PTEN/mTOR/TGF-β1 signaling cascades may be a potential pathway for HUMSC-Exos to deliver miR-29a-3p for tendon healing and implicate a novel therapeutic strategy for tendon regeneration via engineered stem cell-derived exosomes. Graphic abstract

Biodegradation of aromatic and heterocyclic compounds requires an oxidative ring cleavage enzymatic step. Extensive biochemical research has yielded mechanistic insights about catabolism of aromatic substrates; yet much less is known about the reaction mechanisms underlying the cleavage of heterocyclic compounds such as pyridine-ring-containing ones like 2,5-hydroxy-pyridine (DHP). 2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 uses a mononuclear nonheme Fe(II) to catalyze the oxidative pyridine ring cleavage reaction by transforming DHP into N-formylmaleamic acid (NFM). Herein, we report a crystal structure for the resting form of NicX, as well as a complex structure wherein DHP and NFM are trapped in different subunits. The resting state structure displays an octahedral coordination for Fe(II) with two histidine residues (His265 and His318), a serine residue (Ser302), a carboxylate ligand (Asp320), and two water molecules. DHP does not bind as a ligand to Fe(II), yet its interactions with Leu104 and His105 function to guide and stabilize the substrate to the appropriate position to initiate the reaction. Additionally, combined structural and computational analyses lend support to an apical dioxygen catalytic mechanism. Our study thus deepens understanding of non-heme Fe(II) dioxygenases.

A characteristic feature of phytopathogenic Xanthomonas bacteria is the production of yellow membrane-bound pigments called xanthomonadins. Previous studies showed that 3-hydroxybenzoic acid (3-HBA) was a xanthomonadin biosynthetic intermediate and also, that it had a signaling role. The question of whether the structural isomers 4-HBA and 2-HBA (salicylic acid) have any role in xanthomonadin biosynthesis remained unclear. In this study, we have selectively eliminated 3-HBA, 4-HBA, or the production of both by expression of the mhb, pobA, and pchAB gene clusters in the Xanthomonas campestris pv. campestris strain XC1. The resulting strains were different in pigmentation, virulence factor production, and virulence. These results suggest that both 3-HBA and 4-HBA are involved in xanthomonadin biosynthesis. When both 3-HBA and 4-HBA are present, X. campestris pv. campestris prefers 3-HBA for Xanthomonadin-A biosynthesis; the 3-HBA–derived Xanthomonadin-A was predominant over the 4-HBA–derived xanthomonadin in the wild-type strain XC1. If 3-HBA is not present, then 4-HBA is used for biosynthesis of a structurally uncharacterized Xanthomonadin-B. Salicylic acid had no effect on xanthomonadin biosynthesis. Interference with 3-HBA and 4-HBA biosynthesis also affected X. campestris pv. campestris virulence factor production and reduced virulence in cabbage and Chinese radish. These findings add to our understanding of xanthomonadin biosynthetic mechanisms and further help to elucidate the biological roles of xanthomonadins in X. campestris pv. campestris adaptation and virulence in host plants.

Laccase–mediator systems (LMSs) have been intensively investigated in lignin degradation. Although only natural metabolites are available for fungal lignin degradation, mediator molecules from metabolites have received substantially less attention than artificial organic–synthetic compounds. It remains unclear which metabolites can accelerate laccase-catalyzed reactions and how those natural mediators influence lignin degradation. In this work, we evaluated Trametes versicolor laccase-catalyzed reaction kinetics on a lignin guaiacyl subunit model (guaiacylglycerol-β-guaiacyl ether, G-β-GE) in the presence of a group of lignin syringyl subunit molecules: syringaldehyde, acetosyringone, and methyl syringate. We then compare their performance to a well-known synthetic mediator ABTS, 2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulfonic acid). Time-resolved UPLC-TOF-MS revealed that the syringyl mediators were more effective in accelerating the β-O-4 cleavage and Cα-oxidation of G-β-GE than ABTS under laccase-catalysis, despite the syringyl compounds possessing slower individual oxidation rates. In addition, the product profile of polymerization was also promoted dramatically, compared to that of the ABTS/laccase system. The LMS kinetic modeling suggested that mediator–substrate aggregation played a critical role in the laccase–mediator system; in which, the lignin syringyl and guaiacyl subunits likely form a π–π stacking van der Waals complex that can be oxidized faster than the syringyl or guaiacyl monomers by themselves. This syringyl–guaiacyl aggregation hypothesis postulates that the weak interactions in lignin biopolymers are able to accelerate the laccase-catalyzed biodegradation.