Oxidation and cross-link of tyrosine-rich proteins are involved in the periostracum formation of the green mussel Perna viridis (Linnaeus).

Ocean acidification causes severe shell dissolution and threats the survival of marine molluscs. The periostracum in molluscs consists of macromolecules such as proteins and polysaccharides, and protects the inner shell layers from dissolution and microbial erosion. Moreover, it serves as the primary template for shell deposition. However, the chemical composition and formation mechanism of the periostracum is largely unknown. In this study, we applied transcriptomic, proteomics, physical, and chemical analysis to unravel the mysteries of the periostracum formation in the green mussel Perna viridis Linnaeus. FTIR analysis showed that the periostracum layer was an organic membrane mainly composed of polysaccharides, lipids, and proteins, similar to that of the shell matrix. Interestingly, the proteomic study identified components enriched in tyrosine and some enzymes that evolved in tyrosine oxidation, indicating that tyrosine oxidation might play an essential role in the periostracum formation. Moreover, comparative transcriptomics suggested that tyrosine-rich proteins were intensively synthesized in the periostracum groove. After being secreted, the periostracum proteins were gradually tanned by oxidation in the seawater, and the level of crosslink increased significantly as revealed by the ATR-FTIR. Our present study sheds light on the chemical composition and putative tanning mechanism of the periostracum layer in bivalve molluscs. SIGNIFICANCE: The periostracum layer, plays an essential role in the initiation of shell biomineralization, the protection of minerals from dissolution for molluscs and especially ocean acidification conditions in the changing global climate. However, the molecular mechanism underlying the periostracum formation is not fully understood. In this study, we revealed that the oxidation and cross-link of tyrosine-rich proteins by tyrosinase are involved in periostracum formation in the green mussel Perna viridis. This study provides some insights into the first step of mussel shell formation and the robust adaptation of P. viridis to diverse habitats. These findings also help to reveal the potential acclimation of bivalves to the projected acidifying seawater.

Enhanced pyroptosis induction with pore-forming gene delivery for osteosarcoma microenvironment reshaping.

Osteosarcoma is the most common malignant bone tumor without efficient management for improving 5-year event-free survival. Immunotherapy is also limited due to its highly immunosuppressive tumor microenvironment (TME). Pore-forming gasdermins (GSDMs)-mediated pyroptosis has gained increasing concern in reshaping TME, however, the expressions and relationships of GSDMs with osteosarcoma remain unclear. Herein, gasdermin E (GSDME) expression is found to be positively correlated with the prognosis and immune infiltration of osteosarcoma patients, and low GSDME expression was observed. A vector termed as LPAD contains abundant hydroxyl groups for hydrating layer formation was then prepared to deliver the GSDME gene to upregulate protein expression in osteosarcoma for efficient TME reshaping via enhanced pyroptosis induction. Atomistic molecular dynamics simulations analysis proved that the hydroxyl groups increased LPAD hydration abilities by enhancing coulombic interaction. The upregulated GSDME expression together with cleaved caspase-3 provided impressive pyroptosis induction. The pyroptosis further initiated proinflammatory cytokines release, increased immune cell infiltration, activated adaptive immune responses and create a favorable immunogenic hot TME. The study not only confirms the role of GSDME in the immune infiltration and prognosis of osteosarcoma, but also provides a promising strategy for the inhibition of osteosarcoma by pore-forming GSDME gene delivery induced enhanced pyroptosis to reshape the TME of osteosarcoma.