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Purpose: This study aimed to determine how different strain intensities-including normal, moderately increased, and high strain-influence protein expression profiles and related biological processes in human corneal stromal keratocytes.

Methods: A well-established in vitro model using the Flexcell FX-5000 Tension System, which replicates the natural corneal curvature and enables precise strain application to keratocytes, was used. Keratocytes were exposed to three strain levels: 3% (normal), 6% (moderately increased), and 12% (high). Following strain application, cells were collected for liquid chromatography-tandem mass spectrometry-based proteomic analysis to generate protein expression profiles. Differentially expressed proteins (DEPs) among the three groups were identified and subjected to biological pathway enrichment to reveal strain-dependent biological processes. Western blot analysis was performed to validate the expression of selected DEPs.

Results: Keratocytes exhibited strain intensity-dependent responses. Three percent strain maintained keratocytes in a quiescent phenotype, consistent with our previous findings. Six percent strain activated protective and adaptive programs to preserve tissue homeostasis under stress. In contrast, 12% strain suppressed immune-related processes and induced extracellular matrix (ECM) remodeling. Notably, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and cathepsin L (CTSL)-two ECM remodeling-related proteins implicated in fibrotic responses-were significantly upregulated under 12% strain, highlighting a potential link between excessive mechanical stress and stromal fibrosis.

Conclusions: These findings demonstrate that corneal strain regulates keratocyte behavior in an intensity-dependent manner and suggest that high mechanical stress may drive pathologic stromal remodeling and fibrotic responses, offering mechanistic insights that may inspire future therapeutic strategies.

Despite the widespread deployment of current COVID-19 vaccines, significant gaps remain in understanding the complete biological behavior of the SARS-CoV-2 spike (S) protein. Through systematic characterization of mammalian expression systems, this study shows that the full-length S protein exhibits a complex intracellular distribution, predominantly localizing not only to the cell membrane but also to the cytoplasm, nucleus, and extracellular compartments. Comparative analyses revealed distinct subunit-specific trafficking patterns. The S1 subunit showed increased intracellular accumulation and secretion compared to the full-length S protein, although with reduced surface expression. Conversely, the receptor-binding domain (RBD) and S2 domains were mainly associated with cytoskeletal (CS) structures. Notably, the signal sequence-enhanced RBD (SS-RBD) construct engineered in this study demonstrated dramatically enhanced extracellular accumulation, approximately 100-fold higher than the full-length S protein and 10-fold greater than S1, as measured by proximity extension assay (PEA). Signal peptide modification effectively redirected RBD from CS retention to efficient secretion, significantly improving detection sensitivity. PEA outperformed conventional methods such as flow cytometry (FACS), Western blotting (WB), and immunofluorescence, offering sensitivities several orders of magnitude higher. Consequently, these findings provide: (1) a structural framework for rational antigen design by distinguishing essential versus dispensable domains; (2) experimental support for SS-RBD as a promising vaccine candidate due to its high secretion efficiency and preservation of neutralizing epitopes; and (3) a robust platform using PEA for high-sensitivity antigen characterization. This study enhances the fundamental understanding of spike protein biology and offers actionable insights for developing next-generation vaccines targeting SARS-CoV-2 and related coronaviruses.

Keratocytes are the primary resident cells in the corneal stroma. They play an essential role in maintaining corneal physiological function. Studying the factors that affect the phenotype and behavior of keratocytes offers meaningful perspectives for improving the understanding and treatment of corneal injuries. In this study, 3% strain was applied to human keratocytes using the Flexcell® Tension Systems. Real-time quantitative PCR (RT-qPCR) and western blot were used to investigate the influence of strain on the expression of intracellular aldehyde dehydrogenase 3A1 (ALDH3A1). ALDH3A1 knockdown was achieved using double-stranded RNA-mediated interference (RNAi). Immunofluorescence (IF) staining was employed to observe the impact of changes in ALDH3A1 expression on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation. Keratocyte proliferation and migration were assessed by bromodeoxyuridine (BrdU) assay and scratch wound healing assay, respectively. Mouse injury models and single-cell RNA sequencing of keratocytes from keratoconus patients were used to assess how strain influenced ALDH3A1 in vivo. Our results demonstrate that 3% strain suppresses keratocyte proliferation and increases ALDH3A1. Increased ALDH3A1 inhibits NF-κB nuclear translocation, a key step in the activation of the NF-κB signaling pathway. Conversely, ALDH3A1 knockdown promotes NF-κB nuclear translocation, ultimately enhancing keratocyte proliferation and migration. Elevated ALDH3A1 levels were also observed in mouse injury models with increased corneal strain and keratoconus patients. These findings provide valuable insights for further research into the role of corneal strain and its connection to corneal injury repair.