Retinal Proteome Analysis Reveals a Region-Specific Change in the Rabbit Myopia Model.

Uncovering region-specific changes in the myopic retina can provide clues to the pathogenesis of myopia progression. After imposing form deprivation myopia in the right eye of 6-week-old rabbits, we investigated the proteome profile of each retinal region (central, mid-periphery, and far-periphery retina), using accurate high-resolution mass spectrometry. Protein expression was analyzed using gene ontology and network analysis compared with that of the control, the left eyes. Among 2065 proteins detected from whole retinal samples, 249 differentially expressed proteins (DEPs) were identified: 164 DEPs in the far-periphery, 39 in the mid-periphery, and 83 in the central retina. In network analysis, the far-periphery retina showed the most significant connectivity between DEPs. The regulation of coagulation was the most significant biological process in upregulated DEPs in the far-periphery retina. Proteasome was the most significant Kyoto Encyclopedia of Genes and Genomes pathway in downregulated DEPs in the central retina. Antithrombin-III, fibrinogen gamma chain, and fibrinogen beta chain were identified as hub proteins for myopia progression, which were upregulated in the far-periphery retina. Proteomic analysis in this study suggested that oxidative stress can be the primary pathogenesis of myopia progression and that the far-periphery retina plays a role as the key responder.

Integrated Analysis of Transcriptome and Proteome of the Human Cornea and Aqueous Humor Reveal Novel Biomarkers for Corneal Endothelial Cell Dysfunction.

Earlier studies have reported that elevated protein levels in the aqueous humor (AH) are associated with corneal endothelial cell dysfunction (CECD), but the details of the underlying mechanism as well as specific biomarkers for CECD remain elusive. In the present study, we aimed to identify protein markers in AH directly associated with changes to corneal endothelial cells (CECs), as AH can be easily obtained for analysis. We carried out an in-depth proteomic analysis of patient-derived AH as well as transcriptomic analysis of CECs from the same patients with bullous keratopathy (BK) resulting from CECD. We first determined differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) from CECs and AH in CECD, respectively. By combining transcriptomic and proteomic analyses, 13 shared upregulated markers and 22 shared downregulated markers were observed between DEGs and DEPs. Among these 35 candidates from biomarker profiling, three upregulated markers were finally verified via data-independent acquisition (DIA) proteomic analysis using additional individual AH samples, namely metallopeptidase inhibitor 1 (TIMP1), Fc fragment of IgG binding protein (FCGBP), and angiopoietin-related protein 7 (ANGPTL7). Furthermore, we confirmed these AH biomarkers for CECD using individual immunoassay validation. Conclusively, our findings may provide valuable insights into the disease process and identify biofluid markers for the assessment of CEC function during BK development.

The Protective Role of Exosome-Derived MicroRNAs and Proteins from Human Breast Milk against Infectious Agents.

Human breast milk (HBM)-derived exosomes contain various biological and immunological components. However, comprehensive immune-related and antimicrobial factor analysis requires transcriptomic, proteomic, and multiple databases for functional analyses, and has yet to be conducted. Therefore, we isolated and confirmed HBM-derived exosomes by detecting specific markers and examining their morphology using western blot and transmission electron microscopy. Moreover, we implemented small RNA sequencing and liquid chromatography-mass spectrometry to investigate substances within the HBM-derived exosomes and their roles in combating pathogenic effects, identifying 208 miRNAs and 377 proteins associated with immunological pathways and diseases. Integrated omics analyses identified a connection between the exosomal substances and microbial infections. In addition, gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that HBM-derived exosomal miRNA and proteins influence immune-related functions and pathogenic infections. Finally, protein-protein interaction analysis identified three primary proteins (ICAM1, TLR2, and FN1) associated with microbial infections mediating pro-inflammation, controlling infection, and facilitating microbial elimination. Our findings determine that HBM-derived exosomes modulate the immune system and could offer therapeutic strategies for regulating pathogenic microbial infection.

Tissue extracellular matrix hydrogels as alternatives to Matrigel for culturing gastrointestinal organoids.

Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration.

Proteome alterations in the aqueous humor reflect structural and functional phenotypes in patients with advanced normal-tension glaucoma.

Previous reports have shown possible association between altered protein levels in aqueous humor (AH) and normal-tension glaucoma (NTG), but the underlying pathogenetic mechanism as well as specific molecular biomarkers for NTG remains still elusive. Here, we aimed to identify novel biomarkers for advanced NTG by analyzing the proteome of patient-derived AH and their correlation with various functional and structural parameters from the visual field test (VF), optical coherence tomography (OCT), and OCT angiography (OCTA). We determined differentially expressed proteins (DEPs) of the AH of patients with advanced NTG (n = 20) using label-free quantitative (LFQ) proteomics with pooled samples and data-independent acquisition (DIA) analysis with individual samples, and the roles of AH DEPs in biological pathways were evaluated using bioinformatics. We identified 603 proteins in the AH of patients with advanced NTG, and 61 of them were selected as DEPs via global proteome LFQ profiling. Individual DIA analyses identified a total of 12 DEPs as biomarker candidates, seven of which were upregulated, and five were downregulated. Gene ontology enrichment analysis revealed that those DEPs were mainly involved in the immune response. Moreover, IGFBP2, ENO1, C7, B2M, AMBP, DSP, and DCD showed a significant correlation with the mean deviation of VF and with peripapillary and macular parameters from OCT and OCTA. The present study provides possible molecular biomarkers for the diagnosis of advanced NTG.

The Dopaminergic Neuronal System Regulates the Inflammatory Status of Mouse Lacrimal Glands in Dry Eye Disease.

Purpose: Comparison of the parasympathetic and sympathetic neurons, including the dopaminergic neural system, in dry eye (DE)-induced pathophysiology has not been elucidated well. This study investigated the presence of dopamine receptors (DRs) and their functional roles in the lacrimal glands (LGs) of DE-induced mice. Methods: After DE was induced in B6 mice for 2 weeks, the expression of tyrosine hydroxylase (TH), dopamine, and DRs (DR1, DR2, etc.) in the LGs and corneas were measured by quantitative RT-PCR, immunoblot, and ELISA. Using flow cytometry and ELISA, immune cell infiltration and inflammatory cytokine expression were determined in DE-induced LGs with or without DR blockers, SCH-23390 (DR1i), or melperone (DR2i). Corneal erosion scores were also investigated. Results: The mRNA and protein levels of TH significantly increased in DE-induced LGs. The dopamine concentration of LGs was 9.51 pmol in DE (versus naive: 1.39 pmol; P < 0.001). Both DR1 and DR2 mRNA expression were significantly enhanced in desiccating stress compared with those in naive (3.7- and 2.1-fold, P < 0.001). Interestingly, DR1 and DR2 immunostaining patterns stained independently in DE-induced LGs. CD3+ and CD19+ cell infiltration was significantly increased by DR2i (P < 0.001) but not by DR1i. Furthermore, IFN-γ, IL-17, and TNF-α were significantly upregulated by DR2i compared with the blow-only condition. The severity of corneal erosion and inflammation was also aggravated by DR2i. Conclusions: Upregulation of DR1 and DR2 was observed in DE-induced mouse LGs. As the inflammatory conditions are aggravated by the inhibition of DRs, especially DR2, their activity may be an important factor preserving ocular surface homeostasis.

A loss of antitumor therapeutic activity of CEA DNA vaccines is associated with the lack of tumor cells' antigen presentation to Ag-specific CTLs in a colon cancer model.

Human colon cancers express carcinoembryonic antigen (CEA). Thus, CEA has been considered as a potential vaccine target for immune therapy against colon cancer. In this study, CEA DNA vaccines plus anti-4-1BB Abs treatment was found to increase Ag-specific CTL activity and antitumor protective responses to MC32 cells. However, CEA DNA vaccines alone displayed few antitumor therapeutic effects while significantly inducing Ag-specific CTL responses. Anti-4-1BB Abs alone displayed antitumor therapeutic effects. Intratumoral electroporation with IL-12 cDNA also showed antitumor therapeutic activity against MC32 cells in a CD8+ T cell-dependent and CEA-non-specific manner, suggesting that established MC32 cells are still susceptible to CTL-mediated killing. Finally, our in vitro assays (Western blot assay, IFN-γ, CTL and apoptosis assays, FACS analysis) and animal studies demonstrated that a lack of antitumor therapeutic activity of CEA DNA vaccines might result from acquisition of tumor cell resistance to Ag-specific CTL-mediated killing through the loss of tumor cells' antigen presentation to Ag-specific CTLs. Taken together, these data show that MC32 cells may resist CEA DNA vaccination by their loss of antigen presentation to CEA-specific CTLs in the therapeutic model.

DNA vaccines targeting human papillomavirus-associated diseases: progresses in animal and clinical studies.

Human papillomavirus (HPV) infection is a major cause of cervical cancer and its precancerous diseases. Cervical cancer is the second deadliest cancer killer among women worldwide. Moreover, HPV is also known to be a causative agent of oral, pharyngeal, anal and genital cancer. Recent application of HPV structural protein (L1)-targeted prophylactic vaccines (Gardasil® and Cervarix®) is expected to reduce the incidence of HPV infection and cervical cancer, and possibly other HPV-associated cancers. However, the benefit of the prophylactic vaccines for treating HPV-infected patients is unlikely, underscoring the importance of developing therapeutic vaccines against HPV infection. In this regard, numerous types of therapeutic vaccine approaches targeting the HPV regulatory proteins, E6 and E7, have been tested for their efficacy in animals and clinically. In this communication, we review HPV vaccine types, in particular DNA vaccines, their designs and delivery by electroporation and their immunologic and antitumor efficacy in animals and humans, along with the basics of HPV and its pathogenesis.