Stem cell therapies hold great promise as alternative treatments for incurable optic nerve disorders. Although mesenchymal stem cells exhibit various tissue regeneration and recovery capabilities that may serve as valuable therapies, the clinical applications remain limited. Thus, we investigated the utility of extracellular vesicles (EVs) from human placenta-derived mesenchymal stem cells (hPSCs) in this context. Hypoxically preconditioned hPSCs (HPPSCs) were prepared via short-term incubation under 2.2% O₂ and 5.5% CO₂. The EVs were then isolated. R28 cells (retinal precursor cells) were exposed to CoCl₂ and treated with EVs for 24 h. Cell proliferation and regeneration were measured using a BrdU assay and immunoblotting; ATP quantification revealed the extent of the mitochondrial function. The proteome was determined via liquid chromatography-tandem mass spectroscopy. Differentially expressed proteins (DEPs) were detected and their interactions identified. HPPSC_EVs functions were explored using animal models of optic nerve compression. HPPSC_EVs restored cell proliferation and mitochondrial quality control in R28 cells damaged by CoCl₂. We identified DEPs (p < 0.05) that aided recovery. The mitochondrial DEPs included LONP1; PARK7; VDAC1, 2, and 3; HSPD1; and HSPA9. EVs regulated the levels of mitophagic proteins in R28 cells injured by hypoxia; the protein levels did not increase in LONP1 knockdown cells. LONP1 is a key mediator of the mitophagy that restores mitochondrial function after hypoxia-induced optic nerve injury.

Various substances within the aqueous humor (AH) can directly or indirectly impact intraocular tissues associated with intraocular pressure (IOP), a critical factor in glaucoma development. This study aims to investigate individual changes in these AH substances and the interactions among altered components through a multi‐omics approach. LC/MS analysis was conducted on AH samples from patients with exfoliation syndrome (XFS, n = 5), exfoliation glaucoma (XFG, n = 4), primary open‐angle glaucoma (POAG, n = 11), and cataracts (control group, n = 7). Subsequently, differentially expressed proteins and metabolites among groups, alterations in their network interactions, and their biological functions were examined. Both data‐independent acquisition and data‐dependent acquisition methods were employed to analyze the AH proteome and metabolome, and the results were integrated for a comprehensive analysis. In the proteomics analysis, proteins upregulated in both the XFG and POAG groups were associated with lipid metabolism, complement activation, and extracellular matrix regulation. Metabolomic analysis highlighted significant changes in amino acids related to antioxidant processes in the glaucoma groups. Notably, VTN, APOA1, C6, and L‐phenylalanine exhibited significant alterations in the glaucoma groups. Integration of individual omics analyses demonstrated that substances associated with inflammation and lipid metabolism, altered in the glaucoma groups, showed robust interactions within a complex network involving PLG, APOA1, and L‐phenylalanine or C3, APOD, and L‐valine. These findings offer valuable insights into the molecular mechanisms governing IOP regulation and may contribute to the development of new biomarkers for managing glaucoma.

Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

Post-mortem interval (PMI) estimation is a critical task in forensic science. In this study, we used maggots collected from pig carcasses and applied an integrated proteomics and metabolomics approach to determine potential candidate substances for the estimation of PMI. After methanol precipitation, the supernatant containing metabolites and the protein pellet were separated and subjected to metabolomic and proteomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS/MS data were analyzed for identification and quantification using Proteome Discoverer and Compound Discoverer software. A total of 573 metabolites and more than 800 porcine proteins were identified in maggots. This is the first dataset of proteins and metabolites in maggots collected from porcine carcasses. In this study, guanosine monophosphate, xanthine, inosine, adenosine, and guanine were detected with a similar tendency to increase during early days of maggot development and then decreased gradually. We broadly profiled various biomolecules through analysis in the spot of incident. Especially, we confirmed that proteome and metabolome profiling could be performed directly and indirectly.

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increases in ischemia and expression of genes linked to neuronal regeneration. Post NPC injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, the proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved the mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/β-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/β-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Emiliania huxleyi is one of the most abundant marine planktons, and it has a crucial feature in the carbon cycle. However, proteomic analyses of Emiliania huxleyi have not been done extensively. In this study, a three-dimensional liquid chromatography (3D-LC) system consisting of strong cation exchange, high- and low-pH reversed-phase liquid chromatography was established for in-depth proteomic profiling of Emiliania huxleyi. From tryptic proteome digest, 70 fractions were generated and analyzed using liquid chromatography-tandem mass spectrometry. In total, more than 84,000 unique peptides and 10,000 proteins groups were identified with a false discovery rate of ≤0.01. The physicochemical properties of the identified peptides were evaluated. Using ClueGO, approximately 700 gene ontology terms and 15 pathways were defined from the identified protein groups with p-value ≤0.05, covering a wide range of biological processes, cellular components, and molecular functions. Many biological processes associated with CO₂ fixation, photosynthesis, biosynthesis, and metabolic process were identified. Various molecular functions relating to protein binding and enzyme activities were also found. The 3D-LC strategy is a powerful approach for comparative proteomic studies on Emiliania huxleyi to reveal changes in its protein level and related mechanism.