Pao-Chi Liao

Background: Chitinase-3-like-protein 1 (CHI3L1), a low-abundance protein in normal tissues, is markedly upregulated in the pancreatic tumor microenvironment (TME), where it promotes fibrosis and immune evasion. Spatial transcriptomics revealed enrichment of CHI3L1 at tumor-stroma interfaces, suggesting that cell-cell interactions regulate CHI3L1 expression. Methods: We employed 2D and 3D multicellular culture models, incorporating pancreatic cancer cells, macrophages, and pancreatic stellate cells (PSCs), to recapitulate TME-mediated CHI3L1 induction. Mechanistic insights were gained through secretome profiling, chromatin immunoprecipitation (ChIP), and methylation-specific PCR. The therapeutic potential of targeting identified axes was evaluated in an orthotopic pancreatic adenocarcinoma (PDAC) mouse model. Results: We identified CCL2, CXCL10, and SPARC as key secreted mediators during tumor-stroma crosstalk that cooperatively induced CHI3L1 expression in cancer cells via activation of NF-κB and STAT3. Specifically, inflammatory cytokines within the TME recruited macrophages to induce CCL2, which, in concert with PSCs, enhances CXCL10 and SPARC secretion. Mechanistically, NF-κB directly bound the CHI3L1 promoter, while STAT3 reduced DNA methyltransferase 1 levels, leading to promoter hypomethylation and epigenetic activation of CHI3L1. Clinically, high CHI3L1 expression correlated with the upregulated stromal CCL2, CXCL10, and SPARC levels, and the combined four-marker signature was an independent prognostic factor for both overall and disease-free survival in multivariable analysis. In vivo, blockade of the upstream stromal pathways (CCR2/CXCR3/TGFβR1) coupled with CHI3L1 neutralization reduced tumor growth and remodeled the immunosuppressive microenvironment in immunocompetent PDAC models. Conclusions: Our findings uncover a cooperative multicellular signaling network that orchestrates CHI3L1 expression in cancer via transcriptional and epigenetic reprogramming. Disrupting these upstream stromal pathways concurrently with CHI3L1 neutralization was associated with reduced T-cell exhaustion, offering a promising combinatorial approach for PDAC immunotherapy.

Carbon monoxide poisoning (COP) induces systemic hypoxia and oxidative stress-related injury, leading to myocardial injury and persistent cardiac dysfunction. However, reliable biomarkers for monitoring long-term cardiac sequelae and therapeutic responses remain lacking. Extracellular vesicles (EVs), which reflect the molecular status of their cells of origin, may serve as candidate biomarkers for organ-specific injury. This study investigated whether cardiac EV proteins capture COP-induced myocardial and mitochondrial dysfunction and reflect the therapeutic effects of hyperbaric oxygen (HBO) therapy. A rat model of COP was established with or without HBO treatment. Cardiac function was assessed by echocardiography, and myocardial injury was evaluated using histological, ultrastructural, and biochemical analyses. Cardiac-enriched EVs isolated from ex vivo whole-heart perfusate were used for global proteomic profiling. Candidate differentially abundant proteins were analyzed with emphasis on pathways related to mitochondrial dynamics, mitochondrial energy metabolism, calcium handling, and myocardial contractility. Key EV-associated and tissue proteins were further validated, and selected candidates were examined in serum-derived EVs as preliminary targeted circulating EV validation. COP induced significant cardiac dysfunction, as evidenced by reduced ejection fraction and fractional shortening, together with histological myocardial injury, all of which were attenuated by HBO treatment. Proteomic analysis demonstrated that COP reshaped the cardiac EV proteome in a manner consistent with mitochondrial abnormalities, altered calcium-handling protein profiles, and impaired myocardial contractile function. These EV proteomic alterations were enriched in pathways related to mitochondrial dynamics, calcium signaling, and cardiac contractile regulation. Specifically, COP was associated with dysregulation of mitochondrial dynamics regulators, including optic atrophy type 1 (Opa1) and mitochondrial fission protein 1 (FIS1), as well as calcium-handling proteins such as ryanodine receptor 2 (Ryr2) and phospholamban (Pln). Ultrastructural and biochemical analyses showed mitochondrial cristae disruption, altered mitochondrial fusion-fission protein profiles, mitophagy-related protein changes, and pyroptosis-associated signaling in cardiac tissue following COP, whereas HBO mitigated these abnormalities. Notably, EV-associated Opa1 and FIS1 were associated with COP-related alterations in mitochondrial dynamic balance, whereas EV-associated Ryr2 and Pln were associated with impaired myocardial contractile parameters. Additional analysis of EV proteins related to mitochondrial function and ATP energy production further supported COP-associated mitochondrial energy metabolism-related protein remodeling. Targeted analysis of serum-derived EVs further showed that selected calcium-handling proteins, including Ryr2 and Pln, were detectable in circulating EVs and exhibited COP-associated changes consistent with cardiac tissue alterations. These findings support selected cardiac-enriched EV proteins as candidate molecular readouts of COP-associated myocardial, mitochondrial, and contractile abnormalities. Cardiac EV proteomic remodeling reflects COP-associated mitochondrial and contractile abnormalities and captures the therapeutic effects of HBO. These findings identify cardiac-enriched EV proteins as candidate molecular readouts of myocardial injury and treatment response, providing a cardiac-enriched EV discovery framework for future blood-based biomarker development. The observed alterations in mitochondrial dynamics-, mitochondrial energy metabolism-, and calcium-handling-related proteins provide hypothesis-generating insight into molecular pathways associated with COP-induced cardiac dysfunction. Further validation using circulating EV proteomics, biomarker classifier analyses, and dedicated redox proteomics will be required to establish clinical utility and redox-regulated mechanistic relevance.

Glutathione conjugation is a key metabolic detoxification pathway, and its downstream products, mercapturic acid conjugates (MACs), may serve as urinary biomarkers of toxicant exposure. However, MAC profiling is hindered by incomplete filtering coverage and the lack of comprehensive spectral and structural databases. To address these limitations, we developed an analytical workflow integrating novel enzymatic deacetylation with ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS)-based neutral loss filtering. The aminoacylase-1-mediated deacetylation provides an orthogonal approach to detect MACs that lack the characteristic neutral loss of 129.0426 Da. Additionally, an in-house MAC library of 734,170 putative structures, combined with an in silico structure annotation tool, facilitated efficient MAC identification. The strategy was validated using 11 MAC standards spiked into urine and applied to urine samples from 15 participants before and after consuming deep-fried foods. A total of 847 features were mapped to at least one MAC structure, exceeding the ∼100 reported in previous studies, with 581 absent from the Human Metabolome Database (HMDB) or PubChem. Postconsumption, 59 MACs increased and 11 decreased, reflecting exposure-related shifts in reactive aldehydes, lipid oxidation products, acylcarnitines, and acyl-CoA intermediates. These findings demonstrate the utility of the workflow for capturing urinary MAC changes associated with deep-fried food consumption and for prioritizing candidate exposure indicators of reactive chemical species for future validation studies.

Per- and polyfluoroalkyl substances (PFAS) represent a critical class of persistent environmental contaminants with significant ecological and human health implications. However, the rapid emergence of novel PFAS has far outpaced the development of reference mass spectral databases. Here, Neural Per- and Polyfluoroalkyl Substances Mass Spectrometry (NPFAS-MS), a transfer learning-based neural network model, was developed to predict PFAS-specific high-resolution mass spectra. NPFAS-MS was fine-tuned from a pretrained model using PFAS tandem mass (MS/MS) spectra. NPFAS-MS outperformed other in silico spectral prediction models for PFAS spectra prediction across multiple spectral similarity metrics. In library searching tasks, libraries generated by other spectral prediction models showed top-1 recall between 42.1% and 55.4%, while NPFAS-MS demonstrated 71.1%. Applying the virtual PFAS mass spectral library generated with NPFAS-MS using 10,553 PFAS structures from the U.S. EPA and NORMAN databases to groundwater and aqueous film-forming foam (AFFF) samples revealed more potential PFAS than other mass spectral databases. Specifically, 38 potential PFAS were annotated in AFFF products and 40 in groundwater samples. NPFAS-MS enabled characterization of emerging PFAS, including ultrashort-chain, unsaturated, and substituted derivatives in environmental matrices. This advancement enables comprehensive environmental monitoring of rapidly evolving PFAS contamination. NPFAS-MS and associated resources were deployed as a web-based tool at https://cosbi10.ee.ncku.edu.tw/NPFAS_MS/, enabling both structure-to-spectrum prediction and library searching against 31,659 predicted PFAS spectra.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are widely utilized in food contact materials (FCMs) due to their water- and oil-repellent properties, yet their potential migration into food raises significant health concerns. This study employs high-resolution mass spectrometry (HRMS) to quantify the migration of 40 PFAS from microwave popcorn bags and assess the associated health risks. HRMS offers high mass accuracy and resolution, enabling precise detection of a broad spectrum of PFASs, including those with low migration levels. Migration experiments were conducted using 10% ethanol and 50% ethanol as food simulants at 70 °C for 2 h. The results indicate that when risk assessment is based solely on the European Food Safety Authority’s (EFSA) tolerable weekly intake (TWI) for four PFAS, hazard ratio (HR) values range from 0.01 to 0.8, suggesting minimal risk. However, when all PFAS are converted into perfluorooctanoic acid equivalents (PEQs) and compared against the U.S. Environmental Protection Agency’s (EPA’s) reference dose (RfD), HR values range from 0.3 to 142.3, indicating a significantly elevated health risk. These findings emphasize the necessity of comprehensive risk assessments incorporating the cumulative effects of all PFAS to better understand potential human exposure and inform regulatory policies.