Biomarker Development and Application, 2nd Edition

Background: Peritoneal metastasis (PM) is a lethal and often occult event in advanced gastric cancer (GC), and its preoperative detection remains difficult. The role of peripheral NK and NKT−like cells in predicting PM has not been well defined. Methods: We retrospectively analyzed 433 patients who underwent surgery for GC from 2016 to 2020. Patients were stratified by PM status and randomly assigned to training and held−out internal validation cohorts. Flow cytometry−derived lymphocyte subset percentages were treated as compositional data, and NK and NKT−like cell variables were entered into the model after log−ratio transformation. Candidate variables were selected using LASSO logistic regression in the training cohort and further assessed by multivariable logistic regression. Model performance was evaluated using AUROC, AUPRC, and confusion matrices in the validation cohort. Nomogram and SHAP analyses were used for model interpretation. Results: Seven predictors were retained in the final model: log−ratio−transformed NKT−like cells, direct bilirubin, prealbumin, lymphocyte count, lactate dehydrogenase, CA125, and log−ratio−transformed NK cells. The model achieved an AUROC of 0.879 (95% CI: 0.805–0.936) and an AUPRC of 0.698 (95% CI: 0.524–0.848) in the validation cohort. At the conventional threshold of 0.50, the model achieved an accuracy of 0.915, sensitivity of 0.821, specificity of 0.941, PPV of 0.793, and NPV of 0.950. At the Youden−optimal threshold of 0.101, sensitivity, specificity, accuracy, PPV, and NPV were 1.000, 0.892, 0.915, 0.718, and 1.000, respectively. Conclusions: A preoperative model integrating log−ratio−transformed NK/NKT−like cell variables and routine hematological parameters showed good ability to identify PM in GC and may help select patients for diagnostic laparoscopy or closer preoperative evaluation. Full article

Background: Although advances in critical care have improved short-term outcomes, sepsis survivors continue to face substantial chronic morbidity and impaired long-term survival. Conventional threshold-based tools such as the Sequential Organ Failure Assessment (SOFA) and Modified Early Warning Score (MEWS) show moderate and variable discrimination across cohorts. Reported areas under the receiver operating characteristic curve (AUROCs) must therefore be interpreted in relation to the population, prediction horizon, and outcome used in each study rather than as direct head-to-head comparisons. Objectives: This review evaluates how artificial intelligence (AI) could be linked with dynamic plasma metabolites, particularly citrulline and β-hydroxybutyrate (3-HB), to support biologically informed sepsis phenotyping, while critically examining mechanistic evidence, clinical limitations, and translational readiness. Data Synthesis: Machine-learning and natural language processing architectures have shown promising discrimination in many early-detection studies, with pooled AUROCs near 0.87 and reported prediction windows extending to 48 h. However, performance estimates vary with cohort composition, outcome definition, and validation design, and they should not be ranked against unrelated biomarker studies. Human sepsis studies generally associate low or persistently low citrulline with impaired intestinal function and organ injury, but no sepsis-specific decision cutoff has been externally validated. For 3-HB, an AUROC of 0.8429 for septic liver injury was derived from a cohort of 57 patients and has not been shown to add value beyond routine liver tests or illness-severity measures. Murine experiments provide mechanistic hypotheses for ketone-mediated organ protection, but model-specific and sometimes opposing nutritional effects limit direct translation. These metabolites are therefore best considered candidate longitudinal features for multimodal phenotyping rather than stand-alone clinical triggers. Conclusions: Biologically informed algorithmic surveillance is a promising direction, but clinical implementation requires prospective serial sampling, explicit adjustment for renal, hepatic and nutritional confounders, head-to-head comparison with routine markers, and external validation of calibration and clinical utility. Until these requirements are met, citrulline and 3-HB should support research phenotyping rather than direct treatment selection. Full article

CircRNAs are covalently closed ncRNAs originating through back splicing; their expression is finely regulated, displaying specific patterns across different cell types, tissues, and developmental stages. While the molecular functions of circRNAs are not completely elucidated, their regulatory involvement in physiological processes is well established, alongside their dysregulation in several human disorders. These features, together with their higher stability compared to other ncRNAs, make this class of molecules promising theragnostic agents, particularly in biomarker discovery. Accordingly, it is crucial to develop and standardize experimental strategies that improve circRNA analysis, ensuring accurate and effective isolation of these molecules. In biomarker discovery, selecting the appropriate biological matrix is critical; whole blood is often preferred for its accessibility and minimally invasive collection. Because circRNAs are present in human peripheral blood and show promise as disease theragnostic biomarkers, we established a preliminary workflow tailored to isolate and analyze circRNAs from whole blood. The promising effectiveness and robustness of this workflow were demonstrated by qPCR analysis, suggesting highly reproducible detection and reliability in isolating and analyzing circRNAs. This, together with their stability and specific expression profiles, supports the utility of circRNAs in biomarker discovery and advanced circRNA research and contributes to accelerating their future integration into theragnostic applications in clinical settings. Full article

Background: Head and neck squamous cell carcinoma (HNSCC) remains a major global health challenge due to the lack of effective and non-invasive diagnostic tools, often resulting in late-stage detection of cancer. Small extracellular vesicles (sEVs) have emerged as promising biomarkers for early cancer detection and disease monitoring due to their omnipresence and stability in bodily fluids, such as blood plasma. In addition, cancer-derived sEVs specifically carry cargo reflective of oncogene-derived molecular alterations. In summary, these characteristics position sEVs as a potential platform for non-invasive testing of HNSCC. Methods: Plasma-derived sEVs from HNSCC patients (n = 71) and benign subjects (n = 25) were isolated using size exclusion chromatography. Proteomic profiling via liquid chromatography-tandem mass spectrometry identified potential candidate biomarkers, followed by ELISA validation. Results: Proteomic analyses revealed a significant enrichment of multiple proteins in HNSCC-derived sEVs compared to sEVs derived from non-cancer individuals. The von Willebrand factor (vWF) was significantly higher in HNSCC patient-derived sEVs compared to those derived from benign individuals. A validation cohort confirmed that sEV-associated vWF (sEV-vWF) effectively distinguished HNSCC patients from benign subjects, demonstrating strong diagnostic performance, specifically in laryngeal HNSCC (AUC = 0.82) and oropharyngeal HNSCC (AUC = 0.96) patient cohorts. Moreover, postoperative reductions and recurrence-associated increases in sEV-vWF levels corresponded with clinical outcomes, indicating its potential as a dynamic disease indicator. Conclusions: These findings highlight sEV-vWF as a novel and non-invasive biomarker with potential applications in early detection and real-time monitoring of HNSCC, supporting advancement toward precision liquid biopsy strategies in head and neck oncology. Full article

Liver fibrosis is a chronic pathological process driven by the activation of hepatic stellate cell (HSC) and characterized by the excessive deposition of extracellular matrix (ECM) components in response to persistent liver injury. This condition can lead to progressive hepatic dysfunction, cirrhosis, and ultimately liver failure. Growth differentiation factor 15 (GDF15) has emerged as a pivotal regulator in the initiation and progression of liver fibrosis, exhibiting context-dependent profibrotic and antifibrotic effects. GDF15 modulates multiple cellular processes, including HSC activation and macrophage polarization, as well as the functions of T cells, natural killer cells, B cells and mesenchymal stem cells. This review provides a comprehensive overview of the role of GDF15 in regulating HSC activation and immune cell responses and elaborates on its immunomodulatory functions in attenuating liver fibrosis. Furthermore, we discuss the therapeutic potential of targeting GDF15 for the treatment of liver fibrosis. Ultimately, this review aims to provide a theoretical foundation and propose novel intervention strategies for the early diagnosis and targeted therapy of liver fibrosis. Full article

Polyphenols are natural compounds with antioxidant properties that help prevent chronic diseases. Red onion (Allium cepa) peels are an underutilized source of polyphenols, offering a sustainable opportunity for the valorization of agricultural by-products. Polyphenol-rich extracts were obtained from red onion peels using subcritical water extraction and adsorption resin chromatography. Their biological effects were evaluated in Caenorhabditis elegans under oxidative stress conditions. A multi-omics approach integrating transcriptomics, metabolomics, and lipidomics was applied to assess molecular responses. The extract exhibited a high total phenolic content (>400 mg GAE/g) and strong antioxidant capacity, supporting a highly enriched polyphenolic profile. Survival assays confirmed the absence of toxicity at 100 µg/mL GAE. Transcriptomic analysis revealed 158 differentially expressed genes associated with stress response and metabolic regulation. Metabolomic profiling indicated a systematic reduction in amino acid levels alongside an increase in betaine in treated worms, suggesting adaptive metabolic reprogramming. Lipidomic analysis revealed significant lipid remodeling, characterized by decreased triglycerides, increased diacylglycerols, and changes in membrane phospholipids, indicating alterations in membrane composition and cellular responses associated with oxidative stress adaptation. Together, these results support a model in which red onion peel extracts induce coordinated transcriptional and metabolic adaptations associated with cellular resilience and stress adaptation. This study highlights the potential of agro-industrial by-products as functional bioactive ingredients and demonstrates the value of multi-omics approaches for uncovering system-level responses. Full article

The anion exchanger 1 (AE1), traditionally known for its role in erythrocyte anion transport and acid–base homeostasis, has recently been identified in non-erythroid tissues, suggesting broader physiological functions. In the present study, we investigated for the first time the expression and potential role of AE1 in human ovarian granulosa cells (GCs) obtained from women with endometriosis (ENDO-GCs) or male factor infertility controls (MF-GCs). Cells were cultured in the presence of follicular fluid derived from control (FF-MF) or endometriosis patients (FF-ENDO), and DIDS-sensitive anion exchange activity was pharmacologically inhibited using 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). AE1 expression was evaluated at both protein and transcript levels together with markers of proliferation, inflammation, and steroidogenesis. The results demonstrate that AE1 is constitutively expressed in GCs and may contribute to granulosa cell homeostasis. Inhibition of DIDS-sensitive anion exchange activity inhibited cell proliferation, shifted cell morphology toward a fibroblast-like phenotype, and reduced estradiol and progesterone secretion and inflammatory (IL-6) gene transcription. Notably, MF-GCs cultured in FF-MF exhibited compensatory upregulation of AE1, whereas ENDO-GCs and cells exposed to FF-ENDO showed impaired adaptive responses and generalized transcriptional suppression. These findings provide preliminary evidence supporting a role for DIDS-sensitive anion exchange activity in granulosa cell physiology and warrant further studies to clarify the specific contribution of AE1 to follicular dysfunction associated with endometriosis. However, the specific mechanistic contribution of AE1 could not be established and will require further functional and genetic investigations. Full article

Background: Young stroke (age ≤ 45) is a growing global health concern often driven by metabolic factors, with insulin resistance (IR) and dyslipidemia being key contributors. Novel metabolic indices, the Cholesterol–High-Density Lipoprotein–Glucose (CHG) index and the Remnant Cholesterol Inflammatory Index (RCII), have emerged as promising tools, yet their prognostic value in young stroke populations remains unexplored. Methods: We retrospectively analyzed 541 young stroke patients (age ≤ 45 years) between January 2019 and December 2021. The associations between CHG and RCII indices and poor functional outcomes at 90 days and discharge, infarct size, and hemorrhagic transformation were evaluated using multivariate logistic regression, restricted cubic spline (RCS) analysis, and receiver operating characteristic (ROC) curves. Subgroup, interaction, and sensitivity analyses were performed to assess the stability and robustness of the findings. Results: A total of 541 young stroke patients were included (median age 37 years). Patients with poor 90-day functional outcome (modified Rankin Scale ≥ 2) exhibited significantly higher CHG (5.2 vs. 5.1, p = 0.018) and RCII (2.8 vs. 1.7, p < 0.001) levels. After multivariable adjustment, both indices were independently associated with poor 90-day outcome (CHG: OR 1.31 per SD, 95% CI 1.07–1.61; RCII: OR 1.23 per SD, 95% CI 1.04–1.46) and poor discharge outcome. The highest RCII quartile exhibited more than threefold increased odds of a poor 90-day outcome (OR 3.43, 95% CI 1.90–6.20). Both indices were associated with larger lesion size, and RCII was additionally associated with hemorrhagic transformation (Q4 vs. Q1: OR 2.42, 95% CI 1.23–4.75). Restricted cubic spline analyses demonstrated no evidence of non-linearity. Adding RCII to a base clinical model significantly improved discrimination (AUC 0.645 vs. 0.564, DeLong p = 0.004). No significant sex interaction was observed; RCII showed significant interactions with age and TOAST subtype. Conclusions: Both CHG and RCII indices are independently associated with poor functional outcomes, larger infarct size, and—for RCII—hemorrhagic transformation in young patients with acute ischemic stroke. These readily calculable biomarkers may provide complementary prognostic information in this population, though their clinical utility requires further prospective validation. Full article

Chicken coccidiosis, a severe intestinal parasitic disease caused by Eimeria protozoa, causes substantial annual economic losses to the global poultry industry. This study focused on investigating the role of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) in modulating chicken innate immune responses against Eimeria tenella (E. tenella) infections. Here, we show that TRAF6 is sensitive to the process of E. tenella infection, and cecal tissue responds to the early infection of E. tenella by up-regulating the expression of TRAF6. Specifically, TRAF6 overexpression enhances E. tenella-induced activation of the NF-κB pathway (a core innate immune signaling cascade), thereby promoting host inflammatory cytokines production and cell apoptosis, while TRAF6 knockdown mitigates these pathological effects. Mechanistically, TRAF6-mediated regulation of NF-κB pathway activation and inflammatory responses during E. tenella infection can be specifically targeted by key microRNAs (miRNA), gga-miR-7b, in chickens. Taken together, this study identifies that TRAF6 plays an important regulatory role in innate immune response against E. tenella infection, providing novel insights into host–parasite interactions and potential targets for coccidiosis control. Full article

Background: Chronic viral hepatitis is a major global health problem associated with progressive liver injury and an increased risk of cirrhosis and hepatocellular carcinoma. The identification of novel biomarkers may improve disease monitoring and diagnostic accuracy. Methods: In this prospective case–control study, a total of 90 participants were included: 20 patients with chronic hepatitis B (CHB); 20 with chronic hepatitis C (CHC); 20 with HBeAg-negative chronic infection (HCI); and 30 age-, sex-, and body mass index-matched healthy controls. Serum irisin and nesfatin-1 levels were measured using enzyme-linked immunosorbent assays (ELISAs). Group comparisons were performed using multivariate analysis of variance (MANOVA) followed by Scheffé post hoc tests. Receiver operating characteristic (ROC) curve analysis was used to evaluate diagnostic performance. Results: Significant differences were observed among groups in terms of irisin, nesfatin-1, total bilirubin, and platelet counts (p ≤ 0.05). Nesfatin-1 levels were significantly higher in all patient groups compared with healthy controls (p < 0.001). Irisin levels were only significantly lower in the HCI group (p < 0.001). ROC analysis indicated that nesfatin-1 may have the potential to discriminate between infected patients and healthy individuals; however, the generalizability of this finding is limited by the study design and sample size. Conclusions: Nesfatin-1 may represent a potential biomarker for chronic viral hepatitis, whereas alterations in irisin levels may be more specific to the inactive carrier phase. Full article