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PdIr bimetallic nanozyme engineered metal-organic frameworks integrated dual-mode sensor toward Stx2 detection in food.

Shiga toxin II (Stx2) has attracted extensive attention due to its toxicity and pathogenicity, making the development of sensitive detection methods urgent. This study constructed a dual-mode sensing platform for the sensitive detection of Stx2 in food. Composite material UIO-66@PdIr with peroxidase-like activity and fluorescent properties was synthesized and combined with cDNA as the signal probe, while aptamer-modified magnetic beads served as the capture probe. Specific binding of Stx2 to the aptamer triggered the release of the signal probe, enabling colorimetric and fluorescence signal readout. The colorimetric mode showed a linear range of 0.05-100 ng/mL with an LOD of 0.039 ng/mL, and the fluorescence mode exhibited 0.01-1000 ng/mL with an LOD of 0.0097 ng/mL. Additionally, this method was successfully applied to the detection of Stx2 in food, and the recovery rates were 94.33% ∼ 102.20%. It indicated that the constructed sensor holds great practical potential for Stx2 detection.

Food Contamination

MET-Aberrant non-small cell lung cancer: from kinase dependence to cell-surface targetability-mechanistic basis and biomarker framework for bispecific antibodies and antibody-drug conjugates.

MET-aberrant non-small cell lung cancer (NSCLC) is not a uniform therapeutic entity. Its biology, diagnostic pathways, and treatment sensitivity differ across MET exon 14 skipping alteration (METex14), MET amplification, and MET overexpression. This heterogeneity cannot be fully explained by conventional event-based classification and is reflected in the distinct clinical activity of MET tyrosine kinase inhibitors (MET-TKIs), bispecific antibodies (BsAbs), and antibody-drug conjugates (ADCs). With the emergence of antibody-based therapies, MET has evolved from a signaling driver to a cell-surface target for receptor modulation and payload delivery. We therefore propose a clinically anchored two-dimensional framework for interpreting therapeutic relevance in MET-aberrant NSCLC: kinase dependence and cell-surface targetability. Neither dimension should be regarded as a directly measurable binary variable. Kinase dependence is inferred from genomic and treatment-contextual proxies, most strongly METex14 and, more conditionally, high-level focal MET amplification. Cell-surface targetability is approximated by drug-specific IHC assessment of assay-defined c-MET protein expression; however, receptor internalization, intracellular trafficking, and payload delivery capacity remain incompletely measurable in routine clinical practice. Within this framework, MET-TKIs have the most evidence-supported established role in tumors with evidence of MET-driven kinase dependence. EGFR × MET BsAbs have demonstrated clinical activity in broad post-osimertinib EGFR-mutant NSCLC, while EGFR/MET co-dependence or MET-mediated bypass activation provides a mechanistic rationale for their use; MET-defined preferential benefit remains to be prospectively established. MET-directed antibody-drug conjugates (MET-ADCs) are supported in drug- and assay-defined populations with high c-MET protein overexpression, although the predictive relevance of delivery-related factors remains hypothesis-generating. Accordingly, MET testing should shift from single-event detection to platform-oriented stratification: next-generation sequencing (NGS) for driver alterations and resistance profiles, fluorescence in situ hybridization (FISH) for high-level focal amplification, and immunohistochemistry (IHC) for surface expression relevant to antibody-based therapies. This framework is intended to organize current biological and clinical evidence rather than to replace drug-specific companion diagnostics, regulatory indications, or prospectively validated treatment-selection algorithms. Precision treatment of MET-aberrant NSCLC is thus moving from event-based drug selection toward mechanism-based therapeutic matching. Future priorities include standardizing biomarkers, defining optimal target populations, and aligning biological subtypes, diagnostic strategies, and therapeutic platforms.

Antibody-drug conjugate

Dual signal-enhanced immunochromatographic test strip based on Au@PtNPs: From sensitive detection of thiamethoxam to multiplex pesticide screening in vegetables.

Immunochromatographic test strip (ICTS) is a rapid analytical technique widely used in environmental and food detection owing to its merits of simple operation and short analysis time. Herein, three-dimensional nanoflower-structured gold‑platinum nanoparticles (Au@PtNPs) were synthesized via a seed-growth method. Compared with conventional gold nanoparticles (AuNPs), Au@PtNPs exhibited stronger signal intensity, excellent catalytic performance, and efficient antibody binding efficiency. Colorimetric Au@PtNPs-ICTS and catalytic colorimetric Au@PtNPs-ICTS were developed for the sensitive detection of thiamethoxam (THI) in vegetables. The limits of detection (LODs) for colorimetric Au@PtNPs-ICTS and catalytic colorimetric Au@PtNPs-ICTS quantitative analysis were 0.18 ng/mL and 0.093 ng/mL, respectively, representing approximately 3-fold and 6-fold improvement compared to AuNPs-ICTS (0.56 ng/mL). Furthermore, highly sensitive detection of multiple pesticide residues (chlorpyrifos, acetamiprid, and imidacloprid) was achieved by replacing the corresponding target antigens and antibodies, which further verified the universality of this immunochromatographic strategy.

Thiamethoxam

In vivo genome-wide CRISPR screens identify FOXR1 as a suppressor of CD8+ T cell antitumor immunity.

T cell dysfunction critically limits the efficacy of T cell-based immunotherapies in solid tumors, yet the intrinsic regulators of T cell dysfunction remain incompletely understood. Through an in vivo genome-wide CRISPR screen in tumor-infiltrating CD8+ T cells, we identified Forkhead Box R1 (FOXR1) as a potent transcriptional suppressor of CD8+ T cell effector functions. Genetic ablation of FOXR1 significantly enhanced cytokine production and cytotoxic capacity in both murine and human CD8+ T cells, whereas its overexpression impaired T cell activation and effector molecule expression. Mechanistically, multiomics integration of RNA-seq, CUT&Tag-seq, and ATAC-seq revealed that FOXR1 binds directly to promoter regions of key effector genes, including IL2, GZMB, and PRF1, and represses their expression. Importantly, FOXR1 deletion in human anti-CD19 CAR T cells improved their efficacy against solid tumors, demonstrating that FOXR1 is a checkpoint of T cell effector function and targeting FOXR1 is a promising strategy to enhance CAR T cell efficacy against solid tumors.

Animals

A combined stimulus of acute fasting and exercise modulates hippocampal mitochondrial quality control in healthy mice.

BACKGROUND AND AIMS: Exercise and fasting are recognized for their ability to improve brain health and mitigate neurodegeneration. However, little is known about how these interventions acutely impact mitochondrial quality control mechanisms including mitophagy. METHODS: We examined the effects of a single bout of fasting and exercise (FEx) on hippocampal mitochondrial function and proteomic remodeling in male and female mice. To assess in vivo autophagy dynamics, we combined proteomics with chloroquine (CQ) inhibition of autophagic flux. Mice were assigned to sedentary (Sed), fasting (F), exercise (Ex), or combined FEx groups and received unilateral intrahippocampal injections of CQ or PBS following treatments. Four hours later, hippocampi were collected for analysis. RESULTS: LC3-II levels significantly increased in the FEx group only following CQ treatment, indicating enhanced autophagic flux. Proteomic profiling showed sedentary males failed to mount a robust response to FEx however females exhibited upregulation of proteins involved in the TCA cycle, glutathione metabolism, and oxidative phosphorylation, suggesting greater mitochondrial adaptability. Functional assays supported these findings, females showed increased complex IV activity post-FEx. The mitochondrial DNA / nuclear DNA ratio increased after FEx regardless of sex, and upstream regulator analysis predicted activation of mitochondrial biogenesis. CONCLUSIONS: Together, these data reveal sex-specific mitochondrial remodeling in response to acute fasting and exercise. Defining these normative responses is critical for understanding how mitochondrial adaptability shapes resilience or vulnerability to neurological challenges.

Animals

PPRC1 is a prognostic biomarker and key regulator of mitochondrial oxidative phosphorylation in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) remains an incurable haematological malignancy, underscoring the need for novel prognostic biomarkers and therapeutic targets. This study aimed to investigate the clinical and biological significance of peroxisome proliferator-activated receptor gamma coactivator-related protein 1 (PPRC1) in MM. METHODS: Expression and clinical data were obtained from public databases and an independent local cohort. Kaplan-Meier and Cox regression analyses were performed to evaluate prognostic value. Differential expression analysis, pathway enrichment analysis and single-cell RNA-seq data analysis were used to explore biological functions. PPRC1 was silenced in MM cell lines using siRNA to assess its effects on cell survival and oxidative phosphorylation. RESULTS: PPRC1 was significantly upregulated in MM and was associated with advanced disease stage and poor overall survival. Multivariate Cox analysis identified PPRC1 as an independent prognostic factor. A nomogram incorporating PPRC1 and revised-ISS improved survival prediction. Functional analyses revealed that PPRC1 was positively correlated with oxidative phosphorylation and oncogenic signalling pathways. A potential connection between PPRC1 expression and immune cell infiltration was observed. PPRC1 knockdown inhibited cell proliferation, induced cell cycle arrest and apoptosis and impaired oxidative phosphorylation in MM. CONCLUSIONS: PPRC1 acts as a prognostic biomarker and metabolic regulator in MM by sustaining mitochondrial oxidative phosphorylation. These findings highlight PPRC1 as a potential therapeutic target in MM.

Humans

Protein isolation markedly enhances in vitro digestibility, nutritional quality, and bioactivity of fungal mycelial proteins.

Fungal mycelial proteins are promising sustainable protein sources, yet their nutritional utilization is often limited by structural constraints. This study systematically evaluated the effects of protein isolation on the proteomic composition, gastrointestinal digestion behavior, amino acid utilization, and bioactivity of Pleurotus citrinopileatus mycelial proteins. Quantitative proteomics identified 3591 proteins, of which 3374 were shared between mycelial flour (PCMF) and protein isolate (PCMPI), indicating that PCMPI primarily represents the soluble proteome fraction. In vitro digestion revealed that PCMPI exhibited significantly higher digestibility (93.98%) than PCMF (42.98%) (p&#xa0;<&#xa0;0.05), reaching levels comparable to whey protein isolate. Enhanced enzymatic accessibility in PCMPI promoted rapid peptide generation during the gastric phase and efficient amino acid release during the intestinal phase, resulting in higher peptide (634.76&#xa0;mg/g) and free amino acid levels (341.69&#xa0;mg/g) at the digestion endpoint. Consequently, PCMPI achieved a balanced amino acid profile with a PDCAAS of 1.0. Moreover, its digestion products exhibited stronger antioxidant activity (IC&#x2085;&#x2080;&#xa0;=&#xa0;8.36&#xa0;mg/mL) and ACE inhibitory activity (IC&#x2085;&#x2080;&#xa0;=&#xa0;15.65&#xa0;mg/mL) compared with PCMF. Mechanistically, protein isolation disrupted the cell wall matrix, shifting digestion from a structure-limited to an accessibility-driven regime. Collectively, these findings demonstrate that protein isolation markedly enhances the digestibility, nutritional quality, and functional potential of mycelial proteins, supporting their application as high-value sustainable protein ingredients.

Digestion

Single-cell transcriptomics reveals heterogeneous stress responses and Mg2+-mediated survival mechanisms in Lactobacillus delbrueckii subsp. bulgaricus during freeze-drying and storage.

Maintaining the viability of lactic acid bacteria during dehydration and subsequent storage remains a significant challenge. Here, we employed single-cell RNA sequencing to reveal the heterogeneous stress responses of Lactobacillus delbrueckii subsp. bulgaricus, identifying seven distinct transcriptional clusters across the liquid culture, freeze-drying, and storage phases. The dominant clusters in the freeze-drying and storage were not completely consistent, showing significant functional differentiation. Genomic stability may be important for survival during freeze-drying and storage, while intracellular energy homeostasis appears important for viability during storage. The magnesium transporter mgtB was highly expressed in clusters tolerant to freeze-drying and storage, suggesting a critical role for Mg2+ homeostasis. Further experimental validation confirmed that Mg2+ treatment significantly bolstered stress resistance, increasing immediate post-freeze-drying survival by over 2-fold (up to 92.90%) and post-storage survival by over 5-fold (up to 5.98%). Proteomic data indicated that Mg2+ supplementation correlated with the maintenance of several biological functions potentially relevant to bacterial survival during freeze-drying and storage, including DNA repair, translation, and central carbon metabolism. These findings provide a map of microbial stress resistance through population heterogeneity and offer a potential strategy that may be adapted for enhancing the stability of other industrial lactic acid bacteria products.

Freeze Drying

Functional and Nutritional Potential of Chickpea Protein Hydrolysates: A Systematic Review and Plant-protein Network Analysis.

Chickpea is a protein-rich legume increasingly explored as a substrate for functional plant-based ingredients. Chickpea protein hydrolysates (CPHs) and chickpea-derived peptides (CPs), obtained through enzymatic hydrolysis or simulated gastrointestinal digestion, may provide technological and biological properties while supporting the valorization of chickpea fractions and by-products. This review integrates a network analysis of title-abstract terms from 5,728 unique Scopus and PubMed records on plant protein hydrolysates with a systematic review of 72 studies focused on CPH production, peptide characterization, bioactivity, and translational gaps. The evidence indicates that CPHs and CPs show promising antioxidant, antihypertensive, antidiabetic, anti-inflammatory, lipid-lowering, immunomodulatory, antimicrobial, and anticancer-related activities, mainly supported by biochemical assays, cell models, and animal studies. However, heterogeneous hydrolysis protocols, incomplete peptide characterization, inconsistent bioactivity methods, limited scale-up evidence, and the absence of human intervention trials restrict translation. Future studies should prioritize standardized protocols, mechanistic validation, bioavailability, sensory and regulatory assessment, food-matrix validation, and clinical trials.

Cicer

Intestinal content accelerates muscle protein degradation in red shrimp (Solenocera crassicornis) during refrigeration: Insights from metagenomics and metabolomics.

This study systematically explored the effects of intestinal components on muscle quality deterioration and protein degradation of red shrimp during refrigerated storage. The results demonstrated that refrigeration induced continuous quality degradation and muscle protein breakdown in red shrimp, whereas eliminating intestinal tissues effectively retarded muscle spoilage and protein degradation, and optimized muscle texture. The intestinal microorganisms could secrete extracellular proteases to promote muscle protein degradation were primarily Vibrio, Bacillus, Pseudomonas, Photobacterium, and Shewanella. These microorganisms promote protein degradation by secreting zinc proteases, serine proteases, and aspartyl proteases. This study elucidates the molecular mechanisms of intestinal microbial metabolism influences the muscle protein degradation of red shrimp during refrigeration. The findings provide a theoretical foundation for precise regulation of intestinal-targeted microorganisms, thereby maintaining optimal quality of shrimps during refrigeration.

Animals

Mapping the immune-genetic architecture of Epstein-Barr virus-related phenotypes and multiple sclerosis through a single-cell genetic framework for target prioritization and pharmacologic hypothesis generation.

BACKGROUND: Multiple sclerosis (MS) is a severe neuroinflammatory disease causing substantial long-term disability. Strong epidemiologic evidence links Epstein-Barr virus (EBV) exposure with MS risk, but genetic evidence for immune target prioritization in EBV-related phenotypes remains limited. METHODS: We integrated single-cell cis-eQTL data from 14 immune cell types with GWASs of an EBV-related clinical phenotype and MS using a single-cell Mendelian randomization framework with colocalization analyses. Candidate eGenes were evaluated in independent cohorts. For multi-SNP instruments, we performed heterogeneity, pleiotropy, MR-Egger, weighted median, mode-based, and MR-PRESSO sensitivity analyses. We also conducted phenome-wide association analyses and queried DrugBank to annotate candidate compounds targeting prioritized genes. RESULTS: We prioritized 43 immune-cell-specific candidate eGenes with convergent genetic support, including 6 for the EBV-related phenotype and 37 for MS. SERPINB1 in NK cells was associated with increased risk of the EBV-related phenotype, whereas HLA-G was associated with decreased risk. For MS, APOM and MSH5 showed protective associations, while AHI1 showed cell-type-dependent, bidirectional associations across immune lineages. Colocalization and independent cohort evaluation supported these findings. Among FDR-significant multi-SNP associations, MR-Egger intercept tests did not indicate directional pleiotropy, although a small subset showed heterogeneity or MR-PRESSO signals. Phenome-wide analyses identified no significant adverse phenotypic associations among evaluable genes at the prespecified threshold. DrugBank annotation nominated sodium nitroprusside, fasudil, artenimol, and choline as hypothesis-generating compounds for experimental follow-up. CONCLUSIONS: This study provides a single-cell genetic framework for prioritizing immune-cell-specific candidate targets for EBV-related phenotypes and MS, and nominates genetically supported targets and pharmacologic hypotheses for experimental investigation.

Humans

Deciphering CD8+ T cell exhaustion in human cancers through single-cell and spatial transcriptomics.

Exhausted CD8+ T cells (Tex) within the tumor microenvironment (TME) represents a critical barrier limiting anti-tumor immune responses. Tex cells are characterized by upregulated inhibitory immune checkpoint receptors, reduced cytotoxicity, and functional heterogeneity. Their genomic features and regulatory networks remain poorly defined, and only a minority of patients respond to immune checkpoint blockade (ICB) therapy. Single-cell RNA sequencing (scRNA-seq), through high-resolution transcriptomic profiling, has revealed diverse Tex subpopulations, identified subpopulation-specific marker genes and regulatory pathways. Spatial transcriptomics has further mapped the spatial distribution of Tex and their interaction networks with immune cells, tumor cells, and stromal cells, elucidating the impact of spatial heterogeneity on Tex functionality. Current studies indicate that the exhausted state of Tex is dynamic and modifiable, with functional differences among subpopulations closely associated with tumor progression and therapeutic response. However, the genomic characteristics, epigenetic regulation, and spatial interaction mechanisms of Tex require further exploration. This review summarizes recent advances in high-resolution omics technologies for precisely dissecting Tex heterogeneity, functional features, and interactions with other cells. It emphasizes the central value of optimizing Tex-targeted tumor immunotherapy strategies, providing theoretical foundations and directional guidance for developing more effective anti-tumor immunotherapies.

Humans

Decoding tumor immune microenvironment heterogeneity by single-cell and spatial multi-omics: From immunotherapy resistance to translational biomarkers.

Immune checkpoint blockade has transformed cancer therapy, yet primary and acquired resistance remain major clinical challenges. Increasing evidence indicates that immunotherapy resistance cannot be fully explained by tumor-intrinsic alterations or conventional biomarkers such as PD-L1 expression, tumor mutational burden, or microsatellite instability. Instead, therapeutic response is shaped by the tumor immune microenvironment (TIME) as a heterogeneous, spatially organized, and dynamically evolving ecosystem. Single-cell omics has revealed diverse immune and stromal cell states, including progenitor and terminally exhausted T cells, suppressive myeloid programs, B-cell/TLS-associated immune-reactive states, and CAF-mediated exclusion phenotypes. Spatial transcriptomics, spatial proteomics, and imaging-based approaches further demonstrate that these cell states assemble into distinct immune niches, including immune-inflamed, T-cell-excluded, myeloid-suppressive, metabolic/hypoxic, and TLS-associated niches. These spatial ecosystems determine whether antitumor immune cells can access malignant cells, receive antigen-presenting support, or become restrained by stromal, vascular, metabolic, and myeloid barriers. In this review, we summarize how single-cell and spatial multi-omics redefine TIME heterogeneity in immunotherapy resistance, highlight ligand-receptor communication networks linking cell states to spatial immune dysfunction, and discuss emerging translational biomarkers for patient stratification. We further propose that future immunotherapy biomarkers should evolve from static single-marker assays toward longitudinal, spatially resolved, and interpretable multi-omics models that guide precision combination immunotherapy.

Humans

Single-cell transcriptome revealed the aberrant keratinocytes activation in antigen presentation in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD), a common chronic inflammatory skin disease, has been extensively studied using single-cell genomics. However, keratinocytes, as key effector cells in AD, have underlying mechanisms remain incompletely understood and require further investigation. METHODS: We integrated single-cell transcriptomic data from skin tissues of healthy controls, chronic active AD patients, spontaneously healed AD (SHAD) patients, and an ovalbumin-induced AD mouse model. The study particularly emphasized the gene expression and cellular dynamics of keratinocytes across the different groups, as well as their interactions with immune cells. RESULTS: Compared to healthy controls, we observed significant changes in the keratinocyte transcriptome, cellular state, and keratinocyte-immune cell ligand-receptor interactions in AD skin, particularly the marked activation of genes involved in antigen processing and presentation. Interestingly, such gene activation was not observed in keratinocytes from the ovalbumin-induced AD mouse model, despite its phenotype closely resembling human AD. Furthermore, in SHAD, we identified a recovery of both the ligand-receptor interaction patterns and antigen processing and presentation genes, accompanied by a notable shift in the transcriptome. This involved a significant downregulation of genes related to cytoplasmic transcription and oxidative phosphorylation. Notably, this pattern was not observed in the self-healing mouse model following the removal of ovalbumin stimulation. CONCLUSION: Our results suggest that the persistent activation of antigen processing and presentation pathways in keratinocytes may be a key driver of chronic inflammation in AD. Therefore, redirecting anti-allergic therapeutic strategies from solely targeting immune cells to targeting of keratinocyte-mediated antigen presentation may offer a more effective approach. Furthermore, we raise concerns about the use of ovalbumin-induced mouse models to recapitulate human chronic AD, as the underlying mechanisms may differ significantly.

Dermatitis, Atopic

The circadian clock proteins PRR modulate root hair development via the RHD6/RSL module in Arabidopsis.

Root hairs, derived from trichoblasts, are critical for plant growth and environmental adaptation. Although environmental cues are known to influence root hair development, how endogenous timing systems such as the circadian clock integrate into the core transcriptional network governing root hair formation remains unclear. Here, we show that the circadian clock-associated protein PSEUDO-RESPONSE REGULATOR5 (PRR5) physically interacts with ROOT HAIR DEFECTIVE6 (RHD6) and RHD6 LIKE1 (RSL1), two basic helix-loop-helix transcription factors essential for root hair initiation. Genetic analyses suggest that PRR proteins contribute to root hair development under long-day conditions in Arabidopsis thaliana. Simultaneous disruption of PRR5, PRR7, and PRR9 results in defective root hairs, whereas PRR5 overexpression markedly increases root hair density and length. Transcriptomic and RT-qPCR analyses reveal that PRRs enhance the expression of RHD6, RSL1, and multiple downstream root hair-responsive genes, while modulating their temporal expression patterns. Furthermore, PRR5-mediated root hair promotion requires RHD6/RSL1, and PRR proteins enhance RHD6-dependent activation of the RSL4 promoter. PRRs also contribute to root hair development under phosphate-deficient and salt-stress conditions. Together, these findings establish a molecular framework in which PRR proteins regulate the RHD6/RSL network to coordinate root hair development and environmental responses.

Arabidopsis

Advances in cereal protein applications for infant and Young child nutrition.

BACKGROUND: The increasing use of plant-derived proteins in infant and young child nutrition necessitates tailored amino acid profiles, high digestibility and strict safety controls. Cereal proteins, such as rice, oat, maize, millet, barley, and wheat, are widely used in complementary foods but face intrinsic limitations, notably lysine and tryptophan deficits, antinutritional factors that reduce bioavailability, gluten immunogenicity in wheat/barley, and inorganic arsenic risks in rice. SCOPE AND APPROACH: This review synthesizes recent advances in processing and formulation strategies, including enzymatic hydrolysis, fermentation, germination, extrusion, cereal-legume complementation, and micronutrient fortification. Their impacts on digestibility, techno-functionality, iron and zinc bioavailability, and protein quality, including Protein Digestibility-Corrected Amino Acid Score (PDCAAS) and Digestible Indispensable Amino Acid Score (DIAAS) are critically reviewed using data from in vitro assays, product development, and clinical trials. KEY FINDINGS AND CONCLUSIONS: Processing and blending approaches can substantially improve protein digestibility, amino acid balance and micronutrient availability, and hydrolyzed rice protein holds clinical promise for cow's milk protein allergy (CMPA). However, most evidence is preclinical, reporting of protein quality is inconsistent, and industrial translation is constrained by sensory, shelf-life, contaminant and cost issues. We recommend standardized DIAAS-based reporting, large-scale feeding trials, sensory /stability optimization, and targeted exploration of underutilized grains (e.g., oat, millet) with active allergen monitoring. Prioritizing amino acid-focused formulation coupled with strategies to enhance micronutrient bioavailability will accelerate safe adoption of cereal proteins in early-life nutrition.

Humans

Chemical and sensory profiling of fermented, washed, and artificially flavored coffee beans: Insights into flavour quality, authenticity, and food safety implications.

This study establishes an integrated framework combining chemical profiling, sensory analysis, and molecular mechanism evaluation to compare flavour quality and authenticity among fermented, washed, and artificially flavored coffees. GC&#xa0;&#xd7;&#xa0;GC-TOF-MS and UHPLC-HRMS showed that fermented samples had markedly higher ester and aromatic alcohol levels (total esters 74.5&#xa0;&#xb1;&#xa0;7.8&#xa0;mg&#xa0;kg-1; phenylethanol 27.5&#xa0;&#xb1;&#xa0;3.2&#xa0;mg&#xa0;kg-1, p&#xa0;<&#xa0;0.01), enhancing fruity-floral notes. Washed coffees contained the highest organic acid concentrations (45.2&#xa0;&#xb1;&#xa0;3.8&#xa0;mg&#xa0;kg-1, p&#xa0;<&#xa0;0.01), supporting brightness and umami. Artificially flavored coffees exhibited elevated exogenous aromatics (vanillin 21.5&#xa0;&#xb1;&#xa0;3.1&#xa0;mg&#xa0;kg-1) but significantly fewer Maillard products (p&#xa0;<&#xa0;0.05) and reduced flavour retention (55% after 14 days). Molecular docking revealed higher theoretical binding affinities for naturally generated compounds, suggesting a potential molecular basis for their greater sensory persistence. The framework supports constructing coffee quality fingerprints and verifying flavour authenticity.

Flavoring Agents