Search Results (296)

Peritoneal metastasis confers a dismal prognosis in colorectal cancer (CRC-PM), with median survival rarely exceeding 20 months. Here we employed multi-omic profiling to identify actionable drivers of CRC-PM. We performed whole-exome sequencing (WES) and label-free quantitative proteomics on 13 and 7 matched primary-peritoneal metastasis pairs, respectively. Public single-cell (GSE183916 and GSE132465) and spatial transcriptomic datasets (GSE225857) were integrated to dissect cellular heterogeneity. TCGA cohorts were used for survival analysis, and functional validation was performed in DLD1 cells. GPAA1, a catalytic subunit of the GPI transamidase complex, emerged as the most consistently upregulated protein in peritoneal metastases across discovery and validation proteomic cohorts. High GPAA1 expression correlated with shorter recurrence-free survival (HR = 1.72, p = 3.4 × 10⁻⁵). Single-cell analysis localized GPAA1 to malignant cells exhibiting oxidative phosphorylation and enhanced stromal crosstalk. GPAA1 overexpression promoted proliferation, migration, and EMT via AKT activation, whereas knockdown reversed these phenotypes. Our findings identify GPAA1 as a candidate driver of peritoneal metastasis in CRC, linking GPI anchor biosynthesis to metabolic reprogramming and AKT-mediated EMT. GPAA1 may serve as a prognostic marker in colorectal cancer. Full article

The demand for user-friendly applications to support biologists in analyzing high-throughput proteomics data remains a pressing challenge. Given the complexity and the multiple intermediate steps involved, this process is time-consuming and often requires specialized computational skills. To simplify and accelerate the exploration of proteomics data, we present PiProteline, an R package designed to operate on high-dimensional data matrices assembled from the output of any search engine commonly used in bottom-up proteomics experiments. In addition to data preprocessing and descriptive statistics, PiProteline enables label-free quantitation, functional enrichment, and systems biology analyses. It supports both unweighted and weighted protein–protein interaction (PPI) network topological analyses for the identification of critical nodes, such as hubs and bottlenecks. By integrating multiple computational approaches, PiProteline accelerates the selection of potentially relevant protein signatures, providing insights into the molecular mechanisms characterizing the investigated systems. This information facilitates the formulation of new hypotheses and the design of targeted experiments, ultimately reducing costs and advancing translational medicine. PiProteline is available as an open-source R package via GitHub, as a Shiny application, and via Zenodo. Full article

Snake venomics and antivenomics approaches have increasingly relied on mass spectrometry to characterize the venom components and to evaluate the antivenom neutralization potential. However, accurate quantification remains essential for distinguishing biologically relevant toxins from snake venom and for translating proteomic data into meaningful clinical interpretations. This review article examines the role of quantitative analysis in snake venomics and antivenomics, with special emphasis on massspectrometry-based strategies used to estimate toxin abundance. This review summarizes the main applications and limitations of mass spectrometry in venom research covering bottom-up and top-down proteomics, venom databases, toxin quantification, and how MS-based quantification determines the efficacy of antivenom through antivenomics approaches. This review also explains why abundance alone cannot determine the biological or therapeutic importance of a toxin. The article discusses label and label-free based quantification, absolute quantification approaches, top-down venomics, mass spectrometry-based antivenomics, and the integration of proteo-transcriptomic workflows in snake venom research. These approaches can be affected by several factors including the problem of measuring toxin isoforms that share similar protein structures and toxin components that are important for antivenom recognition within complex venom samples. A conceptual overview is provided of how inaccurate quantification can influence venom proteome comparisons among similar and different species, as well as the interpretation of antivenom binding and neutralization potential. Reliable mass-spectrometry-based quantification will be critical for linking the venom composition with toxicity, antivenom performance evaluations in preclinical settings, and evidence-based strategies for snake venom research and snakebite management. Full article

Within-cohort growth heterogeneity constrains aquaculture productivity, and its molecular correlates in carnivorous teleosts are poorly described. We compared the three heaviest and the three lightest individuals (n = 3 biological replicates per group, one fish per replicate) from a single cohort of mandarin fish (Siniperca chuatsi) at 90 days post-hatch, which differed 3.09-fold in body mass (p = 7.1 × 10⁻⁵), by combining muscle RNA sequencing with label-free quantitative proteomics of muscle and liver. Muscle transcriptomics identified 827 differentially expressed genes. The proteasome (ko03050) was the only KEGG pathway reaching significance, with all 14 contributing subunits being more abundant in slow-growing fish, and GO enrichment recovered proteasome, peptidase and endopeptidase complexes alongside serine-type peptidase activities in the same direction. Proteomics identified 37 differentially abundant proteins in muscle and 24 in liver at a nominal threshold. Protein-level gene set enrichment analysis showed negative enrichment of oxidative phosphorylation (NES = −1.96, adjusted p = 0.001) and of mitochondrial proteins (NES = −1.77, adjusted p = 0.005) in fast-growing muscle. Haemoglobin subunits were more abundant in fast-growing muscle and liver at the protein level, while muscle haemoglobin transcripts showed paralog-specific differences. The cross-sectional extreme-phenotype design identifies molecular signatures associated with the observed growth phenotype without establishing causality. The dataset provides candidate genes and proteins for functional study in S. chuatsi. Full article

Staphylococcus epidermidis biofilms are inherently tolerant to antimicrobial treatment, contributing to the persistence and recurrence of biofilm-associated infections. In this study, we examined the phenotypic and proteomic responses of S. epidermidis ATCC 35984 biofilms following exposure to subinhibitory concentrations (1/8× MIC) of levofloxacin, rifampicin, and their combination. Phenotypic analyses revealed clear differences among the treatments. Rifampicin significantly reduced the number of viable biofilm-associated cells, whereas levofloxacin alone had little effect. The combination of rifampicin and levofloxacin produced the most pronounced antibiofilm activity, significantly reducing both biofilm biomass and viability. These findings were supported by confocal laser scanning microscopy, which showed the greatest loss of viable cells and biofilm integrity in the combination-treated biofilms. Label-free quantitative proteomic analysis demonstrated distinct treatment-specific adaptive responses. Rifampicin, either alone or in combination with levofloxacin, triggered broadly increased abundance of proteins involved in translation, energy production, amino acid and nucleotide metabolism, ATP-binding cassette transport, and quorum sensing. In contrast, levofloxacin predominantly suppressed proteins associated with central metabolism, transcription, translation, and biosynthetic pathways. Hierarchical clustering further showed that the proteomic profile of the combination treatment closely resembled that of rifampicin alone, suggesting that rifampicin was the dominant driver of proteomic remodeling. Expression pattern network analysis identified a conserved stress-response signature comprising Fhs, YdaG, PerR, and YcsE across all treatment conditions. Notably, the multidrug efflux pumps NorA and NorC showed decreased protein abundance across all treatment conditions, indicating a potential reduction in efflux-mediated resistance under sub-MIC antibiotic exposure. Taken together, these findings demonstrate that rifampicin retains substantial antibiofilm activity at subinhibitory concentrations and elicits extensive metabolic adaptation, whereas levofloxacin primarily induces a metabolically repressed state. The enhanced activity of the rifampicin–levofloxacin combination appears to arise from complementary effects on biofilm viability and cellular physiology. This study provides new insights into the adaptive responses of S. epidermidis biofilms to sublethal antibiotic stress and highlights the potential of rifampicin-based combination therapies for the management of biofilm-associated infections. Full article

Ataxia-telangiectasia (A-T) is a multisystemic genetic disorder caused by pathogenic mutations in the ATM gene, with effects extending beyond the canonical DNA damage response. We investigated whether two compact ATM transcript variants, ATM 4-53 (exons 4 and 53 joined) and ATM SINT (an in silico-designed ATM variant), could rescue disease associated with cellular alterations and inform construct selection for future delivery strategies. A-T fibroblasts transduced with either ATM variants or an empty vector were compared with wild-type fibroblasts using complementary label-free and tandem mass tag-based quantitative proteomics. Bioinformatic pathway and network analyses were integrated with biochemical and functional validation assays. A-T fibroblasts displayed coordinated alterations in proteostasis, proteasome function, RNA processing pathways, interferon-associated signaling, extracellular matrix organization, cell migration, and nanomechanical properties. Expression of both ATM variants partially or extensively reversed these abnormalities, restoring protein folding and proteasomal components, reducing protein aggregation, attenuating inflammatory signaling, and normalizing extracellular matrix-related phenotypes. ATM SINT generally produced the broader and more consistent rescue, particularly for proteostasis and RNA processing-associated networks. These findings show that compact ATM variants can recover multiple non canonical ATM dependent cellular functions in A-T fibroblasts and identify ATM SINT as the more effective construct in this model. Proteomic profiling combined with orthogonal functional validation may therefore support rational selection of ATM variants for subsequent preclinical delivery studies. Full article

Carbon monoxide-releasing molecules (CORMs) display promising antibacterial activity, yet their global cellular targets and underlying mechanisms remain incompletely understood. This study aimed to characterize the proteomic response of Escherichia coli MG1655 exposed to a sub-inhibitory concentration (10 µM, 40 min) of the ruthenium-based CORM-3 compared to its inactive counterpart, iCORM-3, in defined minimal medium. Utilizing label-free quantitative LC-MS/MS proteomics combined with differential abundance, functional annotation, and STRING-based network analyses, we explored treatment-associated proteomic signatures and subcellular localization patterns. While iCORM-3 exhibited no antibacterial activity, CORM-3 inhibited growth in a concentration-dependent manner. Although no individual protein reached FDR significance after multiple-testing correction, candidate abundance changes converged on functionally related modules, including envelope/periplasmic stress, sulfur metabolism and transport, redox/metal homeostasis and selected energy-associated functions. These findings support a hypothesis-generating model in which sub-inhibitory CORM-3 exposure is associated with multifactorial bacterial stress adaptation rather than a single dominant protein-level target. Envelope homeostasis, sulfur-containing pathways, redox/metal adaptation, and attenuation of selected energy-associated functions therefore emerge as candidate components of the E. coli response to CORM-3 and require future targeted validation. Full article

Bacterial diseases caused by Flavobacterium covae (Fc), Vibrio harveyi (Vh), Vibrio vulnificus (Vv) and Photobacterium damselae (Pd) seriously constrain Asian seabass aquaculture. Here we dissect the mucosal immune mechanisms engaged by a five-month sequential vaccination strategy that combines nanoemulsion immersion priming with multivalent oral hydrogel boosting. Juvenile seabass were vaccinated, then challenged with F. covae by freshwater immersion and with a Vibrio–Photobacterium (Vh/Vv/Pd) mix by immersion or intraperitoneal injection. Gills were sampled after immersion challenges and intestine after injection, and profiled by RNA sequencing and label-free quantitative proteomics, with selected genes validated by RT-qPCR. Principal component analysis showed clear separation of vaccinated and control fish in all tissues and challenges, indicating a strong and coherent transcriptional reprogramming. Vaccination markedly increased the number of upregulated genes, with Gene Ontology enrichment revealing dominant signatures of ribosome biogenesis, RNA processing, lysosomal organization and immune response. KEGG analysis highlighted cytokine receptor interaction; NOD and Toll-like receptor signaling; oxidative phosphorylation; and phagosome, lysosome and cell adhesion molecule pathways, consistent with heightened antimicrobial readiness. Volcano plots and focused heatmaps showed strong induction of interferon-stimulated genes, cytokines and chemokine receptors, complement components, macrophage mannose receptor, epithelial barrier mediators and numerous immunoglobulin transcripts, with tissue- and challenge-specific patterns. Proteomics corroborated these trends, demonstrating a higher abundance of immunoglobulin heavy chains, complement proteins, cathepsins, heat shock and redox chaperones, ribosomal proteins and cytoskeletal and adhesion regulators in vaccinated mucosae. Integrated pathway mapping linked endothelial adhesion molecules and leukocyte integrins with T cell costimulation networks and an intestinal immune network for immunoglobulin production, including enhanced pIgR-mediated transcytosis. Overall, the sequential vaccination regimen was associated with coordinated transcriptomic and proteomic signatures related to epithelial responses, innate immunity, and humoral immune functions across gill and intestinal tissues. These molecular patterns were accompanied by improved survival following bacterial challenge; however, the present data do not directly demonstrate the functional activity of the inferred immune mechanisms in Asian seabass. Full article

Background: Seed dormancy is crucial for plant survival and agricultural productivity, yet its molecular mechanisms, particularly the role of maternal effects, remain poorly understood. Methods: In this study, we applied a SWATH-based, label-free, quantitative shotgun proteomic mass spectrometry approach to investigate the temporal dynamics of dormancy establishment in Xanthium strumarium, a wild plant with two seeds in one burr that, despite sharing the same genetic and environmental conditions, exhibit distinct dormancy states. Results: Our data show that dormant seeds undergo coordinated metabolic suppression, marked by a decrease in energy metabolism, cell cycle arrest, and auxin signaling, explaining their smaller size. Simultaneously, dormant seeds exhibit metabolic re-prioritization towards fatty acid desaturation, cell wall modification, and an active epigenetic program stabilized by dormancy-promoting factors alongside a transcriptionally quiescent state in early–mid development. However, in the late developmental stage, molecular signaling pathways showed a recalibration distinguished by changes in seed metabolism (such as carbon–nitrogen reallocation, sulfur assimilation, and GABA production), hormonal fluctuations, and epigenetic regulation. Notably, previously reported high DOG1 transcript abundance, together with the absence of detectable DOG1 protein in the proteomic dataset, suggests that post-transcriptional mechanisms may contribute to DOG1 regulation. Conclusions: Based on these findings and the available literature, we propose a framework whereby dormancy establishment occurs in two phases: an early DOG1-mediated priming phase followed by a temperature-sensitive plasticity phase during seed maturation. Full article

Background: Large yellow tea (LYT), a distinctive variety made from mature leaves, has recently gained attention for its remarkable health benefits. However, whether these benefits can be transmitted from mother to offspring remains unexplored. Purpose: This study investigated whether maternal LYT consumption confers intergenerational protection against metabolic and behavioral disorders induced by perinatal bisphenol A (BPA) exposure in F1 offspring. Methods: A mouse model of perinatal BPA exposure (0.03% in diet) was established with or without maternal LYT supplementation (2.5% in diet). Metabolic parameters were assessed through biochemical assays and gene expression analysis (RT-PCR). Energy expenditure and spontaneous activity were monitored using a Comprehensive Lab Animal Monitoring System (CLAMS). Hippocampal proteomic profiling was performed using label-free quantitative proteomics. Results: LYT significantly reduced maternal BPA body burden, potentially via limiting absorption, enhancing glucuronidation metabolism, and promoting excretion. Notably, LYT exhibited bidirectional metabolic regulation, alleviating gestational hyperglycemia in dams while restoring hypoglycemia in offspring, and normalizing underweight and hypolipidemia. Mechanistically, the SIRT6 (sirtuin 6)/FOXO1 and SIRT6/SREBP1 pathways may be involved in regulating gluconeogenesis and lipogenesis. Concurrently, LYT rectified BPA-induced hyperactivity and reduced excessive energy expenditure. Proteomic analysis revealed that LYT partially restores BPA-induced dysregulation of cholesterol metabolism and glutamatergic/GABAergic synaptic pathways, which may contribute to rebalancing synaptic homeostasis. Conclusions: These findings suggest that maternal LYT supplementation confers intergenerational protection against BPA-induced metabolic and behavioral disorders in mice, potentially acting through enhanced toxin clearance, bidirectional metabolic regulation, behavioral normalization, and partial restoration of hippocampal synaptic homeostasis. This study provides a theoretical basis for developing natural dietary interventions to mitigate developmental toxicant-induced health risks. Full article

Jing Guan Fang (JGF) is a traditional multi-herb formula used for hyperinflammatory conditions associated with severe viral infections; however, its protein-level effects in macrophage-mediated inflammation remain incompletely characterised. In this study, we combined ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)-based phytochemical profiling, functional anti-inflammatory assays, and discovery-level proteomic and secretome analyses to investigate the anti-inflammatory effects of JGF in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Phytochemical profiling revealed predominantly flavonoid- and phenolic-related annotated features, together with selected terpenoid- and alkaloid-related annotations. Functionally, JGF treatment was associated with reduced nitric oxide production and differential effects on pro-inflammatory cytokine release, with a clearer concentration-dependent reduction in interleukin-6 (IL-6) and a more variable tumour necrosis factor-α (TNF-α) response, without reducing cell viability. Label-free quantitative proteomics suggested that JGF treatment was associated with modulation of LPS-induced protein changes, including reduced abundance of inflammation-associated proteins such as myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (NOS2), together with partial restoration of selected regulatory proteins such as AIMP1 and AIMP2. Secretome analysis further suggested that JGF reduced extracellular inflammatory and tissue-remodelling-related mediators, including SERPINE1/PAI-1. Exploratory pathway analysis indicated that JGF treatment was associated with changes in inflammation-related and oxidative stress-related signalling networks. Collectively, these findings provide discovery-level molecular insights into the anti-inflammatory effects of JGF in LPS-stimulated macrophages and support further targeted validation of its pharmacological activity as a candidate multi-component anti-inflammatory formula. Full article

Multiomics is increasingly valued as a strategy for investigating the regulatory mechanisms by which plants respond to adverse stress conditions. Currently, information on the molecular processes underlying plant responses to Pb stress, particularly those observed through an approach that combines proteomics and metabolomics, is lacking. Therefore, in this study, we aimed to explore functional correlations between Pb-responsive proteins and metabolites under Pb stress by performing label-free quantitative proteomics and untargeted metabolomics on the roots of the Pb hyperaccumulator Pogonatherum crinitum (Thunb.) Kunth. The selected Pb stress-responsive proteins were functionally verified using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and parallel reaction monitoring (PRM). Under Pb stress, 397 upregulated and 431 downregulated proteins were identified through proteomic analysis. Metabolomic analysis identified 478 upregulated and 354 downregulated metabolites. Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes revealed that differentially expressed proteins and metabolites were involved in pathways linked to heavy metal stress, such as starch and sucrose metabolism and plant hormone signal transduction. Through integrated proteomic and metabolomics analyses, we uncovered the coordinated regulatory interplay between glutathione (GSH) metabolism and jasmonic acid signaling. GSH reductase and 12-oxophytodienoate reductase drive the accumulation of GSH and jasmonic acid, respectively. The synergistic enhancement of these components is critical for maintaining cellular redox homeostasis and activating hormone-mediated defense signaling. These downstream metabolites were upregulated under Pb stress. RT-qPCR validation revealed that the transcriptional change trends were consistent with those of the proteomics analysis. Further quantitative validation of the target protein using PRM revealed significant upregulation under Pb stress. In conclusion, the P. crinitum root system upregulated the activity of key enzymes in the antioxidant system and plant hormone synthesis under Pb stress, thereby regulating the accumulation of GSH, glutamate, and metabolites for jasmonic acid synthesis. This integrated regulatory network provides promising candidate targets for breeding Pb-tolerant hyperaccumulators to remediate Pb-contaminated farmland and mining soil. Full article

Cell surface markers are essential for lineage tracing and for understanding cellular mechanisms of regeneration, yet they remain largely unavailable for echinoderms. Using the starfish Asterias rubens as a model for physiological coelomocyte renewal, we combined Percoll density-gradient fractionation with label-free LC-MALDI proteomics to identify candidate surface proteins that distinguish subpopulations of coelomocytes and coelomic epithelium (CE) cells. However, LC-MALDI is prone to signal variability, sample depletion and nonlinear response. To address the quantitative limitations of LC-MALDI, we implemented a chi-square test with an empirically derived probability density function, which we validated on control mixtures, achieving high precision. Four fractions enriched in distinct cell types were analyzed: coelomocyte fractions 1 (roundish cells) and 4 (large agranulocytes that form cell nets) and CE fractions 1 (type 2 small CE cells) and 4 (ciliated cells), together with a weakly attached CE subpopulation enriched in small putative progenitor cells. LC-MALDI identified 714 protein groups, of which 160 showed significant differential abundance across fractions. Functional enrichment revealed actin cytoskeleton and integrin signaling in coelomocytes, and catenin-related terms in CE-W. Among the 101 predicted membrane proteins, 31 candidate cell surface markers were selected. These include integrins α8β1 and α9β1, three-finger proteins, scavenger receptors, and low-density lipoprotein receptor-related proteins, as well as metallopeptidases. This work provides the first proteome-scale set of candidate surface markers for starfish cell subpopulations, enabling future lineage-tracing studies to resolve the origin of coelomocytes from the coelomic epithelium. Full article

Coronary heart disease (CHD) is a global cardiovascular disease with high morbidity and mortality, and its complex pathological mechanism poses great challenges to clinical prevention and treatment. Paeoniflorin (PA), a monoterpene glycoside active ingredient from Ranunculaceae plants, has shown potential in cardiovascular protection, but its specific anti-CHD molecular targets and systematic regulatory networks remain unclear. In this study, a mouse model of CHD was established, and a multi-omics strategy combining label-free quantitative proteomics and metabolomics was adopted to explore the mechanism of PA in treating CHD. The results showed that PA significantly improved cardiac function, alleviated myocardial pathological injury and fibrosis, and regulated lipid metabolism in CHD model mice, with the high-dose group showing the optimal effect. Proteomic analysis identified 51 key differentially expressed proteins (DEPs) reversed by PA, which were mainly enriched in complement and coagulation cascades, and neutrophil extracellular trap formation pathways, with the C3-Cfd-C3aR signaling axis as the core hub. Further verification confirmed that PA could downregulate the expression of C3, Cfd, C3aR, and their downstream molecule BTK, thereby inhibiting myocardial inflammatory response and cardiomyocyte apoptosis. In addition, PA downregulated the expression of platelet activation markers ITGA2B/ITGB3. Metabolomic analysis revealed that PA reversed 57 abnormal metabolites in CHD mice, which were enriched in GABAergic synapse, retrograde endocannabinoid signaling and other pathways. Molecular docking confirmed that PA could stably bind to C3, Cfd, C3aR, BTK, and ITGA2B/ITGB3 with strong binding activity. In conclusion, PA exerts anti-CHD effects through a multi-target and multi-pathway synergism, mainly by targeting the C3-Cfd-C3aR axis to inhibit inflammation, apoptosis and platelet activation, and regulating metabolic disorders. This study provides experimental evidence and theoretical support for the clinical application of PA as a multi-target therapeutic drug for CHD. Full article

Background: Processed extracellular lipid–protein preparations require rigorous analytical characterization to determine whether their compositional profile, processing stability, and short-term cellular compatibility are preserved across storage and handling conditions. Methods: In this study, we quantitatively characterized a processed phospholipoproteic secretome-derived formulation under fresh, concentrated, cryopreserved, and lyophilized conditions. Results: Label-free quantitative proteomic analyses performed using timsTOF Pro mass spectrometry coupled to dia-PASEF acquisition identified 574 human proteins across all experimental conditions following predefined analytical quality criteria. Comparative analyses demonstrated preservation of the overall structural proteomic profile following processing and storage procedures, with retention of membrane-associated and extracellular structural proteins consistently exceeding 90% relative to the fresh reference condition. Quantitative reproducibility remained high across all experimental groups, with coefficients of variation ranging from 3.0% to 4.5% and strong inter-replicate Pearson correlations. Principal component analysis, hierarchical clustering, peptide/protein overlap analyses, and differential expression profiling demonstrated limited proteomic divergence while preserving the majority of quantified proteins within conserved abundance ranges. Complementary real-time live-cell kinetic imaging performed in non-malignant dermal-derived cells using the IncuCyte^® S3 platform demonstrated stable short-term confluence kinetics and cellular viability exceeding 92% over 48 h across all evaluated formulations. No sustained proliferative suppression or detectable morphological evidence of cytotoxicity was observed. Collectively, these findings support the preservation of compositional stability, analytical reproducibility, and short-term ex vivo cellular compatibility across defined processing and storage conditions. These integrated proteomic and kinetic datasets provide a quantitative framework for the analytical evaluation of processed extracellular phospholipoproteic preparations, while functional barrier activity, membrane incorporation, lipid raft engagement, and long-term tissue-level effects remain to be addressed in dedicated future studies. Full article