A new approach for treating AD: Guifu Dihuang Pills improves brain insulin resistance by promoting NrCAM to activate the EGFR/PI3K/Akt signaling pathway.

Mass spectrometry-based major phytochemicals and dose dependent cytotoxic and nephrotoxic effects of Commiphora leptophloeos stem bark extract in preclinical in vitro and in vivo models.

Exploring the anti-Helicobacter pylori activity and mechanism of Shouhui Tongbian through chemical composition analysis and network pharmacology.

Er-Chen decoction alleviates spermatogenic dysfunction in obese mice by tuning the SIRT1/p53 axis.

Gassericins K7 are heterodimeric bacteriocins produced by Lactobacillus paragasseri K7. Their biological characterisation has been limited by challenges in obtaining these compounds in sufficient purity and quantity. In this study, we used a preparative isolation and purification workflow for gassericins K7, combining ammonium sulphate precipitation, Amberlite XAD-16 extraction, and reversed-phase chromatography. Mass spectrometry confirmed the presence of all four peptide components GasK7A α, GasK7A β, GasK7B α, and GasK7B β in the pooled preparation, including GasK7A β, identified here for the first time in natural isolates of this strain. The purified bacteriocin preparation exhibited strong antibacterial activity against indicator strain Latilactobacillus sakei NCDO 2714. Cytotoxicity testing in RAW 264.7 macrophages revealed minimal effects at concentrations up to 50µg/ml, with only a modest reduction in cell viability observed at 100µg/ml. Subsequent evaluation of immunomodulatory properties demonstrated a clear dose-dependent anti-inflammatory effect in LPS-stimulated macrophages. Treatment with gassericins K7 reduced nitric oxide (NO) production, downregulated inducible nitric oxide synthase (iNOS) expression, and suppressed secretion of the pro-inflammatory cytokines TNF-α and IL-6. Importantly, exposure of macrophages to gassericins alone did not elicit any inflammatory response. These findings provide the first evidence of anti-inflammatory activity of purified gassericins K7 in eukaryotic immune cells and highlight their potential as postbiotic components or biotherapeutic candidates for inflammatory disorders. The preparative method used in this study enables the production of gassericins K7 in quantities sufficient for advanced in vitroand in vivo investigations, facilitating future development of bacteriocin-based therapeutics.

Seven new saponins (1-7) isolated from Stenocereus eruca (Cactaceae) and four new saponins (8-11) isolated from Polaskia chichipe (Cactaceae) are described. Their structures were elucidated using high-resolution mass spectrometry and nuclear magnetic resonance analysis including 1H and 13C NMR, DEPT, HMQC, HMBC, H2BC, DQF-COSY, HSQC-TOCSY, phase sensitive TOCSY, 1D-TOCSY, phase sensitive NOESY, and ROESY experiments. One saponin required J-resolved spectroscopy experiments due to its complex 1H NMR spectrum. The inhibitory activity of Aβ aggregation, and protective effects on SH-SY5Y neuroblastoma cells against Aβ toxicity, were evaluated. Only 7 showed weak inhibitory activity of Aβ aggregation at 100 µM compared to the control group. No compounds showed obvious protective effects, but 8 possessed a very weak protective effect on SH-SY5Y cells. The acetylation of the C-16, C-22, and C-30 hydroxyl groups could be important for inhibitory activity of Aβ aggregation and protective effects on SH-SY5Y cells against Aβ toxicity.

EZH2 (enhancer of zeste homologue 2) inhibitors are an emerging class of drugs that target epigenetic regulation. However, their efficacy in solid tumours has been limited, partly due to drug-induced upregulation of fatty acid synthesis. Combining lipid metabolic modulation with EZH2 inhibition may offer a promising strategy to enhance antitumor activity. We conducted a screen of clinically approved lipid-lowering drugs to identify candidates that could enhance the efficacy of EZH2 inhibitors and found that fenofibrate significantly potentiated the antitumor effects of EZH2 inhibition. Mechanistic studies revealed that this synergistic effect was associated with the degradation of EZH2 protein. To uncover the underlying regulatory pathway, we performed mass spectrometry analysis, which identified the E3 ubiquitin ligase TRIM21 and the deubiquitinase OTUD4 as key mediators of fenofibrate-induced EZH2 degradation. Fenofibrate significantly enhanced the antitumor effects of EZH2 inhibitors in melanoma, independent of its conventional lipid-lowering function. TRIM21 and OTUD4 were identified as critical mediators of this synergistic effect. Fenofibrate disrupted the non-canonical functions of EZH2 by promoting its destabilization, thereby exerting dual effects-inhibiting EZH2 enzymatic activity and accelerating its degradation. Combination therapy with fenofibrate and EZH2 inhibitors resulted in a potent synergistic suppression of tumour growth. Our findings reveal a previously unrecognized role for fenofibrate in augmenting EZH2-targeted therapy. This study provides a novel strategy to improve the efficacy of epigenetic therapies in cancer by combining EZH2 inhibitors with fenofibrate, offering potential clinical benefits for precision oncology.

Mass spectrometry imaging (MSI) can directly detect surface analytes, enabling spatial mapping of metabolite synthesis and migration within tissues. However, MSI often lacks the sensitivity for routine analysis of low exposure, low molecular weight endogenous metabolites. Amine-containing metabolites, such as methylamine, dimethylamine, trimethylamine, lysine, dopamine, and 5-hydroxytryptamine, are widely used as medical biomarkers due to their potential biological activity or toxicity. Herein, we innovatively developed a universal in situ derivatization workflow combined with desorption electrospray ionization MS/MS (DESI-MS/MS) for mapping endogenous amine-containing metabolites, with trimethylamine (TMA) selected as a case study. For the first time, tert-butyl bromoacetate (TBBA) was used as an in situ derivatization reagent, which improved sensitivity and reproducibility by overcoming the strong matrix effect and signal instability of low molecular weight compounds in MS analysis. Our data suggested that the derivatization efficiency was affected by the alkalinity of the tissue surface, while spray/extraction solvent significantly affected the sensitivity for TMA-TBBA. Notably, DESI-MS/MS exhibited good linearity, reproducibility, and stability in the analysis of amine-containing metabolites derived from TBBA. The optimized workflow was utilized for the visual analysis of endogenous TMA distribution in the brain and serum of mice, revealing the enrichment characteristics of TMA in the cortex and hippocampus, as well as the severe accumulation of TMA in aged animals. Therefore, this targeted DESI-MS/MS-MSI approach, combined with in situ derivatization, offers novel avenues for the routine quantitation of intratissue amine-containing metabolites. In principle, this assay can be extended to a wide variety of metabolites in different biological samples.

Sodium-ion batteries (SIBs) are promising next-generation batteries as a sustainable alternative to lithium-ion systems, yet an understanding of the solid electrolyte interphase (SEI) is far from sufficient. Here, we develop a probing approach using redox mediator molecules to characterize subnanometric SEI pores, revealing that Na+ transport occurs through diffusion channels. By electrochemical analysis, differential electrochemical mass spectrometry, and theoretical calculations, the influences of solvent salts on SEI architecture have been studied. These findings offer fundamental knowledge beyond classical SEI models and provide both a powerful characterization tool and principles for electrolyte choice for SIBs.

Mass spectrometry-based spatial omics is a powerful approach for visualizing the spatial organization of proteins, metabolites, lipids, and other biomolecules in situ, combining the molecular depth of mass spectrometry with spatially resolved imaging. This systematic review traces the rapid technological and computational evolution of this field, including innovations in mass spectrometry imaging (MSI), labeling-based approaches, and proximity labeling techniques. It also highlights recent advances that enhance spatial resolution, expand molecular coverage, and enable deep molecular characterization and review analytical pipelines that integrate deep learning, cross-modality registration, and cloud-optimized data formats. From the multimodal and practical perspective, the integration of MSI with other spatial omics platforms and its transformative applications in tumor microenvironment profiling, neurodegenerative disease, developmental biology, biomarker discovery, and precision medicine are discussed. Finally, this review outlines challenges and opportunities, emphasizing the need for standardization, clinical validation, and interpretable artificial intelligence to enable broader adoption. These advances position MS-based spatial omics as a foundational pillar for multimodal spatial biology and personalized healthcare.

Background Inflammatory bowel diseases (IBD) are associated with elevated levels of proinflammatory cytokines exerting a detrimental effect on the intestinal epithelium. Since contradictory studies regarding time-dependent actions on epithelial secretion exist, intestinal organoids were treated with a mix of proinflammatory cytokines. Methods Long-term (1–5 days) effects of a cytokine mix consisting of tumor necrosis factor α, interleukin-1β and interferon-γ on murine small intestinal epithelial organoids were investigated using live cell imaging, immunofluorescent staining, qPCR, scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI), and liquid-chromatography tandem mass spectrometry (UHPLC MS/MS). Results Treatment with the cytokine mix resulted in an enlarged organoid lumen. Stimulation of Cl – secretion seems to underly this swelling since it was inhibited by bumetanide, a blocker of Na + -K + -2Cl – cotransporters. Ca 2+ imaging experiments revealed that the cytokine mix time-dependently enhanced the response to carbachol, a Ca 2+ -dependent secretagogue, which would favor Ca 2+ -dependent Cl − secretion. This was concomitant with an upregulated gene expression of inositol-1,4,5-trisphosphate receptor type 1 (IP 3 R1) and stromal interaction molecule 2 (STIM2), which is involved in store-operated Ca 2+ entry. The cytokines induced apoptosis in organoids confirmed by an increase in caspase 3-immunopositive cells. Lipidomic analysis using UHPLC MS/MS revealed an upregulation of ceramides, sphingomyelins and ether-linked lipids within the epithelial cells. AP-SMALDI MSI showed an accumulation of ceramide-positive (apoptotic) cells within the organoid lumen. Conclusion Proinflammatory cytokines induce fluid secretion and transiently upregulate Ca 2+ signaling concomitant with the induction of an apoptotic lipidome in small intestinal organoids.

Mass spectrometry (MS)-based protein footprinting and, more specifically, fast photochemical oxidation of proteins (FPOP) are methods that have been found to be important for studying proteins, their structures, and their relationships to other proteins or ligands. In-cell FPOP (IC-FPOP) was developed to study proteins in their native environment. Initial work with IC-FPOP has been performed using a platform incubator with an XY movable stage (PIXY). However, low throughput and a six-well plate format restricted the experiment by limiting the number of technical replicates that can be analyzed at one time and requiring large amounts of samples per experiment. Here, we introduce an improved, higher throughput platform that allows IC-FPOP to be run on a fully automated XY stage (AXYS) using 24-well plates. Comparison with the PIXY system results shows that this platform can successfully modify more proteins in less time. AXYS also increases the types of biological samples that can be analyzed by IC-FPOP.

Hendra virus (HeV) is a bat-adapted zoonotic henipavirus belonging to the Paramyxoviridae family. It is classified as a biosafety level 4 (BSL-4) pathogen owing to its broad host range and high fatality rate. Currently, no vaccines or therapeutics are approved for human use. Viral entry is mediated by the attachment (G) and fusion (F) glycoproteins; the heavily glycosylated G protein is responsible for receptor binding. The extracellular domain of HeV-G was expressed in Expi293F cells and its glycosylation sites and glycan composition were identified by mass spectrometry. A series of functional assays-including viral entry, receptor binding, cell-cell membrane fusion, antibody neutralization and immunogenicity-were performed to delineate the role of each N-linked glycosylation site. Glycan profiling of HeV-G identified seven N-linked and multiple O-linked glycosylation sites, revealing that the stalk residues (N72, N159) predominantly carry high-mannose glycans, whereas the head-domain N-glycan sites (N306, N378, N417, N481, N529) are primarily modified with complex glycans. Notably, among the head-domain sites, N481 also harbors a substantial proportion of high-mannose glycans. Functional assays revealed that removal of N-glycans at N159, N306 and N417 markedly reduced membrane fusion. The N159 residue is a key site for fusion triggering, and its function is tolerant to specific amino acid substitutions, which may contribute to stabilizing or facilitating the conformational cascade required for F protein-mediated membrane fusion. The N529Q mutant specifically decreased EB3 binding by 2.6-fold, correlating with reduced infectivity. Binding assays with neutralizing antibodies showed that most N-glycan deletions had negligible effects, except that N159Q and N481Q reduced affinity to nAH1.3. Immunization studies in mice demonstrated that N-glycans had minimal impact on humoral immunity, with only minor site-specific differences. These findings provide a comprehensive characterization of HeV-G glycosylation, reveal site-specific roles of N-glycans in viral entry, receptor binding and membrane fusion, and offer new insights for vaccine and antibody development.

GABAA receptors (GABAARs) mediate fast inhibitory neurotransmission in the brain and are assembled from 19 subunit isoforms into multiple pentameric assemblies. Although α1-containing GABAARs are broadly expressed and are pharmacologically important, the molecular diversity of native α1-based assemblies in specific brain regions remains incompletely understood. Here, we use immunofluorescence, mass spectrometry, and cryogenic electron microscopy (cryo-EM) to characterize the spatial distribution, subunit composition, and structural architecture of native α1-containing GABAARs in the rat cerebellum. Confocal microscopy reveals robust colocalization of α1 and γ2 subunits across cerebellar layers, including prominent labeling at glomerular synapses. Biochemical purification and proteomic analysis identify a range of α, β, and γ subunits, along with abundant α6 and δ subunits. Using cryo-EM and automated subunit identification, we resolve eight α1-containing receptor assemblies, including the first structure of α6-containing receptors. We further determine the binding mode of the α6-selective pyrazoloquinolinone modulator PZ-II-029 at the α+/γ- interface, showing ligand-induced expansion of the entire extracellular domain (ECD). Together, our study defines the structure and subunit composition of the α1-containing cerebellar GABAARs and elaborates the molecular interactions between native receptors and pyrazoloquinolinone, thereby laying the groundwork for brain region and subunit-specific pharmacology.

Antibody-drug conjugates (ADCs) are modern biopharmaceuticals that combine the therapeutic effects of small-molecule drugs with the outstanding selectivity of monoclonal antibodies (mAbs). Since their introduction in the biomedical field, research has focused on elucidating the structure, stability, and mode of action of ADCs. Nevertheless, standard characterization methods for ADCs heavily rely on disruptive techniques like mass spectrometry in a non-physiological environment. Here, we present an NMR approach combining 1H-13C ALSOFAST-HMQC and T2-edited 1H CPMG experiments, which together provide information on: i. the fingerprint and higher-ordered structure (HOS) of mAbs and ADCs and ii. the properties of the bound linker-payload fragment. In this study, we chose Trastuzumab as a well-known mAb and a Remdesivir-derived fragment as a linker-payload model system to validate our approach.

The widespread presence of pharmaceuticals, including antibiotics, in our aquatic environment raises important societal concerns. When studying their environmental fate, stable isotope analysis of nitrogen and carbon at natural abundance offers unique insight into source fingerprinting and degradation-associated kinetic isotope effects. Here, we synthesized compound-specific reference standards to enable electrospray ionization (ESI) Orbitrap mass spectrometry (MS) for fragment-specific carbon and nitrogen isotope analysis (Δδ13C and Δδ15N) of sulfamethoxazole (SMX), a most frequently detected antibiotic. Fragment-specific isotope analysis relied on fragmentation of SMX ions in the collision cell, resulting in two fragment ions representing the aniline part (m/z = 92, F92) and the 3-amino-5-methylisoxazole ring (m/z = 99, F99) of SMX. Reference materials were prepared (i) through total synthesis of SMX from labeled precursors that resulted in specific positions labeled with 13C and 15N, (ii) followed by the mixing of labeled SMX with SMX at natural abundance. The bulk isotope values of these in-house standards were determined by elemental analysis isotope ratio mass spectrometry and used for calibration of the ESI-Orbitrap-MS method. Injecting standards directly into the ESI-Orbitrap-MS resulted in 95% confidence intervals (CIs) of 0.7‰ and 3.4‰ for Δδ13C and Δδ15N in F92, respectively, and 1.3‰ and 2.9‰ for Δδ13C and Δδ15N in F99, for quintuplicate measurements of standards. A proof-of-principle demonstration shows that this approach could indeed successfully quantify changes in fragment-specific isotopic signatures, Δδ13C and Δδ15N, during degradation of SMX.

Data-independent acquisition (DIA) mass spectrometry is a technique used in proteomics to identify and quantify proteins in complex biological samples. While this comprehensive approach yields more complete and reproducible protein profiles than data-independent acquisition (DDA), the resulting data are substantially larger and more complex, presenting significant challenges for data analysis and interpretation. These challenges can be effectively addressed using dedicated workflow managers that support parallel execution of complex analysis pipelines on high-performance computing infrastructure. Nextflow, in particular, is well-suited for streamlining data analysis, as it automates key aspects of workflow management, allowing researchers to efficiently analyze large-scale data sets with minimal manual intervention. Here, we describe glaDIAtor-nf, a Nextflow version of our software package glaDIAtor for untargeted analysis of DIA mass spectrometry proteomics data. We first demonstrate its technical accuracy through rigorous testing on gold standard data sets. Building on this, we then reveal known proteome patterns from public breast cancer data that remained hidden in the processed data of the original study. This illustrates the potential of reanalyzing the accumulating public data, but also highlights the need for convenient tools to facilitate such reanalysis in large-scale.

Calotropis procera, commonly referred to as the apple of Sodom, is a perennial shrub, notable for its ecological role and extensive use in traditional medicine. This study aims to explore the phytochemical variability among different organs' extracts of C. procera, specifically stems, leaves, flowers, fruits, and seeds, using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and multivariate statistical techniques. A comprehensive analysis identified 112 distinct phytoconstituents, with unique profiles observed across the various organs' extracts. The study further assessed the anti-inflammatory potential of these extracts by examining their effects on the gene expression of key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and INF-γ) in lipopolysaccharide (LPS)-stimulated human white blood cells. Results indicate that the extracts from flowers and fruits significantly downregulated IL-1β expression to levels lower than those exhibited by piroxicam, indicating their proficient inflammation inhibitory efficacy. Orthogonal Projection to Latent Structures (OPLS) coefficient plots unraveled that methyldimethoxycinnamate, vinyloxybutanol, butanediol dibenzoate, pyroterebic acid and ursenone are the key biomarkers accountable for this efficacy. This research underscores C. procera's pharmacological significance, providing a scientific foundation for its traditional medicinal applications. The findings highlight the necessity for further investigations into the mechanistic pathways of its bioactive compounds and their implications for developing novel anti-inflammatory therapies.

The skin and oral environment represent complex microbial ecosystems that host diverse bacterial communities with potential health-promoting properties beyond the gastrointestinal tract (GIT). In this study, four bacterial and three yeast isolates were obtained from saliva (S1, S3, S5, and S6) and human skin (A1, A2, and A3) and subjected to identification and functional characterization. Phenotypic identification by API and MALDI-TOF mass spectrometry identified bacterial isolates as Limosilactobacillus sp. (S1) and Lacticaseibacillus rhamnosus (S3, S5, and S6), while the yeasts were identified as Saccharomyces cerevisiae (A1, A2, and A3). The isolates were evaluated for their functional properties, including antimicrobial activity, autoaggregation, antioxidative potential, resistance to freeze-drying, survival in simulated saliva and GIT conditions, adhesion to Caco-2 and HaCaT cell lines, and biofilm-forming ability. Lcb. rhamnosus S3 demonstrated the highest probiotic potential, characterized by strong inhibition of S. aureus, high autoaggregation capacity, substantial survival following freeze-drying, and good tolerance to simulated saliva and GIT conditions. Limosilactobacillus sp. (S1) demonstrated the strongest antimicrobial activity against C. acnes and the highest adhesion capacity to HaCaT cells, indicating its suitability for topical dermatological applications. Although S. cerevisiae isolates did not exhibit antimicrobial activity, they showed strong autoaggregation and notable antioxidant capacity. However, their low resistance to freeze-drying limits their applicability in probiotic formulation development.

This study presents a dynamic multiple reaction monitoring (dMRM) method for the simultaneous analysis of 122 polycyclic aromatic compounds (PACs), including polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, halogenated PAHs, heterocyclic PACs, and halogenated HPACs using gas chromatography-tandem mass spectrometry (GC-MS/MS). Conventional MRM methods for these complex mixtures (including the one used as our benchmark) require multiple GC injections and time segments to maintain sufficient MS dwell and cycle times. The dMRM developed here captured all our analytes in a single GC-injection. The analytical performance characteritsitc of the dMRM method was compared to our conventional time-segmented MRM methods using matrices of increasing complexity, including calibration standards, certified sediment and mussel reference materials, and an in-house fortified egg reference material. Instrument detection limits were similar for both methods and ranged from 0.1 to 1.3 pg µL-1. The dMRM method achieved comparable or improved precision and accuracy compared to conventional MRM for less complex matrices, such as standard solutions and biota. Negative systematic biases were observed for a subset of analytes in the sediment matrix for both approaches and are attributed to to non-exhaustive extractions rather than limitations of the MS methods. Provided that sample preparation is carefully optimized for challenging matrices, the dMRM technique offers a powerful tool for high-throughput environmental analysis of PACs, enabling a single GC injection to streamline laboratory workflows and enhance analytical efficiency.