Methods in molecular biology (Clifton, N.J.)Natália G Salomão, Kíssila Rabelo
Histopathological analysis of tissues infected with Chikungunya virus (CHIKV), including biopsy and autopsy specimens, can provide valuable insights into the pathogenesis of atypical and fatal cases. By examining tissue architecture and cellular alterations under the microscope, it is possible to identify patterns of injury, inflammation, and cellular degeneration. These morphological findings provide evidence of how the pathogen interacts with host cells and tissues. Moreover, immunohistochemistry may be performed in tissue sections for the detection of viral antigens. Importantly, this technique allows the characterization of diverse biologically relevant targets in tissue samples, such as distinct immune cell subsets, cytokines, and additional molecular markers. This is accomplished by the use of antibodies selected for their specificity toward the antigen of interest. In this chapter, we present a concise overview of how histological analysis and immunohistochemical approaches can enhance the understanding of CHIKV-associated pathological mechanisms.
Methods in molecular biology (Clifton, N.J.)Lorenzo Fontanelli, Carolina Ferri, Adrian Florentin Suman, Elisa Ceccherini, Giovanni Signore, Fabio Anastasio Recchia, Silvia Rocchiccioli
Cardiac organoids are increasingly used to model human cardiac development and disease, but their small size often limits molecular characterization, especially if different approaches and protocols are necessary to extract and quantify metabolites and lipids. Here, we present a multistep workflow for combined targeted free amino-acid (FAA) based metabolomic and lipidomic profiling from a single pooled cardiac organoid sample. The protocol covers organoid harvesting, detergent-assisted lysis, and a modified Folch extraction that generates an organic phase for lipid analysis and an aqueous phase for FAA metabolite analysis. Lipids are quantified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) in Multiple Reaction Monitoring (MRM) mode using class-matched external standards and an internal standard to support calibration and reduce technical variability. Free amino acids and derivatives are analyzed from the same sample after filtration, drying, and AccQ-Tag derivatization, with norvaline as an internal standard. Together, this approach maximizes information yield from limited material and enables integrated analysis of metabolic and lipid pathways within the same biological specimen, facilitating organoid-based studies of cardiac maturation, disease modeling, and pharmacological responses.
Methods in molecular biology (Clifton, N.J.)Gijs Wilbrink, Roman Pleskot, Peter Grones
The plasma membrane (PM) is the primary interface between plant cells and their environment, and its resident proteins mediate key processes such as extracellular signal perception and downstream cellular reprogramming. Yet, PM proteins are typically underrepresented in total protein extracts, and existing enrichment strategies are often laborious and require extensive optimization. Here, a simple and robust workflow is described for enriching PM proteins from Arabidopsis thaliana seedlings using total microsomal membranes obtained by differential centrifugation as starting material. Sequential low- and high-speed spins are used to isolate total microsomal membranes and progressively deplete contaminating organelles, thereby increasing the relative abundance of PM proteins. Coupled with the rich genetic toolkit available in Arabidopsis, this protocol provides an accessible platform for systematic characterization of the PM proteome.
Methods in molecular biology (Clifton, N.J.)Zhenlan Yao, Melody Li
Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.
Global genomic surveillance has emerged as a foundational pillar of public health in the twenty-first century, enabling real-time tracking of pathogen evolution and informing outbreak response. This chapter examines the strategic architecture of global genomic surveillance, focusing on its application to arboviruses such as chikungunya virus (CHIKV). It explores the integration of genomic data with epidemiological, clinical, and environmental information within a One Health framework, while addressing critical challenges in governance, equity, and interoperability. The discussion covers the entire genomic surveillance workflow, from sample collection and sequencing to bioinformatic analysis and phylogenetic inference, and highlights the transformative role of artificial intelligence (AI) in predictive surveillance. By analyzing global initiatives, operational barriers, and emerging technologies, this chapter underscores the necessity of sustainable, equitable, and interoperable genomic systems to proactively address current and future infectious disease threats.
Methods in molecular biology (Clifton, N.J.)Mark Roosjen, Dolf Weijers
Mass spectrometry-based proteomics allows the unbiased identification and quantification of proteins and phosphopeptides in biological materials. The nature of walled plant cells requires specific protocols for effective and efficient protein isolation, and, in general, the plant sciences can benefit from more accessible, optimized proteomics workflows. Advances in MS instrumentation now allow the measurement of large numbers of samples, shifting constraints in proteomics toward the accurate, high-throughput preparation of samples. Here, we describe a high-throughput (phospho)proteomics protocol that enables processing of samples using different filter types in a 96-well format.
Single-cell transcriptomics has revolutionized our understanding of cellular heterogeneity by enabling high-resolution gene expression profiling at the individual cell level. However, traditional single-cell RNA sequencing (scRNA-seq) lacks direct protein quantification, limiting comprehensive immunophenotyping. Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) overcomes this limitation by integrating antibody-derived tag (ADT) quantification with scRNA-seq, allowing simultaneous measurement of surface protein and gene expression from the same cell. This multimodal approach enhances immune cell characterization, revealing new functional states and rare subpopulations in complex biological systems. Here, we provide a detailed protocol for performing CITE-seq, from sample preparation to sequencing and data analysis. We highlight key experimental considerations, discuss challenges related to antibody selection and batch effects, and provide troubleshooting strategies to ensure robust and reproducible results. The integration of transcriptomic and proteomic data through CITE-seq provides unparalleled insights into cellular function, with broad applications in immunology, oncology, and systems biology.
Studying the transcriptome and the proteome of cells is essential for gaining a detailed understanding of cellular behavior, development, drug action, and disease progression. Spatial biology emerges to advance our ability to study the expression of molecules within tissues while preserving their natural spatial context. These cutting-edge technologies enable the mapping of thousands of individual cells in their original environment by detecting the location and biological quantity of cellular contents, such as RNAs and proteins. Here, we present a protocol to combine spatial transcriptomics (Xenium) and spatial proteomics (PhenoCycler-Fusion) within 8 days on the same tissue section to successfully study the expression of hundreds of RNAs and tens of proteins simultaneously. The combination of these two technologies and consequent integration of the two data layers together with high-resolution H&E images allows for the extraction of a maximum of information from a single tissue section. Application of this protocol and the resulting integrated data will help researchers to understand complex biological processes and disease mechanisms, supporting more nuanced research in molecular biology and pathology.
Proteomics, the large-scale study of proteins, enables the identification, quantification, and functional characterization of proteins, revealing post-translational modifications and protein interactions that are not apparent from transcriptomic data. Human organoids, which recapitulate the structural and functional complexity of native epithelial tissues, provide powerful tools to study disease mechanisms and personalize therapies. However, their culture poses challenges for efficient protein extraction and reproducible analysis. Here, we present a proteomics workflow optimized to maximize protein recovery from Matrigel-encased organoids. Samples were processed using S-Trap microcolumns to minimize losses, followed by liquid chromatography-mass spectrometry (LC-MS) in data-independent acquisition (DIA/SWATH-MS) mode for comprehensive, untargeted quantification. Library-free computational analysis using DIA-NN, combined with differential expression analysis, enabled sensitive detection of key proteins in intestinal organoids.
Methods in molecular biology (Clifton, N.J.)Samna Sagadevan, Paloma Ordóñez-Morán
Intestinal stem cell (ISC)-derived organoids provide a physiologically relevant 3D culture system to model intestinal biology, regeneration, and disease mechanisms. Their complex architecture, recapitulating crypt structures, poses challenges for downstream histological and immunohistochemical analyses, necessitating optimized processing, and staining protocols. Here, we describe robust workflows for the fixation, embedding, sectioning, and staining of ISC-derived organoids. Each method is detailed stepwise, with emphasis on preserving morphology and antigenicity. We discuss reagent choices, critical technical considerations, and troubleshooting tips to maximize data reproducibility and quality. We have integrated recent advances and best practices, serving as a comprehensive guide for researchers working with intestinal organoids.
Methods in molecular biology (Clifton, N.J.)Almudena Escobar-Niño, Olivier Coste, Maria Victoria Calcis-Marzán, Francisco Javier Fernandez-Acero
Proteomics has been revealed as a key set of technologies that provide a detailed description of the molecular processes involved in the development of a specific phenotype. "Omics" technologies can collect an incredible amount of information. Among them, proteomics is an invaluable tool for defining specific biological information by studying the complete set of proteins under specific conditions, the proteome; or specific subsets of proteins, the subproteome. It is a crucial instrument for describing protein post-translational modifications, the functional annotation of the genome, and the detection of orphan genes. Protein extraction procedures are necessary to obtain B. cinerea protein extracts of sufficient quality to be analyzed by LC-MS/MS, avoiding contaminants that interfere with the identification process. After experimental design, collect the samples and replicates as defined in each experimental approach; we will describe protocols and procedures for the next steps of proteome and subproteome extraction and LC-MS analysis.
Methods in molecular biology (Clifton, N.J.)Shivam Patel, Paloma Ordóñez-Morán
Human intestinal organoids (HIOs) recapitulate the architecture and cell diversity of intestinal epithelium, hence providing a system for modeling processes like regeneration and tumorigenesis. Here, we describe a detailed whole-mount immunostaining protocol for HIOs to visualize proliferative cell population. Briefly, this protocol includes preparation of HIOs, fixation and permeabilization of tissue, blocking of non-specific binding, antibody crosslinking, and visualization using a confocal microscope. Additionally, the protocol is broadly adaptable for investigating different antibodies, enabling the exploration of other cell signaling processes in HIO models. By eliminating embedding and sectioning steps, this method is both time-efficient and preserves spatial tissue architecture.
Rapid communications in mass spectrometry : RCMCiara J Sallowicz, Thiago Mattos, Rahul R Deshpande, Bashar Amer, Susan S Bird
RATIONALE: Untargeted lipidomics is commonly performed at analytical flow rates, which consume more solvent and may require higher on-column sample loads when sensitivity is limited by analyte abundance. Nano flow separations use lower flow rates and sample loads, reducing solvent consumption and facilitating improved electrospray ionization. We present here practical considerations for implementing a routine nano flow lipidomics workflow. METHODS: Bovine liver total lipid extract was spiked with SPLASH Lipidomix internal standards and analyzed by nano flow and high flow liquid chromatography coupled to a high-resolution accurate mass Orbitrap-based mass spectrometer. Full-scan polarity switching was used for untargeted profiling, and the AcquireX Deep Scan workflow was applied to support data-dependent MS/MS acquisition in the complex matrix. A SPLASH dilution series was analyzed in triplicate injections to compare analytical response across on-column loads between the two workflows. RESULTS: At 25 ng on-column, nano flow yielded 1266 total lipid annotations and 835 high-quality annotations, compared with 919 total and 518 high-quality annotations for high flow at 100 ng. Unintentional fragmentation decreased under nano flow conditions by 21%-31% across the representative lipid species evaluated. The use of nano flow allowed for the detection of lower on-column loads across several standards, extending the lower end of the response range by up to 40-fold compared with high flow analyses. CONCLUSIONS: Nano flow lipidomics improved sensitivity for untargeted analysis by increasing the number of lipid annotations, reducing unintentional fragmentation, and extending the analytical response to lower on-column loads. Together with practical guidance around sample preparation, injection volume, washing, and equilibration, these results support nano flow chromatography as a sensitive and reliable approach for sample-limited untargeted lipidomics.
Rapid communications in mass spectrometry : RCMMarco Rolando Aronés Jara, Kirianova Godoy Bautista, Freshsia Ingrid Ortiz Pérez, Edgar Cárdenas Landeo, Edith Eveling Conislla Cáceres, Hugo Roberto Luna Mole…
RATIONALE: Lepidoceras peruvianum Kuijt is an understudied hemiparasitic species endemic to the Peruvian Andes that lacks comprehensive phytochemical characterization. Investigating its metabolome is important for understanding its chemotaxonomic relevance and potential as a source of bioactive compounds. METHODS: Leaves and fruits of L. peruvianum were subjected to untargeted metabolomic profiling using high-resolution UHPLC-ESI-Orbitrap-MS/MS in positive and negative ionization modes. Metabolites were annotated based on accurate mass measurements, isotopic patterns, collision-induced dissociation (CID) fragmentation data, and spectral matching with the Global Natural Products Social Molecular Networking (GNPS), METLIN, and MassBank databases. Structural elucidation employed diagnostic fragmentation pathways, including retro-Diels-Alder (RDA), heterocyclic ring fission (HRF), quinone methide (QM), and benzofuran-forming (BFF) cleavages. Antioxidant activity was evaluated using DPPH●, ABTS●+, and FRAP assays, while antimicrobial activity and toxicity were assessed through antibacterial testing and the Artemia salina lethality assay. RESULTS: Leaves contained 34 metabolites, mainly flavan-3-ols, proanthocyanidins, flavonols, lignans, and isoquinoline alkaloids, whereas fruits were characterized by anthocyanins, organic acids, and polar lipids. Total phenolic content was higher in leaves (292.4 mg GAE/g) than in fruits (216.8 mg GAE/g), corresponding to stronger antioxidant activity. Leaves extracts exhibited greater antibacterial activity against Staphylococcus aureus, while fruit extracts showed stronger effects against Gram-negative bacteria. LC50 values in the A. salina assay were 181.8 μg/mL for leaves and 475.9 μg/mL for fruits. CONCLUSIONS: This study provides the first comprehensive metabolomic characterization of L. peruvianum, revealing organ-specific chemical diversity and notable bioactive properties. The findings demonstrate the utility of HRMS/MS fragmentation analysis for metabolite annotation and identify this endemic species as a promising source of bioactive natural products.
Rapid communications in mass spectrometry : RCMJianping Liu, Ningxing Duan, Tian Xin, Yang Cao, Haiyan Qian
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a leading cause of death, underscoring the need for improved therapies. Xuanfei Heji (XFHJ), a hospital-prepared herbal formula, has been used clinically in the treatment of COPD. However, its mechanisms remain unclear. METHODS: XFHJ constituents were profiled using UHPLC-HRMS. COPD was induced in rats by intratracheal lipopolysaccharide instillation and cigarette smoke exposure. Treatment effects were assessed using pulmonary function, lung histopathology, and proinflammatory cytokines. Untargeted serum metabolomics and fecal 16S rRNA gene sequencing were performed; associations among differential metabolites, microbial taxa, and inflammatory markers were evaluated using Spearman's rank correlation analysis. RESULTS: Chemical profiling tentatively identified 374 constituents. XFHJ improved pulmonary function and attenuated lung histopathological injury and inflammation. Tryptophan and glycerophospholipid metabolism were the principal treatment-associated pathways. XFHJ also altered gut microbial diversity and composition, with enrichment of potentially beneficial taxa such as Bifidobacterium, Roseburia, and several Clostridia-related taxa. Treatment-responsive taxa correlated positively with indole-related metabolites, which correlated inversely with pulmonary inflammatory markers. CONCLUSIONS: XFHJ exhibited significant therapeutic effects on COPD rats, and its mechanism may be correlated with regulating the intestinal microbiota structure and metabolic profiles of COPD rats, thereby attenuating lung histopathological injury and pulmonary inflammation.
Veterinary medicine and scienceHossein Lak, Amir Amniattalab
BACKGROUND: Mammary tumours are the most common neoplasm in female dogs. Canine mammary tumours (CMTs) are appropriate models for human studies. OBJECTIVES: The present study aimed to diagnose CMTs pathologically and compare different tumour types by immunohistochemistry (IHC). METHODS: Histological features of various tumours were recorded. Moreover, haematological, biochemical and immunohistochemical changes were evaluated in affected animals. RESULTS: Among 130 mastectomized dogs in veterinary hospitals in Tehran, Iran, 74 dogs were affected by various types of mammary tumours. The highest frequency of malignant tumours was related to carcinoma types (38/74; 51.4%) and the highest frequency of benign tumours was related to fibroadenoma (6/74; 8.1%). In malignant tumours, anaemia and monocytosis were observed compared to benign tumours. Serum ALT and ALP levels were also significantly increased in malignant tumours (p < 0.05) compared to benign tumours. The IHC showed the profile of adenoma (ductal and intraductal papillary) ER+/PR-/HER2-, benign mixed tumour ER-/PR-/HER2-, carcinoma (mucinous, anaplastic) ER-/PR-/HER2- and metastatic mast cell tumour ER-/PR+/HER2-. The highest expression of αSMA was recorded in benign tumours and the highest expression of Ki67 was recorded in metastatic mast cell tumour, benign mixed tumour and adenoma (p < 0.05). CONCLUSION: Together, although the dog is a good model for studying mammary tumours, the variability of results in CMTs requires further studies in this field. An interesting finding of this study was the triple-negative profile in the benign mixed tumour, which had previously been reported only for malignant mammary tumours. In addition, the significant positivity of Ki67 expression (34%-66%) in benign CMTs is noted for the first time.
Biomedical chromatography : BMCGuangliang Zhang, Wentao Shao, Junbin Shu, Jianfeng Tong, Jing Zhang, Yi Tao
Brucea javanica oil oral emulsion (BJOE) serves as an adjunctive therapeutic agent for glioblastoma multiforme (GBM); however, the inhibitory effects of its serum metabolites remain unexplored. In this study, ultrahigh performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was employed to analyze serum metabolites in rats following BJOE administration. The inhibitory effects of the serum on GBM were assessed using U251 cell bioassays, and the relationship between serum metabolites and cellular inhibitory activity was examined. Six prototype components and six metabolites were identified in the serum, including linoleic acid, linoleic acid-M1, linoleic acid-M2, linoleic acid-M3, oleic acid, oleic acid-M1, oleic acid-M2, oleic acid-M3, sphingosine, palmitoleic acid, oleamide, and 1-stearoyl-rac-glycerol. Rat serum collected 2 h postdosing significantly inhibited U251 cell proliferation. Gray relationship analysis indicated that oleic acid, oleic acid-M1, linoleic acid, linoleic acid-M1, linoleic acid-M2, sphingosine, palmitoleic acid, oleamide, and 1-stearoyl-rac-glycerol were highly correlated with the inhibition rate of U251 cell proliferation, with oleic acid-M1 showing the highest correlation coefficient (0.9806). These findings suggest that BJOE and its metabolites may possess therapeutic potential against GBM, particularly in inhibiting glioma cell proliferation, with oleic acid and its metabolites likely playing a significant role.
Journal of inherited metabolic diseasePatryk Lipiński, Jaak Jaeken
Congenital disorders of glycosylation (CDG) are a rapidly expanding group of inherited metabolic diseases affecting glycoconjugate glycan biosynthesis and attachment. This review provides a structured overview of major advances in human CDG reported from 2023 to 2026, including newly recognized CDG, emerging biochemical and pathophysiological insights, advances in diagnostic strategies and biomarkers, and progress in treatment. Recent developments include the recognition of RPN1-CDG, UGGT1-CDG, and DHRSX-CDG, refinement of phenotypes in established disorders, including PMM2-CDG, ALG13-CDG, ALG8-CDG, GMPPA-CDG, TRAPPC11-CDG, and PGAP3-CDG, and increasing use of glycomics, glycoproteomics, cellular models, and multi-omics approaches. Therapeutic progress remains uneven: no FDA/EMA-approved disease-modifying therapy is currently available for any CDG, and most interventions remain off-label, experimental, or supported by limited observational evidence. Despite substantial progress, most CDG still lack disease-modifying treatment, validated biomarkers, and prospective natural history data.
GliaRobert W Lewis, Breana C Dogan, Amy L Stanek, Elliot B Evans, Madelyn G Coble, Hayli E Spence-Osorio, Karen L G Farizatto, Angie L Mordant, C Allie Mills, Laur…
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy characterized by early-onset macrocephaly, white matter edema, seizures, and motor and cognitive decline. Approximately 25% of MLC patients carry HEPACAM pathogenic variants, many of which are dominant missense variants causing remitting MLC Type 2b. HEPACAM encodes hepatic and glial cell adhesion molecule (hepaCAM), also known as GlialCAM, an astrocyte-enriched transmembrane protein with important roles in astrocyte territory establishment, gap junction coupling, branching organization, synaptic function, and development of the gliovascular unit. The molecular mechanisms through which pathogenic variants in HEPACAM alter hepaCAM protein function in vivo and facilitate MLC pathogenesis during brain development remain largely unknown. Here, we used new viral tools and proximity-based proteomics to examine how three different dominant pathogenic variants alter hepaCAM subcellular localization and protein interactome in astrocytes of the developing mouse cortex. We found dramatic changes in hepaCAM distribution throughout the astrocyte, which were common to all mutants tested. We also observed significant changes in protein interactome between wild type and mutant hepaCAM, including decreased association with previously described hepaCAM-interacting proteins Connexin 43 and CLC-2. Moreover, we identified the epilepsy-associate potassium channel KCNQ2 as a novel hepaCAM interaction partner and found reduced association between KCNQ2 and pathogenic variants. Collectively, our data provide new insights into hepaCAM protein function in astrocytes during brain development, reveal altered protein dynamics of pathogenic variants, and provide a new resource to explore the molecular underpinnings of MLC pathogenesis.
Epidermal growth factor receptor (EGFR), cellular mesenchymal-epithelial transition factor (c-MET), and B7 homolog 3 (B7H3) are important targets for antibody-based drug development in colorectal cancer (CRC). To overcome the limitations of single-target antibody therapies - such as limited efficacy, widespread resistance, narrow patient populations, and treatment-related toxicities - various bispecific antibodies or antibody-drug conjugate (ADCs) targeting two targets are being extensively validated in clinical settings. A deeper understanding of their expression profiles and co-expression patterns of actionable therapeutic targets may guide more effective treatment strategies. We evaluated the immunohistochemical expression of B7H3, c-MET, and EGFR in a cohort of 193 CRC patients. Among which, B7H3 exhibited the highest positivity rate (80.83%) in CRC tissues, followed by c-MET (77.20%) and EGFR (47.15%). The positive rates of B7H3 showed no significant differences across patient gender, age, TNM stage, differentiation grade, or tumor site. In contrast, the c-MET positivity rate was elevated in patients with stage I-II disease, and the expression of EGFR was significantly higher in female patients than in male patients. Concurrent assessment of the three targets in metastatic lesions revealed that B7H3 expression levels were comparable between primary and metastatic tumors, whereas c-MET expression was significantly higher in primary lesions. Co-expression analysis indicated that the group with the highest patient coverage was the B7H3/c-MET dual-positive group, followed by the B7H3/EGFR, and c-MET/EGFR combinations, and the proportion of patients with at least one positive target exceeded 80%. Finally, mRNA profile from TIMER database reveals that CD276 and MET expression in CRC tumor tissues were higher than those in the majority of normal tissues. Therefore, utilizing B7H3/c-MET combination therapies that integrate intracellular signaling inhibition with immune checkpoint modulation could potentially extend therapeutic benefits to a wider range of CRC patients.
Acta physiologica (Oxford, England)Jorge Andrés Narváez Pardo, Sofía Bajicoff, Leonie Gerling, Ernest Oppong-Danquah, Zahraa Jaber, Anke Schumann, Luciana Hannibal, Fernando Pablo Dominici
AIM: Despite increasing evidence for the cardiometabolic protective effects of angiotensin II Type 2 receptor (AT2R) activation, the underlying molecular mechanisms remain incompletely understood. This study aimed to determine how AT2R activation modulates cellular metabolism and to identify pathways relevant to its tissue-protective actions. METHODS: 3T3-L1 adipocytes, endothelial cells, and renal proximal tubular epithelial cells were treated with the specific AT2R agonist Compound 21 (C21). Targeted liquid chromatography-tandem mass spectrometry was used to quantify amino acids and metabolites involved in energy metabolism and redox homeostasis. Functional analyses included nitrate/nitrite determination and cystathionine β-synthase (CBS) activity. RESULTS: Compound 21 induced shared and cell type-specific metabolic changes. Arginine, citrulline, glucose, and glyceraldehyde 3-phosphate increased consistently across all cell types, whereas citrate decreased. Sulfur-containing metabolites and glutathione-related intermediates increased predominantly in adipocytes and renal cells. Pathway analysis highlighted three metabolic axes affected by C21: arginine biosynthesis, central carbon metabolism, and amino acid-linked redox pathways. Functionally, C21 increased nitrate/nitrite accumulation in BAECs and CBS activity in RPTECs. CONCLUSION: Treatment with C21 was associated with a conserved metabolic signature across adipocytes, renal epithelial cells, and endothelial cells involving arginine biosynthesis, central carbon metabolism, and amino acid-linked redox homeostasis. While the response differed among cell types, coordinated changes in arginine, citrulline, glucose, glyceraldehyde 3-phosphate, citrate, and GSH-related metabolites suggest that AT2R signaling is associated with cellular pathways involved in nitric oxide metabolism, energy metabolism, and redox homeostasis. Increased NOx production in BAECs and CBS activity in RPTECs support the cell type-specific metabolic effects of C21.
Huoxue Huayu Gel Plaster (HXHYGP) is a topical traditional Chinese medicine preparation used for oxaliplatin-induced peripheral neuropathy (OIPN), but its locally exposed bioactive components remain unclear. This study developed an integrated strategy combining pharmacodynamic evaluation, UHPLC-HRMS-based tissue pharmacochemistry, untargeted metabolomics, network pharmacology, molecular docking, and qRT-PCR to identify effective components and explore potential mechanisms. In OIPN rats, HXHYGP improved mechanical and cold sensory dysfunction; reduced plantar tissue injury and inflammatory-cell infiltration; increased nerve growth factor levels; decreased TNF-α, IL-6, and IL-1β levels; and enhanced nerve conduction velocity. A total of 496 constituents were detected in skin tissue, including 20 tissue-migrant constituents attributed to HXHYGP. Untargeted metabolomics identified 53 OIPN-related differential metabolites, of which 42 were significantly reversed after treatment. Correlation analysis and network pharmacology further identified 14 effective components and 98 common targets, with EGFR, PIK3CA, MTOR, RELA, and MAPK14 as key nodes. Molecular docking supported potential component-target interactions, while qRT-PCR confirmed reduced mRNA expression of these targets after HXHYGP treatment. These findings indicate that HXHYGP may alleviate OIPN by modulating the proposed EGFR/PI3K/AKT/mTOR/NF-κB signaling network and MAPK14-associated inflammation. This strategy provides an analytical basis for identifying pharmacodynamic substances and potential quality-marker candidates in topical traditional Chinese medicine preparations.
Rapid communications in mass spectrometry : RCMTing-Yu Wei, Joao A Paulo
RATIONALE: Multiplexed quantitative proteomics enables simultaneous analysis of multiple biological samples, increasing throughput while reducing instrument time and sample requirements. However, integrating sample multiplexing with data-independent acquisition (DIA) remains challenging. We present a TMTpro plexDIA strategy leveraging MS1-level mass differences between nonisobaric TMTpro reagent variants to enable multiplexed quantification without compromising DIA sensitivity. METHODS: Three Saccharomyces cerevisiae deletion strains (Δmet6, Δpfk2, and Δura2) were labeled with TMTproZero (light), TMTpro16 (standard), and super-heavy TMTpro (heavy), mixed in three permutations, and analyzed by narrow-window DIA (2 m/z isolation, 300 scan events) on an Orbitrap Astral mass spectrometer. Database searching was performed using FragPipe/MSFragger with plex-DIA quantification enabled. RESULTS: Over 2000 protein groups and over 20 000 precursors were identified per channel per mixture, with closely matched identification rates across all three channels. All nine expected deletion patterns were correctly identified, with channel-specific depletion reproduced consistently across precursor charge states (2+, 3+, and 4+). CONCLUSIONS: TMTpro plex-DIA enables accurate, multiplexed quantitative proteomics through MS1-level mass separation of the nonisobaric TMTpro isotopologs. The characteristic deletion patterns observed for each knockout strain serve as intrinsic molecular barcodes, validating sample identity and demonstrating the broad utility of plex-DIA for high-throughput, multiplexed proteomics applications.
Salt stress is a major environmental factor limiting crop growth and productivity. We found a long-chain acyl-CoA synthetase (LACS) gene ZmLACS9 that was associated with salt tolerance traits in maize (Zea mays). Mutation of ZmLACS9 impaired plant growth, while overexpression of ZmLACS9 improved plant growth vigour under salt stress conditions. Metabolome and transcriptome analyses indicated that ZmLACS9 may regulate the expression of lipid trafficking-related genes, thereby influencing the concentrations of galactolipids and phospholipid in the photosynthetic membrane under salt stress. Under salt stress conditions, ultrastructural observation and chlorophyll fluorescence parameters showed that the mutation of ZmLACS9 led to significant impairment of the number of thylakoid layer structures and PSII activity, while overexpression of ZmLACS9 markedly improved the number of chloroplast thylakoid grana lamella and PSII activity. Furthermore, yeast one-hybrid and LUC transient expression assays found that ZmWRKY17 could bind to the promoter of ZmLACS9. Collectively, our study provides candidate genes for breeding maize varieties with higher stress resistance.
BACKGROUND: Claudin 18.2 (CLDN18.2) is a promising therapeutic target in gastric/esophagogastric junction adenocarcinoma (GC/EGJC), but its dynamic expression during systemic therapy is unclear. METHODS: CLDN18.2 expression by immunohistochemistry in paired tumor samples from 155 patients with advanced GC/EGJC was retrospectively evaluated before and after systemic therapy. CLDN18.2-positive was defined as ≥75% of viable tumor cells exhibiting moderate-to-strong (2+ or 3+) membranous staining. RESULTS: Among all patients, 54 (34.84%) were CLDN18.2-positive. Following treatment, the proportion of CLDN18.2-positive patients increased significantly to 61 (39.35%) (p < .001). Among patients who were CLDN18.2-negative at baseline, 57 (56.44%) showed increased expression after treatment, including 22 (21.78%) who converted to positive status. In contrast, among those CLDN18.2-positive at baseline, expression decreased in 35 patients (64.81%), with 15 (27.78%) converting to negative status (p < .001). Among patients receiving chemotherapy alone (n = 25), 1 (4.00%) converted to negative and 5 (20.00%) converted to positive, whereas among those receiving chemotherapy combined with immunotherapy (n = 130), 14 (10.77%) converted to negative and 17 (13.08%) converted to positive (p = .45). A total of 131 patients (84.52%) demonstrated changes in CLDN18.2 expression following therapy, encompassing both conversions across the positivity threshold and fluctuations within the same classification. CONCLUSION: CLDN18.2 expression in GC/EGJC is dynamic and frequently altered after systemic therapy. These findings support reassessment of CLDN18.2 status before initiating CLDN18.2-targeted therapies.
Rapid communications in mass spectrometry : RCMEssy Harnelly, Alya Munira, Ayyagari Ramlal, Sreeramanan Subramaniam, Khairan Khairan
INTRODUCTION: Patchouli is an aromatic medicinal plant. The essential oil from patchouli is widely used in the perfume and pharmaceutical industries. In vitro culture systems provide a sustainable platform for increasing biomass and secondary metabolite exploration; however, the role of light quality in regulating these responses in Tapak Tuan patchouli callus remains underexplored. OBJECTIVE: This study compared the effects of blue, red, and white light-emitting diodes (LEDs) with fluorescent light as the control on patchouli callus cultures. RESULTS: After 8 weeks, blue LEDs produced the highest callus fresh weight (7.34 ± 0.29) and promoted early organogenic responses, with shoot primordia observed in all explants, although complete plantlets were not obtained during the culture period. Red LED treatment showed the strongest qualitative flavonoid reaction based on phytochemical screening. Further GC-MS-based metabolite profiling, expressed as relative peak area (%), indicated that red LED-treated callus had the highest number of detected compounds, including several treatment-associated compounds, whereas blue LED-treated callus showed the presence of pogostone, a pharmacologically relevant metabolite. These results indicate that different light treatments, representing both spectral and PPFD differences, were associated with distinct callus growth responses and metabolite profiles in patchouli callus. CONCLUSION: The integration of qualitative phytochemical screening and descriptive GC-MS profiling provides preliminary insights into LED-associated variation in patchouli callus cultures and highlights the potential use of targeted lighting strategies for optimizing biomass production and metabolite profiling. Future studies using standardized PPFD, replicated GC-MS analysis, internal standards, and quantitative biochemical assays are needed to validate these findings and clarify the underlying mechanisms.
Chembiochem : a European journal of chemical biologyStefania De Chiara, Angela Di Somma, Valentina Mazziotti, Serena Coppola, Franca Oglio, Lidia Tammaro, Maria Pia Riccio, Carmela Bravaccio, Antonio Molinaro, R…
The etiology of pediatric autism spectrum disorder (ASD) has been increasingly linked to alterations in the gut-brain axis, highlighting the intricate bidirectional communication between gut microbiota and central nervous system. The molecular mechanisms of this communication are poorly understood. Here we investigated whether ASD-associated gut microbiota could exhibit altered inflammatory molecular outputs by integrating chemistry-driven profiling of fecal lipopolysaccharides (LPS), functional bacterial proteomics, and neuroimmune cellular assays. Structural analyses revealed that LPS from non-autistic healthy donors (NASD) displayed highly conserved carbohydrate and lipid A signatures dominated by hypo-acylated mono-phosphorylated species typically associated with immunomodulatory Bacteroides-derived LPS. In contrast, LPS from ASD children exhibited increased structural heterogeneity. These molecular alterations were functionally reflected in human HMC3 microglial cells, where ASD-derived fecal LPS induced stronger IL-6 and IL-8 release compared with NASD-derived LPS, indicating enhanced neuroinflammatory potential. Functional proteomic profiling disclosed broadly comparable microbial compositions but marked metabolic divergence. These findings suggest that gut microbiota in ASD children are associated with a functionally heterogeneous microbial ecosystem characterized by altered immunostimulatory molecular outputs despite the absence of massive taxonomic shifts, with potential relevance for neuroimmune dysregulation.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseasesC Z Zhang, S S Lyu, X D Han, H Wang, Y Sun, X D Liu
Objective: To investigate the molecular epidemiological characteristics, antimicrobial resistance mechanisms, and clinical risk factors for infection with carbapenem-resistant Klebsiella pneumoniae (CRKP), and to examine the relationship between antimicrobial resistance and virulence. Methods: A total of 528 Klebsiella pneumoniae (KP) isolates and corresponding clinical data were collected from hospitalized patients at Qingdao Municipal Hospital between November 2016 and October 2023. Based on antimicrobial susceptibility testing results, 80 patients with CRKP infection were assigned to the CRKP group. Patients with carbapenem-susceptible K. pneumoniae (CSKP) isolates recovered within 7 days before or after the CRKP isolation date were selected as matched controls for each patient in the CRKP group. If insufficient eligible controls were available, the matching window was expanded to 30 days before or after the isolation date. Ultimately, 160 matched patients were included in the CSKP group. Whole-genome sequencing was performed on all included isolates to systematically characterize their molecular epidemiological features and resistance and virulence gene profiles. A multivariable logistic regression model was used to identify independent risk factors for CRKP infection. Spearman rank correlation analysis was performed to assess the correlation between resistance and virulence gene scores. Results: Multivariable logistic regression analysis showed that a history of antimicrobial agent use in the 30 days before infection (OR=4.411, 95%CI: 1.749-11.128; Wald χ²=9.882; P<0.01) and invasive abdominal procedures in 30 days before infection (OR=6.846, 95%CI: 3.141-14.922; Wald χ²=23.411; P<0.01) were independent risk factors for CRKP infection. ST11 was the predominant sequence type among CRKP isolates (80.0%), and the blaKPC-2 gene was the predominant resistance determinant (88.8%). The predominant CRKP clone, ST11, underwent a serotype transition from ST11-K25/O5 to ST11-K64/O2a. At the overall population level, the antimicrobial resistance gene score was negatively correlated with the virulence gene score (Spearman ρ=-0.321, P<0.001). However, subgroup analysis showed no significant correlation between the two scores within the same ST11 clone (Spearman ρ=0.020, P=0.872). These findings suggest that the overall negative correlation was a population-level artifact caused by inherent phenotypic differences among distinct clonal groups, rather than an evolutionary trade-off in which the acquisition of antimicrobial resistance within the same clone resulted in reduced virulence. The intensive care unit (ICU) was the central hub for nosocomial clonal transmission of CRKP. The predominant clone may have disseminated from the ICU to general wards through patient transfers, healthcare workers, or medical equipment, thereby forming interdepartmental transmission chains. Conclusions: A history of antimicrobial agent use and invasive abdominal procedures in the 30 days before CRKP infection were independent risk factors for CRKP infection. CRKP isolates frequently carried plasmids coharboring multiple antimicrobial resistance determinants and exhibited extensive antimicrobial resistance.
Life science allianceLaura Alunno, Ilaria Massignani, Meriem Hadjer Hamadou, Fabio Mazza, Daniele Peroni, Romina Belli, Erik Dassi, Alessandro Romanel, Alberto Inga
To prioritize germline genetic variants affecting mRNA fate at the translational level, we used sucrose gradient-based isolation of 80S monosomes and polysomes, followed by RNA sequencing in RPE-1 cells, under mock and nutlin treatment conditions. Differential gene expression analysis confirmed a canonical p53 response. Heterozygous SNPs and SNVs were identified from the RNA-sequencing data, and allelic fractions were calculated for total, monosomal, and polysomal mRNAs. Variants showing reproducible allelic fraction differences across fractions beyond experimental variability were defined as tranSNPs. Among over 7,000 heterozygous variants analyzable in polysomal RNA and over 5,000 in mRNAs associated with monosome-enriched fractions, 1,247 in 1,015 genes displayed a significant imbalance. Reporter assays performed in RPE-1 and HCT116 cells validated allelic or haplotype effects for 17 selected variants in UTRs and coding regions, confirming differences in 15 cases, with evidence of cell line-specific responses. Proteomic analysis supported allelic imbalance for a few selected missense variants. Monosome-enriched fractions improved sensitivity in measuring allelic imbalances without introducing a tranSNP positional bias, suggesting that 80S profiling enhances detection of allele-specific translational regulation in RPE-1 cells.
Life science allianceSarah L Pashley, Molly Hair, Chiamaka V Ukegbu, Mohammad Zeeshan, Akancha Mishra, Declan Brady, Robert Markus, Sue Vaughan, Carla Pasquarello, Anthony A Holder…
Plasmodium parasites rely on diverse protein kinases to regulate critical processes, yet many remain uncharacterised. Protein kinase 2 (PK2) is required for asexual blood-stage proliferation and merozoite invasion, but its role in sexual stages and transmission is unknown. Here, using live-cell and super-resolution imaging, ultrastructural analysis, functional phenotyping, and quantitative phosphoproteomics, we define the role of PK2 in Plasmodium berghei transmission. PK2 was expressed in merozoites, ookinetes, and sporozoites: the three invasive forms of the parasite. Conditional depletion of PK2 impaired ookinete motility, disrupted apical microneme positioning, abolished oocyst formation, and prevented mosquito-to-host transmission. Direct haemocoel injection of mutant ookinetes failed to restore sporozoite production, demonstrating an additional requirement for PK2 downstream of midgut traversal. Although global protein abundance was largely preserved, phosphoproteomic profiling identified altered phosphorylation of proteins associated with cytoskeletal organisation, host-cell interaction, midgut invasion, and gene regulation. These findings highlight PK2's critical role across multiple invasive stages, advancing understanding of malaria parasite transmission and identifying putative PK2-regulated pathways as potential targets for transmission-blocking interventions.
The global prevalence of Metabolic dysfunction-associated steatotic liver disease (MASLD) among persons with HIV (PWHIV) is estimated at 34%. This study examined the influence of gut microbial and metabolic alterations in PWHIV with MASLD. We conducted 16S rRNA sequencing on 60 fecal samples (24 from HIV group and 36 from HIV-MASLD group) to assess microbial composition, and employed LC/MS-based metabolomics on both fecal and plasma samples to identify variations in metabolites. An exploratory analysis was performed to compare plasma cytokines and oxidative stress markers between the two groups. Sequencing in the HIV-MASLD group showed lower Faecalibacterium and Anaerobutyricum, and higher Klebsiella, Escherichia, and Enterococcus. Metabolomic analysis revealed a reduction in anti-inflammatory metabolites (Dodecanedioic acid, 5-Phenylvaleric acid, Kynurenic acid) and antioxidant metabolites, coupled with an increase in the pro-inflammatory metabolite Gamma-Glutamyl-L-Putrescine in the HIV-MASLD group. And plasma analysis indicated a decline in 20-carboxy arachidonic acid. Additionally, Faecalibacterium and Anaerobutyricum correlated positively with anti-inflammatory metabolites, while Klebsiella, Enterobacter, and Citrobacter correlated negatively. Furthermore, Faecalibacterium and Anaerobutyricum also showed a moderate negative correlation with MDA levels. A random forest model using plasma metabolites showed the optimal discriminative ability for HIV-MASLD (AUC = 0.819 ± 0.0059). Differences in gut microbiota and metabolites in PWHIV may affect MASLD progression by influencing inflammation and oxidative stress. Modulating gut microbiota, such as adding Faecalibacterium and Anaerobutyricum, could be a treatment strategy for HIV-MASLD. Plasma metabolites have potential as noninvasive markers.
Aging cellShangshu Zhao, Chia-Ling Kuo, Eric J Lenze, Julie L Wetherell, Laura Haynes, Perla El-Ahmad, Richard Fortinsky, George Kuchel, Trevor Harris, Breno S Diniz
The accumulation of senescent cells is a recognized hallmark of biological aging and is associated with the onset of multiple chronic medical conditions. Senescent cells exhibit a distinct secretory profile, known as the senescence-associated secretory phenotype (SASP), which can propagate cellular senescence to neighboring and distant tissues. Measuring SASP factors in blood serves as a practical proxy for cellular senescence burden and may help track disease states and intervention outcomes. We developed and validated a composite SASP Score by integrating large-scale population proteomics data with a semi-supervised deep learning framework. The analytical workflow included: (1) selection of biologically curated SASP proteins; (2) development of a Guided autoencoder with Transformer (GAET) model using data from the UK Biobank Pharma Proteomics Project (UKB-PPP); (3) internal evaluation and association analyses within the UK Biobank; and (4) external validation and longitudinal assessment in an independent randomized clinical trial cohort. The deep learning-based SASP Score was a strong, independent predictor of mortality risk and incident serious, chronic medical conditions (e.g., dementia, COPD, myocardial infarction, stroke). In an independent cohort, multimodal exercise significantly changed the SASP Score trajectory over 18 months. Our findings support the potential of a deep learning-derived SASP Score as a biomarker for systemic cellular senescence burden. Our statistical approach can offer enhanced interpretability and cross-platform utility, providing a valuable tool for aging research and the evaluation of geroscience-guided interventions.
The clinical teacherOrlando Carlos Conceição-Neto, Frederico Medeiros Rosas da Silva, Erika Verissimo Villela, Paula Borges de Andrade Lemes, João Pedro Gotti Ferreira, Chiara Gur…
BACKGROUND: Medical biochemistry and molecular genetics stand out as some of the most demanding courses for first-year medical students, who often require repeated exposure to complex pathways and molecular processes. To provide additional support in this early stage of training, we created PodBases, a peer-led educational podcast integrated into an academic mentoring program and made available through a widely accessible global audio-streaming platform. The goal was to offer brief, clinically oriented episodes that reinforced key concepts and complemented traditional teaching. APPROACH: The initiative followed a defined workflow involving close collaboration between student tutors and faculty. Tutors prepared scripts aligned with the weekly course content, which were subsequently reviewed for accuracy. Recordings were made using smartphones and edited with freely accessible audio-editing tools and published on a podcast-hosting service. We documented the entire process to create a practical tutorial that facilitates replication in similar educational settings. EVALUATION: Between May 2023 and January 2026, 30 episodes were published, reaching 13,050 streams across 16 countries. Student feedback highlighted the usefulness of the podcast during commutes and study sessions. Mentors reported skill development in communication, simplification of content and teamwork. IMPLICATIONS: PodBases demonstrates that student-generated podcasts can effectively complement traditional teaching by reinforcing core topics, supporting learner autonomy, enhancing comprehension and developing tutors' pedagogical skills. The experience suggests that peer-led digital resources can enhance comprehension while promoting engagement and professional growth in the foundational years of training. TRIAL REGISTRATION: Not applicable.
Life science allianceZsolt Szilagyi, Pauline Michon, Carlos Pardo-Hernández, Mãdãlina Sãcultanu, Javier Miralles Fusté, Andrew M Griffin, Paul S Charifson, Cindy Phan, Yonghong Shi…
Lon protease 1 (LONP1) is a conserved hexameric protease implicated in mitochondrial disorders and cancer progression. In this study, we present PZL-26, a potent and selective small-molecule inhibitor that targets LONP1 without affecting the proteasome, leading to selective accumulation of mitochondrial proteins. Using PZL-26 in a whole-genome CRISPR-Cas9 screen, we identified genes essential for cell survival under protease inhibition, supporting a role for LONP1 in key mitochondrial processes, including complex I biogenesis, mitochondrial transcription, and translation. Our CRISPR screen results are consistent with proteomics analysis, with both approaches converging on the same mitochondrial pathways and highlighting functional interactions between LONP1 and other mitochondrial proteases, including potential compensatory mechanisms. These findings establish PZL-26 as an effective tool for exploring LONP1 function and pave the way for future therapeutic strategies targeting LONP1 in mitochondrial diseases and cancer.
Journal of medical virologyNicole Lardini Freitas, Yago Côrtes Pinheiro Gomes, Fábio César Sousa Nogueira, Juliana Echevarria-Lima, João Victor Carvalho Deus, Carlos Otávio Brandão, Cris…
Pathophysiological mechanisms of neurological manifestations in COVID-19 are not fully known yet. In this case-control study, proteomic analysis was used to characterize cerebrospinal fluid (CSF) samples from COVID-19 patients with acute neurological manifestations primarily categorized as: isolated refractory headache (n = 12), encephalopathy (n = 24), and inflammatory neurological diseases (IND) (n = 13). Individuals with non-inflammatory, non-infectious neurological conditions (n = 8) were included as controls. Differentially expressed proteins (DEPs) in neuro-COVID-19 were associated with the complement system, function/activation of microglia/macrophages, inflammatory responses, and neuronal function/homeostasis. Functional enrichment analysis of DEPs and/or proteins specifically detected in neuro-COVID-19 groups indicated activation of complement and coagulation cascades, and synapse pruning in encephalopathy and IND. Upregulated complement activation was confirmed by increased CSF levels of C3a and soluble C5b-9. Biological processes of neuroinflammation, migration of immune cells into the central nervous system, activation of glia cells (microglia and astrocytes), and tissue remodeling were also present in IND. In turn, processes associated with disturbed organization/formation of neuron projections were related to encephalopathy and isolated headache. CSF from patients with isolated refractory headache was enriched for proteins related to neurons and to the cerebral cortex, cerebellum, and basal ganglia. Meanwhile, CSF from encephalopathy and IND patients was mostly enriched for proteins mapped to the cerebral cortex, choroid plexus, and thalamus. Moreover, CSF from IND patients had an increased proportion of microglia-related proteins. These findings support a model in which cases of IND and encephalopathy are associated with blood-brain barrier disruption potentially induced by complement activation and glia-promoted neuroinflammation, resulting in the involvement of distinct brain structures in COVID-19.
The genus Salvia, widely known as sage, represents the largest group within the Lamiaceae family and includes numerous species with traditional uses. This study investigates the chemical composition, antioxidant activity, enzyme inhibition, and antibacterial activity of the aerial parts and roots of two Salvia species, Salvia heldreichiana, and Salvia tomentosa. For chemical analysis, ultraperformance liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) was used. LC-QTOF-MS analysis revealed that the extracts from the aerial parts of S. tomentosa had a higher number of metabolites than those of S. heldreichiana, including amino and organic acids, various phenolic compounds, and unknown metabolites. S. heldreichiana aerial parts water extract possessed the highest antioxidant activity in CUPRAC > FRAP > ABTS > DPPH > MCA > PBD. For the enzyme inhibition, S. heldreichiana root extracts exhibited the highest anti-AChE activity, particularly in Ethanol extracts (2.88 mg GALAE/g), while the highest anti-BChE activity was the ethanol extract of the aerial part (3.65 mg GALAE/g). Consequently, the ethanol/water aerial part extracts, with their superior antioxidant, phenolic, and multienzyme inhibitory profile, present themselves as best candidates of S. heldreichiana and S. tomentosa for further in vivo pharmacological evaluation and development.
CNS neuroscience & therapeuticsKai Fricke, Diego Manuel Baur, Kapil Dev Singh, Felix Schmidt, Kathrin Müsch, Noriane Andrina Sievi, Marco Di Donato, Anton Schmick, Silvia Ulrich, Pablo Sinue…
PURPOSE: Achieving seizure control without intolerable adverse effects remains a major challenge in adult epilepsy, and serum concentrations of antiseizure medications correlate poorly with clinical outcomes. Metabolic processes influencing treatment response are not captured by blood monitoring. This study evaluates whether exhaled breath metabolomic profiles reflect treatment response and adverse effects in adults receiving antiseizure therapy, using epilepsy as a model for pharmacodynamic phenotyping. MATERIALS AND METHODS: A single-center observational cohort of 117 adults provided 127 breath samples during stable antiseizure treatment. Exhaled breath samples were analyzed using secondary electrospray ionization high-resolution mass spectrometry. Machine-learning models with nested cross-validation were used to classify treatment response and adverse effects, and pathway-level analyses were performed to provide biological context. Findings were compared with an independent, predominantly pediatric cohort analyzed using the same workflow. RESULTS: Breath metabolomic profiles distinguished responders from nonresponders and individuals with and without adverse effects, whereas serum drug concentrations showed no meaningful association. Classification models achieved areas under the curve of 0.84 for treatment response and 0.88 for adverse effects, with good calibration. Pathway-level alterations were directionally consistent across cohorts. CONCLUSION: Breath metabolomics supports a non-invasive approach to pharmacodynamic phenotyping that captures treatment-related metabolic states not reflected by serum drug concentrations.
Bacteria-derived carotenoids have attracted increasing attention as natural bioactive compounds with potential pharmaceutical applications, particularly in cancer therapy. However, the chemical composition and anticancer mechanisms of carotenoids from Micrococcus terreus remain largely unexplored. This study aimed to characterize the carotenoid extract from the locally isolated M. terreus SK34 strain (GenBank: PX765938) and to investigate its anticancer activity and underlying mechanisms using integrated in vitro and in silico approaches. Under nonoptimized culture conditions, strain SK34 produced 128 mg/L of carotenoid-rich pigment extract. LC-Q-TOF-MS analysis identified eight carotenoids, including phytoene, β-carotene, astaxanthin, zeaxanthin, β-cryptoxanthin, α-carotene, lycopene, and sarcinaxanthin. The extract exerted selective cytotoxicity against different cancer cell lines (IC50 = 61.91-243.76 μg/mL), particularly the neuroblastoma (SH-SY5Y) cancer cell line, while showing minimal cytotoxicity toward normal human dermal fibroblasts. Gene expression analysis demonstrated the significant upregulation of proapoptotic genes (p53, BAX, CASP3, CASP8, and CASP9) in the SH-SY5Y cell line, together with the downregulation of antiapoptotic genes (BCL-2 and AKT1), whereas autophagy-related genes were not significantly affected. ELISA and flow cytometry analyses further confirmed apoptosis in the SH-SY5Y cell line. Molecular docking analyses revealed that β-carotene, astaxanthin, and zeaxanthin exhibited strong binding affinities toward apoptosis-related proteins, supporting the experimental findings. This study provides the first comprehensive characterization of carotenoids from M. terreus SK34 and highlights their potential as selective apoptosis-inducing agents for future anticancer therapy.
GliaAlba M Lucart-Sanchez, Edward Sellés-Climent, Jorge Navarro-Calvo, Guillem Pont-Espinós, Jose A Gomez-Sanchez, Raúl Estévez, Luis M Valor, Rocío Pérez-González
Extracellular vesicles (EVs) mediate intercellular communication in the central nervous system (CNS) and are emerging as biomarkers of brain health and disease. However, the molecular composition of cell type-specific brain EVs, particularly astrocyte-derived EVs (ADEVs), remains poorly defined. We performed comparative proteomic analysis of neuronal (NDEVs), microglial (MDEVs), and ADEVs from mouse brain using magnetic immunocapture and LC-MS/MS proteomic profiling. Each EV subtype displayed distinct molecular fingerprints. NDEVs were enriched in synaptic and neurogenesis-related proteins (e.g., APP, SNAP25, GPR158, and BDNF), whereas MDEVs contained immune and phagocytic markers (e.g., TMEM119, CX3CR1, CD11b). Strikingly, the ADEV proteome closely mirrored the recently characterized GlialCAM interactome from leukodystrophy research, encompassing GlialCAM/MLC1 and associated partners involved in ion and water homeostasis (EAAT1/2, AQP4, GJA1), together with GPCRs such as GPRC5B. This overlap suggests that ADEVs encapsulate a molecular scaffold characteristic of astrocytic endfeet, potentially extending their signaling functions to the extracellular space. In conclusion, our study provides a detailed comparative proteomic characterization of brain cell type-specific EVs, revealing that ADEVs contain the GlialCAM/MLC1 network and GPCRs. These findings identify candidate molecular signatures that support the future investigation of ADEVs for biomarker development and provide a proteomic framework for exploring their relationship with astrocytic endfoot biology, blood-brain barrier (BBB)-associated pathways, and neurodegenerative disorders.
Anaerobic digestion (AD) of organic wastes by microorganisms into biomethane contributes significantly towards bioenergy generation. However, the bioprocess remains challenging due to its complex microbial ecology, dynamic biochemical pathways and sensitivity to perturbations. Over the past decade, using meta-omics like metataxonomics, metagenomics, metatranscriptomics, metaproteomics and metabolomics to diagnose its functioning has become necessary and common. However, a single meta-omics method can only provide limited biological understanding of the bioprocess. This led to the use of multi-meta-omics analyses in tandem; however, most studies still examine and interpret each meta-omics separately, limiting their ability to uncover functional interactions across molecular levels. Currently, the frontier is integrated meta-omics, where multi-meta-omics and process data are systematically combined into a single and interpretable system to unlock mechanistic understanding, diagnostic and predictive control of AD. This review critically examines specific application of meta-omics in AD, discussing their strengths, limitations and distinct position in integrated meta-omics approach. Importantly, it explores the computational frameworks, methodologies and challenges of integrated meta-omics. Discovering diagnostic biomarkers with high predictive power and transferability across AD systems through cross-omics validation is highlighted. As workflows standardise and technologies mature, integrated meta-omics would significantly contribute to the advancement of AD bioprocess for bioenergy.