Liquid Chromatography-high Resolution Mass Spectrometry Research Articles

Enhanced N-Glycan Profiling of Therapeutic Monoclonal Antibodies through the Application of Upper-Hinge Middle-Up Level LC-HRMS Analysis

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up (UHMU)-level ultra-high-performance liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis to improve N-glycan profiling of mAbs. Two specific enzymes, known as IgG degradation enzymes (IGDEs), were used to selectively cleave therapeutic mAbs above the hinge region to separate antibody subunits for further Fc glycan analysis by means of the UHMU/LC-HRMS workflow. The complexity of the mass spectra of IGDEs-digested mAbs was significantly reduced compared to the intact MS level, enabling reliable assignment and relative quantitation of paired Fc glycoforms. The results of the UHMU/LC-HRMS analysis of nine approved therapeutics highlight the significance of this approach for in-depth glycoform profiling.

Phytochemical Characterization and Bioactivity of Extracts from Different Fruit Parts of Opuntia leucotricha DC.: A Comparison between a Conventional Organic Solvent and Green Natural Deep Eutectic Solvents

The objective of this study was to analyze the chemical profile of extracts from different fruit parts of the perennial arborescent cactus Opuntia leucotricha by applying ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) and to evaluate their biological properties (antioxidant and enzyme inhibitory capacities). Extracts were obtained from the fruit pulp (without or with seeds), seeds, and peel by ultrasound-assisted extraction (UAE) using the conventional solvent methanol 50% and two Natural Deep Eutectic Solvents (NADESs) (glycerol:urea, 1:1 and citric acid:sorbitol, 1:2). A total of 33 compounds were identified, including phenolics, fatty acids, and others. Phenolic acids were the most abundant class of phenolics identified in all fruit parts, with the highest concentration observed in the methanol extracts from peel (593.02 µg/gDW), followed by seed-containing pulp (69.03 µg/gDW), pulp (57.83 µg/gDW), and seeds (39.97 µg/gDW). The second most effective extractant was NADES 1 (glycerol:urea, 1:1), which was also successful in extracting compounds with antioxidant capacity. Overall, the extracts demonstrated considerable enzyme inhibitory activities, with the greatest effects observed against α-amylase and α-glucosidase. The results indicate that O. leucotricha fruits could be a promising source of bioactive compounds, and NADES a viable alternative to organic solvents for their industrial exploitation.

Quantification of additives in beached plastic debris from Aotearoa New Zealand

Plastics have become an essential part of modern society. Their properties can be easily manipulated by incorporating additives to impart desirable attributes, such as colour, flexibility, or stability. However, many additives are classified as hazardous substances. To better understand the risk of plastic pollution within marine ecosystems, the type and concentration of additives in plastic debris needs to be established. We report the quantification of thirty-one common plastic additives (including plasticisers, antioxidants, and UV stabilisers) in beached plastic debris collected across Aotearoa New Zealand. Additives were isolated from the plastic debris by solvent extraction and quantified using high-resolution liquid chromatography-mass spectrometry. Twenty-five of the target additives were detected across 200 items of debris, with plasticisers detected at the highest frequency (99 % detection frequency). Additives were detected in all samples, with a median of four additives per debris item. A significantly higher number of additives were detected per debris item for polyvinyl chloride (median = 7) than polyethylene or polypropylene (median = 4). The additives bis(2-ethylhexyl) phthalate, diisononyl phthalate, diisodecyl phthalate, and antioxidant 702 were detected at the highest concentrations (up to 196,930 μg/g). The sum concentration of additives per debris item (up to 320,325 μg/g) was significantly higher in polyvinyl chloride plastics (median 94,716 μg/g) compared to other plastic types, primarily due to the presence of phthalate plasticisers. Non-target analysis was consistent with the targeted analysis, indicating a higher number and concentration of additives in polyvinyl chloride debris items compared to all other polymer types. Feature identification indicated the presence of more additives than previously detected in the targeted analysis, including plasticisers (phthalate and non-phthalate), processing aids, and nucleating agents. This study highlights phthalates and polyvinyl chloride as key targets for consideration in ecotoxicology and risk assessments, and the development of policies to reduce the impacts of plastic pollution.

Metabolism study of 3-chloromethcathinone (3-CMC) by dried blood spot (DBS) sampling after controlled administration using a murine model.

The metabolism of 3-chloromethcathinone (3-CMC) was studied after controlled administration in a murine model using the dried blood spot (DBS) technique for the sampling, storage and purification of blood samples. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was used for the identification of metabolites and investigation of their fragmentation pattern. Subsequently, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for their identification and 3-CMC quantification in routine workload. The main metabolites identified were two stereoisomers of dihydro-CMC, N-demethyl-CMC, and dihydro-N-demethyl-CMC. The stability of 3-CMC and of its metabolites deposited on DBS was evaluated by replicate analyses after 30, 50, and 90 days, demonstrating a decrease in concentration. It was more pronounced for 3-CMC, with -67% and -82% percentage deviation from the initial concentrations, and for N-demethyl 3-CMC (decrease comprised between -48% and -88%) than for the di-hydro metabolites, ranging from -5% to -37%. Regardless, all of them were detectable till 90 days after deposition as DBS. The possibility of identifying 3-CMC and its metabolites with high sensitivity is an invaluable tool for the diagnosis of exposure to the substance, also in low doses or after some hours, and for various applications in clinical and forensic toxicology, such as driving under the influence, drug-facilitated crimes, and addiction to intoxications. DBS demonstrated to be a reliable technique for the sampling, storage, and purification of the blood specimen for 3-CMC and metabolite detection.

Naringin and temozolomide combination suppressed the growth of glioblastoma cells by promoting cell apoptosis: network pharmacology, in-vitro assays and metabolomics based study

Introduction: Glioblastoma, which affects a large number of patients every year and has an average overall lifespan of around 14.6 months following diagnosis stands out as the most lethal primary invasive brain tumor. Currently, surgery, radiation, and chemotherapy with temozolomide (TMZ) are the three major clinical treatment approaches. However, the ability to treat patients effectively is usually limited by TMZ resistance. Naringin, a bioflavonoid with anti-cancer, antioxidant, metal-chelating, and lipid-lowering effects, has emerged as a promising therapeutic option.Methods: To explore the targets and pathways of naringin and TMZ in glioblastoma network pharmacology, cell line-based ELISA, flow cytometry, immunocytochemistry, western blotting, and LC-HRMS based metabolomics study were used.Results: The findings through the network pharmacology suggested that the key targets of naringin in the chemosensitization of glioblastoma would be Poly [ADP-ribose] polymerase 1 (PARP-1), O-6-Methylguanine-DNA Methyltransferase (MGMT), and caspases. The functional enrichment analysis revealed that these targets were significantly enriched in important pathways such as p53 signaling, apoptosis, and DNA sensing. Further, the results of the in-vitro study in U87-MG and T98-G glioblastoma cells demonstrated that TMZ and naringin together significantly reduced the percentage of viability and inhibited the DNA repair enzymes PARP-1 and MGMT, and PI3K/AKT which led to chemosensitization and, in turn, induced apoptosis, which was indicated by increased p53, caspase-3 expression and decreased Bcl2 expression. Additionally, a metabolomics study in T98-G glioblastoma cells using liquid chromatography high-resolution mass spectrometry (LC-HRMS) revealed downregulation of C8-Carnitine (−2.79), L-Hexanoylcarnitine (−4.46), DL-Carnitine (−2.46), Acetyl-L-carnitine (−3.12), Adenine (−1.3), Choline (−2.07), Propionylcarnitine (−1.69), Creatine (−1.33), Adenosine (−0.84), Spermine (−1.42), and upregulation of Palmitic Acid (+1.03) and Sphingosine (+0.89) in the naringin and TMZ treatment groups.Discussion: In conclusion, it can be said that naringin in combination with TMZ chemosensitized TMZ antiglioma response and induced apoptosis in tumor cells.

Development and validation of general plasma screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

The screening of drugs in plasma and urine often requires initial extraction (such as liquid-liquid extraction and solid-phase extraction) before the samples are submitted to instrumental analyses. These extraction procedures are often laborious and time-consuming. In this manuscript, a high-throughput automated assay based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 600-μL plasma aliquot, which is subjected to solid phase extraction (SPE) using OASIS HLB 96-well SPE with Biotage Extrahera system, evaporation, and reconstitution in a 96-well collection plate. LC-HRMS analyses were carried out on a Thermo Q-Exactive Mass spectrometer coupled with Thermo UHPLC system equipped with Thermo Accela ALS 2.4.0 autosampler linked to ACE Excel column. Drug targets were detected by retention time and accurate mass, with a mass tolerance window of 5ppm in positive and negative ionization mode. The screening method was validated for over 300 drug targets in a 13-min run. Validation data including sensitivity, specificity, extraction recovery, and precision are presented. As the method employs full-scan mass spectrometry, unlimited number of drug targets can theoretically be incorporated into this method.

Lipidomics by Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography–High-Resolution Mass Spectrometry in Osteosarcoma: A Pilot Study

Cancer is a complex disease that can also affect the younger population; however, it is responsible for a relatively high mortality rate of children and youth, especially in low- and middle-income countries (LMICs). Besides that, lipidomic studies in this age range are scarce. Therefore, we analyzed blood serum samples from young patients (12 to 35 years) with bone sarcoma (osteosarcoma) and compared their lipidomics to the ones from the control group of samples, named healthy control (HC group), using NMR and LC-MS techniques. Furthermore, differences in the lipidomic profiles between OS patients with and without metastasis indicate higher glycerophosphocholine (GPC) and glycerophospholipid (GPL) levels in osteosarcoma and increased cholesterol, choline, polyunsaturated fatty acids (PUFAs), and glycerols during the metastasis. These differences, detected in the peripheral blood, could be used as biomarkers for liquid biopsy.

Serum Metabolites as Potential Markers and Predictors of Depression-like Behavior and Effective Fluoxetine Treatment in Chronically Socially Isolated Rats

Metabolic perturbation has been associated with depression. An untargeted metabolomics approach using liquid chromatography-high resolution mass spectrometry was employed to detect and measure the rat serum metabolic changes following chronic social isolation (CSIS), an animal model of depression, and effective antidepressant fluoxetine (Flx) treatment. Univariate and multivariate statistics were used for metabolic data analysis and differentially expressed metabolites (DEMs) determination. Potential markers and predictive metabolites of CSIS-induced depressive-like behavior and Flx efficacy in CSIS were evaluated by the receiver operating characteristic (ROC) curve, and machine learning (ML) algorithms, such as support vector machine with linear kernel (SVM–LK) and random forest (RF). Upregulated choline following CSIS may represent a potential marker of depressive-like behavior. Succinate, stachydrine, guanidinoacetate, kynurenic acid, and 7-methylguanine were revealed as potential markers of effective Flx treatment in CSIS rats. RF yielded better accuracy than SVM–LK (98.50% vs. 85.70%, respectively) in predicting Flx efficacy in CSIS vs. CSIS, however, it performed almost identically in classifying CSIS vs. control (75.83% and 75%, respectively). Obtained DEMs combined with ROC curve and ML algorithms provide a research strategy for assessing potential markers or predictive metabolites for the designation or classification of stress-induced depressive phenotype and mode of drug action.

Discrepancies between the stated contents and analytical findings for electronic cigarette liquid products: Identification of the new cannabinoid, Δ9-tetrahydrocannabihexol acetate.

A number of synthetic cannabinoids have been appearing in the recreational drug market for more than a decade. Recent additions are so-called semi-synthetic cannabinoids, and they structurally closely resemble the main psychoactive component of cannabis, Δ9-tetrahydrocannabinol. Knowledge of new (semi-)synthetic cannabinoids is essential to help identify them in authentic forensic case samples. Therefore, the aim of the study was to examine two commercially available electronic cigarette liquid products claiming to contain cannabinoids and characterize the structures of the main compounds. The liquid products were analyzed by gas chromatography-mass spectrometry (GC-MS), GC-quadrupole time-of-flight mass spectrometry (GC-QTOF-MS), and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). In product A, typical cannabinoids (cannabidiol, cannabigerol, and cannabinol) and terpenes (α-caryophyllene and β-caryophyllene) were identified by comparison with reference materials. An unknown peak was isolated by semi-preparative high-performance LC, analyzed by nuclear magnetic resonance (NMR) spectroscopy, and identified to be Δ9-tetrahydrocannabihexol acetate (Δ9-THCH-O). To the authors' knowledge, this is the first report of the identification of Δ9-THCH-O in commercially available products. Another compound estimated as cannabihexol acetate was also detected. In product B, cannabidiol, cannabinol, α-caryophyllene, and β-caryophyllene were identified, while two unknown peaks were estimated as tetrahydrocannabidiol isomers. Despite products A and B being labeled to contain "60% HHCPM" and "80% 10-OH-HHC," respectively, no such compounds were detected. The findings of this study could help detect Δ9-THCH-O in case samples and highlight the need to keep monitoring commercial products to identify new drugs, while warning that the package labels cannot be trusted.

Exploring the therapeutic potential of Thai medicinal plants: in vitro screening and in silico docking of phytoconstituents for novel anti-SARS-CoV-2 agents

BackgroundThe high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking.MethodsTwenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Molecular docking analyses of these compounds against the main protease (Mpro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation.ResultsThe water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC50 of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 Mpro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-β-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~ − 9 kcal/mol. The stability of each ligand–protein complex of these compounds with the Mpro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.

Elucidating the antimicrobial and anticarcinogenic potential of methanolic and water extracts of edible Tragopogon coelesyriacus Boiss.

AbstractTragopogon coelesyriacus is a pharmacotherapeutic herbaceous plant belonging to the Asteraceae family and consumed as a vegetable. Here, the methanolic and water extracts of T. coelesyriacus were obtained from its aboveground parts (stem, leaves, and flowers), and the phytochemical potentials were investigated by LC‐HRMS (liquid chromatography–high resolution mass spectrometry) analysis for the first time. The antibacterial, antifungal, and anticarcinogenic activities of T. coelesyriacus extracts were investigated using experimental and in silico methods. T. coelesyriacus methanol extract revealed remarkable inhibitory activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumonia (MICs = 0.83, 1.67, and 1.67 mg/mL, respectively) compared to Escherichia coli and Enterobacter aerogenes (MIC = 53.3 mg/mL). Inhibitory effects of T. coelesyriacus methanolic extracts were also observed in all Candida species tested, with the highest inhibition on Candida krusei (MIC = 0.83 mg/mL), whereas no inhibitory effect was identified from the water extract. Additionally, both T. coelesyriacus methanolic (IC50 = 86 μg/mL) and water (IC50 = 92 μg/mL) extracts revealed significant selective anticarcinogenic effects on AR42J pancreatic cancer cells. HeLa and MDA‐MB‐231 cells were, however, more resilient to methanol and water extract, respectively. In silico analyses further elucidated the noteworthy antibacterial potential of keracyanin chloride on S. aureus MurB enzyme and the remarkable inhibitory potential of naringin on FYN kinase specific for pancreatic cancer (AR42J) development. In conclusion, T. coelesyriacus phytochemicals with antibacterial, antifungal, and anticancer properties were revealed for the first time, and molecular docking studies on potential targets confirmed good agreement with experimental findings. Therefore, the current studies on T. coelesyriacus provide the basis for investigating new pharmaceutical potentials of other Tragopogon members.

Unraveling Grewia bilamellata Gagnep. Role in cerebral ischemia: Comprehensive in vivo and in silico studies.

The present study investigated the neuroprotective properties of whole plants of Grewia bilamellata Gagnep. extract (GBEE) against cerebral ischemia by harnessing both In vivo studies in a rat model and In silico studies focusing on nitric oxide synthase (NOS) inhibition. High-resolution liquid chromatography‒mass spectrometry (HR LC‒MS) analysis identified 32 phytochemicals in the GBEE, 15 of which adhered to Lipinski's rule of five. These compounds exhibited diverse physicochemical properties and high binding affinity to NOS, with cleomiscosin D showing the greatest potential. In vivo, GBEE had significant neuroprotective effects on bilateral common carotid artery occlusion/reperfusion (BCCAO/R) in rats, especially at doses of 200mg/kg and 400mg/kg body weight. GBEE treatment improved brain function, as evidenced by EEG normalization, substantial reductions in cerebral infarction size, mitigated neuronal loss, and the restoration of regular histological arrangement in the CA1 hippocampal area of the brain. Furthermore, GBEE enhanced antioxidant defenses by augmenting the activity of catalase (CAT) and superoxide dismutase (SOD), reducing malondialdehyde (MDA) levels, and restoring reduced glutathione (GSH) levels. These effects were accompanied by a decrease in nitric oxide (NO) levels, indicative of attenuated oxidative and nitrosative stress. Collectively, our findings suggest that GBEE is a promising natural therapeutic agent that may prevent or alleviate ischemic brain injury through a multifaceted mechanism involving NOS inhibition and attenuation of the oxidative stress response. This study highlights the therapeutic potential of GBEE and warrants further research into its mechanism of action and possible clinical applications.

Metabolomics Panel Associated with Cystic Fibrosis-Related Diabetes toward Biomarker Discovery.

The most prevalent comorbidity among cystic fibrosis (CF) patients is cystic fibrosis-related diabetes (CFRD). CFRD has been linked to one of the worse clinical outcomes and a higher mortality. Improved clinical results have been related to earlier diagnosis and treatment of CFRD. Therefore, the present study aimed to investigate the metabolome of human serum of patients with CFRD. This might aid in identifying novel biomarkers linked with the pathophysiology of CFRD and its diagnosis. The liquid chromatography-high-resolution mass spectrometry (LC-HRMS) metabolomics approach was utilized for serum samples from patients with CF (n = 36) and healthy controls (n = 36). Nine patients in the CF group had CFRD, and 27 were non-CFRD patients (nCFRD). A total of 2328 metabolites were significantly altered in CF compared with the healthy control. Among those, 799 significantly dysregulated metabolites were identified between CFRD and nCFRD. Arachidonic acid (AA), ascorbate, and aldarate metabolism were the most common metabolic pathways dysregulated in CF. l-Homocysteic acid (l-HCA) levels were significantly reduced in CF and CFRD compared to the control and nCFRD, respectively. In addition, gamma-glutamylglycine and l-5-hydroxytryptophan (5-HTP) had the highest discrimination between CFRD and nCFRD with AUC (0.716 and 0.683, respectively). These biomarkers might serve as diagnostic biomarkers and aid in understanding potential metabolic changes linked to CF and CFRD.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) β-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG β-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Detection of the selective androgen receptor modulator S-23 and its metabolites in equine urine and plasma following oral administration.

S-23 is an arylpropionamide selective androgen receptor modulator that has been investigated in animal models for use as a male hormonal contraceptive but is not yet available therapeutically. S-23 is available alongside other selective androgen receptor modulators (SARMs) to purchase online via uncontrolled sites, sold as supplement products. It has been detected in several human doping cases, highlighting the importance of identifying the best analytical targets for equine doping control. The purpose of this study was to investigate the detection of S-23 and its phase I metabolites in equine urine and plasma following a multiple dose oral administration to two Thoroughbred racehorses. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification, and liquid chromatography-tandem mass spectrometry was used for full sample analysis and generation of urine and plasma profiles. S-23 and seven phase I metabolites were observed in urine following enzyme hydrolysis and solvolysis. The most abundant analyte detected was the hydroxylated 4-amino-2-(trifluoromethyl)benzonitrile metabolite, which also allowed the longest duration of detection in urine from both horses, for up to 360 h following administration. The data suggest that this metabolite was likely to be highly conjugated with both sulphate and glucuronide moieties. In plasma, S-23 and two phase I metabolites were observed. S-23 was the most abundant analyte detected for both horses, allowing detection for up to 143 h post-administration. To the best of the authors' knowledge, this is the first report of S-23 and metabolites in equine urine and plasma samples.

Deciphering Transcriptomic and Metabolomic Wood Responses to Grapevine Trunk Disease-Associated Fungi

Esca is one of the main grapevine trunk diseases affecting vineyards worldwide. Phaeoacremonium minimum and Phaeomoniella chlamydospora are thought to be two of the main causal agents of this disease. However, the molecular mechanisms underlying plant defense responses in the grapevine trunk against esca-associated pathogens are poorly understood. To provide a first glimpse at the trunk responses to P. minimum and P. chlamydospora, transcriptomic and metabolomic analyses were performed to compare and contrast host responses to these pathogens. Transcriptomic analysis revealed different gene expression reprogramming in the trunk in response to each fungus. The main significant differences were found among genes associated with secondary metabolism, signaling, and hormone signaling. An untargeted liquid chromatography–high resolution mass spectrometry metabolomic approach performed 3 weeks after inoculation was used, and dereplication mainly highlighted flavonoids and stilbenes as plant defense metabolites in the infected trunk. Some metabolites were overproduced with both fungi, but specific responses were also observed. Particularly, a lipophilic flavonoid cluster was emphasized after P. minimum inoculation. The assessment of fungal infection 6 weeks postinfection showed more copies of P. minimum than P. chlamydospora. This dissimilarity in the level of colonization could be linked to the metabolomic responses observed. Our results reveal both different gene expression reprogramming and metabolomic-specific signatures depending on the wood pathogen. Altogether, these observations suggest that grapevine trunks can differently perceive and respond to P. minimum and P. chlamydospora. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Non-target liquid chromatography high-resolution mass spectrometry screening to prioritize unregulated micropollutants that persist through domestic wastewater treatment

Efforts to regulate and monitor emerging contaminants are insufficient because new chemicals are continually brought to market, and many are unregulated and potentially harmful. Domestic wastewater treatment plants are not designed to remove micropollutants and are important sources of emerging contaminants in the aquatic environment. In this study, non-target screening, an unbiased method for analyzing compounds without prior information, was used to identify compounds that may be emitted in wastewater treatment plant effluent and should be monitored. Nine wastewater treatment plants using different treatment methods were studied, and a non-target screening data-processing method was used. The frequencies at which the contaminants were detected and contaminant persistence through the treatment processes were considered, and then the contaminants were prioritized. The predicted no-effect concentration of each prioritized contaminant was used to determine whether further analysis and monitoring of the contaminant was necessary. Quantitative analyses of five compounds (amantadine, atenolol, benzotriazole, diphenhydramine, and sulpiride) were performed using reference standards. Probable molecular formulae and structures were proposed for 17 contaminants, and the risks posed by the contaminants were estimated using predicted no-effect concentrations. The results provide valuable insights into how unregulated micropollutants can be identified and prioritized for monitoring in future studies.

Enhancing Molecular Characterization of Dissolved Organic Matter by Integrative Direct Infusion and Liquid Chromatography Nontargeted Workflows.

Dissolved organic matter (DOM) in aquatic systems is a highly heterogeneous mixture of water-soluble organic compounds, acting as a major carbon reservoir driving biogeochemical cycles. Understanding DOM molecular composition is thus of vital interest for the health assessment of aquatic ecosystems, yet its characterization poses challenges due to its complex and dynamic chemical profile. Here, we performed a comprehensive chemical analysis of DOM from highly urbanized river and seawater sources and compared it to drinking water. Extensive analyses by nontargeted direct infusion (DI) and liquid chromatography (LC) high-resolution mass spectrometry (HRMS) through Orbitrap were integrated with novel computational workflows to allow molecular- and structural-level characterization of DOM. Across all water samples, over 7000 molecular formulas were calculated using both methods (∼4200 in DI and ∼3600 in LC). While the DI approach was limited to molecular formula calculation, the downstream data processing of MS2 spectral information combining library matching and in silico predictions enabled a comprehensive structural-level characterization of 16% of the molecular space detected by LC-HRMS across all water samples. Both analytical methods proved complementary, covering a broad chemical space that includes more highly polar compounds with DI and more less polar ones with LC. The innovative integration of diverse analytical techniques and computational workflow introduces a robust and largely available framework in the field, providing a widely applicable approach that significantly contributes to understanding the complex molecular composition of DOM.

Plasma metabolomic and lipidomic profiles accurately classify mothers of children with congenital heart disease: an observational study

IntroductionCongenital heart disease (CHD) is the most common congenital anomaly, representing a significant global disease burden. Limitations exist in our understanding of aetiology, diagnostic methodology and screening, with metabolomics offering promise in addressing these.ObjectiveTo evaluate maternal metabolomics and lipidomics in prediction and risk factor identification for childhood CHD.MethodsWe performed an observational study in mothers of children with CHD following pregnancy, using untargeted plasma metabolomics and lipidomics by ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). 190 cases (157 mothers of children with structural CHD (sCHD); 33 mothers of children with genetic CHD (gCHD)) from the children OMACp cohort and 162 controls from the ALSPAC cohort were analysed. CHD diagnoses were stratified by severity and clinical classifications. Univariate, exploratory and supervised chemometric methods were used to identify metabolites and lipids distinguishing cases and controls, alongside predictive modelling.Results499 metabolites and lipids were annotated and used to build PLS-DA and SO-CovSel-LDA predictive models to accurately distinguish sCHD and control groups. The best performing model had an sCHD test set mean accuracy of 94.74% (sCHD test group sensitivity 93.33%; specificity 96.00%) utilising only 11 analytes. Similar test performances were seen for gCHD. Across best performing models, 37 analytes contributed to performance including amino acids, lipids, and nucleotides.ConclusionsHere, maternal metabolomic and lipidomic analysis has facilitated the development of sensitive risk prediction models classifying mothers of children with CHD. Metabolites and lipids identified offer promise for maternal risk factor profiling, and understanding of CHD pathogenesis in the future.

Optimisation of corn silk tea production, and its antioxidant profile

Following COVID-19 pandemic, functional foods and beverages continue to develop, especially those with high antioxidant activity. The functional beverage from corn silk can be processed into brewed tea. The critical points in the making process of corn silk tea are the drying equipment, drying temperature, and drying time. With efficient method, corn silk tea can produce high antioxidant activity. The present work optimised the temperature and drying method based on antioxidant activity. The results of the present work are expected to be used in micro/small/medium enterprises (MSMEs) to produce corn silk herbal tea. The present work used Randomised Block Design (RBD) with two factors; drying temperatures (45, 55, and 65°C) and drying equipment (cabinet and oven) for 5 h. The analysis carried out was antioxidant activity and profiling using LC-HRMS (liquid chromatography high-resolution mass spectrometry) that ran in a positive (+) mode. The processing of corn silk tea using cabinet drying at 65°C for 5 h produced antioxidant activity of 82.00 ± 0.75%. This was higher than commercial black tea (81.71 ± 0.15%) and commercial green tea (78.37 ± 0.43%). The analysis using LC-HRMS showed that the corn silk tea contained betaine (N,N,N-trimethylglycine) (RT 0.928), 6-methylquinoline (RT 4.741), hesperidin (3’,5,7-trihydroxy-4’-methoxyflavanone) (RT 302.07817), luvangetin (10-methoxy-2,2-dimethylpyrano[3,2-g]chromen-8-one) (RT 258.08837), embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) (RT 13.074), and eucalyptol (1,8-cineole) (RT 13.325).