Liquid Chromatography-tandem Mass Spectrometry Analysis Research Articles

Prediction of Flavor Potential of Ocimum basilicum L. Side-Stream Phytoconstituents, Using Liquid Chromatography–Tandem Mass Spectrometry Analysis and In Silico Techniques

Although post-distillation side-streams of basil (Ocimum basilicum L.) pose significant economic and environmental challenges, they also bring forth new opportunities in the flavor industry. Thus, the objective of the current study was to assess the phenolic profile of basil side-stream extracts to identify key compounds and to evaluate their taste properties, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, flavor prediction tools and molecular docking. In particular, 52 phytoconstituents, mainly phenolic acids, salvianolic acids, flavonoids and fatty acids derivatives, were elucidated in the side-streams of two different basil varieties (Minimum and Genovese) harvested and distilled in early and late autumn, highlighting the effect of pre-harvest factors on basil’s phenolic fingerprint. Furthermore, the results of tests undertaken using taste prediction tools showed that most of the identified compounds were very likely to taste bitter, while six of them (caffeoylferuloyltartaric acid, isoquercetin, lithospermic acid A, sagerinic acid, salvianolic acids C and F) presented a high bitterant capacity (70–90%). Moreover, according to molecular docking studies, these compounds exhibited a stronger binding affinity to the hTAS2R46 bitter receptor compared to its known agonist, strychnine. This outcome and consequently their bitterness were mainly attributed to interactions with Glu265, Thr180 and/or Trp88 through the formation of direct hydrogen bonds. Therefore, the present results provide insights into the taste profiles of basil side-streams, leading to more sustainable and innovative uses of aromatic herbs residues.

Squaraine-Linked Magnetic Covalent Organic Framework as a Solid-Phase Extraction Absorbent to Determine Trace Phenylpyrazoles.

Phenylpyrazoles are widely used pesticides in the food industry. It is highly desirable to develop efficient pre-treatment and analysis methods to extract and detect phenylpyrazoles in complex food matrices. Herein, the study reports novel squaraine-linked zwitterionic core-shell magnetic covalent organic frameworks (MCOFs), which are found to be excellent pretreatment materials for the detection of trace phenylpyrazoles in samples. By coupling MCOFs to magnetic solid-phase extraction (MSPE) with Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) analysis, the detection of phenylpyrazoles (fipronil, fipronil sulfone, fipronil sulfide, fipronil de-sulfoxide, fipronil desulfinyl, ethiprole, and flufiprole) is achieved and shows good linearity at concentrations of 1-800µg L-1 (R2 ≥ 0.9930). The limit of detection (LOD), limit of quantification (LOQ), and recovery rates are 0.01-0.50µg kg-1, 0.04-1.72µg kg-1, and 70.96-115.32%, respectively. More importantly, this method is successfully applied to determine the phenylpyrazoles in commercial egg, poultry, milk, jujube, cabbage, tea, and rice with a detection rate of ≈0.04%. Therefore, the developed method may contribute to a new strategy for the purification and multi-target extraction of complex food matrices.

Determination of ten bisphenols and five parabens in urine by solid supported liquid-liquid extraction and liquid chromatography-tandem mass spectrometry

Bisphenols (BPs) and parabens (PBs) are of great concern for environmental pollution and human health because of their endocrine-disrupting effects and potential health hazards. Urinary biomonitoring of BPs and PBs can provide basic data for human internal exposure evaluation, which is a prerequisite for accurately assessing their health risks. In this study, we developed a new pretreatment procedure based on solid supported liquid-liquid extraction (SLE) for the simultaneous separation of ten BPs and five PBs in human urine, followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. In the instrumental analysis, the HPLC conditions and MS/MS parameters were comprehensively optimized. Accurate qualitative and quantitative determination of ten BPs and five PBs was achieved by introducing a ternary gradient elution system of water, methanol, and acetonitrile for LC separation. During sample pretreatment, the extraction solvent and elution volume were optimized. Specifically, urine samples were held at room temperature and centrifuged at 3000 r/min for 10 min. The supernatant (2 mL) was then transferred to a glass tube, and the pH was adjusted to 5.0 using HCl (0.5 mL; 0.1 mol/L) and NaAc-HAc buffer (1.5 mL). Thereafter, β-glucuronidase-arylsulfatase (20 μL) and surrogate standard solutions (10 ng;13C12-BPS,13C12-BPAF,13C6-MeP, and 13C6-BuP) were added, and the mixture was incubated in a shaker bath in the dark at 37 ℃ for 16 h. After incubation, the hydrolyzed sample (4 mL) was loaded onto an SLE cartridge and equilibrated for a minimum of 5 min to ensure the solution was completely absorbed by the packing material. Subsequently, the target chemicals were eluted with a mixed ethyl acetate/n-hexane solution (3∶7, v/v; 15 mL). Separation of the targets was performed on a ZORBAX SB-C18 reversed-phase column (250 mm×4.6 mm, 5 μm) using an acetonitrile-methanol-water system as the mobile phase. The method was verified by spiking mixed urine samples at three levels (1, 5, and 50 μg/L), with the recoveries ranging from 84.3% to 119.8%. Except for bisphenols (BPS), whose matrix effect was calculated as -21.8%, the matrix effects of other analytes were lower than 20%, indicating low matrix interference. The linear ranges of the analytes varied from 0.1-500 μg/L to 1-500 μg/L, with correlation coefficients higher than 0.995. The method limits of quantification for target chemicals ranged from 0.03 to 0.30 μg/L, and the relative standard deviations of intra- and inter-day experiments were 1.4%-8.4% and 5.7%-14.6%, respectively, suggesting high stability and reproducibility. The method was successfully applied to the determination of ten BPs and five PBs in 10 urine samples from a general population. The concentrations of target chemicals in the human urine samples varied. Methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), and bisphenol A (BPA) were detected in all samples, with median mass concentrations of 1.10, 0.60, 0.21, and 0.55 μg/L, respectively. The detection rates of the other chemicals were less than 50%, which may be related to the production and use of specific chemicals, their bioavailability, and biological metabolism in humans.

Riboflavin-meditated interspecies electron transfer in a H2-based denitrifying biofilm

H2-based membrane biofilm reactors (MBfR) are effective for nitrogen removal, yet the interactions of microorganisms within the MBfR biofilms have received limited attention. In this study, we explored a flavin-mediated electron transport mechanism occurring between hydrogenotrophic microorganisms and non‑hydrogenotrophic denitrifying microorganisms in H2-based MBfR biofilms. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and electrochemical analysis confirmed riboflavin act as the sole potential redox mediator present in the system. Batch experiments demonstrated that riboflavin accelerates the denitrification process and that microorganisms can use reduced flavins as the sole electron donor to reduce nitrate. Metagenomic and metatranscriptomic analyses revealed increased expression of genes related to riboflavin biosynthesis and transport in hydrogenotrophic microorganisms, indicating endogenous riboflavin secretion into the medium. Our findings reveal the potential existence of a novel flavin-mediated electron transfer mechanism in the H₂-based denitrification process, enriching the understanding of interspecies interactions within MBfR biofilms. This study also provides a theoretical basis for efficient nitrogen removal through regulating interspecies electron transfer.

Quantification of florfenicol and its metabolites in fillets of Nile tilapia: Synthesis of metabolites and validation of an on-line solid-phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry method

This study concerns the synthesis of the florfenicol (FF) metabolites florfenicol amine (FFA), florfenicol alcohol (FFOH), and monochloroflorfenicol (FFCl), for their subsequent use as reference standards in On-line solid-phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (SPE-UHPLC-MS/MS) analysis. The metabolites were characterized using 1H and 13C NMR, as well as HRMS, and their purities were confirmed by quantitative NMR to ensure analytical reliability. Validation of the developed analytical method showed that it presented acceptable performance, with linearity >0.99 for all the target analytes, accuracies within ±10 % of nominal concentrations, and intra- and inter-day precisions within 15 %. Application of this method to fillets from fish that had been treated with florfenicol (dose of 10 mg/kg bw daily) demonstrated its effectiveness in consistently detecting FF and its metabolites throughout the treatment. The results emphasized the utility of the method for enhancing pharmacokinetic and residue depletion research. The ability to precisely monitor the drug and its metabolites in treated fish provides important insights into florfenicol metabolism, laying the groundwork for further comprehensive profiling studies of metabolites in fish tissue.

Efficacy of BRAF/MEK-inhibitor therapy for epithelioid glioblastoma with a novel BRAFV600 mutation

Epithelioid glioblastoma (eGB), a very aggressive and rare brain tumour, is associated with a dismal median overall survival. Effective therapies for patients with eGB, particularly with leptomeningeal dissemination, are still lacking. Here, we describe a case of a 25-year-old male diagnosed with an intramedullary cervical tumour with subsequent leptomeningeal disease. Histopathology identified a highly necrotising, epithelioid-type tumour with high cell density, most compatible with the diagnosis of an eGB. DNA analysis revealed an unprecedented B-Raf protooncogene, serine/threonine kinase (BRAF) gene variant in exon 15 (ENST00000288602.6, c.1799_1810delinsATG, p.(V600_W604delinsDG)), triggering activation of the mitogen-activated protein kinase (MAPK) pathway. Consequently, we initiated MAPK inhibitor (MAPKi) therapy, utilizing a combination of BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors. Liquid chromatography–tandem mass spectrometry analysis confirmed the drugs’ presence in the patient’s cerebrospinal fluid, indicating their capacity to cross the blood-brain barrier. Remarkably, the patient responded very well to therapy and transitioned from a near-comatose state to significantly improved health, sustained for over three months. This study highlights that MAPKi, particularly targeted towards novel BRAFV600 mutations, might offer promising advancements in eGB treatment strategies.

Identification of the Metabolic Signature of Aging Retina.

This study aims to explore the metabolic signature of aging retina and identify the potential metabolic biomarkers for the diagnosis of retinal aging. Retinal samples were collected from both young (two months) and aging (14 months) mice to conduct an unbiased metabolic profiling. Liquid chromatography-tandem mass spectrometry analysis was conducted to screen for the metabolic biomarkers and altered signaling pathways associated with retinal aging. We identified 166 metabolites differentially expressed between young and aged retinas using a threshold of orthogonal projection to latent structures discriminant analysis variable importance in projection >1 and P < 0.05. These metabolites were significantly enriched in several metabolic pathways, including purine metabolism, citrate cycle, phenylalanine, tyrosine and tryptophan biosynthesis, glycerophospholipid metabolism, and alanine, aspartate and glutamate metabolism. Among these significantly enriched pathways, glycerophospholipid metabolites emerged as promising candidates for retinal aging biomarkers. We assessed the potential of these metabolites as biomarkers through an analysis of their sensitivity and specificity, determined by the area under the receiver-operating characteristic (ROC) curves. Notably, the metabolites like PC (15:0/22:6), PC (17:0/14:1), LPC (P-16:0), PE (16:0/20:4), and PS (17:0/16:1) demonstrated superior performance in sensitivity, specificity, and accuracy in predicting retinal aging. This study sheds light on the molecular mechanisms underlying retinal aging by identifying distinct metabolic profiles and pathways. These findings provide a valuable foundation for developing future clinical applications in diagnosing, identifying, and treating age-related retinal degeneration. This study sheds light on novel metabolic profiles and biomarkers in aging retinas, potentially paving the way for targeted interventions in preventing, diagnosing, and treating age-related retinal degeneration and other retinal diseases.

Bacterial infection-biased abundance of proteins in the skin mucus of obscure puffer (Takifugu Obscurus)

The skin mucus of fish is equipped with immunological and antimicrobial peptides that confer protection against invading pathogens. The skin mucus has been studied in fish however information regarding its immunological roles in bacterial infection is rare. This study highlighted the proteins and peptides in the skin mucus of Obscure puffer Takifugu obscurus that quantitatively altered against Aeromonas hydrophila infection. We infected the fish through bath immersion, intraperitonially, and treated with PBS (control) then compared the level of proteins in the skin mucus among the groups using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The Tandem Mass Tag (TMT) based quantification showed that 4896 proteins were Deferentially Quantified Proteins (DQPs), based on 19,751 unique peptides. Of which 170 were depleted (decreased in abundance) and 69 were abundant in comparison of Bath Treated (BT) vs Control (C) groups. Similarly, 76 DQPs were depleted and 70 were abundant in comparison of Treated (T) vs BT groups. Further, 126 DQPs were depleted, and 34 were abundant in comparison to T vs C groups. The DQPs we report were mostly immunological and were involved in unique biological functions and pathways. The interesting protein we report, where some of the proteins are for the first time in fish, shows the protein-rich structure of the mucus of fish, which may act as a biomarker to be targeted for bacterial disease therapy in fish and ultimately hint to the way of making resistance in fish against bacterial pathogens.

Development and validation of a multiresidue method for the determination of antibiotic residues in crawfish using LCMS/MS

ABSTRACT This study developed a rapid multi-class method for determining 29 antibiotic residues in crawfish using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A quick and affordable extraction method was employed, and sample preparation was performed using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique. The extraction process involved adding acetonitrile to crawfish samples. The sample was homogenised with a solution of sodium citrate buffer and sodium EDTA, followed by a cleanup step. The method was validated according to European Union (EU) guidelines 2002/657/EC. The limit of quantitation (LOQ) for all antibiotics was 5 µg/kg, except for flumequine and trimethoprim, which had LOQs of 2.5 µg/kg. The LOQ for the tetracycline group was 25 µg/kg, and for chloramphenicol, it was 0.015 µg/kg. The average recoveries at different concentration levels varied between 60–120%. The precision, expressed as repeatability and reproducibility, was calculated to be less than 16%. Thirty crawfish samples collected from local Egyptian market were monitored for antibiotic residues. The results revealed contamination with antibiotics belonging to three different groups. Of the thirty samples, 27% were contaminated with ciprofloxacin and enrofloxacin, 17% with chlortetracycline and oxytetracycline, 7% with oxolinic acid, and 3% with sulphamethazine. The applicability of the developed method was proven through successful proficiency testing. The developed method met performance criteria and is suitable for monitoring these antibiotics in crawfish.

Exploring the Potential Mechanisms of Action of Gentiana Veitchiorum Hemsl. Extract in the Treatment of Cholestasis using UPLC-MS/MS, Systematic Network Pharmacology, and Molecular Docking.

Gentiana veitchiorum Hemsl. (GV) has a long history in Tibetan medicine for treating hepatobiliary disease cholestasis. However, the mechanisms mediating its efficacy in treating cholestasis have yet to be determined. To elucidate the mechanisms of action of GV in the treatment of cholestasis, an integrated approach combining ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with network pharmacology was established. A comprehensive analysis of the chemical composition of GV was achieved by UPLC-MS/MS. Subsequently, a network pharmacology method that integrated target prediction, a protein-protein interaction (PPI) network, gene set enrichment analysis, and a component- target-pathway network was established, and finally, molecular docking and experiments in vitro were conducted to verify the predicted results. Twenty compounds that were extracted from GV were identified by UPLC-MS/MS analysis. Core proteins such as AKT1, TNF, and IL6 were obtained through screening in the Network pharmacology PPI network. The Kyoto Encyclopedia of the Genome (KEGG) pathway predicted that GV could treat cholestasis by acting on signaling pathways such as TNF/IL-17 / PI3K-Akt. Network pharmacology suggested that GV might exert a therapeutic effect on cholestasis by regulating the expression levels of inflammatory mediators, and the results were further confirmed by the subsequent construction of an LPS-induced RAW 264.7 cell model. In this study, UPLC-MS/MS analysis, network pharmacology, and experiment validation were used to explore potential mechanisms of action of GV in the treatment of cholestasis.

Biochemical foundation of the aroma and antioxidant activity of Indian traditional rice landrace Maharaji and the effect of radiation-induced mutagenesis on its metabolome

Maharaji rice, an aromatic variety with medium slender grains, is traditionally cultivated in the central regions of India. This study aimed to identify the biochemical compounds responsible for Maharaji rice's distinctive fragrance and enhance its agro-morphological traits through mutation breeding. Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis, forty major metabolites were identified which may be responsible for its characteristic aroma. The bioactive compounds included terpenes, flavonoids, and amino acids. Maharaji brown rice extract exhibited potent radical scavenging activity. Radiation-induced mutation breeding improved the agro-morphological traits and also triggered biochemical diversification in different mutants. Maharaji Mutant-2 exhibited improved aroma due to higher abundance of aromatic compounds, improved yield and morphological characters as compared to the parent. This study, for the first time identifies the compounds associated with the characteristic aroma of Maharaji rice. Global metabolomics may, therefore, expedite the selection of mutants with suitable aroma and desirable biological properties.

Novel Fe2O3/Bi2O3/CeO2 heterojunction photocatalyst for wastewater treatment: Characterization, photocatalytic performance and mechanism study

A novel ternary Fe2O3/Bi2O3/CeO2 (FeB/CeO2) heterojunction nanocomposite with different Fe2O3/Bi2O3 amounts on CeO2 (5, 10, 20, 30 and 40 wt%) was synthesized through a simple wet impregnation process. The properties of the synthesized nanocomposite were validated thorough XPS, XRD, FE-SEM, TEM, and EDS-Mapping analyses. This photocatalyst exhibited exceptional efficiency in the of Tetracycline’s (TC) photodegradation in comparison to individual Fe2O3, Bi2O3, and CeO2. The effect of Fe2O3/Bi2O3 loading on CeO2 and the influence of operational factors on TC decomposition including photocatalyst dosage (0.05–0.2 g/L), TC concentration (10–40 mg/L), and pH (1–11) were examined with the optimal conditions yielding an experimental degradation efficiency of 99.35 %. Moreover, scavenger tests showed that O2− and OH radicals served as the main oxidative radical species. The study further delved into proposing potential degradation pathways of TC in accordance with liquid chromatography tandem-mass spectrometry analysis. Notably, the stability of the synthesized photocatalyst was assessed through five degradation cycles, affirming its sustained effectiveness.

Combined matrix-assisted laser desorption/ionisation-mass spectrometry imaging with liquid chromatography-tandem mass spectrometry for observing spatial distribution of lipids in whole Caenorhabditis elegans.

Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for biomolecule detection (e.g., lipids), within tissue sections across various biological species. However, despite its utility in many applications, the nematode Caenorhabditis elegans is not routinely used in combination with MALDI-MSI. The lack of studies exploring spatial distribution of biomolecules in nematodes is likely due to challenges with sample preparation. This study developed a sample preparation method for whole intact nematodes, evaluated using cryosectioning of nematodes embedded in a 10% gelatine solution to obtain longitudinal cross sections. The slices were then subjected to MALDI-MSI, using a RapifleX Tissuetyper in positive and negative polarities. Samples were also prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Exploris 480 coupled to a HPLC Vanquish system to confirm the MALDI-MSI results. An optimised embedding method was developed for longitudinal cross-sectioning of individual worms. To obtain longitudinal cross sections, nematodes were frozen at -80°C so that all worms were rod shaped. Then, the samples were defrosted and transferred to a 10% gelatine matrix in a cryomold; the worms aligned, and the whole cryomold submerged in liquid nitrogen. Using MALDI-MSI, we were able to observe the distribution of lipids within C. elegans, with clear differences in their spatial distribution at a resolution of 5μm. To confirm the lipids from MALDI-MSI, age-matched nematodes were subjected to LC-MS/MS. Here, 520 lipids were identified using LC-MS/MS, indicating overlap with MALDI-MSI data. This optimised sample preparation technique enabled (un)targeted analysis of spatially distributed lipids within individual nematodes. The possibility to detect other biomolecules using this method thus laid the basis for prospective preclinical and toxicological studies on C. elegans.

Near UV and Visible Light-Induced Degradation of Bovine Serum Albumin and a Monoclonal Antibody Mediated by Citrate Buffer and Fe(III): Reduction vs Oxidation Pathways.

Light exposure during manufacturing, storage, and administration can lead to the photodegradation of therapeutic proteins. This photodegradation can be promoted by pharmaceutical buffers or impurities. Our laboratory has previously demonstrated that citrate-Fe(III) complexes generate the •CO2- radical anion when photoirradiated under near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) [Subelzu, N.; Schöneich, C. Mol. Pharmaceutics 2020, 17 (11), 4163-4179; Zhang, Y. Mol. Pharmaceutics 2022, 19 (11), 4026-4042]. Here, we evaluated the impact of citrate-Fe(III) on the photostability and degradation mechanisms of disulfide-containing proteins (bovine serum albumin (BSA) and NISTmAb) under pharmaceutically relevant conditions. We monitored and localized competitive disulfide reduction and protein oxidation by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis depending on the reaction conditions. These competitive pathways were affected by multiple factors, including light dose, Fe(III) concentration, protein concentration, the presence of oxygen, and light intensity.

Characterization of atmospheric nitroaromatic compounds in Southwest China by direct injection liquid chromatography-tandem mass spectrometry analysis of aerosol extracts

Nitroaromatic compounds (NACs) in ambient particles are of great concern due to their adverse effects on human health and climate. However, investigations on the characteristics and potential sources of NACs in Southwest China are still scarce. In this study, a field sampling campaign was carried out in the winter of 2022 at a suburban site in Mianyang, Southwest China. A direct injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to rapidly determine 10 NACs in fine particulate matter (PM2.5) extracts. The method was sensitive for the quantification of the NACs, with a limit of quantification (LOQ) in the range of 0.092–0.52 ng mL−1. Then, the developed method was applied to determine the concentrations of nitrophenols (NPs), nitrocatechols (NCs), nitrosalicylic acids (NSAs), and nitronaphthol in PM2.5 in Mianyang. The average concentration of total NACs was 78.2 ± 31.2 ng m−3, with daily concentrations ranging from 20.7 to 127.9 ng m−3. Among the measured NACs, 4-nitrocatechol was the most abundant, accounting for 57.8% of the NACs in winter. The five NPs compounds together contributed to 14% of the NACs, which was lower than in other Chinese cities due to the warm climate in winter in Southwest China. NSAs and nitronaphthol each accounted for less than 5% of the NACs. Three major sources of NACs were identified based on the principal component analysis, including vehicle emissions, biomass burning, and secondary formation. The significant correlation between individual NACs and NO2 supported their secondary formation sources. The good correlation between NPs and cloud amount further suggested that gas-phase oxidation was the possible NPs formation mechanism. Our findings revealed the important role of nitrocatechols in NACs in Southwest China, implying that more measures should be taken to control biomass burning and aromatic volatile organic compounds emissions to reduce the level of NACs.

Metabolomics-based profiling of five Salvia L. (Lamiaceae) species using untargeted data analysis workflow.

The genus Salvia L., a member of the family Lamiaceae, is a keystone genus with a wide range of medicinal properties. It possesses a rich metabolite source that has long been used to treat different disorders. Due to a deficiency of untargeted metabolomic profiling in the genus Salvia, this work attempts to investigate a comprehensive mass spectral library matching, computational data annotations, exclusive biomarkers, specific chemotypes, intraspecific metabolite profile variation, and metabolite enrichment by a case study of five medicinal species of Salvia. Aerial parts of each species were subjected to QTRAP liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis workflow based on untargeted metabolites. A comprehensive and multivariate analysis was acquired on the metabolite dataset utilizing MetaboAnalyst 6.0 and the Global Natural Products Social Molecular Networking (GNPS) Web Platform. The untargeted approach empowered the identification of 117 metabolites by library matching and 92 nodes annotated by automated matching. A machine learning algorithm as substructural topic modeling, MS2LDA, was further implemented to explore the metabolite substructures, resulting in four Mass2Motifs. The automated library newly discovered a total of 23 metabolites. In addition, 87 verified biomarkers of library matching, 58 biomarkers of GNPS annotations, and 11 specific chemotypes were screened. Integrative spectral library matching and automated annotation by the GNPS platform provide comprehensive metabolite profiling through a workflow. In addition, QTRAP LC-MS/MS with multivariate analysis unveiled reliable information about inter and intraspecific levels of differentiation. The rigorous investigation of metabolite profiling presents a large-scale overview and new insights for chemotaxonomy and pharmaceutical studies.

Biocontrol of Botrytis cinerea by Streptomyces noursei C27 and preliminary identification of antimicrobial metabolites

A wide variety of soluble bioactive secondary metabolites and volatile organic compounds are produced by species of Streptomyces, making them potential candidates for controlling phytopathogenic microorganisms. In the present study, Streptomyces noursei C27, isolated from soil, was found to exhibit broad spectrum inhibitory activity against several phytopathogenic fungi. Furthermore, volatile organic compounds (VOCs) produced by S. noursei C27 exhibited total inhibition of Botrytis cinerea. An analysis of the complete genome sequence confirmed the identification of C27 as Streptomyces noursei. Liquid chromatography-tandem mass spectrometry analysis was used to characterize antimicrobial metabolites present in the fermentation broth of Streptomyces C27, that were identified as gougerotin, natamycin, nystatin, and anisomycin. Streptomyces C27 was shown to control gray mold on grape leaves and fruit in vitro, inhibiting infections by 72.5% and 71.9%, respectively. Gas chromatography-mass spectrometry analysis revealed that thirty alkenes, alcohols, esters, and alkanes were among the VOCs secreted by Streptomyces C27 and contributed to the inhibition of B. cinerea growth and development in inoculated grape leaves and fruit. The biocontrol efficacy of a wettable powder formulation of Streptomyces C27 against gray mold was tested on tomato seedlings in pot experiments and greenhouse experiment. Results indicated 61.86% and 67.25% control in the pot and field tests, respectively. In summary, Streptomyces noursei C27 has excellent potential to be a commercial biocontrol agent of plant diseases caused by pathogenic fungi, especially gray mold infections of vegetables and fruits caused by B. cinerea.

Beyond PSA: a multi-modal pilot study identifying novel proteomic biomarkers in paraffin-embedded prostate cancer tissue

Abstract Background Prostate cancer (PCa) is a common and complex disease in men, often progressing from localized to aggressive meta-static stages requiring advanced therapies. Early detection of PCa relies primarily on multiparametric tests, with limitations, like over-diagnosis and lack of specificity. Advances in molecular profiling, particularly proteomics, could enhance patient stratification and personalized therapies. Methods We conducted an analysis using Formalin-Fixed Paraffin-Embedded (FFPE) samples from 23 patients diagnosed with prostatic adenocarcinoma. Proteins were extracted from tissues, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The data were processed to identify proteins and subsequent bioinformatics analysis was performed to uncover significant pathways and potential biomarkers panel. Validation of identified biomarkers was carried out through Western blotting (WB). Results Proteomic profiling identified 1,159 proteins, including 176 significantly elevated in tumor tissues. Enrichment analysis highlighted their involvement in stress response, protein metabolism, and signaling pathways associated with PCa progression. Key pathways included mTORC1 signaling, Myc signaling, and focal adhesion. A biomarker panel consisting of KLK3, GDF15, MIF, and AZGP1 was proposed based on their discriminatory power in distinguishing tumor from normal tissues. WB confirmed the tumor-specific expression of these candidates, particularly GDF15 and KLK3. Conclusions The present study shows for the first time a multi-modal approach that combines a panel of PCa tissue proteins, as a new tool of PCa in patients. The identified biomarker panel offers promise for improving detection and tailoring personalized treatment strategies in PCa management. Further validation in larger cohorts and clinical settings is warranted to establish these findings.

The synergistic effect and mechanism of different adjuvants on pinoxaden efficacy against Lolium multiflorum Lam

Adjuvants play a crucial role in optimizing and improving the efficacy of herbicides. Therefore, adding appropriate adjuvants to herbicides is an important part of weed control. The aim of this study was to investigate the synergistic effect and underlying mechanism of action of three commercial adjuvants (Hasuteng, Penwubao, and Zhenxiao IV) in conjunction with pinoxaden for controlling Lolium multiflorum Lam. The results of synergic effect, which were determined by dose‒response assay showed that the addition of Hasuteng and Penwubao could significantly enhance the efficacy of pinoxaden and decrease the ED50 (the herbicide dose resulting in 50% weed control) on L. multiforum while being safe to wheat. Hasuteng and Penwubao were further selected to investigate the underlying mechanisms by which adjuvants enhanced the efficacy of pinoxaden. The results indicated that the addition of Hasuteng and Penwubao significantly reduced the surface tension and shortened the drying time of the pinoxaden solution; moreover, it significantly increased both expanded diameter and maximum retention. Additionally, ultra high performance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS) analysis was used to evaluate the recovery rate, absorption level, and rainfastness. These two adjuvants could improve the absorption and metabolism levels of L. multiforum to pinoxaden. However, Hasuteng and Penwubao did not improve the rainfastness of pinoxaden. In summary, Hasuteng and Penwubao enhance the efficacy of pinoxaden by improving its physical properties and absorption level. The studies present a novel approach for enhancing the utilization rate of pinoxaden and provide theoretical guidance toward effectively reducing herbicide application in wheat fields.

Antioxidant and Alpha-Glucosidase Inhibitory Activity of Durio zibethinus L. Clone 175 (Durian Udang Merah) Shell and UHPLC-ORBITRAP-MS Profiling of the Extract

Durio zibethinus or ‘durian’ is a well-known seasonal fruit of Southeast Asia and has been called the ‘King of Fruit’. The popularity of durian has led to large production of this crop in Malaysia, consequently creating a huge agricultural waste including the shell. Despite illness remedies from various parts of durian, the information on phytochemical constituents and bioactivities of durian shells remained scarce. Therefore, this study aimed to evaluate the total phenolic content (TPC), antioxidant, and α-glucosidase inhibitory activities of D. zibethinus shell extracted with different ethanol percentage (0%, 50%, and 100%). Results showed that the 100% ethanolic extract exhibited the highest 2,2-dipheny-1-picrylhydrazyl (DPPH) free radical scavenging activity with an IC50 value of 96.91 ± 1.09 µg/mL. Furthermore, the 50% ethanol extract exhibited the highest TPC with 130.57 ± 1.92 mg GAE/g crude extract, and nitric oxide (NO) scavenging activity with an IC50 value of 435.30 ± 3.41 µg/mL. Both 50% and 100% ethanolic extracts of D. zibethinusshell exhibited great potential in α-glucosidase inhibitory activity with IC50 values of 1.99 ± 0.90 and 4.53 ± 0.21 µg/mL, respectively. Thus, the bioactive compounds in 100% ethanolic extract of D. zibethinus were profiled by ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. A total of 20 compounds were tentatively identified, including flavonoids, alkaloids, benzofurans, terpenoids, pentose phosphate, organosulfur compounds, organooxygen compounds, polyketides, carotene, carboxylic acid, coumarin, and stigmasterol. In conclusion, durian shell exhibits potential for future applications driven by its inherent pharmacological benefits, consequently contributing to waste reduction.