Search Results (52)

Background: Liquid chromatography-mass spectrometry (LC-MS), widely used in metabolomics, is often limited by low ionization efficiency and ion suppression, which reduce overall metabolite detectability and quantification accuracy. To address these challenges, chemical isotope labeling (CIL) LC-MS has emerged as a powerful approach, offering high sensitivity, accurate quantification, and broad metabolome coverage. This method enables comprehensive profiling by targeting multiple submetabolomes. Specifically, amine-/phenol- and hydroxyl-containing metabolites are labeled using dansyl chloride under distinct reaction conditions. While this strategy provides extensive coverage, the sequential analysis of each submetabolome reduces throughput. To overcome this limitation, we propose a two-channel mixing strategy to improve analytical efficiency. Methods: In this approach, samples labeled separately for the amine/phenol and hydroxyl submetabolomes are combined prior to LC-MS analysis, leveraging the common use of dansyl chloride as the labeling reagent. This integration effectively doubles throughput by reducing LC-MS runtime and associated costs. The method was evaluated using human urine and serum samples, focusing on peak pair detectability and metabolite identification. A proof-of-concept study was also conducted to assess the approach’s applicability in putative biomarker discovery. Results: Results demonstrate that the two-channel mixing strategy enhances throughput while maintaining analytical robustness. Conclusions: This method is particularly suitable for large-scale studies that require rapid sample processing, where high efficiency is essential. Full article

Background: Scientific and rational fertilizer management can not only improve the yield and quality of hazelnut (Corylus heterophylla × Corylus avellana) but also reduce the negative impact on the environment. Methods: Liquid Chromatography–tandem Mass Spectrometry (LC-MS/MS) technology was used to reveal the contents of various metabolites in hazelnut seedlings, and differential metabolites were screened by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Results: The results showed that a total of 178 up-regulated differential metabolites (Fold change > 1) and 175 down-regulated differential metabolites (Fold change < 1) were detected in 6 comparison groups (DWF0 vs. DWF4, DWF0 vs. DWF5, DWF0 vs. DWF6, DWF4 vs. DWF5, DWF4 vs. DWF6, DWF5 vs. DWF6). Interestingly, the flavonoid metabolic pathway was dramatically enriched, and it was involved in each fertilization combination. The metabolites of the flavonoid pathway in different fertilized and unfertilized groups were compared and analyzed, which displayed that metabolites tricetin, eriodictyol, garbanzol, apigenin, and biochanin A were significantly up-regulated, while garbanzol and astraglin were significantly down-regulated. More interestingly, the determination of flavonoid content and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) displayed that the application of trace element water-soluble fertilizer could significantly enhance the flavonoid content and the expression of genes related to the flavonoid biosynthesis pathway, such as phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H), with the DWF4 treatment displaying the most significant values. Conclusions: Overall, the application of trace element water-soluble fertilizer (especially the DWF4 treatment) markedly affected the changes in key metabolites of the flavonoid pathway and the expression levels of key genes, thus promoting the growth and development of the hazelnut, which offers an important starting point for future analysis through genetic engineering. Full article

Lesion mimic mutants provide unique tools to investigate plant–pathogen interactions, often exhibiting hypersensitive responses in the absence of biotic or abiotic stresses. The overexpression of the S-domain receptor-like kinase gene, SPL11 cell-death suppressor 2 (SDS2), in rice leads to constitutive programmed cell death and enhanced resistance to fungal and bacterial pathogens. However, the mechanisms underlying this broad-spectrum resistance remain unclear. This study integrates transcriptomic and metabolomic analyses of the SDS2-ACT mutant to uncover gene expression and metabolic shifts associated with disease resistance. To identify SDS2-specific physiological changes related to pathogen resistance, leaf tissues from the SDS2-ACT mutant and the Kitkaake WT line were subjected to both transcriptomic and non-targeted metabolic profiling. Transcriptomic analyses identified 1497 differentially expressed genes (DEGs), including up-regulated genes involved in terpenoid and flavonoid biosynthesis, phytohormone signaling, and defense-related pathways (including pathogenesis-related [PR] genes). Metabolomic profiling revealed significant alterations in the accumulation of several compound classes, including putative: terpenoids, phenylpropanoids, phytohormones, fatty acids, and sugars. These changes are likely correlated with the observed cell death and resistance phenotypes in the SDS2-ACT mutant. This study provides an overall landscape of the transcriptomic and metabolomic alterations in a lesion mimic mutant, identifying candidate defense-related genes and metabolites for functional analysis in rice. Full article

Background/Objectives: Diabetes mellitus is often accompanied by mental health complications, including anxiety, depression, and cognitive decline. Recent research suggested that capsaicin, the active component of chili peppers, may influence mental health. This study aimed to determine the effect of dietary capsaicin on mental disorders in a type 1 diabetes (T1D) mouse model, while also exploring the potential involvement of the microbiota-gut-brain axis. Methods: We induced T1D in mice using streptozotocin (STZ) and administered a diet supplemented with 0.005% capsaicin for five weeks. Behavioral assessments, including the open field test (OFT), tail suspension test (TST), forced swimming test (FST), elevated plus maze (EPM) test, and Morris water maze (MWM) test, were conducted to evaluate depressive and anxiety-like behaviors as well as cognitive function. Targeted and untargeted metabolomics analyses were performed to assess neurotransmitter levels in the hippocampus and serum metabolites, while 16S rRNA sequencing was utilized to analyze gut microbiota composition. Intestinal barriers were determined using western blot detection of the tight junction proteins ZO-1 and occludin. Results: Dietary capsaicin exacerbated anxiety and depressive-like behaviors along with cognitive declines in T1D mice. Capsaicin reduced gut microbiota diversity and levels of beneficial bacteria, while broad-spectrum antibiotic treatment further intensified anxiety and depression behaviors. Metabolomic analysis indicated that capsaicin disrupted metabolic pathways related to tryptophan and phenylalanine, leading to decreased neuroprotective metabolites, such as kynurenic acid, hippurate, and butyric acid. Additionally, capsaicin diminished the expression of ZO-1 and occludin, indicating increased intestinal permeability. Conclusions: Dietary capsaicin aggravates gut microbiota and metabolic disturbances in diabetic mice, thereby worsening anxiety, depression, and cognitive decline. Full article

Lettuce (Lactuca sativa L.) is a popular leafy vegetable valued for its dietary fiber, antioxidants, and beneficial vitamins. This study presents a comprehensive spatio-temporal analysis of the lettuce metabolome, revealing complex dynamics in metabolite accumulation influenced by plant age, leaf position, proximodistal distribution within a leaf, and head closure. Samples were collected from plants at five maturity stages (ranging from baby leaf to full commercial maturity and eventually to bolting) and from five leaf positions (from the apex to the base of each plant). A widely targeted metabolomics approach identified 1905 compounds, with flavonoids, phenolic acids, and lipids as the largest classes. Younger plants exhibited higher levels of flavonoids, while older plants accumulated more saccharides and amino acids. Metabolites showed distinct proximodistal distributions, with flavonoids and vitamins concentrated at leaf tips and terpenoids declining from base to tip. Head closure significantly reduced levels of flavonoids, retinol (vitamin A1), and riboflavin (vitamin B2), while it was associated with increased content of other beneficial vitamins, such as thiamine (B1), pantothenate (B5), and pyridoxine (B6). Broad-sense heritability (H²) estimates for metabolites yielded mean H² values of 0.648 and 0.743 for plants at baby-leaf and commercial maturity stages, respectively. The overall highest heritability was observed in tannins (H² = 0.909) in younger plants and chalcones (H² = 0.894) in older plants, suggesting strong genetic control over specific metabolite classes and subclasses. These findings offer a robust framework for optimizing lettuce’s nutritional profile by linking metabolite distributions to developmental processes, plant architecture, and genetic regulation. By leveraging these insights, breeders and producers can develop targeted strategies to enhance metabolite content through optimized breeding and harvesting strategies. Full article

Spinosad is an efficient and broad-spectrum environmentally friendly biopesticide, but its low yield in wild-type Saccharopolyspora spinosa limits its further application. ARTP/NTG compound mutagenesis was used in this study to improve the spinosad titer of S. spinosa and obtain a high-yield mutant—NT24. Compared with the wild-type strain, the fermentation cycle of NT24 was shortened by 2 days and its maximum titer of spinosad reached 858.3 ± 27.7 mg/L, which is 5.12 times more than for the same-period titer of the wild-type strain. In addition, RT-qPCR, resequencing, and targeted metabolomics showed that the upregulation of the key differential genes accD6, fadD, sdhB, oadA, and gntZ caused increased metabolic flux in the tricarboxylic acid cycle and pentose phosphate pathway, suggesting that the accumulation of pyruvate and short-chain acyl-CoA was the primary cause of spinosad accumulation in NT24. This study demonstrates the effectiveness of ARTP mutagenesis in S. spinosa, and provides new insights for the mechanism of spinosad biosynthesis and metabolic engineering in S. spinosa. Full article

Inonotus hispidus (Bull.) P. Karst., is a medicinal fungus, which parasitizes broad-leaved tree such as Morus alba L., Fraxinus mandshurica Rupr., and Ulmus macrocarpa Hance. To elucidate the internal relationship between I. hispidus and its hosts, this study analyzed endophytic bacteria and fungi in the roots of M. alba and F. mandshurica growing I. hispidus using the 16S rDNA and ITS high-throughput sequencing technologies; and conducted widely targeted metabolomics research using UPLC-MS/MS. The results showed that Cyanobacteria and unidentified chloroplasts had the highest relative abundance at the phylum and genus levels, respectively. For endophytic fungi, Ascomycota was dominant at the phylum level, while Pleosporales gen Incertae sedis and Oncopodiella were the dominant genera in the roots of M. alba and F. mandshurica, respectively. Widely targeted metabolomics identified 562 differential metabolites and 46 metabolic pathways. Correlation analysis revealed that Xanthobacteraceae, Pseudorhodoplanes, and Bauldia were potential regulators of phenolic acids and phenylpropanoids biosynthesis. Additionally, the genus Oncopodiella was primarily associated with the enrichment of lipids, amino acids, sugars, phenolic acids, and other compounds. This result provides significant insights into the size of the fruiting body, resource development, and active ingredients of I. hispidus from different tree sources. Full article

Oleum cinnamomi (OCM) is a volatile component of the Cinnamomum cassia Presl in the Lauraceae family, which displays broad-spectrum antibacterial properties. It has been found that OCM has a significant inhibitory effect against Cutibacterium acnes (C. acnes), but the precise target and molecular mechanism are still not fully understood. In this study, the antibacterial activity of OCM against C. acnes and its potential effect on cell membranes were elucidated. Metabolomics methods were used to reveal metabolic pathways, and proteomics was used to explore the targets of OCM inhibiting C. acnes. The yield of the OCM was 3.3% (w/w). A total of 19 compounds were identified, representing 96.213% of the total OCM composition, with the major constituents being phenylpropanoids (36.84%), sesquiterpenoids (26.32%), and monoterpenoids (15.79%). The main component identified was trans-cinnamaldehyde (85.308%). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of OCM on C. acnes were 60 µg/mL and 180 µg/mL, respectively. The modified proteomics results indicate that cinnamaldehyde was the main bioactive ingredient within OCM, which covalently modifies the ABC transporter adenosine triphosphate (ATP)-binding protein and nicotinamide adenine dinucleotide (NADH)-quinone oxidoreductase, hindering the amino acid transport process, and disrupting the balance between NADH and nicotinamide adenine dinucleoside phosphorus (NAD⁺), thereby hindering energy metabolism. We have reported for the first time that OCM exerts an antibacterial effect by covalent binding of cinnamaldehyde to target proteins, providing potential and interesting targets to explore new control strategies for gram-positive anaerobic bacteria. Full article

Nepeta nuda L., a notable medicinal species in the tradition of the Balkan region, is a rich source of bioactive iridoids and phenolics previously described as high-resolution taxonomical classifiers for the genus Nepeta. However, their potential in investigating intra-species differentiation is here described for the first time. The aim was to recognize the sources of natural chemical diversity and their association with the genetic variability both within and among N. nuda populations in the Central Balkans. Chemical diversity was assessed from methanol extracts and essential oils through untargeted and targeted metabolomics using state-of-the-art analytical tools, covering a broad spectrum of compounds that represent the N. nuda metabolome. We found that chemodiversity primarily resides within populations of N. nuda, and similar results were obtained at the DNA level using microsatellite markers. The low genetic and chemical differentiation of the studied N. nuda populations implies that their metabolomic profiles may be less influenced by geographic distance and variable environmental conditions within the Central Balkans, as they are under the pivotal control of their genetic backgrounds. Screening the distribution of the major bioactive compounds belonging to phenolics (phenolic acids and flavonoids) and iridoids (both aglycones and glycosylated forms), within and among N. nuda populations, is able to guarantee mass spectrometry-based tools for the selection of elite representative genotypes with practical importance. The knowledge acquired will allow us to delve deeper into the molecular background of N. nuda chemical diversity, which is the course of our further work. Full article

Orbitrap mass spectrometry in full scan mode enables the simultaneous detection of hundreds of metabolites and their isotope-labeled forms. Yet, sensitivity remains limiting for many metabolites, including low-concentration species, poor ionizers, and low-fractional-abundance isotope-labeled forms in isotope-tracing studies. Here, we explore selected ion monitoring (SIM) as a means of sensitivity enhancement. The analytes of interest are enriched in the orbitrap analyzer by using the quadrupole as a mass filter to select particular ions. In tissue extracts, SIM significantly enhances the detection of ions of low intensity, as indicated by improved signal-to-noise (S/N) ratios and measurement precision. In addition, SIM improves the accuracy of isotope-ratio measurements. SIM, however, must be deployed with care, as excessive accumulation in the orbitrap of similar m/z ions can lead, via space-charge effects, to decreased performance (signal loss, mass shift, and ion coalescence). Ion accumulation can be controlled by adjusting settings including injection time and target ion quantity. Overall, we suggest using a full scan to ensure broad metabolic coverage, in tandem with SIM, for the accurate quantitation of targeted low-intensity ions, and provide methods deploying this approach to enhance metabolome coverage. Full article

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool for the activation of biosynthetic gene clusters (BGCs) to generate new metabolites, as well as to increase the yield of respective target metabolites. As part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, we selected the co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophyted in mangrove Rhizophora mangle considering the impart of the taxonomic criteria and ecological data. The competition interaction of the two strains was investigated through morphology observation and scanning electron microscopy (SEM), and it was found that the mycelia of the DHS-48 and DHS-11 compacted and tangled with each other with an interwoven pattern in the co-culture system. A new approach that integrates HPLC chromatogram, ¹HNMR spectroscopy, UPLC-MS-PCA, and molecular networking enabled the targeted isolation of the induced metabolites, including three new dimeric xanthones phomoxanthones L-N (1–3), along with six known analogs (4–9). Their planar structures were elucidated by an analysis of their HRMS, MS/MS, and NMR spectroscopic data and the absolute configurations based on ECD calculations. These metabolites showed broad cytotoxic activity against the cancer cells assessed, of which compounds 7–9 displayed significant cytotoxicity towards human liver cells HepG-2 with IC₅₀ values ranging from 4.83 μM to 12.06 μM. Compounds 1–6 exhibited weak immunosuppressive activity against the proliferation of ConA-induced (T-cell) and LPS-induced (B-cell) murine splenic lymphocytes. Therefore, combining co-cultivation with a metabolomics-guided strategy as a discovery tool will be implemented as a systematic strategy for the quick discovery of target bioactive compounds. Full article

Atherosclerosis and resulting cardiovascular disease are the leading causes of death in the US. Hyperhomocysteinemia (HHcy), or the accumulation of the intermediate amino acid homocysteine, is an independent risk factor for atherosclerosis, but the intricate biological processes mediating this effect remain elusive. Several factors regulate homocysteine levels, including the activity of several enzymes and adequate levels of their coenzymes, including pyridoxal phosphate (vitamin B6), folate (vitamin B9), and methylcobalamin (vitamin B12). To better understand the biological influence of HHcy on the development and progression of atherosclerosis, apolipoprotein-E-deficient (apoE^−/− mice), a model for human atherosclerosis, were fed a hyperhomocysteinemic diet (low in methyl donors and B vitamins) (HHD) or a control diet (CD). After eight weeks, the plasma, aorta, and liver were collected to quantify methylation metabolites, while plasma was also used for a broad targeted metabolomic analysis. Aortic plaque burden in the brachiocephalic artery (BCA) was quantified via 14T magnetic resonance imaging (MRI). A severe accumulation of plasma and hepatic homocysteine and an increased BCA plaque burden were observed, thus confirming the atherogenic effect of the HHD. Moreover, a decreased methylation capacity in the plasma and aorta, indirectly assessed by the ratio of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) was detected in HHD mice together with a 172-fold increase in aortic cystathionine levels, indicating increased flux through the transsulfuration pathway. Betaine and its metabolic precursor, choline, were significantly decreased in the livers of HHD mice versus CD mice. Widespread changes in the plasma metabolome of HHD mice versus CD animals were detected, including alterations in acylcarnitines, amino acids, bile acids, ceramides, sphingomyelins, triacylglycerol levels, and several indicators of dysfunctional lipid metabolism. This study confirms the relevance of severe HHcy in the progression of vascular plaque and suggests novel metabolic pathways implicated in the pathophysiology of atherosclerosis. Full article

Reuterin is a dynamic small-molecule complex produced through glycerol fermentation by Limosilactobacillus reuteri and has potential as a food biopreservative. Despite its broad-spectrum antimicrobial activity, the underlying mechanism of action of reuterin is still elusive. The present paper aimed to explore the antibacterial mechanism of reuterin and its effects on membrane damage and the intracellular metabolome of S. aureus. Our results showed that reuterin has a minimum inhibitory concentration of 18.25 mM against S. aureus, based on the 3-hydroxypropionaldehyde level. Key indicators such as extracellular electrical conductivity, membrane potential and permeability were significantly increased, while intracellular pH, ATP and DNA were markedly decreased, implying that reuterin causes a disruption to the structure of the cell membrane. The morphological damage to the cells was confirmed by scanning electron microscopy. Subsequent metabolomic analysis identified significant alterations in metabolites primarily involved in lipid, amino acid, carbohydrate metabolism and phosphotransferase system, which is crucial for cell membrane regulation and energy supply. Consequently, these findings indicated that the antibacterial mechanism of reuterin initially targets lipid and amino acid metabolism, leading to cell membrane damage, which subsequently results in energy metabolism disorder and, ultimately, cell death. This paper offers innovative perspectives on the antibacterial mechanism of reuterin, contributing to its potential application as a food preservative. Full article

Long-term cognitive dysfunction, or “chemobrain”, has been observed in cancer patients treated with chemotherapy. Mitoxantrone (MTX) is a topoisomerase II inhibitor that binds and intercalates with DNA, being used in the treatment of several cancers and multiple sclerosis. Although MTX can induce chemobrain, its neurotoxic mechanisms are poorly studied. This work aimed to identify the adverse outcome pathways (AOPs) activated in the brain upon the use of a clinically relevant cumulative dose of MTX. Three-month-old male CD-1 mice were given a biweekly intraperitoneal administration of MTX over the course of three weeks until reaching a total cumulative dose of 6 mg/kg. Controls were given sterile saline in the same schedule. Two weeks after the last administration, the mice were euthanized and their brains removed. The left brain hemisphere was used for targeted profiling of the metabolism of glutathione and the right hemisphere for an untargeted metabolomics approach. The obtained results revealed that MTX treatment reduced the availability of cysteine (Cys), cysteinylglycine (CysGly), and reduced glutathione (GSH) suggesting that MTX disrupts glutathione metabolism. The untargeted approach revealed metabolic circuits of phosphatidylethanolamine, catecholamines, unsaturated fatty acids biosynthesis, and glycerolipids as relevant players in AOPs of MTX in our in vivo model. As far as we know, our study was the first to perform such a broad profiling study on pathways that could put patients given MTX at risk of cognitive deficits. Full article

Plant-parasitic nematodes (PPNs) pose a threat to global food security in both the developed and developing worlds. PPNs cause crop losses worth a total of more than USD 150 billion worldwide. The sedentary root-knot nematodes (RKNs) also cause severe damage to various agricultural crops and establish compatible relationships with a broad range of host plants. This review aims to provide a broad overview of the strategies used to identify the morpho-physiological and molecular events that occur during RKN parasitism. It describes the most current developments in the transcriptomic, proteomic, and metabolomic strategies of nematodes, which are important for understanding compatible interactions of plants and nematodes, and several strategies for enhancing plant resistance against RKNs. We will highlight recent rapid advances in molecular strategies, such as gene–silencing technologies, RNA interference (RNAi), and small interfering RNA (siRNA) effector proteins, that are leading to considerable progress in understanding the mechanism of plant–nematode interactions. We also take into account genetic engineering strategies, such as targeted genome editing techniques, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system, and quantitative trait loci (QTL), to enhance the resistance of plants against nematodes. Full article