To investigate the role of the previously little-studied tyrosine (Tyr), we have used stable isotope-assisted metabolomics to compare its metabolism to the well-studied phenylalanine (Phe) in wheat. Flowering wheat ears were treated either with ¹³C₉-Phe or ¹³C₉-Tyr as metabolic tracers under both control and stress conditions (addition of 0.2 mg per ear of the Fusarium mycotoxin and virulence factor deoxynivalenol (DON)). Overall, 115 Phe- and Tyr-derived wheat metabolites were detected. For ¹³C₉-Tyr, the overall uptake and metabolization of the initially applied tracers (0.5 mg each) was about 40 % compared to 80 % of Phe, and 48 downstream metabolites were shared between both tracers. About one-third of the detected metabolites were found to be significantly induced by DON treatment (DON+). Those belonged to the classes of hydroxycinnamic acids (HCAs), HCA-amides, -glycosides, -quinates and lignans, suggesting their role as precursors for cell wall reinforcement and antifungal compounds. Evaluation of ¹³C isotopolog signals revealed that Tyr was incorporated into common downstream metabolites at a rate of approximately 1/6 (mock) and 1/4 (DON) of that compared to Phe. Tyr incorporation was induced upon DON stress compared to control conditions, whereas Phe showed indifferent median incorporation rates. This study highlights the added value and complementarity of using labeled Tyr and Phe as tracers in isotope-assisted metabolomics to improve annotation confidence and biological interpretation of plant metabolic responses to stress.

Objectives: This study presents a versatile, AI-guided workflow for the targeted isolation and characterization of prenylated flavonoids from Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae). Methods: The approach integrates established extraction and chromatography-based fractionation protocols with LC-UV-HRMS/MS analysis and supervised machine-learning (ML) custom-trained classification models, which predict prenylated flavonoids from LC-HRMS/MS spectra based on the recently developed Python package AnnoMe (v1.0). Results: The workflow effectively reduced the chemical complexity of plant extracts and enabled efficient prioritization of fractions and compounds for targeted isolation. From the pre-fractionated plant extracts, 2687 features were detected, 42 were identified using reference standards, and 214 were annotated via spectra library matching (public and in-house). Furthermore, ML-trained classifiers predicted 1805 MS/MS spectra as derived from prenylated flavonoids. LC-UV-HRMS/MS data of the most abundant presumed prenyl-flavonoid candidates were manually inspected for coelution and annotated to provide dereplication. Based on this, one putative prenylated (C5) dihydroflavonol (1) and four geranylated (C10) flavanones (2–5) were selected and successfully isolated. Structural elucidation employed UV spectroscopy, HRMS, and 1D as well as 2D NMR spectroscopy. Compounds 1 and 5 were isolated from a natural source for the first time and were named 6-prenyl-4′-O-methyltaxifolin and 3′,4′-O-dimethylpaulodiplacone A, respectively. Conclusions: This study highlights the combination of machine learning with analytical techniques to streamline natural product discovery via MS/MS and AI-guided pre-selection, efficient prioritization, and characterization of prenylated flavonoids, paving the way for a broader application in metabolomics and further exploration of prenylated constituents across diverse plant species.

Short non-coding RNAs, specifically microRNAs (miRNAs), are of a great interest due to their presumed function in genome regulation. Moreover, miRNAs are currently perceived as potential biomarkers for numerous diseases; a variety of detection methods and sensing systems have therefore been studied. We present a magnetic-bead-based assay for specific miRNA isolation coupled with sensitive electrophoretic analysis with fluorescence detection. The magnetic separation step involves creating a duplex with targeted miR-141, which is subsequently cleaved from the magnetic bead surface with a specific endonuclease. The duplex is then determined using capillary electrophoresis with laser-induced fluorescence detection in the presence of the fluorescent dye PicoGreen for quantitating double-stranded DNA. The benefits of using microcolumn separation technique coupled with sensitive detection over traditionally used determination by fluorescence spectrometry include the fact that there is no need for a specific pre-labeled fluorescent probe. This significantly simplifies the method and reduces the costs. Cross-reactivity with mismatched oligonucleotides (3 and 5 mismatched bases) and different miRNAs (miR-124 and miR-150) was tested, demonstrating the specificity of the developed method for miRNA-141. This magnetic extraction method was demonstrated for the direct isolation and determination of miR-141 at different concentration levels from urine samples and the achieved nanomolar detection limit.

Brown algae such as Ascophyllum nodosum (AN) and Fucus vesiculosus (FV) are gaining considerable attention as functional feed additives due to their health-beneficial properties. This study evaluated the antioxidant potential of AN and FV extracts in intestinal epithelial cells and the in vivo model Caenorhabditis elegans (C. elegans). Aqueous AN and FV extracts were characterized for total phenolic content (TPC), antioxidant capacity (TEAC, FRAP), and phlorotannin composition using LC-HRMS/MS. Antioxidant effects were assessed in vitro, measuring AAPH-induced ROS production in Caco-2 and IPEC-J2 cells via H₂DCF-DA, and in vivo, evaluating the effects of paraquat-induced oxidative stress and AN or FV treatment on worm motility, GST-4::GFP reporter expression, and gene expression in C. elegans. FV exhibited higher total phenolic content, antioxidant capacity (TEAC, FRAP), and a broader phlorotannin profile (degree of polymerization [DP] 2–9) than AN (DP 2–7), as determined by LC-HRMS/MS. Both extracts attenuated AAPH-induced oxidative stress in epithelial cells, with FV showing greater efficacy. In C. elegans, pre-treatment with AN and FV significantly mitigated a paraquat-induced motility decline by 22% and 11%, respectively, compared to PQ-stressed controls. Under unstressed conditions, both extracts enhanced nematode healthspan, with significant effects observed at 400 µg/g for AN and starting at 100 µg/g for FV. Gene expression analysis indicated that both extracts modulated antioxidant pathways in unstressed worms. Under oxidative stress, pre-treatment with AN and FV significantly reduced GST-4::GFP expression. In the nematode, AN was more protective under acute stress, whereas FV better supported physiological function in the absence of stressors. These findings demonstrate that AN and FV counteract oxidative stress in intestinal epithelial cells and in C. elegans, highlighting their potential as stress-reducing agents in animal feed.