Anthropogenic pollutants known as xenobiotics, such as pharmaceuticals, pesticides, or human metabolites, primarily originate on land and accumulate near populated coasts, where rainfall further increases their transport and harmful impacts on marine ecosystems. However, empirical evidence for such dynamics remains limited. Here, we investigate spatial and seasonal patterns of terrigenous xenobiotics along the land-sea continuum of Curaçao. Using solid-phase extraction and untargeted LC-HR-MS/MS metabolomics, we detected approximately 30,000 molecular features, including 1,394 xenobiotics that were most abundant in terrestrial waters, declined offshore, and correlated strongly with other terrestrial input indicators (fDOM). Results from three cross-seasonal campaigns (2021-2022) revealed that rainfall and increased water residence times were dominant drivers for xenobiotic abundances along the shore, with 3.5 times higher abundances during the 2022 wet season compared to drier periods. Additional factors were local landscape features, distance to fluvial runoff, hydrodynamic connectivity, and, for specific substance groups (e.g., human metabolites or personal care products), human activities in proximity to sampling locations. Our findings show that terrigenous xenobiotics spread island-wide, making them a broader concern beyond discharge sites. This first island-wide, seasonally resolved assessment provides a baseline for managing emerging contaminants and understanding their wider impact on Caribbean coastal ecosystems.
The single-electron reduction of redox-active drugs and xenobiotics (quinones, aromatic nitrocompounds, and N-oxides) by flavoenzymes, which initiates redox cycling and oxidative stress, is an important factor in their therapeutic/toxic effects. This review summarizes information on the action of mitochondrial flavoenzymes from various organisms in these processes, emphasizing the kinetic and mechanistic aspects. The flavoenzymes discussed also include those of which only a fraction is localized in mitochondria. According to kinetic data, the most effective generator of free radicals of xenobiotics is respiratory Complex I. However, it is unclear to what extent these reactions can compete with the rapid reduction of ubiquinone in normally functioning mitochondria. In specific cases, a very active free radical generator can be the NADPH:adrenodoxin reductase-adrenodoxin complex. The properties of other dehydrogenases-electrontransferases (succinate:ubiquinone reductase, fatty acid oxidation system) are less well characterized. Due to its high catalytic capacity, a potential but poorly studied source of free radicals of xenobiotics may be NADH:cytochrome b5 reductase and its complex with cytochrome b5. Flavoenzyme disulfide reductases, with the possible exception of Plasmodium falciparum thioredoxin reductase, are less active free radical generators. Importantly, in most cases, flavoenzymes perform the mixed single- and two-electron reduction of xenobiotics. According to the available data, the reactivity of redox cyclers depends mostly on their standard single-electron reduction potential and is little influenced by their structure. Therefore, in order to intensify these processes or achieve some structural specificity, it is necessary to focus on the selective accumulation of compounds in mitochondria.
The gut microbiota is implicated in adverse effects associated with low-calorie sweeteners. Yet, the direct impact of sweeteners on gut bacteria remains largely uncharacterized. Here, we report interactions between 25 phylogenetically diverse gut bacterial strains and 39 commercially used sweeteners. We tested these sweeteners individually and in combination with four commonly co-consumed compounds, viz., advantame, caffeine, vanillin, and duloxetine. Three-quarters of the tested sweeteners individually impacted the growth of at least one tested bacterial strain. Further, over 100 interactions were found between sweeteners and the four co-consumed compounds. Isosteviol, a commonly used sweetener-component, and duloxetine, an antidepressant, synergistically inhibited Roseburia intestinalis, a bacterium previously linked to glucose homeostasis, and Parabacteroides merdae, a prevalent commensal linked to healthy microbiota. Proteomic, metabolomic, and genetic analyses indicate altered small molecule transport underpinning this sweetener-drug synergy. The isosteviol-duloxetine combination also modulated metabolism of a synthetic gut bacterial community, leading to increased toxicity to HeLa cells and altered secretion of inflammation-modulatory cytokines IL-6 and IL-8 by Caco-2 cells. Our data warrant further studies on interactions between low-calorie sweeteners and common xenobiotics.
The aim of the study was to describe and evaluate the biotechnological potential of a novel species of Nocardia isolated from underground coal mine water. Nocardia strains represent important human pathogens, but are also common in soil and water environments. Recently, they have gained attention for their biosynthetic potential and vast metabolic abilities to degrade aromatic compounds, including xenobiotics. In this work, we established the taxonomic status of strain MW-W600-9T. Phylogenetic analysis based on 16 S rRNA gene and whole genome sequencing revealed close relatedness to Nocardia rhizosphaerihabitans and Nocardia asteroides. This is further supported by phenotyping tests, chemotaxonomic analyses and MALDI-ToF profiling. We therefore propose that strain MW-W600-9T (= DSM 120649T =PCM 3565T) constitutes a new species, Nocardia fodinahabitans sp. nov. The strain has 31 putative biosynthetic gene clusters, 12 showing low or no similarity to the clusters of known compounds, indicating its potential to produce novel bioactive compounds. The strain MW-W600-9T shows degradation potential of aromatic xenobiotics, tested for haloorganic compounds and bisphenols. The concentration of 4-chlorophenol and bisphenol A was decreasing significantly during the strain culture, and the results suggest co-metabolic removal of iohexol. This work shows the biodegradation ability of aromatic xenobiotic compounds by a new species of Nocardia, highlighting the potential use of the strain in bioremediation applications. It also shows for the first time the degradation of bisphenol by a member of the Nocardia genus, along with potential co-metabolic degradation of environmentally persistent haloorganic compounds.
The rapid emergence of new psychoactive substances (NPS) and their extensive biotransformation challenge the reliability and interpretability of chemical measurements in forensic toxicology. Targeted analytical workflows offer high selectivity but frequently fail when parent compounds are present at low concentrations or are absent from biological matrices, limiting metabolite coverage and evidential interpretation. In this study, an untargeted LC-HRMS measurement and data-analysis framework was evaluated to improve metabolite annotation and structural contextualization in complex forensic samples. Blood and urine collected from a suspected driving under the influence of drugs (DUID) case were analyzed using high-resolution full-scan and data-dependent MS/MS acquisition as a representative test system. Data were processed in Compound Discoverer using a customized workflow combining spectral library matching (mzCloud and in-house spectral database built from HighResNPS.com, containing over 2400 high-resolution spectra of drugs of abuse and NPS), rule-based metabolite prediction (MetID), and transformation-aware molecular networking. The transformation-aware molecular networking strategy integrates MS/MS spectral similarity with predicted phase I and phase II biotransformations, enabling relational organization of parent compounds, metabolites, and structurally related features. Compared to rule-based metabolite prediction alone, this approach increased the number of metabolite-related features associated with detected xenobiotics and supported reconstruction of chemically consistent metabolic families, including cases in which parent compounds were not observed in the measured sample. Taken together, the results show that transformation-aware molecular networking provides an effective means of organizing and interpreting untargeted LC-HRMS data by linking spectral similarity with biotransformation relationships. This framework supports a more contextual interpretation of metabolite-related features in forensic and toxicological investigations involving NPS and other xenobiotics.
Influenza and SARS-CoV-2 can cause severe respiratory failure, but the metabolic pathways that lead to clinical deterioration are not fully uncovered. Tryptophan catabolism has been linked to disease progression and adverse outcomes. We aimed to find out whether the link between tryptophan catabolism and disease progression is shared by the 2 viral infections and, in an exploratory manner, to assess other metabolic pathways. Adults hospitalized due to influenza or SARS-CoV-2 from 3 prospective studies were pooled in a nested case-control study design. Cases were defined by disease progression: an increase in oxygen supplementation, intensive care unit admission, or death within 28 days. Cases were matched 1:2 to nonprogressors by pathogen and initial disease severity. We tested associations of plasma kynurenine, tryptophan, and the kynurenine/tryptophan ratio with disease progression. Metabolic profiles were investigated by unsupervised clustering-based, pathway-resolved methods. We included 303 patients hospitalized with influenza or SARS-CoV-2. Higher levels of kynurenine and higher kynurenine/tryptophan ratios were associated with disease progression (odds ratio per log2 increase [95% CI], 1.81 [1.21-2.70] and 1.89 [1.26-2.84], respectively) independent of pathogen. Two metabolite modules were associated with disease progression. One module contained multiple amino acids, including kynurenine and 10 other tryptophan catabolism metabolites. The other module contained mainly lipids and xenobiotics. Several groups of metabolites were associated with disease progression independent of the pathogen. This indicates that biological mechanisms related to disease severity are shared in influenza and COVID-19. These mechanisms could be used for risk stratification of patients for potential disease-modifying treatments.
Drug-induced liver injury (DILI) refers to hepatotoxicity caused by conventional chemical drugs or xenobiotics, whereas herb-induced liver injury (HILI) is attributed to herbal and dietary supplements. Both these conditions pose diagnostic challenges, particularly when concurrent etiologies such as acute viral hepatitis are present. Hurler syndrome (mucopolysaccharidosis Type I) causes hepatocyte and Kupffer cell vacuolization and can predispose to DILI. Early diagnosis is critical given the high fatality rates associated with DILI. We present a case of a 28-year-old male with Hurler syndrome who presented with acute onset of nausea, vomiting, and jaundice. Liver function tests (LFTs) revealed markedly elevated liver enzymes. Serological workup identified newly acquired acute hepatitis C virus (HCV) infection. The patient had recent amoxicillin use and was taking hibiscus tea daily. Causality assessment using the updated Roussel Uclaf Causality Assessment Method (RUCAM) of 2016 yielded a score of 6 (probable DILI). Liver biopsy confirmed DILI. The patient showed clinical improvement with N-acetylcysteine and corticosteroids, with progressive normalization of liver enzymes. This case highlights the importance of differentiating DILI from acute viral hepatitis: strong clinical suspicion, temporal relation with offending drug, liver biopsy, and treatment response assessment. Clinicians should have a high index of suspicion for DILI even in the presence of concurrent acute HCV infection, especially in patients with underlying hepatic dysfunction such as Hurler syndrome in our case.
The detection of doping in sport has historically relied on the direct identification of exogenous substances or their metabolites in urine and blood. While effective for xenobiotics, this strategy is not always applicable for detection of pseudo-endogenous compounds such as testosterone or recombinant human erythropoietin (rhEPO), where endogenous production adds a layer of complexity to interpretation. To overcome these limitations, the Athlete Biological Passport (ABP) was developed, shifting the focus toward individualized, longitudinal monitoring of biological responses. The ABP currently includes hematological, steroidal, and endocrine modules, each targeting distinct classes of prohibited substances and methods. Despite its improved sensitivity, the ABP remains constrained by a limited repertoire of biomarkers, susceptibility to confounding factors, and the sophistication of emerging doping strategies. Recent advances in molecular biology have highlighted RNA-based biomarkers such as messenger RNAs (mRNAs) and microRNAs (miRNAs) as promising complementary tools for ABP. Blood transcriptomics provides sensitive indicators of erythropoietic stimulation and blood manipulation, with genes such as ALAS2 and CA1 that have shown strong responses to rhEPO and transfusion. The introduction of dried blood spots (DBS) as sample matrix has further enhanced feasibility by simplifying collection, storage, and transport, while preserving RNA integrity. However, before integration into the ABP, inter-laboratory harmonization and validation, quality control systems, and regulatory framework are essential. Beyond human testing, RNA biomarkers also demonstrate translational potential in equine anti-doping and clinical contexts such as treatment of anemia. Together, these developments underline the potential of RNA profiling to expand the ABP framework, improve sensitivity, and strengthen global anti-doping programs through a next generation of biomarkers.
Polystyrene exposure poses an increasing threat to reproductive health, and effective interventions remain limited. Jinkui Shenqi pills (JSP), a traditional Chinese medicine used to treat reproductive disorders, may protect against such damage. This study integrated network pharmacology with experimental validation to explore the mechanisms by which JSP counteracts polystyrene-induced reproductive toxicity. Potential targets related to polystyrene toxicity and bioactive components of JSP were retrieved from relevant databases. Overlapping targets were identified using VENNY 2.1. Protein-protein interaction networks were constructed using STRING, and core targets were identified with Cytoscape. Enrichment analyses were performed using DAVID. Molecular docking was used to evaluate ligand-target binding. A polystyrene-exposed mouse model was established, and tissue injury and recovery were assessed by hematoxylin-eosin (HE) staining and Western blotting (WB). In-depth screening identified 111 overlapping targets, and three key molecules-AKT serine/threonine kinase 1 (AKT1), caspase-3 (CASP3), and B-cell lymphoma 2 (BCL2)-were selected for further investigation. Gene Ontology analysis highlighted phosphorylation-related processes and responses to xenobiotics, while Kyoto Encyclopedia of Genes and Genomes analysis indicated enrichment in pathways related to lipid metabolism, atherosclerosis, cancer, and phosphatidylinositol-3-kinase (PI3K)-AKT signaling. Molecular docking confirmed effective binding of sitosterol, alisol B monoacetate, acetate, and alisol to these targets. HE staining revealed polystyrene-induced damage in testicular and ovarian tissues, which was effectively alleviated by JSP administration. WB results further demonstrated that JSP upregulated the expression of core target proteins, supporting its protective role against polystyrene-induced reproductive toxicity. JSP mitigates polystyrene-induced reproductive toxicity by modulating AKT1, CASP3, and BCL2, supporting its potential as a protective agent.
Pesticide mixtures are prevalent in aquatic systems within agricultural regions, but their transport and removal mechanisms in the presence of submerged macrophytes remain unclear. This study investigated changes in water quality, plant physiology, epiphytic microbial communities, and pesticide distribution in wetland substrates during 56 days of repeated exposure to a mixture of 12 pesticides at environmental (0.5 and 1 μg/L) and high (5 and 10 μg/L) concentrations. The average removal rates of total pesticides ranged from 58.61% to 75.52% and decreased with increasing pesticide addition and exposure time. Bioconcentration factors in plants were significantly higher than sediment-water partition coefficients for all pesticides. Notably, triazole pesticides (paclobutrazol, tebuconazole and hexaconazole) tended to accumulate in the system compared to neonicotinoid insecticides (dinotefuran, imidacloprid, and thiamethoxam). Pesticide addition reduced nutrient removal efficiency and induced oxidative damage in V. natans leaves. A total of 3, 14, and 17 biomarkers were identified in Con, PG1, and PG10, respectively, suggesting that PG1 and PG10 disturbed the epiphytic bacterial community. Co-occurrence network analysis revealed potential associations between pesticide residues and epiphytic bacterial taxa in PG10. PICRUSt2-based functional prediction indicated that compared to control, the predicted potential for xenobiotics biodegradation and metabolism was higher in PG1, whereas predicted genes related to starch and hemicellulose decomposition increased and denitrification-related predicted genes decreased in PG10. These data highlight that V. natans can accumulate pesticides and survive repeated pesticide exposure during the 56-day experiment, although high concentration pesticides had adverse effects on V. natans-epiphytic bacterial community.
Gene delivery for neurodegenerative cerebral disorders faces formidable structural and practical challenges. The blood-brain, blood- cerebrospinal fluid (CSF), and arachnoid barriers tightly regulate molecular traffic, restrict paracellular diffusion, and actively clear xenobiotics, limiting brain penetration and retention of large molecules and nucleic acid therapeutics. Additional barriers include heterogeneous and diffuse pathology, the need for precise anatomical targeting, vector dose-limiting toxicities, pre-existing and therapy-induced immunity to viral capsids, and procedural risks of neurosurgical or intrathecal administration. These constraints have slowed translation, reflected by the small number of approved central nervous system (CNS)-directed gene therapies. Against this backdrop, a diverse therapeutic landscape has emerged. In vivo strategies are dominated by adeno-associated virus (AAV)-9 and AAV2 vectors delivered intravenously, intrathecally (including intracisternal and intracerebroventricular routes), or via image-guided intraparenchymal and intraputaminal infusions, alongside intrathecal antisense oligonucleotides and RNA interference therapeutics. Concurrently, emerging approaches, including engineered AAV capsids, receptor- and transporter-mediated transcytosis, nanoparticle platforms, and focused ultrasound with microbubbles, have demonstrated compelling yet preclinical proof-of-concept. Future progress will likely depend on convergent advances in machine learning-guided capsid, more controllable blood-brain barrier modulation, rational route selection tailored to disease topology, and optimized ex vivo and cell-mediated delivery strategies. These innovations could enable a more predictable therapeutic paradigm for cerebral neurodegeneration.
Intracellular oxidative stress is a common mechanism of cellular dysregulation as a result of supraphysiological levels of reactive oxygen species (ROS). Imbalances in redox homeostasis underly adverse responses arising from exposure to a wide variety of xenobiotics and environmental exposures. Observing oxidative cellular events in real time poses multiple analytical challenges, requiring sensitive and specific methodologies that are capable of detecting transient events with high spatiotemporal resolution. We review here the advantages that live-cell imaging offers as a non-destructive approach that is well suited for redox toxicology studies. The effectiveness of this approach is heavily reliant on the use of fluorescent redox sensitive probes, such as small molecule and genetically encoded sensors that report on specific ROS and redox couples. We discuss a variety of small molecule and genetically encoded sensors that are used in redox toxicology, as well as the caveats and limitations posed by their use.
Antimicrobial resistance (AMR) continues to outpace the development of new anti-infective agents, particularly against priority bacterial pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, as well as clinically relevant fungi including Candida auris. In this scenario, biotransformation has emerged as a complementary innovation strategy for antimicrobial discovery because it expands the chemical space around bioactive scaffolds through selective enzymatic or whole-cell modification. Among the available biocatalysts, fungi are especially attractive due to their metabolic plasticity and broad enzymatic repertoire, including cytochrome P450 monooxygenases, unspecific peroxygenases, laccases, peroxidases, and hydrolases. Current evidence shows that fungal systems can mediate regio- and stereoselective transformations of xenobiotics, aromatics, steroids, terpenes, and lipids, generating structurally refined metabolites of pharmacological and biotechnological interest. This narrative review discusses where fungal biotransformation currently stands as a platform for antimicrobial innovation, highlighting representative enzyme-characterized examples, the main fungal groups and catalytic systems involved, and the experimental workflows used to evaluate these processes. Particular emphasis is given to assay design with growing cells, resting cells, and isolated enzymes, as well as to analytical monitoring by time-course sampling, LC-HRMS/MS, dereplication, molecular networking, isolation, and structural elucidation. Overall, fungal biotransformation is presented as a discovery-enabling platform that links biodiversity, enzymatic catalysis, analytical chemistry, and biological prioritization in the search for new anti-infective molecules.
Mercury (Hg) is a major neurotoxicant and public health concern and gold mining is a significant source of Hg contamination in the Amazon. There, Indigenous peoples are vulnerable to this exposure. Individual susceptibility influences both internal mercury levels and related clinical outcomes. In this context, the GSTP1 gene stands out due to its role in detoxification of xenobiotics. The objectives were to assess the associations between: (1) Hg levels and neurotoxicity signs; (2) the GSTP1 rs1695 polymorphism and Hg levels; and (3) whether the GSTP1 rs1695 polymorphism modifies the effect of mercury on neurotoxicity signs. A cross-sectional study was conducted between April and May 2023, with 113 Paiter-Suruí Indigenous people. Sociodemographic and clinical data were collected using a validated methodology. Hair and oral mucosa cells were collected to assess Hg levels and the GSTP1 rs1695 polymorphism. Hg levels ranged from 0.1 μg/g to 6.5 μg/g (median = 1 μg/g, IQR = 1.43). Individuals with impaired memory and muscle strength had significantly higher mercury levels (β = 4.39 and β = 1.24). Carriers of the GSTP1AA genotype showed a 0.46-point reduction for each 1 μg/g increase in mean Hg levels, compared to individuals with the GSTP1GG genotype (β = -0.46). These results may support public policies by identifying priority groups for intervention based on genetic profiles.
Serum and plasma are the most widely used matrices in metabolomics and human biomonitoring studies; however, the optimal matrix for integrated non-targeted analysis (NTA) workflows combining metabolomics and exposomics has not been systematically evaluated. This pilot study applied parallel NTA workflows to paired serum and plasma samples from five individuals to characterize matrix-dependent differences and provide an empirical basis for matrix selection in integrated studies. Three analytical methods were employed: one metabolomic method (Method 1) using Hydrophilic Interaction Liquid Chromatography (HILIC) and Reversed-Phase Liquid Chromatography (RPLC) columns and one exposomics (Method 2) method using an RPLC column, each analyzed in both electrospray ionization (ESI) positive and negative modes. Overall, serum and plasma showed broad similarity, with substantial overlap in detected features and strong linear correlations between paired samples (R2 = 0.70-0.87). However, PCA revealed systematic differences between the two matrices along PC1 and PC2, likely attributable to matrix effects arising from coagulation-related compositional changes in serum. For metabolomics, glycerophospholipids, sphingolipids, and acylcarnitines were consistently enriched in serum, attributable to platelet activation and phospholipase release during blood coagulation, consistent with prior reports. In contrast, oxidized fatty acid species were predominantly elevated in plasma, warranting caution in oxylipin-focused studies using serum. For exogenous chemical profiling, the two matrices performed comparably, with 32 out of 36 annotated features showing no significant matrix-dependent differences (p > 0.05), including PFAS, pharmaceuticals, and diverse xenobiotics. These findings support the interchangeability of serum and plasma for broad exposomics studies. Overall, while both matrices provided broadly comparable global coverage, plasma may represent a more appropriate matrix for integrated NTA workflows, as it better preserves in vivo metabolite composition and minimizes coagulation-induced confounding, though validation in larger cohorts is needed.
Diet plays a critical role in shaping the composition of gut microbiota in insects. Samia ricini, an economically important Lepidoptera insect, is a polyphagous herbivore that offers a useful model for studying dietary effects on the animal gut microbiome. Here, we fed S. ricini larvae with different food plants, Ricinus communis, Ailanthus altissima, and Manihot esculenta leaves to investigate how host plant species influence growth performance, digestive enzyme activities, and the gut microbial community. Our results showed that the Ricinus group exhibited better growth performance. Regarding digestive enzymes, the midgut lipase activity was significantly higher in the Ricinus group than in the Ailanthus group, while no significant differences were observed in α-amylase, cellulase, or trypsin activities among the three groups. Compared to the Manihot group, the Ricinus group showed increased bacterial richness, while the Ailanthus group showed increased bacterial diversity. β-diversity analysis further revealed distinct microbial community structures among all three dietary groups. Specifically, Acinetobacter, Mammaliicoccus, Roseateles, Methylobacterium, Agrobacterium, Faecalibacterium, and Segatella were the dominant bacterial genera. Functional prediction revealed that gut microbes enriched in the Ricinus group were associated with terpenoid/polyketide metabolism, xenobiotics biodegradation, and glycan biosynthesis, whereas those involved in carbohydrate metabolism and biosynthesis of other secondary metabolites were higher in the Manihot group. Spearman correlation analysis indicated that Methylobacterium, Methylorubrum, and Agrobacterium were significantly positively correlated with larval weight, while Staphylococcus and Cyanothece_PCC-7424 exhibited negative correlations. Collectively, these findings suggest a potential association between different plant-derived diets, gut microbiota composition, and host growth performance, highlighting the pivotal role of diet in shaping insect gut microbial communities.
Artificial sweeteners (ASs) are low-calories additives which utilization has increased in the last decades, reaching an economic revenue of ca. 26 billion US dollars in 2024. Through direct discharge or inadequate treatment of wastewater, they can spread in soil and reach the marine environment. Although the concrete risk of pollution deriving from these substances, their influence on coastal benthic communities is still unknown. We tested the effects of two ASs (aspartame and saccharin) through a chronic toxicity test on the cnidarian model species Aurelia coerulea polyps. We exposed polyps to a solution of aspartame and saccharine with filtered seawater (100 mg/l according to the REACH legislation for xenobiotics (Regulation 1272, Supplement No. 1, 2008), and measured their health status, asexual reproduction, somatic growth and mortality rates at 3-days intervals for ca. 60 days. Spectrophotometric analysis showed the degradation of aspartame, while saccharine remained unvaried indicating stability in water solution. Saccharin induced a moderate slowdown of growth, not impacting reproduction and health, while aspartame affected polyps by arresting growth, asexual reproduction, and triggering regression process. Afterwards, a full regeneration was reported with registered values similar to control group, suggesting acclimatization. Even though no mortality was observed, a delay in budding and growth rates could result in a carry-over effect on ephyrae production and adult populations. Since polyps are recognized as primary responsible driving jellyfish outbreaks, their altered physiological processes could affect the population dynamics of planktonic life-stages and consequently the trophic relationships of the habitat both at small and large scale.
Forest musk deer (Moschus berezovskii) is a globally endangered species, and its conservation has long been a matter of concern. Wild populations are scarce, while artificially captive populations are also constrained by health issues such as digestive system diseases. To reveal the differences in gut microbiota between captive and wild forest musk deer from different geographical regions, fecal samples were collected from captive individuals in Nanyang, Henan (HN) and Gaoping, Shanxi (SX), as well as wild individuals in Baotianman, Henan (YS), with 5 samples per group. High-throughput 16S rRNA sequencing was employed to analyze the microbial community structure and function. The sequencing revealed Firmicutes, Bacteroidota, and Proteobacteria as the dominant phyla across all three groups, with Actinobacteriota exhibiting a significantly higher abundance in the YS wild group (11.13%) than in the HN (1.29%) captive and SX (6.12%) captive groups. There were no significant differences in α-diversity among the groups. However, β-diversity analysis (PCoA and NMDS) indicated a clear separation in microbial community structure between captive and wild groups, with some individuals in the SX captive group clustering with the wild group. LEfSe analysis identified 36 differential biomarkers: the YS wild group was enriched in genera including Bacillus, Arthrobacter, and Microbacterium, whereas the HN captive group was enriched in Bacteroides, Clostridium, and Eubacterium, while the SX captive group was enriched in Skermanella (genus) and Cytophagales (order). Functional prediction analysis revealed that the gut microbiota of the wild group was significantly enriched in the pathways of xenobiotics biodegradation and metabolism as well as lipid metabolism, whereas the captive groups showed higher activity in the translation and nucleotide metabolism pathways. This study reveals the impacts of rearing methods and geographical factors on the gut microbial community structure and function of forest musk deer. These findings can serve as a theoretical foundation for promoting healthy breeding of captive populations and as a reference for evaluating the health status of wild populations.
Glycine betaine transport systems are widely exploited by bacteria to survive osmotic stress and represent potential entry routes for antimicrobial delivery. Here, we investigate the bactericidal glycine betaine analog Tox-GB and its uptake, intracellular fate, and antimicrobial activity in Escherichia coli K-12 under osmotic stress. We show that the xenobiotic enters cells via a hierarchical uptake route involving the osmotically regulated compatible solute transporters ProU and ProP, ABC- and MFS-type transporters, respectively. ProU functions as the primary high-affinity transporter at low concentrations, whereas ProP provides a secondary uptake route at somewhat higher substrate levels. Loss of either transporter confers partial resistance, while simultaneous inactivation of both systems causes full resistance, underscoring their functional redundancy and the robustness of Tox-GB import. Intracellularly, Tox-GB undergoes oxygen-dependent degradation, yielding 4-nitrobenzaldehyde and dimethylglycine. While 4-nitrobenzaldehyde contributes to toxicity under aerobic conditions, Tox-GB remains bactericidal under anaerobic conditions, indicating additional oxygen-independent mechanisms involving either the parent compound or unidentified metabolites. These findings suggest a complex intracellular fate and multifactorial mode of action. Despite initial promise as a Trojan horse antimicrobial strategy, the use of Tox-GB for practical applications faces key limitations. Resistance readily emerges via transporter inactivation, and intrinsic resistance occurs in species lacking appropriate compatible solute uptake systems. Structural constraints in glycine betaine transporters further restrict design flexibility. Osmotic regulation limits activity to specific niches, and potential host toxicity stemming from reactive metabolites raises safety concerns. Collectively, these findings highlight the mechanistic complexity and translational challenges faced by glycine betaine-derived xenobiotics as antimicrobial agents.
Plants have evolved abundant defensive secondary metabolites to resist insect herbivores. Lycorine is an alkaloid with insecticidal activity from Amaryllidaceae plants, which the destructive pest Spodoptera litura naturally avoids. Cytochrome P450 enzymes are central to xenobiotic detoxification in insects, but the mechanism by which lycorine acts against S. litura remains unknown. This study aimed to reveal the toxic mechanism of lycorine focusing on P450-mediated detoxification. Lycorine exhibited substantial toxicity to first-instar S. litura larvae (LD50 = 0.55 μg larva-1). Subsequently, when fifth-instar larvae were exposed to a sublethal dose (LD30) of lycorine, Lyc disrupted metabolic pathways, damaged Malpighian tubules, and induced oxidative stress. Furthermore, lycorine strongly repressed a CYP6AE gene cluster (CYP6AE47, CYP6AE50, CYP6AE70, CYP6AE138 and CYP6AE139) and decreased total P450 activity to 45% in the Malpighian tubules. RNAi co-silencing of these cluster genes increased larval mortality (+30%) under lycorine treatment. Finally, molecular docking and microscale thermophoresis analyses further confirmed direct binding between Lyc and this CYP6AE gene cluster, with the strongest affinity observed for CYP6AE47 (Kd = 518.5 nM). A key residue, ARG170, may be vital for the interaction between Lyc and CYP6AE47. These results demonstrate that the insecticidal mechanism of Lyc involves suppressing the expression and function of a CYP6AE gene cluster, thereby impairing detoxification capacity, which leads to Lyc accumulation and larval mortality. Elucidation of the detoxification system-targeted mechanism for this plant-derived compound provides a foundation for developing novel, sustainable pest management strategies against S. litura and potentially other noctuid pests. © 2026 Society of Chemical Industry.