Abstract This study investigates human exposure to unintentionally produced persistent organic pollutants (POPs) through geophagy involving edible clays and soil in Africa. The concentration of PCDD/Fs, PCBs, PCNs, PeCB, HCB, and HCBD were determined in 18 edible clay and termite mound soil samples from Nigeria and Botswana using HRGC–HRMS, GC–MS/MS, and GC–MS. PCDD/Fs were detected in all samples. Only two edible clays from Ogun State (0.03–0.04 pg WHO2022-TEQ/g) were below the European regulatory limit for minerals in feed additives (0.75 pg TEQ/g), while other clays ranged from 10.8 to 75.7 pg WHO2022-TEQ/g. Termite mound soils showed 1.8–1.9 pg WHO2022-TEQ/g. Consumption of some clay even exceeded European health guidelines over 200-fold. Other POPs contributed minimally to overall toxicity. Edible clay consumption is an important exposure pathway for PCDD/Fs, posing considerable health risks, particularly to pregnant women and infants. Urgent systematic monitoring, source control, and risk-based regulation are warranted.
Abstract α-Amylase inhibitors often exhibit markedly inconsistent activities across substrate systems, yet the molecular basis remains poorly understood. Here, using epigallocatechin gallate (EGCG) as a model inhibitor, we compared corn starch, a continuous α-1,4-glucan, with the small, structurally defined chromogenic substrate GalG2CNP, demonstrating that substrate structural organization, rather than the inhibitor itself, governs apparent inhibitory outcomes. Their distinct enzyme affinities produced opposing responses: low-affinity GalG2CNP was readily inhibited by EGCG, whereas high-affinity starch resisted inhibition even with excess inhibitor. Multimodal biophysics, molecular docking, and molecular dynamics simulations confirmed that EGCG occupies the α-amylase active pocket without substantially perturbing enzyme structure. Ternary system analyses further revealed that high-affinity starch displaced prebound EGCG, whereas GalG2CNP induced only partial re-equilibration. We therefore propose a substrate-affinity-driven tug-of-war model and a two-tier screening strategy, using artificial substrates for primary screening and starch for physiological verification, providing a mechanistic framework for more reliably evaluating candidate dietary α-amylase inhibitors.
Abstract Sichuan pepper (Zanthoxylum bungeanum) is widely consumed in China, but its allergenicity raises food-safety concerns. In this study, the 11S globulin allergen Zan b 2 was identified as the major allergen and characterized through structural and immunological analyses. Integrated immunoinformatics, molecular docking, and molecular dynamics simulations were used to predict B-cell epitopes and screen proteases capable of specifically cleaving these regions. Prediction results indicate that proteinase K targets the following epitopes: Glu13, Thr100, Tyr149, Glu164, Glu229, Glu231, Leu239, Glu241, Thr245, Val328, Glu368, Ala381, and thermolysin targets Arg182, Lys247, Ser273, Asn294, Gln332, and Arg380. Experimental validation using enzymatic hydrolysis, SDS-PAGE, indirect ELISA, LC–MS/MS, and proteomics showed that combined treatment with proteinase K and thermolysin markedly reduced the IgE-binding activity. These findings demonstrate that simulation-assisted targeted enzymatic hydrolysis is an effective strategy for reducing Zan b 2 immunoreactivity and supports the development of hypoallergenic Sichuan pepper products.
Abstract A growing body of literature reports the health benefits of the jellyfish collagen hydrolysate. However, although hydroxyproline (Hyp)-containing oligopeptides absorbed into the bloodstream are considered responsible for the biological functions of collagen hydrolysate, the active ingredient specific to jellyfish collagen remains unclear. Here, we revealed that a collagenase-digested cannonball jellyfish collagen hydrolysate is highly enriched with a bioactive tripeptide, Gly-3Hyp-4Hyp (15.6 mg/g). In humans, oral ingestion of the jellyfish collagen hydrolysate resulted in a 7.8-fold higher blood absorption of Gly-3Hyp-4Hyp than that of ingesting bovine collagen hydrolysate. Remarkably, its concentration surpassed that of Pro-Hyp, which is conventionally the predominant blood-absorbable peptide. Furthermore, in mice, Gly-3Hyp-4Hyp was efficiently delivered to the skin after jellyfish collagen hydrolysate administration, showing a 19-fold greater accumulation compared with the bovine group, whereas other Hyp-containing oligopeptides exhibited minimal transport. This remarkable bioavailability and effective tissue delivery suggest that Gly-3Hyp-4Hyp is a major candidate driving the biological functions of the jellyfish collagen hydrolysate.
Abstract The invasive pest Spodoptera frugiperda threatens global agriculture. Understanding its growth regulation is key for developing novel control strategies. This study reveals that the transcription factor Cubitus interruptus (SfCi) of S. frugiperda, an unstable nuclear protein with conserved C2H2 zinc finger domains, is directly targeted and negatively regulated by miR-7-5p. Knocking down Sfci via RNA interference induces metabolic reprogramming: it promotes triglyceride storage by enhancing adipocyte differentiation while inhibiting lipolysis, and simultaneously increases glucose utilization to fuel larval development. Molecularly, this shift upregulates adipogenesis genes and suppresses cell proliferation genes. These findings establish the miR-7-5p/Ci regulatory module as a core regulator balancing lipid and glucose metabolism to coordinate energy allocation and development in S. frugiperda.
Abstract 3-Hydroxy-3-methylbutyrate (HMB) is an important nutritional supplement for managing sarcopenia. This study engineered an HMB biosynthetic pathway in the probiotic Escherichia coli Nissle 1917 (EcN). The initial engineered strain exhibited low HMB titers, as carbon flux was primarily diverted toward acetate formation. Deleting the pyruvate oxidase gene (poxB) reduced acetate but caused significant pyruvate accumulation, revealing a severe kinetic bottleneck in endogenous pyruvate dehydrogenase (PDH) activity. Supplementing pantothenic acid (Vitamin B5) profoundly expanded the intracellular coenzyme A (CoA) pool, providing the essential backbone for pathway intermediates. This strategy enhanced PDH flux and improved HMB production by 5.5-fold. Through fed-batch fermentation, the engineered strain achieved an HMB titer of 8.6 g/L with a yield of 0.14 g/g glucose. The results of this study provided a probiotic strain that can synthesize HMB de novo and demonstrated the importance of pantothenic acid availability for EcN metabolic engineering.
In this study, a novel β-N-acetylhexosaminidase (AuHex92) was expressed in Escherichia coli. AuHex92 could utilize both p-nitrophenyl-N-acetylglucosamine (pNP-GlcNAc) and N-acetyl chitobiose [(GlcNAc)2] as donors. pNP-GlcNAc as the donor afforded an exceptional N-acetyl chitotriose [(GlcNAc)3] yield (74.5%), whereas (GlcNAc)2 gave a yield of 12.6%. AuHex92 featured a broad entrance, large internal volume, and good flexibility in its catalytic groove, which enabled utilization of both donors, while the hydrophobicity and π-π stacking interactions in the groove improved its transglycosylation activity, with these functional contributions further validated by site-directed mutagenesis. Molecular dynamics simulation results indicated that AuHex92-pNP-GlcNAc exhibited a stable, compact conformation, which enhanced its substrate affinity and transglycosylation activity. AuHex92-(GlcNAc)2 achieved both-donor usage by utilizing a flexible catalytic groove region to guide the entry of the larger (GlcNAc)2 donor. The unique biochemical properties and structural features of AuHex92 establish a foundation for producing (GlcNAc)3 in the food, chemical, and pharmaceutical industries.
Abstract RpedOBP17, an odorant-binding protein highly expressed in adult Riptortus pedestris antennae, was systematically investigated using prokaryotic expression, fluorescence competitive binding, molecular docking, site-directed mutagenesis, and RNA interference. Binding assays revealed affinity to six soybean volatile compounds. Molecular docking and mutagenesis identified Ile46 and Phe129 as critical residues for ligand recognition, since alanine substitutions markedly reduced binding. RNAi-mediated knockdown significantly impaired olfactory chemotaxis, with electroantennogram recordings showing attenuated responses to (Z)-3-hexenyl acetate, (E)-2-hexenyl acetate, and 4-ethylbenzaldehyde in both sexes, along with sex-specific changes to 2-methyl-1-butanol (females) and (Z)-3-hexenyl propionate (males). Y-tube olfactometer tests further confirmed decreased attraction to (E)-2-hexenyl acetate. These results elucidate the molecular interaction mechanism and provide a theoretical foundation for developing novel olfactory disruption-based control agents targeting RpedOBP17.
Abstract The calcium-sensing receptor (CaSR) is a nutrient-sensing class C G protein-coupled receptor implicated in intestinal hormone release, yet the signaling pharmacology of the porcine receptor and its relevance to epithelial homeostasis remain poorly defined. Here, a stable Flp-In-293 reporter platform expressing porcine CaSR was combined with structure-based docking, molecular dynamics simulations, secretin tumor cell line-1 (STC-1) enteroendocrine assays, and an STC-1/IPEC-J2 co-culture system challenged with peptidoglycan. Extracellular Ca2+ activated porcine CaSR through NFAT, SRE, SRF, and cAMP readouts, whereas l-tryptophan and α-casein (90–95) differentially potentiated Ca2+-dependent signaling and docked within the extracellular Venus flytrap domain. Both ligands stimulated CaSR-sensitive glucagon-like peptide-1 (GLP-1) secretion and recruited ERK1/2, IP1, and cAMP signaling. In co-culture, ligand exposure attenuated inflammatory transcription, preserved selected transporter and tight-junction programs, and improved barrier function under peptidoglycan challenge. Together, these data identify food-derived ligands that couple porcine CaSR signaling to enteroendocrine–epithelial responses.
Abstract Bitterness limits the development of palatable foods containing bioactive ingredients. This study investigated whether long-chain fatty acids (LCFAs) modulate the human bitter taste receptor TAS2R16 using a HEK293T cell-based calcium assay with salicin as an agonist. None of the tested LCFAs directly activated TAS2R16. Under co-treatment, only oleic acid inhibited salicin-induced calcium responses, shifting the salicin EC50 from 1.82 ± 0.11 mM to 3.55 ± 1.22 mM at 30 μM (p < 0.01). Pretreatment with all LCFAs inhibited TAS2R16 concentration-dependently. Reliable IC50 values were obtained for palmitic acid (8.67 ± 1.44 μM), stearic acid (0.082 ± 0.002 μM), and oleic acid (0.41 ± 0.12 μM), whereas lower plateaus were not reached for myristic, linoleic, and α-linolenic acids. LCFA treatment did not consistently change membrane fluidity, and inhibition was attenuated in the TAS2R16 N96T mutant. These findings demonstrate compound- and treatment-dependent modulation of TAS2R16 by food-derived LCFAs.
Abstract Tomato (Solanum lycopersicum) β-mannanase is known as playing an important role in mannan degradation; however, its biological functions and transcriptional regulation remain poorly understood. Here, we characterized the roles of mannan-1,4-β-mannanase 2 (SlMAN2) in tomato development and salt stress tolerance. Results showed that SlMAN2 was preferentially expressed in seeds, stems, stamens, and stigmas. SlMAN2 overexpression significantly increased fruit and seed size, accelerated seed germination, and enhanced salt tolerance under 100 mM NaCl, whereas knockout mutants exhibited the opposite phenotypes. Notably, dual-luciferase assays and electrophoretic mobility shift assay (EMSA) demonstrated that the transcription factor SlDOF3.1 directly binds to the SlMAN2 promoter and activates its transcription, but SlMAN2 overexpression suppressed SlDOF3.1 expression, suggesting the existence of a negative feedback regulatory loop. Collectively, our findings reveal that SlMAN2 functions as a pleiotropic regulator coordinating tomato fruit and seed development with salt stress tolerance and uncover a SlDOF3.1–SlMAN2 regulatory module underlying these processes.
Abstract Aristoloxazines (AXs) represent a newly identified class of neurotoxins and genotoxins found in plants of the Asarum and Aristolochia families, many of which are traditionally used as herbal remedies. Recently, they have also been detected in high abundance in some herbal plant cultivation fields. We revealed in this study that AX-C and AX-A exhibit significant cytotoxicity in both HEK293 and L02 cells, with LC50s < 5 μM. Furthermore, AX-C generates adducts that, based on mass spectral and chromatographic data, are identical or very similar to those of the well-characterized human carcinogen aristolochic acid I (AA-I), in DNA samples from both cultured human cells and internal organs of mice exposed to AXs. Given that AA-I DNA adducts have been shown to induce high frequencies of AT → TA transversions and are believed to be responsible for the observed toxicity of AA-I, our results highlight a previously unrecognized group of potent genotoxins that pose an ongoing risk to human health.
Abstract Food spoilage and product counterfeiting demand unified smart packaging solutions. This work reports a super-tough and highly adhesive hydrogel integrating anticounterfeiting and real-time freshness monitoring. The hydrogel, constructed from a polyacrylamide-grafted-chitosan network physically crosslinked with whey protein, exhibited a 2035% breaking elongation and 1.13 MJ/m3 toughness. It demonstrated exceptional defect tolerance (7.9 kJ/m2 tearing energy) and dynamic broad-spectrum adhesion (∼21 kPa on glass). When doped with nitrogen-doped carbon dots, the hydrogel displayed pH- and ammonia-responsive dual-mode fluorescence, enabling rewritable texts and smartphone-readable QR codes for high-security anticounterfeiting. Furthermore, it serves as a real-time indicator for monitoring chicken breast freshness. It provided accurate visual alerts synchronized with spoilage thresholds at 25 and 4 °C, exhibiting remarkable logistic correlation (R2 > 0.98) with TVB-N and pH levels. Overall, this work provides an intelligent platform integrating structural resilience, real-time food quality tracking, and data security for next-generation smart packaging.
Abstract When investigating honey bee suspected poisoning events, traditional targeted methods may overlook relevant compounds. Thus, a suspect screening workflow combining two complementary and platform-independent strategies in parallel, an exact mass screening tool and a combinatorial analysis of tandem mass (MS2) spectra, was developed to enhance exposure coverage. Its performance was assessed across three distinct high-resolution mass spectrometers (HRMS), using well-defined quality assurance and quality control metrics to ensure transparent and reproducible reporting of nontargeted analyses. True positive rates exceeded 80% across instruments during exact mass feature detection. Incorporating MS2-based annotation further improved identification confidence, with few false positives (≤9). Applied to incurred bee samples, the workflow identified atrazine and two transformation products, deethylatrazine and deisopropylatrazine. Overall, this workflow provides a comprehensive framework for investigating bee poisoning incidents and represents an important step toward improving the transfer of suspect screening workflows and the comparability of HRMS data across laboratories.
Abstract The resistance to pyrethroid insecticides is becoming increasingly severe, while the number of insecticides with negative cross-resistance (NCR) remains extremely limited. Thus, the development of novel targets and candidate compounds is urgently required. To exploit NCR insecticides targeting the voltage-gated sodium channel (VGSC), structural modifications of the B and C rings were performed on the lead compound 6i, leading to the synthesis of a series of derivatives. Bioassays, electrophysiological experiments and cytotoxicity assays demonstrated that the derivative LC-2 exhibited insecticidal activity against multiple insect pests. Notably, LC-2 showed significant NCR against pyrethroid-resistant Aedes aegypti (resistance ratio = 0.12). Its mode of action involves targeting the insect VGSC and impairing the function of the channel’s fast inactivation gate. Meanwhile, this compound presented low toxicity to mammalian cells. In conclusion, this study identified a novel compound LC-2 with NCR to pyrethroids, multi-pest insecticidal activity, which provides a theoretical basis and material support for insecticide resistance management and the development of low-toxic NCR insecticides.
Abstract Yarrowia lipolytica is a promising industrial host, yet its metabolic engineering potential remains limited by insufficient genetic tools. Here, we engineered a synthetic Transcription Activation Toolkit (TAT) based on LacI–VPRH, a chimeric protein fusing the prokaryotic DNA-binding domain LacI with the eukaryotic activation domain VPRH. Systematic optimization of LacO copy numbers and core promoter composition achieved up to 205-fold gene activation. The TAT platform was further expanded to construct bidirectional expression systems and enable multiplexed gene control. Applied to resveratrol biosynthesis via a “push-pull” strategy, CRISPR/Cas9-mediated integration of TAT-controlled synthetic promoters upregulated the shikimate pathway genes aroM10 and aroC alongside the rate-limiting enzyme ST1, achieving a shake-flask titer of 2.715 g/L─the highest reported to date. Additionally, an IPTG-inducible “turn-on” system (TAT-2.0) incorporating the antiLacI9 mutant was developed for small-molecule-responsive transcriptional control. Collectively, the modular TAT system provides a versatile strategy for precise metabolic pathway optimization in Y. lipolytica.
Abstract Ethanol-induced gastric mucosal injury (AGMI) is intrinsically linked to oxidative stress, inflammatory responses, and compromised mucosal defense. This study aimed to isolate bioactive peptides from yeast protein (YP) and elucidate these in vitro protective mechanisms against AGMI. Activity-guided multi-technique separation, nanoLC-MS/MS, and bioinformatic screening were used to identify two novel peptides, ISPALLDKL and TAADLRYF. In ethanol-injured GES-1 cells, these peptides (240 and 320 μg/mL) exhibited potent cytoprotective activity comparable to omeprazole. Peptide pre-treatment attenuated reactive oxygen species (from 31.79 to 9.52 and 10.02), restored mitochondrial membrane potential (ΔΨm increased from 0.5 to 2.78 and 2.11), and reduced apoptosis (from 28.55 to 10.52% and 12.58%). Furthermore, these peptides associated with the suppression of pro-inflammatory cytokines, decreasing TNF-α (24.9 and 21.9%), IL-1β (30.1 and 26.3%), and IL-6 (41.9 and 37.6%), while markedly enhancing the anti-inflammatory cytokine IL-10 (110.8 and 91.0%), respectively; these effects were accompanied by inhibition of NF-κB and JAK2–STAT3 phosphorylation. Collectively, ISPALLDKL and TAADLRYF protect against ethanol-induced gastric epithelial cell injury by simultaneously modulating oxidative stress, apoptosis, and inflammation, positioning them as promising natural candidates for gastric mucosal protection.
Abstract Fungal diseases remain a major constraint on global crop production, driving the search for fungicides with broader efficacy and improved selectivity. Confronting the dual challenges of structural rigidity and declining antifungal performance in conventional succinate dehydrogenase inhibitors (SDHIs), this study developed novel syringic acid derivatives. Notably, W11 exhibited remarkable broad-spectrum potency against eight phytopathogenic fungi and two oomycete species, with EC50 values of 0.679, 0.424, and 0.384 μg/mL against Gibberella zeae, Alternaria solani, and Colletotrichum fructicola, respectively. This activity significantly surpassed that of the commercial fungicide boscalid (EC50 >25.0, 0.826, and >25.0 μg/mL). Interestingly, W11 exhibited excellent succinate dehydrogenase inhibitory activity, causing a marked increase in intracellular reactive oxygen species (ROS), severe mitochondrial damage, and ultimately cell death. Overall, this work presents a structure-driven strategy that expands the antifungal spectrum of SDHIs and offers a promising scaffold for developing eco-friendly agrochemicals to address both limited antifungal activity and emerging oomycete challenges in sustainable agriculture.
Abstract The gem-difluoroalkylthio (−SCF2−) moiety has achieved great success in medicinal chemistry, but its application in agrochemicals remains underexplored because of synthetic challenges. Herein, we report a series of 1,2,4-triazole derivatives bearing the gem-difluoromethylthio group and evaluate their antifungal activities against ten phytopathogenic strains. Compound 7n exhibits potent broad-spectrum activity, with EC50 values of 0.007, 0.121, 0.122, and 0.068 μg/mL against Botrytis cinerea, Phytophthora capsici, Curvularia lunata, and Alternaria solani, respectively, outperforming tebuconazole. In vivo, 7n shows protective and curative efficacy comparable to tebuconazole on apple fruits, tomato fruits, and Suzhou Qing leaves. Mechanistic studies, including scanning electron microscopy (SEM), transcriptomics, qRT-PCR, and molecular docking, indicate that 7n disrupts mycelial morphology, interferes with membrane-associated processes, and inhibits sterol biosynthesis by targeting CYP51. These findings highlight the −SCF2– group as a promising scaffold for novel agricultural fungicides.
Shell ginger (Alpinia zerumbet) is a perennial species widely utilized as both an edible spice and a medicinal plant. Phytochemical investigation of its rhizomes yielded 33 terpenoids, of which 12 are previously undescribed compounds, comprising both diterpenes and sesquiterpenes. Notably, compound 1 represents a rare, highly oxygenated isospongiane-type diterpenoid with a novel skeleton. The anti-inflammatory potential of all isolates was assessed using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and BV2 microglial cells. Subsequent mechanism-oriented assays demonstrated that compounds 2 and 4 attenuated neuroinflammation in BV2 cells through an effect attributable to inhibition of nuclear factor kappa-B (NF-κB) nuclear translocation, reduction of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and signal transducer and activator of transcription 3 (STAT3) protein expression, and downregulation of the mRNA levels of iNOS, interleukin-1β (IL-1β), COX-2, and tumor necrosis factor-α (TNF-α).
