A capture systematic evolution of ligands by exponential enrichment (Capture-SELEX) was described to discover novel aptamers specific for 5-hydroxymethylfurfural (5-HMF), and a biosensor based on molecular beacon was constructed to detect 5-HMF. The ssDNA library was immobilized to streptavidin (SA) resin to select the specific aptamer. The selection progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing (HTS). Candidate and mutant aptamers were selected and identified by Isothermal Titration Calorimetry (ITC). The FAM-aptamer and BHQ1-cDNA were designed as the quenching biosensor to detect 5-HMF in milk matrix. After the 18th round selection, the Ct value decreased from 9.09 to 8.79, indicating that the library was enriched. The HTS results indicated that the total sequence numbers for 9th, 13th, 16th, and 18th were 417054, 407987, 307666, and 259867, but the number of sequences for the top 300 sequences was gradually increased from 9th to 18th, and the ClustalX2 analysis showed that there were four families with high homology rate. ITC results indicated that the K_d values of H1 and its mutants H1-8, H1-12, H1-14, and H1-21 were 2.5 μM, 1.8 μM, 1.2 μM, 6.5 μM, and 4.7 μM. The linear range of the quenching biosensor was from 0 μM to 75 μM, and it had a similar linear range in the 0.1% milk matrix. This is the first report to select a novel aptamer specific for 5-HMF and develop quenching biosensor for the rapid detection of 5-HMF in milk matrix.

Microcystin-LR (MCLR) poses a significant threat to aquatic ecosystems and public health. This study investigated the protective effects of the probiotic Lactobacillus rhamnosus against MCLR-induced developmental toxicity in zebrafish larvae. Zebrafish larvae were exposed to various concentrations of MCLR (0, 0.9, 1.8, and 3.6 mg/L) with or without L. rhamnosus from 72 to 168 h post-fertilization (hpf). Probiotic supplementation significantly improved survival, hatching, and growth rates and reduced malformation rates in MCLR-exposed larvae. L. rhamnosus alleviated MCLR-induced oxidative stress by reducing reactive oxygen species (ROS) levels and enhancing glutathione (GSH) content and catalase (CAT) activity. Probiotics also mitigated MCLR-induced lipid metabolism disorders by regulating key metabolites (triglycerides, cholesterol, bile acids, and free fatty acids) and gene expression (ppara, pparb, srebp1, and nr1h4). Moreover, 16S rRNA sequencing revealed that L. rhamnosus modulated the gut microbiome structure and diversity in MCLR-exposed larvae, promoting beneficial genera like Shewanella and Enterobacter and inhibiting potential pathogens like Vibrio. Significant correlations were found between gut microbiota composition and host antioxidant and lipid metabolism parameters. These findings suggest that L. rhamnosus exerts protective effects against MCLR toxicity in zebrafish larvae by alleviating oxidative stress, regulating lipid metabolism, and modulating the gut microbiome, providing insights into probiotic-based strategies for mitigating MCLR toxicity in aquatic organisms.

Lipid peroxidation products can induce oxidative modification of myofibrillar proteins (MPs), thereby compromising their water-holding capacity (WHC), but the underlying molecular mechanism remains unclear. This study systematically investigated the mechanism of MDA-induced WHC deterioration in bovine myofibrillar proteins (MPs) using multispectral techniques, redox proteomics, and molecular docking. Results demonstrated that low MDA concentrations caused relatively limited water release. Structural alterations became evident at 2 mM, whereas pronounced WHC deterioration occurred at 5–10 mM. At these higher concentrations, centrifugal loss increased by up to 48.80%, and immobilized water migrated to free water (p < 0.05). This functional decline was accompanied by substantial structural remodeling, characterized by a transition from α-helix to β-sheet conformations, decreased hydrogen bonding, and enhanced disulfide-associated cross-linking. Furthermore, redox proteomics identified 581 differential cysteine redox sites, including 343 increased and 238 decreased sites. Among these sites, 57 markedly decreased sites associated with myofibrillar and cytoskeletal proteins were further characterized, including sites in actin, α-actinin, myosin, and LIM-domain-containing proteins. Motif analysis further revealed a characteristic cysteine-rich C-x-x-C-x-C sequence pattern surrounding responsive oxidation sites. Molecular docking of 12 representative cysteine sites supported the spatial feasibility of MDA pre-association near these cysteine-containing regions, with actin C258 exhibiting the most negative docking score among the examined sites (−3.3 kcal/mol). These findings reveal that cysteine redox remodeling was associated with structural reorganization and increased water mobility, thereby contributing to WHC deterioration.