Triple-helix molecular-switch-actuated rolling circle amplification and catalytic hairpin assembly multistage signal amplified fluorescent aptasensor for detection of aflatoxin B1.

BACKGROUND: Mycotoxins, a class of secondary metabolites produced by molds, are widely distributed in nature and are very common in food contamination. Aflatoxin B1 (AFB1) is a highly stable natural mycotoxin, and many agricultural products are easily contaminated by AFB1, it is important to establish a sensitive and efficient AFB1 detection method for food safety. The fluorescence aptamer sensor has shown satisfactory performance in AFB1 detection, but most of the fluorescence aptasensors are not sensitive enough, so improving the sensitivity of the aptasensor becomes the focus of this work. RESULTS: Herein, an innovative fluorescent aptasensor for AFB1 detection which is based on catalytic hairpin assembly (CHA) and rolling circle amplification (RCA) driven by triple helix molecular switch (THMS) is proposed. A functional single-strand with an AFB1 aptamer, here called an APF, is first designed to lock onto the signal transduction probe (STP), which separates from THMS when target AFB1 is present. Subsequently, STP initiates the RCA reaction along the circular probe, syntheses macro-molecular mass products through repeated triggering sequences, triggers the CHA reaction to produce a large number of H1-H2 structures, which causes FAM to move away from BHQ-1 and recover its fluorescence signal. The fluorescence signal from FAM at 520 nm was collected as the signal output of aptasensor in this work. With high amplification efficiency of RCA and CHA of the fluorescence sensor, resulting in a low LOD value of 2.95 pg mL-1(S/N = 3). SIGNIFICANCE: The successful establishment of the sensor designed in this work shows that the cascade amplification reaction is perfectly applied in the fluorescent aptamer sensor, and the signal amplification through the reaction between DNA strands is a simple and efficient method. In addition, it's also important to remember that the aptasensor can detect other targets only by changing the sequence of the aptamer, without redesigning other DNA sequences in the reaction system.

A biosensor integrating the electrochemical and fluorescence strategies for detection of aflatoxin B1 based on a dual-functionalized platform.

BACKGROUND: Aflatoxin B1 (AFB1), is a potent hepatic carcinogen which causes cancer by inducing DNA changes in the liver cells. Variety of methods have been developed for detection of AFB1 which are based on single mode detection strategy. Fabrication of novel platform which are compatible for multimodal detection of AFB1 provide robust performance for reliable detection of AFB1. In this study, we aimed to develop a robust biosensing platform that combines electrochemical and fluorescence techniques for the sensitive and specific detection of Aflatoxin B1. RESULTS: The sensing platform includes the magnetic core-shell Fe3O4@AuNPs and zeolitic imidazolate framework-8 (ZIF-8). In electrochemical mode, the applied voltametric approach was used through functionalization of glassy carbon electrode and exhibited a linear range between 0.5 and 10000 pg mL-1 with LOD of 0.32 pg mL-1. Fluorescence analysis was based on the FRET on/off status of FAM-functionalized aptamer deposited on the same platform. The FAM emission recovered by the addition of AFB1 concentration in the range of 6-60 fg mL-1 with the LOD of 0.20 fg mL-1. The real sample analysis demonstrated satisfactory relative recoveries in the range of 92.81-105.32 % and 91.66-106.66 % using the electrochemical and fluorescence methods, respectively, and its reliability was confirmed by the HPLC technique. SIGNIFICANCE: The experimental results affirm that the proposed aptasensor serves as a sensitive, efficient, and precise platform for monitoring AFB1 in both electrochemical and fluorescence detection approaches. Proposed strategy showed efficient selectivity among different analytes and was reproducible. Furthermore, the applicability of biosensor was confirmed in food and biological samples.

X-ray Irradiation Reduces Live Aspergillus flavus Viability but Not Aflatoxin B1 in Naturally Contaminated Maize.

Food crops around the world are commonly contaminated with Aspergillus flavus, which can produce the carcinogenic mycotoxin aflatoxin B1 (AFB1). The objective of this study is to test an X-ray irradiation sterilization method for studying AFB1 in contaminated maize samples in the laboratory. Maize that had been naturally contaminated with 300 ppb AFB1 by the growth of aflatoxigenic A. flavus was ground and then irradiated at 0.0, 1.0, 1.5, 2.0, 2.5, and 3.0 kGy. A. flavus was quantified by dilution plating on potato dextrose agar (PDA) and modified Rose Bengal media (MDRB) for viability and qPCR for gene presence. AFB1 was quantified by HPLC and ELISA. A. flavus viability, but not gene copies, significantly decreased with increasing doses of radiation (PDA: p < 0.001; MDRB: p < 0.001; qPCR: p = 0.026). AFB1 concentration did not significantly change with increasing doses of radiation (HPLC: p = 0.153; ELISA: p = 0.567). Our results imply that X-ray irradiation is an effective means of reducing viable A. flavus without affecting AFB1 concentrations. Reducing the hazard of fungal spores and halting AFB1 production at the targeted dose are important steps to safely and reproducibly move forward research on the global mycotoxin challenge.

Antioxidative and Antimycotoxigenic Efficacies of Thunbergia laurifolia Lindl. for Addressing Aflatoxicosis in Cherry Valley Ducks.

This study aimed to assess the effectiveness of aflatoxin B1 (AFB1) and Thunbergia laurifolia extract (TLE) in the diets of Cherry Valley ducklings. Our investigation covered growth indicators, blood biochemical indices, meat quality, intestinal morphology, immune response, and CP450 enzyme-related gene expression. We conducted the study with 180 seven-day-old Cherry Valley ducks, randomly divided into five dietary treatments. These treatments included a basal diet without AFB1 (T1 group), TLE, or a commercial binder; the basal diet containing 0.1 mg AFB1/kg (T2 group), 0.1 mg AFB1/kg and 100 mg TLE/kg (T3 group), 0.1 mg AFB1/kg and 200 mg TLE/kg (T4 group), and 0.1 mg AFB1/kg and 0.5 g/kg of a commercial binder (T5 group), respectively. Ducklings fed with the T2 diet exhibited lower final body weight (BW), average body weight gain (ADG), and poor feed conversion ratio (FCR) during the 42-day trials. However, all ducklings in the T3, T4, and T5 groups showed significant improvements in final BW, ADG, and FCR compared to the T2 group. Increased alanine transaminase (ALT) concentration and increased expression of CYP1A1 and CYP1A2 indicated hepatotoxicity in ducklings fed the T2 diet. In contrast, ducklings fed T3, T4, and T5 diets all showed a decrease in the expression of CYP1A1 and CYP1A2, but only the T4 treatment group showed improvement in ALT concentration. AFB1 toxicity considerably raised the crypt depth (CD) in both the duodenum and jejunum of the T2 group, while the administration of 200 mg TLE/kg (T4) or a commercial binder (T5) effectively reduced this toxicity. Additionally, the villus width of the jejunum in the T2 treatment group decreased significantly, while all T3, T4, and T5 groups showed improvement in this regard. In summary, T. laurifolia extract can detoxify aflatoxicosis, leading to growth reduction and hepatic toxicosis in Cherry Valley ducklings.

Mitigation of Deoxynivalenol (DON)- and Aflatoxin B1 (AFB1)-Induced Immune Dysfunction and Apoptosis in Mouse Spleen by Curcumin.

In the context of the potential immunomodulatory properties of curcumin in counteracting the detrimental effects of concurrent exposure to Deoxynivalenol (DON) and Aflatoxin B1 (AFB1), a comprehensive 28-days trial was conducted utilizing 60 randomly allocated mice divided into four groups. Administration of curcumin at a dosage of 5 mg/kg body weight in conjunction with DON at 0.1 mg/kg and AFB1 at 0.01 mg/kg body weight was undertaken to assess its efficacy. Results indicated that curcumin intervention demonstrated mitigation of splenic structural damage, augmentation of serum immunoglobulin A (IgA) and immunoglobulin G (IgG) levels, elevation in T lymphocyte subset levels, and enhancement in the mRNA expression levels of pro-inflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-6. Furthermore, curcumin exhibited a suppressive effect on apoptosis in mice, as evidenced by decreased activity of caspase-3 and caspase-9, reduced expression levels of pro-apoptotic markers Bax and Cytochrome-c (Cyt-c) at both the protein and mRNA levels, and the maintenance of a balanced expression ratio of mitochondrial apoptotic regulators Bax and Bcl-2. Collectively, these findings offer novel insights into the therapeutic promise of curcumin in mitigating immunosuppression and apoptotic events triggered by mycotoxin co-exposure.

Effect of Bioactive Ingredients on Urinary Excretion of Aflatoxin B1 and Ochratoxin A in Rats, as Measured by Liquid Chromatography with Fluorescence Detection.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are highly toxic mycotoxins present in food and feed, posing serious health risks to humans and animals. This study aimed to validate an efficient and cost-effective analytical method for quantifying AFB1 and OTA in rat urine using immunoaffinity column extraction and liquid chromatography with fluorescence detection (IAC-LC-FD). Additionally, the study evaluated the effect of incorporating fermented whey and pumpkin into the feed on the urinary excretion of these mycotoxins. The limits of detection and quantification were determined to be 0.1 µg/kg and 0.3 µg/kg, respectively, for both mycotoxins in feed, and 0.2 ng/mL and 0.6 ng/mL, respectively, in urine. The method demonstrated robust recovery rates ranging from 74% to 119% for both AFB1 and OTA in both matrices. In feed samples, the levels of AFB1 and OTA ranged from 4.3 to 5.2 µg/g and from 5.4 to 8.8 µg/g, respectively. This validated method was successfully applied to analyze 116 urine samples from rats collected during the fourth week of an in vivo trial. The results indicated that the addition of fermented whey and pumpkin to the feed influenced mycotoxin excretion in urine, with variations observed based on the sex of the rats, type of mycotoxin, and exposure dosage.

Aflatoxin B1 Control by Various Pseudomonas Isolates.

The climate-change-coupled fungal burden in crop management and the need to reduce chemical pesticide usage highlight the importance of finding sustainable ways to control Aspergillus flavus. This study examines the effectiveness of 50 Pseudomonas isolates obtained from corn rhizospheres against A. flavus in both solid and liquid co-cultures. The presence and quantity of aflatoxin B1 (AFB1) and AFB1-related compounds were determined using high-performance liquid chromatography-high resolution mass spectrometry analysis. Various enzymatic- or non-enzymatic mechanisms are proposed to interpret the decrease in AFB1 production, accompanied by the accumulation of biosynthetic intermediates (11-hydroxy-O-methylsterigmatocystin, aspertoxin, 11-hydroxyaspertoxin) or degradation products (the compounds C16H10O6, C16H14O5, C18H16O7, and C19H16O8). Our finding implies the upregulation or enhanced activity of fungal oxidoreductases and laccases in response to bacterial bioactive compound(s). Furthermore, non-enzymatic reactions resulted in the formation of additional degradation products due to acid accumulation in the fermented broth. Three isolates completely inhibited AFB1 or any AFB1-related compounds without significantly affecting fungal growth. These bacterial isolates supposedly block the entire pathway for AFB1 production in the fungus during interaction. Apart from identifying effective Pseudomonas isolates as potential biocontrol agents, this work lays the foundation for exploring new bacterial bioactive compounds.

Guide DNA dephosphorylation-modulated Pyrococcus furiosus Argonaute fluorescence biosensor for the detection of alkaline phosphatase and aflatoxins B1.

Foodborne hazardous factors pose a significant risk to public health, emphasizing the need for the development of sensitive and user-friendly detection strategies to effectively manage and control these risks in the food supply chain. Pyrococcus furiosus argonaute (PfAgo)-based biosensing approaches have been extensively explored due to its built-in signal amplification. However, the property that PfAgo is a DNA-guided DNA endonuclease has enabled almost all the existing PfAgo-based reports to be used for the detection of nucleic acids. To lend PfAgo toolbox to extended non-nucleic acid detection, we systematically investigated the mechanism characteristic of PfAgo' preference for guide DNA (gDNA) and proposed a gDNA dephosphorylation-modulated PfAgo sensor for the detection of non-nucleic acid targets. Our results indicated that PfAgo exhibits preference for 5'-phosphorylated gDNA at a specific ratio of PfAgo to gDNA concentration. Leveraging this PfAgo' preference and the dephosphorylation activity of alkaline phosphatase (ALP), ALP could be detected as low as 2.7 U/L. Furthermore, the PfAgo was coupled with immunolabelled ALP to develop a PfAgo-based fluorescence immunosensor, which achieves aflatoxins B1 detection with a detection limit of 29.89 pg/mL and exhibits satisfactory recoveries in wheat and maize samples. The developed method broadens the application scope of PfAgo toolbox, and provides a simple, sensitive, and universal detection platform for a variety targets.

Widespread distribution of the DyP-carrying bacteria involved in the aflatoxin B1 biotransformation in Proteobacteria and Actinobacteria.

Aflatoxin is one of the most notorious mycotoxins, of which aflatoxin B1 (AFB1) is the most harmful and prevalent. Microbes play a crucial role in the environment for the biotransformation of AFB1. In this study, a bacterial consortium, HS-1, capable of degrading and detoxifying AFB1 was obtained. Here, we combined multi-omics and cultivation-based techniques to elucidate AFB1 biotransformation by consortium HS-1. Co-occurrence network analysis revealed that the key taxa responsible for AFB1 biotransformation in consortium HS-1 mainly belonged to the phyla Proteobacteria and Actinobacteria. Moreover, metagenomic analysis showed that diverse microorganisms, mainly belonging to the phyla Proteobacteria and Actinobacteria, carry key functional enzymes involved in the initial step of AFB1 biotransformation. Metatranscriptomic analysis indicated that Paracoccus-related bacteria were the most active in consortium HS-1. A novel bacterium, Paracoccus sp. strain XF-30, isolated from consortium HS-1, contains a novel dye-decolorization peroxidase (DyP) enzyme capable of effectively degrading AFB1. Taxonomic profiling by bioinformatics revealed that DyP, which is involved in the initial biotransformation of AFB1, is widely distributed in metagenomes from various environments, primarily taxonomically affiliated with Proteobacteria and Actinobacteria. The in-depth examination of AFB1 biotransformation in consortium HS-1 will help us to explore these crucial bioresources more sensibly and efficiently.

Characterization of a Novel Monoclonal Antibody with High Affinity and Specificity against Aflatoxins: A Discovery from Rosetta Antibody-Ligand Computational Simulation.

Aflatoxin B1 (AFB1) accumulates in crops, where it poses a threat to human health. To detect AFB1, anti-AFB1 monoclonal antibodies have been developed and are widely used. While the sensitivity and specificity of these antibodies have been extensively studied, information regarding the atomic-level docking of AFB1 (and its derivatives) with these antibodies is limited. Such information is crucial for understanding the key interactions that are required for high affinity and specificity in aflatoxin binding. First, a 3D comparative model of anti-AFB1 antibody (Ab-4B5G6) was predicted from the sequence using RosettaAntibody. We then utilized RosettaLigand to dock AFB1 onto ten homology models, producing a total of 10,000 binding modes. Interestingly, the best-scoring mode predicted strong interactions involving four sites within the heavy chain: ALA33, ASN52, HIS95, and TRP99. Importantly, these strong binding interactions exclusively involve the variable domain of the heavy chain. The best-scoring mode with AFB1 was also obtained through AF multimer combined with RosettaLigand, and two interactions at TRP and HIS were consistent with those found by Rosetta antibody-ligand computational simulation. The role of tryptophan in π interactions in antibodies was confirmed through mutation experiments, and the resulting mutant (W99A) exhibited a >1000-fold reduction in binding affinity for AFB1 and analogs, indicating the effect of tryptophan on the stability of CDR-H3 region. Additionally, we evaluated the binding of two glycolic acid-derived molecular derivatives (with impaired hydrogen bonding potential), and these derivatives (AFB2-GA and AFG2-GA) demonstrated a very weak binding affinity for Ab-4B5G6. The heavy chain was successfully isolated, and its sensitivity and specificity were consistent with those of the intact antibody. The homology models of variable heavy (VH) single-domain antibodies were established by RosettaAntibody, and the docking analysis revealed the same residues, including Ala, His, and Trp. Compared to the potential binding mode of fragment variable (FV) region, the results from a model of VH indicated that there are seven models involved in hydrophobic interaction with TYR32, which is usually referred to as polar amino acid and has both hydrophobic and hydrophilic features depending on the circumstances. Our work encompasses the entire process of Rosetta antibody-ligand computational simulation, highlighting the significance of variable heavy domain structural design in enhancing molecular interactions.

Colorimetric and ECL dual-mode aptasensor for smartphone-based onsite sensitive detection of aflatoxin B1 in combination with ZnO@MWCNTs/g-C3N4 nanosheets and CuO@CuPt nanocomposites.

The development of dual-mode strategies with superior sensitivity and accuracy have garnered increasing attention for researchers in Aflatoxin B1 (AFB1) analysis. Herein, a colorimetric-electrochemiluminescence (ECL) dual-mode biosensor was constructed for onsite and ultrasensitive determination of AFB1. The multi-wall carbon nanotubes (MWCNTs) were integrated with the ZnO metal organic frameworks (MOFs) to accelerate the electron transfer and boost the ECL intensity of g-C3N4 nanoemitters. Through the aptamer-based DNA sandwich assay, the CuO@CuPt nanocomposites were introduced onto the electrode and acted as the dual functional signal nanoprobes. Due to the good spectrum overlap between the CuO@CuPt nanoprobes and g-C3N4 nanosheets, ECL signal could be efficiently quenched. Additionally, the CuO@CuPt nanoprobes show superior catalytic properties towards the TMB and H2O2 colorimetric reactions, and an obvious color alteration from colorless to blue can be observed using the smartphone. Under optimized conditions, a sensitive and accurate dual-mode analysis of the AFB1 was accomplished with the colorimetric detection limit of 3.26 fg/mL and ECL detection limit of 0.971 fg/mL (S/N = 3). This study combines innovative nanomaterial properties of ZnO@MWCNTs, g-C3N4 and CuO@CuPt for ultrasensitive dual-mode detection, which offers new opportunities for the innovative engineering of the dual-mode sensors and demonstrates significant potential in food safety analysis.

Biodegradation Characteristics and Mechanism of Aflatoxin B1 by Bacillus amyloliquefaciens from Enzymatic and Multiomics Perspectives.

Aflatoxin B1 (AFB1), a mycotoxin and natural carcinogen, commonly contaminates cereals and animal feeds, posing serious health risks to human and animal. In this study, Bacillus amyloliquefaciens ZG08 isolated from kimchi could effectively remove 80.93% of AFB1 within 72 h at 37 °C and pH 7.0. Metabolome and transcriptome analysis showed that metabolic processes including glycerophospholipid metabolism and amino acid metabolism were most affected in B. amyloliquefaciens ZG08 exposed to AFB1. The adaptation mechanism likely involved activation of the thioredoxin system to restore intracellular redox equilibrium. The key genes, tpx and gldA, overexpressed in Escherichia coli BL21, achieved degradation rates of 60.15% and 47.16% for 100 µg/kg AFB1 under optimal conditions of 37 °C and pH 8.0 and 45 °C and pH 7.0, respectively. The degradation products, identified as AFD1, were less cytotoxic than AFB1 in HepG2 cells. These findings suggest potential strategies for utilizing probiotics and engineered enzymes in AFB1 detoxification.

Characterization, Mechanism, and Application of Dipeptidyl Peptidase III: An Aflatoxin B1-Degrading Enzyme from Aspergillus terreus HNGD-TM15.

Aflatoxin B1 is a notorious mycotoxin with mutagenicity and carcinogenicity, posing a serious hazard to human and animal health. In this study, an AFB1-degrading dipeptidyl-peptidase III mining from Aspergillus terreus HNGD-TM15 (ADPP III) with a molecular weight of 79 kDa was identified. ADPP III exhibited optimal activity toward AFB1 at 40 °C and pH 7.0, maintaining over 80% relative activity at 80 °C. The key amino acid residues that affected enzyme activity were identified as H450, E451, H455, and E509 via bioinformatic analysis and site-directed mutagenesis. The degradation product of ADPP III toward AFB1 was verified to be AFD1. The zebrafish hepatotoxicity assay verified the toxicity of the AFB1 degradation product was significantly weaker than that of AFB1. The result of this study proved that ADPP III presented a promising prospect for industrial application in food and feed detoxification.

Aflatoxin B1 as a complicit in intestinal damage caused by Eimeria ovinoidalis in lambs: Novel insights to reveal parasite-gut battle.

Mycotoxins, unavoidable contaminants in feed and feed ingredients, have the potential to influence the incidence and severity of various diseases upon ingestion. Sheep coccidiosis is an enteric disease caused by protozoa of Eimeria spp. However, the extent to which the presence of aflatoxin b1 (AFB1) synergistically exacerbates damage to intestinal health in lambs with Eimeria remains unclear. 50-day-old female lambs were randomly assigned to a 2 × 2 factorial arrangement of treatments for 15 days to assess the impact of AFB1 exposure on lambs with or without Eimeria (E.) ovinoidalis infection. Our findings reveal that AFB1 synergistically intensifies damage to intestinal health in lambs challenged by E. ovinoidalis. This is evidenced by disruptions to the intestinal microbiota and reductions in the production of short-chain fatty acids. AFB1 further aggravates damage to the cecal mechanical barrier. Additionally, AFB1 contributes to the entry of lipopolysaccharide into the bloodstream, activating the inflammatory response. Interestingly, AFB1 exposure history results in an early peak of oocyst excretion and a decreased number of oocyst excretion in E. ovinoidalis infected lambs. This may be closely linked to the destruction of the intestinal epithelial cell structure and its apoptosis, as indicated by a decreased ratio of Bcl-2 to Bax and increased caspase-3 levels. Mechanistically, proteomics analysis identified mitochondrial dysfunction (inhibition of the oxidative phosphorylation pathway) as the primary factor intensifying intestinal epithelial cell destruction caused by coccidia, exacerbated by AFB1 through the inhibiting the conversion of NADH to NAD+ in the cecum of lambs via down-regulation of the PGC-1α/NRF1/TFAM pathway. Overall, these results offer novel insights into the AFB1 complicity in accelerating intestinal damage caused by E. ovinoidalis in lambs. Targeting the mitochondrial oxidative phosphorylation pathway of the intestine may represent a new therapeutic strategy against the detrimental effects of mycotoxin and coccidia.

Bidirectional hybridized hairpin DNA fluorescent aptasensor based on Au-Pd NPs and CDs for ratiometric detection of AFB1.

A novel and simple ratiometric fluorescent aptasensor was developed for the sensitive detection of aflatoxin B1 (AFB1). A hairpin DNA (h-DNA) was independently synthesized as the basic skeleton, and the bidirectional hybridization of h-DNA can increase the load of aptamer and signal probes, thereby realizing signal amplification. The high-efficiency fluorescence resonance energy transfer interaction between gold-palladium nanoparticles (Au-Pd NPs) and the self-synthesized fluorescent probe carbon dots (CDs) was utilized. Moreover, the label-free probe SYBR Green I (SG I) dye was introduced to form a double-signal probe with CDs, and a ratiometric sensor with FCDs/FSG I as a response signal was constructed. The ratio strategy can eliminate the fluctuation of external factors, thus improving the accuracy and reliability of the sensor. The quenching effect of Au-Pd NPs on CDs was 1.4 times that of AuNPs and 3.4 times that of Pd NPs, respectively. In the range 1-100 ng/mL, FCDs/FSG I showed a good linear relationship with the logarithm of the concentration of AFB1, and the limit of detection was as low as 0.07 ng/mL. The sensor was used to detect AFB1 in spiked peanuts and wine samples, and the recovery was between 91 and 115%, indicating that the sensor has high application potential in real sample analysis.

Fluorescence/visual aptasensor based on Au/MOF nanocomposite for accurate and convenient aflatoxin B1 detection.

A dual-mode fluorescence/visual aptasensor was developed for straightforward and accurate determination of aflatoxin B1 (AFB1) based on an Au/metal-organic framework (Au/MOF) composite. Aptamer-modified Au/Fe3O4 (Apt/Au/Fe3O4) served as the recognition element, and Au/MOF modified with complementary chains and 3,3',5,5'-tetramethylbenzidine (cDNA/TMB/Au/MOF) acted as the fluorescence and visual probes. These components are integrated to form conjugates (Apt/Au/Fe3O4-cDNA/TMB/Au/MOF). Upon the introduction of AFB1, some cDNA/TMB/Au/MOF dissociated from Apt/Au/Fe3O4, enabling the use of detached probes for visual detection. The undecomposed conjugates were isolated magnetically for use in fluorescence detection. As the AFB1 concentration increases, the visual signal intensifies and fluorescence intensity diminishes. Thus, the proposed aptasensor achieves the simultaneous fluorescence and visual determination of AFB1, obviating the need for material and reagent substitutions. The detection limits were established at 0.07 ng mL-1 for the fluorescence mode and 0.08 ng mL-1 for the visual mode. The effectiveness of the aptasensor was further validated by quantifying AFB1 in real samples.

Chromatin accessibility and transcriptional landscape in PK-15 cells during early exposure to Aflatoxin B1.

Aflatoxin B1 (AFB1) not only causes significant losses in livestock production but also poses a serious threat to human health. It is the most carcinogenic among known chemicals. Pigs are more susceptible to AFB1 and experience a higher incidence. However, the molecular mechanism of the toxic effect of AFB1 remains unclear. In this study, we used assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA-seq to uncover chromatin accessibility and gene expression dynamics in PK-15 cells during early exposure to AFB1. We observed that the toxic effects of AFB1 involve signaling pathways such as p53, PI3K-AKT, Hippo, MAPK, TLRs, apoptosis, autophagy, and cancer pathways. Basic leucine zipper (bZIP) transcription factors (TFs), including AP-1, Fos, JunB, and Fra2, play a crucial role in regulating the biological processes involved in AFB1 challenge. Several new TFs, such as BORIS, HNF1b, Atf1, and KNRNPH2, represent potential targets for the toxic mechanism of AFB1. In addition, it is crucial to focus on the concentration of intracellular zinc ions. These findings will contribute to a better understanding of the mechanisms underlying AFB1-induced nephrotoxicity and offer new molecular targets.

Dual-template magnetic molecularly imprinted polymers for selective extraction and sensitive detection of aflatoxin B1 and benzo(α)pyrene in environmental water and edible oil.

The coexistence of multiple contaminates in the environment and food is of growing concern due to their extremely hazard as a well-known class I carcinogen, like aflatoxin B1 (AFB1) and benzo(α)pyrene (BaP). AFB1 and BaP are susceptible to coexistence in environmental water and edible oil, posing a significant potential risk to environmental monitoring and food safety. The remaining challenges in detecting multiple contaminates include unsatisfied sensitivity, insufficient targets selectivity, and interferences in complex matrices. Here, we developed dual-template magnetic molecularly imprinted polymers (DMMIPs) for selective extraction of dual targets in complex matrices from the environment and food. The DMMIPs were fabricated by surface imprinting with vinyl-functionalized Fe3O4 as carrier, 5,7-dimethoxycoumarin and pyrene as dummy templates, and methacrylamide as functional monomer. The DMMIPs showed excellent adsorption ability (12.73-15.80 mg/g), imprinting factors (2.01-2.58), and reusability of three adsorption-desorption cycles for AFB1 and BaP. The adsorption mechanism including hydrogen bond, electrostatic interaction and van der Waals force was confirmed by physical characterization and DFT calculation. Applying DMMIPs in magnetic solid phase extraction (MSPE) followed by high-performance liquid chromatography (HPLC) analysis enabled detection limits of 0.134 µg/L for AFB1 and 0.107 µg/L for BaP. Recovery rates for water and edible oil samples were recorded as 86.2%-110.3% with RSDs of 4.1%-11.9%. This approach demonstrates potential for simultaneous identification and extraction of multiple contaminants in environmental and food.

The occurrence and human health risk assessment of total and aflatoxin B1 in selected food commodities in Bhutan.

Aflatoxins are mycotoxins that contaminate staple foods globally and pose a significant health risk. To the best of our knowledge, information on the occurrence of aflatoxins in Bhutanese diets is scarce. This study aimed to estimate the aflatoxin levels in selected foodstuffs in Bhutan and determine the health risk associated with aflatoxin exposure. Ten different types of food commodities were randomly collected from farmers' markets, shelves of supermarkets, and wholesale and retail shops from 20 districts of the country. The samples were subjected to analysis by an enzyme-linked immunosorbent assay for both total aflatoxins (B1, B2, G1 and G2) and aflatoxin B1. Among the 315 samples included, 48.81% and 79.35% were positive for total aflatoxins and aflatoxin B1, respectively. The overall mean total aflatoxin concentration was 11.49 ± 12.83 µg/kg, and that for B1 was 17.62 ± 23.99 µg/kg. The most prevalent food commodity with the highest aflatoxin contamination was chili products. In addition, the estimated daily intake and margin of exposure to aflatoxin B1 via the consumption of chili products ranged from 0.98 to 5.34 ng kg-1 bw day-1 and from 74.90 to 408.10, indicating a risk for public health. The liver cancer risk was estimated to be 0.01 and 0.007 cancers per year per 100,000 population resulting from the consumption of chili products. The present findings revealed the presence of total aflatoxins and aflatoxin B1 in the selected samples. The margin of exposure values was exorbitant, demanding a stringent public health measure. Notably, these results suggest the need for routine monitoring of aflatoxin contamination in the region and stress rigorous safety management strategies to reduce exposure.

Rhein Inhibits Cell Development and Aflatoxin Biosynthesis via Energy Supply Disruption and ROS Accumulation in Aspergillus flavus.

Aspergillus flavus and its carcinogenic secondary metabolites, aflatoxins, not only cause serious losses in the agricultural economy, but also endanger human health. Rhein, a compound extracted from the Chinese herbal medicine Rheum palmatum L. (Dahuang), exhibits good anti-inflammatory, anti-tumor, and anti-oxidative effects. However, its effect and underlying mechanisms against Aspergillus flavus have not yet been fully illustrated. In this study, we characterized the inhibition effect of rhein on A. flavus mycelial growth, sporulation, and aflatoxin B1 (AFB1) biosynthesis and the potential mechanism using RNA-seq analysis. The results indicate that A. flavus mycelial growth and AFB1 biosynthesis were significantly inhibited by 50 µM rhein, with a 43.83% reduction in colony diameter and 87.2% reduction in AFB1 production. The RNA-seq findings demonstrated that the differentially expressed genes primarily participated in processes such as spore formation and development, the maintenance of cell wall and membrane integrity, management of oxidative stress, the regulation of the citric acid cycle, and the biosynthesis of aflatoxin. Biochemical verification experiments further confirmed that 50 µM rhein effectively disrupted cell wall and membrane integrity and caused mitochondrial dysfunction through disrupting energy metabolism pathways, leading to decreased ATP synthesis and ROS accumulation, resulting in impaired aflatoxin biosynthesis. In addition, a pathogenicity test showed that 50 µM rhein inhibited A. flavus spore growth in peanut and maize seeds by 34.1% and 90.4%, while AFB1 biosynthesis was inhibited by 60.52% and 99.43%, respectively. In conclusion, this research expands the knowledge regarding the antifungal activity of rhein and provides a new strategy to mitigate A. flavus contamination.