Thiram (TMTD), a widely used dithiocarbamate fungicide, provokes oxidative hepatotoxicity, yet practical strategies for its detection and immunoprotection remain scarce. Here we develop an antibody-enabled framework integrating detection, therapy, and prevention against TMTD. In the murine model, we characterized the dose-dependent hepatic injury induced by TMTD. Guided by rational C3/C7 hapten design, we then generate a monoclonal antibody with high affinity. Building on this reagent, we create a portable immunodetection platform that couples Au nanoflowers with an Au/Ir@Zn/Cu-MOF probe, achieving an IC50 of 0.246 µg/mL. To evaluate protective efficacy, we demonstrated that passive immunization conferred a clear time-dependent therapeutic benefit: mortality decreased from 37.5% in the TMTD-poisoned group to 0% with treatment at 15 or 30 min and to 12.5% with treatment at 60 min, while improving body weight, liver function, and oxidative-stress markers. Complementarily, active immunization with TMTD-BSA elicited high antibody titers and conferred complete survival with graded histopathological protection. Together, this work offers an integrated detection-to-protection strategy that effectively bridges immunodetection with immune protection against TMTD-induced toxicity.

Sulfamethoxazole (SMX) is a widely used sulfonamide antibiotic, and its residues in animal-derived foods pose significant health risks. In this study, a high-affinity monoclonal antibody (2.65 × 1010 L/mol) against SMX was developed and used to construct an Au/Ir@Zn/Cu-MOF-based lateral flow immunoassay (LFIA). The probe was thoroughly characterized with good stability. The LFIA achieved a visual detection limit of 2.5 μg/mL, with an IC50 of 565.84 ng/mL and a linear detection range of 6.8 ng/mL-47.06 μg/mL. No cross-reactivity to structural analogues or matrix interference was observed in real food samples. Recovery rates (97.92-108%) were consistent with LC-MS results, confirming the accuracy of the method. This LFIA provides a sensitive and reliable tool for rapid SMX screening in food products.

Alternative splicing of pre-mRNA is a crucial mechanism in gene expression regulation. As a core component of the spliceosome, the biological function of the Skip protein in Aspergillus flavus remains unknown. Quantitative real-time PCR (qPCR) analysis revealed the presence of two skip gene copies in A. flavus. Single-copy deletion of Skip resulted in slowed growth, reduced conidiation, abolished sclerotial formation, increased aflatoxin biosynthesis, and diminished crop colonization. Meanwhile, Skip was found to regulate the oxidative stress response by modulating the alternative splicing of yapA. Subsequently, immunoprecipitation and Western blot analyses identified lysine 325 (K325) as the benzoylated site on the Skip protein, which catalyzed by the acyltransferase EsaA. Mutation of benzoylated site K325 directly impaired fungal morphogenesis, pathogenicity, and stress adaptation. These findings established the crucial role of Skip and its benzoylation in A. flavus and suggested a potential target for controlling its infection in important crops.

Acifluorfen (AFF), a widely used diphenyl ether herbicide, raises significant environmental concerns due to its persistence and ecotoxicological risks, but the absence of immunoassay methods hinder efficient monitoring. A hybridoma cell line secreting a monoclonal antibody against AFF was developed via hybridoma technology to address this need. Using this antibody, we developed two lateral flow immunoassays (LFIAs) based on gold nanoparticles (AuNP-LFIA) and gold nanoflowers (AuNF-LFIA). The AuNP-LFIA exhibited a limit of detection (LOD) of 75.01 ng/mL. In contrast, the AuNF-LFIA demonstrated significantly enhanced sensitivity, achieving an LOD as low as 7.79 ng/mL, which represents a 9.6-fold improvement over the AuNP-LFIA. When applied to fruit and vegetable samples, both methods showed spike recovery rates ranging from 92.58% to 108.41%, and the results were in good agreement with those obtained by LC-MS. These findings suggest that the developed assays hold strong potential as reliable tools for the on-site detection of AFF.

Aspergillus flavus, a ubiquitous filamentous fungus, severely compromises global food safety and public health by producing carcinogenic aflatoxins. Heat shock factor 1 (HSF1) orchestrates stress responses in eukaryotes, yet the functional role and regulatory mechanisms of its homolog, HsfA, in A. flavus remain elusive. Here, we demonstrate that A. flavus encodes two hsfA copies, whose knockdown or expression of a dominant-negative variant abrogates spore germination, a prerequisite for fungal development and colonization. Through integrated reverse genetics, ChIP-qPCR, electrophoretic mobility shift assay, and transcriptional profiling, we identify six non-chaperone targets—brlA, fksP, flbC, sntB, velB, and vosA—directly regulated by HsfA via binding to conserved heat shock elements (HSEs) in their promoters. Ectopic expression of each target partially restores germination, developmental progression, and pathogenicity in HsfA-deficient strains, confirming HsfA’s central role in driving these processes via transcriptional activation. Structural divergence between the A. flavus HsfA DNA-binding domain and human HSF1 explains the ineffectiveness of three HSF1 inhibitors against the fungus. Our findings establish HsfA as a pivotal regulator of A. flavus virulence and uncover a novel HSF regulatory pathway, highlighting HsfA as a promising target for mitigating aflatoxin contamination.

INTRODUCTION:Sulfamethazine (SM2) antibiotic residues in food pose a significant risk to human health, including pathogenic bacterial resistance, allergic reactions, and carcinogenicity. Current lateral flow immunoassay (LFIA) typically employing colloidal gold (AuNP) as a signal reporter exhibits unsatisfactory sensitivity. Recently, metal-organic frameworks (MOFs) nanomaterial as a supporting matrix for loading nanoparticles provides a powerful tool to improve the performance of LFIA. OBJECTIVES:This study aims to develop an enhanced immunosensor based on antibody labeled Au/Ir@Cu/Zn-MOF signal amplification nano-composite for sensitive and point-of-care monitoring of SM2 antibiotic residues. METHODS:Firstly, the highly specific monoclonal antibody (mAb) against SM2 belonging to the IgG1 subclass was screened. Subsequently, the Au/Ir@Cu/Zn-MOF nanocomposite was synthesized by incorporating Au/Ir bimetallic nanoparticles into the previously prepared Cu/Zn-MOF composite. The anti-SM2 mAb was then conjugated to Au/Ir@Cu/Zn-MOF nanocomposite via a co-precipitation method to fabricate the mAb-Au/Ir@Cu/Zn-MOF probe. Finally, a lateral flow immunoassay was constructed for the detection of SM2 residues. RESULTS:The vLOD the Au/Ir@Cu/Zn-MOF-based LFIA for SM2 detection was approximately 2.5 ng/mL, which was at least 600 times greater than that of the AuNP-based LFIA (with the vLOD of 1563 ng/mL). Average recovery rates were 80.08-90.8% in milk sample, 88-89.3% in eel sample and 85.3-95.2% in pork sample, respectively. CONCLUSION:The Au/Ir@Cu/Zn-MOF nanocomposite utilized as signal amplification probe demonstrated remarkable sensitivity for the detection of SM2. The developed Au/Ir@Cu/Zn-MOF-based LFIA exhibits significant potential for the rapid and sensitive detection of contaminants in environmental monitoring and food safety.

Lysine benzoylation (Kbz) is a newly identified post-translational modification, which participates in the regulation of a variety of cellular processes. However, the function of Kbz in pathogenic fungi remains unclear. Here, we first identified that FoxA is a benzoylated protein with two benzoylated sites at lysines 425 and 433. Mutations of Kbz sites in FoxA significantly reduced long-chain fatty acid (LCFA) utilization activity and exhibited a phenotype similar to that of foxA gene deletion mutant, including decreased conidiation and aflatoxin production, reduced seed colonization, and increased sclerotia formation. Metabolomic analyses indicated that the deletion of FoxA or interference with its benzoylation could disrupt peroxisomal β-oxidation, resulting in the accumulation of LCFAs. This disruption may inhibit conidiation and aflatoxin production by modulating the synthesis of 15d-PGJ(2)-G. Furthermore, we found that EsaA has benzoyltransferase activity in vitro and in vivo, and its expression influences the Kbz of FoxA and LCFA utilization activity. Notably, mutants at the acetylated sites exhibited phenotypes similar to those of the benzoylated site mutants, suggesting that acetylation also plays a significant role in FoxA protein. Our study uncovers a previously unknown mechanism by which benzoylation and acetylation regulate FoxA activity to affect the development, secondary metabolism, and pathogenicity of A. flavus.IMPORTANCEAs a predominantly plant-pathogenic fungus, Aspergillus flavus not only causes severe crop diseases and economic losses but also poses a global food safety threat due to its production of aflatoxins. Furthermore, under certain conditions, it can act as an opportunistic pathogen, endangering the health of both humans and animals. Numerous studies have shown that protein post-translational modifications, such as acetylation, succinylation, and benzylation, were involved in aflatoxin production; however, the exact mechanism was still unclear. This study reveals, for the first time, the molecular mechanism by which benzoylation regulates the functional execution of multifunctional β-oxidation hydratase/dehydrogenase FoxA proteins and further influences the development and aflatoxin synthesis in Aspergillus flavus. This discovery not only provides new insights for the prevention and control of aflatoxin contamination but also provides theoretical support for the study of secondary metabolism regulation mechanisms of other fungi.

Endocrine-disrupting chemicals (EDCs), such as 17-β-estradiol (E2) and Bisphenol A (BPA), disrupt endocrine function and modulate critical physiological processes in the human body, even at low concentrations. This underscores the urgent need for efficient and rapid screening methods capable of detecting trace amounts of these compounds. Here, we generated a gene circuit to enhance EDC detection in whole-cell assays, utilizing classical nuclear estrogen receptor (ER) response elements and the mScarlet3 red fluorescent protein reporter in human 293T and MCF7 cell lines. To further improve sensitivity, we integrated a galactose-regulated upstream promoter element (GAL4)- upstream activating sequence (UAS) system into these cell-based biosensors, initiating a cascaded amplification platform that achieved a limit of detection (LOD) of 10 pM for both E2 and BPA. Application of these biosensors to environmental samples successfully identified these EDCs at a LOD of 100 pM. The incorporation of cascaded amplifying circuits significantly enhances detection sensitivity and signal output, providing a method for evaluating the toxicity of environmental pollutants and supporting risk assessments.

Aspergillus flavus is the second most prevalent species of Aspergillus causing invasive aspergillosis, but its treatment efforts had been hindered by the continuous emergence of drug-resistant fungal strains, while the underlying mechanisms remain largely unexplored. In this study, we investigated the role of the chromatin remodeling factor Arp9 in A. flavus drug-resistant. We show that Arp9 up-regulates the chromatin accessibility of the Erg3 and Erg6 promoters, thereby increasing their transcription levels and enhancing ergosterol synthesis. Therefore, the absence of Arp9 enhances A. flavus sensitivity to amphotericin B (AMB). Additionally, by down-regulating chromatin accessibility of Erg11A gene promoter, Arp9 leads to the decrease of its transcription level and subsequently reduces A. flavus resistance to voriconazole (VOR). Co-immunoprecipitation analysis revealed that Arp9 exists in both SWI/SNF and RSC complex. Drug susceptibility test results indicated that the drug sensitivity response induced by Arp9 may be unique to Arp9, as neither SWP82 of the SWI/SNF nor Sth1 of the RSC is required. The role of Arp9 in drug-resistance was also confirmed using the Galleria mellonella model. Furthermore, we found that VOR induces aflatoxin B1 (AFB1) biosynthesis in an Arp9-dependent manner at 35°C and 37°C, and the effect is dramatically magnified in the VOR-resistant A. flavus strain. This study demonstrates that Arp9 plays a critical role in regulating fungal drug-resistance in vitro and in vivo and revealed that Arp9 is an important factor in enhancing AFB1 biosynthesis under Mammalian physiological temperatures. This study provides potential new insights for the control of the infections caused by filamentous pathogenic fungi.

Aspergillus flavus is a typical filamentous fungus that poses risk to both humans and animals, as well as for crops. The secondary metabolite aflatoxin B1 produced by A. flavus is also one of the most carcinogenic and toxic natural pollutants discovered so far. Lysine benzoylation (Kbz), which participates in various life activities in different organisms, is an important post-translational modification of proteins. Cystathionine β-synthase (CBS) is a key enzyme in the maintenance of the homocysteine balance in organisms. However, the basic mechanism of CBS in the life activities of A. flavus is still unclear. By knocking out the cbs gene, it was found that compared to the wild-type and complementary strains, the cbs deficient strains had lower growth diameter, spore yield and seed colonization, while the number of sclerotia was increased, and toxin accumulation was increased by TLC. We then validated these results through RT-PCR. We also found the existence of a benzoyl site K109 on CBS by immunoprecipitation. Mutation at K109 abolishes benzoylation, resulting in reduced CBS enzymatic activity, which We found that the phenotype of point mutations is consistent with that of knockout strains. In addition, we found that benzoyltransferase GcnE catalyzes the benzoylation of CBS protein and affects enzyme activity. These results not only give theoretical support for the research of cystathionine β-synthase and benzoyl modification, but also provides fresh ideas for the prevention and control of pathogenic fungus A. flavus.

Rab7 GTPases coordinate late endosome maturation, vesicle trafficking, and vacuole biogenesis across eukaryotes, yet the functional contribution of the Rab7 C-terminal tail to organismal physiology remains incompletely defined. Here we present the crystal structure of Aspergillus flavus Rab7 (AflRab7), which reveals a well-ordered GTPase core, whereas its C-terminal tail is conserved across Aspergillus species yet lacks interpretable electron density, consistent with an intrinsically disordered region (IDR). Biochemical assays show that deletion of this IDR does not alter basal GTP hydrolysis, whereas genetic and cell-biological studies demonstrate that deletion of residues 191-205 causes severe growth retardation, defective conidiation and sclerotium formation, markedly reduces aflatoxin production, and fragmented vacuolar compartments. The IDR-deficient mutants further exhibit pronounced hypersensitivity to cell-wall stress and altered tolerance to membrane, osmotic, and redox challenges. Transcriptome profiling, supported by qPCR validation, reveals extensive transcriptional reprogramming upon loss of the C-terminal region, including dysregulation of pathways linked to development and secondary metabolism. Overall, our findings extend the mechanistic understanding of Rab7 beyond its catalytic pocket and establish the AflRab7 C-terminal IDR as a critical regulatory element that couples vacuolar dynamics to A. flavus development and aflatoxin biosynthesis, highlighting a tractable target for mitigating fungal toxigenic potential.

Iron homeostasis and secondary metabolism are controlled by the basic leucine zipper (bZIP) transcription factor HapX in many fungi. In this study, AflHapX was identified through homology research of the Aspergillus flavus genome with the bZIP protein HapX in Aspergillus fumigatus and subsequent gene deletion. We established that AflHapX functions as a regulator for adaptation to iron-excess and iron-starvation conditions. The AflhapX deletion strain (ΔAflhapX) produces less aflatoxin B1 (AFB1) compared to the wild type (WT) and complementary strain (ΔAflhapXC) on YES medium. While iron typically represses AFB1 biosynthesis in A. flavus, the deletion of AflhapX restores iron’s inhibition of AFB1 production. Furthermore, compared to WT and ΔAflhapXC, ΔAflhapX produced more sclerotia and displayed greater sensitivity to the oxidative reagents H2O2, menadione sodium bisulfite (MSB), and diamide. On peanuts, the ΔAflhapX strain produced fewer conidia and less AFB1 than WT and ΔAflhapXC. Transcriptome analysis data provided evidence that AflHapX globally regulates genes involved in aflatoxin biosynthesis, oxidative stress response, and iron homeostasis. Our results suggest that AflHapX may regulate the effect of iron on aflatoxin production.

Aspergillus flavus, along with its notorious secondary metabolite aflatoxin B1 (AFB1), seriously endangers human health. Histone methyltransferase complex COMPASS (complex of proteins associated with Set1) plays a crucial role in regulating aflatoxin biosynthesis and virulence of A. flavus, but the underlying mechanism is unclear. Here, we find that Bre2, the key subunit of COMPASS, regulates AFB1 biosynthesis, fungal morphogenesis, and virulence through modulation of H3K4 methylation. ChIP-seq and biochemical analyses reveal that chromatin remodeling factor (CRF) Arp9 is directly targeted by Bre2, and Arp9 exerts bio-functions through interacting with the other CRFs such as RSC8, Arp7, and Sth1. ATAC-seq results indicate that Arp9 contributes to fungal pathogenicity by modulating chromatin conformation of genes that are involved in secondary metabolism, morphogenesis, and virulence. The study reveals an epigenetic signaling pathway mediated by chromatin remodeler Arp9 and provides a potential strategy for the control of pathogenic fungi and mycotoxins.

Aspergillus flavus (A. flavus) is a common contaminant of food and feed due to the production of aflatoxin B1, which is susceptible to environmental signals. Nevertheless, how red light plays a role in A. flavus remains unclear. Here, we identified the uncharacterized hypothetical protein G4B84_010091 as a red-light sensor, defined as fungal phytochrome A (FphA), in A. flavus. The fphA knockout strain (ΔfphA) and complementary strain (fphA-com) were successfully constructed to characterize the function of FphA. Our results indicated that aflatoxin B1 biosynthesis was promoted, while the development of conidia and sclerotia as well as the infection of peanuts were impaired in ΔfphA when compared with WT or fphA-com. The FphAΔRR domain deletion mutant exhibited all the phenotypes observed in the ΔfphA strain, indicating that the RR domain is indispensable for the function of FphA. In summary, FphA is involved not only in the formation of spores and sclerotia, but also in aflatoxin B1 biosynthesis and the pathogenicity of A. flavus, which offers a potential target for novel approaches to controlling the dispersal and toxin production of this fungus.

This study evaluates the effects of pyruvate decarboxylase and its lysine succinylation (Ksucc) on the development of Aspergillus flavus and the production of secondary metabolites. Mutant strains, including the knockout (∆pdc1), pdc1 point mutants (K258R and K258E) and complementary (∆pdc1.com) were constructed. The results showed that both Δpdc1 and K258R strains exhibited decreased conidiophore and conidia production and failed to generate sclerotia. The production of aflatoxin B1 (AFB1) was significantly increased in the Δpdc1 and K258R strains, while it decreased in the K258E strain. The results also indicate that pdc1 and its Ksucc are involved in the stress response and pathogenicity of A. flavus. Through GC-MS analysis, Δpdc1 was found to produce several significantly decreased compounds, including n-hexadecanoic acid, 2-bromotetradecane, and palmitic acid, among others. Additionally, different volatile metabolites, such as 1-iodo-decane, n, n-dimethyloctanamide and n, n-dimethyl-7-octynamide, were not detected in the Δpdc1 strain compared to WT and Δpdc1.com. HPLC results showed that the production of pyruvic acid, malic acid and succinic acid increased in both the Δpdc1 and K258R strains when compared to WT and Δpdc1.com strains. Comparative RNA-seq analysis revealed a total of 3,817 differentially expressed genes (DEGs) including 1,913 up-regulated and 1,904 down-regulated genes in Δpdc1 vs. WT. These results suggest that PDC1 and K258 play a crucial role in the biosynthesis of secondary metabolites, development and stress responses of A. flavus.

Pathogenic filamentous fungi pose a significant threat to global food security and human health. The limitations of available antifungal agents, including resistance and toxicity, highlight the need for developing innovative antifungal strategies. Antifungal proteins (AFPs) are a class of secreted small proteins that exhibit potent antifungal activity against filamentous fungi, yet the underlying mechanism remains partially understood. In this study, we investigate the molecular and cellular effects of two AFPs, PgAFP and AfAFP, on Aspergillus flavus, a representative filamentous fungus. These AFPs affect various fungal phenotypes and exert an intracellular effect by interacting with Ntp1, a fungi exclusive protein modulating diverse fungal traits. We find that Ntp1 amino acids 417-588 are critical for AFP binding and play a role in regulating growth, development, sporulation, sclerotia formation, toxin synthesis, and pathogenicity. Results generated from this study will help to control pathogenic fungi.

Flumequine (Flu) is a fluoroquinolone veterinary antibiotic, which is easy to accumulate in animals, and Flu residues may cause a latent risk to human physiological health. In this study, a high-affinity monoclonal antibody (2.09 × 109 M-1) to specifically recognize Flu (anti-Flu mAb) was prepared, and then the lateral flow immunochromatographic strips (LFIS) with excellent sensitivity and specificity were developed by combining mAbs with nanoparticles. The linear detection ranges of LFIS based on gold particles (AuNP-LFIS) and gold nanoflowers (AuNF-LFIS) were 1.95-250 ng/mL and 0.39-100 ng/mL, respectively, and two types of LFIS showed high sensitivity and accuracy in actual sample detection. It is noteworthy that the AuNF-LFIS exhibited a higher sensitivity compared to that of the AuNP-LFIS. The LFIS developed in this study were considered to have great application potential in monitoring food safety because of its superiorities including simple operation, high sensitivity, prompted speed and good specificity.

Given the high toxicity of ochratoxin A (OTA) and its consequent threat to human health, the development of a portable OTA detection device is of great significance. Enzymatic biofuel cells (EBFCs) have been extensively demonstrated as portable self-powered devices without any external power supply, but self-powered detection of OTA by EBFCs has not been reported. Herein, we report a sensitive self-powered OTA sensor by integrating a target-triggered hybridization chain reaction (HCR) with a glucose/oxygen biofuel cell. OTA binding to a hairpin-locked aptamer opens the hairpin structure, exposing an HCR initiator strand. This initiator triggers HCR amplification, generating long double-stranded DNA products with numerous side strands. Subsequent strand displacement between these HCR side strands and glucose oxidase (GOD)-tagged double-stranded DNA releases GOD-tagged single-stranded DNA probes. These DNA probes hybridize with complementary DNA strands immobilized on the electrode surface, facilitating efficient immobilization of GOD molecules on the anode. With increasing OTA concentration, the GOD loading at the bioanode increases, enabling sensitive self-powered OTA detection. The self-powered sensor achieves a linear detection range from 1 pg mL-1 to 5 ng mL-1 and a detection limit of 0.7 pg mL-1. This study provides a promising portable platform for OTA detection.