Metabolic depletion of sphingolipids inhibits agonist-induced endocytosis of the serotonin1A receptor.

G protein-coupled receptors (GPCRs) are vital cellular signaling machinery and currently represent ~40% drug targets. Endocytosis of GPCRs is an important process that allows stringent spatiotemporal control over receptor population on the cell surface. Although the role of proteins in GPCR endocytosis is well addressed, the contribution of membrane lipids in this process is rather unexplored. Sphingolipids are essential functional lipids in higher eukaryotes and are implicated in several neurological functions. To understand the role of sphingolipids in GPCR endocytosis, we subjected cells expressing human serotonin1A receptors (an important neurotransmitter GPCR involved in cognitive and behavioral functions) to metabolic sphingolipid depletion using fumonisin B1 , an inhibitor of sphingolipid biosynthetic pathway. Our results, using flow cytometric analysis and confocal microscopic imaging, show that sphingolipid depletion inhibits agonist-induced endocytosis of the serotonin1A receptor in a concentration-dependent manner, which was restored when sphingolipid levels were replenished. We further show that there was no change in the internalization of transferrin, a marker for clathrin-mediated endocytosis, under sphingolipid-depleted condition, highlighting the specific requirement of sphingolipids for endocytosis of serotonin1A receptors. Our results reveal the regulatory role of sphingolipids in GPCR endocytosis and highlight the importance of neurotransmitter receptor trafficking in health and disease.

Mild endoplasmic reticulum stress alleviates FB1-triggered intestinal pyroptosis via the Sec62-PERK pathway.

Fumonisin B1 (FB1), a water-soluble mycotoxin released by Fusarium moniliforme Sheld, is widely present in corn and its derivative products, and seriously endangers human life and health. Recent studies have reported that FB1 can lead to pyroptosis, however, the mechanisms by which FB1-induced pyroptosis remain indistinct. In the present study, we aim to investigate the mechanisms of pyroptosis in intestinal porcine epithelial cells (IPEC-J2) and the relationship between FB1-induced endoplasmic reticulum stress (ERS) and pyroptosis. Our experimental results showed that the pyroptosis protein indicators in IPEC-J2 were significantly increased after exposure to FB1. The ERS markers, including glucose-regulated Protein 78 (GRP78), PKR-like ER kinase protein (PERK), and preprotein translocation factor (Sec62) were also significantly increased. Using small interfering RNA silencing of PERK or Sec62, the results demonstrated that upregulation of Sec62 activates the PERK pathway, and activation of the PERK signaling pathway is upstream of FB1-induced pyroptosis. After using the ERS inhibitor 4-PBA reduced the FB1-triggered intestinal injury by the Sec62-PERK pathway. In conclusion, we found that FB1 induced pyroptosis by upregulating Sec62 to activate the PERK pathway, and mild ERS alleviates FB1-triggered damage. It all boils down to one fact, the study provides a new perspective for further, and improving the toxicological mechanism of FB1.

FvKex2 is required for development, virulence, and mycotoxin production in Fusarium verticillioides.

Fusarium verticillioides is one of the most important fungal pathogens causing maize ear and stalk rots, thereby undermining global food security. Infected seeds are usually unhealthy for consumption due to contamination with fumonisin B1 (FB1) mycotoxin produced by the fungus as a virulence factor. Unveiling the molecular factors that determine fungal development and pathogenesis will help in the control and management of the diseases. Kex2 is a kexin-like Golgi-resident proprotein convertase that is involved in the activation of some important proproteins. Herein, we identified and functionally characterized FvKex2 in relation to F. verticillioides development and virulence by bioinformatics and functional genomics approaches. We found that FvKex2 is required for the fungal normal vegetative growth, because the growth of the ∆Fvkex2 mutant was significantly reduced on culture media compared to the wild-type and complemented strains. The mutant also produced very few conidia with morphologically abnormal shapes when compared with those from the wild type. However, the kexin-like protein was dispensable for the male role in sexual reproduction in F. verticillioides. In contrast, pathogenicity was nearly abolished on wounded maize stalks and sugarcane leaves in the absence of FvKEX2 gene, suggesting an essential role of Fvkex2 in the virulence of F. verticillioides. Furthermore, high-performance liquid chromatography analysis revealed that the ∆Fvkex2 mutant produced a significantly lower level of FB1 mycotoxin compared to the wild-type and complemented strains, consistent with the loss of virulence observed in the mutant. Taken together, our results indicate that FvKex2 is critical for vegetative growth, FB1 biosynthesis, and virulence, but dispensable for sexual reproduction in F. verticillioides. The study presents the kexin-like protein as a potential drug target for the management of the devastating maize ear and stalk rot diseases. Further studies should aim at uncovering the link between FvKex2 activity and FB1 biosynthesis genes. KEY POINTS: •The kexin-like protein FvKex2 contributes significantly to the vegetative growth of Fusarium verticillioides. •The conserved protein is required for fungal conidiation and conidial morphology, but dispensable for sexual reproduction. •Deletion of FvKEX2 greatly attenuates the virulence and mycotoxin production potential of F. verticillioides.

Gold nanoparticles-doped MXene heterostructure for ultrasensitive electrochemical detection of fumonisin B1 and ampicillin.

Early transition metal carbides (MXene) hybridized by precious metals open a door for innovative electrochemical biosensing device design. Herein, we present a facile one-pot synthesis of gold nanoparticles (AuNPs)-doped two-dimensional (2D) titanium carbide MXene nanoflakes (Ti3C2Tx/Au). Ti3C2Tx MXene exhibits high electrical conductivity and yields synergistic signal amplification in conjunction with AuNPs leading to excellent electrochemical performance. Thus Ti3C2Tx/Au hybrid nanostructure can be used as an electrode platform for the electrochemical analysis of various targets. We used screen-printed electrodes modified with the Ti3C2Tx/Au electrode and functionalized with different biorecognition elements to detect and quantify an antibiotic, ampicillin (AMP), and a mycotoxin, fumonisin B1 (FB1). The ultralow limits of detection of 2.284 pM and 1.617 pg.mL-1, which we achieved respectively for AMP and FB1 are far lower than their corresponding maximum residue limits of 2.8 nM in milk and 2 to 4 mg kg-1 in corn products for human consumption set by the United States Food and Drug Administration. Additionally, the linear range of detection and quantification of AMP and FB1 were, respectively, 10 pM to 500 nM and 10 pg mL-1 to 1 µg mL-1. The unique structure and excellent electrochemical performance of Ti3C2Tx/Au nanocomposite suggest that it is highly suitable for anchoring biorecognition entities such as antibodies and oligonucleotides for monitoring various deleterious contaminants in agri-food products.

ELOVL6 deficiency aggravates allergic airway inflammation through the ceramide-S1P pathway in mice.

BACKGROUND: Elongation of very-long-chain fatty acids protein 6 (ELOVL6), an enzyme regulating elongation of saturated and monounsaturated fatty acids with C12 to C16 to those with C18, has been recently indicated to affect various immune and inflammatory responses; however, the precise process by which ELOVL6-related lipid dysregulation affects allergic airway inflammation is unclear. OBJECTIVES: This study sought to evaluate the biological roles of ELOVL6 in allergic airway responses and investigate whether regulating lipid composition in the airways could be an alternative treatment for asthma. METHODS: Expressions of ELOVL6 and other isoforms were examined in the airways of patients who are severely asthmatic and in mouse models of asthma. Wild-type and ELOVL6-deficient (Elovl6-/-) mice were analyzed for ovalbumin-induced, and also for house dust mite-induced, allergic airway inflammation by cell biological and biochemical approaches. RESULTS: ELOVL6 expression was downregulated in the bronchial epithelium of patients who are severely asthmatic compared with controls. In asthmatic mice, ELOVL6 deficiency led to enhanced airway inflammation in which lymphocyte egress from lymph nodes was increased, and both type 2 and non-type 2 immune responses were upregulated. Lipidomic profiling revealed that the levels of palmitic acid, ceramides, and sphingosine-1-phosphate were higher in the lungs of ovalbumin-immunized Elovl6-/- mice compared with those of wild-type mice, while the aggravated airway inflammation was ameliorated by treatment with fumonisin B1 or DL-threo-dihydrosphingosine, inhibitors of ceramide synthase and sphingosine kinase, respectively. CONCLUSIONS: This study illustrates a crucial role for ELOVL6 in controlling allergic airway inflammation via regulation of fatty acid composition and ceramide-sphingosine-1-phosphate biosynthesis and indicates that ELOVL6 may be a novel therapeutic target for asthma.

Oral Exposure to Low Concentration of Fumonisin B2, but Not Fumonisin B1, Significantly Exacerbates the Pathophysiology of Imiquimod-Induced Psoriasis in Mice.

This study aimed to determine whether oral fumonisin exposure contributes to the development of psoriasis. Oral administration of fumonisin B1 (FB1, 0.1 mg/kg) or fumonisin B2 (FB2, 0.1 mg/kg) was conducted for 10 days, in addition to the induction of psoriatic symptoms through topical application of 5% imiquimod cream from day 6 to day 10 (5 days) in female BALB/c mice. The results demonstrated that oral administration of FB2 significantly exacerbated psoriatic symptoms, including skin thickness, itching behavior, transepidermal water loss, immune cell infiltration in the dermis, and proinflammatory cytokine production. However, no changes were observed following exposure to FB1. Our results confirm that oral exposure to FB2 adversely affects the pathogenesis of psoriasis by increasing skin thickness and impairing barrier function.

The Protective Effect of Quercetin against the Cytotoxicity Induced by Fumonisin B1 in Sertoli Cells.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium species, is prevalent in crops and animal feed, posing significant health risks to livestock and humans. FB1 induces oxidative stress in Sertoli cells, destroys testicular structure, and affects spermatogenesis. However, methods to mitigate the reproductive toxicity of FB1 in testes remain unknown. Quercetin, a natural flavonoid antioxidant, may offer protective benefits. This study investigated the protective effects and mechanisms of quercetin against FB1-induced reproductive toxicity in TM4 cells (a Sertoli cell line). The results indicated that 40 µM quercetin improved cell viability, reduced apoptosis, and preserved cell functions. Quercetin also decreased reactive oxygen species (ROS) levels in TM4 cells exposed to FB1, enhanced the expression of antioxidant genes, and improved mitochondrial membrane potential. Compared with FB1 alone, the combination of quercetin and FB1 increased ATP levels, as well as pyruvate and lactic acid, the key glycolysis products. Furthermore, this combination elevated the mRNA and protein expression of glycolysis-related genes, including glucose-6-phosphate isomerase 1 (Gpi1), hexokinase 2 (Hk2), aldolase (Aldoa), pyruvate kinase, muscle (Pkm), lactate dehydrogenase A (Ldha) and phosphofructokinase, liver, B-type (Pfkl). Quercetin also boosted the activity of PKM and LDHA, two crucial glycolytic enzymes. In summary, quercetin mitigates FB1-induced toxicity in TM4 cells by reducing ROS levels and enhancing glycolysis. This study offers new insights into preventing and treating FB1-induced toxic damage to the male reproductive system and highlights the potential application of quercetin.

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis.

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.

Preharvest Maize Fungal Microbiome and Mycotoxin Contamination: Case of Zambia's Different Rainfall Patterns.

The preharvest maize mycobiome may be crucial in defining the health of the crop in terms of potential disease burden and mycotoxins. We investigated the preharvest maize mycobiome structure, including the influence of weather patterns, in terms of rainfall intensity, on its composition. In addition, we investigated correlation of genera Fusarium and Aspergillus with maize fumonisin-B1 and aflatoxin. Forty maize fields from selected districts in the wetter northern (N) and drier southern (S) agroecological zones of Zambia were sampled twice over two seasons (1 and 2). The defined weather variables over the two seasons were low rainfall with dry spell (S1), low rainfall (S2), and high rainfall (N1 and N2). High-throughput DNA amplicon sequencing of internal transcribed spacer 1 (ITS1) was used to determine the mycobiome structure and the composition in relation to rainfall patterns. We detected 61 genera, with Fusarium and previously unreported Sarocladium in Zambia to have the highest frequency of detection on the maize. There was a significant difference in fungal genera composition between S1 and S2 but no difference between N1 and N2. The weather pattern with dry spell, S1, had a strong proliferation of Meyerozyma and xerophiles Penicillium, Kodamaea, and Aspergillus. The four genera drove the difference in composition between S1 and S2 and the significantly higher fungal diversity in S1 compared to N2. Of the mycotoxin-important fungi, dry conditions (S1) were a key driver for proliferation of Aspergillus, while Fusarium proliferation occurred irrespective of weather patterns. The relative abundance of Aspergillus and Fusarium resonated with maize aflatoxin and fumonisin-B1 levels, respectively. IMPORTANCE Fungi contaminate various crops worldwide. Maize, an important human staple and livestock cereal, is susceptible to contamination with fungi in the field. Fungi are drivers of plant disease and can compromise yield. Some species of fungi are known to produce chemical compounds (mycotoxins), which are cancer-causing agents in humans and impair livestock productivity. It is important to understand the spectrum of fungi on maize and how weather conditions can impact their abundance. This is because the abundance of fungi in the field can have a bearing on the health of the crop as well as potential for mycotoxins contamination. By understanding the spectrum of the preharvest fungi, it becomes possible to know the key fungi adapted to the maize and subsequently the potential for crop disease as well as mycotoxins contamination. The influence of weather conditions on the spectrum of preharvest fungi on maize has not been fully explored.

Antifungal activity of Lysinibacillus macroides against toxigenic Aspergillus flavus and Fusarium proliferatum and analysis of its mycotoxin minimization potential.

BACKGROUND: Toxigenic fungi (Aspergillus and Fusarium) and their metabolites represent the major cause of corn and corn-based products contamination and consequently lead to severe economic and health issues. AIM: Our current study aimed to investigate the efficacy of using L. macroides Bac6 as a biological control agent against the toxigenic fungi; A. flavus f10 and F. proliferatum f30 and their mycotoxins. RESULTS: The results illustrated that A. flavus f10 produced the aflatoxins AFB1 and AFG2 with concentrations of 21.239 and 13.593 ppb, respectively. While F. proliferatum f30 produced fumonisin B1 (9600 ppb). Furthermore, L. macroides showed a high potential for inhibition of toxigenic fungal growth using a dual culture method. F. proliferatum f30 and A. flavus f10 were found to be inhibited by a percentage of 80 and 62.5%, respectively. The results were confirmed using the scanning electron microscope. The antagonistic bacteria, L. macroides, showed chitinase productivity and activity of 26.45 U/L and 0.12 U/mL/min, respectively, which illustrates its potential application as a biocontrol agent. The GC-MS analysis revealed an abundance of Pyrrolo[1,2-a] pyrazine-1,4-dione, Hexahydro in the bacterial supernatant that exhibited antifungal characteristics. L. macroides had a significant reduction of AFB1 and AFG2 produced by A. flavus f10, recording 99.25% and 99% inhibition, respectively. It also showed strong inhibition of fumonisin B1 (90% inhibition) produced by F. proliferatum f30. CONCLUSION: Thus, the current study is a prospective study evaluating for the first time the potential impact of L. macroides Bac6 against the toxigenic fungi and their toxins.

Graphene oxide-Mn3O4nanocomposites for advanced electrochemical biosensor for fumonisin B1 detection.

Occurrence of mycotoxins in food samples threat to its safety issue due to the presence of high toxicity and carcinogenic behavior, thus requiring highly sensitive and selective detection. Herein, the trimanganese tetraoxide (Mn3O4) nanoparticles in combination with graphene oxide (GO) nanocomposite were used to enhance the electrochemical performance for fabrication of electrochemical biosensor for fumonisin B1 (FB1) detection. The various characterization tools were used to validate the fabrication of GOMn3O4nanocomposites. To fabricate the electrochemical biosensor on an indium tin oxide (ITO) coated glass substrate, a thin film of GOMn3O4nanocomposite was prepared using electrophoretic deposition technique, and antibodies (ab-FB1) were immobilized onto the electrode for selective FB1 detection. The differential pulse voltammetry technique was used to observe the sensing performance. The non-binding sites of the ab-FB1 on the immunoelectrode were blocked with bovine serum albumin (BSA). The biosensor electrode was fabricated as BSA/ab-FB1/GOMn3O4/ITO for the detection of FB1. The sensitivity of the biosensor was obtained as 10.08µA ml ng-1cm-2in the detection range of 1 pg ml-1to 800 ng ml-1with a limit of detection of 0.195 pg ml-1. In addition, the recovery of BSA/ab-FB1/GOMn3O4/ITO immunoelectrodes was also performed on sweet corn samples and is calculated to be 98.91%.

Design of a new electrochemical aptasensor based on screen printed carbon electrode modified with gold nanoparticles for the detection of fumonisin B1 in maize flour.

A new aptasensor for detecting fumonisin B1 (FB1) in the maize samples was developed based on DNA- aptamer recognition and electrochemical technique. A thiol-modified single-stranded DNA (ss-HSDNA) was immobilized on a screen printed carbon electrode (SPCE) electrodeposited by gold nanoparticles (AuNPs). The morphology and structure of SPCE and AuNPs/SPCE were evaluated via scanning electron microscopy (SEM) equipped with energy dispersive spectroscopy (EDS). The SEM results demonstrated that the SPCE had a flat sheet-like structure, and the AuNPs were homogeneously electrodeposited on the SPCE. Cyclic voltammetry (CV) experiments in the [Fe(CN)6]- 3/- 4 solution were conducted to investigate each step of electrode modification as well as aptasensor performance. Aptamer-FB1 interaction prevented the electron transfer permitting the determination of FB1 in the range of 0.5-500 ng/mL with a low detection limit (0.14 ng/mL). The designed aptasensor was also shown high selectivity, acceptable repeatability and reproducibility, good long-term stability, and excellent recovery. Furthermore, there was a strong correlation between the findings achieved via the designed aptasensor and high performance liquid chromatography (HPLC). Therefore, a simple construction process and satisfactory electrochemical performance of the proposed aptasensor have a great potential for the detection of FB1 in maize samples.

Sphingolipid Long-Chain Base Signaling in Compatible and Non-Compatible Plant-Pathogen Interactions in Arabidopsis.

The chemical diversity of sphingolipids in plants allows the assignment of specific roles to special molecular species. These roles include NaCl receptors for glycosylinositolphosphoceramides or second messengers for long-chain bases (LCBs), free or in their acylated forms. Such signaling function has been associated with plant immunity, with an apparent connection to mitogen-activated protein kinase 6 (MPK6) and reactive oxygen species (ROS). This work used in planta assays with mutants and fumonisin B1 (FB1) to generate varying levels of endogenous sphingolipids. This was complemented with in planta pathogenicity tests using virulent and avirulent Pseudomonas syringae strains. Our results indicate that the surge of specific free LCBs and ceramides induced by FB1 or an avirulent strain trigger a biphasic ROS production. The first transient phase is partially produced by NADPH oxidase, and the second is sustained and is related to programmed cell death. MPK6 acts downstream of LCB buildup and upstream of late ROS and is required to selectively inhibit the growth of the avirulent but not the virulent strain. Altogether, these results provide evidence that a LCB- MPK6- ROS signaling pathway contributes differentially to the two forms of immunity described in plants, upregulating the defense scheme of a non-compatible interaction.

Isolation of Arabidopsis extracellular ATP binding proteins by affinity proteomics and identification of PHOSPHOLIPASE C-LIKE 1 as an extracellular protein essential for fumonisin B1 toxicity.

ATP is secreted to the extracellular matrix, where it activates plasma membrane receptors for controlling plant growth and stress-adaptive processes. DOES NOT RESPOND TO NUCLEOTIDES 1 (DORN1), was the first plant ATP receptor to be identified but key downstream proteins remain sought after. Here, we identified 120 proteins secreted by Arabidopsis cell cultures and screened them for putative stress-responsive proteins using ATP-affinity purification. We report three Arabidopsis proteins isolated by ATP-affinity: PEROXIDASE 52, SUBTILASE-LIKE SERINE PROTEASE 1.7 and PHOSPHOLIPASE C-LIKE 1. In wild-type Arabidopsis, the expression of genes encoding all three proteins responded to fumonisin B1, a cell death-activating mycotoxin. The expression of PEROXIDASE 52 and PHOSPHOLIPASE C-LIKE 1 was altered in fumonisin B1-resistant salicylic acid induction-deficient (sid2) mutants. Exposure to fumonisin B1 suppressed PHOSPHOLIPASE C-LIKE 1 expression in sid2 mutants, suggesting that the inactivation of this gene might provide mycotoxin tolerance. Accordingly, gene knockout mutants of PHOSPHOLIPASE C-LIKE 1 were resistant to fumonisin B1-induced death. The activation of PHOSPHOLIPASE C-LIKE 1 gene expression by exogenous ATP was not blocked in dorn1 loss-of-function mutants, indicating that DORN1 is not required. Furthermore, exogenous ATP rescued both the wild type and the dorn1 mutants from fumonisin-B1 toxicity, suggesting that different ATP receptor(s) are operational in this process. Our results point to the existence of additional plant ATP receptor(s) and provide crucial downstream targets for use in designing screens to identify these receptors. Finally, PHOSPHOLIPASE C-LIKE 1 serves as a convergence point for fumonisin B1 and extracellular ATP signalling, and functions in the Arabidopsis stress response to fumonisin B1.

Metabolic Depletion of Sphingolipids Does Not Alter Cell Cycle Progression in Chinese Hamster Ovary Cells.

The cell cycle is a sequential multi-step process essential for growth and proliferation of cells comprising multicellular organisms. Although a number of proteins are known to modulate the cell cycle, the role of lipids in regulation of cell cycle is still emerging. In our previous work, we monitored the role of cholesterol in cell cycle progression in CHO-K1 cells. Since sphingolipids enjoy a functionally synergistic relationship with membrane cholesterol, in this work, we explored whether sphingolipids could modulate the eukaryotic cell cycle using CHO-K1 cells. Sphingolipids are essential components of eukaryotic cell membranes and are involved in a number of important cellular functions. To comprehensively monitor the role of sphingolipids on cell cycle progression, we carried out metabolic depletion of sphingolipids in CHO-K1 cells using inhibitors (fumonisin B1, myriocin, and PDMP) that block specific steps of the sphingolipid biosynthetic pathway and examined their effect on individual cell cycle phases. Our results show that metabolic inhibitors led to significant reduction in specific sphingolipids, yet such inhibition in sphingolipid biosynthesis did not show any effect on cell cycle progression in CHO-K1 cells. We speculate that any role of sphingolipids on cell cycle progression could be context and cell-type dependent, and cancer cells could be a better choice for monitoring such regulation, since sphingolipids are differentially modulated in these cells.

Extracellular ATP targets Arabidopsis RIBONUCLEASE 1 to suppress mycotoxin stress-induced cell death.

Extracellular ATP is a purinergic signal with important functions in regulating plant growth and stress-adaptive responses, including programmed cell death. While signalling events proximate to receptor activation at the plasma membrane have been characterised, downstream protein targets and the mechanism of cell death activation/regulation are unknown. We designed a proteomic screen to identify ATP-responsive proteins in Arabidopsis cell cultures exposed to mycotoxin stress via fumonisin B1 (FB1) application. Arabidopsis RIBONUCLEASE 1 (RNS1) was identified by the screen, and transgenic plants overexpressing native RNS1 showed greater susceptibility to FB1, while a gene knockout rns1 mutant and antisense RNS1 transgenic plants were resistant to FB1-induced cell death. Native RNS1 complemented rns1 mutants and restored the cell death response to FB1, while a catalytically inactive version of the ribonuclease could not. The FB1 resistance of salicylic acid (SA)-depleted nahG-expressing plants was abolished by transformation with native RNS1, but not the catalytically dead version. The mechanism of FB1-induced cell death is activation of RNS1-dependent RNA cleavage, which is blocked by ATP via RNS1 suppression, or enhanced by SA through induction of RNS1 expression. Our study reveals RNS1 as a previously unknown convergence point of ATP and SA signalling in the regulation of stress-induced cell death.

Aptasensors for mycotoxin detection: A review.

Mycotoxins are toxic compounds produced by fungi, which represent a risk to the food and feed supply chain, having an impact on health and economies. A high percentage of feed samples have been reported to be contaminated with more than one type of mycotoxin. Systematic, cost-effective and simple tools for testing are critical to achieve a rapid and accurate screening of food and feed quality. In this review, we describe the various aptamers that have been selected against mycotoxins and their incorporation into optical and electrochemical aptasensors, outlining the strategies exploited, highlighting the advantages and disadvantages of each approach. The review also discusses the different materials used and the immobilization methods employed, with the aim of achieving the highest sensitivity and selectivity.

Distinct Function of Mediator Subunits in Fungal Development, Stress Response, and Secondary Metabolism in Maize Pathogen Fusarium verticillioides.

Mediator is a nucleus-localized, multisubunit protein complex highly conserved across eukaryotes. It interacts with RNA polymerase II transcription machinery as well as various transcription factors to regulate gene expression. However, systematic characterization of the Mediator complex has not been performed in filamentous fungi. In our study, the goal was to investigate key biological functions of Mediator subunits in a mycotoxigenic plant pathogen Fusarium verticillioides. Although there is some level of divergence in the constituent subunits, the overall structure was conserved between Saccharomyces cerevisiae and F. verticillioides. We generated 11 Mediator subunit deletion mutants and characterized vegetative growth, conidia formation, environmental stress response, carbon and fatty acid use, virulence, and fumonisin B1 (FB1) biosynthesis. Each Mediator subunit deletion mutant showed deficiencies in at least three of the phenotypes tested, suggesting that each subunit has different principal functions in F. verticillioides development, metabolism, and virulence. The deletion of FvMed1 led to increased FB1 production, and we confirmed that FvMed1 is transported from the nucleus to the cytoplasm under fumonisin-producing conditions. Taken together, our study characterized various important functional roles for Mediator subunits in F. verticillioides and suggests that select subunits can perform unique cytoplasmic functions independent of the core Mediator in fungal nucleus.

Bacillomycin D-C16 inhibits growth of Fusarium verticillioides and production of fumonisin B1 in maize kernels.

Fusarium verticillioides causes ear and kernel rot in maize and produces mycotoxins, like fumonisin B1 (FB1). Bacillomycin D-C16 is a natural antimicrobial lipopeptide produced by Bacillus subtilis. In this study, the inhibitory effects of Bacillomycin D-C16 on the growth of F. verticillioides and on the production of FB1 in maize were investigated. Bacillomycin D-C16 displayed strong fungicidal activity against F. verticillioides, with a minimum inhibitory concentration (MIC) of 32 g/L. Scanning electron microscopy (SEM) showed that Bacillomycin D-C16 altered the morphology of F. verticillioides mycelia. Bacillomycin D-C16 reduced the ergosterol content, increased the release of nucleic acids and proteins, and increased the levels of reactive oxygen species (ROS) in fungal mycelia. Bacillomycin D-C16 also significantly inhibited the production of FB1 by inhibiting mycelial growth and decreasing the levels of fumonisin biosynthetic genes 1 (fum1), fum6 and fum14. The application of Bacillomycin D-C16 on maize kernels prior to storage inhibited the growth of F. verticillioides and the production of FB1. Our results suggested that Bacillomycin D-C16 has a significant antifungal activity that could be used as a potential natural antimicrobial agent to control food contamination and to ensure food safety.

Determination of Fumonisin B1 by Aptamer-Based Fluorescence Resonance Energy Transfer.

Fumonisin FB is produced by Fusarium moniliforme Sheld, of which FB1 is the most common and the most toxic. The establishment of a rapid detection method is an important means to prevent and control FB1 pollution. A highly sensitive fluorescent sensor based on an aptamer for the rapid detection of fumonisin B1 (FB1) in corn was established. In this study, 5-carboxyfluorescein (FAM) was labeled on the aptamer of FB1 (F10). F10 was adsorbed on the surface of graphene oxide (GO) by π-π stacking. The FAM fluorescence signal could be quenched by fluorescence resonance energy transfer between fluorescent molecules and graphene oxide (GO). In the presence of FB1, the binding efficiency of the aptamer to GO was reduced. Therefore, the content of FB1 in corn samples was determined by fluorescence measurements of mixed FAM-labeled F10, GO and corn samples. This method had a good linear relationship in an FB1 concentration range of 0-3000 ng/mL. The equation was y = 0.2576x + 10.98, R2 = 0.9936. The limit of detection was 14.42 ng/mL, and the limit of quantification was 43.70 ng/mL. The recovery of a spiked standard in the corn sample was 89.13-102.08%, and the time of detection was 30 min.