Toxins

13 pages, 1302 KiB

Open AccessEditor’s ChoiceArticle

Impact of Phytochemicals on Viability and Cereulide Toxin Synthesis in Bacillus cereus Revealed by a Novel High-Throughput Method, Coupling an AlamarBlue-Based Assay with UPLC-MS/MS

by Markus Kranzler, Elrike Frenzel, Veronika Walser, Thomas F. Hofmann, Timo D. Stark and Monika Ehling-Schulz

Toxins 2021, 13(9), 672; https://doi.org/10.3390/toxins13090672 - 21 Sep 2021

Cited by 2 | Viewed by 4013

Abstract

Due to its food-poisoning potential, Bacillus cereus has attracted the attention of the food industry. The cereulide-toxin-producing subgroup is of particular concern, as cereulide toxin is implicated in broadscale food-borne outbreaks and occasionally causes fatalities. The health risks associated with long-term cereulide exposure [...] Read more.

Due to its food-poisoning potential, Bacillus cereus has attracted the attention of the food industry. The cereulide-toxin-producing subgroup is of particular concern, as cereulide toxin is implicated in broadscale food-borne outbreaks and occasionally causes fatalities. The health risks associated with long-term cereulide exposure at low doses remain largely unexplored. Natural substances, such as plant-based secondary metabolites, are widely known for their effective antibacterial potential, which makes them promising as ingredients in food and also as a surrogate for antibiotics. In this work, we tested a range of structurally related phytochemicals, including benzene derivatives, monoterpenes, hydroxycinnamic acid derivatives and vitamins, for their inhibitory effects on the growth of B. cereus and the production of cereulide toxin. For this purpose, we developed a high-throughput, small-scale method which allowed us to analyze B. cereus survival and cereulide production simultaneously in one workflow by coupling an AlamarBlue-based viability assay with ultraperformance liquid chromatography–mass spectrometry (UPLC-MS/MS). This combinatory method allowed us to identify not only phytochemicals with high antibacterial potential, but also ones specifically eradicating cereulide biosynthesis already at very low concentrations, such as gingerol and curcumin. Full article

(This article belongs to the Special Issue Foodborne Intoxications and Toxicoinfections—Major Pathogens and Challenges)

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16 pages, 2067 KiB

Open AccessArticle

Neutralizing Concentrations of Anti-Botulinum Toxin Antibodies Positively Correlate with Mouse Neutralization Assay Results in a Guinea Pig Model

by Milan T. Tomic, Shauna Farr-Jones, Emily S. Syar, Nancy Niemuth, Dean Kobs, Michael J. Hackett, Yero Espinoza, Zacchary Martinez, Khanh Pham, Doris M. Snow, James D. Marks and Ronald R. Cobb

Toxins 2021, 13(9), 671; https://doi.org/10.3390/toxins13090671 - 21 Sep 2021

Cited by 8 | Viewed by 3948

Abstract

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency [...] Read more.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency and safety. A three-mAb combination has been developed that specifically neutralizes BoNT serotype A (BoNT/A), and a separate three mAb combination has been developed that specifically neutralizes BoNT serotype B (BoNT/B). A six mAb cocktail, designated G03-52-01, has been developed that combines the anti-BoNT/A and anti-BoNT/B mAbs. The pharmacokinetics and neutralizing antibody concentration (NAC) of G03-52-01 has been determined in guinea pigs, and these parameters were correlated with protection against an inhalation challenge of BoNT/A1 or BoNT/B1. Previously, it was shown that each antibody demonstrated a dose-dependent mAb serum concentration and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intraperitoneal (IP) injection and that a single IM injection of G03-52-01 administered 48 h pre-exposure protected guinea pigs against an inhalation challenge of up to 93 LD₅₀s of BoNT/A1 and 116 LD₅₀s of BoNT/B1. The data presented here advance our understanding of the relationship of the neutralizing NAC to the measured circulating antibody concentration and provide additional support that a single IM or intravenous (IV) administration of G03-52-01 will provide pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A and BoNT/B. Full article

(This article belongs to the Special Issue Occurrence, Detection and Mitigation of Microbial Toxins)

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Concentration–time curves following IV (left) and IM (right) administration of 1.5 mg total antibody XA-a, XA-b, and XA-c; solid lines represent ELISA-measured concentrations and dashed lines represent predicted concentrations based on noncompartmental analysis of the data through 168 h. Full article ">Figure 2
Concentration–time data for IV-administered XA-a, XA-b, and XA-c. Blue line indicates best line fit, based on a two-compartment model using Phoenix WinNonlin. A single point (t = 2 h) represents the distribution phase for XA-c, which biased the model to a large C0 of 66 μg/mL. Full article ">Figure 3
Concentration–time data for IM-administered XA-a, XA-b, and XA-c. Blue line indicates best fit, based on a one-compartment model using Phoenix WinNonlin. Full article ">Figure 4
Concentration–time curves following IV (left) and IM (right) administration of 1.5 mg total antibody XB-a, XB-b, and XB-c; solid lines represent ECL-measured concentrations. Dashed lines represent predicted concentrations, based on noncompartmental analysis of the data through 336 h (IV) or 168 h (IM). Full article ">Figure 5
Predicted and observed concentration–time data for IV-administered XB-a, XB-b, and XB-c, based on a 2-compartment model fit by Phoenix WinNonlin. Full article ">Figure 6
Predicted and observed concentration–time data for IM-administered XB-a, XB-b, and XB-c, based on a 2-compartment model fit by Phoenix WinNonlin. Full article ">Figure 7
Comparison of anti-BoNT/A and /B mAbs average concentrations measured by ELISA or ECL and MNA. Measurements from animals dosed via IM and IV are combined for this analysis. The line was calculated from regression and included the 0, 0 datapoint. Regression and correlation parameters are shown in <a href="#toxins-13-00671-t005" class="html-table">Table 5</a>. Full article ">Figure 8
Comparison of individual anti-BoNT/A and B mAbs concentrations measured by ELISA or ECL and average (avg.) neutralizing antibody concentration (NAC) from the mouse neutralization assay (MNA). Measurements from animals dosed via IM and IV are combined for this analysis. The line was calculated from regression and included the 0, 0 datapoint. Regression and correlation parameters are shown in <a href="#toxins-13-00671-t005" class="html-table">Table 5</a>. Error bars indicate standard deviation. Linear regression and correlation were calculated with Prism v9.0. 0, 0 data point included. Full article ">

14 pages, 1984 KiB

Open AccessEditor’s ChoiceArticle

Naturally Occurring Fusarium Species and Mycotoxins in Oat Grains from Manitoba, Canada

by M. Nazrul Islam, Mourita Tabassum, Mitali Banik, Fouad Daayf, W. G. Dilantha Fernando, Linda J. Harris, Srinivas Sura and Xiben Wang

Toxins 2021, 13(9), 670; https://doi.org/10.3390/toxins13090670 - 18 Sep 2021

Cited by 15 | Viewed by 5410

Abstract

Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during [...] Read more.

Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during 2016–2018 were analyzed for the occurrence of Fusarium head blight and associated mycotoxins. Through morphological and molecular analysis, F. poae was found to be the predominant Fusarium species affecting oat, followed by F. graminearum, F. sporotrichioides, F. avenaceum, and F. culmorum. Deoxynivalenol (DON) and nivalenol (NIV), type B trichothecenes, were the two most abundant Fusarium mycotoxins detected in oat. Beauvericin (BEA) was also frequently detected, though at lower concentrations. Close clustering of F. poae and NIV/BEA, F. graminearum and DON, and F. sporotrichioides and HT2/T2 (type A trichothecenes) was detected in the principal component analysis. Sampling location and crop rotation significantly impacted the concentrations of Fusarium mycotoxins in oat. A phylogenetic analysis of 95 F. poae strains from Manitoba was conducted using the concatenated nucleotide sequences of Tef-1α, Tri1, and Tri8 genes. The results indicated that all F. poae strains belong to a monophyletic lineage. Four subgroups of F. poae strains were identified; however, no correlations were observed between the grouping of F. poae strains and sample locations/crop rotations. Full article

(This article belongs to the Special Issue Selected Papers from the 15th European Fusarium Seminar)

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PCR-based characterization of Fusarium species complex associated with Fusarium head blight on oat in Manitoba (2016–2018). (A): Frequencies of Fusarium DNA detected in commercial oat fields in Manitoba; (B): distribution of Fusarium chemotypes in Manitoba oat fields. Number of samples (n) = 168. Full article ">Figure 2
Principal component analysis of the relationship between the abundance of Fusarium DNA (FP: F. poae, FG: F. graminearum, and FS: F. sporotrichioides) and mycotoxin concentrations (NIV, BEA, DON, T-2, and HT-2) in oat grains. The percentages of variance explained by axes/components 1 and 2 are shown in parentheses. Each point represents the mean of 3-year samples (n = 168). Full article ">Figure 3
The effect of sampling location and crop rotation on the three-year mean concentration of Fusarium mycotoxins in oat samples from Manitoba. Bars (mean ± standard error) followed by the same letter are not significantly different at p = 0.05; (A) The samples locations were divided into fixed crops districts including CMB (Central Manitoba), SWMB (Southwest Manitoba), EMB (Eastern Manitoba), NVMB (Northwest Manitoba), and INMB (Interlake Manitoba). (B) The effect of previous crop on Fusarium mycotoxin content in Manitoba oat samples, collected in 2016, 2017, and 2018. Full article ">Figure 4
Maximum likelihood phylogenetic tree of 95 F. poae strains collected in Manitoba. The phylogenetic tree was inferred from the concatenated sequences of Tef-1α, Tri1, and Tri8 genes. Relevant bootstrap values (expressed as a percentage of 1000 replicates) are shown at branch points. The concatenated sequence of F. graminearum strain PH1 obtained from GenBank was treated as the outgroup. Full article ">

15 pages, 3856 KiB

Open AccessEditor’s ChoiceArticle

Force Mapping Study of Actinoporin Effect in Membranes Presenting Phase Domains

by Katia Cosentino, Edward Hermann, Nicolai von Kügelgen, Joseph D. Unsay, Uris Ros and Ana J. García-Sáez

Toxins 2021, 13(9), 669; https://doi.org/10.3390/toxins13090669 - 18 Sep 2021

Cited by 4 | Viewed by 2944

Abstract

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on [...] Read more.

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins. Full article

(This article belongs to the Special Issue New Frontiers in Pore-Forming Toxins and Related Proteins: Molecular Mechanisms, Functions and Applications)

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Workflow of the Forcemap Analyser program. Detected force curves obtained by the force mapping tool of the AFM were processed by the Forcemap Analyser program to obtain a spatially reconstructed forcemap of the sample. For details see “Automatic force map analysis” in the Methods and Materials section. Full article ">Figure 2
Automatic force mapping analysis. (A) Representative AFM figure of a DOPC/SM/Chol 2:2:1 supported lipid bilayer (SLB). The brighter round areas represent Lo domains whose thickness exceeds the surrounding Ld phase (darker area). In the Figure, the principle of force mapping is illustrated: the imaged area is divided by a grid in small sub-areas. For each of these sub-areas, following the direction indicated by the green arrow, a force spectroscopy measurement is performed. (B) Representative force spectroscopy curves for Lo (gray) and Ld (red) phases measured in correspondence of the gray and red squares, respectively, in (A). The inset depicts the force and thickness values, as measured for each curve with our automatic force mapping tool. (C) The topographical picture of Figure A could be reproduced nicely by our software by spatially plotting the force values. (D) Dot plot of all corresponding force and distance values, coloured according to their affiliation with one cluster determined by the KMeans algorithm. (E) Map of the same 5 μm × 5 μm area with each spot colored according to its affiliation with one cluster determined by the KMeans algorithm. Full article ">Figure 3
Effect of actinoporins on the topology of Lo domains. (A) Representative image of a DOPC/SM/Chol 2:2:1 membrane system presenting lipid phase separation by confocal microscopy. SM/Chol Lo domains are visualized like black round spots surrounded by the Ld-phase enriched in DOPC, labeled with DiD-C18 (magenta). (B) The same sample as in (A) imaged by AFM. Domains are visualized like brighter spots. The representative cross-section corresponds to the green line in the respective AFM image and shows the height mismatch between the two phases. (C–F) Confocal (C,E) and AFM (D,F) representative images of a DOPC/SM/Chol 2:2:1 system prepared from SUVs pre-incubated with EqtII (C,D) or FraC (E,F). Scale bar is 5 µm (A,C) and 2 µm (E). Images are representative of at least three independent experiments. Full article ">Figure 4
Characterization of Lo domains imaged by confocal microscopy in the presence of EqtII. (A) Confocal images of SLB from SUVs not incubated (left panels) or incubated (right panels) with EqtII. Confocal images have been treated with the particle analysis tool of Imagej that allows the estimation of the perimeter, area, and circularity of the domains. The scale bar is 5 µm. (B) Comparison of the perimeter, area, and circularity of the domains without (white bars) and with (black bars) the protein. Histograms are normalized for better comparison (the number of points varies between 600 and 6500 from at least two independent experiments). Full article ">Figure 5
Characterization of Lo domains in the presence of actinoporins by automatic force mapping analysis. (A) (Left) Representative AFM figure of the DOPC/SM/Chol 2:2:1 membrane system prepared from SUVs, presenting Lo domains (brighter areas) whose thickness exceeds the surrounding Ld phase (darker area). (Centre) Force-map reconstructed image by our software. (Right) Map of the same 5 μm × 5 μm area with each spot colored according to its affiliation with one cluster determined by the KMeans algorithm. (B) Same as in (A) for samples prepared from SUVs pre-incubated with FraC. (C) Same as in the left and central panels of (B) for samples prepared from SUVs pre-incubated with EqtII. All images are representative of at least three independent experiments. (D,E) Comparison of force (D) and thickness (E) values of SLBs without (light and dark yellow bars) and in the presence (light and dark gray bars) of the protein. Histograms are normalized for better comparison (the number of points varied from 220 to 450 from at least two independent experiments). Full article ">Figure 6
Confocal microscopy of DOPC/SM/Chol phases in the presence of FraC-488. (A) The Ld phase was stained with 0.1% DID (magenta) and FraCR126C was labelled with Alexa 488 (green). Picture taken immediately (left), 2 min (centre), and 15 min (right) after the addition of the protein. Scale bar is 5 µm. (B) Photobleaching experiments in the Ld phase without (black squares) and with (purple triangles) FraC. FraC does not alter the fluidity of the Ld phase. Dotted lines are for visual purposes only. Normalized fluorescence intensity over time is represented as the average ± SD over three (SLB) and six (FraC-SLB) repetitions. Full article ">

16 pages, 2350 KiB

Open AccessEditor’s ChoiceArticle

Removal of Aflatoxin B₁ by Edible Mushroom-Forming Fungi and Its Mechanism

by Min-Jung Choo, Sung-Yong Hong, Soo-Hyun Chung and Ae-Son Om

Toxins 2021, 13(9), 668; https://doi.org/10.3390/toxins13090668 - 18 Sep 2021

Cited by 10 | Viewed by 4239

Abstract

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B₁ (AFB [...] Read more.

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B₁ (AFB₁), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB₁ removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB₁ removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB₁ analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB₁ removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24–48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB₁ removal activity by whole cells was mainly due to AFB₁ binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB₁ binding process onto cell wall components. Full article

(This article belongs to the Special Issue Determination and Detoxification Strategies of Mycotoxins)

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Time course of pH and AFB1 removal activity during 5 edible mushroom-forming fungal cultures. Each fungal strain was grown in PDB at 25 °C for 14 days with shaking at 100 rpm. (A) The pH and (B) the levels of AFB1 were measured in triplicate. The values are expressed as the mean ± standard deviation. Different letters at the same culture time point indicate statistically significant differences (p < 0.05). Full article ">Figure 2
AFB1 removal activity by cell-free supernatants and cell lysates from 3 mushroom-forming fungal cultures (B. adusta, A. auricular-judae, and L. edodes). Cell-free supernatants or cell lysates from 3 mushroom-forming fungal cultures, which were spiked with AFB1 (final concentration: 1 μg/mL), were incubated for 1 day at 40 °C with shaking at 100 rpm. The levels of AFB1 were measured in triplicate. The data are expressed as the mean ± standard deviation. Different letters indicate statistically significant differences (p < 0.05). Full article ">Figure 3
Effects of different reaction temperatures on AFB1 removal by cell lysates from the 3 mushroom-forming fungal cultures (B. adusta, A. auricular-judae, and L. edodes). Cell lysates from 3 mushroom-forming fungal cultures, which were spiked with AFB1 (final concentration: 1 μg/mL), were incubated for 5 days at 25, 30, 35, and 40 °C with shaking at 100 rpm. The levels of AFB1 were measured in triplicate. The data are expressed as the mean ± standard deviation. Different letters at the same temperature indicate statistically significant differences (p < 0.05). Full article ">Figure 4
Effects of NaIO4 and NADPH on AFB1 removal by cell lysates from the 3 mushroom-forming fungal cultures (B. adusta, A. auricular-judae, and L. edodes). NaIO4- and NADPH-treated cell lysates from 3 mushroom-forming fungal cultures (final concentration: 3 mM and 0.2 mM, respectively) were incubated for 2 days at 40 °C with shaking at 100 rpm after spiked with AFB1 (final concentration: 1 μg/mL). The levels of AFB1 were measured in triplicate. The data are expressed as the mean ± standard deviation. Different letters indicate statistically significant differences (p < 0.05). Full article ">Figure 5
Effects of heat or proteinase treatment on AFB1 removal by whole cells and cell lysates from B. adusta cultures. (A) Heat-treated whole cells, (B) heat-treated cell lysates, and (C) pronase E-treated cell lysates from B. adusta cultures, which were spiked with AFB1 (final concentration: 1 μg/mL), were incubated for 5 days at 40 °C with shaking at 100 rpm. The levels of AFB1 were measured in triplicate. The data are expressed as the mean ± standard deviation. Different letters indicate statistically significant differences (p < 0.05). Full article ">Figure 5 Cont.
Effects of heat or proteinase treatment on AFB1 removal by whole cells and cell lysates from B. adusta cultures. (A) Heat-treated whole cells, (B) heat-treated cell lysates, and (C) pronase E-treated cell lysates from B. adusta cultures, which were spiked with AFB1 (final concentration: 1 μg/mL), were incubated for 5 days at 40 °C with shaking at 100 rpm. The levels of AFB1 were measured in triplicate. The data are expressed as the mean ± standard deviation. Different letters indicate statistically significant differences (p < 0.05). Full article ">Figure 6
AFB1 binding activity by whole cells, cell lysates, cell debris, and exopolysaccharides from B. adusta cultures. Whole cells, cell lysates, cell debris, and exopolysaccharides from B. adusta cultures, which were spiked with AFB1 (final concentration: 1 μg/mL), were incubated for 5 days at 40 °C with shaking at 100 rpm. The levels of AFB1 were measured in triplicate. The data are expressed as the mean ± standard deviation. Different letters indicate statistically significant differences (p < 0.05). Full article ">

16 pages, 3727 KiB

Open AccessArticle

Impact of Season, Region, and Traditional Agricultural Practices on Aflatoxins and Fumonisins Contamination in the Rice Chain in the Mekong Delta, Vietnam

by Lien Thi Kim Phan, Trang Minh Tran, Marthe De Boevre, Liesbeth Jacxsens, Mia Eeckhout and Sarah De Saeger

Toxins 2021, 13(9), 667; https://doi.org/10.3390/toxins13090667 - 18 Sep 2021

Cited by 11 | Viewed by 4001

Abstract

The current study aimed to evaluate the impact of the crop season, cultivation region, and traditional pre- and post-harvest agricultural practices on mycotoxin contamination in the Mekong Delta rice chain of Vietnam. The results showed that aflatoxins (AFs) and fumonisins (FBs) were predominantly [...] Read more.

The current study aimed to evaluate the impact of the crop season, cultivation region, and traditional pre- and post-harvest agricultural practices on mycotoxin contamination in the Mekong Delta rice chain of Vietnam. The results showed that aflatoxins (AFs) and fumonisins (FBs) were predominantly detected in both paddy (n = 91/184, 50%) and white rice (n = 9/46, 20%). Aflatoxin B1 (AFB1)-contaminated paddy samples (n = 3) exceeded the regulatory threshold (5 µg·kg⁻¹). The contamination of paddy with AFs and FBs was not significantly different by growing seasons and cultivation localities. Evidently, in the winter–spring season, fumonisins frequently occurred in paddy planted in Can Tho, while AFs were found in paddy planted in regions Dong Thap and An Giang, and such toxins were absent in Can Tho. Furthermore, the selection of paddy varieties strongly impacted the occurrence of these toxins, especially AFs, for example, line DT8 and Jasmine were susceptible to AFs and FBs. In addition, poor pre- and post-harvest practices (such as crop residue-free fields, fertilizer application, unsanitary means of transport, delayed drying time) had an impact on the AFs and FBs contamination. Our findings can help to understand the dynamics of AFs and FBs in the rice chain in the Vietnamese Mekong Delta, leading to the mitigation of the contamination of AFs and FBs in rice. Full article

(This article belongs to the Section Mycotoxins)

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22 pages, 25705 KiB

Open AccessEditor’s ChoiceArticle

Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?

by Corentin Fournier, Eva Riehle, Daniel R. Dietrich and David Schleheck

Toxins 2021, 13(9), 666; https://doi.org/10.3390/toxins13090666 - 17 Sep 2021

Cited by 9 | Viewed by 4046

Abstract

Recurring blooms of filamentous, red-pigmented and toxin-producing cyanobacteria Planktothrix rubescens have been reported in numerous deep and stratified prealpine lakes, with the exception of Lake Constance. In a 2019 and 2020 Lake Constance field campaign, we collected samples from a distinct red-pigmented biomass [...] Read more.

Recurring blooms of filamentous, red-pigmented and toxin-producing cyanobacteria Planktothrix rubescens have been reported in numerous deep and stratified prealpine lakes, with the exception of Lake Constance. In a 2019 and 2020 Lake Constance field campaign, we collected samples from a distinct red-pigmented biomass maximum below the chlorophyll-a maximum, which was determined using fluorescence probe measurements at depths between 18 and 20 m. Here, we report the characterization of these deep water red pigment maxima (DRM) as cyanobacterial blooms. Using 16S rRNA gene-amplicon sequencing, we found evidence that the blooms were, indeed, contributed by Planktothrix spp., although phycoerythrin-rich Synechococcus taxa constituted most of the biomass (>96% relative read abundance) of the cyanobacterial DRM community. Through UPLC–MS/MS, we also detected toxic microcystins (MCs) in the DRM in the individual sampling days at concentrations of ≤1.5 ng/L. Subsequently, we reevaluated the fluorescence probe measurements collected over the past decade and found that, in the summer, DRM have been present in Lake Constance, at least since 2009. Our study highlights the need for a continuous monitoring program also targeting the cyanobacterial DRM in Lake Constance, and for future studies on the competition of the different cyanobacterial taxa. Future studies will address the potential community composition changes in response to the climate change driven physiochemical and biological parameters of the lake. Full article

(This article belongs to the Special Issue Cyanotoxins in Bloom: Ever-Increasing Occurrence and Global Distribution of Freshwater Cyanotoxins from Planktic and Benthic Cyanobacteria)

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Representative depth profile recorded with a Moldaenke FluoroProbe multichannel fluorimeter, indicating the high abundance of red-pigmented biomass at a water depth below the chlorophyll-a maximum in Lake Constance on 1 July 2019. (A) Absolute fluorescence intensities recorded at the different excitation wavelengths. (B) Abundance of the different algae classes as attributed by FluoroProbe. Red-pigmented cyanobacteria are attributed to ‘cryptophyta’. In this example, the chlorophyll-a maximum was determined at 8–10 m water depth (<a href="#toxins-13-00666-f001" class="html-fig">Figure 1</a>A, 470 nm), and a second maximum, indicating a red-pigmented biomass, at approximately 18–20 m water depth (<a href="#toxins-13-00666-f001" class="html-fig">Figure 1</a>A, 570 nm; 1B, cryptophyta). Full article ">Figure 2
Krona plot of the DRM cyanobacterial community composition, as determined by 16S rRNA gene-amplicon sequencing across the sampling periods in 2019 (A) and 2020 (B). The community analysis was carried out by filtration of DRM water samples, total DNA extraction of the filters and PCR amplicon sequencing of the cyanobacteria-specific 16S rRNA gene region V3–V4 (380 bp length). For this Krona plot, and as an overview, the results shown are based on all samples combined per year. Taxonomic affiliation was carried out using the Greengenes reference database, and all taxonomic ranks are represented in the plot. Amplicon sequence variants (ASVs), as outputs of the Dada2 software package (see <a href="#sec5-toxins-13-00666" class="html-sec">Section 5</a>), distinguish sequence variations by a single nucleotide, giving ASVs a higher resolution than the operational taxonomic units (OTUs) typically used. Therefore, ASVs were used as the deepest taxonomic rank in our study. Total relative abundance was calculated by dividing the number of reads affiliated to an ASV in a sample by the total number of reads in the sample. Full article ">Figure 3
Illustration of the phylogenetic relationship of the Synechococcus spp. taxa observed in 2019 and 2020. The phylogeny is based on the cyanobacteria-specific V3–V4 (380 bp) 16S rRNA gene region. Colors correspond to the origin of the sequences, with green referring to ASVs observed in 2019 and blue referring to ASVs observed in 2020 (sampling year also in brackets). Sequences from Synechococcus spp. taxa, previously isolated from Lake Constance [<a href="#B45-toxins-13-00666" class="html-bibr">45</a>], and sequences of Synechococcus rubescens strain SAG3.81 and Cyanobium gracile PCC 6307 from NCBI were used as references (indicated in black). IQ-TREE was used for phylogenetic inference using maximum likelihood. The model of nucleotide substitutions used was TPM2+F + I [<a href="#B46-toxins-13-00666" class="html-bibr">46</a>], determined as the best-fit model by ModelFinder [<a href="#B47-toxins-13-00666" class="html-bibr">47</a>], based on the Bayesian information criterion scores and weights. Numbers at the internal nodes represent the percentage support of this specific node in 1000 bootstrap testing. The tree was rooted using the sequences of the cyanobacterium Anacystis nidulans PCC 6301 as an outgroup (not shown). The reference sequences are identified by their accession numbers in brackets. Full article ">Figure 4
Dynamics of relative abundance changes for the Synechococcus ASVs observed during the 2019 and 2020 sampling campaigns. Shown are heatmaps across all sampling dates (A,C) and the ASVs’ average relative abundances (B,D) for each year (bars) with each individual data point indicated (grey dots). For the y-axes of heatmaps (A,C), and average relative abundances (B,D), the different ASVs were ordered from the most abundant (top) to the least abundant (bottom). The heatmap data was log10 transformed from the read count matrix, and, to avoid introducing infinity for zero read counts, we added one artificial read to every cell prior to log10 transformation (log10[x + 1]). Color schemes vary between dark blue for low log10 values to bright orange for high log10 values; a higher log10 value means a higher relative abundance. Please note that the x-axes for B and D do not have the same scale. The average relative abundance across all samples of each year was calculated as it was for the Krona plot (see <a href="#toxins-13-00666-f002" class="html-fig">Figure 2</a>). Standard deviation was also calculated and represented for each ASV; if the graphical representation of the standard deviation was below 0, the minimum error bar value was set to 0. Full article ">Figure 5
Relative abundance of the Planktothrix and Microcystis ASVs observed in 2019 and 2020, and of microcystin concentrations for samples taken in 2019. The bar plots represent the total relative abundance observed for the two Planktothrix ASVs (red bars) detected in 2019 (A) and 2020 (B), for the two Microcystis ASVs (blue bars) detected in 2019, and the single Microcystis ASV detected in 2020. The x-axes represent the different sampling dates and the left y-axis the relative abundance (%). The error bars in B represent the standard deviation of the relative abundance, as calculated from biological triplicates (n = 3); no error bars are represented for 2019, as only one sample was collected per sampling date. The right y-axis represents the toxin concentration (ng/L) determined for independently collected DRM filters, for which the microcystin variant concentrations are indicated by dashed lines in black (MC-LR) and grey (MC-YR) (see main text). The text on top of each bar indicates the results of the PCR amplification of the microcystin synthesis gene mcyE (+, detected; −, not detected) and of the phylogenetic affiliation of the mcyE consensus sequence to either Planktothrix or Microcystis, as established by Sanger sequencing of the PCR amplicons (see main text). Full article ">Figure 6
Depth profiles recorded with the FluoroProbe across the sampling campaigns in 2019 and 2020 (A), in comparison to the year 2016, in which P. rubescens blooms were reported in Lake Constance (B). Depicted are the FluoroProbe profiles for ‘cryptophyta’ abundance (cf. <a href="#toxins-13-00666-f001" class="html-fig">Figure 1</a>) (expressed in µg chlorophyll-a per liter), recorded as proxy of red pigment abundance, in the water column from 0–40 m depth at the routine sampling site ‘Wallhausen’, in the Überlingen embayment of Lake Constance. Prominent DRM are highlighted with dates and water depths. Coordinates of the study site: 47.7571°N 9.1273°E; for an illustration, see <a href="#app1-toxins-13-00666" class="html-app">Figure S4</a>. In 2016, blooms of P. rubescens were reported for Lake Constance in September–October at various sampling sites (i.e., of the German, Austrian and Swiss sections of Lake Constance [<a href="#B42-toxins-13-00666" class="html-bibr">42</a>,<a href="#B43-toxins-13-00666" class="html-bibr">43</a>]). For 2016, FluoroProbe data is available for the Überlingen embayment of Lake Constance (B). Full article ">

14 pages, 862 KiB

Open AccessArticle

Adsorbents Reduce Aflatoxin M₁ Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B₁ in Diet and Its Exposure Risk for Humans

by Manqian Cha, Erdan Wang, Yangyi Hao, Shoukun Ji, Shuai Huang, Lihong Zhao, Wei Wang, Wei Shao, Yajing Wang and Shengli Li

Toxins 2021, 13(9), 665; https://doi.org/10.3390/toxins13090665 - 17 Sep 2021

Cited by 4 | Viewed by 3380

Abstract

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human [...] Read more.

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB₁ and adsorbents (CON), CON with 8 μg/kg AFB₁ (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB₁-challenge phase (day 1 to 14) and an AFB₁-withdraw phase (day 15 to 19). Results showed that both AFB₁ and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM₁ concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB₁ into milk AFM₁ (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM₁ in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB₁ in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM₁ residue in milk and its exposure risk for humans. Full article

(This article belongs to the Special Issue Remediation Strategies for Mycotoxin in Animal Feed)

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19 pages, 4878 KiB

Open AccessArticle

A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen

by Ignacio Valenzuela-Chavira, David O. Corona-Martinez, Karina D. Garcia-Orozco, Melissa Beltran-Torres, Filiberto Sanchez-Lopez, Aldo A. Arvizu-Flores, Rocio Sugich-Miranda, Alonso A. Lopez-Zavala, Ramon E. Robles-Zepeda, Maria A. Islas-Osuna, Adrian Ochoa-Leyva, Michael D. Toney, Hugo Serrano-Posada and Rogerio R. Sotelo-Mundo

Toxins 2021, 13(9), 664; https://doi.org/10.3390/toxins13090664 - 17 Sep 2021

Cited by 2 | Viewed by 4392

Abstract

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen [...] Read more.

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708⁺, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis–Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism. Full article

(This article belongs to the Special Issue Vibrio parahaemolyticus Extracellular Toxins: Potential Virulence Factors and Its Role in Disease)

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17 pages, 1455 KiB

Open AccessArticle

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds

by Paula Tejero, Alberto Martín, Alicia Rodríguez, Ana Isabel Galván, Santiago Ruiz-Moyano and Alejandro Hernández

Toxins 2021, 13(9), 663; https://doi.org/10.3390/toxins13090663 - 17 Sep 2021

Cited by 27 | Viewed by 3813

Abstract

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers’ health. Among the safe [...] Read more.

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers’ health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A. flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation. Full article

(This article belongs to the Special Issue Foodborne Filamentous Fungi and Mycotoxins: Detection and Prevention Research)

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Figure 1
The principal component analysis (PCA) score plots (A,C) and loading plots (B,D) using the first three principal components derived from volatile compounds emitted by A. flavus on different sampling days (indicated by numbers), and their confrontations with H. opuntiae L479 (A,B) and H. uvarum L793 (C,D). Full article ">Figure 2
Evolution of the volatile compound profiles of H. opuntiae L479 (A) and H. uvarum L793 (B) in the presence of A. flavus (AF + L479 and AF + L793) throughout the 21-day incubation period. Full article ">Figure 3
Temporal relative expression of the aflR gene by A. flavus in the absence (AF) and presence of H. opuntiae L479 (AF + L479) and H. uvarum L793 (AF + L793) throughout the 21-day incubation period. Calibrators (samples of each batch incubated at 3 days) always take the value of 0. Full article ">Figure 4
Effect of volatile organic compounds produced by H. opuntiae L479 (AF + L479) and H. uvarum L793 (AF + L793) on the relative expression of the aflR gene in Aspergillus flavus throughout the 21-day incubation period. Calibrators (samples of batch AF at each incubation time) always take the value of 0. Full article ">Figure 5
Temporal aflatoxin B1 (A) and B2 (B) production by A. flavus in the absence (AF) and presence of H. opuntiae L479 (AF + L479) and H. uvarum L793 (AF + L793) throughout the 21-day incubation period. Full article ">

11 pages, 3032 KiB

Open AccessArticle

Thermal Stability and Degradation Kinetics of Patulin in Highly Acidic Conditions: Impact of Cysteine

by Enjie Diao, Kun Ma, Hui Zhang, Peng Xie, Shiquan Qian, Huwei Song, Ruifeng Mao and Liming Zhang

Toxins 2021, 13(9), 662; https://doi.org/10.3390/toxins13090662 - 16 Sep 2021

Cited by 7 | Viewed by 2880

Abstract

The thermal stability and degradation kinetics of patulin (PAT, 10 μmol/L) in pH 3.5 of phosphoric-citric acid buffer solutions in the absence and presence of cysteine (CYS, 30 μmol/L) were investigated at temperatures ranging from 90 to 150 °C. The zero-, first-, and [...] Read more.

The thermal stability and degradation kinetics of patulin (PAT, 10 μmol/L) in pH 3.5 of phosphoric-citric acid buffer solutions in the absence and presence of cysteine (CYS, 30 μmol/L) were investigated at temperatures ranging from 90 to 150 °C. The zero-, first-, and second-order models and the Weibull model were used to fit the degradation process of patulin. Both the first-order kinetic model and Weibull model better described the degradation of patulin in the presence of cysteine while it was complexed to simulate them in the absence of cysteine with various models at different temperatures based on the correlation coefficients (R² > 0.90). At the same reaction time, cysteine and temperature significantly affected the degradation efficiency of patulin in highly acidic conditions (p < 0.01). The rate constants (k_T) for patulin degradation with cysteine (0.0036–0.3200 μg/L·min) were far more than those of treatments without cysteine (0.0012–0.1614 μg/L·min), and the activation energy (E_a = 43.89 kJ/mol) was far less than that of treatment without cysteine (61.74 kJ/mol). Increasing temperature could obviously improve the degradation efficiency of patulin, regardless of the presence of cysteine. Thus, both cysteine and high temperature decreased the stability of patulin in highly acidic conditions and improved its degradation efficiency, which could be applied to guide the detoxification of patulin by cysteine in the juice processing industry. Full article

(This article belongs to the Section Mycotoxins)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Michael addition reaction between patulin (PAT) and cysteine (CYS) suggested by Fliege and Metzler [<a href="#B14-toxins-13-00662" class="html-bibr">14</a>]. Full article ">Figure 2
Residual concentrations (A) and degradation efficiencies (B) of patulin in pH 3.5 of solutions with and without cysteine at different temperatures (90–150 °C) (<a href="#app1-toxins-13-00662" class="html-app">Supplementary Data S1</a>). Full article ">Figure 3
Plots of the zero- (A), first- (B), and second-order (C) kinetic models for patulin degradation in pH 3.5 of solutions without cysteine at different temperatures (90–150 °C) (<a href="#app1-toxins-13-00662" class="html-app">Supplementary Data S1</a>). Full article ">Figure 4
Plots of the zero- (A), first- (B), and second-order (C) kinetic models for patulin degradation in pH 3.5 of solutions with cysteine at different temperatures (90–150 °C) (<a href="#app1-toxins-13-00662" class="html-app">Supplementary Data S1</a>). Full article ">Figure 5
Plots of the Weibull kinetic models for patulin degradation in pH 3.5 of solutions without (A) and with cysteine (B) at different temperatures (90–150 °C) (<a href="#app1-toxins-13-00662" class="html-app">Supplementary Data S1</a>). Full article ">Figure 6
Arrhenius plot for patulin degradation in pH 3.5 of solutions without (▲) and with cysteine (●) at different temperatures (90–150 °C) (<a href="#app1-toxins-13-00662" class="html-app">Supplementary Data S1</a>). Full article ">Figure 7
Suggested degradation mechanisms of patulin by cysteine at high-temperature and high-acid conditions. Full article ">

11 pages, 1833 KiB

Open AccessArticle

Bee Venom Alleviated Edema and Pain in Monosodium Urate Crystals-Induced Gouty Arthritis in Rat by Inhibiting Inflammation

by Bonhyuk Goo, Jeeyoun Lee, Chansol Park, Taeyoung Yune and Yeoncheol Park

Toxins 2021, 13(9), 661; https://doi.org/10.3390/toxins13090661 - 16 Sep 2021

Cited by 13 | Viewed by 5598

Abstract

Bee venom (BV) acupuncture has anti-inflammatory and analgesic effects; therefore, it was used as a traditional Korean medicine for various musculoskeletal disorders, especially arthritis. In this study, we investigated the effect of BV on monosodium urate (MSU) crystal-induced acute gouty rats. An intra-articular [...] Read more.

Bee venom (BV) acupuncture has anti-inflammatory and analgesic effects; therefore, it was used as a traditional Korean medicine for various musculoskeletal disorders, especially arthritis. In this study, we investigated the effect of BV on monosodium urate (MSU) crystal-induced acute gouty rats. An intra-articular injection of MSU crystal suspension (1.25 mg/site) was administered to the tibiotarsal joint of the hind paw of Sprague Dawley rats to induce MSU crystal-induced gouty arthritis. Colchicine (30 mg/kg) was orally administered 1 h before MSU crystal injection as a positive control, and BV (0.5 mg/kg) was injected into the tibiotarsal joint immediately after MSU crystal injection. The ankle thickness, mechanical allodynia, and expression of proinflammatory cytokines (TNF-α, IL-1β, IL6, COX2 and iNOS) and chemokines (MIP-1α, MIP-1β, MCP-1, GRO-α, MIP-2α) were then evaluated. BV reduced the expression of proinflammatory cytokines and chemokines, which are important mediators of MSU crystal-induced inflammatory responses. This anti-inflammatory effect was also confirmed histologically to attenuate synovitis and neutrophil infiltration. We demonstrated that BV markedly ameliorated ankle edema and mechanical allodynia in gouty rats. These results suggest that BV acupuncture is a potential clinical therapy for acute gouty management. Full article

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Figure 1
Effects of bee venom (BV) on monosodium urate (MSU) crystal-induced ankle edema in rats. Left hindpaw of rats were intra-articularly injected into the tibiotarsal joint with MSU crystal (1.25 mg/site). Immediately after MSU crystal injection, 50 μL BV (0.5 mg/kg) was injected in the same way. Colchicine (Col; 30 mg/kg) were orally administered 1 h before MSU crystal injection. (A) Representative photographs of hindpaw. Affected side (arrows). (B) Length of ankle circumference according to time after MSU crystal injection (n = 5/group). Data are presented as means ± SEM. * p < 0.05 vs. MSU group (repeated measures of ANOVA). Full article ">Figure 2
Effects of BV on MSU crystal-induced paw pain in rats. Comparison of paw withdrawal thresholds (PWTs) according to time after MSU crystal injection (n = 5/group). Data are presented as mean ± SEM. * p < 0.05 vs. MSU group (repeated measures of ANOVA). Full article ">Figure 3
Effects of BV on infiltration of neutrophil in gouty rat. (A) Representative hematoxylin and eosin (H&E) stained sections of ankle joint from normal, MSU only, MSU with BV, and MSU with Col treated group. Purple dots represent neutrophils infiltration. Bottom panels are a high magnification of boxed area in the upper panels, which is synovial tissue of ankle joint. Scale bars, 50 μm. (B) Immunohistochemistry for myeloperoxidase (MPO) in ankle joint from normal, MSU only, MSU with BV, and MSU with Col treated group. MPO-positive neutrophils were observed in MSU injected rat ankle joint. Note that both BV and Col treatment decreased neutrophil recruitment. Scale bars, 50 μm. Full article ">Figure 4
Effects of BV on proinflammatory cytokines and chemokines in gouty rat. (A) Reverse transcriptase PCR of proinflammatory cytokines (TNF-α, IL-1β, IL6, COX2 and iNOS) and chemokines (MIP-1α, MIP-1β, MCP-1, GRO-α, MIP-2α) (at 24 h) in normal, vehicle-treated, BV-treated, and Col-treated rats (n = 3/group). (B,C) Quantitative analysis of reverse transcriptase PCR. (D) Western blots of iNOS and COX2 at 24 h in normal, vehicle-treated, BV-treated, and Col-treated rats (n = 3/group). (E) Quantitative analyses of western blots. (F) Immunohistochemistry for iNOS and COX2 in synovium tissue of ankle. Scale bar, 50 μm. Data are presented as means ± SD. * p < 0.05 versus vehicle (one way ANOVA). Full article ">

20 pages, 587 KiB

Open AccessFeature PaperReview

Cyanotoxins and the Nervous System

by James S. Metcalf, Maeve Tischbein, Paul Alan Cox and Elijah W. Stommel

Toxins 2021, 13(9), 660; https://doi.org/10.3390/toxins13090660 - 16 Sep 2021

Cited by 29 | Viewed by 5646

Abstract

Cyanobacteria are capable of producing a wide range of bioactive compounds with many considered to be toxins. Although there are a number of toxicological outcomes with respect to cyanobacterial exposure, this review aims to examine those which affect the central nervous system (CNS) [...] Read more.

Cyanobacteria are capable of producing a wide range of bioactive compounds with many considered to be toxins. Although there are a number of toxicological outcomes with respect to cyanobacterial exposure, this review aims to examine those which affect the central nervous system (CNS) or have neurotoxicological properties. Such exposures can be acute or chronic, and we detail issues concerning CNS entry, detection and remediation. Exposure can occur through a variety of media but, increasingly, exposure through air via inhalation may have greater significance and requires further investigation. Even though cyanobacterial toxins have traditionally been classified based on their primary mode of toxicity, increasing evidence suggests that some also possess neurotoxic properties and include known cyanotoxins and unknown compounds. Furthermore, chronic long-term exposure to these compounds is increasingly being identified as adversely affecting human health. Full article

(This article belongs to the Special Issue Health Risk Assessment Related to Cyanotoxins Exposure)

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15 pages, 2565 KiB

Open AccessArticle

Validation of LC-MS/MS Coupled with a Chiral Column for the Determination of 3- or 15-Acetyl Deoxynivalenol Mycotoxins from Fusarium graminearum in Wheat

by Lan Wang, Zheng Yan, Haiyan Zhou, Yingying Fan, Cheng Wang, Jingbo Zhang, Yucai Liao and Aibo Wu

Toxins 2021, 13(9), 659; https://doi.org/10.3390/toxins13090659 - 16 Sep 2021

Cited by 8 | Viewed by 4012

Abstract

The major causal agents Fusarium graminearum (F. graminearum) and Fusarium asiaticum could produce multiple mycotoxins in infected wheat, which threatens the health of humans and animals. Specifically, deoxynivalenol (DON) and its derivatives 3- and 15-acetyldeoxynivalenol (3-ADON and 15-ADON) are commonly detected [...] Read more.

The major causal agents Fusarium graminearum (F. graminearum) and Fusarium asiaticum could produce multiple mycotoxins in infected wheat, which threatens the health of humans and animals. Specifically, deoxynivalenol (DON) and its derivatives 3- and 15-acetyldeoxynivalenol (3-ADON and 15-ADON) are commonly detected mycotoxins in cereal grains. However, the good chromatographic separation of 3-ADON and 15-ADON remains challenging. Here, an LC-MS/MS method for the chemotype determination of Fusarium strains was developed and validated. 3- and 15-ADON could be separated chromatographically in this study with sufficiently low limits of detection (LODs; 4 μg/kg) and limits of quantification (LOQs; 8 μg/kg). The satisfying intraday and interday reproducibility (both %RSD_r and %RSD_R were <20%) of this method indicated good stability. The recoveries of all analytes were in the range of 80–120%. In addition, three F. graminearum complex (FGC) strains, i.e., PH-1 (chemotype 15-ADON), F-1 (chemotype 3-ADON) and 5035 (chemotype 15-ADON), were selected to verify the accuracy of the method in differentiating phenotypes. The validation results showed that this LC-MS/MS method based on sample pretreatment is effective and suitable for the chromatographic separation of 3-ADON and 15-ADON in wheat. Full article

(This article belongs to the Special Issue Mycotoxins Study: Identification and Control)

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Figure 1
Principle required for the chiral separation of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON). Full article ">Figure 2
LC-MS/MS chromatograms of wheat blank samples spiked with 3-ADON and 15-ADON using different columns: (a) waters UPLC BEH amidel (2.1 mm × 100 mm, 1.7 μm); (b) YMC-TRIART diol-HILIC (3.0 mm × 100 mm, 1.9 μm); (c) YMC CHIRAa ART Cellulose-sc (2.0 mm × 100 mm, 3 μm); (d) YMC CHIRAa ART Cellulose-sc (2.0 mm × 100 mm, 3 μm). Eluent A: 5 mM ammonium acetate; eluent B: methanol. Full article ">Figure 3
LC-MS/MS chromatograms of DON, 3-ADON and 15-ADON in method A (a), method B (b) and method C (c). DON and 3-ADON had a concentration of 8 ng/mL, and 15-ADON had a concentration of 16 ng/mL. Full article ">Figure 4
Comparison of the recoveries of DON (a), 15-ADON (b) and 3-ADON (c) in wheat. The high level was 1000 ng; the intermediate level was 500 ng; and the low level was 50 ng. Full article ">Figure 5
Contamination levels of DON, 3-ADON and 15-ADON determined by three analytical methods: (a) strain 5035 cultured in potato dextrose agar (PDA); (b) strain PH-1 cultured in PDA; (c) strain F-1 cultured in PDA; (d) strain 5035 cultured in autoclaved wheat media; (e) strain PH-1 cultured in autoclaved wheat media; (f) strain F-1 cultured in autoclaved wheat media (μg/kg). Full article ">

14 pages, 1866 KiB

Open AccessArticle

Cadherin Protein Is Involved in the Action of Bacillus thuringiensis Cry1Ac Toxin in Ostrinia furnacalis

by Wenzhong Jin, Yuqian Zhai, Yihua Yang, Yidong Wu and Xingliang Wang

Toxins 2021, 13(9), 658; https://doi.org/10.3390/toxins13090658 - 15 Sep 2021

Cited by 13 | Viewed by 2978

Abstract

Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with [...] Read more.

Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with Cry1Ac resistance in several lepidoptera species, including the Asian corn borer, Ostrinia furnacalis, a major pest of maize in Asian–Western Pacific countries. However, the causality of O. furnacalis cadherin (OfCad) with Cry1Ac resistance remains to be clarified. In this study, in vitro and in vivo approaches were employed to examine the involvement of OfCad in mediating Cry1Ac toxicity. Sf9 cells transfected with OfCad showed significant immunofluorescent binding with Cry1Ac toxin and exhibited a concentration-dependent mortality effect when exposed to Cry1Ac. The OfCad knockout strain OfCad-KO, bearing homozygous 15.4 kb deletion of the OfCad gene generated by CRISPR/Cas9 mutagenesis, exhibited moderate-level resistance to Cry1Ac (14-fold) and low-level resistance to Cry1Aa (4.6-fold), but no significant changes in susceptibility to Cry1Ab and Cry1Fa, compared with the original NJ-S strain. The Cry1Ac resistance phenotype was inherited as autosomal, recessive mode, and significantly linked with the OfCad knockout in the OfCad-KO strain. These results demonstrate that the OfCad protein is a functional receptor for Cry1Ac, and disruption of OfCad confers a moderate Cry1Ac resistance in O. furnacalis. This study provides new insights into the mode of action of the Cry1Ac toxin and useful information for designing resistance monitoring and management strategies for O. furnacalis. Full article

(This article belongs to the Special Issue Insect Resistance to Bacillus thuringiensis Toxins)

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Figure 1
Immunolocalization of cadherin and Cry1Ac in non-transfected Sf9 cells (Sf9), Sf9 cells transfected by empty bacmid (EB) or by recombinant OfCad. (A) Immunofluorescence image of OfCad. Fixed Sf9 cells expressing OfCad proteins were probed with anti-His TagMouse monoclonal antibody, and fluorescence was detected by goat anti-mouse antiserum labeled with Alexa Fluor 594 (red). Cell nuclei were stained with DAPI (blue). (B) Immunofluorescence localization of Cry1Ac. The fixed Sf9 cells expressing OfCad proteins were incubated with Cry1Ac (200 nM) and then incubated with rabbit polyclonal anti-Cry1Ac antiserum. Immunofluorescence was detected by goat anti-rabbit antiserum labeled with Alexa Fluor 594 (red). Cells were examined using a Zeiss Laser scanning confocal microscope with 20 objective lens. Scale bars: 100 μm. Full article ">Figure 2
Cytotoxicity of Cry1Ac on non-transfected Sf9 cells (Sf9), Sf9 cells transfected by empty bacmid (EB) or by recombinant OfCad. (A) Imaging of Sf9 cells incubated with activated Cry1Ac (1 μM) or without toxin (Control). The cell lines were treated at 27 °C for 1 h, then stained with 0.4% trypan blue. The nuclei of cells that were live and therefore unstained at time of fixation are light colored, whereas dead or damaged cells dyed blue by the trypan vital stain are colored. A BX60 Olympus light microscope was used to examine cells. Scale bars: 20 μm. (B) Mortality of Sf9 cells exposed to several concentrations of Cry1Ac. Mortality is shown as the mean ± se of three repeated experiments, and a concentration–effect curve was visualized by GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Full article ">Figure 3
CRISPR/Cas9 mediated target mutagenesis of OfCad gene. (A) Diagram of OfCad gene structure, sgRNA targeting site and position of three pairs of primers for allele-specific PCR application. White boxes represent exons, and two sgRNA targeting sites were located at exon 4 and exon 35, respectively. (B) Crossing design for establishing homozygous OfCad gene knockout strain OfCad-KO. (C) Genotyping of individual O. furnacalis sample based on electrophoresis image pattern of allele-specific PCR products. ss, rs and rr represent the banding pattern of wild-type, heterozygous mutant and homozygous mutant, respectively. (D) Partial sequences of wild-type and manipulated mutant OfCad gene. The proto spacer sequence and protospacer adjacent motif (PAM) are shown in blue and red, respectively. Dual sgRNAs introduced 15.4 kb deletion OfCad in the OfCad-KO strain, which was confirmed by directly sequencing PCR products flanking the sgRNAs target regions, as shown in the chromatogram. Full article ">Figure 4
The schematic diagram depicts the mating process for linkage analysis between OfCad knockout and Cry1Ac resistance in the O. furnacalis OfCad-KO strain. rr, ss and rs represent samples bearing homozygous mutant, wild-type and heterozygous mutant OfCad allele, respectively. Full article ">

11 pages, 1139 KiB

Open AccessArticle

Identification of Fish Species and Toxins Implicated in a Snapper Food Poisoning Event in Sabah, Malaysia, 2017

by Ha Viet Dao, Aya Uesugi, Hajime Uchida, Ryuichi Watanabe, Ryoji Matsushima, Zhen Fei Lim, Steffiana J. Jipanin, Ky Xuan Pham, Minh-Thu Phan, Chui Pin Leaw, Po Teen Lim and Toshiyuki Suzuki

Toxins 2021, 13(9), 657; https://doi.org/10.3390/toxins13090657 - 15 Sep 2021

Cited by 6 | Viewed by 3735

Abstract

In the coastal countries of Southeast Asia, fish is a staple diet and certain fish species are food delicacies to local populations or commercially important to individual communities. Although there have been several suspected cases of ciguatera fish poisoning (CFP) in Southeast Asian [...] Read more.

In the coastal countries of Southeast Asia, fish is a staple diet and certain fish species are food delicacies to local populations or commercially important to individual communities. Although there have been several suspected cases of ciguatera fish poisoning (CFP) in Southeast Asian countries, few have been confirmed by ciguatoxins identification, resulting in limited information for the correct diagnosis of this food-borne disease. In the present study, ciguatoxin-1B (CTX-1B) in red snapper (Lutjanus bohar) implicated in a CFP case in Sabah, Malaysia, in December 2017 was determined by single-quadrupole selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS). Continuous consumption of the toxic fish likely resulted in CFP, even when the toxin concentration in the fish consumed was low. The identification of the fish species was performed using the molecular characterization of the mitochondrial cytochrome c oxidase subunit I gene marker, with a phylogenetic analysis of the genus Lutjanus. This is the first report identifying the causative toxin in fish-implicated CFP in Malaysia. Full article

(This article belongs to the Special Issue Monitoring of Marine Biotoxins)

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12 pages, 1980 KiB

Open AccessArticle

Production of Alternaria Toxins in Yellow Peach (Amygdalus persica) upon Artificial Inoculation with Alternaria alternate

by Jiajia Meng, Wenbo Guo, Zhihui Zhao, Zhiqi Zhang, Dongxia Nie, Emmanuel K. Tangni and Zheng Han

Toxins 2021, 13(9), 656; https://doi.org/10.3390/toxins13090656 - 15 Sep 2021

Cited by 12 | Viewed by 3465

Abstract

The yellow peach (Amygdalus persica), an important fruit in China, is highly susceptible to infection by Alternaria sp., leading to potential health risks and economic losses. In the current study, firstly, yellow peaches were artificially inoculated with Alternariaalternate. [...] Read more.

The yellow peach (Amygdalus persica), an important fruit in China, is highly susceptible to infection by Alternaria sp., leading to potential health risks and economic losses. In the current study, firstly, yellow peaches were artificially inoculated with Alternariaalternate. Then, the fruits were stored at 4 °C and 28 °C to simulate the current storage conditions that consumers use, and the Alternaria toxins (ATs) contents from different parts of the fruits were analyzed via ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The results showed that the growth of A. alternate and the ATs production were dramatically affected by the storage temperature. At 28 °C, the fungi grew rapidly and the lesion diameter reached about 4.0 cm within 15 days of inoculation, while, at 4 °C, the fungal growth was noticeably inhibited, with no significant change in the lesion diameter. To our surprise, high contents of ATs were produced under both storage conditions even though the fungal growth was suppressed. With an increase in the incubation time, the amounts of ATs showed a steady tendency to increase in most cases. Remarkably, alternariol monomethyl ether (AME), alternariol (AOH), and tenuazonic acid (TeA) were detected in the rotten tissue and also in the surrounding tissue, while a large amount of TeA could also be found in the healthy tissue. To the best of our knowledge, this is the first report regarding the production of ATs by the infection of Alternaria sp. in yellow peach fruits via artificial inoculation under regulated conditions, and, based on the evidence herein, it is recommended that ATs be included in monitoring and control programs of yellow peach management and food safety administration. Full article

(This article belongs to the Special Issue Mycotoxins, Food Safety and Metrology)

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Figure 1
Chemical structures of alternariol monomethyl ether, AME (A); alternariol, AOH (B); and tenuazonic acid, TeA (C). Full article ">Figure 2
The symptoms of yellow peaches stored at 28 °C (A) and 4 °C (B) upon inoculation with A. alternate, respectively. Full article ">Figure 3
The lesion diameters on the surface of yellow peaches inoculated with A. alternate and stored at 28 °C and 4 °C, respectively. Full article ">Figure 4
Contents of AME (A), AOH (B), and TeA (C) in yellow peaches stored at 28 °C upon inoculation with A. alternate. Note: For each toxin in the same part of the yellow peach, the small letters of a, b, c and d and their combinations were utilized to investigate the differences in the toxin contents with different incubation times. Different letters indicate the significance at p ≤ 0.05, while combinations and individual letters—e.g., ab and a—indicate no significant differences. Hollow squares indicate that the amount of ATs was lower than the LOQ. Full article ">Figure 5
Contents of AME (A), AOH (B), and TeA (C) in yellow peaches stored at 4 °C upon inoculation with A. alternate. Note: For each toxin in the same part of the yellow peach, the small letters of a, b, c, d, and e and their combinations were utilized to investigate the differences in the toxin contents with different incubation times. Different letters indicate the significance at p ≤ 0.05, while combinations and individual letters—e.g., ab and a—indicate no significant difference. Hollow squares indicate that the amount of the ATs was lower than the LOQ. Full article ">Figure 6
Schematic illustration of different parts of yellow peaches for Alternaria toxins (ATs) analysis. Full article ">

11 pages, 628 KiB

Open AccessEditor’s ChoiceReview

Wickerhamomyces Yeast Killer Toxins’ Medical Applications

by Laura Giovati, Tecla Ciociola, Tiziano De Simone, Stefania Conti and Walter Magliani

Toxins 2021, 13(9), 655; https://doi.org/10.3390/toxins13090655 - 15 Sep 2021

Cited by 13 | Viewed by 3563

Abstract

Possible implications and applications of the yeast killer phenomenon in the fight against infectious diseases are reviewed, with particular reference to some wide-spectrum killer toxins (KTs) produced by Wickerhamomyces anomalus and other related species. A perspective on the applications of these KTs in [...] Read more.

Possible implications and applications of the yeast killer phenomenon in the fight against infectious diseases are reviewed, with particular reference to some wide-spectrum killer toxins (KTs) produced by Wickerhamomyces anomalus and other related species. A perspective on the applications of these KTs in the medical field is provided considering (1) a direct use of killer strains, in particular in the symbiotic control of arthropod-borne diseases; (2) a direct use of KTs as experimental therapeutic agents; (3) the production, through the idiotypic network, of immunological derivatives of KTs and their use as potential anti-infective therapeutics. Studies on immunological derivatives of KTs in the context of vaccine development are also described. Full article

(This article belongs to the Special Issue Yeast Killer Toxin)

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19 pages, 5826 KiB

Open AccessArticle

Proteomic Identification and Quantification of Snake Venom Biomarkers in Venom and Plasma Extracellular Vesicles

by Nicholas Kevin Willard, Emelyn Salazar, Fabiola Alejandra Oyervides, Cierra Siobhrie Wiebe, Jack Sutton Ocheltree, Mario Cortez, Ricardo Pedro Perez, Harry Markowitz, Anton Iliuk, Elda Eliza Sanchez, Montamas Suntravat and Jacob Anthony Galan

Toxins 2021, 13(9), 654; https://doi.org/10.3390/toxins13090654 - 15 Sep 2021

Cited by 13 | Viewed by 4778

Abstract

The global exploration of snakebites requires the use of quantitative omics approaches to characterize snake venom as it enters into the systemic circulation. These omics approaches give insights into the venom proteome, but a further exploration is warranted to analyze the venom-reactome for [...] Read more.

The global exploration of snakebites requires the use of quantitative omics approaches to characterize snake venom as it enters into the systemic circulation. These omics approaches give insights into the venom proteome, but a further exploration is warranted to analyze the venom-reactome for the identification of snake venom biomarkers. The recent discovery of extracellular vesicles (EVs), and their critical cellular functions, has presented them as intriguing sources for biomarker discovery and disease diagnosis. Herein, we purified EV’s from the snake venom (svEVs) of Crotalus atrox and C. oreganus helleri, and from plasma of BALB/c mice injected with venom from each snake using EVtrap in conjunction with quantitative mass spectrometry for the proteomic identification and quantification of svEVs and plasma biomarkers. Snake venom EVs from C. atrox and C. o. helleri were highly enriched in 5′ nucleosidase, L-amino acid oxidase, and metalloproteinases. In mouse plasma EVs, a bioinformatic analysis for revealed upregulated responses involved with cytochrome P450, lipid metabolism, acute phase inflammation immune, and heat shock responses, while downregulated proteins were associated with mitochondrial electron transport, NADH, TCA, cortical cytoskeleton, reticulum stress, and oxidative reduction. Altogether, this analysis will provide direct evidence for svEVs composition and observation of the physiological changes of an envenomated organism. Full article

(This article belongs to the Section Animal Venoms)

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15 pages, 766 KiB

Open AccessArticle

Influence of H₂O₂-Induced Oxidative Stress on In Vitro Growth and Moniliformin and Fumonisins Accumulation by Fusarium proliferatum and Fusarium subglutinans

by Davide Ferrigo, Valentina Scarpino, Francesca Vanara, Roberto Causin, Alessandro Raiola and Massimo Blandino

Toxins 2021, 13(9), 653; https://doi.org/10.3390/toxins13090653 - 15 Sep 2021

Cited by 5 | Viewed by 3131

Abstract

Fusarium proliferatum and Fusarium subglutinans are common pathogens of maize which are known to produce mycotoxins, including moniliformin (MON) and fumonisins (FBs). Fungal secondary metabolism and response to oxidative stress are interlaced, where hydrogen peroxide (H₂O₂) plays a pivotal [...] Read more.

Fusarium proliferatum and Fusarium subglutinans are common pathogens of maize which are known to produce mycotoxins, including moniliformin (MON) and fumonisins (FBs). Fungal secondary metabolism and response to oxidative stress are interlaced, where hydrogen peroxide (H₂O₂) plays a pivotal role in the modulation of mycotoxin production. The objective of this study is to examine the effect of H₂O₂-induced oxidative stress on fungal growth, as well as MON and FBs production, in different isolates of these fungi. When these isolates were cultured in the presence of 1, 2, 5, and 10 mM H₂O₂, the fungal biomass of F. subglutinans isolates showed a strong sensitivity to increasing oxidative conditions (27–58% reduction), whereas F. proliferatum isolates were not affected or even slightly improved (45% increase). H₂O₂ treatment at the lower concentration of 1 mM caused an almost total disappearance of MON and a strong reduction of FBs content in the two fungal species and isolates tested. The catalase activity, surveyed due to its crucial role as an H₂O₂ scavenger, showed no significant changes at 1 mM H₂O₂ treatment, thus indicating a lack of correlation with MON and FB changes. H₂O₂ treatment was also able to reduce MON and FB content in certified maize material, and the same behavior was observed in the presence and absence of these fungi, highlighting a direct effect of H₂O₂ on the stability of these mycotoxins. Taken together, these data provide insights into the role of H₂O₂ which, when increased under stress conditions, could affect the vegetative response and mycotoxin production (and degradation) of these fungi. Full article

(This article belongs to the Special Issue Environmental Stress on the Production of Mycotoxins)

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24 pages, 14490 KiB

Open AccessArticle

Ageratina adenophora Disrupts the Intestinal Structure and Immune Barrier Integrity in Rats

by Yujing Cui, Samuel Kumi Okyere, Pei Gao, Juan Wen, Suizhong Cao, Ya Wang, Junliang Deng and Yanchun Hu

Toxins 2021, 13(9), 651; https://doi.org/10.3390/toxins13090651 - 15 Sep 2021

Cited by 21 | Viewed by 3288

Abstract

The aim of this study was to investigate the effects of Ageratina adenophora on the intestines morphology and integrity in rat. Rats were randomly divided into two groups and were fed with 10 g/100 g body weight (BW) basal diet and 10 g/100 [...] Read more.

The aim of this study was to investigate the effects of Ageratina adenophora on the intestines morphology and integrity in rat. Rats were randomly divided into two groups and were fed with 10 g/100 g body weight (BW) basal diet and 10 g/100 g BW experimental diet, which was a mixture of A. adenophora powder and basal diet in a 3:7 ratio. The feeding experiment lasted for 60 days. At days 28 and 60 of the experiment, eight rats/group/timepoint were randomly selected, weighed, and sacrificed, then blood and intestinal tissues were collected and stored for further analysis. The results showed that Ageratina adenophora caused pathological changes and injury in the intestine, elevated serum diamine oxidase (DAO), D-lactate (D-LA), and secretory immunoglobulin A (sIgA) levels, reduced occludin levels in intestinal tissues, as well as increased the count of intraepithelial leukocytes (IELs) and lamina propria leukocytes (LPLs) in the intestine (p < 0.05 or p < 0.01). In addition, the mRNA and protein (ELISA) expressions of pro-inflammation cytokines (IL-1β, IL-2, TNF-α, and IFN-ϒ) were elevated in the Ageratina adenophora treatment groups, whereas anti-inflammatory cytokines such as IL-4 and IL-10 were reduced (p < 0.01 or p < 0.05). Therefore, the results obtained in this study indicated that Ageratina adenophora impaired intestinal function in rats by damaging the intestine structure and integrity, and also triggered an inflammation immune response that led to intestinal immune barrier dysfunction. Full article

(This article belongs to the Collection Toxic and Pharmacological Effect of Plant Toxins)

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16 pages, 2468 KiB

Open AccessEditor’s ChoiceArticle

Validation and Application of a Low-Cost Sorting Device for Fumonisin Reduction in Maize

by William Stafstrom, Julie Wushensky, John Fuchs, Wenwei Xu, Nnenna Ezera and Rebecca J. Nelson

Toxins 2021, 13(9), 652; https://doi.org/10.3390/toxins13090652 - 14 Sep 2021

Cited by 7 | Viewed by 2882

Abstract

Fumonisin mycotoxins are a persistent challenge to human and livestock health in tropical and sub-tropical maize cropping systems, and more efficient methods are needed to reduce their presence in food systems. We constructed a novel, low-cost device for sorting grain, the “DropSort”, and [...] Read more.

Fumonisin mycotoxins are a persistent challenge to human and livestock health in tropical and sub-tropical maize cropping systems, and more efficient methods are needed to reduce their presence in food systems. We constructed a novel, low-cost device for sorting grain, the “DropSort”, and tested its effectiveness on both plastic kernel models and fumonisin-contaminated maize. Sorting plastic kernels of known size and shape enabled us to optimize the sorting performance of the DropSort. The device sorted maize into three distinct fractions as measured by bulk density and 100-kernel weight. The level of fumonisin was lower in the heaviest fractions of maize compared to the unsorted samples. Based on correlations among fumonisin and bulk characteristics of each fraction, we found that light fraction 100-kernel weight could be an inexpensive proxy for unsorted fumonisin concentration. Single kernel analysis revealed significant relationships among kernel fumonisin content and physical characteristics that could prove useful for future sorting efforts. The availability of a low-cost device (materials~USD 300) that can be used to reduce fumonisin in maize could improve food safety in resource-limited contexts in which fumonisin contamination remains a pressing challenge. Full article

(This article belongs to the Section Mycotoxins)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
DropSort rejection rates (three trials) of six plastic kernel model sets at Setting 15 and across three passes of re-sorting the accepted fraction. Within each pass, kernel models’ mean rejection rates were significantly different (1-way ANOVAs: Pass 1 p-value = 9.40 × 10−9, Pass 2 p-value = 1.86 × 10−12, Pass 3 p-value = 1.16 × 10−13). Within each pass, post hoc pairwise t-tests were performed between each kernel model set, and different letters represent significant differences between kernel model groups (p-value < 0.05). Full article ">Figure 2
Differences in rejection rate at Pass 3 and Pass 1 (Delta Rejection Rate (DRR)) for each kernel model group. Group means differed significantly (1-way ANOVA, p-value = 3.7 × 10−7). Post hoc pairwise t-tests were performed between them, and different letters represent significant differences between kernel model groups; the three heaviest/densest kernel models had a lower DRR (p-value < 0.05). Full article ">Figure 3
Cartoon depictions of the six plastic kernel model sets labeled with their respective densities. An arbitrary classification cutoff was applied to further explore sorting performance of the DropSort. The three heaviest/densest models (≥0.96 mg/mm3) were classified as the “to accept” class and the lightest/least dense three kernel models (≤0.84 mg/mm3) were classified as the “to reject” class. Full article ">Figure 4
Specificity and sensitivity rates of the DropSort’s three trials of sorting plastic kernel models at different settings and over three passes. Within each pass, mean sensitivity and specificity rates varied significantly across settings (1-way ANOVAs: sensitivity Pass 1 p-value = 6.30 × 10−11, sensitivity Pass 2 p-value = 1.31 × 10−10, sensitivity Pass 3 p-value = 3.22 × 10−12, specificity Pass 1 p-value = 9.06 × 10−6, specificity Pass 2 p-value = 6.57 × 10−7, specificity Pass 3 p-value = 9.65 × 10−8). Post hoc pairwise t-tests were performed between each group, and different letters (red letters for sensitivity, black letters for specificity) represent significant pairwise differences between settings (p-value < 0.05). Full article ">Figure 5
Differences in sensitivity (Delta Sensitivity) and specificity (Delta Specificity) between Pass 3 and Pass 1 at Settings 13–17. The three heaviest/densest classes of plastic kernel models were classified as “to accept” and the three lightest/least dense classes of plastic kernel models as “to reject”. Across settings, mean differences were significantly different for Delta Specificity (1-way ANOVA, p-value = 5.81 × 10−7), but not for Delta Sensitivity (1-way ANOVA, p-value = 0.98). This suggests that specificity can be improved without a loss of sensitivity by using multiple passes and a higher setting. Different black letters represent significant pairwise differences in Delta Specificity between settings (p-value < 0.05). Full article ">Figure 6
Mean bulk trait values of the unsorted, heavy fraction (HF), medium fraction (MF), and light fraction (LF). (a) Mean 100-kernel weight differed significantly among fractions (1-way ANOVA, p-value = 8.01 × 10−13). LF 100-kernel weight was significantly lower than all other fractions, and MF 100-kernel weight was significantly lower than HF (different letters represent pairwise t-test p-value < 0.05). (b) Bulk density varied significantly among fractions (1-way ANOVA, p-value = 2.20 × 10−16), and LF bulk density was significantly lower than all other fractions (different letters represent pairwise t-test p-value < 0.05). (c) Log10-transformed fumonisin differed significantly among fractions (1-way ANOVA, p-value = 4.63 × 10−13). Dashed line indicates 4 ppm regulatory limit. Compared to unsorted, HF was significantly lower, MF no difference, and LF significantly higher (different letters represent pairwise t-test p-value < 0.05). Full article ">Figure 7
Mass, volume, density and log10-transformed fumonisin distributions for 72 individual maize kernels from the “accepted” heavy fraction (HF; light blue) and 72 kernels from the “rejected” light fraction (LF; orange). Dashed lines indicate group means, and asterisks represent a significant difference between accepted and rejected kernels. (a) Mean HF kernel mass was significantly higher than that of LF kernels (two-sided unpaired t-test, p-value = 2.9 × 10−4). (b) Mean HF kernel volume was significantly higher than that of LF kernels (two-sided unpaired t-test, p-value = 2.2 × 10−3). (c) There was no significant difference in mean density between HF and LF kernels (two-sided unpaired t-test, p-value = 0.33). (d) Mean log10-transformed fumonisin was significantly greater in LF kernels compared to HF kernels (two-sided unpaired t-test, p-value = 2.4 × 10−5), and solid red line indicates 4 ppm regulatory limit. Full article ">Figure 8
Counts of different kernel symptom types in heavy fraction (HF; n = 72) and light fraction (LF; n = 72). Fusarium kernel rot symptom scoring was modified from Morales et al. (2019), and kernels were scored as 1 = asymptomatic, 2 = starburst, 3 = purple, and 4 = mummified. The proportions of symptom types did not differ between HF and LF (chi-squared test, p-value = 0.90). Full article ">Figure 9
Spearman rank correlations among five single kernel traits (mass, volume, density, visually scored symptoms, and log10-transformed fumonisin) for 72 heavy fraction kernels (a) and 72 light fraction kernels (b) (overlaid X indicates p-value ≥ 0.05). Full article ">

19 pages, 2736 KiB

Open AccessArticle

Toward Isolation of Palytoxins: Liquid Chromatography Coupled to Low- or High-Resolution Mass Spectrometry for the Study on the Impact of Drying Techniques, Solvents and Materials

by Antonia Mazzeo, Michela Varra, Luciana Tartaglione, Patrizia Ciminiello, Zita Zendong, Philipp Hess and Carmela Dell’Aversano

Toxins 2021, 13(9), 650; https://doi.org/10.3390/toxins13090650 - 14 Sep 2021

Cited by 4 | Viewed by 2607

Abstract

Palytoxin (PLTX) and its congeners are emerging toxins held responsible for a number of human poisonings following the inhalation of toxic aerosols, skin contact, or the ingestion of contaminated seafood. Despite the strong structural analogies, the relative toxic potencies of PLTX congeners are [...] Read more.

Palytoxin (PLTX) and its congeners are emerging toxins held responsible for a number of human poisonings following the inhalation of toxic aerosols, skin contact, or the ingestion of contaminated seafood. Despite the strong structural analogies, the relative toxic potencies of PLTX congeners are quite different, making it necessary to isolate them individually in sufficient amounts for toxicological and analytical purposes. Previous studies showed poor PLTX recoveries with a dramatic decrease in PLTX yield throughout each purification step. In view of a large-scale preparative work aimed at the preparation of PLTX reference material, we have investigated evaporation as a critical—although unavoidable—step that heavily affects overall recoveries. The experiments were carried out in two laboratories using different liquid chromatography-mass spectrometry (LC-MS) instruments, with either unit or high resolution. Palytoxin behaved differently when concentrated to a minimum volume rather than when evaporated to complete dryness. The recoveries strongly depended on the solubility as well as on the material of the used container. The LC-MS analyses of PLTX dissolved in aqueous organic blends proved to give a peak intensity higher then when dissolved in pure water. After drying, the PLTX adsorption appeared stronger on glass surfaces than on plastic materials. However, both the solvents used to dilute PLTX and that used for re-dissolution had an important role. A quantitative recovery (97%) was achieved when completely drying 80% aqueous EtOH solutions of PLTX under N₂-stream in Teflon. The stability of PLTX in acids was also investigated. Although PLTX was quite stable in 0.2% acetic acid solutions, upon exposure to stronger acids (pH < 2.66), degradation products were observed, among which a PLTX methyl-ester was identified. Full article

(This article belongs to the Section Marine and Freshwater Toxins)

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LC-HRMS response (peak areas of the Extracted Ion Chromatogram at m/z 906.4824) of PLTX (1.0 µg/mL) stored in various solvents mixtures in glass vials. Error bars represent standard deviation (n = 3). Response in 50% aqueous MeOH was taken as reference. * denotes statistically significant difference, p < 0.001. Full article ">Figure 2
Full HRMS spectrum of PLTX (m/z 1510–1900) where PLTX dimer and trimer ion clusters appear dominated by [2M+H+Ca]3+ and [3M+3H+Ca]5+ ions, respectively. Formula assigned to monoisotopic ion peaks are reported together with the isotopic ion patterns of the most intense ions. Full article ">Figure 3
Palytoxin Recoveries after complete drying under Nitrogen stream and Centrifugal Vacuum Concentrator (see <a href="#toxins-13-00650-t001" class="html-table">Table 1</a> for experimental details). Full article ">Figure 4
Recoveries of PLTX (125 ng/mL, 1.0 mL) following complete drying performed with N2-stream in (a) PP and Teflon tubes and (b) normal and silanized glass vials. The residues were re-dissolved in 300 μL of the initial solvent/blend. Recoveries are expressed in % ± RSD as mean of three different replicates. Full article ">Figure 5
Recoveries of PLTX at different initial concentration (initial volume 1.0 mL, in 80% aqueous MeOH) following complete drying performed with N2-stream in Teflon tubes. The samples were re-dissolved in 300 µL or 1.0 mL of 50% aqueous MeOH. Recoveries are expressed in % ± RSD as mean of three different replicates. Full article ">Figure 6
Recoveries of PLTX (125 ng/mL, 1.0 mL) following concentration to 200 µL under N2-stream in (a) PP and Teflon tubes and (b) normal and silanized glass vials. Recoveries are expressed in % ± RSD as mean of three different replicates. Full article ">Figure 7
Recoveries of PLTX (125 ng) in 80% aqueous MeOH with the addition of 0.2% acetic acid (AA), 0.1% trifluoroacetic acid (TFA) and 2% formic acid (FA) and no acid (control) following (a) complete drying in Teflon tubes and subsequent re-dissolution in 1 mL of 80% aqueous MeOH and (b) heat-treatment (60 °C, 1.0 h) in glass tubes. Error bars represent standard deviation from mean of three different replicates. Full article ">Figure 8
LC-HRMS analysis (flow rate 0.4 mL/min) of PLTX in 80% aqueous MeOH with 0.1% TFA (pH 1.20) at day 0 and 14. XICs of m/z 906.4627 (PLTX) and m/z 869.4627 (PLTX derivative) and full HRMS spectrum associated to each peak. Full article ">

15 pages, 594 KiB

Open AccessEditor’s ChoiceArticle

Diversity of Mycobiota in Spanish Grape Berries and Selection of Hanseniaspora uvarum U1 to Prevent Mycotoxin Contamination

by Carolina Gómez-Albarrán, Clara Melguizo, Belén Patiño, Covadonga Vázquez and Jéssica Gil-Serna

Toxins 2021, 13(9), 649; https://doi.org/10.3390/toxins13090649 - 13 Sep 2021

Cited by 17 | Viewed by 4198

Abstract

The occurrence of mycotoxins on grapes poses a high risk for food safety; thus, it is necessary to implement effective prevention methods. In this work, a metagenomic approach revealed the presence of important mycotoxigenic fungi in grape berries, including Aspergillus flavus, Aspergillus [...] Read more.

The occurrence of mycotoxins on grapes poses a high risk for food safety; thus, it is necessary to implement effective prevention methods. In this work, a metagenomic approach revealed the presence of important mycotoxigenic fungi in grape berries, including Aspergillus flavus, Aspergillus niger aggregate species, or Aspergillus section Circumdati. However, A. carbonarius was not detected in any sample. One of the samples was not contaminated by any mycotoxigenic species, and, therefore, it was selected for the isolation of potential biocontrol agents. In this context, Hanseniaspora uvarum U1 was selected for biocontrol in vitro assays. The results showed that this yeast is able to reduce the growth rate of the main ochratoxigenic and aflatoxigenic Aspergillus spp. occurring on grapes. Moreover, H. uvarum U1 seems to be an effective detoxifying agent for aflatoxin B₁ and ochratoxin A, probably mediated by the mechanisms of adsorption to the cell wall and other active mechanisms. Therefore, H. uvarum U1 should be considered in an integrated approach to preventing AFB₁ and OTA in grapes due to its potential as a biocontrol and detoxifying agent. Full article

(This article belongs to the Special Issue Selected Papers from the V Workshop of the Spanish National Network on Mycotoxins and Toxigenic Fungi and Their Decontamination Processes (MICOFOOD), 10–11 December 2020)

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17 pages, 1130 KiB

Open AccessArticle

The Reduction of the Combined Effects of Aflatoxin and Ochratoxin A in Piglet Livers and Kidneys by Dietary Antioxidants

by Roua Gabriela Popescu, Sorin Avramescu, Daniela Eliza Marin, Ionelia Țăranu, Sergiu Emil Georgescu and Anca Dinischiotu

Toxins 2021, 13(9), 648; https://doi.org/10.3390/toxins13090648 - 13 Sep 2021

Cited by 6 | Viewed by 3099

Abstract

The purpose of this study was to investigate the combined effects of aflatoxin B1 and ochratoxin A on protein expression and catalytic activities of CYP1A2, CYP2E1, CYP3A29 and GSTA1 and the preventive effect of dietary byproduct antioxidants administration against these mycotoxin damage. Three [...] Read more.

The purpose of this study was to investigate the combined effects of aflatoxin B1 and ochratoxin A on protein expression and catalytic activities of CYP1A2, CYP2E1, CYP3A29 and GSTA1 and the preventive effect of dietary byproduct antioxidants administration against these mycotoxin damage. Three experimental groups (E1, E2, E3) and one control group (C) of piglets after weaning (TOPIGS-40 hybrid) were fed with experimental diets for 30 days. A basal diet containing normal compound feed for starter piglets was used as a control treatment and free of mycotoxin. The experimental groups were fed as follows: E1—basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal), E2—the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1) and E3—basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform microsomal fraction isolation, followed by protein expression and enzymatic analyses. The protein expressions of CYP2E1 and CYP3A29 were up-regulated in an insignificant manner in liver, whereas in kidney, those of CYP1A2, CYP2E1 and CYP3A29 were down-regulated. The enzymatic activities of CYP1A2, CYP2E1 and CYP3A29 decreased in liver, in a significant manner, whereas in kidney, these increased significantly. The co-presence of the two mycotoxins and the mixture of grape seed and sea buckthorn meal generated a tendency to return to the control values, which suggest that grapeseed and sea buckthorn meal waste represent a promising source in counteracting the harmful effect of ochratoxin A and aflatoxin B. Full article

(This article belongs to the Special Issue The Effects of Mycotoxins on Human and Animal Health—a Special Focus on the Cellular and Molecular Mechanisms Responsible for Mycotoxin Toxicity)

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Figure 1
Relative protein expression and the corresponding quantification of Western blot images for CYP1A2, CYP2E1, CYP3A4 and GSTA1 in the hepatic and renal microsomal fractions of weaned piglets subjected to experimental diets. Calnexin band (70 kDa) was used as reference protein. The control group (C) were fed a basal diet. The experimental groups were fed as follows: the basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal) (E1 group), the basal diet artificially contaminated with AFB1 and OTA (E2 group), and the basal diet containing the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of AFB1 and OTA (E3 group). The data are illustrated as average values of the groups (n = 4) ± standard deviation of the mean (SE) and statistical significance related to the control group level. * E1/E2/E3 vs. C; # E2/E3 vs. E1; *, # p < 0.05; **, ## p < 0.01; *** p < 0.001. Full article ">Figure 2
Enzymatic specific activity in the hepatic and renal microsomal fractions for CYP1A2, CYP2E1, CYP3A29 and GSTA1 of weaned piglets subjected to experimental diets. The control group (C) were fed a basal diet. The experimental groups were fed as follows: the basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal) (E1 group), the basal diet artificially contaminated with AFB1 and OTA (E2 group) and the basal diet containing the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of AFB1 and OTA (E3 group). The data are illustrated as average values of the groups (n = 4) ± standard deviation of the mean (SE) and statistical significance related to the control group level. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Full article ">

12 pages, 1131 KiB

Open AccessArticle

Effect of Botulinum Toxin on Non-Motor Symptoms in Cervical Dystonia

by Matteo Costanzo, Daniele Belvisi, Isabella Berardelli, Annalisa Maraone, Viola Baione, Gina Ferrazzano, Carolina Cutrona, Giorgio Leodori, Massimo Pasquini, Antonella Conte, Giovanni Fabbrini, Giovanni Defazio and Alfredo Berardelli

Toxins 2021, 13(9), 647; https://doi.org/10.3390/toxins13090647 - 12 Sep 2021

Cited by 17 | Viewed by 3578

Abstract

Patients with cervical dystonia (CD) may display non-motor symptoms, including psychiatric disturbances, pain, and sleep disorders. Intramuscular injection of botulinum toxin type A (BoNT-A) is the most efficacious treatment for motor symptoms in CD, but little is known about its effects on non-motor [...] Read more.

Patients with cervical dystonia (CD) may display non-motor symptoms, including psychiatric disturbances, pain, and sleep disorders. Intramuscular injection of botulinum toxin type A (BoNT-A) is the most efficacious treatment for motor symptoms in CD, but little is known about its effects on non-motor manifestations. The aim of the present study was to longitudinally assess BoNT-A’s effects on CD non-motor symptoms and to investigate the relationship between BoNT-A-induced motor and non-motor changes. Forty-five patients with CD participated in the study. Patients underwent a clinical assessment that included the administration of standardized clinical scales assessing dystonic symptoms, psychiatric disturbances, pain, sleep disturbances, and disability. Clinical assessment was performed before and one and three months after BoNT-A injection. BoNT-A induced a significant improvement in dystonic symptoms, as well as in psychiatric disturbances, pain, and disability. Conversely, sleep disorders were unaffected by BoNT-A treatment. Motor and non-motor BoNT-A-induced changes showed a similar time course, but motor improvement did not correlate with non-motor changes after BoNT-A. Non-motor symptom changes after BoNT-A treatment are a complex phenomenon and are at least partially independent from motor symptom improvement. Full article

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14 pages, 362 KiB

Open AccessArticle

Screening for Predictors of Chronic Ciguatera Poisoning: An Exploratory Analysis among Hospitalized Cases from French Polynesia

by Clémence Mahana iti Gatti, Kiyojiken Chung, Erwan Oehler, T. J. Pierce, Matthew O. Gribble and Mireille Chinain

Toxins 2021, 13(9), 646; https://doi.org/10.3390/toxins13090646 - 12 Sep 2021

Cited by 10 | Viewed by 2749

Abstract

Ciguatera poisoning is a globally occurring seafood disease caused by the ingestion of marine products contaminated with dinoflagellate produced neurotoxins. Persistent forms of ciguatera, which prove to be highly debilitating, are poorly studied and represent a significant medical issue. The present study aims [...] Read more.

Ciguatera poisoning is a globally occurring seafood disease caused by the ingestion of marine products contaminated with dinoflagellate produced neurotoxins. Persistent forms of ciguatera, which prove to be highly debilitating, are poorly studied and represent a significant medical issue. The present study aims to better understand chronic ciguatera manifestations and identify potential predictive factors for their duration. Medical files of 49 patients were analyzed, and the post-hospitalization evolution of the disease assessed through a follow-up questionnaire. A rigorous logistic lasso regression model was applied to select significant predictors from a list of 37 patient characteristics potentially predictive of having chronic symptoms. Missing data were handled by complete case analysis, and a survival analysis was implemented. All models used standardized variables, and multiple comparisons in the survival analyses were handled by Bonferroni correction. Among all studied variables, five significant predictors of having symptoms lasting ≥3 months were identified: age, tobacco consumption, acute bradycardia, laboratory measures of urea, and neutrophils. This exploratory, hypothesis-generating study contributes to the development of ciguatera epidemiology by narrowing the list from 37 possible predictors to a list of five predictors that seem worth further investigation as candidate risk factors in more targeted studies of ciguatera symptom duration. Full article

(This article belongs to the Special Issue Ciguatoxins)

20 pages, 3435 KiB

Open AccessArticle

ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo

by Vincent Deruelle, Alice Berry, Stéphanie Bouillot, Viviana Job, Antoine P. Maillard, Sylvie Elsen and Philippe Huber

Toxins 2021, 13(9), 645; https://doi.org/10.3390/toxins13090645 - 11 Sep 2021

Cited by 2 | Viewed by 2884

Abstract

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial [...] Read more.

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples. Full article

(This article belongs to the Section Bacterial Toxins)

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Figure 1
Quantification of ExlA in bacterial secretomes. (A). Scheme of the sandwich ELISA used for quantification of ExlA in liquid samples. The polyclonal antibody ΔCter and the monoclonal antibody 5H6 were used as capture and detection antibodies, respectively. (B). Reproducibility of the standard curve (mean +/− SD, n = 3 independent experiments) and range of the assay. (C). ExlA quantification in bacterial secretomes. Four strains were used: IHMA is a natural exlA-positive strain; IHMAΔexlBA is an isogenic IHMA mutant deficient in ExlA secretion; IHMAΔerfA is an isogenic mutant overproducing ExlA; PAO1 is an exlA-negative strain. LB medium was used as negative control. Secretomes were collected from cultures at OD600 2.0 and supplemented with an anti-protease cocktail. All samples were assayed for ExlA content with the ExlA ELISA in triplicates. Individual values are shown together with the median (bar). Statistical differences were evaluated using Kruskal–Wallis’s test (p = 0.006), followed by Dunn’s test for comparison to control: * p = 0.03; ** p = 0.003; all other values were non-significant. The experiment was reproduced once with similar results. Full article ">Figure 2
ExlA immunolocalization in bacteria. (A,B). Bacteria in exponential phase: IHMA, IHMA exlB-mut (IHMAΔexlB), IHMAΔexlBA and IHMAΔerfA were fixed. Part of the bacteria was permeabilized with 100 µg/mL of polymyxin B (PMB), as indicated. Bacteria were then immunolabeled with monoclonal mouse anti-ExlA antibody (7G10) and subsequently with anti-mouse antibody coupled to Alexa-Fluor® 488 (green). Hoechst labeling (blue) was used to stain the bacterial cytoplasm. FM4-64 staining was used in (B) for membrane labeling (red). (A). ExlA labeling and merge (ExlA + Hoechst) images are shown for all four strains, together with magnifications. (B). Merge immunolabeling images (ExlA + Hoechst + FM4-64) are shown for IHMA and IHMAΔerfA. (C). The percentages of bacteria with ExlA spots were counted on 10–11 images per condition, using MicrobeJ software, and are shown as bars (n = 3302–11,465 cells analyzed in each condition). The Chi2 test was used to establish statistical differences (p < 0.0001), and dual comparisons were calculated using the two-sided Fisher’s exact test: p-values are indicated above the bars. n.s., non-significant. (D). Distribution of the ExlA spots on the bacterial circumference was determined using MicrobeJ. The heat map shows spot density associated with bacteria. Full article ">Figure 3
Cyclic-di-GMP regulates ExlA secretion(A). Intracellular c-di-GMP levels in IHMA::pSW196 (IHMA) and PAO1::pSW196 (PAO1). Both strains contained the chromosomal PcdrA-gfp(ASV)c fusion as fluorescent reporter of c-di-GMP levels. GFP fluorescence was recorded for 16 h and was normalized by OD585 to assess bacterial growth. The results, in arbitrary units (A; U), represent the mean +/− SD of six replicates. (B). The areas under the curves (AUC) were deduced from data shown in (A). Bar: mean. The indicated p-value was calculated with the Student’s test. (C,D). Similar experiment using IHMA::pSW196 (IHMA), IHMA::pSW196-wspR* (c-di-GMP +) and IHMA::pSW196-PA2133 (c-di-GMP −). Gene expression was induced by arabinose 0.025%. Statistical differences between AUC were calculated with ANOVA (p < 0.0001) followed by Dunnett’s test for comparison with IHMA. (E). ExlA contents of secretomes and bacteria for IHMA::pSW196 (WT), IHMA ΔexlBA, IHMA::pSW196-wspR* (c-di-GMP +) and IHMA::pSW196-PA2133 (c-di-GMP −). Secretomes were concentrated 100X and bacterial extracts 10X before Western blot analysis and incubation with ExlA antibodies (Cter and ΔCter). FliC and EFTu were used as loading controls for secretomes and bacterial extracts, respectively. (F). ExlA/control signal ratios in secretomes and bacterial extracts are shown for three independent Western blot experiments (color coded). (G). ExlA was quantified by ELISA in the secretomes of IHMAΔerfA::pSW196 (ΔerfA), IHMAΔerfA::pSW196-wspR* (c-di-GMP +) and IHMAΔerfA::pSW196-PA2133 (c-di-GMP −). Three clones were assayed in triplicates. The dots represent the data for each clone with the mean (bar). Global statistical difference was established with ANOVA (p = 0.0036) and p-values for individual comparisons with IHMAΔerfA (Dunnett’s test) are shown. (H). The three IHMA derivatives described in (C,D) (WT, c-di-GMP + and c-di-GMP −) were analyzed in strains harboring lacZ integrated within exlA gene to measure the transcriptional activity of the exlBA promoter. The β-galactosidase activity was measured in triplicates when bacterial cultures reached OD600 = 1 and expressed in Miller’s units (MU). Results are shown as mean +/− SD. No significant differences were established with ANOVA. (I). ExlA contents in bacterial extracts (concentrated 10X) from strains IHMAexlBmut::pSW196 (ΔexlB), IHMAexlBmut::pSW196-wspR* (ΔexlB c-di-GMP +), IHMAexlBmut::pSW196-PA2133 (ΔexlB c-di-GMP −) were determined by Western blot. EFTu was used as loading control. Full article ">Figure 4
The c-di-GMP holds ExlA at the outer membrane. (A). Cellular fractionation of IHMA ΔerfA containing either pSW196-wspR* (c-di-GMP +) or pSW196-PA1233 (c-di-GMP −): B, total bacterial extract; C, cytosol; P, periplasm; M, membranes; S, secretome. Fractions were analyzed by Western blot to detect ExlA, as well as DsbA and Opr86, as markers for periplasm and membranes, respectively. (B). ExlA signal intensities are shown for two independent Western blot experiments (color coded) in arbitrary units. (C). Accessibility to proteinase K. Bacteria (IHMA or IHMA ΔexlB) were incubated 5 min on ice with increasing concentrations of proteinase K, and total extracts were analyzed by Western blot for detection of ExlA, as well as DsbA to control outer-membrane integrity. (D). ExlA/Dsba signal ratios are shown for two independent experiments (color coded). Full article ">Figure 5
Quantification of ExlA in broncho-alveolar lavage fluids of infected mice. Pneumonia was induced in mice by inhalation of a suspension of IHMA (exlA+; n = 20) or PAO1 (exlA-; n = 4). In parallel, four mice were mock-infected with PBS. BAL fluids (1.5 mL of PBS with anti-proteases) were collected at 18 hpi and centrifuged. (A). ExlA concentration was measured on the supernatants. Individual values are shown together with the median. Data were analyzed by Kruskal–Wallis’s test (p = 0.0004). The p-value of multiple comparisons with Dunn’s test are shown. (B–D). Protein, IL-6 and hemoglobin data are shown in a correlation graph with ExlA concentrations. The linear regression curves are shown, as well as the r2 values. (B,C). Protein and IL-6 concentrations were measured in BAL supernatants. (D). Hemoglobin content was measured in BAL pellets and represented as OD560. Full article ">Figure 6
Proposed model for ExlA secretion regulation. ExlA and ExlB are transported into the periplasm by the Sec machinery. ExlB forms a pore inside the outer membrane, in which ExlA is maintained in conditions of high c-di-GMP levels and points outside. When c-di-GMP levels are low, ExlA is secreted into the extracellular milieu. The indirect pathway that conveys the cytosolic c-di-GMP signal to ExlA in the outer membrane is unknown. Full article ">

15 pages, 2353 KiB

Open AccessEditor’s ChoiceArticle

A Validation System for Selection of Bacteriophages against Shiga Toxin-Producing Escherichia coli Contamination

by Agnieszka Necel, Sylwia Bloch, Bożena Nejman-Faleńczyk, Aleksandra Dydecka, Gracja Topka-Bielecka, Alicja Węgrzyn and Grzegorz Węgrzyn

Toxins 2021, 13(9), 644; https://doi.org/10.3390/toxins13090644 - 11 Sep 2021

Cited by 5 | Viewed by 3189

Abstract

Shiga toxin-producing Escherichia coli (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) [...] Read more.

Shiga toxin-producing Escherichia coli (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) contaminated with cow feces (often due to the use of natural fertilizers in agriculture). Since the use of antibiotics against STEC is controversial, other methods for protection of food against contaminations by these bacteria are required. Here, we propose a validation system for selection of bacteriophages against STEC contamination. As a model system, we have employed a STEC-specific bacteriophage vB_Eco4M-7 and the E. coli O157:H7 strain no. 86-24, bearing Shiga toxin-converting prophage ST2-8624 (Δstx2::cat gfp). When these bacteria were administered on the surface of sliced cucumber (as a model vegetable), significant decrease in number viable E. coli cells was observed after 6 h of incubation. No toxicity of vB_Eco4M-7 against mammalian cells (using the Balb/3T3 cell line as a model) was detected. A rapid decrease of optical density of STEC culture was demonstrated following addition of a vB_Eco4M-7 lysate. However, longer incubation of susceptible bacteria with this bacteriophage resulted in the appearance of phage-resistant cells which predominated in the culture after 24 h incubation. Interestingly, efficiency of selection of bacteria resistant to vB_Eco4M-7 was higher at higher multiplicity of infection (MOI); the highest efficiency was evident at MOI 10, while the lowest occurred at MOI 0.001. A similar phenomenon of selection of the phage-resistant bacteria was also observed in the experiment with the STEC-contaminated cucumber after 24 h incubation with phage lysate. On the other hand, bacteriophage vB_Eco4M-7 could efficiently develop in host bacterial cells, giving plaques at similar efficiency of plating at 37, 25 and 12 °C, indicating that it can destroy STEC cells at the range of temperatures commonly used for vegetable short-term storage. These results indicate that bacteriophage vB_Eco4M-7 may be considered for its use in food protection against STEC contamination; however, caution should be taken due to the phenomenon of the appearance of phage-resistant bacteria. Full article

(This article belongs to the Special Issue Factors Governing the Persistence, Infection, Virulence and Treatment of Shiga Toxin-Encoding E. coli (STEC))

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18 pages, 5872 KiB

Open AccessArticle

Ciguatoxin-Producing Dinoflagellate Gambierdiscus in the Beibu Gulf: First Report of Toxic Gambierdiscus in Chinese Waters

by Yixiao Xu, Xilin He, Wai Hin Lee, Leo Lai Chan, Douding Lu, Pengbin Wang, Xiaoping Tao, Huiling Li and Kefu Yu

Toxins 2021, 13(9), 643; https://doi.org/10.3390/toxins13090643 - 10 Sep 2021

Cited by 9 | Viewed by 3673

Abstract

Ciguatera poisoning is mainly caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs) produced by the benthic dinoflagellates Gambierdiscus and Fukuyoa. China has a long history of problems with ciguatera, but research on ciguatera causative organisms is very limited, [...] Read more.

Ciguatera poisoning is mainly caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs) produced by the benthic dinoflagellates Gambierdiscus and Fukuyoa. China has a long history of problems with ciguatera, but research on ciguatera causative organisms is very limited, especially in the Beibu Gulf, where coral reefs have been degraded significantly and CTXs in reef fish have exceeded food safety guidelines. Here, five strains of Gambierdiscus spp. were collected from Weizhou Island, a ciguatera hotspot in the Beibu Gulf, and identified by light and scanning electron microscopy and phylogenetic analyses based on large and small subunit rDNA sequences. Strains showed typical morphological characteristics of Gambierdiscus caribaeus, exhibiting a smooth thecal surface, rectangular-shaped 2′, almost symmetric 4″, and a large and broad posterior intercalary plate. They clustered in the phylogenetic tree with G. caribaeus from other locations. Therefore, these five strains belonged to G. caribaeus, a globally distributed Gambierdiscus species. Toxicity was determined through the mouse neuroblastoma assay and ranged from 0 to 5.40 fg CTX3C eq cell⁻¹. The low level of toxicity of G. caribaeus in Weizhou Island, with CTX-contaminated fish above the regulatory level in the previous study, suggests that the long-term presence of low toxicity G. caribaeus might lead to the bioaccumulation of CTXs in fish, which can reach dangerous CTX levels. Alternatively, other highly-toxic, non-sampled strains could be present in these waters. This is the first report on toxic Gambierdiscus from the Beibu Gulf and Chinese waters and will provide a basis for further research determining effective strategies for ciguatera management in the area. Full article

(This article belongs to the Special Issue Ciguatoxins)

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Scanning electron microscope images of Gambierdiscus caribaeus GCBG01 strain from Weizhou Island, Beibu Gulf of China. (A,B): apical view, (C,D): antapical view, (E): dorsal view, (F): ventral view. Scale bars: 10 µm. Full article ">Figure 2
Scanning electron microscope images of Gambierdiscus caribaeus GCBG02 strain from Weizhou Island, Beibu Gulf of China. (A,B): apical view, (C,D): antapical view, (E): apical–lateral view, (F): ventral view. Scale bars: 10 µm. Full article ">Figure 3
Scanning electron microscope images of fish-hook apical pore plates (Po) for Gambierdiscus caribaeus strains GCBG01 and GCBG02 from Weizhou Island, Beibu Gulf of China. (A,B): GCBG01, (C,D): GCBG02. Scale bars: 2 µm. Full article ">Figure 4
Phylogenetic tree constructed based on Gambierdiscus D1–D3 large subunit ribosomal DNA (D1–D3 LSU rDNA) sequences. Values at nodes indicate bootstrap values from the maximum likelihood method and posterior probabilities from the Bayesian inference method. Bootstrap values <50 and posterior probabilities <0.50 are not shown. # Indicates the topology; here, the maximum-likelihood tree differs from the Bayesian tree. Full article ">Figure 5
Phylogenetic tree constructed based on Gambierdiscus D8–D10 large subunit ribosomal DNA (D8–D10 LSU rDNA) sequences. Values at nodes indicate bootstrap values from the maximum likelihood method and posterior probabilities from the Bayesian inference method. Bootstrap values <50 and posterior probabilities <0.50 are not shown. # Indicates the topology; here, the maximum-likelihood tree differs from the Bayesian tree. Full article ">Figure 6
Phylogenetic tree constructed based on Gambierdiscus small subunit ribosomal DNA (SSU rDNA). Values at nodes indicate bootstrap values from the maximum likelihood method and posterior probabilities from the Bayesian inference method. Bootstrap values <50 and posterior probabilities <0.50 are not shown. Full article ">Figure 7
Sampling sites. Full article ">

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