Editor’s Choice Articles

Recurrent blooms of Ostreopsis cf. ovata have been reported in Brazil and the Mediterranean Sea with associated ecological, and in the latter case, health impacts. Molecular data based on the D1–D3 and D8–D10 regions of the LSU rDNA and ITS loci, and the morphology of O. cf. ovata isolates and field populations from locations along the Brazilian tropical and subtropical coastal regions and three oceanic islands are presented. Additional ITS sequences from three single cells from the tropical coast are provided. Toxin profiles and quantities of PLTX and their analogues; OVTXs; contained in cells from two clonal cultures and two field blooms from Rio de Janeiro were investigated. Morphology was examined using both light and epifluorescence microscopy. Morphometric analysis of different strains and field populations from diverse locations were compared. Molecular analysis showed that six of the seven sequences grouped at the large “Atlantic/Mediterranean/Pacific” sub-clade, while one sequence branched in a sister clade with sequences from Madeira Island and Greece. The toxin profile of strains and bloom field samples from Rio de Janeiro were dominated by OVTX-a and -b, with total cell quotas (31.3 and 39.3 pg cell⁻¹) in the range of that previously reported for strains of O. cf. ovata. Full article

The objective of the study was to explore the incidence of patulin (PAT) mycotoxin in mango and orange fruits and derived products marketed in Pakistan. A total of 274 samples, including 70 mango fruits, 63 mango-based products (juices, pulp, and jam), 77 orange fruits, and 64 orange-based products, were collected. PAT was determined by reverse-phase high-performance liquid chromatography (HPLC) with UV-Vis detector (276 nm). Linear detector response was observed (R² > 0.99), the limit of detection (LOD) was 5 µg/kg and recovery percentage was 97.4%. The incidence of PAT in mango samples was 61.7%, and the concentration ranged from <LOD to 6415 µg/kg with a mean of 110.9 µg/kg. Our results showed the high susceptibility of mango fruits to patulin, and it was observed that decayed mango fruits were most contaminated with PAT. Among the mango samples, PAT concentration was higher in fruits than in processed products such as mango juice, pulp, and jam. Toxin incidence in orange samples was 52.5% with concentrations from <LOD to 61 µg/kg and a mean of 6.3 µg/kg. As much as 29 samples of mango (21.8%) contained PAT concentration above the regulatory limit (50 µg/kg), whereas there was only one exceeding orange sample (0.7%). Our results show that PAT seems to be a problem in fruits, juices, and derived solid products, especially from mango, and needs surveillance on regular basis. Full article

Venomous snakebite is one of the world’s most lethal neglected tropical diseases. Animal-derived antivenoms are the only standardized specific therapies currently available for treating snakebite envenoming, but due to venom variation, often this treatment is not effective in counteracting all clinical symptoms caused by the multitude of injected toxins. In this study, the coagulopathic toxicities of venoms from the medically relevant snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus and Echis ocellatus were assessed. The venoms were separated by liquid chromatography (LC) followed by nanofractionation and parallel mass spectrometry (MS). A recently developed high-throughput coagulation assay was employed to assess both the pro- and anticoagulant activity of separated venom toxins. The neutralization capacity of antivenoms on separated venom components was assessed and the coagulopathic venom peptides and enzymes that were either neutralized or remained active in the presence of antivenom were identified by correlating bioassay results with the MS data and with off-line generated proteomics data. The results showed that most snake venoms analyzed contained both procoagulants and anticoagulants. Most anticoagulants were identified as phospholipases A₂s (PLA₂s) and most procoagulants correlated with snake venom metalloproteinases (SVMPs) and serine proteases (SVSPs). This information can be used to better understand antivenom neutralization and can aid in the development of next-generation antivenom treatments. Full article

Dispersive magnetic solid-phase extraction (DMSPE) has received growing attention for sample treatment preconcentration prior to the separation of analytes due to its many advantages. In the present work, the potential of DMSPE for the determination of emergent mycotoxins (enniatins A, A1, B and B1, and beauvericin) is investigated for the first time. Different magnetic nanoparticles were tested and a magnetic multiwalled carbon nanotube (Fe₃O₄@MWCNT) composite was selected for the extraction and preconcentration of the five target mycotoxins in human urine samples before their analysis by ultrahigh performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). The nanocomposite was characterized by energy dispersive X-ray spectrometry, scanning electron microscopy, Fourier transform infrared spectrophotometry, and X-ray diffraction. Several parameters affecting the adsorption and desorption of DMSPE steps were optimized and the method was fully validated. Due to a matrix effect, matrix-matched calibration curves were necessary to carry out quantification. In this way, limits of quantification of between 0.04 and 0.1 μg/L, relative standard deviation values lower than 12% and recoveries between 89.3% and 98.9% were obtained. Finally, a study of the reuse of the Fe₃O₄@MWCNT composite was carried out, confirming that it can be reused at least four times. Full article

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B₁ (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U g⁻¹ dry matter) and low MnP activity (0.4 U g⁻¹ dry matter) were present; (2) after 1 d of incubation at 25 °C, the SMSE was able to degrade more than 50% of AFB1, whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively; (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable. Full article

T-2 toxin, as a highly toxic mycotoxin to humans and animals, induces oxidative stress and apoptosis in various cells and tissues. Apoptosis and mitochondrial fusion/fission are two tightly interconnected processes that are crucial for maintaining physiological homeostasis. However, the role of mitochondrial fusion/fission in apoptosis of T-2 toxin remains unknown. Hence, we aimed to explore the putative role of mitochondrial fusion/fission on T-2 toxin induced apoptosis in normal human liver (HL-7702) cells. T-2 toxin treatment (0, 0.1, 1.0, or 10 μg/L) for 24 h caused decreased cell viability and ATP concentration and increased production of (ROS), as seen by a loss of mitochondrial membrane potential (∆Ψm) and increase in mitochondrial fragmentation. Subsequently, the mitochondrial dynamic imbalance was activated, evidenced by a dose-dependent decrease and increase in the protein expression of mitochondrial fusion (OPA1, Mfn1, and Mfn2) and fission (Drp1 and Fis1), respectively. Furthermore, the T-2 toxin promoted the release of cytochrome c from mitochondria to cytoplasm and induced cell apoptosis triggered by upregulation of Bax and Bax/Bcl-2 ratios, and further activated the caspase pathways. Taken together, these results indicate that altered mitochondrial dynamics induced by oxidative stress with T-2 toxin exposure likely contribute to mitochondrial injury and HL-7702 cell apoptosis. Full article

Ochratoxin A (OTA) is a common mycotoxin contaminant in animal feed. When absorbed from the gastrointestinal tract, OTA has a propensity for pathological effects on animal health and deposition in animal tissues. In this study, the potential of yeast cell wall extracts (YCWE) to adsorb OTA was evaluated using an in vitro method in which consecutive animal digestion events were simulated. Low pH markedly increased OTA binding to YCWE, which was reversed with a pH increased to 6.5. Overall, in vitro analysis revealed that 30% of OTA was adsorbed to YCWE. Additional computational molecular modelling revealed that change in pH alters the OTA charge and modulates the interaction with the YCWE β-d-glucans. The effectiveness of YCWE was tested in a 14-day broiler chicken trial. Birds were subjected to five dietary treatments; with and without OTA, and OTA combined with YCWE at three dosages. At the end of the trial, liver OTA deposition was evaluated. Data showed a decrease of up to 30% in OTA deposits in the liver of broilers fed both OTA and YCWE. In the case of OTA, a tight correlation between the mitigation efficacy of YCWE between in vitro and in vivo model could be observed. Full article

Traditional medicinal herbs are widely used and may be contaminated with mycotoxigenic fungi during cultivation, harvesting, and storage, causing spoilage and mycotoxin production. We evaluated the predominant mycoflora and extent of mycotoxin contaminations in 48 contaminated samples of 13 different medicinal herbs. In total, 70.8% of herbs were slightly contaminated with aflatoxins (<5 μg kg⁻¹). Codonopsis radix samples contained ochratoxin A (OTA) (360–515 μg kg⁻¹), and Scutellariae radix samples contained OTA (49–231 μg kg⁻¹) and citrinin (15–53 μg kg⁻¹). Forty samples (83.3%) contained fungal contamination. Sixty-nine strains were characterized via morphological and molecular identification. The predominant mycoflora comprised four genera, Aspergillus spp. (26.1%), Penicillium spp. (24.6%), Rhizopus spp. (14.5%), and Trichoderma spp. (11.6%). Aflatoxins, OTA, and citrinin were detected in 37 cultures by high-performance liquid chromatography-tandem mass spectrometry. Approximately 21.6% of Aspergillus and Penicillium isolates produced mycotoxins. One Penicillium polonicum strain isolated from Scutellariae radix synthesized citrinin. Multiplex PCR analysis showed that three Aspergillus flavus strains harbored aflatoxin biosynthesis genes. One Aspergillus flavus strain isolated from Amomi fructus produced AFB₁ and AFB₂. To the best of our knowledge, the citrinin production by Aspergillus chevalieri and Penicillium sacculum was first reported in this study, which poses a potential risk of mycotoxin contamination in medicinal herbs. Full article

Toxins have been proposed to facilitate fungal root infection by creating regions of readily-penetrated necrotic tissue when applied externally to intact roots. Isolates of the charcoal rot disease fungus, Macrophomina phaseolina, from soybean plants in Mississippi produced a phytotoxic toxin, (−)-botryodiplodin, but no detectable phaseolinone, a toxin previously proposed to play a role in the root infection mechanism. This study was undertaken to determine if (−)-botryodiplodin induces toxic responses of the types that could facilitate root infection. (±)-Botryodiplodin prepared by chemical synthesis caused phytotoxic effects identical to those observed with (−)-botryodiplodin preparations from M. phaseolina culture filtrates, consistent with fungus-induced phytotoxicity being due to (−)-botryodiplodin, not phaseolinone or other unknown impurities. Soybean leaf disc cultures of Saline cultivar were more susceptible to (±)-botryodiplodin phytotoxicity than were cultures of two charcoal rot-resistant genotypes, DS97-84-1 and DT97-4290. (±)-Botryodiplodin caused similar phytotoxicity in actively growing duckweed (Lemna pausicostata) plantlet cultures, but at much lower concentrations. In soybean seedlings growing in hydroponic culture, (±)-botryodiplodin added to culture medium inhibited lateral and tap root growth, and caused loss of root caps and normal root tip cellular structure. Thus, botryodiplodin applied externally to undisturbed soybean roots induced phytotoxic responses of types expected to facilitate fungal root infection. Full article

Hydrogen peroxide (H₂O₂) can be used as an emergency method to selectively suppress cyanobacterial blooms in lakes and drinking water reservoirs. However, it is largely unknown how environmental parameters alter the effectiveness of H₂O₂ treatments. In this study, the toxic cyanobacterial strain Microcystis aeruginosa PCC 7806 was treated with a range of H₂O₂ concentrations (0 to 10 mg/L), while being exposed to different light intensities and light colors. H₂O₂ treatments caused a stronger decline of the photosynthetic yield in high light than in low light or in the dark, and also a stronger decline in orange than in blue light. Our results are consistent with the hypothesis that H₂O₂ causes major damage at photosystem II (PSII) and interferes with PSII repair, which makes cells more sensitive to photoinhibition. Furthermore, H₂O₂ treatments caused a decrease in cell size and an increase in extracellular microcystin concentrations, indicative of leakage from disrupted cells. Our findings imply that even low H₂O₂ concentrations of 1–2 mg/L can be highly effective, if cyanobacteria are exposed to high light intensities. We therefore recommend performing lake treatments during sunny days, when a low H₂O₂ dosage is sufficient to suppress cyanobacteria, and may help to minimize impacts on non-target organisms. Full article

Fungal spoilage is an important issue for the food industry, leading to food sensory defects, food waste, economic losses and public health concern through the production of mycotoxins. Concomitantly, the search for safer natural products has gained importance since consumers began to look for less processed and chemically treated foods. In this context, the aim of this study was to evaluate the antifungal and antimycotoxigenic effect of seven strains of Lactobacillus plantarum. Lactic acid bacteria (LAB) were grown on Man Rogosa Sharpe (MRS) broth at 37 °C in anaerobic conditions. After that, the cell-free supernatant (CFS) were recovered to determine its antifungal activity by halo diffusion agar test. In addition, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was determined for each L. plantarum CFS by 96-well microplates method. Additionally, CFS was used as a natural biocontrol agent on corn kernels and corn ears contaminated with Aspergillus flavus and Fusarium verticillioides, respectively. The L. plantarum CECT 749 CFS showed the highest antifungal effect against all essayed strains. Moreover, the employment of this CFS in food reduced the mycotoxin production at a percentage ranging from 73.7 to 99.7%. These results suggest that the L. plantarum CECT 749 CFS could be promising for the biocontrol of corn. Full article

Background: Snake venom phospholipases A₂ (PLA₂s) have hemolytic, anticoagulant, myotoxic, oedematogenic, bactericidal, and inflammatory actions. BthTX-I, a Lys49-PLA₂ isolated from Bothrops jararacussu venom, is an example of Lys49-PLA₂ that presents such actions. NLRP3 is a cytosolic receptor from the NLR family responsible for inflammasome activation via caspase-1 activation and IL-1β liberation. The study of NLRs that recognize tissue damage and activate the inflammasome is relevant in envenomation. Methods: Male mice (18–20 g) received an intramuscular injection of BthTX-I or sterile saline. The serum was collected for creatine-kinase (CK), lactate dehydrogenase (LDH), and interleukin-1β (IL-1β) assays, and muscle was removed for inflammasome activation immunoblotting and qRT-PCR expression for nucleotide and oligomerization domain, leucine-rich repeat-containing protein family, pyrin-containing domain 3 receptor (NLRP3) inflammasome components. Results: BthTX-I-induced inflammation and myonecrosis, shown by intravital microscope, and LDH and CK release, respectively. Mouse treatment with A438079, a P2X7 receptor antagonist, did not modify these effects. BthTX-I induced inflammasome activation in muscle, but P2X7R participation in this effect was not observed. Conclusion: Together, the results showed for the first time that BthTX-I in gastrocnemius muscle induces inflammation and consequently, inflammasome activation via NLRP3 with caspase-1 activation and IL-1β liberation. Full article

Cyanobacteria are photosynthetic organisms that produce a large diversity of natural products with interesting bioactivities for biotechnological and pharmaceutical applications. Cyanobacterial extracts exhibit toxicity towards other microorganisms and cancer cells and, therefore, represent a source of potentially novel natural products for drug discovery. We tested 62 cyanobacterial strains isolated from various Brazilian biomes for antileukemic and antimicrobial activities. Extracts from 39 strains induced selective apoptosis in acute myeloid leukemia (AML) cancer cell lines. Five of these extracts also exhibited antifungal and antibacterial activities. Chemical and dereplication analyses revealed the production of nine known natural products. Natural products possibly responsible for the observed bioactivities and five unknown, chemically related chlorinated compounds present only in Brazilian cyanobacteria were illustrated in a molecular network. Our results provide new information on the vast biosynthetic potential of cyanobacteria isolated from Brazilian environments. Full article

Arterial stiffness (AS) has an important impact on the outcomes of patients on hemodialysis (HD), and p-cresyl sulfate (PC) can mediate the process of vascular damage. We aimed to investigate the relationship between carotid–femoral pulse wave velocity (cfPWV) and the level of PCs in HD patients. Serum PCs were quantified using liquid chromatography mass spectrometry. Patients who were on standard HD for more than 3 months were enrolled and categorized according to the cfPWV into the high AS (>10 m/s) and control (≤10 m/s) groups. Forty-nine (41.5%) patients belonged to the high AS group and had a higher incidence of diabetes mellitus (DM) and increased systolic blood pressure, serum C-reactive protein, and PC levels but had lower creatinine, compared with those in the control group. In HD patients, the risk for developing high AS increased in the presence of DM (OR 4.147, 95% confidence interval (CI) 1.497–11.491) and high PCs (OR 1.067, 95% CI 1.002–1.136). Having DM (r = 0.446) and high PC level (r = 0.174) were positively associated with cfPWV. The most optimal cutoff value of PC for predicting AS was 18.99 mg/L (area under the curve 0.661, 95% CI 0.568–0.746). We concluded that DM and PCs were promising predictors of high AS in patients on maintenance HD. Full article

Background: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. Methods: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-H_N or BoNT/F LC-H_N. Results: 21 unique BoNT/H LC-H_N mAbs were obtained; 15 from the BoNT/H LC-H_N immunized library (K_D 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (K_D 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-H_N. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. Conclusions: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities. Full article

Ciguatera poisoning (CP) is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa. The toxin production and toxin profiles were explored in four clones of G. polynesiensis originating from different islands in French Polynesia with contrasted CP risk: RIK7 (Mangareva, Gambier), NHA4 (Nuku Hiva, Marquesas), RAI-1 (Raivavae, Australes), and RG92 (Rangiroa, Tuamotu). Productions of CTXs, maitotoxins (MTXs), and gambierone group analogs were examined at exponential and stationary growth phases using the neuroblastoma cell-based assay and liquid chromatography–tandem mass spectrometry. While none of the strains was found to produce known MTX compounds, all strains showed high overall P-CTX production ranging from 1.1 ± 0.1 to 4.6 ± 0.7 pg cell⁻¹. In total, nine P-CTX analogs were detected, depending on strain and growth phase. The production of gambierone, as well as 44-methylgamberione, was also confirmed in G. polynesiensis. This study highlighted: (i) intraspecific variations in toxin production and profiles between clones from distinct geographic origins and (ii) the noticeable increase in toxin production of both CTXs, in particular CTX4A/B, and gambierone group analogs from the exponential to the stationary phase. Full article

The aim of this study was to investigate the effects of deoxynivalenol (DON) exposure on the inflammatory injury nuclear factor kappa-B (NF-κB) pathway in intestinal epithelial cells (IPEC-J2 cells) of pig. The different concentrations of DON (0, 125, 250, 500, 1000, 2000 ng/mL) were added to the culture solution for treatment. The NF-κB pathway inhibitor pyrrolidine dithiocarbamate (PDTC) was used as a reference. The results showed that when the DON concentration increased, the cell density decreased and seemed damaged. With the increase of DON concentration in the culture medium, the action of diamine oxidase (DAO) in the culture supernatant also increased. The activities of IL-6, TNF-α, and NO in the cells were increased with the increasing DON concentration. The relative mRNA expression of IL-1β and IL-6 were increased in the cells. The mRNA relative expression of NF-κB p65, IKKα, and IKKβ were upregulated with the increasing of DON concentration, while the relative expression of IκB-α mRNA was downregulated. At the same time, the expression of NF-κB p65 protein increased gradually in the cytoplasm and nucleus with a higher concentration of DON. These results showed that DON could change the morphology of IPEC-J2 cells, destroy its submicroscopic structure, and enhance the permeability of cell membrane, as well as upregulate the transcription of some inflammatory factors and change the expression of NF-κB-related gene or protein in cells. Full article

Resveratrol, a natural polyterpenoid, can scavenge reactive oxygen species in vivo to carry out the functions of antioxidation and antiaging. Resveratrol’s anti-cancer capability has attracted widespread attention, but its molecular mechanism has not been systematically explained. In this study, by comparing the activity of normal cell lines and cancer cell lines after treating with resveratrol, it was found that resveratrol has more significant cytotoxicity in cancer cell lines. Resveratrol could play a toxic role through inducing apoptosis of the cancer cell in a time- and concentration-dependent manner. A total of 330 significantly differential genes were identified through large-scale transcriptome sequencing, among which 103 genes were upregulated and 227 genes were downregulated. Transcriptome and qRT-PCR data proved that a large number of genes related to cell cycle were differentially expressed after the treatment of resveratrol. The changes of cell cycle phases at different time points after treating with resveratrol were further detected, and it was found that the cells were arrested in the S phase because of the percentage of cells in S phase increased and cells in G1/G0 phase decreased. In conclusion, resveratrol can inhibit the proliferation of 4T1 cancer cells by inhibiting cell cycle and inducing apoptosis. Full article

Microcystins (MC) and nodularin (NOD) are toxins released by cyanobacteria during harmful algal blooms. They are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A) and cause a variety of adverse symptoms in humans and animals if ingested. More than 250 chemically diverse congeners of MCs have been identified, but certified reference materials are only available for a few. A diagnostic test that does not require each reference material for detection is necessary to identify human exposures. To address this need, our lab has developed a method that uses an antibody to specifically isolate MCs and NOD from urine prior to detection via a commercially available PP2A kit. This assay quantitates the summed inhibitory activity of nearly all MCs and NOD on PP2A relative to a common MC congener, microcystin-LR (MC-LR). The quantitation range for MC-LR using this method is from 0.050–0.500 ng/mL. No background responses were detected in a convenience set of 50 individual urines. Interday and intraday % accuracies ranged from 94%–118% and relative standard deviations were 15% or less, meeting FDA guidelines for receptor binding assays. The assay detected low levels of MCs in urines from three individuals living in close proximity to harmful algal blooms (HABs) in Florida. Full article

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g⁻¹ tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure–activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean. Full article

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 °C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples. Full article

Harmful cyanobacteria and their toxic metabolites constitute a big challenge for the production of safe drinking water. Microcystins (MC), chemically stable hepatotoxic heptapeptides, have often been involved in cyanobacterial poisoning incidents. A desirable solution for cyanobacterial management in lakes and ponds would eliminate both excess cyanobacteria and the MC that they potentially produce and release upon lysis. Hydrogen peroxide (H₂O₂) has recently been advocated as an efficient means of lysing cyanobacteria in lakes and ponds, however H₂O₂ (at least when used at typical concentrations) cannot degrade MC in environmental waters. Therefore, mesocosm experiments combining the cyanobacteria-lysing effect of H₂O₂ and the MC-degrading capacity of the enzyme MlrA were set up in the highly eutrophic Lake Ludoš (Serbia). The H₂O₂ treatment decreased the abundance of the dominant cyanobacterial taxa Limnothrix sp., Aphanizomenon flos-aquae, and Planktothrix agardhii. The intracellular concentration of MC was reduced/eliminated by H₂O₂, yet the reduction of the extracellular MC could only be accomplished by supplementation with MlrA. However, as H₂O₂ was found to induce the expression of mcyB and mcyE genes, which are involved in MC biosynthesis, the use of H₂O₂ as a safe cyanobacteriocide still requires further investigation. In conclusion, the experiments showed that the combined use of H₂O₂ and MlrA is promising in the elimination of both excess cyanobacteria and their MC in environmental waters. Full article

Food and feed can be naturally contaminated by several mycotoxins, and concern about the hazard of exposure to mycotoxin mixtures is increasing. In this study, more than 800 metabolites were analyzed in 524 finished pig feed samples collected worldwide. Eighty-eight percent of the samples were co-contaminated with deoxynivalenol (DON) and other regulated/emerging mycotoxins. The Top 60 emerging/regulated mycotoxins co-occurring with DON in pig feed shows that 48%, 13%, 8% and 12% are produced by Fusarium, Aspergillus, Penicillium and Alternaria species, respectively. Then, the individual and combined toxicity of DON and the 10 most prevalent emerging mycotoxins (brevianamide F, cyclo-(L-Pro-L-Tyr), tryptophol, enniatins A1, B, B1, emodin, aurofusarin, beauvericin and apicidin) was measured at three ratios corresponding to pig feed contamination. Toxicity was assessed by measuring the viability of intestinal porcine epithelial cells, IPEC-1, at 48-h. BRV-F, Cyclo and TRPT did not alter cell viability. The other metabolites were ranked in the following order of toxicity: apicidin > enniatin A1 > DON > beauvericin > enniatin B > enniatin B1 > emodin > aurofusarin. In most of the mixtures, combined toxicity was similar to the toxicity of DON alone. In terms of pig health, these results demonstrate that the co-occurrence of emerging mycotoxins that we tested with DON does not exacerbate toxicity. Full article

Within mega-diverse Hymenoptera, non-aculeate parasitic wasps represent 75% of all hymenopteran species. Their ovipositor dual-functionally injects venom and employs eggs into (endoparasitoids) or onto (ectoparasitoids) diverse host species. Few endoparasitoid wasps such as Pimpla turionellae paralyze the host and suppress its immune responses, such as encapsulation and melanization, to guarantee their offspring’s survival. Here, the venom and its possible biology and function of P. turionellae are characterized in comparison to the few existing proteo-transcriptomic analyses on parasitoid wasp venoms. Multiple transcriptome assembly and custom-tailored search and annotation strategies were applied to identify parasitoid venom proteins. To avoid false-positive hits, only transcripts were finally discussed that survived strict filter settings, including the presence in the proteome and higher expression in the venom gland. P. turionella features a venom that is mostly composed of known, typical parasitoid enzymes, cysteine-rich peptides, and other proteins and peptides. Several venom proteins were identified and named, such as pimplin2, 3, and 4. However, the specification of many novel candidates remains difficult, and annotations ambiguous. Interestingly, we do not find pimplin, a paralytic factor in Pimpla hypochondriaca, but instead a new cysteine inhibitor knot (ICK) family (pimplin2), which is highly similar to known, neurotoxic asilid1 sequences from robber flies. Full article

Aspergillus flavus is a renowned plant, animal and human pathogen. areA is a global nitrogen regulatory gene of the GATA transcription factor family, shown to be the major nitrogen regulator. In this study, we identified areA in A. flavus and studied its function. The AreA protein contained a signatory zinc finger domain, which is extremely conserved across fungal species. Gene deletion (ΔareA) and over-expression (OE::areA) strains were constructed by homologous recombination to elucidate the role of areA in A. flavus. The ΔareA strain was unable to efficiently utilize secondary nitrogen sources for growth of A. flavus, and it had poorly developed conidiophores, when observed on complete medium, resulting in the production of significantly less conidia than the wild-type strain (WT). Aflatoxin B1 (AFB1) production was reduced in ΔareA compared with the WT strain in most conditions tested, and ΔareA had impaired virulence in peanut seeds. areA also played important roles in the sensitivity of A. flavus to osmotic, cell wall and oxidative stresses. Hence, areA was found to be important for the growth, aflatoxin production and pathogenicity of A. flavus. This work sheds light on the function of areA in the regulation of the nitrogen metabolism of A. flavus, and consequently aims at providing new ways for controlling the crossover pathogen, A. flavus. Full article

A knowledge, attitude and practice (KAP) study was conducted in three districts of Malawi to test whether the training had resulted in increased knowledge and adoption of recommended pre- and post-harvest crop management practices, and their contribution to reducing aflatoxin contamination in groundnut, maize and sorghum. The study was conducted with 900 farmers at the baseline and 624 farmers at the end-line, while 726 and 696 harvested crop samples were collected for aflatoxin testing at the baseline and end-line, respectively. Results show that the knowledge and practice of pre- and post-harvest crop management for mitigating aflatoxin were inadequate among the farmers at the baseline but somewhat improved after the training as shown at the end-line. As a result, despite unfavorable weather, the mean aflatoxin contamination level in their grain samples decreased from 83.6 to 55.8 ppb (p < 0.001). However, it was also noted that increased knowledge did not significantly change farmers’ attitude toward not consuming grade-outs because of economic incentive incompatibility, leaving potential for improving the practices further. This existing gap in the adoption of aflatoxin mitigation practices calls for approaches that take into account farmers’ needs and incentives to attain sustainable behavioral change. Full article

Eighty-seven samples of malt from several Polish malting plants and 157 beer samples from the beer available on the Polish market (in 2018) were tested for Fusarium mycotoxins (deoxynivalenol (DON), nivalenol (NIV)), and their modified forms ((deoxynivalenol-3-glucoside (DON-3G), nivalenol-3-glucoside (NIV-3G), 3-acetyldeoxynivalenol (3-AcDON)). DON and its metabolite, DON-3G, were found the most, among the samples analyzed; DON and DON-3G were present in 90% and 91% of malt samples, and in 97% and 99% of beer samples, respectively. NIV was found in 24% of malt samples and in 64% of beer samples, and NIV-3G was found in 48% of malt samples and 39% of beer samples. In the malt samples, the mean concentration of DON was 52.9 µg/kg (range: 5.3–347.6 µg/kg) and that of DON-3G was 74.1 µg/kg (range: 4.4–410.3 µg/kg). In the beer samples, the mean concentration of DON was 12.3 µg/L (range: 1.2–156.5 µg/L) and that of DON-3G was 7.1 µg/L (range: 0.6–58.4 µg/L). The concentrations of other tested mycotoxins in the samples of malt and beer were several times lower. The risk of exposure to the tested mycotoxins, following the consumption of beer in Poland, was assessed. The corresponding probable daily intakes (PDIs) remained a small fraction of the tolerable daily intake (TDI). However, in the improbable worst-case scenario, in which every beer bottle consumed would be contaminated with mycotoxins present at the highest level observed among the analyzed beer samples, the PDI would exceed the TDI for DON and its metabolite after the consumption of a single bottle (0.5 L) of beer. Full article

Acute hepatopancreatic necrosis disease (AHPND), a newly emergent farmed penaeid shrimp bacterial disease originally known as early mortality syndrome (EMS), is causing havoc in the shrimp industry. The causative agent of AHPND was found to be a specific strain of bacteria, e.g., Vibrio and Shewanella sps., that contains pVA1 plasmid (63–70 kb) encoding the binary PirA^VP and PirB^VP toxins. The PirAB^VP and toxins are the primary virulence factors of AHPND-causing bacteria that mediates AHPND and mortality in shrimp. Hence, in this study using a germ-free brine shrimp model system, we evaluated the PirAB^VP toxin-mediated infection process at cellular level, including toxin attachment and subsequent toxin-induced damage to the digestive tract. The results showed that, PirAB^VP toxin binds to epithelial cells of the digestive tract of brine shrimp larvae and produces characteristic symptoms of AHPND. In the PirAB^VP-challenged brine shrimp larvae, shedding or sloughing of enterocytes in the midgut and hindgut regions was regularly visualized, and the intestinal lumen was filled with moderately electron-dense cells of variable shapes and sizes. In addition, the observed cellular debris in the intestinal lumen of the digestive tract was found to be of epithelial cell origin. The detailed morphology of the digestive tract demonstrates further that the PirAB^VP toxin challenge produces focal to extensive necrosis and damages epithelial cells in the midgut and hindgut regions, resulting in pyknosis, cell vacuolisation, and mitochondrial and rough endoplasmic reticulum (RER) damage to different degrees. Taken together, our study provides substantial evidence that PirAB^VP toxins bind to the digestive tract of brine shrimp larvae and seem to be responsible for generating characteristic AHPND lesions and damaging enterocytes in the midgut and hindgut regions. Full article

Snake venom evolution is typically considered to be predominantly driven by diet-related selection pressures. Most evidence for this is based on lethality to prey and non-prey species and on the identification of prey specific toxins. Since the broad toxicological activities (e.g., neurotoxicity, coagulotoxicity, etc.) sit at the interface between molecular toxinology and lethality, these classes of activity may act as a key mediator in coevolutionary interactions between snakes and their prey. Indeed, some recent work has suggested that variation in these functional activities may be related to diet as well, but previous studies have been limited in geographic and/or taxonomic scope. In this paper, we take a phylogenetic comparative approach to investigate relationships between diet and toxicological activity classes on a global scale across caenophidian snakes, using the clinically oriented database at toxinology.com. We generally find little support for specific prey types selecting for particular toxicological effects except that reptile-feeders are more likely to be neurotoxic. We find some support for endothermic prey (with higher metabolic rates) influencing toxic activities, but differently from previous suggestions in the literature. More broadly, we find strong support for a general effect of increased diversity of prey on the diversity of toxicological effects of snake venom. Hence, we provide evidence that selection pressures on the toxicological activities of snake venom has largely been driven by prey diversity rather than specific types of prey. These results complement and extend previous work to suggest that specific matching of venom characteristics to prey may occur at the molecular level and translate into venom lethality, but the functional link between those two is not constrained to a particular toxicological route. Full article

The ecological conditions during the ripening of dry-cured ham favour the development of moulds on its surface, being frequently the presence of Penicillium nordicum, a producer of ochratoxin A (OTA). Biocontrol using moulds and yeasts usually found in dry-cured ham is a promising strategy to minimize this hazard. The aim of this work is to evaluate the effect of previously selected Debaryomyces hansenii and Penicillium chrysogenum strains on growth, OTA production, and relative expression of genes involved in the OTA biosynthesis by P. nordicum. P. nordicum was inoculated against the protective cultures individually and combined on dry-cured ham for 21 days at 20 °C. None of the treatments reduced the growth of P. nordicum, but all of them decreased OTA concentration. The lower production of OTA could be related to significant repression of the relative expression of otapksPN and otanpsPN genes of P. nordicum. The efficacy of the combined protective cultures was tested in 24 dry-cured hams in industrial ripening (an 8 month-long production). OTA was detected in nine of the 12 dry-cured hams in the batch inoculated only with P. nordicum. However, in the batch inoculated with both P. nordicum and the combined protective culture, a considerable reduction of OTA contamination was observed. In conclusion, although the efficacy of individual use P. chrysogenum is great, the combination with D. hansenii enhances its antifungal activity and could be proposed as a mixed protective culture to control the hazard of the presence of OTA in dry-cured ham. Full article

Mycotoxins are secondary metabolites produced by certain filamentous fungi, causing human and animal health issues upon the ingestion of contaminated food and feed. Among the safest approaches to the control of mycotoxigenic fungi and mycotoxin detoxification is the application of microbial biocontrol agents. Burkholderia cepacia is known for producing metabolites active against a broad number of pathogenic fungi. In this study, the antifungal potential of a Qatari strain of Burkholderia cepacia (QBC03) was explored. QBC03 exhibited antifungal activity against a wide range of mycotoxigenic, as well as phytopathogenic, fungal genera and species. The QBC03 culture supernatant significantly inhibited the growth of Aspergillus carbonarius, Fusarium culmorum and Penicillium verrucosum in PDA medium, as well as A. carbonarius and P. verrucosum biomass in PDB medium. The QBC03 culture supernatant was found to dramatically reduce the synthesis of ochratoxin A (OTA) by A. carbonarius, in addition to inducing mycelia malformation. The antifungal activity of QBC03’s culture extract was retained following thermal treatment at 100 °C for 30 min. The findings of the present study advocate that QBC03 is a suitable biocontrol agent against toxigenic fungi, due to the inhibitory activity of its thermostable metabolites. Full article

Accidental contact with caterpillar bristles causes local symptoms such as severe pain, intense heat, edema, erythema, and pruritus. However, there is little functional evidence to indicate a potential mechanism. In this study, we analyzed the biological characteristics of the crude venom from the larval stage of Latoia consocia living in South-West China. Intraplantar injection of the venom into the hind paws of mice induced severe acute pain behaviors in wild type (WT) mice; the responses were much reduced in TRPV1-deficit (TRPV1 KO) mice. The TRPV1-specific inhibitor, capsazepine, significantly attenuated the pain behaviors. Furthermore, the crude venom evoked strong calcium signals in the dorsal root ganglion (DRG) neurons of WT mice but not those of TRPV1 KO mice. Among the pain-related ion channels we tested, the crude venom only activated the TRPV1 channel. To better understand the venom components, we analyzed the transcriptome of the L. consocia sebaceous gland region. Our study suggests that TRPV1 serves as a primary nociceptor in caterpillar-induced pain and forms the foundation for elucidating the pain-producing mechanism. Full article

The venom of predators should be under strong selection pressure because it is a costly substance and prey may potentially become resistant. Particularly in prey-specialized predators, venom should be selected for its high efficiency against the focal prey. Very effective venom paralysis has been observed in specialized predators, such as spiders preying on dangerous prey. Here, we compared the toxicity of the venoms of two prey-specialized species, araneophagous Palpimanus sp. and myrmecophagous Zodarion nitidum, and their related generalist species. We injected different venom concentrations into two prey types—the prey preferred by a specialist and an alternative prey—and observed the mortality and the paralysis of the prey within 24 h. We found that the venoms of specialists were far more potent towards the preferred prey than alternative prey. The venoms of generalists were similarly potent towards both prey types. In addition, we tested the efficacy of two venom fractions (smaller and larger than 10 kDa) in araneophagous Palpimanus sp. Compounds larger than 10 kDa paralyzed both prey types, but smaller compounds (<10 kDa) were effective only on preferred prey, suggesting the presence of prey-specific compounds in the latter fraction. Our results confirm that prey-specialized spiders possess highly specific venom that allows them to subdue dangerous prey. Full article

Consumption of fruit juice is becoming trendy for consumers seeking freshness and high vitamin and low caloric intake. Mycotoxigenic moulds may infect fruits during crop growth, harvest, and storage leading to mycotoxin production. Many mycotoxins are resistant to food processing, which make their presence in the final juice product very likely expected. In this way, the presence of 30 mycotoxins including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), alternariol (AOH), alternariol monomethyl ether (AME), Ochratoxin A (OTA), fumonisin B1 (FB1), fumonisin B2 (FB2), enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1), beauvericin (BEA), sterigmatocystin (STG), zearalenone (ZEA), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON), diacetoxyscirpenol (DAS), nivalenol (NIV), fusarenon-X (FUS-X), neosolaniol (NEO), patulin (PAT), T-2 toxin and HT-2 toxin was evaluated in 80 juice samples collected from Valencia retail Market. An efficient Dispersive Liquid-Liquid Microextraction method (DLLME) was carried out before their trace level determination by chromatographic techniques coupled to tandem mass spectrometry. The results obtained revealed the presence of nine mycotoxins namely AOH, AME, PAT, OTA, AFB1, AFB2, AFG2, β-ZAL, and HT2 in the analyzed samples, with incidences ranging from 3 to 29% and mean contents between 0.14 and 59.52 µg/L. Considerable percentages of TDIs were reached by children when 200 mL was considered as daily fruit juice intake. Full article

Human poisoning by microcystin has been recorded in many countries, including Brazil, where fatal cases have already occurred. The Amazon River is the main source of drinking water in municipalities such as Macapá, where there is no monitoring of cyanobacteria and cyanotoxins. This study investigated the presence of cyanobacteria and cyanotoxins in samples from a drinking water treatment plant (DWTP) that catches water from the Amazon River. The toxin analyses employed ELISA, LC/MS, and molecular screening for genes involved in the production of cyanotoxins. The sampling was carried out monthly from April 2015 to April 2016 at the intake (raw water) and exit (treated water) of the DWTP. This study reports the first detection of microcystin-LR (MC-LR) in the Amazon River, the world’s largest river, and in its treated water destined for drinking water purposes in Macapá, Brazil. The cyanobacterial density and MC-LR concentration were both low during the year. However, Limnothrix planctonica showed a density peak (± 900 cells mL⁻¹) in the quarter of June–August 2015, when MC-LR was registered (2.1 µg L⁻¹). Statistical analyses indicate that L. planctonica may produce the microcystin. Full article

Deoxynivalenol (DON) is highly toxic to animals and humans, but pigs are most sensitive to it. The porcine mucosal injury related mechanism of DON is not yet fully clarified. Here, we investigated DON-induced injury in the intestinal tissues of piglet. Thirty weanling piglets [(Duroc × Landrace) × Yorkshire] were randomly divided into three groups according to single factor experimental design (10 piglets each group). Piglets were fed a basal diet in the control group, while low and high dose groups were fed a DON diet (1300 and 2200 μg/kg, respectively) for 60 days. Scanning electron microscopy results indicated that the ultrastructure of intestinal epithelial cells in the DON-treated group was damaged. The distribution and optical density (OD) values of zonula occludens 1 (ZO-1) protein in the intestinal tissues of DON-treated groups were decreased. At higher DON dosage, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α mRNA levels were elevated in the intestinal tissues. The mRNA and protein levels of NF-κB p65, IκB-α, IKKα/β, iNOS, and COX-2 in the small intestinal mucosa were abnormally altered with an increase in DON concentration. These results indicate that DON can persuade intestinal damage and inflammatory responses in piglets via the nuclear factor-κB signaling pathway. Full article

Contamination of animal feed with multiple mycotoxins is an ongoing and growing issue, as over 60% of cereal crops worldwide have been shown to be contaminated with mycotoxins. The present study was carried out to assess the efficacy of commercial feed additives sold with multi-mycotoxin binding claims. Ten feed additives were obtained and categorised into three groups based on their main composition. Their capacity to simultaneously adsorb deoxynivalenol (DON), zearalenone (ZEN), fumonisin B1 (FB1), ochratoxin A (OTA), aflatoxin B1 (AFB1) and T-2 toxin was assessed and compared using an in vitro model designed to simulate the gastrointestinal tract of a monogastric animal. Results showed that only one product (a modified yeast cell wall) effectively adsorbed more than 50% of DON, ZEN, FB1, OTA, T-2 and AFB1, in the following order: AFB1 > ZEN > T-2 > DON > OTA > FB1. The remaining products were able to moderately bind AFB1 (44–58%) but had less, or in some cases, no effect on ZEN, FB1, OTA and T-2 binding (<35%). It is important for companies producing mycotoxin binders that their products undergo rigorous trials under the conditions which best mimic the environment that they must be active in. Claims on the binding efficiency should only be made when such data has been generated. Full article

An intercollaborative study was organized to evaluate the performance characteristics of a liquid chromatography tandem mass spectrometry procedure for the simultaneous determination of 12 mycotoxins in food, which were ochratoxin A, aflatoxins B1, B2, G1, G2, and M1, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins. The method combined the simplicity of the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) approach with the efficiency of immunoaffinity column cleanup (the step used to enhance sensitivity and sample cleanup for some matrices only). Twenty-three entities were enrolled and were European reference laboratories for mycotoxin analysis, U.S. and European service laboratories, and Nestlé laboratories. Each participant analyzed 28 incurred and/or spiked blind samples composed of spices, nuts, milk powder, dried fruits, cereals, and baby food using the protocol given. Method performances were assessed according to ISO 5725-2. Relative standard deviations of repeatability and reproducibility and trueness values for each of the 115 mycotoxin/sample combinations ranged from 5% to 23%, 7% to 26%, and 85% to 129%, respectively, in line with requirements defined in EC 401/2006. The overall set of data gathered demonstrated that the method offered a unique platform to ensure compliance with EC 1881/2006 and EC 165/2013 regulations setting maximum limits for mycotoxins in food samples, even at low regulated levels for foods intended for infants and young children. The method was applicable regardless of the food, the regulated mycotoxin, and the concentration level, and thus is an excellent candidate for future standardization. Full article

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, results in major economic losses in soybean production in southern USA. M. phaseolina has been proposed to use the toxin (-)-botryodiplodin in its root infection mechanism to create a necrotic zone in root tissue through which fungal hyphae can readily enter the plant. The majority (51.4%) of M. phaseolina isolates from plants with charcoal rot disease produced a wide range of (-)-botryodiplodin concentrations in a culture medium (0.14–6.11 µg/mL), 37.8% produced traces below the limit of quantification (0.01 µg/mL), and 10.8% produced no detectable (-)-botryodiplodin. Some culture media with traces or no (-)-botryodiplodin were nevertheless strongly phytotoxic in soybean leaf disc cultures, consistent with the production of another unidentified toxin(s). Widely ranging (-)-botryodiplodin levels (traces to 3.14 µg/g) were also observed in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. This is the first report of (-)-botryodiplodin in plant tissues naturally infected with charcoal rot disease. No phaseolinone was detected in M. phaseolina culture media or naturally infected soybean tissues. These results are consistent with (-)-botryodiplodin playing a role in the pathology of some, but not all, M. phaseolina isolates from soybeans with charcoal rot disease in southern USA. Full article

Aflatoxin (AF) contamination of maize is a major concern for food safety. The use of chemical fungicides is controversial, and it is necessary to develop new effective methods to control Aspergillus flavus growth and, therefore, to avoid the presence of AFs in grains. In this work, we tested in vitro the effect of six essential oils (EOs) extracted from aromatic plants. We selected those from Satureja montana and Origanum virens because they show high levels of antifungal and antitoxigenic activity at low concentrations against A. flavus. EOs are highly volatile compounds and we have developed a new niosome-based encapsulation method to extend their shelf life and activity. These new formulations have been successfully applied to reduce fungal growth and AF accumulation in maize grains in a small-scale test, as well as placing the maize into polypropylene woven bags to simulate common storage conditions. In this latter case, the antifungal properties lasted up to 75 days after the first application. Full article

Gut microbiota-dependent Trimethylamine-N-oxide (TMAO) has been reported to be strongly linked to renal function and to increased cardiovascular events in the general population and in Chronic Kidney Disease (CKD) patients. Considering the lack of data assessing renal handling of TMAO, we conducted this study to explore renal excretion and mechanisms of accumulation of TMAO during CKD. We prospectively measured glomerular filtration rate (mGFR) with gold standard methods and plasma concentrations of trimethylamine (TMA), TMAO, choline, betaine, and carnitine by LC-MS/MS in 124 controls, CKD, and hemodialysis (HD) patients. Renal clearance of each metabolite was assessed in a sub-group of 32 patients. Plasma TMAO was inversely correlated with mGFR (r² = 0.388, p < 0.001), confirming elevation of TMAO plasma levels in CKD. TMAO clearances were not significantly different from mGFR, with a mean ± SD TMAO fractional excretion of 105% ± 32%. This suggests a complete renal excretion of TMAO by glomerular filtration with a negligible participation of tubular secretion or reabsorption, during all stages of CKD. Moreover, TMAO was effectively removed within 4 h of hemodiafiltration, showing a higher fractional reduction value than that of urea (84.9% ± 6.5% vs. 79.2% ± 5.7%, p = 0.04). This study reports a strong correlation between plasma TMAO levels and mGFR, in CKD, that can be mainly related to a decrease in TMAO glomerular filtration. Clearance data did not support a significant role for tubular secretion in TMAO renal elimination. Full article

Mycotoxins are produced by fungi and are potentially toxic to pigs. Yeast cell wall extract (YCWE) is known to adsorb mycotoxins and improve gut health in pigs. One hundred and twenty growing (56 kg; experiment 1) and 48 nursery piglets (6 kg; experiment 2) were assigned to four dietary treatments in a 2 × 2 factorial design for 35 and 48 days, respectively. Factors were mycotoxins (no addition versus experiment 1: 180 μg/kg aflatoxins and 14 mg/kg fumonisins; or experiment 2: 180 μg/kg aflatoxins and 9 mg/kg fumonisins, and 1 mg/kg deoxynivalenol) and YCWE (0% versus 0.2%). Growth performance, blood, gut health and microbiome, and apparent ileal digestibility (AID) data were evaluated. In experiment 1, mycotoxins reduced ADG and G:F, and duodenal IgG, whereas in jejunum, YCWE increased IgG and reduced villus width. In experiment 2, mycotoxins reduced BW, ADG, and ADFI. Mycotoxins reduced ADG, which was recovered by YCWE. Mycotoxins reduced the AID of nutrients evaluated and increased protein carbonyl, whereas mycotoxins and YCWE increased the AID of the nutrients and reduced protein carbonyl. Mycotoxins reduced villus height, proportion of Ki-67-positive cells, and increased IgA and the proportion of bacteria with mycotoxin-degrading ability, whereas YCWE tended to increase villus height and reduced IgA and the proportion of pathogenic bacteria in jejunum. The YCWE effects were more evident in promoting gut health and growth in nursery pigs, which showed higher susceptibility to mycotoxin effects. Full article

Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94–0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events Full article

Parasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with the crosses of two lines of Leptopilina boulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance/susceptibility to the parental parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE electrophoresis protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results made evident a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened. Full article

(1) Background. Snake venom phosphodiesterases (SVPDEs) are among the least studied venom enzymes. In envenomation, they display various pathological effects, including induction of hypotension, inhibition of platelet aggregation, edema, and paralysis. Until now, there have been no 3D structural studies of these enzymes, thereby preventing structure–function analysis. To enable such investigations, the present work describes the model-based structural and functional characterization of a phosphodiesterase from Crotalus adamanteus venom, named PDE_Ca. (2) Methods. The PDE_Ca structure model was produced and validated using various software (model building: I-TESSER, MODELLER 9v19, Swiss-Model, and validation tools: PROCHECK, ERRAT, Molecular Dynamic Simulation, and Verif3D). (3) Results. The proposed model of the enzyme indicates that the 3D structure of PDE_Ca comprises four domains, a somatomedin B domain, a somatomedin B-like domain, an ectonucleotide pyrophosphatase domain, and a DNA/RNA non-specific domain. Sequence and structural analyses suggest that differences in length and composition among homologous snake venom sequences may account for their differences in substrate specificity. Other properties that may influence substrate specificity are the average volume and depth of the active site cavity. (4) Conclusion. Sequence comparisons indicate that SVPDEs exhibit high sequence identity but comparatively low identity with mammalian and bacterial PDEs. Full article

Cyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 μg/L and 50 μg/L, or CYN/MC mixture at 5 + 5 μg/L and 25 + 25 μg/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50µg/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 µg/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxins. Full article

Spiders rely on venom to catch prey and few species are even capable of capturing vertebrates. The majority of spiders are generalist predators, possessing complex venom, in which different toxins seem to target different types of prey. In this study, we focused on the trophic ecology and venom toxicity of Phoneutria boliviensis F. O. Pickard-Cambridge, 1897, a Central American spider of medical importance. We tested the hypothesis that its venom is adapted to catch vertebrate prey by studying its trophic ecology and venom toxicity against selected vertebrate and invertebrate prey. We compared both trophic ecology (based on acceptance experiments) and toxicity (based on bioassays) among sexes of this species. We found that P. boliviensis accepted geckos, spiders, and cockroaches as prey, but rejected frogs. There was no difference in acceptance between males and females. The venom of P. boliviensis was far more efficient against vertebrate (geckos) than invertebrate (spiders) prey in both immobilization time and LD50. Surprisingly, venom of males was more efficient than that of females. Our results suggest that P. boliviensis has adapted its venom to catch vertebrates, which may explain its toxicity to humans. Full article

In this study, magnetic graphene nanocomposite Fe₃O₄/rGO was synthesized by facile one-pot solvothermal method. The nanocomposite was successfully used as magnetic solid phase extraction (MSPE) adsorbents for the determination of aflatoxins in edible vegetable oils through the π–π stacking interactions. MSPE parameters including the amount of adsorbents, extraction and desorption time, washing conditions, and the type and volume of desorption solvent were optimized. Under optimal conditions, good linear relationships were achieved. Limits of detection of this method were as low as 0.02 µg/kg and 0.01 µg/kg for aflatoxin B₁ and B₂, respectively. Finally, the magnetic graphene nanocomposite was successfully applied to aflatoxin analysis in vegetable oils. The results indicated that the recoveries of the B-group aflatoxins ranged from 80.4% to 106.0%, whereas the relative standard deviations (RSDs) were less than 8.1%. Owing to the simplicity, rapidity and efficiency, Fe₃O₄/rGO magnetic solid phase extraction coupled with high-performance liquid chromatography fluorescence with post-column photochemical derivatization (Fe₃O₄/rGO MSPE-HPLC-PCD-FLD) is a promising analytical method for routine and accurate determination of aflatoxins in lipid matrices. Full article

Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu¹, Ser⁷] MC-HtyR, [Asp³]MC-RHar, AP731, and AP803). The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L⁻¹. To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems. Full article

Cyanobacterial blooms increasingly impair inland waters, with the potential for a concurrent increase in cyanotoxins that have been linked to animal and human mortalities. Microcystins (MCs) are among the most commonly detected cyanotoxins, but little is known about the distribution of different MC congeners despite large differences in their biomagnification, persistence, and toxicity. Using raw-water intake data from sites around the Great Lakes basin, we applied multivariate canonical analyses and regression tree analyses to identify how different congeners (MC-LA, -LR, -RR, and -YR) varied with changes in meteorological and nutrient conditions over time (10 years) and space (longitude range: 77°2′60 to 94°29′23 W). We found that MC-LR was associated with strong winds, warm temperatures, and nutrient-rich conditions, whereas the equally toxic yet less commonly studied MC-LA tended to dominate under intermediate winds, wetter, and nutrient-poor conditions. A global synthesis of lake data in the peer-reviewed literature showed that the composition of MC congeners differs among regions, with MC-LA more commonly reported in North America than Europe. Global patterns of MC congeners tended to vary with lake nutrient conditions and lake morphometry. Ultimately, knowledge of the environmental factors leading to the formation of different MC congeners in freshwaters is necessary to assess the duration and degree of toxin exposure under future global change. Full article