Calf diarrhea is one of the considerable infectious diseases in calves, which results in tremendous economic losses globally. To determine the prevalence of Shiga-toxigenic E. coli (STEC) and Enterotoxigenic E. coli (ETEC) incriminated in calf diarrhea, with special reference to Shiga- toxins genes (stx1 and stx2) and enterotoxins genes (lt and sta) that govern their pathogenesis, as well as the virulence genes; eaeA (intimin) and f41(fimbrial adhesion), and the screening of their antibiogram and antimicrobial resistance genes; aadB, sul1, and bla-TEM, a total of 274 fecal samples were collected (April 2018–Feb 2019) from diarrheic calves at different farms in El-Sharqia Governorate, Egypt. The bacteriological examination revealed that the prevalence of E. coli in diarrheic calves was 28.8%. The serotyping of the isolated E. coli revealed 7 serogroups; O₂₆, O₁₂₈, O₁₁₁, O₁₂₅, O₄₅, O₁₁₉ and O₉₁. Furthermore, the Congo red binding test was carried out, where 89.8% of the examined strains (n = 71) were positive. The antibiogram of the isolated strains was investigated; the majority of E. coli serotypes exhibit multidrug resistance (MDR) to four antimicrobial agents; neomycin, gentamycin, streptomycin, and amikacin. Polymerase chain reaction (PCR) was used to detect the prevalence of the virulence genes; stx1, stx2 lt, sta, f41 and eaeA, as well as the antimicrobial resistance genes; aadB, sul1, and bla-TEM. The prevalence of STEC was 20.2% (n = 16), while the prevalence of ETEC was 30.4% (n = 24). Briefly, the Shiga toxins genes; stx1 and stx2, are the most prevalent virulence genes associated with STEC, which are responsible for the pathogenesis of the disease and helped by the intimin gene (eaeA). In addition, the lt gene is the most prevalent enterotoxin gene accompanied by the ETEC strains, either alone or in combination with sta and/or f41 genes. The majority of pathogenic E. coli incriminated in calf diarrhea possesses the aadB resistance gene, followed by the sul1 gene. Enrofloxacin, florfenicol, amoxicillin-clavulanic acid, and ampicillin-sulbactam, are the most effective antimicrobial agents against the isolated STEC and ETEC strains. Full article

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 μM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites. Full article

The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalis ABCC2 (OfABCC2) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis. Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin. Full article

Beauvericin (BEA) and zearalenone derivatives, α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL), are produced by several Fusarium species. Considering the impact of various mycotoxins on human’s health, this study determined and evaluated the cytotoxic effect of individual, binary, and tertiary mycotoxin treatments consisting of α-ZEL, β-ZEL, and BEA at different concentrations over 24, 48, and 72 h on SH-SY5Y neuronal cells, by using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide). Subsequently, the isobologram method was applied to elucidate if the mixtures produced synergism, antagonism, or additive effects. Ultimately, we determined the amount of mycotoxin recovered from the media after treatment using liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry (LC–ESI–qTOF-MS). The IC₅₀ values detected at all assayed times ranged from 95 to 0.2 μM for the individual treatments. The result indicated that β-ZEL was the most cytotoxic mycotoxin when tested individually. The major effect detected for all combinations assayed was synergism. Among the combinations assayed, α-ZEL + β-ZEL + BEA and α-ZEL + BEA presented the highest cytotoxic potential with respect to the IC value. In individual treatment, α-ZEL was the most recovered mycotoxin; while, this was observed for BEA in binary combination α-ZEL + BEA. Full article

Prey-selective venoms and toxins have been documented across only a few species of snakes. The lack of research in this area has been due to the absence of suitably flexible testing platforms. In order to test more species for prey specificity of their venom, we used an innovative taxonomically flexible, high-throughput biolayer interferometry approach to ascertain the relative binding of 29 α-neurotoxic venoms from African and Asian elapid representatives (26 Naja spp., Aspidelaps scutatus, Elapsoidea boulengeri, and four locales of Ophiophagus hannah) to the alpha-1 nicotinic acetylcholine receptor orthosteric (active) site for amphibian, lizard, snake, bird, and rodent targets. Our results detected prey-selective, intraspecific, and geographical differences of α-neurotoxic binding. The results also suggest that crude venom that shows prey selectivity is likely driven by the proportions of prey-specific α-neurotoxins with differential selectivity within the crude venom. Our results also suggest that since the α-neurotoxic prey targeting does not always account for the full dietary breadth of a species, other toxin classes with a different pathophysiological function likely play an equally important role in prey immobilisation of the crude venom depending on the prey type envenomated. The use of this innovative and taxonomically flexible diverse assay in functional venom testing can be key in attempting to understanding the evolution and ecology of α-neurotoxic snake venoms, as well as opening up biochemical and pharmacological avenues to explore other venom effects. Full article

A survey on 120 cereal samples (barley, maize, rice and wheat) from Algerian markets has been carried out to evaluate the presence of 15 mycotoxins (ochratoxin A, deoxynivalenol, fumonisin B1 and B2, T-2 and HT-2 toxins, zearalenone, fusarenon X, citrinin, sterigmatocystin, enniatins A, A1, B and B1, and beauvericin). With this purpose, a QuEChERS-based extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) were used. Analytical results showed that 78 cereal samples (65%) were contaminated with at least one toxin, while 50% were contaminated with three to nine mycotoxins. T-2 toxin, citrinin, beauvericin and deoxynivalenol were the most commonly found mycotoxins (frequency of 50%, 41.6%, 40.8% and 33.3%, respectively). Fumonisins (B1 + B2), enniatins B and B1, deoxynivalenol and zearalenone registered high concentrations (289–48878 µg/kg, 1.2–5288 µg/kg, 15–4569 µg/kg, 48–2055 µg/kg and 10.4–579 µg/kg, respectively). Furthermore, concentrations higher than those allowed by the European Union (EU) were observed in 21, 8 and 1 samples for fumonisins, zearalenone and deoxinivalenol, respectively. As a conclusion, the high levels of fumonisins (B1 + B2) in maize and deoxynivalenol, zearalenone and HT-2 + T-2 toxins in wheat, represent a health risk for the average adult consumer in Algeria. These results pointed out the necessity of a consistent control and the definition of maximum allowed levels for mycotoxins in Algerian foodstuffs. Full article

Aspergillus flavus is the main producer of aflatoxin B1, one of the most toxic contaminants of food and feed. With global warming, climate conditions have become favourable for aflatoxin contamination of agricultural products in several European countries, including Serbia. The infection of maize with A. flavus, and aflatoxin synthesis can be controlled and reduced by application of a biocontrol product based on non-toxigenic strains of A. flavus. Biological control relies on competition between atoxigenic and toxigenic strains. This is the most commonly used biological control mechanism of aflatoxin contamination in maize in countries where aflatoxins pose a significant threat. Mytoolbox Af01, a native atoxigenic A. flavus strain, was obtained from maize grown in Serbia and used to produce a biocontrol product that was applied in irrigated and non-irrigated Serbian fields during 2016 and 2017. The application of this biocontrol product reduced aflatoxin levels in maize kernels (51–83%). The biocontrol treatment had a highly significant effect of reducing total aflatoxin contamination by 73%. This study showed that aflatoxin contamination control in Serbian maize can be achieved through biological control methods using atoxigenic A. flavus strains. Full article

Mycotoxins found in randomly selected commercial milk thistle dietary supplement were evaluated for their toxicity in silico and in vitro. Using in silico methods, the basic physicochemical, pharmacological, and toxicological properties of the mycotoxins were predicted using ACD/Percepta. The in vitro cytotoxicity of individual mycotoxins was determined in mouse macrophage (RAW 264.7), human hepatoblastoma (HepG2), and human embryonic kidney (HEK 293T) cells. In addition, we studied the bioavailability potential of mycotoxins and silibinin utilizing an in vitro transwell system with differentiated human colon adenocarcinoma cells (Caco-2) simulating mycotoxin transfer through the intestinal epithelial barrier. The IC₅₀ values for individual mycotoxins in studied cells were in the biologically relevant ranges as follows: 3.57–13.37 nM (T-2 toxin), 5.07–47.44 nM (HT-2 toxin), 3.66–17.74 nM (diacetoxyscirpenol). Furthermore, no acute toxicity was obtained for deoxynivalenol, beauvericin, zearalenone, enniatinENN-A, enniatin-A1, enniatin-B, enniatin-B1, alternariol, alternariol-9-methyl ether, tentoxin, and mycophenolic acid up to the 50 nM concentration. The acute toxicity of these mycotoxins in binary combinations exhibited antagonistic effects in the combinations of T-2 with DON, ENN-A1, or ENN-B, while the rest showed synergistic or additive effects. Silibinin had a significant protective effect against both the cytotoxicity of three mycotoxins (T-2 toxin, HT-2 toxin, DAS) and genotoxicity of AME, AOH, DON, and ENNs on HEK 293T. The bioavailability results confirmed that AME, DAS, ENN-B, TEN, T-2, and silibinin are transported through the epithelial cell layer and further metabolized. The bioavailability of silibinin is very similar to mycotoxins poor penetration. Full article

The presence of mycotoxins in cereal grain is a very important food safety issue with the occurrence of masked mycotoxins extensively investigated in recent years. This study investigated the variation of different Fusarium metabolites (including the related regulated, masked, and emerging mycotoxin) in maize from various agriculture regions of South Africa. The relationship between the maize producing regions, the maize type, as well as the mycotoxins was established. A total of 123 maize samples was analyzed by a LC-MS/MS multi-mycotoxin method. The results revealed that all maize types exhibited a mixture of free, masked, and emerging mycotoxins contamination across the regions with an average of 5 and up to 24 out of 42 investigated Fusarium mycotoxins, including 1 to 3 masked forms at the same time. Data obtained show that fumonisin B_1, B_2, B_3, B₄, and A₁ were the most prevalent mycotoxins and had maximum contamination levels of 8908, 3383, 990, 1014, and 51.5 µg/kg, respectively. Deoxynivalenol occurred in 50% of the samples with a mean concentration of 152 µg/kg (max 1380 µg/kg). Thirty-three percent of the samples were contaminated with zearalenone at a mean concentration of 13.6 µg/kg (max 146 µg/kg). Of the masked mycotoxins, DON-3-glucoside occurred at a high incidence level of 53%. Among emerging toxins, moniliformin, fusarinolic acid, and beauvericin showed high occurrences at 98%, 98%, and 83%, and had maximum contamination levels of 1130, 3422, and 142 µg/kg, respectively. Significant differences in the contamination pattern were observed between the agricultural regions and maize types. Full article

In the last decade, several outbreaks of ciguatera fish poisoning (CFP) have been reported in the Canary Islands (central northeast Atlantic Ocean), confirming ciguatera as an emerging alimentary risk in this region. Five Gambierdiscus species, G. australes, G. excentricus, G. silvae, G. carolinianus and G. caribaeus, have been detected in macrophytes from this area and are known to produce the ciguatoxins (CTXs) that cause CFP. A characterization of the toxicity of these species is the first step in identifying locations in the Canary Islands at risk of CFP. Therefore, in this study the toxicity of 63 strains of these five Gambierdiscus species were analysed using the erythrocyte lysis assay to evaluate their maitotoxin (MTX) content. In addition, 20 of the strains were also analysed in a neuroblastoma Neuro-2a (N2a) cytotoxicity assay to determine their CTX-like toxicity. The results allowed the different species to be grouped according to their ratios of CTX-like and MTX-like toxicity. MTX-like toxicity was especially high in G. excentricus and G. australes but much lower in the other species and lowest in G. silvae. CTX-like toxicity was highest in G. excentricus, which produced the toxin in amounts ranging between 128.2 ± 25.68 and 510.6 ± 134.2 fg CTX1B equivalents (eq) cell⁻¹ (mean ± SD). In the other species, CTX concentrations were as follows: G. carolinianus (100.84 ± 18.05 fg CTX1B eq cell⁻¹), G. australes (31.1 ± 0.56 to 107.16 ± 21.88 fg CTX1B eq cell⁻¹), G. silvae (12.19 ± 0.62 to 76.79 ± 4.97 fg CTX1B eq cell⁻¹) and G. caribaeus (<LOD to 90.37 ± 15.89 fg CTX1B eq cell⁻¹). Unlike the similar CTX-like toxicity of G. australes and G. silvae strains from different locations, G. excentricus and G. caribaeus differed considerably according to the origin of the strain. These differences emphasise the importance of species identification to assess the regional risk of CFP. Full article

The phospholipase A₂ (PLA₂) inhibitor Varespladib (LY315920) and its orally bioavailable prodrug, methyl-Varespladib (LY333013) inhibit PLA₂ activity of a wide variety of snake venoms. In this study, the ability of these two forms of Varespladib to halt or delay lethality of potent neurotoxic snake venoms was tested in a mouse model. The venoms of Notechis scutatus, Crotalus durissus terrificus, Bungarus multicinctus, and Oxyuranus scutellatus, all of which have potent presynaptically acting neurotoxic PLA₂s of variable quaternary structure, were used to evaluate simple dosing regimens. A supralethal dose of each venom was injected subcutaneously in mice, followed by the bolus intravenous (LY315920) or oral (LY333013) administration of the inhibitors, immediately and at various time intervals after envenoming. Control mice receiving venom alone died within 3 h of envenoming. Mice injected with O. scutellatus venom and treated with LY315920 or LY333013 survived the 24 h observation period, whereas those receiving C. d. terrificus and B. multicinctus venoms survived at 3 h or 6 h with a single dose of either form of Varespladib, but not at 24 h. In contrast, mice receiving N. scutatus venom and then the inhibitors died within 3 h, similarly to the control animals injected with venom alone. LY315920 was able to reverse the severe paralytic manifestations in mice injected with venoms of O. scutellatus, B. multicinctus, and C. d. terrificus. Overall, results suggest that the two forms of Varespladib are effective in abrogating, or delaying, neurotoxic manifestations induced by some venoms whose neurotoxicity is mainly dependent on presynaptically acting PLA₂s. LY315920 is able to reverse paralytic manifestations in severely envenomed mice, but further work is needed to understand the significance of species-specific differences in animal models as they compare to clinical syndromes in human and for potential use in veterinary medicine. Full article

Vip3Aa, a soluble protein produced by certain Bacillus thuringiensis strains, is capable of inducing apoptosis in Sf9 cells. However, the apoptosis mechanism triggered by Vip3Aa is unclear. In this study, we found that Vip3Aa induces mitochondrial dysfunction, as evidenced by signs of collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, release of cytochrome c, and caspase-9 and -3 activation. Meanwhile, our results indicated that Vip3Aa reduces the ability of lysosomes in Sf9 cells to retain acridine orange. Moreover, pretreatment with Z-Phe-Tyr-CHO (a cathepsin L inhibitor) or pepstatin (a cathepsin D inhibitor) increased Sf9 cell viability, reduced cytochrome c release, and decreased caspase-9 and -3 activity. In conclusion, our findings suggested that Vip3Aa promotes Sf9 cell apoptosis by mitochondrial dysfunction, and lysosomes also play a vital role in the action of Vip3Aa. Full article

Cannabidiol (CBD) food supplements made of Cannabis sativa L. extracts have quickly become popular products due to their health-promoting effects. However, potential contaminants, such as mycotoxins and pesticides, can be coextracted during the manufacturing process and placed into the final product. Accordingly, a novel methodology using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was developed to quantify 16 mycotoxins produced by major C. sativa fungi, followed by a post-target screening of 283 pesticides based on a comprehensive spectral library. The validated procedure was applied to ten CBD-based products. Up to six different Fusarium mycotoxins were found in seven samples, the most prevalent being zearalenone (60%) and enniatin B1 (30%), both found at a maximum level of 11.6 ng/g. Co-occurrence was observed in four samples, including one with enniatin B1, enniatin A and enniatin A1. On the other hand, 46 different pesticides were detected after retrospective analysis. Ethoxyquin (50%), piperonyl butoxide (40%), simazine (30%) and cyanazine (30%) were the major residues found. These results highlight the necessity of monitoring contaminants in food supplements in order to ensure a safe consumption, even more considering the increase trend in their use. Furthermore, the developed procedure is proposed as a powerful analytical tool to evaluate the potential mycotoxin profile of these particular products. Full article

Ruminants are generally considered to be less susceptible to the effects of mycotoxins than monogastric animals as the rumen microbiota are capable of detoxifying some of these toxins. Despite this potential degradation, mycotoxin-associated subclinical health problems are seen in dairy cows. In this research, the disappearance of several mycotoxins was determined in an in vitro rumen model and the effect of realistic concentrations of those mycotoxins on fermentation was assessed by volatile fatty acid production. In addition, two hypotheses were tested: (1) a lower rumen pH leads to a decreased degradation of mycotoxins and (2) rumen fluid of lactating cows degrade mycotoxins better than rumen fluid of non-lactating cows. Maize silage was spiked with a mixture of deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROQ-C) and zearalenone (ZEN). Fresh rumen fluid of two lactating cows (L) and two non-lactating cows (N) was added to a buffer of normal pH (6.8) and low pH (5.8), leading to four combinations (L6.8, L5.8, N6.8, N5.8), which were added to the spiked maize substrate. In this study, mycotoxins had no effect on volatile fatty acid production. However, not all mycotoxins fully disappeared during incubation. ENN B and ROQ-C disappeared only partially, whereas MPA showed almost no disappearance. The disappearance of DON, NIV, and ENN B was hampered when pH was low, especially when the inoculum of non-lactating cows was used. For ZEN, a limited transformation of ZEN to α-ZEL and β-ZEL was observed, but only at pH 6.8. In conclusion, based on the type of mycotoxin and the ruminal conditions, mycotoxins can stay intact in the rumen. Full article

Snake venom metalloproteinases (SVMPs) play an important role in local tissue damage of snakebite patients, mostly by hydrolysis of basement membrane (BM) components. We evaluated the proinflammatory activity of SVMPs Atroxlysin-Ia (ATXL) and Batroxrhagin (BATXH) from Bothrops atrox venom and their hydrolysis products of Matrigel. BALB/c mice were injected with SVMPs (2 μg), for assessment of paw edema and peritoneal leukocyte accumulation. Both SVMPs induced edema, representing an increase of ~70% of the paw size. Leukocyte infiltrates reached levels of 6 × 10⁶ with ATXL and 5 × 10⁶ with BATXH. TNF-α was identified in the supernatant of BATXH—or venom-stimulated MPAC cells. Incubation of Matrigel with the SVMPs generated fragments, including peptides from Laminin, identified by LC–MS/MS. The Matrigel hydrolysis peptides caused edema that increased 30% the paw size and promoted leukocyte accumulation (4–5 × 10⁶) to the peritoneal cavity, significantly higher than Matrigel control peptides 1 and 4 h after injection. Our findings suggest that ATXL and BATXH are involved in the inflammatory reaction observed in B. atrox envenomings by direct action on inflammatory cells or by releasing proinflammatory peptides from BM proteins that may amplify the direct action of SVMPs through activation of endogenous signaling pathways. Full article

[D-Leu¹]MC-LY (1) ([M + H]⁺ m/z 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from Microcystis aeruginosa strain CPCC-464. The compound was characterized by ¹H and ¹³C NMR spectroscopy, liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by ¹H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of 1 in a protein phosphatase 2A inhibition assay was essentially the same as for MC-LR (2). Related microcystins, [D-Leu¹]MC-LR (3) ([M + H]⁺ m/z 1037.6041, Δ 1.0 ppm), [D-Leu¹]MC-M(O)R (6) ([M + H]⁺ m/z 1071.5565, Δ 2.0 ppm) and [D-Leu¹]MC-MR (7) ([M + H]⁺ m/z 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu¹]MC-M(O₂)R (8) ([M + H]⁺ m/z 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC–HRMS/MS experiments. The relative abundances of 1, 3, 6, 7 and 8 in a freshly extracted culture in the positive ionization mode LC–HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu¹]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC–MS/MS screening methods. Full article

Venoms are best known for their ability to incapacitate prey. In predatory groups, venom potency is predicted to reflect ecological and evolutionary drivers relating to diet. While venoms have been found to have prey-specific potencies, the role of diet breadth on venom potencies has yet to be tested at large macroecological scales. Here, using a comparative analysis of 100 snake species, we show that the evolution of prey-specific venom potencies is contingent on the breadth of a species’ diet. We find that while snake venom is more potent when tested on species closely related to natural prey items, we only find this prey-specific pattern in species with taxonomically narrow diets. While we find that the taxonomic diversity of a snakes’ diet mediates the prey specificity of its venom, the species richness of its diet was not found to affect these prey-specific potency patterns. This indicates that the physiological diversity of a species’ diet is an important driver of the evolution of generalist venom potencies. These findings suggest that the venoms of species with taxonomically diverse diets may be better suited to incapacitating novel prey species and hence play an important role for species within changing environments. 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

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

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

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

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

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

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

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

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

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

Aflatoxin B₁ (AFB₁) and zearalenone (ZEN) exert deleterious effects to human and animal health. In this study, the ability of a CotA laccase from Bacillus subtilis (BsCotA) to degrade these two mycotoxins was first investigated. Among the nine structurally defined chemical compounds, methyl syringate was the most efficient mediator assisting BsCotA to degrade AFB₁ (98.0%) and ZEN (100.0%). BsCotA could also use plant extracts, including the Epimedium brevicornu, Cucumis sativus L., Lavandula angustifolia, and Schizonepeta tenuifolia extracts to degrade AFB₁ and ZEN. Using hydra and BLYES as indicators, it was demonstrated that the degraded products of AFB₁ and ZEN using the laccase/mediator systems were detoxified. Finally, a laccase of fungal origin was also able to degrade AFB₁ and ZEN in the presence of the discovered mediators. The findings shed light on the possibility of using laccases and a mediator, particularly a natural plant-derived complex mediator, to simultaneously degrade AFB₁ and ZEN contaminants in food and feed. Full article

Escherichia coli O157:H7 is the predominant cause of diarrhea-associated hemolytic uremic syndrome (HUS) worldwide. Its cardinal virulence traits are Shiga toxins, which are encoded by stx genes, the most common of which are stx1a, stx2a, and stx2c. The toxins these genes encode differ in their in vitro and experimental phenotypes, but the human population-level impact of these differences is poorly understood. Using Shiga toxin-encoding bacteriophage insertion typing and real-time polymerase chain reaction, we genotyped isolates from 936 E. coli O157:H7 cases and verified HUS status via chart review. We compared the HUS risk between isolates with stx2a and those with stx2a and another gene and estimated additive interaction of the stx genes. Adjusted for age and symptoms, the HUS incidence of E. coli O157:H7 containing stx2a alone was 4.4% greater (95% confidence interval (CI) −0.3%, 9.1%) than when it occurred with stx1a. When stx1a and stx2a occur together, the risk of HUS was 27.1% lower (95% CI −87.8%, −2.3%) than would be expected if interaction were not present. At the population level, temporal or geographic shifts toward these genotypes should be monitored, and stx genotype may be an important consideration in clinically predicting HUS among E. coli O157:H7 cases. Full article

The binding of compounds to nicotinic acetylcholine receptors is of great interest in biomedical research. However, progress in this area is hampered by the lack of a high-throughput, cost-effective, and taxonomically flexible platform. Current methods are low-throughput, consume large quantities of sample, or are taxonomically limited in which targets can be tested. We describe a novel assay which utilizes a label-free bio-layer interferometry technology, in combination with adapted mimotope peptides, in order to measure ligand binding to the orthosteric site of nicotinic acetylcholine receptor alpha-subunits of diverse organisms. We validated the method by testing the evolutionary patterns of a generalist feeding species (Acanthophis antarcticus), a fish specialist species (Aipysurus laevis), and a snake specialist species (Ophiophagus hannah) for comparative binding to the orthosteric site of fish, amphibian, lizard, snake, bird, marsupial, and rodent alpha-1 nicotinic acetylcholine receptors. Binding patterns corresponded with diet, with the Acanthophis antarcticus not showing bias towards any particular lineage, while Aipysurus laevis showed selectivity for fish, and Ophiophagus hannah a selectivity for snake. To validate the biodiscovery potential of this method, we screened Acanthophis antarcticus and Tropidolaemus wagleri venom for binding to human alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-6, alpha-7, alpha-9, and alpha-10. While A. antarcticus was broadly potent, T. wagleri showed very strong but selective binding, specifically to the alpha-1 target which would be evolutionarily selected for, as well as the alpha-5 target which is of major interest for drug design and development. Thus, we have shown that our novel method is broadly applicable for studies including evolutionary patterns of venom diversification, predicting potential neurotoxic effects in human envenomed patients, and searches for novel ligands of interest for laboratory tools and in drug design and development. Full article

Harmful algal blooms (HABs) are increasing in magnitude, frequency, and duration globally. Even though a limited number of phytoplankton species can be toxic, they are becoming one of the greatest water quality threats to public health and ecosystems due to their intrinsic toxicity to humans and the numerous interacting factors that undermine HAB forecasting. Here, we show that the carbon:nitrogen:phosphorus (C:N:P) stoichiometry of a common toxic phytoplankton species, Microcystis, regulates toxin quotas during blooms through a tradeoff between primary and secondary metabolism. Populations with optimal C:N (< 8) and C:P (< 200) cellular stoichiometry consistently produced more toxins than populations exhibiting stoichiometric plasticity. Phosphorus availability in water exerted a strong control on population biomass and C:P stoichiometry, but N availability exerted a stronger control on toxin quotas by regulating population biomass and C:N:P stoichiometry. Microcystin-LR, like many phytoplankton toxins, is an N-rich secondary metabolite with a C:N stoichiometry that is similar to the optimal growth stoichiometry of Microcystis. Thus, N availability relative to P and light provides a dual regulatory mechanism that controls both biomass production and cellular toxin synthesis. Overall, our results provide a quantitative framework for improving forecasting of toxin production during HABs and compelling support for water quality management that limit both N and P inputs from anthropogenic sources. Full article

The present investigation studied the chemical composition of the essential oils extracted from Dracocephalum integrifolium Bunge growing in three different localities in northwest China and evaluated the phytotoxic, antimicrobial and insecticidal activities of the essential oils as well as their major constituents, i.e., sabinene and eucalyptol. GC/MS analysis revealed the presence of 21–24 compounds in the essential oils, representing 94.17–97.71% of the entire oils. Monoterpenes were the most abundant substances, accounting for 85.30–93.61% of the oils; among them, sabinene (7.35–14.0%) and eucalyptol (53.56–76.11%) were dominant in all three oils, which occupied 67.56–83.46% of the total oils. In general, phytotoxic bioassays indicated that the IC₅₀ values of the oils and their major constituents were below 2 μL/mL (1.739–1.886 mg/mL) against Amaranthus retroflexus and Poa annua. Disc diffusion method demonstrated that the oils and their major constituents possessed antimicrobial activity against Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Saccharomyces cerevisiae, and Candida albicans, with MIC values ranging from 5–40 μL/mL (4.347–37.712 mg/mL). The oils, sabinene and eucalyptol also exhibited significant pesticidal activity, with the mortality rates of Aphis pomi reaching 100% after exposing to 10 μL oil/petri dish (8.694–9.428 mg/petri dish) for 24 h. To the best of our knowledge, this is the first report on the chemical composition, phytotoxic, antimicrobial and insecticidal activity of the essential oils extracted from D. integrifolium; it is noteworthy to mention that this is also the first report on the phytotoxicity of one of the major constituents, sabinene. Our results imply that D. integrifolium oils and sabinene have the potential value of being further exploited as natural pesticides. Full article

Indoxyl sulfate (IS), a product metabolized from tryptophan, is negatively correlated with renal function and cardiovascular diseases in patients with chronic kidney disease (CKD). We investigated the association between serum IS levels and endothelial function in patients with CKD. Fasting blood samples were obtained from 110 patients with stages 3–5 CKD. The endothelial function, represented by vascular reactivity index (VRI), was measured non-invasively using digital thermal monitoring. Serum IS levels were determined using liquid chromatography–mass spectrometry. Twenty-one (19.1%), 36 (32.7%), and 53 (48.2%) patients had poor (VRI < 1.0), intermediate (1.0 ≤ VRI < 2.0), and good (VRI ≥ 2.0) vascular reactivity. By univariate linear regression analysis, a higher prevalence of smoking, advanced age, higher systolic, and diastolic blood pressure (DBP), elevated levels of serum phosphorus, blood urea nitrogen, creatinine, and IS were negatively correlated with VRI values, but estimated glomerular filtration rate negatively associated with VRI values. After being adjusted by using multivariate stepwise linear regression analysis, DBP and IS levels were significantly negatively associated with VRI values in CKD patients. We concluded that IS level associated inversely with VRI values and had a modulating role in endothelial function in patients with stages 3–5 CKD. Full article

Forages are important components of dairy cattle rations but might harbor a plethora of mycotoxins. Ruminants are considered to be less susceptible to the adverse health effects of mycotoxins, mainly because the ruminal microflora degrades certain mycotoxins. Yet, impairment of the ruminal degradation capacity or high ruminal stability of toxins can entail that the intestinal epithelium is exposed to significant mycotoxin amounts. The aims of our study were to assess (i) the mycotoxin occurrence in maize silage and (ii) the cytotoxicity of relevant mycotoxins on bovine intestinal cells. In total, 158 maize silage samples were collected from European dairy cattle farms. LC-MS/MS-based analysis of 61 mycotoxins revealed the presence of emerging mycotoxins (e.g., emodin, culmorin, enniatin B1, enniatin B, and beauvericin) in more than 70% of samples. Among the regulated mycotoxins, deoxynivalenol and zearalenone were most frequently detected (67.7%). Overall, 87% of maize silages contained more than five mycotoxins. Using an in vitro model with calf small intestinal epithelial cells B, the cytotoxicity of deoxynivalenol, nivalenol, fumonisin B1 and enniatin B was evaluated (0–200 µM). Absolute IC50 values varied in dependence of employed assay and were 1.2–3.6 µM, 0.8–1.0 µM, 8.6–18.3 µM, and 4.0–6.7 µM for deoxynivalenol, nivalenol, fumonisin B1, and enniatin B, respectively. Results highlight the potential relevance of mycotoxins for bovine gut health, a previously neglected target in ruminants. Full article

The manifestation of aflatoxins in feed and food is a major issue in the world as its presence leads to some health problems. This study investigates the incidence of aflatoxin M₁ (AFM₁) contamination in raw milk samples which were collected from Punjab, Pakistan. The Cluster Random Sampling technique was used to collect 960 milk samples from five different regions, and samples were collected every month. The AFM₁ level in raw milk was analyzed by the ELISA technique. The findings demonstrate that 70% of samples exceeded the United States permissible maximum residue limits (MRL 0.50 µg/L), with an overall AFM₁ level that ranged from 0.3 to 1.0 µg/L. AFM₁ contamination varied with the season: The highest average contamination was detected in winter (0.875 µg/L), followed by autumn (0.751 µg/L), spring (0.654 µg/L), and summer (0.455 µg/L). The Eastern region exhibited the highest average AFM₁ contamination (0.705 µg/L). Milk samples from the Northern region were found to be widely contaminated, as 86.9% samples exceeded the US MRL, followed by the Eastern region, with 72.3% samples being contaminated with >0.5 µg/L AFM₁. The study indicated that the raw milk supply chain was heavily contaminated. Recommendations and remedial measures need to be developed by regulatory authorities to improve the raw milk quality. Full article

Alkaloids have protective functions for plants and can play an important role in living organisms. Alkaloids may have a wide range of pharmacological activities. Many of them have cytotoxic activity. Nowadays, cancer has become a serious public health problem. Searching for effective drugs with anticancer activity is one of the most significant challenges of modern scientific research. The aim of this study was the investigation of cytotoxic activity of extracts obtained from Corydalis lutea root and herb, Dicentra spectabilis root and herb, Fumaria officinalis, Macleaya cordata leaves and herb, Mahonia aquifolia leaves and cortex, Meconopsis cambrica root and herb on FaDu, SCC-25, MCF-7, and MDA-MB-231 cancer cell lines. The cytotoxic activity of these extracts has not been previously tested for these cell lines. The aim was also to quantify selected alkaloids in the investigated extracts by High Performance Liquid Chromatography (HPLC). The analyses of alkaloid content were performed using HPLC in reversed phase (RP) mode using Polar RP column and mobile phase containing acetonitrile, water and ionic liquid (IL). Cytotoxic effect of the tested plant extracts and respective alkaloid standards were examined using human pharyngeal squamous carcinoma cells (FaDu), human tongue squamous carcinoma cells (SCC-25), human breast adenocarcinoma cell line (MCF-7), human triple-negative breast adenocarcinoma cell line (MDA-MB-231). All investigated plant extracts possess cytotoxic activity against tested cancer cell lines: FaDu, SCC-25, MCF-7, and MDA-MB-231. The highest cytotoxic activity against FaDu, SCC-25, and MCF-7 cell lines was estimated for Macleaya cordata leaf extract, while the highest cytotoxic activity against MDA-MB-231 cell line was obtained for Macleaya cordata herb extract. Differences in cytotoxic activity were observed for extracts obtained from various parts of investigated plants. In almost all cases the cytotoxic activity of investigated plant extracts, especially at the highest concentration against tested cell lines was significantly higher than the activity of anticancer drug etoposide. Our investigations exhibit that these plant extracts can be recommended for further in vivo experiments to confirm their anticancer activity. Full article

Enzymatic treatment is an attractive method for mycotoxin detoxification, which ideally prefers the use of one or a few enzymes. However, this is challenged by the diverse structures and co-contamination of multiple mycotoxins in food and feed. Lignin-degrading fungi have been discovered to detoxify organics including mycotoxins. Manganese peroxidase (MnP) is a major enzyme responsible for lignin oxidative depolymerization in such fungi. Here, we demonstrate that eight MnPs from different lignocellulose-degrading fungi (five from Irpex lacteus, one from Phanerochaete chrysosporium, one from Ceriporiopsis subvermispora, and another from Nematoloma frowardii) could all degrade four major mycotoxins (aflatoxin B₁, AFB₁; zearalenone, ZEN; deoxynivalenol, DON; fumonisin B₁, FB₁) only in the presence of a dicarboxylic acid malonate, in which free radicals play an important role. The I. lacteus and C. subvermispora MnPs behaved similarly in mycotoxins transformation, outperforming the P. chrysosporium and N. frowardii MnPs. The large evolutionary diversity of these MnPs suggests that mycotoxin degradation tends to be a common feature shared by MnPs. MnP can, therefore, serve as a candidate enzyme for the degradation of multiple mycotoxins in food and feed if careful surveillance of the residual toxicity of degradation products is properly carried out. Full article

Fusarium head blight (FHB) epidemics in wheat and contamination with Fusarium mycotoxins has become an increasing problem over the last decades. This prompted the need for non-invasive and non-destructive techniques to screen cereal grains for Fusarium infection, which is usually accompanied by mycotoxin contamination. This study tested the potential of hyperspectral imaging to monitor the infection of wheat kernels and flour with three Fusarium species. Kernels of two wheat varieties inoculated at anthesis with F. graminearum, F. culmorum, and F. poae were investigated. Hyperspectral images of kernels and flour were taken in the visible-near infrared (VIS-NIR) (400–1000 nm) and short-wave infrared (SWIR) (1000–2500 nm) ranges. The fungal DNA and mycotoxin contents were quantified. Spectral reflectance of Fusarium-damaged kernels (FDK) was significantly higher than non-inoculated ones. In contrast, spectral reflectance of flour from non-inoculated kernels was higher than that of FDK in the VIS and lower in the NIR and SWIR ranges. Spectral reflectance of kernels was positively correlated with fungal DNA and deoxynivalenol (DON) contents. In the case of the flour, this correlation exceeded r = −0.80 in the VIS range. Remarkable peaks of correlation appeared at 1193, 1231, 1446 to 1465, and 1742 to 2500 nm in the SWIR range. Full article

Alternaria alternata is a critical phytopathogen that causes foodborne spoilage and produces a polyketide mycotoxin, alternariol (AOH), and its derivative, alternariol monomethyl ether (AME). In this study, the inhibitory effects of the essential oil citral on the fungal growth and mycotoxin production of A. alternata were evaluated. Our findings indicated that 0.25 μL/mL (222.5 μg/mL) of citral completely suppressed mycelial growth as the minimum inhibitory concentration (MIC). Moreover, the 1/2MIC of citral could inhibit more than 97% of the mycotoxin amount. Transcriptomic profiling was performed by comparative RNA-Seq analysis of A. alternata with or without citral treatment. Out of a total of 1334 differentially expressed genes (DEGs), 621 up-regulated and 713 down-regulated genes were identified under citral stress conditions. Numerous DEGs for cell survival, involved in ribosome and nucleolus biogenesis, RNA processing and metabolic processes, and protein processing, were highly expressed in response to citral. However, a number of DEGs responsible for the metabolism of several carbohydrates and amino acids, sulfate and glutathione metabolism, the metabolism of xenobiotics and transporter activity were significantly more likely to be down-regulated. Citral induced the disturbance of cell integrity through the disorder of gene expression, which was further confirmed by the fact that exposure to citral caused irreversibly deleterious disruption of fungal spores and the inhibition of ergosterol biosynthesis. Citral perturbed the balance of oxidative stress, which was likewise verified by a reduction of total antioxidative capacity. In addition, citral was able to modulate the down-regulation of mycotoxin biosynthetic genes, including pksI and omtI. The results provide new insights for exploring inhibitory mechanisms and indicate citral as a potential antifungal and antimytoxigenic alternative for cereal storage. Full article

Fumonisins (FBs) are mycotoxins produced by Fusarium species that can contaminate human food and animal feed. Due to the harmful effects of FBs on animals, the European Union (EU) defined a recommendation of a maximum of 5 mg FBs (B1 + B2)/kg for complete feed for swine and 1 µg FBs/kg body weight per day as the tolerable daily intake for humans. The aim of this study was to evaluate the toxicity of dietary exposure to low doses of FBs, including a dose below the EU regulatory limits. Four groups of 24 weaned castrated male piglets were exposed to feed containing 0, 3.7, 8.1, and 12.2 mg/kg of FBs for 28 days; the impact was measured by biochemical analysis and histopathological observations. Dietary exposure to FBs at a low dose (3.7 mg/kg of feed) significantly increased the plasma sphinganine-to-sphingosine ratio. FBs-contaminated diets led to histological modifications in the intestine, heart, lung, lymphoid organs, kidney, and liver. The histological alterations in the heart and the intestine appeared at the lowest dose of FBs-contaminated diet (3.7 mg/kg feed) and in the kidney at the intermediate dose (8.1 mg/kg feed). At the highest dose tested (12.2 mg/kg feed), all the organs displayed histological alterations. This dose also induced biochemical modifications indicative of kidney and liver alterations. In conclusion, our data indicate that FBs-contaminated diets at doses below the EU regulatory limit cause histological lesions in several organs. This study suggests that EU recommendations for the concentration of FBs in animal feed, especially for swine, are not sufficiently protective and that regulatory doses should be modified for better protection of animal health. Full article

The aim of this research was to evaluate the potential protective mechanism of astaxanthin (ASTA) against oxidative damage and inflammation caused by ochratoxin (OTA) in mouse lung. We divided mice into a control group (CG), an OTA group (PG), an astaxanthin group (AG), and an OTA+ASTA group (JG). Oxidative indices (malondialdehyde (MDA), total superoxide dismutase (T-SOD), and reduced glutathione (GSH)) and inflammatory markers (interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α)) were assayed in the lung, and the lung-weight-to-body-weight ratio was calculated. Apoptosis was detected in pathological sections by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Oxidative damage and inflammation were detected in the lung of mice after exposure to OTA. Besides, Nrf2- and NF-κB-pathway-associated proteins were detected by Western blot. In contrast with OTA, ASTA significantly raised the expression of Nrf2, HO-1, and MnSOD, while the expression of other proteins (Keap1, TLR4, and NF-κB) was significantly decreased. These results indicate that ASTA exerted protective effects against OTA-induced oxidative damage and inflammation in the lung by regulating the Nrf2 and NF-κB pathways. Full article

Feed samples are frequently contaminated by a wide range of chemically diverse natural products, which can be determined using highly sensitive analytical techniques. Next to already well-investigated mycotoxins, unknown or unregulated fungal secondary metabolites have also been found, some of which at significant concentrations. In our study, 1141 pig feed samples were analyzed for more than 800 secondary fungal metabolites using the same LC-MS/MS method and ranked according to their prevalence. Effects on the viability of the 28 most relevant were tested on an intestinal porcine epithelial cell line (IPEC-J2). The most frequently occurring compounds were determined as being cyclo-(L-Pro-L-Tyr), moniliformin, and enniatin B, followed by enniatin B1, aurofusarin, culmorin, and enniatin A1. The main mycotoxins, deoxynivalenol and zearalenone, were found only at ranks 8 and 10. Regarding cytotoxicity, apicidin, gliotoxin, bikaverin, and beauvericin led to lower IC₅₀ values, between 0.52 and 2.43 µM, compared to deoxynivalenol (IC₅₀ = 2.55 µM). Significant cytotoxic effects were also seen for the group of enniatins, which occurred in up to 82.2% of the feed samples. Our study gives an overall insight into the amount of fungal secondary metabolites found in pig feed samples compared to their cytotoxic effects in vitro. Full article

Bacillus thuringiensis Cry1Ac toxin binds to midgut proteins, as cadherin (CAD) and ABCC2 transporter, to form pores leading to larval death. In cell lines, co-expression of CAD and ABCC2 enhance Cry1Ac toxicity significantly, but the mechanism remains elusive. Here, we show that the expression of Helicoverpa armigera CAD (HaCAD-GFP) in Hi5 cells induces susceptibility to Cry1Ac and enhanced Cry1Ac toxicity when co-expressed with H. armigera ABCC2 (HaABCC2-GFP), since Cry1Ac toxicity increased 735-fold compared to Hi5 cells expressing HaCAD-GFP alone or 28-fold compared to HaABCC2-GFP alone. In contrast, the expression of the Spodoptera litura CAD (SlCAD-GFP) in Hi5 cells did not induce susceptibility to Cry1Ac nor it potentiated Cry1Ac toxicity with HaABCC2-GFP. To identify the CAD regions involved in the enhancement of Cry1Ac toxicity with ABCC2, the different CAD domains were replaced between SlCAD-GFP and HaCad-GFP proteins, and cytotoxicity assays were performed in Hi5 cells in the absence or presence of HaABCC2-GFP. The HaCAD toxin-binding region (TB), specifically the CAD repeat-11, was necessary to enhance Cry1Ac toxicity with ABCC2. We propose that CAD TB is involved in recruiting Cry1Ac to localize it in a good position for its interaction with the ABCC2, resulting in efficient toxin membrane insertion enhancing Cry1Ac toxicity. Full article

Cardiotoxins (CTXs) are suggested to exert their cytotoxicity through cell membrane damage. Other studies show that penetration of CTXs into cells elicits mitochondrial fragmentation or lysosome disruption, leading to cell death. Considering the role of AMPK-activated protein kinase (AMPK) in mitochondrial biogenesis and lysosomal biogenesis, we aimed to investigate whether the AMPK-mediated pathway modulated Naja atra (Taiwan cobra) CTX3 cytotoxicity in U937 human leukemia cells. Our results showed that CTX3 induced autophagy and apoptosis in U937 cells, whereas autophagic inhibitors suppressed CTX3-induced apoptosis. CTX3 treatment elicited Ca²⁺-dependent degradation of the protein phosphatase 2A (PP2A) catalytic subunit (PP2Acα) and phosphorylation of AMPKα. Overexpression of PP2Acα mitigated the CTX3-induced AMPKα phosphorylation. CTX3-induced autophagy was via AMPK-mediated suppression of the Akt/mTOR pathway. Removal of Ca²⁺ or suppression of AMPKα phosphorylation inhibited the CTX3-induced cell death. CTX3 was unable to induce autophagy and apoptosis in U937 cells expressing constitutively active Akt. Met-modified CTX3 retained its membrane-perturbing activity, however, it did not induce AMPK activation and death of U937 cells. These results conclusively indicate that CTX3 induces autophagy and apoptosis in U937 cells via the Ca²⁺/PP2A/AMPK axis, and suggest that the membrane-perturbing activity of CTX3 is not crucial for the cell death signaling pathway induction. Full article

The selective and sensitive analysis of mycotoxins in highly complex feed matrices is a great challenge. In this study, the suitability of Orbitrap^TM-based high-resolution mass spectrometry (HRMS) for routine mycotoxin analysis in complex feeds was demonstrated by the successful validation of a full MS/data-dependent MS/MS acquisition method for the quantitative determination of eight Fusarium mycotoxins in forage maize and maize silage according to the Commission Decision 2002/657/EC. The required resolving power for accurate mass assignments (<5 ppm) was determined as 35,000 full width at half maximum (FWHM) and 70,000 FWHM for forage maize and maize silage, respectively. The recovery (R_A), intra-day precision (RSD_r), and inter-day precision (RSD_R) of measurements were in the range of 94 to 108%, 2 to 16%, and 2 to 12%, whereas the decision limit (CCα) and the detection capability (CCβ) varied from 11 to 88 µg/kg and 20 to 141 µg/kg, respectively. A set of naturally contaminated forage maize and maize silage samples collected in northern Germany in 2017 was analyzed to confirm the applicability of the HRMS method to real samples. At least four Fusarium mycotoxins were quantified in each sample, highlighting the frequent co-occurrence of mycotoxins in feed. Full article

Enzymatic detoxification has become a promising approach for control of mycotoxins postharvest in grains through modification of chemical structures determining their toxicity. In the present study fumonisin esterase FumD (EC 3.1.1.87) (FUMzyme^®; BIOMIN, Tulln, Austria), hydrolysing fumonisin (FB) mycotoxins by de-esterification, was utilised to develop an enzymatic reduction method in a maize kernel enzyme incubation mixture. Efficacy of the FumD FB reduction method in “low” and “high” FB contaminated home-grown maize was compared by monitoring FB₁ hydrolysis to the hydrolysed FB₁ (HFB₁) product utilising a validated LC-MS/MS analytical method. The method was further evaluated in terms of enzyme activity and treatment duration by assessing enzyme kinetic parameters and the relative distribution of HFB₁ between maize kernels and the residual aqueous environment. FumD treatments resulted in significant reduction (≥80%) in “low” (≥1000 U/L, p < 0.05) and “high” (100 U/L, p < 0.05; ≥1000 U/L, p < 0.0001) FB contaminated maize after 1 h respectively, with an approximate 1:1 µmol conversion ratio of FB₁ into the formation of HFB₁. Enzyme kinetic parameters indicated that, depending on the activity of FumD utilised, a significantly (p < 0.05) higher FB₁ conversion rate was noticed in “high” FB contaminated maize. The FumD FB reduction method in maize could find application in commercial maize-based practices as well as in communities utilising home-grown maize as a main dietary staple and known to be exposed above the tolerable daily intake levels. Full article

Ruminants are less susceptible to the effects of mycotoxins than monogastric animals as their rumen microbiota are claimed to degrade and/or deactivate at least some of these toxic compounds. However, the mycotoxin degradation is not well-known yet. For this, a sensitive, specific, and accurate analytical method is needed to determine mycotoxins in the rumen fluid. This study aims to develop and thoroughly validate an ultra-performance liquid chromatography tandem mass spectrometry method for the quantitative determination in the rumen fluid of some of the most relevant mycotoxins found in maize silage in Western Europe: deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), mycophenolic acid (MPA), roquefortine C (ROQ-C) and enniatin B (ENN B), as well as their metabolites deepoxy-deoxynivalenol (DOM-1), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). As feed is often present in the rumen fluid samples, the potential interaction of feed particles with the mycotoxin extraction and analysis was investigated. Extraction recovery and matrix effects were determined in the rumen fluid with and without maize silage. Differences in those parameters between rumen fluid alone and rumen fluid with maize silage highlight the importance of using matrix-matched calibration curves for the quantification of mycotoxins in rumen fluid samples. A cross-validation of the method with rumen fluid and maize silage demonstrates that this analytical method can be applied in research on rumen fluid samples to investigate the degradation of the reported mycotoxins by rumen microbiota if matrix-matched calibration is performed. Full article

Filamentous fungi, although producing noxious molecules such as mycotoxins, have been used to produce numerous drugs active against human diseases such as paclitaxel, statins, and penicillin, saving millions of human lives. Cyclodepsipeptides are fungal molecules with potentially adverse and positive effects. Although these peptides are not novel, comparative studies of their antimicrobial activity, toxicity, and mechanism of action are still to be identified. In this study, the fungal cyclohexadepsipeptides enniatin (ENN) and beauvericin (BEA) were assessed to determine their antimicrobial activity and cytotoxicity against human cells. Results showed that these peptides were active against Gram-positive bacteria, Mycobacterium, and fungi, but not against Gram-negative bacteria. ENN and BEA had a limited hemolytic effect, yet were found to be toxic at low doses to nucleated human cells. Both peptides also interacted with bacterial lipids, causing low to no membrane permeabilization, but induced membrane depolarization and inhibition of macromolecules synthesis. The structure–activity analysis showed that the chemical nature of the side chains present on ENN and BEA (either iso-propyl, sec-butyl, or phenylmethyl) impacts their interaction with lipids, antimicrobial action, and toxicity. Full article

β-methylamino-L-alanine (BMAA) is a non-protein amino acid that has been implicated as a risk factor for motor neurone disease (MND). BMAA is produced by a wide range of cyanobacteria globally and by a small number of marine diatoms. BMAA is commonly found with two of its constitutional isomers: 2,4-diaminobutyric acid (2,4-DAB), and N-(2-aminoethyl)glycine (AEG). The isomer 2,4-DAB, like BMAA, has neurotoxic properties. While many studies have shown BMAA production by cyanobacteria, few studies have looked at other algal groups. Several studies have shown BMAA production by marine diatoms; however, there are no studies examining freshwater diatoms. This study aimed to determine if some freshwater diatoms produced BMAA, and which diatom taxa are capable of BMAA, 2,4-DAB and AEG production. Five axenic diatom cultures were established from river and lake sites across eastern Australia. Cultures were harvested during the stationary growth phase and intracellular amino acids were extracted. Using liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS), diatom extracts were analysed for the presence of both free and protein-associated BMAA, 2,4-DAB and AEG. Of the five diatom cultures analysed, four were found to have detectable BMAA and AEG, while 2,4-DAB was found in all cultures. These results show that BMAA production by diatoms is not confined to marine genera and that the prevalence of these non-protein amino acids in Australian freshwater environments cannot be solely attributed to cyanobacteria. Full article

Staphylococcus aureus is a major human pathogen causing a wide range of nosocomial infections including pulmonary, urinary, and skin infections. Notably, the emergence of bacterial strains resistant to conventional antibiotics has prompted researchers to find new compounds capable of killing these pathogens. Nature is undoubtedly an invaluable source of bioactive molecules characterized by an ample chemical diversity. They can act as unique platform providing new scaffolds for further chemical modifications in order to obtain compounds with optimized biological activity. A class of natural compounds with a variety of biological activities is represented by alkaloids, important secondary metabolites produced by a large number of organisms including bacteria, fungi, plants, and animals. In this work, starting from the screening of 39 alkaloids retrieved from a unique in-house library, we identified a heterodimer β-carboline alkaloid, nigritanine, with a potent anti-Staphylococcus action. Nigritanine, isolated from Strychnos nigritana, was characterized for its antimicrobial activity against a reference and three clinical isolates of S. aureus. Its potential cytotoxicity was also evaluated at short and long term against mammalian red blood cells and human keratinocytes, respectively. Nigritanine showed a remarkable antimicrobial activity (minimum inhibitory concentration of 128 µM) without being toxic in vitro to both tested cells. The analysis of the antibacterial activity related to the nigritanine scaffold furnished new insights in the structure–activity relationships (SARs) of β-carboline, confirming that dimerization improves its antibacterial activity. Taking into account these interesting results, nigritanine can be considered as a promising candidate for the development of new antimicrobial molecules for the treatment of S. aureus-induced infections. Full article

Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways. Full article

Cladosporium species are endophytic fungi that grow on organic matter and are considered food contaminants. The anti-microbial and anti-tumor naphthoquinones fusarubin (FUS) and anhydrofusarubin (AFU) were isolated using column chromatography from a Cladosporium species residing inside Rauwolfia leaves. The impact of FUS and AFU on cell growth was assessed in acute myeloid leukemia (OCI-AML3) and other hematologic tumor cell lines (HL-60, U937, and Jurkat). Treatment with FUS or AFU reduced the number of OCI-AML3 cells as evaluated by a hemocytometer. Flow cytometry analyses showed that this effect was accompanied by diverse impairments in cell cycle progression. Specifically, FUS (20 or 10 μg/mL significantly decreased the percentage of cells in S phase and increased the percentage of cells in G2/M phase, whereas AFU increased the percentage of cells in G0/G1 phase (50 and 25 μg/mL) and decreased the percentage of cells in S (50 μg/mL) and G2/M (50 and 25 μg/mL) phases. Both substances significantly increased apoptosis at higher concentrations. The effects of FUS were more potent than those of AFU, with FUS up-regulating p21 expression in a p53-dependent manner, as detected by Western blot analyses, likely the consequence of decreased ERK phosphorylation and increased p38 expression (both of which increase p21 stability). FUS also decreased Akt phosphorylation and resulted in increased Fas ligand production and caspase-8/3-dependent apoptosis. These results suggest that FUS and AFU inhibit proliferation and increase apoptosis in cell lines derived from hematological cancers. Full article

Peanuts are widely consumed in many local dishes in southeast Asian countries, especially in Malaysia which is one of the major peanut-importing countries in this region. Therefore, Aspergillus spp. and aflatoxin contamination in peanuts during storage are becoming major concerns due to the tropical weather in this region that favours the growth of aflatoxigenic fungi. The present study thus aimed to molecularly identify and characterise the Aspergillus section Flavi isolated from imported peanuts in Malaysia. The internal transcribed spacer (ITS) and β-tubulin sequences were used to confirm the species and determine the phylogenetic relationship among the isolates, while aflatoxin biosynthesis genes (aflR, aflP (omtA), aflD (nor-1), aflM (ver-1), and pksA) were targeted in a multiplex PCR to determine the toxigenic potential. A total of 76 and one isolates were confirmed as A. flavus and A. tamarii, respectively. The Maximum Likelihood (ML) phylogenetic tree resolved the species into two different clades in which all A. flavus (both aflatoxigenic and non-aflatoxigenic) were grouped in the same clade and A. tamarii was grouped in a different clade. The aflatoxin biosynthesis genes were detected in all aflatoxigenic A. flavus while the non-aflatoxigenic A. flavus failed to amplify at least one of the genes. The results indicated that both aflatoxigenic and non-aflatoxigenic A. flavus could survive in imported peanuts and, thus, appropriate storage conditions preferably with low temperature should be considered to avoid the re-emergence of aflatoxigenic A. flavus and the subsequent aflatoxin production in peanuts during storage. Full article

The diffusion of Panton–Valentine leukocidin (PVL)–positive methicillin-resistant S. aureus (MRSA) is a health problem in Algeria. The objectives of the study were to investigate the global distribution of methicillin-susceptible S. aureus (MSSA) and MRSA isolates in different ecological niches in this country. In total, 2246 samples were collected from humans, livestock, wild animals, pets, food products and the aquatic environment, from 12 Algerian provinces. A total of 312 S. aureus were detected from 2446 samples (12.7%) in the screened niches. We observed the emergence of toxinogenic S. aureus representing 41% of the isolates. Among them, we noted the diffusion of ST80-IV CA-MRSA PVL + strains isolated in human, animals, and food and genetic diversity of MSSA PVL + isolates. This study suggests an alarming dissemination of MRSA-ST80 PVL + in both human and extra-human sources in Algeria. Moreover, MSSA may become a permanent reservoir of the PVL genes necessary for human infections. Full article