Toxins, Volume 11, Issue 5 (May 2019) – 66 articles

Snake venoms are attractive natural sources for drug discovery and development, with a number of substances either in clinical use or in research and development. These drugs were developed based on RGD-containing snake venom disintegrins, which efficiently antagonize fibrinogen activation of αIIbβ3 integrin (glycoprotein GP IIb/IIIa). Typical examples of anti-platelet drugs found in clinics are Integrilin (Eptifibatide), a heptapeptide derived from Barbourin, a protein found in the venom of the American Southeastern pygmy rattlesnake and Aggrastat (Tirofiban), a small molecule based on the structure of Echistatin, and a protein found in the venom of the saw-scaled viper. Using a similar drug discovery approach, linear and cyclic peptides containing the sequence K(R)TS derived from VP12, a C-type lectin protein found in the venom of Israeli viper venom, were used as a template to synthesize Vipegitide, a novel peptidomimetic antagonist of α2β1 integrin, with anti-platelet activity. This review focus on drug discovery of these anti-platelet agents, their indications for clinical use in acute coronary syndromes and percutaneous coronary intervention based on several clinical trials, as well as their adverse effects. Full article

Indole-diterpenes are an important class of chemical compounds which can be unique to different fungal species. The highly complex lolitrem compounds are confined to Epichloë species, whilst penitrem production is confined to Penicillium spp. and Aspergillus spp. These fungal species are often present in association with pasture grasses, and the indole-diterpenes produced may cause toxicity in grazing animals. In this review, we highlight the unique structural variations of indole-diterpenes that are characterised into subgroups, including paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines. A detailed description of the unique biological activities has been documented where even structurally related compounds have displayed unique biological activities. Indole-diterpene production has been reported in two classes of ascomycete fungi, namely Eurotiomycetes (e.g., Aspergillus and Penicillium) and Sordariomycetes (e.g., Claviceps and Epichloë). These compounds all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from tryptophan. Structure diversity is generated from the enzymatic conversion of different sites on the basic indole-diterpene structure. This review highlights the wide-ranging biological versatility presented by the indole-diterpene group of compounds and their role in an agricultural and pharmaceutical setting. Full article

Zearalenone (ZEA) is an estrogenic and ochratoxin A (OTA) is a hepatotoxic Fusarium mycotoxin commonly seen in cereals and fruits products. No previous investigation has studied on a single platform for the multi degradation mycotoxin. The current study aimed to investigate the bifunctional activity of a novel fusion recombinant. We have generated a recombinant fusion enzyme (ZHDCP) by combining two single genes named zearalenone hydrolase (ZHD) and carboxypeptidase (CP) in frame deletion by crossover polymerase chain reaction (PCR). We identified enzymatic properties and cell cytotoxicity assay of ZHDCP enzyme. Our findings have demonstrated that ZEA was completely degraded to the non-toxic product in 2 h by ZHDCP enzyme at an optimum pH of 7 and a temperature of 35 °C. For the first time, it was found out that ZEA 60% was degraded by CP degrades in 48 h. Fusion ZHDCP and CP enzyme were able to degrade 100% OTA in 30 min at pH 7 and temperature 30 °C. ZEA- and OTA-induced cell death and increased cell apoptosis rate and regulated mRNA expression of Sirt1, Bax, Bcl2, Caspase3, TNFα, and IL6 genes. Our novel findings demonstrated that the fusion enzyme ZHDCP possess bifunctional activity (degrade OTA and ZEA), and it could be used to degrade more mycotoxins. Full article

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kaštela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 × 10⁴ cells L⁻¹ in seawater, while on macroalgae, it was estimated to be up to 2.67 × 10⁶ cells g⁻¹ of macroalgae fresh weight and 14.4 × 10⁶ cells g⁻¹ of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography–high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell⁻¹), while the isobaric PLTX concentration was very low (0.1 pg cell⁻¹). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell⁻¹), followed by OVTX-b (1.3 pg cell⁻¹), OVTX-d (1.1 pg cell⁻¹), and OVTX-c (0.2 pg cell⁻¹). Full article

Species interactions are fundamental ecological forces that can have significant impacts on the evolutionary trajectories of species. Nonetheless, the contribution of predator-prey interactions to genetic and phenotypic divergence remains largely unknown. Predatory marine snails of the family Conidae exhibit specializations for different prey items and intraspecific variation in prey utilization patterns at geographic scales. Because cone snails utilize venom to capture prey and venom peptides are direct gene products, it is feasible to examine the evolution of genes associated with changes in resource utilization. Here, we compared feeding ecologies and venom duct transcriptomes of individuals from three populations of Conus miliaris, a species that exhibits geographic variation in prey utilization and dietary breadth, in order to determine the extent to which dietary differences are correlated with differences in venom composition, and if expanded niche breadth is associated with increased variation in venom composition. While populations showed little to no overlap in resource utilization, taxonomic richness of prey was greatest at Easter Island. Changes in dietary breadth were associated with differences in expression patterns and increased genetic differentiation of toxin-related genes. The Easter Island population also exhibited greater diversity of toxin-related transcripts, but did not show increased variance in expression of these transcripts. These results imply that differences in dietary breadth contribute more to the structural and regulatory differentiation of venoms than differences in diet. Full article

Bacterial lipopolysaccharide (LPS) could result in poor lactation performance in dairy cows. High methylation of DNA is associated with gene repression. However, it is unclear whether LPS could suppress the expression of lactation-related genes by inducing DNA methylation. Therefore, the objective of this study was to investigate the impact of LPS on genome-wide DNA methylation, using methylated DNA immunoprecipitation with high-throughput sequencing (MeDIP-seq) and on the promoter methylation of lactation-related genes using MassArray analysis in bovine mammary epithelial cells. The bovine mammary epithelial cell line MAC-T cells were treated for 48 h with LPS at different doses of 0, 1, 10, 100, and 1000 endotoxin units (EU)/mL (1 EU = 0.1 ng). The results showed that the genomic methylation levels and the number of methylated genes in the genome as well as the promoter methylation levels of milk genes increased when the LPS dose was raised from 0 to 10 EU/mL, but decreased after further increasing the LPS dose. The milk gene mRNA expression levels of the 10 EU/mL LPS treatment were significantly lower than these of untreated cells. The results also showed that the number of hypermethylated genes was greater than that of hypomethylated genes in lipid and amino acid metabolic pathways following 1 and 10 EU/mL LPS treatments as compared with control. By contrast, in the immune response pathway the number of hypomethylated genes increased with increasing LPS doses. The results indicate LPS at lower doses induced hypermethylation of the genome and promoters of lactation-related genes, affecting milk gene mRNA expression. However, LPS at higher doses induced hypomethylation of genes involved in the immune response pathway probably in favor of immune responses. Full article

Fusarium species are among the most important fungal pathogens of maize, where they cause severe reduction of yield and accumulation of a wide range of harmful mycotoxins in the kernels. In order to identify the Fusarium species and their mycotoxin profiles associated to maize ear rot and kernel contamination in Iran, a wide sampling was carried out from field in ten major maize-producing provinces in Iran, during 2015 and 2016. From 182 samples of maize kernels, 551 strains were isolated and identified as belonging to Fusarium genus. Among the 234 representative strains identified at species level by translation elongation factor (EF-1α) sequences, the main Fusarium species were F. verticillioides and F. proliferatum, together representing 90% of the Iranian Fusarium population, and, to a lesser extent, F. incarnatum equiseti species complex (FIESC), F. thapsinum and F. redolens. Fumonisin (FBs) production by F. verticillioides and F. proliferatum representative strains was analysed, showing that all strains produced FB₁. None of F. verticillioides strains produced FB₂ nor FB₃, while both FB₂ and FB₃ were produced only by F. proliferatum. Total mean of FBs production by F. verticillioides was higher than F. proliferatum. The occurrence of different Fusarium species on Iranian maize is reason of great concern because of the toxigenic risk associated to these species. Moreover, the diversity of the species identified increases the toxigenic risk associated to Fusarium contaminated maize kernels, because of the high possibility that a multi-toxin contamination can occur with harmful consequences on human and animal health. Full article

Zearalenone is a frequent contaminant of cereals and their by-products in regions with a temperate climate. This toxic molecule is produced naturally by Fusarium fungi in crops. The aim of this study was to determine the influence of low zearalenone doses (LOAEL, NOAEL and MABEL) on the intestinal microbiome of gilts on different days of exposure (days 7, 21 and 42). Intestinal contents were sampled from the duodenal cap, the third part of the duodenum, jejunum, caecum and the descending colon. The experiment was performed on 60 clinically healthy gilts with average BW of 14.5 ± 2 kg, divided into three experimental groups and a control group. Group ZEN5 animals were orally administered ZEN at 5 μg /kg BW, group ZEN10—10 μg ZEN/kg BW and group ZEN15—15 µg ZEN/kg BW. Five gilts from every group were euthanized on analytical dates 1, 2 and 3. Differences in the log values of microbial counts, mainly Escherichia coli and Enterococcus faecalis, were observed between the proximal and distal segments of the intestinal tract on different analytical dates as well as in the entire intestinal tract. Zearalenone affected the colony counts of intestinal microbiota rather than microbiome diversity, and its effect was greatest in groups ZEN10 and ZEN15. Microbial colony counts were similar in groups ZEN5 and C. In the analysed mycobiome, ZEN exerted a stimulatory effect on the log values of yeast and mould counts in all intestinal segments, in particular in the colon, and the greatest increase was noted on the first analytical date. Full article

Fusarium graminearum is a notorious pathogen that causes Fusarium head blight (FHB) in cereal crops. It produces secondary metabolites, such as deoxynivalenol, diminishing grain quality and leading to lesser crop yield. Many strategies have been developed to combat this pathogenic fungus; however, considering the lack of resistant cultivars and likelihood of environmental hazards upon using chemical pesticides, efforts have shifted toward the biocontrol of plant diseases, which is a sustainable and eco-friendly approach. Fengycin, derived from Bacillus amyloliquefaciens FZB42, was purified from the crude extract by HPLC and further analyzed by MALDI-TOF-MS. Its application resulted in structural deformations in fungal hyphae, as observed via scanning electron microscopy. In planta experiment revealed the ability of fengycin to suppress F. graminearum growth and highlighted its capacity to combat disease incidence. Fengycin significantly suppressed F. graminearum, and also reduced the deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) production in infected grains. To conclude, we report that fengycin produced by B. amyloliquefaciens FZB42 has potential as a biocontrol agent against F. graminearum and can also inhibit the mycotoxins produced by this fungus. Full article

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations. Full article

Domoic acid (DA)-producing harmful algal blooms (HABs) have been present at unprecedented geographic extent and duration in recent years causing an increase in contamination of seafood by this common environmental neurotoxin. The toxin is responsible for the neurotoxic illness, amnesic shellfish poisoning (ASP), that is characterized by gastro-intestinal distress, seizures, memory loss, and death. Established seafood safety regulatory limits of 20 μg DA/g shellfish have been relatively successful at protecting human seafood consumers from short-term high-level exposures and episodes of acute ASP. Significant concerns, however, remain regarding the potential impact of repetitive low-level or chronic DA exposure for which there are no protections. Here, we report the novel discovery of a DA-specific antibody in the serum of chronically-exposed tribal shellfish harvesters from a region where DA is commonly detected at low levels in razor clams year-round. The toxin was also detected in tribal shellfish consumers’ urine samples confirming systemic DA exposure via consumption of legally-harvested razor clams. The presence of a DA-specific antibody in the serum of human shellfish consumers confirms long-term chronic DA exposure and may be useful as a diagnostic biomarker in a clinical setting. Adverse effects of chronic low-level DA exposure have been previously documented in laboratory animal studies and tribal razor clam consumers, underscoring the potential clinical impact of such a diagnostic biomarker for protecting human health. The discovery of this type of antibody response to chronic DA exposure has broader implications for other environmental neurotoxins of concern. Full article

The detection of mycotoxins is crucial because of their toxicity in plants, animals, and humans. It is very important to determine whether food products are contaminated with mycotoxins such as ochratoxin A (OTA), as mycotoxins can survive heat treatments and hydrolysis. In this study, we designed a fluorescence resonance energy transfer (FRET)-based system that exploits antibody-antigen binding to detect mycotoxins more rapidly and easily than other currently available methods. In addition, we were able to effectively counteract the matrix effect in the sample by using a nitrocellulose membrane that enabled fluorescence measurement in coffee samples. The developed FRET on lateral flow immunoassay (FRET-LFI) system was used to detect OTA at a limit of detection (LOD) of 0.64 ng∙mL⁻¹, and the test can be completed in only 30 min. Moreover, OTA in coffee samples was successfully detected at a LOD of 0.88 ng∙mL⁻¹_, overcoming the matrix effect, owing to the chromatographic properties of the capillary force of the membrane. We believe that the developed system can be used as a powerful tool for the sensitive diagnosis of harmful substances such as mycotoxins and pesticides for environmental and food quality control monitoring. Full article

Possible contamination by Staphylococcus aureus of the production environment and of the meat of a canned meat production factory was analysed. A total of 108 samples were taken from nine critical control points, 13 of them were positive for S. aureus. None of the isolates produced enterotoxins. To determine how much time can elapse between can seaming and sterilisation in the autoclave without any risk of enterotoxin production by S. aureus, the growth and enterotoxin production of three enterotoxin A producing strains of S. aureus (one ATCC strain and two field strains) in canned meat before sterilisation was investigated at three different temperatures (37, 20 and 10 °C). Two types of meat were used, one with and one without sodium nitrite. In the canned products, the spiked bacteria spread throughout the meat and reached high levels. Enterotoxin production was shown to start 10 hours after incubation at 37 °C and after 48 h after incubation at 20 °C; the production of enterotoxin was always detected in the transition between the exponential and the stationary growth phase. At 10 °C, the enterotoxin was never detected. The statistical analysis of the data showed that the difference between the two different types of meat was not statistically significant (p value > 0.05). Since it is well known that following heat treatment, staphylococcal enterotoxins, although still active (in in vivo assays), can be undetectable (loss of serological recognition) depending on the food matrix and pH, it is quite difficult to foresee the impact of heat treatment on enterotoxin activity. Therefore, although the bacteria are eliminated, the toxins may remain and cause food poisoning. The significance of the results of this study towards implementing good manufacturing practices and hazard analysis critical control points in a canned meat factory are discussed with reference to the management of pre-retorting steps after seaming. Full article

Today, we have been witnessing a steady tendency in the increase of global demand for maize, wheat, soybeans, and their products due to the steady growth and strengthening of the livestock industry. Thus, animal feed safety has gradually become more important, with mycotoxins representing one of the most significant hazards. Mycotoxins comprise different classes of secondary metabolites of molds. With regard to animal feed, aflatoxins, fumonisins, ochratoxins, trichothecenes, and zearalenone are the more prevalent ones. In this review, several constraints posed by these contaminants at economical and commercial levels will be discussed, along with the legislation established in the European Union to restrict mycotoxins levels in animal feed. In addition, the occurrence of legislated mycotoxins in raw materials and their by-products for the feeds of interest, as well as in the feeds, will be reviewed. Finally, an overview of the different sample pretreatment and detection techniques reported for mycotoxin analysis will be presented, the main weaknesses of current methods will be highlighted. Full article

Fusarium proliferatum can infect many crops and then produce fumonisins that are very harmful to humans and animals. Previous study indicates that carbon sources play important roles in regulating the fumonisin biosynthesis. Unfortunately, there is limited information on the effects of carbon starvation in comparison with the carbon sources present in the host of fumonisin production in F. proliferatum. Our results indicated that F. proliferatum cultivated in the Czapek’s broth (CB) medium in the absence of sucrose could greatly induce production of fumonisin, while an additional supplementation of sucrose to the culture medium significantly reduced the fumonisin production. Furthermore, cellulose and hemicellulose, and polysaccharide extracted from banana peel, which replaced sucrose as the carbon source, can reduce the production of fumonisin by F. proliferatum. Further work showed that these genes related to the synthesis of fumonisin, such as FUM1 and FUM8, were significantly up-regulated in the culture medium in the absence of sucrose. Consistent with fumonisin production, the expressions of FUM gene cluster and ZFR1 gene decreased after the addition of sucrose. Moreover, these genes were also significantly down-regulated in the presence of cellulose, hemicellulose or polysaccharide extracted from peel. Altogether, our results suggested that fumonisin production was regulated in F. proliferatum in response to different carbon source conditions, and this regulation might be mainly via the transcriptional level. Future work on these expressions of the fumonisin biosynthesis-related genes is needed to further clarify the response under different carbon conditions during the infection of F. proliferatum on banana fruit hosts. The findings in this study will provide a new clue regarding the biological effect of the fumonisin production in response to environmental stress. Full article

Most cyanobacterial organisms included in the genus Microcystis can produce a wide repertoire of secondary metabolites. In the mid-2010s, summer cyanobacterial blooms of Microcystis sp. occurred regularly in Lake Balaton. During this period, we investigated how the alimentary tract of filter-feeding bigheaded carps could deliver different chemotypes of viable cyanobacteria with specific peptide patterns. Twenty-five Microcystis strains were isolated from pelagic plankton samples (14 samples) and the hindguts of bigheaded carp (11 samples), and three bloom samples were collected from the scums of cyanobacterial blooms. An LC-MS/MS-based untargeted approach was used to analyze peptide patterns, which identified 36 anabaenopeptin, 17 microginin, and 13 microcystin variants. Heat map clustering visualization was used to compare the identified chemotypes. A lack of separation was observed in peptide patterns of Microcystis that originated from hindguts, water samples, and bloom-samples. Except for 13 peptides, all other congeners were detected from the viable and cultivated chemotypes of bigheaded carp. This finding suggests that the alimentary tract of bigheaded carps is not simply an extreme habitat, but may also supply the cyanobacterial strains that represent the pelagic chemotypes. Full article

The usage of insects as model organisms is becoming more and more common in toxicological, pharmacological, genetic and biomedical research. Insects, such as fruit flies (Drosophila melanogaster), locusts (Locusta migratoria), stick insects (Baculum extradentatum) or beetles (Tenebrio molitor) are used to assess the effect of different active compounds, as well as to analyse the background and course of certain diseases, including heart disorders. The goal of this study was to assess the influence of secondary metabolites extracted from Solanaceae and Brassicaceae plants: Potato (Solanum tuberosum), tomato (Solanum lycopersicum), black nightshade (Solanum nigrum) and horseradish (Armoracia rusticana), on T. molitor beetle heart contractility in comparison with pure alkaloids. During the in vivo bioassays, the plants glycoalkaloid extracts and pure substances were injected at the concentration 10⁻⁵ M into T. molitor pupa and evoked changes in heart activity. Pure glycoalkaloids caused mainly positive chronotropic effects, dependant on heart activity phase during a 24-h period of recording. Moreover, the substances affected the duration of the heart activity phases. Similarly, to the pure glycoalkaloids, the tested extracts also mainly accelerated the heart rhythm, however S. tuberosum and S. lycopersicum extracts slightly decreased the heart contractions frequency in the last 6 h of the recording. Cardioacceleratory activity of only S. lycopersicum extract was higher than single alkaloids whereas S. tubersoum and S. nigrum extracts were less active when compared to pure alkaloids. The most cardioactive substance was chaconine which strongly stimulated heart action during the whole recording after injection. A. rusticana extract which is composed mainly of glucosinolates did not significantly affect the heart contractions. Obtained results showed that glycoalkaloids were much more active than glucosinolates. However, the extracts depending on the plant species might be more or less active than pure substances. Full article

Citrullus colocynthis L. Schrader is an annual plant belonging to the Cucurbitaceae family, widely distributed in the desert areas of the Mediterranean basin. Many pharmacological properties (anti-inflammatory, anti-diabetic, analgesic, anti-epileptic) are ascribed to different organs of this plant; extracts and derivatives of C. colocynthis are used in folk Berber medicine for the treatment of numerous diseases—such as rheumatism arthritis, hypertension bronchitis, mastitis, and even cancer. Clinical studies aimed at confirming the chemical and biological bases of pharmacological activity assigned to many plant/herb extracts used in folk medicine often rely on results obtained from laboratory preliminary tests. We investigated the biological activity of some C. colocynthis stem, leaf, and root extracts on the mycotoxigenic and phytopathogenic fungus Aspergillus flavus, testing a possible correlation between the inhibitory effect on aflatoxin biosynthesis, the phytochemical composition of extracts, and their in vitro antioxidant capacities. Full article

The research presented in this manuscript concerns the evaluation of the effectiveness of microstrainers, which are designed to reduce the amount of plankton in treated surface water. The efficiency of microstrainer filtration analysis is very important for the proper course of the water-treatment process not only in the Water-Treatment Plant (WTP) in Zielona Góra (central western Poland) but also in other WTPs around the world. The qualitative and quantitative monitoring of the abundance of plankton including cyanobacteria during the particle-filtration process allows not only for the assessment of the potential cyanotoxic risk in surface water providing a source of drinking water, but also allows the evaluation of the action and the prevention of adverse impacts of microstrainers. Over four years of research, it was observed that the largest amount of cyanobacteria before microstrainer filtration took place in May. The dominant species was Limnothrix redeckei. The microstrainer removal of plankton and cyanobacteria was statistically significant. The quantity of removed plankton increased with its increasing content in raw water. The particle-filtration process, by reducing the amount of cyanobacteria, contributes to a decrease in intracellular microcystins. Full article

Moraea pallida Bak. (yellow tulp) poisoning is the most important cardiac glycoside-induced intoxication in ruminants in South Africa. The toxic principle, 1α, 2α-epoxyscillirosidine, is a bufadienolide. To replace the use of sentient animals in toxicity testing, the aim of this study was to evaluate the cytotoxic effects of epoxyscillirosidine on rat embryonic cardiomyocytes (H9c2 cell line). This in vitro cell model can then be used in future toxin neutralization or toxico-therapy studies. Cell viability, evaluated with the methyl blue thiazol tetrazolium (MTT) assay, indicated a hormetic dose/concentration response, characterized by a biphasic low dose stimulation and high dose inhibition. Increased cell membrane permeability and leakage, as expected with necrotic cells, were demonstrated with the lactate dehydrogenase (LDH) assay. The LC50 was 382.68, 132.28 and 289.23 μM for 24, 48, and 72 h respectively. Numerous cytoplasmic vacuoles, karyolysis and damage to the cell membrane, indicative of necrosis, were observed at higher doses. Ultra-structural changes suggested that the cause of H9c2 cell death, subsequent to epoxyscillirosidine exposure, is necrosis, which is consistent with myocardial necrosis observed at necropsy. Based on the toxicity observed, and supported by ultra-structural findings, the H9c2 cell line could be a suitable in vitro model to evaluate epoxyscillirosidine neutralization or other therapeutic interventions in the future. Full article

Erectile dysfunction (ED) is a highly prevalent condition with a variety of possible risk factors and/or etiologies. Despite significant advances regarding ED pharmacological management, there are still insufficient responders to existing pharmacological treatments e.g., approximately 30% of patients are insufficient responders to phosphodiesterase type 5 inhibitors (PDE5-Is). It has been recently proposed that botulinum toxin A intracavernosally (IC) delivered could be effective in these patients. Data from a retrospective uncontrolled single center study of 47 ED patients, consecutively recruited, insufficient responders to existing pharmacological treatments e.g., PDE5-Is or IC PGE1 injections treated with IC abobotulinumtoxinA 250 or 500 U as free combination with their existing treatment have been analyzed. Response rate, according to the International Index of Erectile Function-Erectile Function domain score, 6 weeks following IC abobotulinumtoxinA in combination with prior pharmacological treatment, was 54%. Two patients have reported mild penile pain on injection or during the 3 days following injection. Therapeutic efficacy did not seem to be influenced by the etiologies and/or risk factors for ED. Conversely, the less severe ED, the higher the response rate. Preliminary evidence for the therapeutical potential with acceptable safety of IC abobotulinumtoxinA as add-on therapy for ED not sufficiently responsive to standard therapy should be confirmed in randomized clinical trials. Full article

The aim of this study was to determine the potential for accumulation of deoxynivalenol (DON) in yellow mealworm larvae (Tenebrio molitor) reared on high DON Fusarium-infected wheat and investigate the effects on production, survival and nutritional traits. Wheat containing 200 μg/kg DON was used as the control diet. A different source of wheat was sorted into six fractions and mixed to obtain low (2000 μg/kg), medium (10,000 μg/kg) and high (12,000 μg/kg) levels of DON. Each diet was replicated five times with 300 or 200 mealworms per replicate for the feeding and breeding trials, respectively. Trial termination occurred when the first two pupae were observed (32–34 days). There was no difference in the concentrations of DON detected in the larvae between diets that ranged from 122 ± 19.3 to 136 ± 40.5 μg/kg (p = 0.88). Excretion of DON was 131, 324, 230 and 742 μg/kg for control, low, medium and high, respectively. Nutritional analysis of larvae showed maximum crude protein of 52% and crude fat of 36%. Ash, fiber, chitin, fatty-acids and amino-acid content were consistent across diets. Survival was greater than 96% for all life stages and average daily gain ranged from 1.9 ± 0.1 to 2.1 ± 0.1 mg/day per mealworm. Larvae accumulated low levels of DON from Fusarium-infected wheat diets suggesting contaminated wheat could be used to produce a sustainable, safe protein source. Full article

A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L₁ and L₂. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L₁ hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L₂:L₁:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L₁ - L₂. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L₁ and L₂ occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L₁ enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related. Full article

Enzyme-linked immunosorbent assay (ELISA) test kits have been widely used for the determination of mycotoxins in agricultural products and foods, however, this test uses toxin standards with high toxicity and carcinogenicity that seriously threaten human health. In this work, the anti-idiotypic nanobody VHH 2-24 was first developed and then, using it as a surrogate standard, a toxin-free enzyme immunoassay for ochratoxin A (OTA) was established. The IC₅₀ value of the VHH 2-24 surrogate standard-based ELISA was 0.097 µg/mL, with a linear range of 0.027–0.653 µg/mL. The average recoveries were tested by spike-and-recovery experiments, and ranged from 81.8% to 105.0%. The accuracy of the developed ELISA for detecting OTA was further verified by using the high performance liquid chromatography (HPLC) method, and an excellent correlation was observed. In summary, the toxin-free ELISA established in this study proves the latent use of the anti-idiotypic VHH as a surrogate calibrator for other mycotoxins and highly toxic small molecule analysis to improve assay properties for highly sensitive analyte determination in agricultural products. Full article

High serum levels of gut-derived uremic toxins, especially p-cresyl sulfate (pCS), indoxyl sulfate (IS) and indole acetic acid (IAA), have been linked to adverse outcomes in patients with chronic kidney disease (CKD). Sevelamer carbonate could represent an interesting option to limit the elevation of gut-derived uremic toxins. The aim of the present study was to evaluate the adsorptive effect of sevelamer carbonate on different gut-derived protein-bound uremic toxins or their precursors in vitro, and its impact on the serum levels of pCS, IS and IAA in patients with CKD stage 3b/4. For the in vitro experiments, IAA, p-cresol (precursor of pCS) and indole (precursor of IS), each at a final concentration of 1 or 10 µg/mL, were incubated in centrifugal 30 kDa filter devices with 3 or 15 mg/mL sevelamer carbonate in phosphate-buffered saline at a pH adjusted to 6 or 8. Then, samples were centrifuged and free uremic toxins in the filtrates were analyzed. As a control experiment, the adsorption of phosphate was also evaluated. Additionally, patients with stage 3b/4 CKD (defined as an eGFR between 15 and 45 mL/min per 1.73 m²) were included in a multicenter, double-blind, placebo-controlled, randomized clinical trial. The participants received either placebo or sevelamer carbonate (4.8 g) three times a day for 12 weeks. The concentrations of the toxins and their precursors were measured using a validated high-performance liquid chromatography method with a diode array detector. In vitro, regardless of the pH and concentration tested, sevelamer carbonate did not show adsorption of indole and p-cresol. Conversely, with 10 µg/mL IAA, use of a high concentration of sevelamer carbonate (15 mg/mL) resulted in a significant toxin adsorption both at pH 8 (mean reduction: 26.3 ± 3.4%) and pH 6 (mean reduction: 38.7 ± 1.7%). In patients with CKD stage 3b/4, a 12-week course of treatment with sevelamer carbonate was not associated with significant decreases in serum pCS, IS and IAA levels (median difference to baseline levels: −0.12, 0.26 and −0.06 µg/mL in the sevelamer group vs. 1.97, 0.38 and 0.05 µg/mL in the placebo group, respectively). Finally, in vitro, sevelamer carbonate was capable of chelating a gut-derived uremic toxin IAA but not p-cresol and indole, the precursors of pCS and IS in the gut. In a well-designed clinical study of patients with stage 3b/4 CKD, a 12-week course of treatment with sevelamer carbonate was not associated with significant changes in the serum concentrations of pCS, IS and IAA. Full article

Drinking water treatment plants throughout the world are increasingly facing the presence of toxic cyanobacteria in their source waters. During treatment, the oxidation of cyanobacteria changes cell morphology and can potentially lyse cells, releasing intracellular metabolites. In this study, a combination of techniques was applied to better understand the effect of oxidation with chlorine, ozone, potassium permanganate, and hydrogen peroxide on two cell cultures (Microcystis, Dolichospermum) in Lake Champlain water. The discrepancy observed between flow cytometry cell viability and cell count numbers was more pronounced for hydrogen peroxide and potassium permanganate than ozone and chlorine. Liquid chromatography with organic carbon and nitrogen detection was applied to monitor the changes in dissolved organic matter fractions following oxidation. Increases in the biopolymer fraction after oxidation with chlorine and ozone were attributed to the release of intracellular algal organic matter and/or fragmentation of the cell membrane. A novel technique, Enhanced Darkfield Microscopy with Hyperspectral Imaging, was applied to chlorinated and ozonated samples. Significant changes in the peak maxima and number of peaks were observed for the cell walls post-oxidation, but this effect was muted for the cell-bound material, which remained relatively unaltered. Full article

Fungal non-ribosomal peptide synthetase (NRPS) clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one NRPS. However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in Fusarium graminearum. Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase kmt6, which induced the production of fusaoctaxin A. Full article

Vip3A proteins are important for the control of spodopteran pests in crops, including Spodoptera frugiperda (fall armyworm). Native Vip3Ab1 controls S. frugiperda, but it is ineffective against S. eridania (southern armyworm), a major pest of soybean in South America. Recently, a Vip3Ab1 chimera with a modified C-terminus was described, Vip3Ab1-740, which has increased potency against S. eridania while maintaining activity against S. frugiperda. As S. frugiperda and S. eridania are differentially susceptible to Vip3Ab1, experiments were conducted to identify and understand the mechanism by which this expanded potency is conferred. The role of protein stability, processing, and in vivo effects of Vip3Ab1 and Vip3Ab1-740 in both of these species was investigated. Biochemical characterization of the midgut fluids of these two species indicated no obvious differences in the composition and activity of digestive enzymes, which protease inhibitor studies indicated were likely serine proteases. Histological examination demonstrated that both proteins cause midgut disruption in S. frugiperda, while only Vip3Ab1-740 affects S. eridania. Immunolocalization indicated that both proteins were present in the midgut of S. frugiperda, but only Vip3Ab1-740 was detected in the midgut of S. eridania. We conclude that the gain of toxicity of Vip3Ab1-740 to S. eridania is due to an increase in protein stability in the midgut, which was conferred by C-terminal modification. Full article

In this review, we discuss the role of the endocannabinoid (eCB) system in regulating energy and metabolic homeostasis. Endocannabinoids, via activating the cannabinoid type-1 receptor (CB₁R), are commonly known as mediators of the thrifty phenotype hypothesis due to their activity in the central nervous system, which in turn regulates food intake and underlies the development of metabolic syndrome. Indeed, these findings led to the clinical testing of globally acting CB₁R blockers for obesity and various metabolic complications. However, their therapeutic potential was halted due to centrally mediated adverse effects. Recent observations that highlighted the key role of the peripheral eCB system in metabolic regulation led to the preclinical development of various novel compounds that block CB₁R only in peripheral organs with very limited brain penetration and without causing behavioral side effects. These unique molecules, which effectively ameliorate obesity, type II diabetes, fatty liver, insulin resistance, and chronic kidney disease in several animal models, are likely to be further developed in the clinic and may revive the therapeutic potential of blocking CB₁R once again. Full article

Seafood is an emerging health food, and interest in improving the quality of seafood is increasing. Saxitoxin (STX) is a neurotoxin produced by marine dinoflagellates that is accumulated in seafood. It can block the neuronal transmission between nerves and muscle cell membranes, resulting in the disturbance of neuromuscular transmission and subsequent voluntary muscle paralysis. Here, we developed a new aptamer for the detection of STX using graphene oxide–systematic evolution of ligands by exponential enrichment (GO-SELEX). Furthermore, we confirmed sensitivity and selectivity of the developed aptamer specific to STX using a localized surface plasmon resonance (LSPR) sensor. The sensing chip was fabricated by fixing the new STX aptamer immobilized on the gold nanorod (GNR) substrate. The STX LSPR aptasensor showed a broad, linear detection range from 5 to 10,000 μg/L, with a limit of detection (LOD) of 2.46 μg/L (3σ). Moreover, it was suitable for the detection of STX (10, 100, and 2000 μg/L) in spiked mussel samples and showed a good recovery rate (96.13–116.05%). The results demonstrated that the new STX aptamer-modified GNR chip was sufficiently sensitive and selective to detect STX and can be applied to real samples as well. This LSPR aptasensor is a simple, label-free, cost-effective sensing system with a wide detectable range. Full article