Humans are chronically exposed to the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), as indicated by their widespread presence in foods and occasional exposure in the workplace. This exposure is confirmed by human biomonitoring (HBM) studies on (metabolites of) these mycotoxins in human matrices. We evaluated the exposure–health relationship of the mycotoxins in humans by reviewing the available literature. Since human studies did not allow the identification of unequivocal chronic health effects upon exposure to DON and FB1, the adverse outcome pathway (AOP) framework was used to structure additional mechanistic evidence from in vitro and animal studies on the identified adverse effects. In addition to a preliminary AOP for DON resulting in the adverse outcome (AO) ‘reduced body weight gain’, we developed a more elaborated AOP for FB1, from the molecular initiating event (MIE) ‘inhibition of ceramide synthases’ leading to the AO ‘neural tube defects’. The mechanistic evidence from AOPs can be used to support the limited evidence from human studies, to focus FB1- and DON-related research in humans to identify related early biomarkers of effect. In order to establish additional human exposure–health relationships in the future, recommendations are given to maximize the information that can be obtained from HBM. Full article

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples. Full article

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A${}_2$ (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes. Full article

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and—at least bitiscetin-1—induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein—bitiscetin-3—were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit—which differs from that of both bitiscetin-3 subunits—the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen. Full article

The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis, the earliest diverging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently diverged cone snail lineages. Full article

Vibrio cholerae uses cholera toxin (CT) to cause cholera, a severe diarrheal disease in humans that can lead to death within hours of the onset of symptoms. The catalytic activity of CT in target epithelial cells increases cellular levels of 3′,5′-cyclic AMP (cAMP), leading to the activation of the cystic fibrosis transmembrane conductance regulator (CFTR), an apical ion channel that transports chloride out of epithelial cells, resulting in an electrolyte imbalance in the intestinal lumen and massive water loss. Here we report that when administered perorally, benzopyrimido-pyrrolo-oxazinedione, (R)-BPO-27), a potent small molecule inhibitor of CFTR, blocked disease symptoms in a mouse model for acute diarrhea caused by toxigenic V. cholerae. We show that both (R)-BPO-27 and its racemic mixture, (R/S)-BPO-27, are able to protect mice from CT-dependent diarrheal disease and death. Furthermore, we show that, consistent with the ability of the compound to block the secretory diarrhea induced by CT, BPO-27 has a measurable effect on suppressing the gut replication and survival of V. cholerae, including a 2010 isolate from Haiti that is representative of the most predominant ‘variant strains’ that are causing epidemic and pandemic cholera worldwide. Our results suggest that BPO-27 should advance to human Phase I studies that could further address its safety and efficacy as therapeutic or preventative drug intervention for diarrheal syndromes, including cholera, that are mediated by CFTR channel activation. Full article

The Vietnamese Mekong Delta is predicted to be one of the regions most impacted by climate change, causing increased temperature and salinity in inland waters. We hypothesized that the increase in temperature and salinity may impact the microcystin (MC) production of two Microcystis strains isolated in this region from a freshwater pond (strain MBC) and a brackish water pond (strain MTV). The Microcystis strains were grown at low (27 °C), medium (31 °C), high (35 °C) and extremely high (37 °C) temperature in flat photobioreactors (Algaemist). At each temperature, when cultures reached a stable state, sea salt was added to increase salinity to 4‰, 8‰, 12‰ and 16‰. MC concentrations and cell quota were reduced at high and extremely high temperatures. Salinity, in general, had comparable effects on MC concentrations and quota. At a salinity of 4‰ and 8‰, concentrations of MC per mL of culture and MC cell quota (based on chlorophyll, dry-weight and particle counts) were higher than at 0.5‰, while at the highest salinities (12‰ and 16‰) these were strongly reduced. Strain MBC produced five MC variants of which MC-RR and MC-LR were most abundant, followed by MC-YR and relatively low amounts of demethylated variants dmMC-RR and dmMC-LR. In strain MTV, MC-RR was most abundant, with traces of MC-YR and dmMC-RR only in cultures grown at 16‰ salinity. Overall, higher temperature led to lower MC concentrations and cell quota, low salinity seemed to promote MC production and high salinity reduced MC production. Hence, increased temperature and higher salinity could lead to less toxic Microcystis, but since these conditions might favour Microcystis over other competitors, the overall biomass gain could offset a lower toxicity. Full article

The assessment of aflatoxin B₁ (AFB₁) exposure using isotope-dilution liquid chromatography-mass spectrometry (LCMS) of AFB₁-lysine adducts in human serum albumin (HSA) has proven to be a highly productive strategy for the biomonitoring of AFB₁ exposure. To compare samples across different individuals and settings, the conventional practice has involved the normalization of raw AFB₁-lysine adduct concentrations (e.g., pg/mL serum or plasma) to the total circulating HSA concentration (e.g., pg/mg HSA). It is hypothesized that this practice corrects for technical error, between-person variance in HSA synthesis or AFB₁ metabolism, and other factors. However, the validity of this hypothesis has been largely unexamined by empirical analysis. The objective of this work was to test the concept that HSA normalization of AFB₁-lysine adduct concentrations effectively adjusts for biological and technical variance and improves AFB₁ internal dose estimates. Using data from AFB₁-lysine and HSA measurements in 763 subjects, in combination with regression and Monte Carlo simulation techniques, we found that HSA accounts for essentially none of the between-person variance in HSA-normalized (R² = 0.04) or raw AFB₁-lysine measurements (R² = 0.0001), and that HSA normalization of AFB₁-lysine levels with empirical HSA values does not reduce measurement error any better than does the use of simulated data (n = 20,000). These findings were robust across diverse populations (Guatemala, China, Chile), AFB₁ exposures (10⁵ range), HSA assays (dye-binding and immunoassay), and disease states (healthy, gallstones, and gallbladder cancer). HSA normalization results in arithmetic transformation with the addition of technical error from the measurement of HSA. Combined with the added analysis time, cost, and sample consumption, these results suggest that it may be prudent to abandon the practice of normalizing adducts to HSA concentration when measuring any HSA adducts—not only AFB₁-lys adducts—when using LCMS in serum/plasma. Full article

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A₂ (PLA₂) that hydrolyses host cell membranes, and P. aeruginosa strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA₂-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA₂-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA₂ activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions. Full article

Fumonisins, a group of highly prevalent and toxic mycotoxins, are suspected to be causal agents of several diseases in animals and humans. In the animal feed industry, fumonisin esterase is used as feed additive to prevent mycotoxicosis caused by fumonisins. In humans, a popular dosage form for dietary supplements, with high patient acceptance for oral intake, is capsule ingestion. Thus, fumonisin esterase provided in a capsule could be an effective strategy against fumonisin intoxication in humans. To determine the efficacy of fumonisin esterase through capsule ingestion, two modes of application were compared using piglets in a small-scale preliminary study. The enzyme was administered intraorally (in-feed analogue) or intragastrically (capsule analogue), in combination with fumonisin B1 (FB1). Biomarkers for FB1 exposure; namely FB1, hydrolysed FB1 (HFB1) and partially hydrolysed forms (pHFB1a and pHFB1b), were measured both in serum and faeces using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and toxicokinetic parameters were calculated. Additionally, the serum sphinganine/sphingosine (Sa/So) ratio, a biomarker of effect, was determined using LC-MS/MS. A significantly higher Sa/So ratio was shown in the placebo group compared to both esterase treatments, demonstrating the efficacy of the esterase. Moreover, a significant decrease in serum FB1 area under the concentration-time curve (AUC) and an increase of faecal HFB1 AUC were observed after intraoral esterase administration. However, these effects were not observed with statistical significance after intragastric esterase administration with the current sample size. Full article

Cheese represents a dairy product extremely inclined to fungal growth and mycotoxin production. The growth of fungi belonging to Aspergillus, Penicillium, Fusarium, Claviceps, Alternaria, and Trichoderma genera in or on cheese leads to undesirable changes able to affect the quality of the final products. In the present investigation, a total of 68 types of commercial and traditional Slovak cheeses were analyzed to investigate the occurrence of fungal metabolites. Altogether, 13 fungal metabolites were identified and quantified. Aflatoxin M1, the only mycotoxin regulated in milk and dairy products, was not detected in any case. However, the presence of metabolites that have never been reported in cheeses, such as tryptophol at a maximum concentration level from 13.4 to 7930 µg/kg (average: 490 µg/kg), was recorded. Out of all detected metabolites, enniatin B represents the most frequently detected mycotoxin (0.06–0.71 µg/kg) in the analyzed samples. Attention is drawn to the lack of data on mycotoxins’ origin from Slovak cheeses; in fact, this is the first reported investigation. Our results indicate the presence of fungal mycotoxin contamination for which maximum permissible levels are not established, highlighting the importance of monitoring the source and producers of contamination in order to protect consumers’ health. Full article

Epidemiology of Fusarium Head Blight (FHB) of spring barley is relatively little understood. In a five-year study, we assessed quantitative resistance to FHB in an assortment of 17 spring barley genotypes in the field in southern Germany. To this end, we used soil and spray inoculation of plants with F. culmorum and F. avenaceum. This increased disease pressure and provoked genotypic differentiation. To normalize effects of variable weather conditions across consecutive seasons, we used a disease ranking of the genotypes based on quantification of fungal DNA contents and multiple Fusarium toxins in harvested grain. Together, this allowed for assessment of stable quantitative FHB resistance of barley in several genotypes. Fungal DNA contents were positively associated with species-specific Fusarium toxins in single years and over several years in plots with soil inoculation. In those plots, plant height limited FHB; however, this was not observed after spray inoculation. A multiple linear regression model of recorded weather parameter and fungal DNA contents over five years identified time periods during the reproductive phase of barley, in which weather strongly influenced fungal colonization measured in mature barley grain. Environmental conditions before heading and late after anthesis showed strongest associations with F. culmorum DNA in all genotypes, whereas for F. avenaceum, this was less consistent where we observed weather-dependent associations, depending on the genotype. Based on this study, we discuss aspects of practical resistance breeding in barley relevant to improve quantitative resistance to FHB and associated mycotoxin contaminations. Full article

Blooms of harmful cyanobacteria Microcystis aeruginosa lead to an adverse effect on freshwater ecosystems, and thus extensive studies on the control of this cyanobacteria’s blooms have been conducted. Throughout this study, we have found that the two bacteria Aeromonas bestiarum HYD0802-MK36 and Pseudomonas syringae KACC10292^T are capable of killing M. aeruginosa. Interestingly, these two bacteria showed different algicidal modes. Based on an algicidal range test using 15 algal species (target and non-target species), HYD0802-MK36 specifically attacked only target cyanobacteria M. aeruginosa, whereas the algicidal activity of KACC10292^T appeared in a relatively broad algicidal range. HYD0802-MK36, as a direct attacker, killed M. aeruginosa cells when direct cell (bacterium)-to-cell (cyanobacteria) contact happens. KACC10292^T, as an indirect attacker, released algicidal substance which is located in cytoplasm. Interestingly, algicidal activity of KACC10292^T was enhanced according to co-cultivation with the host cyanobacteria, suggesting that quantity of algicidal substance released from this bacterium might be increased via interaction with the host cyanobacteria. Full article

Aim of this study was to investigate the long-term results of botulinum toxin A (BoNT-A) injections for the treatment of benign essential blepharospasm (BEB) and to report our experience with (ultra-)long-term treatment with onabotulinumtoxin-A. We conducted a retrospective cross-sectional analysis at a university hospital. Patients with BEB and BoNT-A treatment were assigned to the Total Blepharospasm Group, patients with ≥21 onabotulinumtoxin-A injections to the Ona Long-Term Group. The Total Blepharospasm Group (n = 1940) included 33,933 BoNT-A injections. The age of patients at symptom onset was (mean ± SD) 58.0 ± 13.1 years, and 70.4% were female. The Ona long-term group (n = 234) included 10,632 onabotulinumtoxin-A injections. In this group, patients received 45.4 ± 22.9 injections with a mean dose of 22.2 IU ± 0.5. The duration of treatment was 12.6 ± 5.4 years, ranging from 2.9 to 30.0 years. The effect–duration–dose quotient did not change during long-term treatment. The observed side effects were comparable in type and frequency to other studies, even with the (ultra-)long treatment with onabotulinumtoxin-A. Our results, based on one of the largest patient populations and a treatment duration of up to 30 years, impressively demonstrate that onabotulinumtoxin-A is a safe and effective therapy for essential blepharospasm, even in the ultra-long term. Full article

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural environment. Among the affected hosts are economically important cultured fish, such as sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), yellowtail (Seriola quinqueradiata), and cobia (Rachycentron canadum). The best characterized virulence factor of Phdp is the Apoptosis-Inducing Protein of 56 kDa (AIP56), a secreted AB-type toxin that has been shown to induce apoptosis of sea bass phagocytes during infection. AIP56 has an A subunit that displays metalloprotease activity against NF-kB p65 and a B subunit that mediates binding and internalization of the A subunit in susceptible cells. Despite the fact that the aip56 gene is highly prevalent in Phdp isolates from different fish species, the toxicity of AIP56 has only been studied in sea bass. In the present study, the toxicity of AIP56 for sea bream was evaluated. Ex vivo assays showed that sea bream phagocytes are resistant to AIP56 cytotoxicity and that resistance was associated with an inefficient internalization of the toxin by those cells. Accordingly, in vivo intoxication assays revealed that sea bream is much more resistant to AIP56-induced lethality than sea bass. These findings, showing that the effect of AIP56 is different in sea bass and sea bream, set the basis for future studies to characterize the effects of AIP56 and to fully elucidate its virulence role in different Phdp susceptible hosts. Full article

A total of 209 samples of various cereal crops (maize, wheat, barley, rye and oats) grown in Croatian fields during 2016 and 2017 were collected to analyze and determine the occurrence and co-occurrence of EU regulated mycotoxins in cereals (AFB1, AFB2, AFG1, AFG2, DON, FB1, FB2, ZEA, T-2, HT-2 and OTA). The analysis, performed by a validated confirmatory LC-MS/MS method based on a dilute and shoot principle, highlighted Fusarium mycotoxins as the main contaminants, often co-occurring in samples from both years (50.0% in 2016 and 33.7% in 2017). DON was found to be the most frequent mycotoxin, present in 72.5% of the 2016 samples and 32.6% of the 2017 samples, while maize proved to be the most contaminated cereal type of both years with FUM as the most abundant mycotoxins, with an average concentration of 1180 µg/kg. Moderate temperatures with periods of high humidity favored the accumulation of DON in wheat samples instead of other Fusarium mycotoxins, while similar conditions favored maize contamination with FUM. A total of 8.3% of all the 2016 harvest samples and 7.9% of the 2017 harvest samples were assessed as non-compliant, containing mycotoxins in concentrations higher than the levels set by the EU legislation for food. Full article

Fumonisin B1 (FB1) is a widespread mycotoxin produced by fungal Fusarium species—mainly in maize, one of the plants most commonly used for food and feed. Pigs and horses are the animal species most susceptible to this mycotoxin. FB1 exposure can cause highly diverse clinical symptoms, including hepatotoxicity, immunotoxicity, and intestinal barrier function disturbance. Inhibition of ceramide synthetase is a well-understood ubiquitous molecular mechanism of FB1 toxicity, but other more tissue-specific effects remain to be elucidated. To investigate the effects of FB1 in different exposed tissues, we cross-analyzed the transcriptomes of fours organs: liver, jejunum, jejunal Peyer’s patches, and spleen. During a four-week study period, pigs were fed a control diet or a FB1-contaminated diet (10 mg/kg feed). In response to oral FB1 exposure, we observed common biological processes in the four organs, including predominant and recurrent processes (extracellular matrix organization, integrin activation, granulocyte chemotaxis, neutrophil migration, and lipid and sterol homeostasis), as well as more tissue-specific processes that appeared to be related to lipid outcomes (cell cycle regulation in jejunum, and gluconeogenesis in liver). Full article

Corn (Zea mays) is a worldwide crop subjected to infection by toxigenic fungi such as Fusarium verticillioides during the pre-harvest stage. Fusarium contamination can lead to the synthesis of highly toxic mycotoxins, such as Fumonisin B₁ (FB₁) and Fumonisin B₂ (FB₂), which compromises human and animal health. The work aimed to study the antifungal properties of fermented yellow and oriental mustard extracts using nine lactic acid bacteria (LAB) in vitro. Moreover, a chemical characterization of the main phenolic compounds and organic acids were carried out in the extracts. The results highlighted that the yellow mustard, fermented by Lactiplantibacillus plantarum strains, avoided the growth of Fusarium spp. in vitro, showing Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values, ranging from 7.8 to 15.6 g/L and 15.6 to 31.3 g/L, respectively. Then, the lyophilized yellow mustard fermented extract by L. plantarum TR71 was applied through spray-on corn ears contaminated with F. verticillioides to study the antimycotoxigenic activity. After 14 days of incubation, the control contained 14.71 mg/kg of FB₁, while the treatment reduced the content to 1.09 mg/kg (92.6% reduction). Moreover, no FB₂ was observed in the treated samples. The chemical characterization showed that lactic acid, 3-phenyllactic acid, and benzoic acid were the antifungal metabolites quantified in higher concentrations in the yellow mustard fermented extract with L. plantarum TR71. The results obtained confirmed the potential application of fermented mustard extracts as a solution to reduce the incidence of mycotoxins in corn ears. Full article

Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data. Full article

Botulinum Toxin injections into salivary glands (SG) up to a total dose of 100 units IncobotulinumtoxinA (IncoA) represent the treatment of choice for sialorrhea. However, BTX might also protect SG against sialotoxic radioligand cancer therapies. The radioligand Actinium-225-PSMA effectively targets Prostate Cancer (PCa) metastases but inevitably destroys SG due to unintended gland uptake. A preliminary case series with regular-dose IncoA failed to reduce SG PSMA-radioligand uptake. We therefore increased IncoA dosage in combination with transdermal scopolamine until a clinically relevant SG PSMA-radioligand uptake reduction was achieved. Ten consecutive men with metastasized PCa refractory to all other cancer therapies received gradually increasing IncoA dosages as part of a compassionate use PSMA-radioligand-therapy trial. The parotid gland received six and the submandibular gland three injection points under ultrasound control, up to a maximum of 30 units IncoA per injection point. A maximum total dose of 250 units IncoA was applied with up to 170 units per parotid and 80 units per submandibular gland. Treatment was well tolerated and all side-effects were non-serious. The most frequent side-effect was dry mouth of mild severity. No dysphagia, facial weakness, chewing difficulties or systemic side-effects were observed. SG injections with IncoA up to a total dose of 250 units are safe when distributed among several injection-points under ultrasound control by an experienced physician. These preliminary findings lay the basis for future trials including BTX as major component for SG protection in established as well as newly emerging radioligand cancer therapies. Full article

Fusarium head blight (FHB) is an economically important plant disease. Some Fusarium species produce mycotoxins that cause food safety concerns for both humans and animals. One especially important mycotoxin-producing fungus causing FHB is Fusarium graminearum. However, Fusarium species form a disease complex where different Fusarium species co-occur in the infected cereals. Effective management strategies for FHB are needed. Development of the management tools requires information about the diversity and abundance of the whole Fusarium community. Molecular quantification assays for detecting individual Fusarium species and subgroups exist, but a method for the detection and quantification of the whole Fusarium group is still lacking. In this study, a new TaqMan-based qPCR method (FusE) targeting the Fusarium-specific elongation factor region (EF1α) was developed for the detection and quantification of Fusarium spp. The FusE method was proven as a sensitive method with a detection limit of 1 pg of Fusarium DNA. Fusarium abundance results from oat samples correlated significantly with deoxynivalenol (DON) toxin content. In addition, the whole Fusarium community in Finnish oat samples was characterized with a new metabarcoding method. A shift from F. culmorum to F. graminearum in FHB-infected oats has been detected in Europe, and the results of this study confirm that. These new molecular methods can be applied in the assessment of the Fusarium community and mycotoxin risk in cereals. Knowledge gained from the Fusarium community analyses can be applied in developing and selecting effective management strategies for FHB. Full article

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) naturally co-occur in several foods, but no studies have followed the fate of mycotoxins’ interactions along the gastrointestinal tract using in vitro digestion models. This study used a novel semi-dynamic model that mimics gradual acidification and gastric emptying, coupled with a static colonic fermentation phase, in order to monitor mycotoxins’ bioaccessibility by the oral route. AFB1 and OTA bioaccessibility patterns differed in single or co-exposed scenarios. When co-exposed (MIX meal), AFB1 bioaccessibility at the intestinal level increased by ~16%, while OTA bioaccessibility decreased by ~20%. Additionally, a significant increase was observed in both intestinal cell viability and NO production. With regard to mycotoxin–probiotic interactions, the MIX meal showed a null effect on Lactobacillus and Bifidobacterium strain growth, while isolated AFB1 reduced bacterial growth parameters. These results were confirmed at phylum and family levels using a gut microbiota approach. After colonic fermentation, the fecal supernatant did not trigger the NF-kB activation pathway, indicating reduced toxicity of mycotoxins. In conclusion, if single exposed, AFB1 will have a significant impact on intestinal viability and probiotic growth, while OTA will mostly trigger NO production; in a co-exposure situation, both intestinal viability and inflammation will be affected, but the impact on probiotic growth will be neglected. Full article

Anemonia viridis is an abundant and widely distributed temperate sea anemone that can form dense congregations of individuals. Despite the potential severity of its sting, few detailed cases have been reported. We report a case of a severe toxic reaction following an A. viridis sting in a 35-year-old oceanographer. She developed severe pain, itching, redness, and burning sensation, which worsened one week after treatment with anti-inflammatories, antihistamines and corticosteroids. Prompted by this event, and due to the insufficient risk prevention, lack of training for marine-environment users, and lack of research into sting-specific first-aid protocols, we evaluated the cnidocyst response to five different compounds commonly recommended as rinse solutions in first-aid protocols (seawater, vinegar, ammonia, baking soda, and freshwater) by means of the Tentacle Solution Assay. Vinegar and ammonia triggered an immediate and massive cnidocyst discharge after their application and were classified as activator solutions. Baking soda and freshwater were also classified as activator solutions, although with a lower intensity of discharge. Only seawater was classified as a neutral solution and therefore recommended as a rinse solution after A. viridis sting, at least until an inhibitory solution is discovered. Full article

Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular. Full article

Ergot alkaloid mycotoxins interfere in many functions associated with serotonergic neurotransmitters. Therefore, the objective was to evaluate whether the association of serotonin (5-hydroxytryptamine, 5-HT) and ergot alkaloids during a 24 h pre-incubation could affect the vascular contractile response to ergot alkaloids. To evaluate the effects of 24 h exposure to 5-HT and ergot alkaloids (ergovaline, ERV), two assays were conducted. The first assay determined the half-maximal inhibitory concentration (IC₅₀) following the 24 h pre-exposure period, while the second assay evaluated the effect of IC₅₀ concentrations of 5-HT and ERV either individually or in combination. There was an interaction between previous exposure to 5-HT and ERV. Previous exposure to 5-HT at the IC₅₀ concentration of 7.57 × 10⁻⁷ M reduced the contractile response by more than 50% of control, while the exposure to ERV at IC₅₀ dose of 1.57 × 10⁻¹⁰ M tended to decrease (p = 0.081) vessel contractility with a response higher than 50% of control. The 24 h previous exposure to both 5-HT and ERV did not potentiate the inhibitory response of blood vessels in comparison with incubation with each compound alone. These results suggest receptor competition between 5-HT and ERV. More studies are necessary to determine the potential of 5-HT to treat toxicosis caused by ergot alkaloids. Full article

Collagen is a major component of the extracellular matrix (ECM) and has an imminent role in fibrosis, in, among others, chronic kidney disease (CKD). Collagen alpha-1(I) (col1a1) is the most abundant collagen type and has previously been underlined for its contribution to the disease phenotype. Here, we examined 5000 urinary peptidomic datasets randomly selected from healthy participants or patients with CKD to identify urinary col1a1 fragments and study their abundance, position in the main protein, as well as their correlation with renal function. We identified 707 col1a1 peptides that differed in their amino acid sequence and/or post-translational modifications (hydroxyprolines). Well-correlated peptides with the same amino acid sequence, but a different number of hydroxyprolines, were combined into a final list of 503 peptides. These 503 col1a1 peptides covered 69% of the full col1a1 sequence. Sixty-three col1a1 peptides were significantly and highly positively associated (rho > +0.3) with the estimated glomerular filtration rate (eGFR), while only six peptides showed a significant and strong, negative association (rho < −0.3). A similar tendency was observed for col1a1 peptides associated with ageing, where the abundance of most col1a1 peptides decreased with increasing age. Collectively the results show a strong association between collagen peptides and loss of kidney function and suggest that fibrosis, potentially also of other organs, may be the main consequence of an attenuation of collagen degradation, and not increased synthesis. Full article

Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial–mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD. Full article

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (β-PFT) produced by Clostridium perfringens type C. It plays a central role in the pathogenesis of necro-hemorrhagic enteritis in young animals and humans via targeting intestinal endothelial cells. We recently identified the membrane protein CD31 (PECAM-1) as the receptor for CPB on mouse endothelial cells. We now assess the role of CD31 in CPB cytotoxicity against human endothelial and monocytic cells using a CRISPR/Cas9 gene knockout and an antibody blocking approach. CD31 knockout human endothelial and monocytic cells were resistant to CPB and CPB oligomers only formed in CD31-expressing cells. CD31 knockout endothelial and monocytic cells could be selectively enriched out of a polyclonal cell population by exposing them to CPB. Moreover, antibody mediated blocking of the extracellular Ig6 domain of CD31 abolished CPB cytotoxicity and oligomer formation in endothelial and monocytic cells. In conclusion, this study confirms the role of CD31 as a receptor of CPB on human endothelial and monocytic cells. Specific interaction with the CD31 molecule can thus explain the cell type specificity of CPB observed in vitro and corresponds to in vivo observations in naturally diseased animals. Full article

Venoms of solitary wasps are utilized for prey capture (insects and spiders), paralyzing them with a stinger injection to be offered as food for their larvae. Thus, the identification and characterization of the components of solitary wasp venoms can have biotechnological application. In the present study, the venom components profile of a solitary scoliid wasp, Campsomeriella annulata annulata, was investigated through a comprehensive analysis using LC-MS and -MS/MS. Online mass fingerprinting revealed that the venom extract contains 138 components, and MS/MS analysis identified 44 complete sequences of the peptide components. The peptides are broadly divided into two classes: bradykinin-related peptides, and linear α-helical peptides. Among the components of the first class, the two main peptides, α-campsomerin (PRLRRLTGLSPLR) and β-campsomerin (PRLRRLTGLSPLRAP), had their biological activities evaluated. Both peptides had no effects on metallopeptidases [human neprilysin (NEP) and angiotensin-converting enzyme (ACE)] and acetylcholinesterase (AChE), and had no cytotoxic effects. Studies with PC12 neuronal cells showed that only α-campsomerin was able to enhance cell viability, while β-campsomerin had no effect. It is noteworthy that the only difference between the primary structures from these peptides is the presence of the AP extension at the C-terminus of β-campsomerin, compared to α-campsomerin. Among the linear α-helical peptides, annulatin (ISEALKSIIVG-NH₂) was evaluated for its biological activities. Annulatin showed histamine releasing activity from mast cells and low hemolytic activity, but no antimicrobial activities against all microbes tested were observed. Thus, in addition to providing unprecedented information on the whole components, the three peptides selected for the study suggest that molecules present in solitary scoliid wasp venoms may have interesting biological activities. Full article

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea. Full article

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work. Full article

Infant botulism is a rare and underdiagnosed disease caused by BoNT-producing clostridia that can temporarily colonize the intestinal lumen of infants less than one year of age. The diagnosis may be challenging because of its rareness, especially in patients showing atypical presentations or concomitant coinfections. In this paper, we report the first infant botulism case associated with Cytomegalovirus coinfection and transient hypogammaglobulinemia and discuss the meaning of these associations in terms of risk factors. Intending to help physicians perform the diagnosis, we also propose a practical clinical and diagnostic criteria checklist based on the revision of the literature. Full article

Epithelial–mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer. Full article

Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis. We performed an in-depth bioinformatics analysis of aegerolysins in entomopathogenic fungi and selected a candidate aegerolysin, beauveriolysin A (BlyA) from Beauveria bassiana. BlyA was expressed as a recombinant protein in Escherichia coli, and purified to further determine its functional and structural properties, including lipid-binding ability. Aegerolysins were found to be encoded in genomes of entomopathogenic fungi, such as Beauveria, Cordyceps, Metarhizium and Ophiocordyceps. Detailed bioinformatics analysis revealed that they are linked to MACPF-like genes in most genomes. We also show that BlyA interacts with an insect-specific membrane lipid. These results were placed in the context of other fungal and bacterial aegerolysins and their partner proteins. We believe that aegerolysins play a role in promoting the entomopathogenic and antagonistic activity of B. bassiana, which is an active ingredient of bioinsecticides. Full article

Chronic kidney disease (CKD) is characterized by gut dysbiosis with a decrease in short-chain fatty acid (SCFA)-producing bacteria. Levels of protein-bound uremic toxins (PBUTs) and post-translational modifications (PTMs) of albumin increase with CKD, both risk factors for cardiovascular morbidity and mortality. The relationship between fecal metabolites and plasma concentrations of PBUTs in different stages of CKD (n = 103) was explored. Estimated GFR tends to correlate with fecal butyric acid (BA) concentrations (r_s = 0.212; p = 0.032), which, in its turn, correlates with the abundance of SCFA-producing bacteria. Specific SCFAs correlate with concentrations of PBUT precursors in feces. Fecal levels of p-cresol correlate with its derived plasma UTs (p-cresyl sulfate: r_s = 0.342, p < 0.001; p-cresyl glucuronide: r_s = 0.268, p = 0.006), whereas an association was found between fecal and plasma levels of indole acetic acid (r_s = 0.306; p = 0.002). Finally, the albumin symmetry factor correlates positively with eGFR (r_s = 0.274; p = 0.005). The decreased abundance of SCFA-producing gut bacteria in parallel with the fecal concentration of BA and indole could compromise the intestinal barrier function in CKD. It is currently not known if this contributes to increased plasma levels of PBUTs, potentially playing a role in the PTMs of albumin. Further evaluation of SCFA-producing bacteria and SCFAs as potential targets to restore both gut dysbiosis and uremia is needed. Full article

Snake venom metalloproteinases (SVMP) are involved in local inflammatory reactions observed after snakebites. Based on domain composition, they are classified as PI (pro-domain + proteolytic domain), PII (PI + disintegrin-like domains), or PIII (PII + cysteine-rich domains). Here, we studied the role of different SVMPs domains in inducing the expression of adhesion molecules at the microcirculation of the cremaster muscle of mice. We used Jararhagin (Jar)—a PIII SVMP with intense hemorrhagic activity, and Jar-C—a Jar devoid of the catalytic domain, with no hemorrhagic activity, both isolated from B. jararaca venom and BnP-1—a weakly hemorrhagic P1 SVMP from B. neuwiedi venom. Toxins (0.5 µg) or PBS (100 µL) were injected into the scrotum of mice, and 2, 4, or 24 h later, the protein and gene expression of CD54 and CD31 in the endothelium, and integrins (CD11a and CD11b), expressed in leukocytes were evaluated. Toxins induced significant increases in CD54, CD11a, and CD11b at the initial time and a time-related increase in CD31 expression. In conclusion, our results suggest that, despite differences in hemorrhagic activities and domain composition of the SVMPs used in this study, they behave similarly to the induction of expression of adhesion molecules that promote leukocyte recruitment. Full article

Ergot fungi (Claviceps spp.) are infamous for producing sclerotia containing a wide spectrum of ergot alkaloids (EA) toxic to humans and animals, making them nefarious villains in the agricultural and food industries, but also treasures for pharmaceuticals. In addition to three classes of EAs, several species also produce paspaline-derived indole diterpenes (IDT) that cause ataxia and staggers in livestock. Furthermore, two other types of alkaloids, i.e., loline (LOL) and peramine (PER), found in Epichloë spp., close relatives of Claviceps, have shown beneficial effects on host plants without evidence of toxicity to mammals. The gene clusters associated with the production of these alkaloids are known. We examined genomes of 53 strains of 19 Claviceps spp. to screen for these genes, aiming to understand the evolutionary patterns of these genes across the genus through phylogenetic and DNA polymorphism analyses. Our results showed (1) varied numbers of eas genes in C. sect. Claviceps and sect. Pusillae, none in sect. Citrinae, six idt/ltm genes in sect. Claviceps (except four in C. cyperi), zero to one partial (idtG) in sect. Pusillae, and four in sect. Citrinae, (2) two to three copies of dmaW, easE, easF, idt/ltmB, itd/ltmQ in sect. Claviceps, (3) frequent gene gains and losses, and (4) an evolutionary hourglass pattern in the intra-specific eas gene diversity and divergence in C. purpurea. Full article

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as additional biocontrol mechanisms and deserves further exploration to improve biocontrol formulations. Full article

Ecologically divergent selection can lead to the evolution of reproductive isolation through the process of ecological speciation, but the balance of responsible evolutionary forces is often obscured by an inadequate assessment of demographic history and the genetics of traits under selection. Snake venoms have emerged as a system for studying the genetic basis of adaptation because of their genetic tractability and contributions to fitness, and speciation in venomous snakes can be associated with ecological diversification such as dietary shifts and corresponding venom changes. Here, we explored the neurotoxic (type A)–hemotoxic (type B) venom dichotomy and the potential for ecological speciation among Timber Rattlesnake (Crotalus horridus) populations. Previous work identified the genetic basis of this phenotypic difference, enabling us to characterize the roles geography, history, ecology, selection, and chance play in determining when and why new species emerge or are absorbed. We identified significant genetic, proteomic, morphological, and ecological/environmental differences at smaller spatial scales, suggestive of incipient ecological speciation between type A and type B C. horridus. Range-wide analyses, however, rejected the reciprocal monophyly of venom type, indicative of varying intensities of introgression and a lack of reproductive isolation across the range. Given that we have now established the phenotypic distributions and ecological niche models of type A and B populations, genome-wide data are needed and capable of determining whether type A and type B C. horridus represent distinct, reproductively isolated lineages due to incipient ecological speciation or differentiated populations within a single species. Full article

Maize expressing Cry1Ab insecticidal toxin (Bt maize) is an effective method to control Sesamia nonagrioides and Ostrinia nubilalis, the most damaging corn borers of southern Europe. In this area, maize is prone to Fusarium infections, which can produce mycotoxins that pose a serious risk to human and animal health, causing significant economic losses in the agrifood industry. To investigate the influence of corn borer damage on the presence of Fusarium species and their mycotoxins, Bt maize ears and insect-damaged ears of non-Bt maize were collected from commercial fields in three Bt maize growing areas in Spain, and differences in contamination were assessed. Additionally, larvae of both borer species were collected to evaluate their role as vectors of these molds. Non-Bt maize ears showed significantly higher presence of F. verticillioides, F. proliferatum, and F. subglutinans than Bt maize ears. For the first time, Fusarium species have been isolated from larvae of the two species. The most frequently found mycotoxins in ears were fumonisins, with non-Bt ears being significantly more contaminated than those of Bt maize. High levels of fumonisins were shown to correlate with the occurrence of corn borers in the ear and the presence of F. verticillioides and F. proliferatum. Full article

Modeling ciguatoxin (CTX) trophic transfer in marine food webs has significant implications for the management of ciguatera poisoning, a circumtropical disease caused by human consumption of CTX-contaminated seafood. Current models associated with CP risk rely on modeling abundance/presence of CTX-producing epi-benthic dinoflagellates, e.g., Gambierdiscus spp., and are based on studies showing that toxin production is site specific and occurs in pulses driven by environmental factors. However, food web models are not yet developed and require parameterizing the CTX exposure cascade in fish which has been traditionally approached through top-down assessment of CTX loads in wild-caught fish. The primary goal of this study was to provide critical knowledge on the kinetics of C-CTX-1 bioaccumulation and depuration in the marine omnivore Lagodon rhomboides. We performed a two-phase, 17 week CTX feeding trial in L. rhomboides where fish were given either a formulated C-CTX-1 (n = 40) or control feed (n = 37) for 20 days, and then switched to a non-toxic diet for up to 14 weeks. Fish were randomly sampled through time with whole muscle, liver, and other pooled viscera dissected for toxin analysis by a sodium channel-dependent MTT-based mouse neuroblastoma (N2a) assay. The CTX levels measured in all tissues increased with time during the exposure period (days 1 to 20), but a decrease in CTX-specific toxicity with depuration time only occurred in viscera extracts. By the end of the depuration, muscle, liver, and viscera samples had mean toxin concentrations of 189%, 128%, and 42%, respectively, compared to fish sampled at the start of the depuration phase. However, a one-compartment model analysis of combined tissues showed total concentration declined to 56%, resulting in an approximate half-life of 97 d (R² = 0.43). Further, applying growth dilution correction models to the overall concentration found that growth was a major factor reducing C-CTX concentrations, and that the body burden was largely unchanged, causing pseudo-elimination and a half-life of 143–148 days (R² = 0.36). These data have important implications for food web CTX models and management of ciguatera poisoning in endemic regions where the frequency of environmental algal toxin pulses may be greater than the growth-corrected half-life of C-CTX in intermediate-trophic-level fish with high site fidelity. Full article

Fumonisins (FB) are mycotoxins known to exert most of their toxicity by blocking ceramide synthase, resulting in disruption of sphingolipid metabolism. Although the effects of FB on sphinganine (Sa) and sphingosine (So) are well documented in poultry, little information is available on their other effects on sphingolipids. The objective of this study was to analyze the effects of FB on the hepatic and plasma sphingolipidome in chickens. The first concern of this analysis was to clarify the effects of FB on hepatic sphingolipid levels, whose variations can lead to numerous toxic manifestations. The second was to specify the possible use of an alteration of the sphingolipidome as a biomarker of exposure to FB, in addition to the measurement of the Sa:So ratio already widely used. For this purpose, we developed an UHPLC MS/MS method that enabled the determination of 82 SL, including 10 internal standards, in chicken liver and plasma. The validated method was used to measure the effects of FB administered to chickens at a dose close to 20 mg FB1 + FB2/kg feed for 9 days. Significant alterations of sphingoid bases, ceramides, dihydroceramides, glycosylceramides, sphingomyelins and dihydrosphingomyelins were observed in the liver. In addition, significant increases in plasma sphinganine 1-phosphate, sphingosine 1-phosphate and sphingomyelins were observed in plasma. Interestingly, partial least-squares discriminant analysis of 11 SL in plasma made it possible to discriminate exposed chickens from control chickens, whereas analysis of Sa and So alone revealed no difference. In conclusion, our results show that the effects of FB in chickens are complex, and that SL profiling enables the detection of exposure to FB when Sa and So fail. Full article

Uremic pruritus is a distressful complication of chronic kidney disease and results in impaired quality of life and higher mortality rates. Intravenous sodium thiosulfate has been reported to alleviate pruritus in hemodialysis patients. We performed a systematic review and meta-analysis to estimate the efficacy of intravenous sodium thiosulfate in patients with uremic pruritus. A systematic search of electronic databases up to June 2021 was conducted for randomized controlled trials that evaluated the clinical effects of sodium thiosulfate in the management of patients with uremic pruritus. Two reviewers selected eligible articles and evaluated the risk of bias; the results of pruritus assessment and uremic pruritus-related laboratory parameters in selected studies were analyzed. There are four trials published between 2018 and 2021, which include 222 participants. The sodium thiosulfate group displayed significant decrease in the pruritus score (standardized mean difference = −3.52, 95% confidence interval = −5.63 to −1.41, p = 0.001), without a significant increase in the adverse effects (risk ratio = 2.44, 95% confidence interval = 0.37 to 15.99, p = 0.35) compared to the control group. Administration of sodium thiosulfate is found to be a safe and efficacious complementary therapy in improving uremic pruritus in patients with chronic kidney disease. Full article

Prevalence, impact on shellfish resources and interspecific, spatial, and temporal variabilities of domoic acid (DA) in bivalves from Galicia (NW Spain) have been studied based on more than 25 years of monitoring data. The maximum prevalence (samples in which DA was detected) (100%) and incidence (samples with DA levels above the regulatory limit) (97.4%) were recorded in Pecten maximus, and the minimum ones in Mytilus galloprovincialis (12.6 and 1.1%, respectively). The maximum DA concentrations were 663.9 mg kg⁻¹ in P. maximus and 316 mg kg⁻¹ in Venerupis corrugata. After excluding scallop P. maximus data, DA was found (prevalence) in 13.3% of bivalve samples, with 1.3% being over the regulatory limit. In general, the prevalence of this toxin decreased towards the North but not the magnitude of its episodes. The seasonal distribution was characterized by two maxima, in spring and autumn, with the later decreasing in intensity towards the north. DA levels decreased slightly over the studied period, although this decreasing trend was not linear. A cyclic pattern was observed in the interannual variability, with cycles of 4 and 11 years. Intoxication and detoxification rates were slower than those expected from laboratory experiments, suggesting the supply of DA during these phases plays an important role. Full article

Fusarium head blight (FHB) is one of the most serious diseases of small-grain cereals worldwide, resulting in yield reduction and an accumulation of the mycotoxin deoxynivalenol (DON) in grain. Weather conditions are known to have a significant effect on the ability of fusaria to infect cereals and produce toxins. In the past 10 years, severe outbreaks of FHB, and grain DON contamination exceeding the EU health safety limits, have occurred in countries in the Baltic Sea region. In this study, extensive data from field trials in Sweden, Poland and Lithuania were analysed to identify the most crucial weather variables for the ability of Fusarium to produce DON. Models were developed for the prediction of DON contamination levels in harvested grain exceeding 200 µg kg⁻¹ for oats, spring barley and spring wheat in Sweden and winter wheat in Poland, and 1250 µg kg⁻¹ for spring wheat in Lithuania. These models were able to predict high DON levels with an accuracy of 70–81%. Relative humidity (RH) and precipitation (PREC) were identified as the weather factors with the greatest influence on DON accumulation in grain, with high RH and PREC around flowering and later in grain development and ripening correlated with high DON levels. High temperatures during grain development and senescence reduced the risk of DON accumulation. The performance of the models, based only on weather variables, was relatively accurate. In future studies, it might be of interest to determine whether inclusion of variables such as pre-crop, agronomic factors and crop resistance to FHB could further improve the performance of the models. Full article

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5–3 month periods (2018–2019). Concentrations of TTX ranged from ‘not detected’ to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex. Full article

As aflatoxins are a global risk for humans and animals, testing methods for rapid on-site screening are increasingly needed alongside the standard analytical laboratory tools. In the presented study, lateral flow devices (LFDs) for rapid total aflatoxin screening were thoroughly investigated with respect to their matrix effects, cross-reactivity, their performance under harsh conditions in Sub-Saharan Africa (SSA), and their stability, as well as when compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To analyze the matrix effects, qualitative test kits offering a certain cutoff level were used to screen different nut samples. In addition, these tests were challenged on their cross-reactivity with 230 fungal toxins and metabolites. Furthermore, the resulting measurements performed under harsh tropical conditions (up to 38.4 °C and 91% relative humidity) in SSA, specifically Burkina Faso and Mozambique, were compared with the results from a well-established and validated LC-MS/MS-based reference method. The comparison of the on-site LFD results with the reference method showed a good agreement: 86.4% agreement, 11.8% non-agreement, and 1.8% invalid test results. To test the robustness of the cutoff tests, short- and long-term stability testing was carried out in Mozambique and Nigeria. For both experiments, no loss of test performance could be determined. Finally, a subset of African corn samples was shipped to Austria and analyzed under laboratory conditions using semiquantitative aflatoxin tests. A good correlation was found between the rapid strip tests and the LC-MS/MS reference method. Overall, the evaluated LFDs showed satisfying results regarding their cross-reactivity, matrix effects, stability, and robustness. Full article

Salinity is an important factor for regulating metabolic processes in aquatic organisms; however, its effects on toxicity and STX biosynthesis gene responses in dinoflagellates require further elucidation. Herein, we evaluated the physiological responses, toxin production, and expression levels of two STX synthesis core genes, sxtA4 and sxtG, in the dinoflagellate Alexandrium pacificum Alex05 under different salinities (20, 25, 30, 35, and 40 psu). Optimal growth was observed at 30 psu (0.12 cell division/d), but cell growth significantly decreased at 20 psu and was irregular at 25 and 40 psu. The cell size increased at lower salinities, with the highest size of 31.5 µm at 20 psu. STXs eq was highest (35.8 fmol/cell) in the exponential phase at 30 psu. GTX4 and C2 were predominant at that time but were replaced by GTX1 and NeoSTX in the stationary phase. However, sxtA4 and sxtG mRNAs were induced, and their patterns were similar in all tested conditions. PCA showed that gene transcriptional levels were not correlated with toxin contents and salinity. These results suggest that A. pacificum may produce the highest amount of toxins at optimal salinity, but sxtA4 and sxtG may be only minimally affected by salinity, even under high salinity stress. Full article

The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7–6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish. Full article

We evaluated the effects of a treatment diet contaminated with 1.7 mg deoxynivalenol and 3.5 mg fumonisins (B1, B2 and B3) per kg ration on immune status and peripheral blood gene expression profiles in finishing-stage Angus steers. The mycotoxin treatment diet was fed for a period of 21 days followed by a two-week washout period during which time all animals consumed the control diet. Whole-blood leukocyte differentials were performed weekly throughout the experimental and washout period. Comparative profiles of CD4⁺ and CD8⁺ T cells, along with bactericidal capacity of circulating neutrophils and monocytes were evaluated at 0, 7, 14, 21 and 35 days. Peripheral blood gene expression was measured at 0, 7, 21 and 35 days via RNA sequencing. Significant increases in the percentage of CD4⁻CD8⁺ T cells were observed in treatment-fed steers after two weeks of treatment and were associated with decreased CD4:CD8 T-cell ratios at this same timepoint (p ≤ 0.10). No significant differences were observed as an effect of treatment in terms of bactericidal capacity at any timepoint. Dietary treatments induced major changes in transcripts associated with endocrine, metabolic and infectious diseases; protein digestion and absorption; and environmental information processing (inhibition of signaling and processing), as evaluated by dynamic impact analysis. DAVID analysis also suggested treatment effects on oxygen transport, extra-cellular signaling, cell membrane structure and immune system function. These results indicate that finishing-stage beef cattle are susceptible to the immunotoxic and transcript-inhibitory effects of deoxynivalenol and fumonisins at levels which may be realistically encountered in feedlot situations. Full article