Toxins, Volume 11, Issue 10 (October 2019) – 65 articles

Shiga toxin-producing Escherichia coli (STEC) infections are the product of the interaction between bacteria, phages, animals, humans, and the environment. In the late 1980s, Alberta had one of the highest incidences of STEC infections in North America. Herein, we revisit and contextualize the epidemiology of STEC O157 human infections in Alberta for the period 2009–2016. STEC O157 infections were concentrated in large urban centers, but also in rural areas with high cattle density. Hospitalization was often required when the Shiga toxin genotype stx2a stx2c was involved, however, only those aged 60 years or older and infection during spring months (April to June) independently predicted that need. Since the late 1980s, the rate of STEC O157-associated hemolytic uremic syndrome (HUS) in Alberta has remained unchanged at 5.1%, despite a marked drop in the overall incidence of the infection. While Shiga toxin genotypes stx1a stx2c and stx2a stx2c seemed associated with HUS, only those aged under 10 years and infection during spring months were independently predictive of that complication. The complexity of the current epidemiology of STEC O157 in Alberta highlights the need for a One Health approach for further progress to be made in mitigating STEC morbidity. Full article

Galicia is an area with a strong mussel aquaculture industry in addition to other important bivalve mollusc fisheries. Between 2014 and 2017, 18,862 samples were analyzed for EU regulated marine lipophilic toxins. Okadaic acid (OA) was the most prevalent toxin and the only single toxin that produced harvesting closures. Toxin concentrations in raft mussels were generally higher than those recorded in other bivalves, justifying the use of this species as an indicator. The Rías of Pontevedra and Muros were the ones most affected by OA and DTX2 and the Ría of Ares by YTXs. In general, the outer areas of the Rías were more affected by OA and DTX2 than the inner ones. The OA level reached a maximum in spring, while DTX2 was almost entirely restricted to the fall–winter season. YTXs peaked in August–September. The toxins of the OA group were nearly completely esterified in all the bivalves studied except mussels and queen scallops. Risk of intoxication with the current monitoring system is low. In less than 2% of cases did the first detection of OA in an area exceed the regulatory limit. In no case, could any effect on humans be expected. The apparent intoxication and depuration rates were similar and directly related, suggesting that the rates are regulated mainly by oceanographic characteristics. Full article

This review gives an overview on the development of research on spider venoms with a focus on structure and function of venom components and techniques of analysis. Major venom component groups are small molecular mass compounds, antimicrobial (also called cytolytic, or cationic) peptides (only in some spider families), cysteine-rich (neurotoxic) peptides, and enzymes and proteins. Cysteine-rich peptides are reviewed with respect to various structural motifs, their targets (ion channels, membrane receptors), nomenclature, and molecular binding. We further describe the latest findings concerning the maturation of antimicrobial, and cysteine-rich peptides that are in most known cases expressed as propeptide-containing precursors. Today, venom research, increasingly employs transcriptomic and mass spectrometric techniques. Pros and cons of venom gland transcriptome analysis with Sanger, 454, and Illumina sequencing are discussed and an overview on so far published transcriptome studies is given. In this respect, we also discuss the only recently described cross contamination arising from multiplexing in Illumina sequencing and its possible impacts on venom studies. High throughput mass spectrometric analysis of venom proteomes (bottom-up, top-down) are reviewed. Full article

An analytical procedure is proposed for determining three cyanotoxins (microcystin RR, microcystin LR, and nodularin) and two phycotoxins (domoic and okadaic acids) in seawater and algae-based food supplements. The toxins were first isolated by a salting out liquid extraction procedure. Since the concentration expected in the samples was very low, a dispersive liquid–liquid microextraction procedure was included for preconcentration. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (80 mg) was used as green extractant solvent and acetonitrile as disperser solvent (0.5 mL) for a 10 mL sample volume at pH 1.5, following the principles of green analytical chemistry. Liquid chromatography with electrospray ionization and quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) was used. The selectivity of the detection system, based on accurate mass measurements, allowed the toxins to be unequivocally identified. Mass spectra for quadrupole time of flight-mass spectrometry (Q-TOF-MS) and Q-TOF-MS/MS were recorded in the positive ion mode and quantification was based on the protonated molecule. Retention times ranged between 6.2 and 18.3 min using a mobile phase composed by a mixture of methanol and formic acid (0.1%). None of the target toxins were detected in any of the seawater samples analyzed, above their corresponding detection limits. However, microcystin LR was detected in the blue green alga sample. Full article

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

The pathophysiological mechanisms of migraine transformation are debated. Modifications of plasma oxidative stress biomarkers have been described in chronic migraine. OnabotulintoxinA (BoNT/A) treatment, approved for chronic migraine prophylaxis, possibly reduces pain neurotransmitters release and oxidative stress products. Aims of our study were to investigate differences in the levels of selected plasmatic oxidative stress biomarkers (Advanced Oxidation Protein Products (AOPP), Ferric Reducing Antioxidant Power (FRAP), Thiolic Groups (SH)) comparing chronic migraineurs (CM) and healthy controls (HC). We also explored possible clinical and biochemical modifications in the CM group after six months of treatment with BoNT/A. At the baseline, we found higher values of AOPP (p < 0.001), and lower values of SH (p < 0.001) and FRAP (p = 0.005) in the CM group. At the six-month follow-up we found a reduction of AOPP (p < 0.001) and an increase of FRAP (p < 0.001) and SH (p = 0.023) within the CM group. BoNT/A treatment improved migraine symptoms in the CM group. We confirmed previous reports of imbalanced antioxidant mechanisms in chronic migraine showing lower antioxidant capacities in patients than controls. BoNT/A improved the levels of plasma oxidative stress biomarkers and confirmed its role as an effective prophylactic treatment for CM. Other studies should investigate the potential antioxidant properties of BoNT/A treatment. Full article

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

Fusarium graminearum is a pervasive plant pathogenic fungal species. Biological control agents employ various strategies to weaken their targets, as shown by Bacillus species, which adopt various mechanisms, including the production of bioactive compounds, to inhibit the growth of F. graminearum. Various efforts to uncover the antagonistic mechanisms of Bacillus against F. graminearum have been undertaken and have yielded a plethora of data available in the current literature. This perspective article attempts to provide a unified record of these interesting findings. The authors provide background knowledge on the use of Bacillus as a biocontrol agent as well as details on techniques and tools for studying the antagonistic mechanism of Bacillus against F. graminearum. Emphasizing its potential as a future biological control agent with extensive use, the authors encourage future studies on Bacillus as a useful antagonist of F. graminearum and other plant pathogens. It is also recommended to take advantage of the newly invented analytical platforms for studying biochemical processes to understand the mechanism of action of Bacillus against plant pathogens in general. Full article

A fluorescent immunochromatographic test strip (FICTS) based on the use of europium nanoparticles (EuNPs) was developed and applied to detect citrinin (CIT) in Monascus fermented food. The sensitivity of the immunoassay to detect CIT was greatly improved by the use of a specific monoclonal antibody to attach EuNPs to form a probe. Under optimum conditions, the visual detection limit was 2.5 ng/mL, and the detection limit of the instrument was 0.05 ng/mL. According to the results, the IC₅₀ was 0.4 ng/mL. Matrix interference from various Monascus fermented foods was investigated in food sample detection. The immunosensor also demonstrated high recoveries (86.8–113.0%) and low relative standard deviations (RSDs) (1.8–15.3%) when testing spiked Monascus fermented food. The detection results of this method showed a good correlation (R² > 0.98) with high-performance liquid chromatography (HPLC). The results showed that the FICTS method could be used as a rapid, sensitive method to detect CIT in Monascus fermented food. Full article

Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system. Full article

α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure–activity relationship (SAR) of EI revealed that the “Arg1–Asn2–Hyp3” residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue ^△1–3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue ^△1–3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs. Full article

Clays are known to have promising adsorbing characteristics, and are used as feed additives to overcome the negative effects of mycotoxicosis in livestock farming. Modification of clay minerals by heat treatment, also called calcination, can alter their adsorption characteristics. Little information, however, is available on the effect of calcination with respect to mycotoxin binding. The purpose of this study was to characterize a Tunisian clay before and after calcination (at 550 °C), and to investigate the effectiveness of the thermal treatment of this clay on its aflatoxin B1 (AFB1), G1 (AFG1), B2 (AFB2), G2 (AFG2), and zearalenone (ZEN) adsorption capacity. Firstly, the purified clay (CP) and calcined clay (CC) were characterized with X-ray Fluorescence (XRF), X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR-IR), cation exchange capacity (CEC), specific surface area (S_BET), and point of zero charge (pH_PZC) measurements. Secondly, an in vitro model that simulated the pH conditions of the monogastric gastrointestinal tract was used to evaluate the binding efficiency of the tested clays when artificially mixed with aflatoxins and zearalenone. The tested clay consisted mainly of smectite and illite. Purified and calcined clay had similar chemical compositions. After heat treatment, however, some changes in the mineralogical and textural properties were observed. The calcination decreased the cation exchange capacity and the specific surface, whereas the pore size was increased. Both purified and calcined clay had a binding efficacy of over 90% for AFB1 under simulated poultry GI tract conditions. Heat treatment of the clay increased the adsorption of AFB2, AFG1, and AFG2 related to the increase in pore size of the clay by the calcination process. ZEN adsorption also increased by calcination, albeit to a more stable level at pH 3 rather than at pH 7. In conclusion, calcination of clay minerals enhanced the adsorption of aflatoxins and mostly of AFG1 and AFG2 at neutral pH of the gastrointestinal tract, and thus are associated with protection against the toxic effects of aflatoxins. Full article

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

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

Mycotoxin removers include enzymes and adsorbents that may be used in animal feeds to eliminate the toxic effects of mycotoxins. This study aimed to determine the removability of two different types of mycotoxin removers, adsorbents and enzyme degradation reagents (EDRs), in the simulated gastrointestinal conditions of pigs and poultry. Seven commercial mycotoxin removers, including five EDRs and two adsorbents, were tested in vitro. In this study, the supplemented dosages of mycotoxin removers used in pig and poultry feeds were the commercial recommendation ranging from 0.05% to 0.2%. For pigs, the in vitro gastric and small intestinal simulations were performed by immersing the mycotoxin-tainted feed in artificial gastric juice (AGJ) at pH 2.5 for 5 h or in artificial intestinal juice (AIJ) at pH 6.5 for 2 h to mimick in vivo conditions. For poultry, mycotoxin-tainted feeds were immersed in AGJ for 2 h at pH 4.5 and 0.5 h at pH of 2.5, respectively, to simulate crop/glandular stomach and gizzard conditions; the small intestinal simulation was in AIJ for 2 h at pH 6.5. For the pig, EDRs and adsorbents had deoxynivalenol (DON) removability (1 mg/kg) of 56% to 100% and 15% to 19%, respectively. Under the concentration of 0.5 mg/kg, the zearalenone (ZEN) removability by EDRs and adsorbents was 65% to 100% and 0% to 36%, respectively. For the simulation in poultry, the removability of DON by EDRs and adsorbents (5 mg/kg) was 56% to 79% and 1% to 36%, respectively; for the concentration of 0.5 mg/kg, the removability of ZEN by EDRs and adsorbents was 38% to 69% and 7% to 9%, respectively. These results suggest that EDRs are more effective in reducing DON and ZEN contamination compared to the adsorbent methods in the simulated gastrointestinal tracts of pig and poultry. The recoveries of DON and ZEN of pig in vitro gastrointestinal simulations were higher than 86.4% and 84.7%, respectively, with 88.8% and 85.9%, respectively, in poultry. These results demonstrated the stability and accuracy of our mycotoxin extraction process and in vitro simulation efficiency. Full article

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

Atrial fibrillation (AF) is the most prevalent arrhythmia in the general population. There is a close association between chronic kidney disease (CKD) and AF. In recent years, attention has been focused on the relationship between AF and uremic toxins, including indoxyl sulfate (IS). Several animal studies have shown that IS promotes the development and progression of AF. IS has been shown to cause fibrosis and inflammation in the myocardium and exacerbate AF by causing oxidative stress and reducing antioxidative defense. Administration of AST-120, an absorbent of uremic toxins, decreases uremic toxin-induced AF in rodents. We have recently reported that patients with a higher serum IS level exhibit a higher rate of AF recurrence after catheter ablation, with serum IS being a significant predictor of AF recurrence. In this review, we discuss the possible mechanisms behind the AF-promoting effects of uremic toxins and summarize the reported clinical studies of uremic toxin-induced AF. Full article

Marine algal toxins, highly toxic secondary metabolites, have significant influences on coastal ecosystem health and mariculture safety. The occurrence and environmental control factors of lipophilic marine algal toxins (LMATs) in the surface seawater of the Changjiang estuary (CJE) and the adjacent East China Sea (ECS) were investigated. Pectenotoxin-2 (PTX2), okadaic acid (OA), dinophysistoxin-1(DTX1), and gymnodimine (GYM) were detected in the CJE surface seawater in summer, with concentration ranges of not detected (ND)–105.54 ng/L, ND–13.24 ng/L, ND–5.48 ng/L, and ND–12.95 ng/L, respectively. DTX1 (ND–316.15 ng/L), OA (ND–16.13 ng/L), and PTX2 (ND–4.97 ng/L) were detected in the ECS during spring. LMATs formed a unique low-concentration band in the Changjiang diluted water (CJDW) coverage area in the typical large river estuary. PTX2, OA, and DTX1 in seawater were mainly derived from Dinophysis caudate and Dinophysis rotundata, while GYM was suspected to be from Karenia selliformis. Correlation analyses showed that LMAT levels in seawater were positively correlated with dissolved oxygen and salinity, but negatively correlated with temperature and nutrients, indicating that the hydrological condition and nutritional status of seawater and climatic factors exert significant effects on the distribution of LMATs. Full article

The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions. Full article

Doenjang, a Korean fermented soybean paste, is vulnerable to contamination by mycotoxins because it is directly exposed to environmental microbiota during fermentation. A method that simultaneously determines 20 mycotoxins in doenjang, including aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), and fumonisins (FBs) with an immunoaffinity column cleanup was optimized and validated in doenjang using LC-MS/MS. The method showed good performance in the analysis of 20 mycotoxins in doenjang with good linearity (R² > 0.999), intra- and inter-day precision (<16%), recovery (72–112%), matrix effect (87–104%), and measurement uncertainty (<42%). The validated method was applied to investigate mycotoxin contamination levels in commercial and homemade doenjang. The mycotoxins that frequently contaminated doenjang were AFs, OTA, ZEN, and FBs and the average contamination level and number of co-occurring mycotoxins in homemade doenjang were higher than those in commercially produced doenjang. Full article

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo. Full article

Dianthin enzymes belong to ribosome-inactivating proteins (RIPs) of type 1, i.e., they only consist of a catalytic domain and do not have a cell binding moiety. Dianthin-30 is very similar to saporin-S3 and saporin-S6, two RIPs often used to design targeted toxins for tumor therapy and already tested in some clinical trials. Nevertheless, dianthin enzymes also exhibit differences to saporin with regard to structure, efficacy, toxicity, immunogenicity and production by heterologous expression. Some of the distinctions might make dianthin more suitable for targeted tumor therapies than other RIPs. The present review provides an overview of the history of dianthin discovery and illuminates its structure, function and role in targeted toxins. It further discusses the option to increase the efficacy of dianthin by endosomal escape enhancers. Full article

Helicobacter pylori (H. pylori) has been identified as a leading cause of gastric cancer, which is one of the most frequent and malignant types of tumor. It is characterized by its rapid progression, distant metastases, and resistance to conventional chemotherapy. A number of receptor tyrosine kinases and non-receptor tyrosine kinases have been implicated in H. pylori-mediated pathogenesis and tumorigenesis. In this review, recent findings of deregulated EGFR, c-Met, JAK, FAK, Src, and c-Abl and their functions in H. pylori pathogenesis are summarized. Full article

Although provisional maximum tolerable daily intake and recommended guidelines have been established for fumonisins (FB) in food, few data are available concerning levels of FB in edible animal tissues. Such data are of particular interest in avian species that can tolerate relatively high levels of fumonisins in their feed. Also, even if multiple contamination of animal feed by toxins produced by Fusarium is very frequent, little is known about the consequences of multiple contamination for FB levels in tissues. The aim of this study was to analyze the concentrations of FB in the muscle and liver of chickens and turkeys fed with FB alone and with FB combined with deoxynivalenol (DON), and with zearalenone (ZEN). Experimental diets were formulated by incorporating ground cultured toxigenic Fusarium strains in corn-soybean based feeds. Control diets were free of mycotoxins, FB diets contained 20 mg FB1+FB2/kg, and FBDONZEN diets contained 20, 5, and 0.5 mg/kg of FB1+FB2, DON, and ZEN, respectively. Animals were reared in individual cages with free access to water and feed. The feed was distributed to male Ross chickens from the 1st to the 35th day of age and to male Grade Maker turkeys from the 55th to the 70th day of age. On the last day of the study, the birds were starved for eight hours, killed, and autopsied for tissues sampling. No sign of toxicity was observed. A UHPLC-MS/MS method with isotopic dilution and immunoaffinity clean-up of samples has been developed for analysis of FB in muscle (n = 8 per diet) and liver (n = 8 per diet). Only traces of FB that were below the LOQ of 0.25 µg/kg were found in most of the samples of animals fed with the control diets. Mean concentrations of FB1, FB2, and FB3 in muscle were 17.5, 3.39, and 1.26 µg/kg, respectively, in chickens, and 5.77, 1.52, and 0.54 µg/kg in turkeys, respectively. In the liver, the respective FB1, FB2, and FB3 concentrations were 44.7, 2.61, and 0.79 µg/kg in chickens, and 41.47, 4.23, and 1.41 µg/kg, in turkeys. Cumulated level of FB1+FB2+FB3 in the highly contaminated samples were above 60 and 100 µg/kg in muscle and liver, respectively. The concentrations of FB in the tissues of animals fed the FBDONZEN diet did not greatly differ from the concentrations measured in animals fed the diet containing only FB. Full article

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

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn²⁺, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn²⁺, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays. Full article

Lake Winnipeg (Manitoba, Canada), the world’s 12th largest lake by area, is host to yearly cyanobacterial harmful algal blooms (cHABs) dominated by Aphanizomenon and Dolichospermum. cHABs in Lake Winnipeg are primarily a result of eutrophication but may be exacerbated by the recent introduction of dreissenid mussels. Through multiple methods to monitor the potential for toxin production in Lake Winnipeg in conjunction with environmental measures, this study defined the baseline composition of a Lake Winnipeg cHAB to measure potential changes because of dreissenid colonization. Surface water samples were collected in 2013 from 23 sites during summer and from 18 sites in fall. Genetic data and mass spectrometry cyanotoxin profiles identified microcystins (MC) as the most abundant cyanotoxin across all stations, with MC concentrations highest in the north basin. In the fall, mcyA genes were sequenced to determine which species had the potential to produce MCs, and 12 of the 18 sites were a mix of both Planktothrix and Microcystis. Current blooms in Lake Winnipeg produce low levels of MCs, but the capacity to produce cyanotoxins is widespread across both basins. If dreissenid mussels continue to colonize Lake Winnipeg, a shift in physicochemical properties of the lake because of faster water column clearance rates may yield more toxic blooms potentially dominated by microcystin producers. Full article

Chronic prostatitis/chronic pelvic pain syndrome (CP/ CPPS) has a negative impact on the quality of life, and its etiology still remains unknown. Although many treatment protocols have been evaluated in CP/CPPS, the outcomes have usually been disappointing. Botulinum neurotoxin A (BoNT-A), produced from Clostridium botulinum, has been widely used to lower urinary tract dysfunctions such as detrusor sphincter dyssynergia, refractory overactive bladder, interstitial cystitis/bladder pain syndromes, benign prostatic hyperplasia, and CP/ CPPS in urology. Here, we review the published evidence from animal models to clinical studies for inferring the mechanism of action underlying the therapeutic efficacy of BoNT in CP/CPPS. Animal studies demonstrated that BoNT-A, a potent inhibitor of neuroexocytosis, impacts the release of sensory neurotransmitters and inflammatory mediators. This pharmacological action of BoNT-A showed promise of relieving the pain of CP/CPPS in placebo-controlled and open-label BoNT-A and has the potential to serve as an adjunct treatment for achieving better treatment outcomes in CP/CPPS patients. Full article

In this work, we evaluate the effect of two peptides Sa12b (EDVDHVFLRF) and Sh5b (DVDHVFLRF-NH₂) on Acid-Sensing Ion Channels (ASIC). These peptides were purified from the venom of solitary wasps Sphex argentatus argentatus and Isodontia harmandi, respectively. Voltage clamp recordings of ASIC currents were performed in whole cell configuration in primary culture of dorsal root ganglion (DRG) neurons from (P7-P10) CII Long-Evans rats. The peptides were applied by preincubation for 25 s (20 s in pH 7.4 solution and 5 s in pH 6.1 solution) or by co-application (5 s in pH 6.1 solution). Sa12b inhibits ASIC current with an IC₅₀ of 81 nM, in a concentration-dependent manner when preincubation application was used. While Sh5b did not show consistent results having both excitatory and inhibitory effects on the maximum ASIC currents, its complex effect suggests that it presents a selective action on some ASIC subunits. Despite the similarity in their sequences, the action of these peptides differs significantly. Sa12b is the first discovered wasp peptide with a significant ASIC inhibitory effect. Full article

Flaviviruses are single-stranded RNA viruses predominantly transmitted by the widely distributed Aedes mosquitoes in nature. As important human pathogens, the geographic reach of Flaviviruses and their threats to public health are increasing, but there is currently no approved specific drug for treatment. In recent years, the development of peptide antivirals has gained much attention. Natural host defense peptides which uniquely evolved to protect the hosts have been shown to have antiviral properties. In this study, we firstly collected the venom of the Alopecosa nagpag spider from Shangri-La County, Yunnan Province. A defense peptide named Av-LCTX-An1a (Antiviral-Lycotoxin-An1a) was identified from the spider venom, and its anti-dengue serotype-2 virus (DENV2) activity was verified in vitro. Moreover, a real-time fluorescence-based protease inhibition assay showed that An1a functions as a DENV2 NS2B–NS3 protease inhibitor. Furthermore, we also found that An1a restricts zika virus (ZIKV) infection by inhibiting the ZIKV NS2B–NS3 protease. Together, our findings not only demonstrate that An1a might be a candidate for anti-flavivirus drug but also indicate that spider venom is a potential resource library rich in antiviral precursor molecules. Full article