Toxins, Volume 12, Issue 2 (February 2020) – 79 articles

Deoxynivalenol is one of the most ubiquitous mycotoxins in the Western diet through its presence in cereals and cereal products. A vast amount of studies indicate the worrying level of exposure to this toxin, while even high percentages of the population exceed the tolerable daily intake. To evaluate and assess dietary exposure, analysis of urinary levels of deoxynivalenol and its glucuronides has been proposed as a reliable methodology. An indirect preliminary method was used based on the cleavage of deoxynivalenol glucuronides through the use of enzymes (β-glucuronidase) and subsequent determination of "total deoxynivalenol" (sum of free and released mycotoxins by hydrolysis). Next, a direct procedure for quantification of deoxynivalenol-3-glucuronide and deoxynivalenol-15-glucuronide was developed. As deoxynivalenol glucuronides reference standards are not commercially available, the indirect method is widely applied. However, to not underestimate the total deoxynivalenol exposure in urine, the direct and indirect methodologies need to be compared. Urinary samples (n = 96) with a confirmed presence of deoxynivalenol and/or deoxynivalenol glucuronides were analysed using both approaches. The indirect method clarified that not all deoxynivalenol glucuronides were transformed to free deoxynivalenol during enzymatic treatment, causing an underestimation of total deoxynivalenol. This short communication concludes on the application of direct or indirect assessment of urinary deoxynivalenol. Full article

Zearalenone (ZEN) and ochratoxin A (OTA) are key concerns of the food industry because of their toxicity and pollution scope. This study investigated the effects of ozone and electron beam irradiation (EBI) on the degradation of ZEN and OTA. Results demonstrated that 2 mL of 50 μg/mL ZEN was completely degraded after 10 s of treatment by 2.0 mg/L ozone. The degradation rate of 1 μg/mL ZEN by 16 kGy EBI was 92.76%. Methanol was superior to acetonitrile in terms of degrading ZEN when the irradiation dose was higher than 6 kGy. The degradation rate of 2 mL of 5 μg/mL OTA by 50 mg/L ozone at 180 s was 34%, and that of 1 μg/mL OTA by 16 kGy EBI exceeded 90%. Moreover, OTA degraded more rapidly in acetonitrile. Ozone performed better in the degradation of ZEN, whereas EBI was better for OTA. The conclusions provide theoretical and practical bases for the degradation of different fungal toxins. Full article

Destruxin A (DA), a cyclodepsipeptidic mycotoxin produced by entomopathogenic fungus Metarhizium anisopliae, has good insecticidal activity and potential to be a new pesticide. However, the mechanism of action is still obscure. Our previous experiments showed that DA was involved in regulation of transcription and protein synthesis and suggested that silkworms’ arginine tRNA synthetase (BmArgRS), Lamin-C Proteins (BmLamin-C) and ATP-dependent RNA helicase PRP1 (BmPRP1) were candidates of DA-binding proteins. In this study, we employed bio-layer interferometry (BLI), circular dichroism (CD), cellular thermal shift assay (CETSA), and other technologies to verify the interaction of DA with above three proteins in vitro and in vivo. The results of BLI indicated that BmArgRS and BmLamin-C were binding-protein of DA with K_D value 5.53 × 10⁻⁵ and 8.64 × 10⁻⁵ M, but not BmPRP1. These interactions were also verified by CD and CETSA tests. In addition, docking model and mutants assay in vitro showed that BmArgRS interacts with DA at the pocket including Lys228, His231, Asp434 and Gln437 in its enzyme active catalysis region, while BmLamin-C binds to DA at His524 and Lys528 in the tail domain. This study might provide new insight and evidence in illustrating molecular mechanism of DA in breaking insect. Full article

Type-B aflatoxins (AFB₁ and AFB₂) frequently contaminate food, especially nuts and fried figs, and seriously threaten human health; hence, it is necessary for the newly rapid and sensitive detection methods to prevent the consumption of potentially contaminated food. Here, a lateral flow aptasensor for the detection of type-B aflatoxins was developed. It is based on the use of fluorescent dye Cy5 as a label for the aptamer, and on the competition between type-B aflatoxins and the complementary DNA of the aptamer. This is the first time that the complementary strand of the aptamer has been used as the test line (T-line) to detect type-B aflatoxins. In addition, the truncated aptamer was used to improve the affinity with type-B aflatoxins in our study. Therefore, the lengths of aptamer and cDNA probe were optimized as key parameters for higher sensitivity. In addition, binding buffer and organic solvent were investigated. The results showed that the best pair for achieving improved sensitivity and accuracy in detecting AFB₁ was formed by a shorter aptamer (32 bases) coupled with the probe complementary to the AFB₁ binding region of the aptamer. Under the optimal experimental conditions, the test strip showed an excellent linear relationship in the range from 0.2 to 20 ng/mL with a limit of detection of 0.16 ng/mL. This aptamer-based strip was successfully applied to the determination of type-B aflatoxins in spiked and commercial peanuts, almonds, and dried figs, and the recoveries of the spiked samples were from 93.3%−112.0%. The aptamer-complementary strand-based lateral flow test strip is a potential alternative tool for the rapid and sensitive detection of type-B aflatoxins in nuts and dried figs. It is of help for monitoring aflatoxins to avoid the consumption of unsafe food. Full article

Impaired physical performance is common in patients on hemodialysis (HD) and is associated with poor prognosis. A patient relevant marker of adequacy of dialysis is lacking. Previous studies evaluated uremic toxicity by assessing the impact of different uremic toxins separately. However, such an approach is most likely not reflective of true uremic toxicity. Therefore, this cross-sectional study aimed to examine if the uremic syndrome, estimated as one composite of different uremic toxins (facilitated by ridge regression method) to reflect the kinetic behavior during dialysis, is associated with physical performance in patients on HD. Levels of p-cresyl glucuronide and sulfate, indole-acetic acid, indoxyl sulfate, uric acid, hippuric acid, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid were assessed and associated by ridge regression to muscle strength, functional exercise capacity, and measures of balance and coordination. 75 HD patients were included (mean age 68 years, 57% male). The composite of different uremic toxins (i.e., uremic load) explained 22% of the variance in handgrip strength. Although there was an association between full body muscle strength and the composite uremic load independent of nutritional status, age and gender, the predictive power of composite uremic load for muscle weakness is limited. Single uremic toxins as well as composite uremic load were not associated with exercise capacity, coordination, and balance, indicating that the degree of uremia does not predict physical performance in patients on HD. Full article

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

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins. Full article

Herein, we review the characteristics of the six predominant venomous snakes in Taiwan and the effects of traditional Chinese medicine on the long-term outcomes of snakebite venom. We electronically searched databases, including PubMed, ClinicalKey, China National Knowledge Infrastructure, National Digital Library of Theses and Dissertations in Taiwan, and Airiti Library, from their inception to November 2019 by using the following Medical Subject Headings’ keywords: snakebite, long-term, chronic, Chinese medicine, CAM, herb, and Taiwan. The most common long-term effects of snakebite envenomation include “migraine-like syndrome”, brain injuries caused by hypoxia or intracranial hemorrhage, and chronic kidney disease. In addition, hypopituitarism is also worth mentioning. Traditional Chinese medicine can potentially be used in a complementary or alternative treatment for these effects, but additional studies are needed. Full article

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

A wide range of pet food types are available on the market; the dominant type is dry food formulated in croquets. One of the most common ingredients of dry food are cereals—vectors of harmful mycotoxins posing the risk to pet health. In this study, 38 cat and dog dry food samples available on the Polish market were investigated. Morphological and molecular methods were applied to identify fungal genera present in pet food. Quantification of ergosterol and Fusarium mycotoxins: Fumonisin B₁, deoxynivalenol, nivalenol, and zearalenone were performed using high performance liquid chromatography. Obtained results indicated five genera of mycotoxigenic fungi: Alternaria sp., Aspergillus sp., Cladosporium sp., Penicillium sp., and Fusarium sp., including Fusarium verticillioides and Fusarium proliferatum. Ergosterol and mycotoxins of interest were detected in both cat and dog food samples in the amounts ranging from 0.31 to 4.05 µg/g for ergosterol and 0.3–30.3, 1.2–618.4, 29.6–299.0, and 12.3–53.0 ng/g for zearalenone, deoxynivalenol, nivalenol, and fumonisin B₁, respectively. The conclusion is the presence of mycotoxins in levels much lower than recommended by EU regulations does not eliminate the risk and caution is advised concerning that long-term daily intake of even small doses of mycotoxins can slowly damage pet’s health. Full article

Intravesical botulinum toxin (BoNT) injection is effective in reducing urgency and urinary incontinence. It temporarily inhibits the detrusor muscle contraction by blocking the release of acetylcholine (Ach) from the preganglionic and postganglionic nerves in the efferent nerves. BoNT-A also blocks ATP release from purinergic efferent nerves in the detrusor muscle. In afferent nerves, BoNT-A injection markedly reduces the urothelial ATP release and increases nitric oxide (NO) release from the urothelium. BoNT-A injection in the urethra or bladder has been developed in the past few decades as the treatment method for detrusor sphincter dyssyndergia, incontinence due to neurogenic or idiopathic detrusor overactivity, sensory disorders, including bladder hypersensitivity, overactive bladder, and interstitial cystitis/chronic pelvic pain syndrome. Although the FDA only approved BoNT-A injection treatment for neurogenic detrusor overactivity and for refractory overactive bladder, emerging clinical trials have demonstrated the benefits of BoNT-A treatment in functional urological disorders. Cautious selection of patients and urodynamic evaluation for confirmation of diagnosis are crucial to maximize the successful outcomes of BoNT-A treatment. Full article

The American Urological Association guidelines for the management of non-neurogenic overactive bladder (OAB) recommend the use of OnabotulinumtoxinA, sacral neuromodulation (SNM), and peripheral tibial nerve stimulation (PTNS) as third line treatment options with no treatment hierarchy. The current study used network meta-analysis to compare the efficacy of these three modalities for managing adult OAB syndrome. We performed systematic literature searches of several databases from January 1995 to September 2019 with language restricted to English. All randomized control trials that compared any dose of OnabotulinumtoxinA, SNM, and PTNS with each other or a placebo for the management of adult OAB were included in the study. Overall, 17 randomized control trials, with a follow up of 3–6 months in the predominance of trials (range 1.5–24 months), were included for analysis. For each trial outcome, the results were reported as an average number of episodes of the outcome at baseline. Compared with the placebo, all three treatments were more efficacious for the selected outcome parameters. OnabotulinumtoxinA resulted in a higher number of complications, including urinary tract infection and urine retention. Compared with OnabotulinumtoxinA and PTNS, SNM resulted in the greatest reduction in urinary incontinence episodes and voiding frequency. However, comparison of their long-term efficacy was lacking. Further studies on the long-term effectiveness of the three treatment options, with standardized questionnaires and parameters are warranted. Full article

In many countries, 400 units (U) is the maximum dose of onabotulinumtoxinA available to treat upper limb spasticity, but few studies have demonstrated the optimal use of this dose. In the double-blind phase of this randomized, controlled trial, we compared the efficacy and safety of 400 vs. 240 U onabotulinumtoxinA in patients with post-stroke upper limb spasticity. Both groups received 240 U onabotulinumtoxinA injected in the forearm. An additional 160 U onabotulinumtoxinA (400 U group) or placebo (240 U group) was injected in the elbow flexors. Both groups showed similar muscle tone reduction in the wrist, fingers, and thumb; muscle tone reduction in the elbow flexors was greater in the group treated with onabotulinumtoxinA (400 U group) compared to placebo (240 U group). Functional disabilities improved in both groups. No substantial difference was found in safety profiles. In the subsequent open-label phase, all participants received repeat injections of 400 U onabotulinumtoxinA (target muscles and doses per muscle determined by the physician). Similar efficacy and safety outcomes, as with the 400 U group in the double-blind phase, were confirmed. This final report demonstrates that injection of onabotulinumtoxinA 400 U relieves muscle tone in a wide range of areas and improves functional disabilities; generally, it was well-tolerated, and no new safety concerns were identified. The dosing data in the open-label phase will inform optimal use of onabotulinumtoxinA in clinical practice (ClinicalTrials.gov: NCT03261167). Full article

Six phytotoxins were obtained from the culture filtrates of the ascomycete Neofusicoccum batangarum, the causal agent of the scabby canker of cactus pear (Opuntia ficus-indica L.) in minor Sicily islands. The phytotoxins were identified as (−)-(R)-mellein (1); (±)-botryoisocoumarin A (2); (−)-(3R,4R)- and (−)-(3R,4S)-4-hydroxymellein (3 and 4); (−)-terpestacin (5); and (+)-3,4-dihydro-4,5,8-trihydroxy-3-methylisocoumarin, which we named (+)-neoisocoumarin (6). This identification was done by comparing their spectral and optical data with those already reported in literature. The absolute configuration (3R,4S) to (+)-neoisocoumarin (6) was determined using the advanced Mosher method. All six metabolites were shown to have phytotoxicity on the host (cactus pear) and non-host (tomato) plants, and the most active compounds were (±)-botryoisocoumarin A (2), (−)-terpestacin (5), and (+)-neoisocoumarin (6). Full article

Citreoviridin (CTV) is a mycotoxin that is produced by Aspergillus terreus, Eupenicillium ochrosalmoneum and Penicillium citreonigrum, and CTV has been detected in a wide range of cereal grains throughout the world. Furthermore, it is especially a serious problem in regions where rice is consumed as a staple food. Moreover, CTV is a well-known yellow rice toxin, and outbreaks of beriberi have occurred due to consumption of rice that is contaminated by CTV even in the recent years. Although CTV biosynthetic genes of A. terreus have been described, those of P. citreonigrum remain unclear, which is concerning since P. citreonigrum is the main cause of CTV contamination in rice. In the present study, we determined the draft genome of the P. citreonigrum strain IMI92228 and revealed the presence of all four genes that form a gene cluster and that are homologous to the CTV biosynthesis genes of A. terreus. The expression of these four homologous genes were highly correlated with CTV production, suggesting that they may play an important role in CTV biosynthesis in P. citreonigrum. We concluded that the gene cluster is a CTV biosynthesis cluster of P. citreonigrum. The findings will contribute to the understanding of the biosynthetic pathway of CTV and will ultimately lead to improvements in the CTV management of agricultural products. Full article

Black spot caused by Alternaria alternata is one of the important diseases of pear fruit during storage. Isothiocyanates are known as being strong antifungal compounds in vitro against different fungi. The aim of this study was to assess the antifungal effects of the volatile compound 2-phenylethyl isothiocyanate (2-PEITC) against A. alternata in vitro and in pear fruit, and to explore the underlying inhibitory mechanisms. The in vitro results showed that 2-PEITC significantly inhibited spore germination and mycelial growth of A. alternata—the inhibitory effects showed a dose-dependent pattern and the minimum inhibitory concentration (MIC) was 1.22 mM. The development of black spot rot on the pear fruit inoculated with A. alternata was also significantly decreased by 2-PEITC fumigation. At 1.22 mM concentration, the lesion diameter was only 39% of that in the control fruit at 7 days after inoculation. Further results of the leakage of electrolyte, increase of intracellular OD₂₆₀, and propidium iodide (PI) staining proved that 2-PEITC broke cell membrane permeability of A. alternata. Moreover, 2-PEITC treatment significantly decreased alternariol (AOH), alternariolmonomethyl ether (AME), altenuene (ALT), and tentoxin (TEN) contents of A. alternata. Taken together, these data suggest that the mechanisms underlying the antifungal effect of 2-PEITC against A. alternata might be via reduction in toxin content and breakdown of cell membrane integrity. Full article

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of β-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography–mass spectrometry (LC-MS) methods. Full article

Knowledge of the genetic diversity detected among fungal species belonging to the genus Aspergillus is of key importance for explaining their important ecological role in the environment and agriculture. The current study aimed to identify Aspergillus species occurring in the rhizosphere of sugarcane in the South of Iran, and to investigate their mycotoxin profiles. One-hundred and twenty-five Aspergillus strains were isolated from the soil of eight major sugarcane-producing sites, and were molecularly identified using sequences of partial -tubulin (benA) and partial calmodulin (CaM) genes. Our molecular and phylogenetic results showed that around 70% of strains belonged to the Aspergillus section Nigri, and around 25% of species belonged to the Aspergillus section Terrei. Species belonging to both sections are able to produce different mycotoxins. The production of mycotoxins was measured for each species, according to their known mycotoxin profile: patulin (PAT) and sterigmatocystin (STG) for Aspergillus terreus; ochratoxin A (OTA) and fumonisins for Aspergillus welwitschiae; and OTA alone for Aspergillus tubingensis. The data showed that the production of OTA was detected in only 4 out of 10 strains of A. welwitschiae, while none of the A. tubingensis strains analyzed produced the mycotoxin. Fumonisins were produced by 8 out of 10 strains of A. welwitschiae. Finally, none of the 23 strains of A. terreus produced STG, while 13 of them produced PAT. The occurrence of such mycotoxigenic plant pathogens among the fungal community occurring in soil of sugarcane fields may represent a significant source of inoculum for the possible colonization of sugarcane plants, since the early stages of plant growth, due to the mycotoxin production capability, could have worrisome implications in terms of both the safety and loss of products at harvest. Full article

Mycotoxins are toxic fungal secondary metabolites that pose a major threat to the safety of food and feed. Mycotoxins are usually converted into less toxic or non-toxic metabolites through biotransformation that are often made by living organisms as well as the isolated enzymes. The conversions mainly include hydroxylation, oxidation, hydrogenation, de-epoxidation, methylation, glycosylation and glucuronidation, esterification, hydrolysis, sulfation, demethylation and deamination. Biotransformations of some notorious mycotoxins such as alfatoxins, alternariol, citrinin, fomannoxin, ochratoxins, patulin, trichothecenes and zearalenone analogues are reviewed in detail. The recent development and applications of mycotoxins detoxification through biotransformation are also discussed. Full article

Abrin is a highly potent and naturally occurring toxin produced in the seeds of Abrus precatorius (Rosary Pea) and is of concern as a potential bioterrorism weapon. There are many rapid and specific assay methods to detect this toxic plant protein, but few are based on detection of toxin activity, critical to discern biologically active toxin that disables ribosomes and thereby inhibits protein synthesis, producing cytotoxic effects in multiple organ systems, from degraded or inactivated toxin which is not a threat. A simple and low-cost CCD detector system was evaluated with colorimetric and fluorometric cell-based assays for abrin activity; in the first instance measuring the abrin suppression of mitochondrial dehydrogenase in Vero cells by the MTT-formazan method and in the second instance measuring the abrin suppression of green fluorescent protein (GFP) expression in transduced Vero and HeLa cells. The limit of detection using the colorimetric assay was 10 pg/mL which was comparable to the fluorometric assay using HeLa cells. However, with GFP transduced Vero cells a hundred-fold improvement in sensitivity was achieved. Results were comparable to those using a more expensive commercial plate reader. Thermal inactivation of abrin was studied in PBS and in milk using the GFP-Vero cell assay. Inactivation at 100 °C for 5 min in both media was complete only at the lowest concentration studied (0.1 ng/mL) while treatment at 63 °C for 30 min was effective in PBS but not milk. Full article

This review assessed the molecular characterization of the methicillin-resistant Staphylococcus aureus (MRSA)-ST80 clone with an emphasis on its proportion of total MRSA strains isolated, PVL production, spa-typing, antibiotic resistance, and virulence. A systematic review of the literature was conducted on MRSA-ST80 clone published between 1 January 2000 and 31 August 2019. Citations were chosen for a review of the full text if we found evidence that MRSA-ST80 clone was reported in the study. For each isolate, the country of isolation, the sampling period, the source of isolation (the type of infection, nasal swabs, or extra-human), the total number of MRSA strains isolated, number of MRSA-ST80 strains, antibiotic resistance patterns, PVL production, virulence genes, and spa type were recorded. The data from 103 articles were abstracted into an Excel database. Analysis of the data showed that the overall proportion of MRSA-ST80 has been decreasing in many countries in recent years. The majority of MRSA-ST80 were PVL positive with spa-type t044. Only six reports of MRSA-ST80 in extra-human niches were found. This review summarizes the rise of MRSA-ST80 and the evidence that suggests that it could be in decline in many countries. Full article

Zearalenone and alternariol are mycotoxins produced by Fusarium and Alternaria species, respectively, that present estrogenic activity and consequently are classified as endocrine disruptors. To estimate the exposure of the Portuguese population to these two mycotoxins at a national level, a modelling approach, based on data from 94 Portuguese volunteers, was developed considering as inputs: i) the food consumption data generated within the National Food and Physical Activity Survey; and ii) the human biomonitoring data used to assess the exposure to the referred mycotoxins. Six models of association between mycoestrogens urinary levels (zearalenone, total zearalenone and alternariol) and food items (meat, cheese, and fresh-cheese, breakfast cereals, sweets) were established. Applying the obtained models to the consumption data (n = 5811) of the general population, the median estimates of the probable daily intake revealed that a fraction of the Portuguese population might exceed the tolerable daily intake defined for zearalenone. A reference intake value for alternariol is still lacking, thus the characterization of risk due to the exposure to this mycotoxin was not possible to perform. Although the unavoidable uncertainties, these results are important contributions to understand the exposure to endocrine disruptors in Portugal and the potential Public Health consequences. Full article

The estrogen-like mycotoxin zearalenone (ZEN) is one of the most widely distributed contaminants especially in maize and its commodities, such as corn oil. ZEN degrading enzymes possess the potential for counteracting the negative effect of ZEN and its associated high safety risk in corn oil. Herein, we targeted enhancing the secretion of ZEN degrading enzyme by Pichia pastoris through constructing an expression plasmid containing three optimized expression cassettes of zlhy-6 codon and signal peptides. Further, we explored various parameters of enzymatic detoxification in neutralized oil and analyzed tocopherols and sterols losses in the corn oil. In addition, the distribution of degraded products was demonstrated as well by Agilent 6510 Quadrupole Time-of-Flight mass spectrometry. P. pastoris GSZ with the glucoamylase signal was observed with the highest ZLHY-6 secretion yield of 0.39 mg/mL. During the refining of corn oil, ZEN in the crude oil was reduced from 1257.3 to 13 µg/kg (3.69% residual) after neutralization and enzymatic detoxification. Compared with the neutralized oil, no significant difference in the total tocopherols and sterols contents was detected after enzymatic detoxification. Finally, the degraded products were found to be entirely eliminated by washing. This study presents an enzymatic strategy for efficient and safe ZEN removal with relatively low nutrient loss, which provides an important basis for further application of enzymatic ZEN elimination in the industrial process of corn oil production. Full article

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

Mycotoxins are an important class of pollutants that are toxic and hazardous to animal and human health. Consequently, various methods have been explored to abate their effects, among which adsorbent has found prominent application. Liquid chromatography tandem mass spectrometry (LC–MS/MS) has recently been applied for the concurrent evaluation of multiple mycotoxins. This study investigated the optimization of the simultaneous removal of mycotoxins in palm kernel cake (PKC) using chitosan. The removal of 11 mycotoxins such as aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), fumonisins (FB1 and FB2) and trichothecenes (deoxynivalenol (DON), HT-2 and T-2 toxin) from palm kernel cake (PKC) was studied. The effects of operating parameters such as pH (3–6), temperature (30–50 °C) and time (4–8 h) on the removal of the mycotoxins were investigated using response surface methodology (RSM). Response surface models obtained with R² values ranging from 0.89–0.98 fitted well with the experimental data, except for the trichothecenes. The optimum point was obtained at pH 4, 8 h and 35 °C. The maximum removal achieved with chitosan for AFB₁, AFB₂, AFG₁, AFG₂, OTA, ZEA, FB₁ and FB₂ under the optimized conditions were 94.35, 45.90, 82.11, 84.29, 90.03, 51.30, 90.53 and 90.18%, respectively. Full article

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

The mycotoxin zearalenone (ZEN), which frequently contaminates cereal-based human food and animal feed, is known to have an estrogenic effect. The biological response associated with exposure to ZEN has rarely been reported in organs other than the reproductive system. In the intestine, several studies suggested that ZEN might stimulate molecular changes related to the activation of early carcinogenesis, but the molecular mechanisms behind these events are not yet known. In this study, we investigated gene expression and changes in protein abundance induced by acute exposure to ZEN in the jejunum of castrated male pigs using an explant model. Our results indicate that ZEN induces the accumulation of ERα but not ERβ, modulates Wnt/β-catenin and TGF-β signaling pathways, and induces molecular changes linked with energy sensing and the antimicrobial activity without inducing inflammation. Our results confirm that the intestine is a target for ZEN, inducing changes that promote cellular proliferation and could contribute to the onset of intestinal pathologies. Full article

Introduction: Botulinum neurotoxin (BoNT) is a potent biological toxin and powerful therapeutic tool for a growing number of clinical orofacial applications. BoNT relaxes striated muscle by inhibiting acetylcholine’s release from presynaptic nerve terminals, blocking the neuromuscular junction. It also has an antinociceptive effect on sensory nerve endings, where BoNT and acetylcholine are transported axonally to the central nervous system. In dentistry, controlled clinical trials have demonstrated BoNT’s efficiency in pathologies such as bruxism, facial paralysis, temporomandibular joint (TMJ) disorders, neuropathic pain, sialorrhea, dystonia and more. Aim: This study’s aim was to conduct a systematic literature review to assess the most recent high-level clinical evidence for BoNT’s efficacy and for various protocols (the toxin used, dilution, dosage and infiltration sites) used in several orofacial pathologies. Materials and methods: We systematically searched the MedLine database for research papers published from 2014 to 2019 with randomly allocated studies on humans. The search included the following pathologies: bruxism, dislocation of the TMJ, orofacial dystonia, myofascial pain, salivary gland disease, orofacial spasm, facial paralysis, sialorrhea, Frey syndrome and trigeminal neuralgia. Results: We found 228 articles, of which only 20 met the inclusion criteria: bruxism (four articles), orofacial dystonia (two articles), myofascial pain (one article), salivary gland disease (one article), orofacial spasm (two articles), facial paralysis (three articles), sialorrhea (four articles) or trigeminal neuralgia (three articles). Discussion: The clinical trials assessed showed variations in the dosage, application sites and musculature treated. Thus, applying BoNT can reduce symptoms related to motor muscular activity in the studied pathologies efficiently enough to satisfy patients. We did not identify the onset of any important side effects in the literature reviewed. We conclude that treatment with BoNT seems a safe and effective treatment for the reviewed pathologies. Full article

Selected species of cyanobacteria and green algae have been reported to produce lipophilic polymethoxy-1-alkenes (PMAs) which were shown to exhibit in vivo teratogenicity. Considering that information on PMAs in Arthospira sp. (known commercially as Spirulina) and Chlorella sp. cultivated for food supplement production was essentially lacking, the present study screened Chlorella (n = 10) and Spirulina (n = 13) food supplements registered in the European Union. Mass spectrometry analysis of column fractionated extracts was performed. None of the four variants previously reported in some cyanobacteria and green algae, nor any potentially related structures were detected in the studied samples. Since the isolated lipophilic fractions contained various compounds, they were further screened for in vivo teratogenicity in Danio rerio embryo, and for the potential to induce oxidative stress and genotoxicity in the liver and neurotoxicity in the brain of adult zebrafish. None of the tested food supplements had detectable levels of PMAs or any potentially related structures. No teratogenicity was revealed except for spinal curvature induced by fractions obtained from two Chlorella products. Selected fractions revealed cytotoxicity as indicated by an increased level of reactive oxygen species, catalase activity, lipid peroxidation and increased frequency of DNA strand breaks in hepatic tissue. The majority (60%) of Chlorella fractions induced an increase in cholinesterase activity in zebrafish brain homogenate while exposure to 61.5% of Spirulina fractions was associated with its decrease. The present study confirms that Chlorella and Spirulina food supplements are free of teratogenic PMAs, although the observed in vivo toxicities raise questions regarding the quality of selected products. Full article

Chronic pelvic pain (CPP) is defined as chronic pain and inflammation in the pelvic organs for more than six months. There are wide ranges of clinical presentations, including pelvic pain, painful intercourse, irritable bowel syndrome, and pain during urinating. Chronic pelvic pain syndrome (CPPS) is a subdivision of CPP, and the pain syndrome may be focused within a single organ or more than one pelvic organ. As there is uncertain pathogenesis, no standard treatment is currently available for CPPS. Botulinum toxin A (BoNT-A) is a potent neurotoxin that blocks acetylcholine release to paralyze muscles. Intravesical BoNT-A injection can reduce bladder pain in patients with interstitial cystitis/bladder pain syndrome. BoNT-A injected into the pelvic floor muscles of women has also been reported to improve chronic pain syndrome. Due to the reversible effect of BoNT-A, repeated injection appears to be necessary and effective in reducing symptoms. Adverse effects of BoNT-A may worsen the preexisting conditions, including constipation, stress urinary incontinence, and fecal incontinence. This review summarizes the evidence of BoNT-A treatment for CPPS in animal studies and clinical studies regarding the therapeutic effects of BoNT-A for CPPS in female patients. Full article