Toxins, Volume 10, Issue 11 (November 2018) – 64 articles

There is an increasing demand for products from natural sources, which includes a growing market for naturally-produced colorants. Filamentous fungi produce a vast number of chemically diverse pigments and are therefore explored as an easily accessible source. In this study we examine the positive regulatory effect of the transcription factor AurR1 on the aurofusarin gene cluster in Fusarium graminearum. Proteomic analyses showed that overexpression of AurR1 resulted in a significant increase of five of the eleven proteins belonging to the aurofusarin biosynthetic pathway. Further, the production of aurofusarin was increased more than threefold in the overexpression mutant compared to the wild type, reaching levels of 270 mg/L. In addition to biosynthesis of aurofusarin, several yet undescribed putative naphthoquinone/anthraquinone analogue compounds were observed in the overexpression mutant. Our results suggest that it is possible to enhance the aurofusarin production through genetic engineering. Full article

Experiments were carried out to evaluate the effectiveness of three different biosorbents (banana peel, Pyracantha leaves, and Aloe powder) in removing aflatoxin B₁ (AFB₁). A noncommercial mycotoxin binder (zeolite) was used as a reference material. A laboratory model that simulated the in vivo conditions of the poultry gastrointestinal tract was utilized to prove the removal efficiency of the biosorbents when added to AFB₁-contaminated diet (100 µg/kg). The concentration of AFB₁ was determined using antibody-based immunoaffinity column and spectrofluorometry methodologies. Z potential (ζ), point of zero charge (pH_pzc), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy with attenuated total reflection (FTIR-ATR), and UV-Vis diffuse reflectance spectroscopy (DRS) techniques were used to further characterize the biosorbents. The addition of the biosorbents (1.5%, w/w) to the diet significantly reduced the bioavailability of AFB₁ in the intestinal section. The highest aflatoxin adsorption values were 69% and 70% using Aloe powder and zeolite, respectively. A moderate biosorption uptake of 46% was achieved using Pyracantha leaves. The biomaterial with the lowest removal capacity was banana peel (28%). In conclusion, Aloe powder could be used as an alternative to conventional systems for AFB₁ removal. Full article

Toxins and venoms produced by different organisms contain peptides that have evolved to have highly selective and potent pharmacological effects on specific targets for protection and predation. Several toxin-derived peptides have become drugs and are used for the management of diabetes, hypertension, chronic pain, and other medical conditions. Despite the similarity in their composition (amino acids as the building blocks), toxin-derived peptide drugs have very profound differences in their structure and conformation, in their physicochemical properties (that affect solubility, stability, etc.), and subsequently in their pharmacokinetics (the processes of absorption, distribution, metabolism, and elimination following their administration to patients). This review summarizes and critically analyzes the pharmacokinetic properties of toxin-derived peptide drugs: (1) the relationship between the chemical structure, physicochemical properties, and the pharmacokinetics of the specific drugs, (2) the major pharmacokinetic properties and parameters of these drugs, and (3) the major pharmacokinetic variability factors of the individual drugs. The structural properties of toxin-derived peptides affect their pharmacokinetics and pose some limitations on their clinical use. These properties should be taken into account during the development of new toxin-derived peptide drugs, and for the efficient and safe use of the clinically approved drugs from this group in the individual patients. Full article

This study investigated effects of modified palygorskite (MPal) on immunity, antioxidant capacity, and intestinal barrier integrity in broiler chickens challenged with permitted feed Fusarium mycotoxin concentrations. One-day-old chicks were allocated into three treatments with eight replicates. Chickens in three groups were fed a basal diet with normal corn (control), contaminated diet containing moldy corn, with Fusarium mycotoxins contents in the diets lower than permitted feed mycotoxin concentrations, and the contaminated diet supplemented with 1 g/kg MPal for 42 days, respectively. Compared with control, moldy corn decreased bursa of Fabricius weight, jejunal secreted immunoglobulin A concentration, ileal superoxide dismutase (SOD) activity, jejunal and ileal villus height (VH) and VH/crypt depth (CD) ratio, and jejunal zonula occludens-1 and mucin 2 mRNA abundances at 42 days as well as ileal VH/CD ratio at 21 days; while they increased jejunal malondialdehyde accumulation at 21 and 42 days, jejunal SOD activity at 21 days, and serum diamine oxidase activity at 42 days, which were almost recovered by MPal. Moreover, dietary MPal upregulated ileal claudin-2 mRNA abundance compared with other two groups. The results indicated that MPal addition exerted protective effects on immunity, oxidative status, and intestinal barrier integrity in chickens challenged with permitted feed Fusarium mycotoxins levels. Full article

In order to investigated current occurrence of major mycotoxins in dietary kelp in Shandong Province in Northern China, a reliable, sensitive, and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of the 7 most frequent mycotoxins, including 3-acetoxy deoxynivalenol (3AcDON), 15-acetoxy deoxynivalenol (15AcDON), Deoxynivalenol (DON), Fusarenon-X (F-X), Nivalenol (NIV), T-2 toxin (T-2), and Zearalenone (ZEA). Based on optimized pretreatment and chromatographic and mass spectrometry conditions, these target analytes could be monitored with mean recoveries from 72.59~107.34%, with intra–day RSD < 9.21%, inter–day RSD < 9.09%, LOD < 5.55 μg kg⁻¹, and LOQ < 18.5 μg kg⁻¹. Approximately 43 kelp samples were detected, 3AcDON/15AcDON ranged from 15.3 to 162.5 μg kg⁻¹ with positive rate of 86% in Shandong Province in Northern China. Considering there were no related investigations about mycotoxin contamination in kelp, the high contamination rate of 3AcDON/15AcDON in kelp showed a neglected mycotoxin exposure pathway, which might lead to high dietary exposure risk to consumers. Full article

Water column bulk Pseudo-nitzschia abundance and the dissolved and particulate domoic acid (DA) concentrations were measured in the Santa Barbara Basin (SBB), California from 2009–2013 and compared to bulk Pseudo-nitzschia cell abundance and DA concentrations and fluxes in sediment traps moored at 147 m and 509 m. Pseudo-nitzschia abundance throughout the study period was spatially and temporally heterogeneous (<200 cells L⁻¹ to 3.8 × 10⁶ cells L⁻¹, avg. 2 × 10⁵ ± 5 × 10⁵ cells L⁻¹) and did not correspond with upwelling conditions or the total DA (tDA) concentration, which was also spatially and temporally diverse (<1.3 ng L⁻¹ to 2.2 × 10⁵ ng L⁻¹, avg. 7.8 × 10³ ± 2.2 × 10⁴ ng L⁻¹). We hypothesize that the toxicity is likely driven in part by specific Pseudo-nitzschia species as well as bloom stage. Dissolved (dDA) and particulate (pDA) DA were significantly and positively correlated (p < 0.01) and both comprised major components of the total DA pool (pDA = 57 ± 35%, and dDA = 42 ± 35%) with substantial water column concentrations (>1000 cells L⁻¹ and tDA = 200 ng L⁻¹) measured as deep as 150 m. Our results highlight that dDA should not be ignored when examining bloom toxicity. Although water column abundance and pDA concentrations were poorly correlated with sediment trap Pseudo-nitzschia abundance and fluxes, DA toxicity is likely associated with senescent blooms that rapidly sink to the seafloor, adding another potential source of DA to benthic organisms. Full article

There is a clear, unmet need for effective, lightweight, shelf-stable and economical snakebite envenoming therapies that can be given rapidly after the time of a snake’s bite and as adjuncts to antivenom therapies in the hospital setting. The sPLA2 inhibitor, LY315920, and its orally bioavailable prodrug, LY333013, demonstrate surprising efficacy and have the characteristics of an antidote with potential for both field and hospital use. The efficacy of the active pharmaceutical ingredient (LY315920) and its prodrug (LY333013) to treat experimental, lethal envenoming by Micrurus fulvius (Eastern coral snake) venom was tested using a porcine model. Inhibitors were administered by either intravenous or oral routes at different time intervals after venom injection. In some experiments, antivenom was also administered alone or in conjunction with LY333013. 14 of 14 animals (100%) receiving either LY315920 (intravenous) and/or LY333013 (oral) survived to the 120 h endpoint despite, in some protocols, the presence of severe neurotoxic signs. The study drugs demonstrated the ability to treat, rescue, and re-rescue animals with advanced manifestations of envenoming. Low molecular mass sPLA2 inhibitors were highly effective in preventing lethality following experimental envenoming by M. fulvius. These findings suggest the plausibility of a new therapeutic approach to snakebite envenoming, in this example, for the treatment of a coral snake species for which there are limitations in the availability of effective antivenom. Full article

Cyanobacteria synthesize neurotoxic β-N-methylamino-l-alanine (BMAA). The roles of this non-protein amino acid in cyanobacterial cells are insufficiently studied. During diazotrophic growth, filamentous cyanobacteria form single differentiated cells, called heterocysts, which are separated by approximately 12–15 vegetative cells. When combined nitrogen is available, heterocyst formation is blocked and cyanobacterial filaments contain only vegetative cells. In the present study, we discovered that exogenous BMAA induces the process of heterocyst formation in filamentous cyanobacteria under nitrogen-replete conditions that normally repress cell differentiation. BMAA treated cyanobacteria form heterocyst-like dark non-fluorescent non-functional cells. It was found that glutamate eliminates the BMAA mediated derepression. Quantitative polymerase chain reaction (qPCR) permitted to detect the BMAA impact on the transcriptional activity of several genes that are implicated in nitrogen assimilation and heterocyst formation in Anabaena sp. PCC 7120. We demonstrated that the expression of several essential genes increases in the BMAA presence under repressive conditions. Full article

Aflatoxins (AFs), deoxynivalenols (DONs), and zearalenones (ZENs) are common mycotoxins that contaminate feedstuff, causing contamination of poultry products. In our study, these mycotoxins were quantified in 152 egg samples collected from markets in Jiangsu (JS), Zhejiang (ZJ), and Shanghai (SH) and in 70 chicken tissue samples (liver, heart, and gizzard) from ZJ in China. The main mycotoxins observed in egg samples were DON, 15-AcDON, and ZEN, although only ZEN family mycotoxins (ZEN, α-ZEL, β-ZEL, and α-ZAL) were detected in chicken tissues. Furthermore, for the first time, we assessed the health risks of exposure of three populations (children, adults, and elder adults) to DONs (DON, 3-AcDON, and 15-AcDON) and ZEN in eggs (from three different areas) and to ZEN in chicken tissues. We show that the mean dietary intake (DI) values and the 97.5th percentile DI values of DON and ZEN through egg ingestion were lower than the provisional maximum tolerable daily intake (PMTDI) (1 μg/kg body weight (BW)/day) for the three populations in the three geographical areas studied. However, eggs contaminated with high levels of DONs and ZEN contributed to a large proportion of the PMTDI of these mycotoxins, especially in children and elder adults. Although ZEN was highly detected in the chicken tissues, no significant health risk was observed. Full article

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation’s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay CANARY^® (Cellular Analysis and Notification of Antigen Risks and Yields) Zephyr detects BoNT/A holotoxin at limits of detection (LOD) of 10.0 ± 2.5 ng/mL in assay buffer. Milk matrices (whole milk, 2% milk and non-fat milk) with BoNT/A holotoxin were detected at similar levels (7.4–7.9 ng/mL). BoNT/A complex was positive in carrot, orange, and apple juices at LODs of 32.5–75.0 ng/mL. The detection of BoNT/A complex in solid complex foods (ground beef, smoked salmon, green bean baby puree) ranged from 14.8 ng/mL to 62.5 ng/mL. Detection of BoNT/A complex in the viscous liquid egg matrix required dilution in assay buffer and gave a LOD of 171.9 ± 64.7 ng/mL. These results show that the CANARY^® Zephyr assay can be a highly useful qualitative tool in environmental and food safety surveillance programs. Full article

Apples and apple-based products are among the most popular foods around the world for their delightful flavors and health benefits. However, the commonly found mold, Penicillium expansum invades wounded apples, causing the blue mold decay and ensuing the production of patulin, a mycotoxin that negatively affects human health. Patulin contamination in apple products has been a worldwide problem without a satisfactory solution yet. A comprehensive understanding of the factors and challenges associated with patulin accumulation in apples is essential for finding such a solution. This review will discuss the effects of the pathogenicity of Penicillium species, quality traits of apple cultivars, and environmental conditions on the severity of apple blue mold and patulin contamination. Moreover, beyond the complicated interactions of the three aforementioned factors, patulin control is also challenged by the lack of reliable detection methods in food matrices, as well as unclear degradation mechanisms and limited knowledge about the toxicities of the metabolites resulting from the degradations. As apple-based products are mainly produced with stored apples, pre- and post-harvest strategies are equally important for patulin mitigation. Before storage, disease-resistance breeding, orchard-management, and elicitor(s) application help control the patulin level by improving the storage qualities of apples and lowering fruit rot severity. From storage to processing, patulin mitigation strategies could benefit from the optimization of apple storage conditions, the elimination of rotten apples, and the safe and effective detoxification or biodegradation of patulin. Full article

Nature endowed snakes with a lethal secretion known as venom, which has been fine-tuned over millions of years of evolution. Snakes utilize venom to subdue their prey and to survive in their natural habitat. Venom is known to be a very poisonous mixture, consisting of a variety of molecules, such as carbohydrates, nucleosides, amino acids, lipids, proteins and peptides. Proteins and peptides are the major constituents of the dry weight of snake venoms and are of main interest for scientific investigations as well as for various pharmacological applications. Snake venoms contain enzymatic and non-enzymatic proteins and peptides, which are grouped into different families based on their structure and function. Members of a single family display significant similarities in their primary, secondary and tertiary structures, but in many cases have distinct pharmacological functions and different bioactivities. The functional specificity of peptides belonging to the same family can be attributed to subtle variations in their amino acid sequences. Currently, complementary tools and techniques are utilized to isolate and characterize the peptides, and study their potential applications as molecular probes, and possible templates for drug discovery and design investigations. Full article

Staphylococcus aureus is a nosocomial pathogen that can cause chronic to persistent infections. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. These systems are frequently studied in Escherichia coli and Mycobacterial species but rarely explored in S. aureus. In the present study, we thoroughly analyzed the S. aureus genome and screened all possible TA systems using the Rasta bacteria and toxin-antitoxin database. We further searched E. coli and Mycobacterial TA homologs and selected 67 TA loci as putative TA systems in S. aureus. The host inhibition of growth (HigBA) TA family was predominantly detected in S. aureus. In addition, we detected seven pathogenicity islands in the S. aureus genome that are enriched with virulence genes and contain 26 out of 67 TA systems. We ectopically expressed multiple TA genes in E. coli and S. aureus that exhibited bacteriostatic and bactericidal effects on cell growth. The type I Fst toxin created holes in the cell wall while the TxpA toxin reduced cell size and induced cell wall septation. Besides, we identified a new TA system whose antitoxin functions as a transcriptional autoregulator while the toxin functions as an inhibitor of autoregulation. Altogether, this study provides a plethora of new as well as previously known TA systems that will revitalize the research on S. aureus TA systems. Full article

Wheat, the main source of carbohydrates worldwide, can be attacked by a wide number of phytopathogenic fungi, included Alternaria species. Alternaria species commonly occur on wheat worldwide and produce several mycotoxins such as tenuazonic acid (TA), alternariol (AOH), alternariol-monomethyl ether (AME), and altenuene (ALT), provided of haemato-toxic, genotoxic, and mutagenic activities. The contamination by Alternaria species of wheat kernels, collected in Tuscany, Italy, from 2013 to 2016, was evaluated. Alternaria contamination was detected in 93 out of 100 field samples, with values ranging between 1 and 73% (mean of 18%). Selected strains were genetically characterized by multi-locus gene sequencing approach through combined sequences of allergen alt1a, glyceraldeyde-3-phosphate dehydrogenase, and translation elongation factor 1α genes. Two well defined groups were generated; namely sections Alternaria and Infectoriae. Representative strains were analyzed for mycotoxin production. A different mycotoxin profile between the sections was shown. Of the 54 strains analyzed for mycotoxins, all strains included in Section Alternaria produced AOH and AME, 40 strains (99%) produced TA, and 26 strains (63%) produced ALT. On the other hand, only a very low capability to produce both AOH and AME was recorded among the Section Infectoriae strains. These data show that a potential mycotoxin risk related to the consumption of Alternaria contaminated wheat is high. Full article

Studies on cyanobacteria in Vietnam are limited and mainly restricted to large reservoirs. Cyanobacterial blooms in small water bodies may pose a health risk to local people. We sampled 17 water bodies in the vicinity of urban settlements throughout the Mekong basin and in southeast Vietnam. From these, 40 water samples were taken, 24 cyanobacterial strains were isolated and 129 fish, 68 snail, 7 shrimp, 4 clam, and 4 duck samples were analyzed for microcystins (MCs). MCs were detected up to 11,039 µg/L or to 4033 µg/g DW in water samples. MCs were detected in the viscera of the animals. MC-LR and MC-RR were most frequently detected, while MC-dmLR, MC-LW, and MC-LF were first recorded in Vietnam. Microcystis was the main potential toxin producer and the most common bloom-forming species. A potential health hazard was found in a duck–fish pond located in the catchment of DauTieng reservoir and in the DongNai river where raw water was collected for DongNai waterwork. The whole viscera of fish and snails must be completely removed during food processing. Cyanobacterial monitoring programs should be established to assess and minimize potential public health risks. Full article

SS1P is an anti-mesothelin immunotoxin composed of a targeting antibody fragment genetically fused to a truncated fragment of Pseudomonas exotoxin A. Delayed responses reported in mesothelioma patients receiving SS1P suggest that anti-tumor immunity is induced. The goal of this study is to evaluate if SS1P therapy renders mesothelioma tumors more sensitive to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) immune checkpoint blockade. We evaluated the ability of SS1P to induce adenosine triphosphate (ATP) secretion and calreticulin expression on the surface of AE17M mouse mesothelioma cells. Both properties are associated with immunogenic cell death. Furthermore, we treated these tumors with intra-tumoral SS1P and systemic CTLA-4. We found that SS1P increased the release of ATP from AE17M cells in a dose and time-dependent manner. In addition, SS1P induced calreticulin expression on the surface of AE17M cells. These results suggest that SS1P promotes immunogenic cell death and could sensitize tumors to anti-CTLA-4 based therapy. In mouse studies, we found that the combination of anti-CTLA-4 with intra-tumoral SS1P induced complete regressions in most mice and provided a statistically significant survival benefit compared to monotherapy. The surviving mice were protected from tumor re-challenge, indicating the development of anti-tumor immunity. These findings support the use of intra-tumoral SS1P in combination with anti-CTLA-4. Full article

Even if cancer represents a burden for human society, an exhaustive cure has not been discovered yet. Low therapeutic index and resistance to pharmacotherapy are two of the major limits of antitumour treatments. Natural products represent an excellent library of bioactive molecules. Thus, tapping into the natural world may prove useful in identifying new therapeutic options with favourable pharmaco-toxicological profiles. Juglans regia, or common walnut, is a very resilient tree that has inhabited our planet for thousands of years. Many studies correlate walnut consumption to beneficial effects towards several chronic diseases, such as cancer, mainly due to the bioactive molecules stored in different parts of the plant. Among others, polyphenols, quinones, proteins, and essential fatty acids contribute to its pharmacologic activity. The present review aims to offer a comprehensive perspective about the antitumour potential of the most promising compounds stored in this plant, such as juglanin, juglone, and the ellagitannin-metabolites urolithins or deriving from walnut dietary intake. All molecules and a chronic intake of the fruit provide tangible anticancer effects. However, the scarcity of studies on humans does not allow results to be conclusive. Full article

Rhabdomyolysis, a condition associated with the consumption of Yellow Knight mushrooms (Tricholoma equestre), was first reported in 2001. In response, some countries began to consider the mushroom as poisonous, whereas in others it is still consumed. In the present study, a nationwide survey of Polish mushroom foragers (n = 1545) was conducted to estimate the frequency of T. equestre consumption. The epidemiological database on mushroom poisonings in Poland was analyzed from the year 2008. Hematological and biochemical parameters were followed for a week in 10 volunteers consuming 300 g of molecularly identified T. equestre. More than half the foragers had consumed T. equestre at least once in their lifetime and a quarter had consumed it consecutively. The frequency of adverse events was low and no rhabdomyolysis was reported. The toxicological database indicated that mushrooms from the Tricholoma genus caused poisonings less frequently than mushrooms with well-established edibility and not a single case of rhabdomyolysis has been reported within the last decade. The volunteers consuming T. equestre revealed no hematological or biochemical alterations and no adverse effects were observed. The findings of this study support the view that T. equestre is edible if consumed in rational amounts by healthy subjects. Full article

Type II toxin-antitoxin (TA) systems are highly prevalent in bacterial genomes and have been extensively studied. These modules involve in the formation of persistence cells, the biofilm formation, and stress resistance, which might play key roles in pathogen virulence. SezAT and yefM-yoeB TA modules in Streptococcus suis serotype 2 (S. suis 2) have been studied, although the other TA systems have not been identified. In this study, we investigated nine putative type II TA systems in the genome of S. suis 2 strain SC84 by bioinformatics analysis and identified three of them (two relBE loci and one parDE locus) that function as typical type II TA systems. Interestingly, we found that the introduction of the two RelBE TA systems into Escherichia coli or the induction of the ParE toxin led to cell filamentation. Promoter activity assays indicated that RelB1, RelB2, ParD, and ParDE negatively autoregulated the transcriptions of their respective TA operons, while RelBE2 positively autoregulated its TA operon transcription. Collectively, we identified three TA systems in S. suis 2, and our findings have laid an important foundation for further functional studies on these TA systems. Full article

Ricin, a highly lethal toxin derived from the seeds of Ricinus communis (castor beans) is considered a potential biological threat agent due to its high availability, ease of production, and to the lack of any approved medical countermeasure against ricin exposures. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this work was to generate anti-ricin antitoxin that confers high level post-exposure protection against ricin challenge. Due to safety issues regarding the usage of ricin holotoxin as an antigen, we generated an inactivated toxin that would reduce health risks for both the immunizer and the immunized animal. To this end, a monomerized ricin antigen was constructed by reducing highly purified ricin to its monomeric constituents. Preliminary immunizing experiments in rabbits indicated that this monomerized antigen is as effective as the native toxin in terms of neutralizing antibody elicitation and protection of mice against lethal ricin challenges. Characterization of the monomerized antigen demonstrated that the irreversibly detached A and B subunits retain catalytic and lectin activity, respectively, implying that the monomerization process did not significantly affect their overall structure. Toxicity studies revealed that the monomerized ricin displayed a 250-fold decreased activity in a cell culture-based functionality test, while clinical signs were undetectable in mice injected with this antigen. Immunization of a horse with the monomerized toxin was highly effective in elicitation of high titers of neutralizing antibodies. Due to the increased potential of IgG-derived adverse events, anti-ricin F(ab’)₂ antitoxin was produced. The F(ab’)₂-based antitoxin conferred high protection to intranasally ricin-intoxicated mice; ~60% and ~34% survival, when administered 24 and 48 h post exposure to a lethal dose, respectively. In line with the enhanced protection, anti-inflammatory and anti-edematous effects were measured in the antitoxin treated mice, in comparison to mice that were intoxicated but not treated. Accordingly, this anti-ricin preparation is an excellent candidate for post exposure treatment of ricin intoxications. Full article

Male Wistar rats were treated intraperitoneally (i.p.) with fumonisin B₁ (FB₁; 0, 20, 50 and 100 mg/kg dietary dose equivalent) for 5 and 10 days (n = 24–24 in each setting) to gain dose- and time-dependent effects on antioxidant status and oxidative stress response, clinical chemical endpoints and liver, kidney and lung histopathology and lymphocyte damage (genotoxicity). FB₁ decreased feed intake, body weight gain and absolute liver weight, irrespective of the toxin dose. Relative kidney weight increased in the 10-day setting. Linear dose response was found for plasma aspartate aminotransferase, alanine aminotransferase, total cholesterol, urea and creatinine, and exposure time-dependence for plasma creatinine level. The latter was coupled with renal histopathological findings, tubular degeneration and necrosis and the detachment of tubular epithelial cells. The pronounced antioxidant response (reduced glutathione accretion, increasing glutathione peroxidase activity) referred to renal cortical response (5–10 days exposure at 50–100 ppm FB₁). Hepatic alterations were moderate, referring to initial phase lipid peroxidation (exposure time dependent difference of conjugated diene and triene concentrations), and slight functional disturbance (↑ total cholesterol). Lymphocyte DNA damage was moderate, supporting a mild genotoxic effect of FB₁. Full article

Deoxynivalenol (DON) is a toxin found in cereals as well as in processed products such as pasta, and causes substantial economic losses for stock breeding as it induces vomiting, reduced feeding, and reduced growth rates in piglets. Oxidative phosphorylation, TCA-cycle, transcription, and translation have been hypothesized to be leading pathways that are affected by DON. We used an application of high and low glucose to examine oxidative phosphorylation and anaerobic glycolysis. A change in the metabolic status of IPEC-J2 was observed and confirmed by microarray data. Measurements of oxygen consumption resulted in a significant reduction, if DON attacks from the basolateral. Furthermore, we found a dose-dependent effect with a significant reduction at 2000 ng/mL. In addition, SLC7A11 and PHB, the genes with the highest regulation in our microarray analyses under low glucose supply, were investigated and showed a variable regulation on protein level. Lactate production and glucose consumption was investigated to examine the impact of DON on anaerobic glycolysis and we observed a significant increase in 2000 bl^high and a decrease in 2000 ap^high. Interestingly, both groups as well as 200 bl^high showed a significant higher de novo protein synthesis when compared to the control. These results indicate the direct or indirect impact of DON on metabolic pathways in IPEC-J2. Full article

A novel, objective, and rapid computed motility inhibition (CMI) assay was developed to identify and assess sublethal injury in toxin-exposed boar spermatozoa and compared with a subjective visual motility inhibition (VMI) assay. The CMI values were calculated from digital micrographic videos using a custom MATLAB^® script by contrasting the motility index values of each experiment with those of the background and control experiments. Following a comparison of the CMI and VMI assays results, it was determined that their agreement depended on the shape of the dose-response curve. Toxins that exhibited a steep slope were indicative of good agreement between the assays. Those depicted by a gentle decline in the slope of the dose-response curve, the CMI assay were shown to be two times more sensitive than the VMI assay. The CMI assay was highly sensitive to the inhibition of mitochondrial function and glucose transport activity by sublethal doses of toxins and to disruption of cellular cation homeostasis by carrier ionophoric toxins, when compared to the cytotoxicity and lethal toxicity assays (i.e., that evaluated the inhibition of cell proliferation in somatic cell lines (FL, PK-15, and MNA cells)) and disruption to spermatozoa membrane integrity. The CMI assay recognized subtle sublethal toxicity changes in metabolism, manifested as a decrease in boar spermatozoa motility. Thus, it was feasible to effectively compare the objectively-measured numerical values for motility inhibition using the CMI assay against those reflecting lethal damage in the spermatozoa cells and somatic cell lines using a cytotoxicity assay. Full article

Immunoaffinity columns (IACs) are most popularly used for mycotoxin clean-up in complex matrices prior to chromatographic analysis. But, their high cost has limited their wide application and the regeneration of IACs for multiple instances of reuse is important. This study aimed to investigate the feasibility of regeneration and reuse of IACs for purification of ochratoxin A (OTA) in spiked raw malt and dried ginger samples followed by high performance liquid chromatography-fluorescence detection. After each use, the IACs were filled with phosphate buffer saline (PBS) as the preservation solution and stored at 8 °C overnight for regeneration and reuse until the recovery rate was <70%. The results showed that matrix type, preparation procedure, and pH value of sample extraction exhibited major effects on the reuse of IACs for OTA clean-up. While, after modifying the sample preparation procedure using water as the diluent and the solution at a pH of 7 to 8, the IACs could be used eight and three times for the spiked raw malt and dried ginger samples with OTA after regeneration. Regarding the traditional procedure recommended in Chinese Pharmacopoeia (2015 edition), the IACs could be used for three and two times for the spiked raw malt and dried ginger samples with OTA, respectively. Therefore, the corresponding experimental cost could be reduced to one-eighth and one-third of the original cost. This is the first study on the regeneration and reuse of IACs for OTA clean-up in complex Chinese herbal medicines, providing a green and economical tool for a large number of samples analysis with low cost. Full article

Antimicrobial peptides (AMPs) are crucial effectors of the innate immune system. They provide the first line of defense against a variety of pathogens. AMPs display synergistic effects with conventional antibiotics, and thus present the potential for combined therapies. Insects are extremely resistant to bacterial infections. Insect AMPs are cationic and comprise less than 100 amino acids. These insect peptides exhibit an antimicrobial effect by disrupting the microbial membrane and do not easily allow microbes to develop drug resistance. Currently, membrane mechanisms underlying the antimicrobial effects of AMPs are proposed by different modes: the barrel-stave mode, toroidal-pore, carpet, and disordered toroidal-pore are the typical modes. Positive charge quantity, hydrophobic property and the secondary structure of the peptide are important for the antibacterial activity of AMPs. At present, several structural families of AMPs from insects are known (defensins, cecropins, drosocins, attacins, diptericins, ponericins, metchnikowins, and melittin), but new AMPs are frequently discovered. We reviewed the biological effects of the major insect AMPs. This review will provide further information that facilitates the study of insect AMPs and shed some light on novel microbicides. Full article

Fusarium graminearum as the main causal agent of Fusarium head blight (FHB) and its ability to produce trichothecenes was investigated by molecular techniques. A total of 37 strains isolated from the wheat, harvested in Serbia in 2005, 2008 and 2015, and previously designated by morphological observation as F. graminearum, were used for trichothecene genotypes characterization. The strains were identified using the species-specific primer set FG16R/FG16F while genotypic characterization was done using specific TRI13 and TRI3 sequences of the trichothecene gene clusters. The PCR assays identified all strains as species of F. graminearum sensu stricto with the DON/15-ADON genotype. The quantification of the mycotoxin (DON) was performed using the biochemical assay. The high levels of DON (>20,000 µg kg⁻¹) were recorded in all of the strains from 2005, four strains from 2008 and two strains from 2015. Weather data of the investigated seasons, showed that the optimal temperature, frequent rains and high relative humidity (RH) was very favourable for the development of F. graminearum, affecting the DON biosynthesis. Full article

During severe bacterial infections, death and disease are often caused by an overly strong immune response of the human host. Acute toxic shock is induced by superantigen toxins, a diverse set of proteins secreted by Gram-positive staphylococcal and streptococcal bacterial strains that overstimulate the inflammatory response by orders of magnitude. The need to protect from superantigen toxins led to our discovery that in addition to the well-known MHC class II and T cell receptors, the principal costimulatory receptor, CD28, and its constitutively expressed coligand, B7-2 (CD86), previously thought to have only costimulatory function, are actually critical superantigen receptors. Binding of the superantigen into the homodimer interfaces of these costimulatory receptors greatly enhances B7-2/CD28 engagement, leading to excessive pro-inflammatory signaling. This finding led to the design of short receptor dimer interface mimetic peptides that block the binding of superantigen and thus protect from death. It then turned out that such a peptide will protect also from Gram-negative bacterial infection and from polymicrobial sepsis. One such CD28 mimetic peptide is advancing in a Phase 3 clinical trial to protect from lethal wound infections by flesh-eating bacteria. These host-oriented therapeutics target the human immune system itself, rendering pathogens less likely to become resistant. Full article

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases. Full article

The identification and quantification of okadaic acid (OA)/dinophysistoxin (DTX) analogues and pectenotoxins (PTXs) in Dinophysis samples collected from coastal locations around Japan were evaluated by liquid chromatography mass spectrometry. The species identified and analyzed included Dinophysis fortii, D. acuminata, D. mitra (Phalacroma mitra), D. norvegica, D. infundibulus, D. tripos, D. caudata, D. rotundata (Phalacroma rotundatum), and D. rudgei. The dominant toxin found in D. acuminata was PTX2 although some samples contained DTX1 as a minor toxin. D. acuminata specimens isolated from the southwestern regions (Takada and Hiroshima) showed characteristic toxin profiles, with only OA detected in samples collected from Takada. In contrast, both OA and DTX1, in addition to a larger proportion of PTX2, were detected in D. acuminata from Hiroshima. D. fortii showed a toxin profile dominated by PTX2 although this species had higher levels of DTX1 than D. acuminata. OA was detected as a minor toxin in some D. fortii samples collected from Yakumo, Noheji, and Hakata. PTX2 was also the dominant toxin found among other Dinophysis species analyzed, such as D. norvegica, D. tripos, and D. caudata, although some pooled picked cells of these species contained trace levels of OA or DTX1. The results obtained in this study re-confirm that cellular toxin content and profiles are different even among strains of the same species. Full article

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies (Lucilia cuprina) produced rapid contractile paralysis (PD₅₀ at 1 min = 3.1 μg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/μL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/β defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom. Full article