Toxins, Volume 14, Issue 6 (June 2022) – 57 articles

Venom is a dynamic trait that has contributed to the success of numerous organismal lineages. Predominantly composed of proteins, these complex cocktails are deployed for predation and/or self-defence. Many non-toxic physiological proteins have been convergently and recurrently recruited by venomous animals into their toxin arsenal. Phospholipase A₂ (PLA₂) is one such protein and features in the venoms of many organisms across the animal kingdom, including snakes of the families Elapidae and Viperidae. Understanding the evolutionary history of this superfamily would therefore provide insight into the origin and diversification of venom toxins and the evolution of novelty more broadly. The literature is replete with studies that have identified diversifying selection as the sole influence on PLA₂ evolution. However, these studies have largely neglected the structural/functional constraints on PLA₂s, and the ecology and evolutionary histories of the diverse snake lineages that produce them. By considering these crucial factors and employing evolutionary analyses integrated with a schema for the classification of PLA₂s, we uncovered lineage-specific differences in selection regimes. Thus, our work provides novel insights into the evolution of this major snake venom toxin superfamily and underscores the importance of considering the influence of evolutionary and ecological contexts on molecular evolution. Full article

Maize and other cereals are the commodities most contaminated with fumonisins. The maize acreage is increasing in Africa, and the maize harvest provides important foods for humans and feeds for domestic animals throughout the continent. In North Africa, high levels of fumonisins have been reported from Algeria and Morocco, while low levels have been detected in the rather few fumonisin analyses reported from Tunisia and Egypt. The West African countries Burkina Faso, Cameroon, Ghana, and Nigeria all report high levels of fumonisin contamination of maize, while the few maize samples analysed in Togo contain low levels. In Eastern Africa, high levels of fumonisin contamination have been reported from the Democratic Republic of Congo, Ethiopia, Kenya, Tanzania, and Uganda. The samples analysed from Rwanda contained low levels of fumonisins. Analysis of maize from the Southern African countries Malawi, Namibia, South Africa, Zambia, and Zimbabwe revealed high fumonisin levels, while low levels of fumonisins were detected in the few analyses of maize from Botswana and Mozambique. Full article

Snakebite envenomation is a neglected tropical disease that causes over 100,000 deaths each year. The only effective treatment consists of antivenoms derived from animal sera, but these have been deemed with highly variable potency and are usually inaccessible and too costly for victims. The production of antivenoms by venom-independent techniques, such as the immunization with multi-epitope constructs, could circumvent those drawbacks. Herein, we present a knowledge-based pipeline to prioritize potential epitopes of therapeutic relevance from toxins of medically important snakes in West Sub-Saharan Africa. It is mainly based on sequence conservation and protein structural features. The ultimately selected 41 epitopes originate from 11 out of 16 snake species considered of highest medical importance in the region and 3 out of 10 of those considered as secondary medical importance. Echis ocellatus, responsible for the highest casualties in the area, would be covered by 12 different epitopes. Remarkably, this pipeline is versatile and customizable for the analysis of snake venom sequences from any other region of the world. Full article

Incobotulinum toxin A (IncoBoNT-A) is effective in preventing ultraviolet B (UVB)-induced hyperpigmentation. This prospective, randomized, controlled study aimed to evaluate the effect of IncoBoNT-A on the treatment of UVB-induced hyperpigmentation in 15 volunteers. Five hyperpigmentation squares (2 × 2 cm) were induced by local UVB on the abdomen at baseline. At Day 7, each site was randomized to receive no treatment (control), normal saline, or intradermal IncoBoNT-A injection with 1:2.5, 1:5, and 1:7.5 dilutions (12, 6, and 4 units, respectively). The mean lightness index (L*), hyperpigmentation improvement score evaluated by blinded dermatologists, and participant satisfaction scores were obtained at Days 21, 28, and 35. At Day 21, improvements in mean L* of 1:2.5, 1:5, and 1:7.5 IncoBoNT-A-treated, saline-treated, and control sites were 14.30%, 12.28%, 6.62%, 0.32%, and 4.98%, respectively (p = 0.86). At Day 28, the improvement in mean L* in IncoBoNT-A-treated groups was superior to that in the other groups. In terms of the hyperpigmentation improvement score, 12 participants (80%) experienced better outcomes with the IncoBoNT-A-injected site compared with the other sites. IncoBoNT-A, especially at higher concentrations, showed some positive effects on the treatment of UVB-induced hyperpigmentation. This may serve as an adjuvant treatment for hyperpigmentary conditions that are aggravated by UVB. Full article

Pistachio nuts can become colonized by mycotoxigenic fungi, especially Aspergillus flavus, resulting in contamination with aflatoxins (AFs). We examined the effect of gaseous O₃ (50–200 ppm; 30 min; 6 L/min) on (a) in vitro germination, (b) mycelial growth, and (c) aflatoxin B1 (AFB₁) production on a milled pistachio nut-based medium at different water activity (a_w) levels and at 30 °C. This was complimented with in situ studies exposing raw pistachio nuts to 50–200 ppm of O₃. Exposure of conidia to gaseous O₃ initially resulted in lower germination percentages at different a_w levels. However, 12 h after treatment, conidial viability recovered with 100% germination after 24–48 h. Growth rates of mycelial colonies were slightly decreased with the increase of the O₃ dose, with significant inhibition only at 0.98 a_w. The production of AFB₁ after O₃ treatment and storage for 10 days was stimulated in A. flavus colonies at 0.98 a_w. Raw pistachio nuts inoculated with A. flavus conidia prior to O₃ exposure showed a significant decrease in population after 20 days of storage. However, AFB₁ contamination was stimulated in most O₃ treatments. The relationship between exposure concentration, time and prevailing a_w levels on toxin control needs to be better understood for these nuts. Full article

Effects of the combined task-oriented trainings with botulinum toxin A (BoNT-A) injection on improving motor functions and reducing spasticity remains unclear. This study aims to investigate effects of 3 task-oriented trainings (robot-assisted therapy (RT), mirror therapy (MT), and active control treatment (AC)) in patients with stroke after BoNT-A injection. Thirty-seven patients with chronic spastic hemiplegic stroke were randomly assigned to receive RT, MT, or AC following BoNT-A injection over spastic upper extremity muscles. Each session of RT, MT, and AC was 75 min, 3 times weekly, for 8 weeks. Outcome measures were assessed at pretreatment, post-treatment, and 3-month follow-up, involving the Fugl-Meyer Assessment (FMA), Modified Ashworth Scale (MAS), Motor Activity Log (MAL), including amount of use (AOU) and quality of movement (QOM), and arm activity level. All 3 combined treatments improved FMA, MAS, and MAL. The AC induced a greater effect on QOM in MAL at the 3-month follow-up than RT or MT. All 3 combined trainings induced minimal effect on arm activity level. Our findings suggest that for patients with stroke who received BoNT-A injection over spastic UE muscles, the RT, MT, or AC UE training that followed was effective in improving motor functions, reducing spasticity, and enhancing daily function. Full article

Acute bovine liver disease (ABLD) is a hepatic disease affecting cattle sporadically in southern Australia, characterised histologically by striking periportal hepatocellular necrosis. The cause of ABLD is unknown; however, the seasonality and acute presentation of outbreaks suggest mycotoxin involvement. We described the geographical and seasonal occurrence of ABLD reports from 2010 to 2020 in Victoria, Australia, and explored potential weather triggers preceding 26 outbreaks occurring across 23 properties using a case-crossover design. Outbreaks occurred most frequently in autumn/early winter and in herds located along the southern coastal plain of Victoria, and occasionally within the low-lying regions of the Great Dividing Range. Lactating adult dairy cattle represented the most reported cases. We observed a significant association between an increase in average daily dewpoint in the 15 days preceding an ABLD outbreak, suggesting that dew formation may be a key determinant for this disease. Our findings support the etiology of a potent hepatotoxic agent that requires moisture for proliferation and/or toxin production. Full article

Background: We investigated the effect of replacing normal corn (NC) or normal wheat bran (NW) with moldy corn (MC) or moldy wheat bran (MW) on growth, ovarian follicular reserves, and oxidative status. Methods: Sixty-three Landrace × Yorkshire gilts were assigned to seven diets formulated by using MC to replace 0% (control), 25% (25% MC), 50% (50% MC), 75% (75% MC), and 100% NC (100% MC), MW to replace 100% NW (100% MW), and MC and MW to replace 100% NC and 100% NW (100% MC + MW), from postnatal day 110 to day 19 of the second estrous cycle. Results: Feeding the gilts with MC or MW induced a lower average daily gain at days 29–56 of the experiment. Age at puberty remained unchanged, but MC inclusion resulted in a linear decrease in antral follicles with diameter >3.0 mm, and control gilts had a 12.7 more large antral follicles than gilts in the 100% MC + MW treatment. MC inclusion linearly decreased the numbers of primordial follicles, growing follicles, and corpora lutea, associated with a lower anti-Müllerian hormone level in serum and 17β-estradiol level in follicular fluid. MC inclusion decreased the serum concentrations of insulin-like growth factor 1 and its mRNA levels in the liver, combined with higher malondialdehyde concentration and lower total superoxide dismutase activities in serum and liver. Conclusion: Chronic exposure to MC-containing diets caused the loss of follicles, even if levels of deoxynivalenol, zearalenone, and aflatoxin B1 were below the levels allowed by China and Europe standards. Full article

Patients with kidney failure (KF) have a high incidence of cardiovascular (CV) disease, partly driven by insufficient clearance of uremic toxins. Recent investigations have questioned the accepted effects of adverse lipid profile and CV risk in uremic patients. Therefore, we related a panel of uremic toxins previously associated with CV morbidity/mortality to a full lipid profile in a large, tri-national, cross-sectional cohort. Total, high-density lipoprotein (HDL), non-HDL, low-density lipoprotein (LDL), and remnant cholesterol, as well as triglyceride, levels were associated with five uremic toxins in a cohort of 611 adult KF patients with adjustment for clinically relevant covariates and other patient-level variables. Univariate analyses revealed negative correlations of total, non-HDL, and LDL cholesterol with all investigated uremic toxins. Multivariate linear regression analyses confirmed independent, negative associations of phenylacetylglutamine with total, non-HDL, and LDL cholesterol, while indole-3 acetic acid associated with non-HDL and LDL cholesterol. Furthermore, trimethylamine-N-Oxide was independently and negatively associated with non-HDL cholesterol. Sensitivity analyses largely confirmed findings in the entire cohort. In conclusion, significant inverse associations between lipid profile and distinct uremic toxins in KF highlight the complexity of the uremic milieu, suggesting that not all uremic toxin interactions with conventional CV risk markers may be pathogenic. Full article

We treated a 51-year-old woman with refractory Complex Regional Pain Syndrome type I (CRPS-I) involving her left hand and forearm with subcutaneous injections of BoNT/A. The injections were performed every 3 months, with a total of six treatments. Each treatment was able to effectively improve pain and motor impairment; however, the duration of the effect was limited to only a few months. BoNT/A could improve patients’ quality of life with CRPS; however, extensive clinical studies are needed to determine its role in clinical practice. Full article

Freshwater bodies and, consequently, drinking water treatment plants (DWTPs) sources are increasingly facing toxic cyanobacterial blooms. Even though conventional treatment processes including coagulation, flocculation, sedimentation, and filtration can control cyanobacteria and cell-bound cyanotoxins, these processes may encounter challenges such as inefficient removal of dissolved metabolites and cyanobacterial cell breakthrough. Furthermore, conventional treatment processes may lead to the accumulation of cyanobacteria cells and cyanotoxins in sludge. Pre-oxidation can enhance coagulation efficiency as it provides the first barrier against cyanobacteria and cyanotoxins and it decreases cell accumulation in DWTP sludge. This critical review aims to: (i) evaluate the state of the science of cyanobacteria and cyanotoxin management throughout DWTPs, as well as their associated sludge, and (ii) develop a decision framework to manage cyanobacteria and cyanotoxins in DWTPs and sludge. The review identified that lab-cultured-based pre-oxidation studies may not represent the real bloom pre-oxidation efficacy. Moreover, the application of a common exposure unit CT (residual concentration × contact time) provides a proper understanding of cyanobacteria pre-oxidation efficiency. Recently, reported challenges on cyanobacterial survival and growth in sludge alongside the cell lysis and cyanotoxin release raised health and technical concerns with regards to sludge storage and sludge supernatant recycling to the head of DWTPs. According to the review, oxidation has not been identified as a feasible option to handle cyanobacterial-laden sludge due to low cell and cyanotoxin removal efficacy. Based on the reviewed literature, a decision framework is proposed to manage cyanobacteria and cyanotoxins and their associated sludge in DWTPs. Full article

The disease severity and mycotoxin DON content in grains caused by fusarium head blight (FHB) have been two prioritized economical traits in wheat. Reliable phenotyping is a prerequisite for genetically improving wheat resistances to these two traits. In this study, three inoculation methods: upper bilateral floret injection (UBFI), basal bilateral floret injection (BBFI), and basal rachis internode injection (BRII), were applied in a panel of 22 near-isogenic lines (NILs) contrasting in Fhb1 alleles. The results showed that inoculation methods had significant influence on both disease severity and mycotoxin accumulation in grains, and the relationship between them. UBFI method caused chronic FHB symptom characterized as slow progress of the pathogen downward from the inoculation site, which minimized the difference in disease severity of the NILs, but, unexpectedly, maximized the difference in DON content between them. The BBFI method usually caused an acute FHB symptom in susceptible lines characterized as premature spike death (PSD), which maximized the difference in disease severity, but minimized the difference in DON content in grains between resistant and susceptible lines. The BRII method occasionally caused acute FHB symptoms for susceptible lines and had relatively balanced characteristics of disease severity and DON content in grains. Therefore, two or more inoculation methods are recommended for precise and reliable evaluation of the overall resistance to FHB, including resistances to both disease spread within a spike and DON accumulation in grains. Full article

Spider venoms constitute a trove of novel peptides with biotechnological interest. Paucity of next-generation-sequencing (NGS) data generation has led to a description of less than 1% of these peptides. Increasing evidence supports the underestimation of the assembled genes a single transcriptome assembler can predict. Here, the transcriptome of the venom gland of the spider Pamphobeteus verdolaga was re-assembled, using three free access algorithms, Trinity, SOAPdenovo-Trans, and SPAdes, to obtain a more complete annotation. Assembler’s performance was evaluated by contig number, N50, read representation on the assembly, and BUSCO’s terms retrieval against the arthropod dataset. Out of all the assembled sequences with all software, 39.26% were common between the three assemblers, and 27.88% were uniquely assembled by Trinity, while 27.65% were uniquely assembled by SPAdes. The non-redundant merging of all three assemblies’ output permitted the annotation of 9232 sequences, which was 23% more when compared to each software and 28% more when compared to the previous P. verdolaga annotation; moreover, the description of 65 novel theraphotoxins was possible. In the generation of data for non-model organisms, as well as in the search for novel peptides with biotechnological interest, it is highly recommended to employ at least two different transcriptome assemblers. Full article

Biocides based on chemical synthetic compounds have been commonly used to counteract damages caused by microorganisms on stone cultural heritage. However, in the last few years, the use of commercial and traditional biocides has been banned and/or limited due to their dangerous profile for the environment, as well as human and animal health. Natural products could be used as suitable alternatives for cultural heritage purposes, as they have low toxicity and stability compared with synthetic pesticides. Even if most of the investigated solutions have already shown promising results, their efficiency, ecotoxicological, and chemical features are poorly investigated. In this manuscript, we aimed to evaluate the ecotoxicological profile of four fungal metabolites—namely, cavoxin, epi-epoformin, seiridin, and sphaeropsidone—with potential antimicrobial properties for monumental artworks. A battery of ecotoxicological tests using Aliivibrio fischeri (bacterium), Raphidocelis subcapitata (alga), Raphanus sativus L. (macrophyte), Daphnia magna (crustacean), and Caenorhabditis elegans (nematode) revealed a relative lower toxicity of these compounds, especially when compared with Preventol^® and Rocima^®, commercial biocides mainly used for the conservation of cultural heritage. Full article

The normal biological wound healing process consists of three precisely and highly programmed phases that require optimal conditions including internal and external factors. Any negative factors that disrupt the sequence or time frame of the healing mechanism can result in a non-healing wound or chronic ulcers. Botulinum neurotoxin A (BoNT-A) which is generally known as anti-contraction of muscles has been reported as a successful treatment in various types of chronic ulcers. The aim of this study is to review the outcome of treatment with BoNT-A for chronic skin ulcers. The results demonstrated some positive effects of BoNT-A on chronic ulcers. Ischemic ulcers secondary to Raynaud’s phenomenon seem to be the most promising type of ulcers that have benefited from BoNT-A. The rationale behind using BoNT-A to fasten the wound healing process is also discussed. Further clinical trial studies should be conducted to affirm the efficacy of wound healing using BoNT-A administration. Full article

The present interlaboratory comparison study involved nine laboratories located throughout the world that tested for 24 regulated and non-regulated mycotoxins by applying their in-house LC-MS/MS multi-toxin method to 10 individual lots of 4 matrix commodities, including complex chicken and swine feed, soy and corn gluten. In total, more than 6000 data points were collected and analyzed statistically by calculating a consensus value in combination with a target standard deviation following a modified Horwitz equation. The performance of each participant was evaluated by a z-score assessment with a satisfying range of ±2, leading to an overall success rate of 70% for all tested compounds. Equal performance for both regulated and emerging mycotoxins indicates that participating routine laboratories have successfully expanded their analytical portfolio in view of potentially new regulations. In addition, the study design proved to be fit for the purpose of providing future certified reference materials, which surpass current analyte matrix combinations and exceed the typical scope of the regulatory framework. Full article

Fungal infections in cereals lead to huge economic losses in the food and agriculture industries. This study was designed to investigate the occurrence of toxigenic fungi and their mycotoxins in marketed cereals and explore the effect of the antagonistic yeast Cyberlindnera jadinii volatiles against key toxigenic fungal strains. Aspergillus spp. were the most frequent contaminating fungi in the cereals, with an isolation frequency (Fr) of 100% in maize, followed by wheat (88.23%), rice (78.57%) and oats (14.28%). Morphological and molecular identification confirmed the presence of key toxigenic fungal strains in cereal samples, including A. carbonarius, A. flavus, A. niger, A. ochraceus and A. parasiticus. Aflatoxins (AFs) were detected in all types of tested cereal samples, with a significantly higher level in maize compared to wheat, rice, oats and breakfast cereals. Ochratoxin A (OTA) was only detected in wheat, rice and maize samples. Levels of mycotoxins in cereals were within EU permissible limits. The volatiles of Cyberlindnera jadinii significantly inhibited the growth of A. parasiticus, A. niger and P. verrucosum. The findings of this study confirm the presence of toxigenic fungi and mycotoxins in cereals within the EU permissible limits and the significant biocontrol ability of Cyberlindnera jadinii against these toxigenic fungi. Full article

rRNA N-glycosylases (EC 3.2.2.22) remove a specific adenine (A₄₃₂₄, rat 28S rRNA) in the sarcin ricin loop (SRL) involved into ribosome interaction with elongation factors, causing the inhibition of translation, for which they are known as plant ‘ribosome inactivating proteins’ (RIPs). However, protein synthesis inactivation could be the result of other enzymes, which often have rRNA as the target. In this scenario, Endo’s assay is the most used method to detect the enzymes that are able to hydrolyze a phosphodiester bond or cleave a single N-glycosidic bond (rRNA N-glycosylases). Indeed, the detection of a diagnostic fragment from rRNA after enzymatic action, with or without acid aniline, allows one to discriminate between the N-glycosylases or hydrolases, which release the β-fragment after acid aniline treatment or α-fragment without acid aniline treatment, respectively. This assay is of great importance in the mushroom kingdom, considering the presence of enzymes that are able to hydrolyze phosphodiester bonds (e.g., ribonucleases, ribotoxins and ribotoxin-like proteins) or to remove a specific adenine (rRNA N-glycosylases). Thus, here we used the β-fragment experimentally detected by Endo’s assay as a hallmark to revise the literature available on enzymes from mushrooms and other fungi, whose action consists of protein biosynthesis inhibition. Full article

In patients with severe kidney disease, renal clearance is compromised, resulting in the accumulation of a plethora of endogenous waste molecules that cannot be removed by current dialysis techniques, the most often applied treatment. These uremic retention solutes, also named uremic toxins, are a heterogeneous group of organic compounds of which many are too large to be filtered and/or are protein-bound. Their renal excretion depends largely on renal tubular secretion, by which the binding is shifted towards the free fraction that can be eliminated. To facilitate this process, kidney proximal tubule cells are equipped with a range of transport proteins that cooperate in cellular uptake and urinary excretion. In recent years, innovations in dialysis techniques to advance uremic toxin removal, as well as treatments with drugs and/or dietary supplements that limit uremic toxin production, have provided some clinical improvements or are still in progress. This review gives an overview of these developments. Furthermore, the role protein-bound uremic toxins play in inter-organ communication, in particular between the gut (the side where toxins are produced) and the kidney (the side of their removal), is discussed. Full article

Pinnatoxins (PnTX) and Portimines (Prtn), two toxins produced by the benthic dinoflagellate Vulcanodinium rugosum, are known to be lethal to mice after intraperitoneal or oral administration. They are also known to accumulate in shellfish such as mussels and clams, but their effect on fish and the upper food chain remains unknown. In this work, juveniles of the fish Liza ramada (Mullet) were exposed to a strain of V. rugosum producing PnTX G and Prtn A. The fishes’ viability and contamination were recorded at times interval. Results showed that L. ramada juveniles were able to feed on V. rugosum and that their tissues could be contaminated by PnTX G and Prtn A without impact on fish viability. Furthermore, the microalgae temporary cysts survived and germinated after fish gut passage. This study showed the potential of L. ramada to transfer PnTX and Prtn toxins to the upper food chain and to disseminate V. rugosum in environment. Full article

Liver S9 fraction is usually employed in mutagenicity/genotoxicity in vitro assays, but some genotoxic compounds may need another type of bioactivation. In the present work, an alternative S9 fraction from the kidneys was used for the genotoxicity assessment of 12 mycotoxins with the SOS/umu test. The results were compared with liver S9 fraction, and 2–4 independent experiments were performed with each mycotoxin. The expected results were obtained with positive controls (4-nitroquinoline-N-oxide and 2-aminoanthracene) without metabolic activation or with liver S9, but a potent dose-dependent effect with 4-nitroquinoline-N-oxide and no activity of 2-aminoanthracene with kidney S9 were noticed. Aflatoxin B1 was genotoxic with metabolic activation, the effect being greater with liver S9. Sterigmatocystin was clearly genotoxic with liver S9 but equivocal with kidney S9. Ochratoxin A, zearalenone and fumonisin B1 were negative in all conditions. Trichothecenes were negative, except for nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 and HT-2 toxins, which showed equivocal results with kidney S9 because a clear dose-response effect was not observed. Most of the mycotoxins have been assessed with kidney S9 and the SOS/umu test for the first time here. The results with the positive controls and the mycotoxins confirm that the organ used for the S9 fraction preparation has an influence on the genotoxic activity of some compounds. Full article

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) or high-resolution MS (LC–HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC–MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard’s reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC–MS/MS and LC–HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1–GRT derivatization strategy mitigates many of the shortcomings of current LC–MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes. Full article

The problem of residues of toxic contaminants in food products has assumed considerable importance in terms of food safety. Naturally occurring contaminants, such as mycotoxins, are monitored routinely in the agricultural and food industries. Unfortunately, the consequences of the presence of mycotoxins in foodstuffs are evident in livestock farms, where both subacute and chronic effects on animal health are observed and could have non-negligible effects on human health. Ochratoxin A (OTA) is a common mycotoxin that contaminates food and feeds. Due to its thermal stability, the eradication of OTA from the food chain is very difficult. Consequently, humans and animals are frequently exposed to OTA in daily life. In this review article, we will devote time to highlighting the redox-based nephrotoxicity that occurs during OTA intoxication. In the past few decades, the literature has improved on the main molecules and enzymes involved in the redox signaling pathway as well as on some new antioxidant compounds as therapeutic strategies to counteract oxidative stress. The knowledge shown in this work will address the use of nutraceutical substances as dietary supplements, which would in turn improve the prophylactic and pharmacological treatment of redox-associated kidney diseases during OTA exposure, and will attempt to promote animal feed supplementation. Full article

Phytoplankton are photosynthetic microorganisms in aquatic environments that produce many bioactive substances. However, some of them are toxic to aquatic organisms via filter-feeding and are even poisonous to humans through the food chain. Human poisoning from these substances and their serious long-term consequences have resulted in several health threats, including cancer, skin disorders, and other diseases, which have been frequently documented. Seafood poisoning disorders triggered by phytoplankton toxins include paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), ciguatera fish poisoning (CFP), and azaspiracid shellfish poisoning (AZP). Accordingly, identifying harmful shellfish poisoning and toxin-producing species and their detrimental effects is urgently required. Although the harmful effects of these toxins are well documented, their possible modes of action are insufficiently understood in terms of clinical symptoms. In this review, we summarize the current state of knowledge regarding phytoplankton toxins and their detrimental consequences, including tumor-promoting activity. The structure, source, and clinical symptoms caused by these toxins, as well as their molecular mechanisms of action on voltage-gated ion channels, are briefly discussed. Moreover, the possible stress-associated reactive oxygen species (ROS)-related modes of action are summarized. Finally, we describe the toxic effects of phytoplankton toxins and discuss future research in the field of stress-associated ROS-related toxicity. Moreover, these toxins can also be used in different pharmacological prospects and can be established as a potent pharmacophore in the near future. Full article

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (K_d = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell. Full article

Fungi belonging to the genus Stachybotrys are frequently detected in water-damaged indoor environments, and a potential correlation between emerging health problems of inhabitants of affected housing and the fungi is controversially discussed. Secondary metabolites (i.e., mycotoxins) produced by Stachybotrys, such as the highly toxic macrocyclic trichothecenes (MCTs), are of potential concern to human health. The present study, however, focused on the potential effects of the more broadly and abundantly formed group of phenylspirodrimanes (PSDs). The phase I and II metabolism of four structurally different PSDs were investigated in vitro using hepatic models in combination with high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS) analysis. In addition to metabolite detection by HRMS, isolation and structure elucidation by nuclear magnetic resonance spectroscopy (NMR) was part of the conducted study as well. Full article

The silkworm’s Cat L-like gene, which encodes a lysosomal cathepsin L-like cysteine protease, is thought to be part of the insect’s innate immunity via an as-yet-undetermined mechanism. Assuming that the primary function of Cat L-like is microbial degradation in mature phagosomes, we hypothesise that the suppression of the Cat L-like gene expression would increase Bacillus thuringiensis (Bt) bacteraemia and toxicity in knockdown insects. Here, we performed a functional analysis of Cat L-like in larvae that were fed mulberry leaves contaminated with a commercial biopesticide formulation based on Bt kurstaki (Btk) (i.e., Dipel) to investigate its role in insect defence against a known entomopathogen. Exposure to sublethal doses of Dipel resulted in overexpression of the Cat L-like gene in insect haemolymph 24 and 48 h after exposure. RNA interference (RNAi)-mediated suppression of Cat L-like expression significantly increased the toxicity of Dipel to exposed larvae. Moreover, Btk replication was higher in RNAi insects, suggesting that Cat L-like cathepsin may be involved in a bacterial killing mechanism of haemocytes. Finally, our results confirm that Cat L-like protease is part of the antimicrobial defence of insects and suggest that it could be used as a target to increase the insecticidal efficacy of Bt-based biopesticides. Full article

Simultaneous removal of mycotoxins has been poorly addressed, and a limited number of studies have reported the efficacy of feed additives in sequestering a large spectrum of mycotoxins. In this study, a new mycotoxin-adsorbing agent was obtained by properly mixing a tri-octahedral smectite with a lignocellulose-based material. At a dosage of 1 mg mL⁻¹, these materials simultaneously adsorbed frequently occurring mycotoxins and did not exert a cytotoxic effect on intestinal cells. Chyme samples obtained by a simulated GI digestion did not affect the viability of Caco-2TC7 cells as measured by the MTT test. In addition, the chyme of the lignocellulose showed a high content of polyphenols (210 mg mL⁻¹ catechin equivalent) and good antioxidant activity. The properties of the individual constituents were maintained in the final composite, and were unaffected by their combination. When tested with a pool of seven mycotoxins at 1 µg mL⁻¹ each and pH 5, the composite (5 mg mL⁻¹) simultaneously sequestered AFB₁ (95%), FB₁ (99%), ZEA (93%), OTA (80%), T-2 (63%), and DON (22%). HT-2 adsorption did not occur. Mycotoxin adsorption increased exponentially as dosage increased, and occurred at physiological pH values. AFB₁, ZEA and T-2 adsorption was not affected by pH in the range 3–9, whereas OTA and FB₁ were adsorbed at pH values of 3–5. The adsorbed amount of AFB₁, ZEA and T-2 was not released when pH rose from 3 to 7. FB₁ and OTA desorption was less than 38%. Langmuir adsorption isotherms revealed high capacity and affinity for adsorption of the target mycotoxins. Results of this study are promising and show the potential of the new composite to remove mycotoxins in practical scenarios where several mycotoxins can co-occur. Full article

VVA2 (volvatoxin A chain 2) is a cardiotoxic protein purified from Volvariella volvacea. Its biological activities include hemolysis, writhing reaction, neurotoxicity, and ventricular systolic arresting activity. The cytotoxicity of VVA2 was mainly considered due to its pore-forming activity. Here we report a novel biological activity of its variants VVA2 I82E/K86K as a duplex-specific nuclease. Recombinant VVA2 variant I82E/L86K (Re-VVA2 I82E/L86K), deprived of the oligomerization property, shows increased nuclease activity compared to VVA2. Re-VVA2 I82E/L86K converts supercoiled DNA (Replicative form I, RF I) into nicked form (RF II) and linear form (RF III) in the presence of Mg²⁺ or Mn²⁺. Besides plasmid DNA, it also exhibits nuclease activity on E. coli genomic DNA rather than ssDNA or RNA. Re-VVA2 I82E/L86K preferentially cleaves dG-dC-rich dsDNA regions and shows the best performance at pH 6–9 and 55 °C. Our structure–function study has revealed amino acid E111 may take an active part in nuclease activity through interacting with metal ions. Based on the sequences of its cleavage sites, a “double-hit” mechanism was thereby proposed. Given that Re-VVA2 I82E/L86K did not exhibit the conserved nuclease structure and sequence, it is considered an atypical duplex-specific nuclease. Full article

Renal impairment in Multiple Myeloma (MM) represents one of the most important factors that influences patient survival. In fact, before the introduction of modern chemotherapy, less than 25% of patients with acute kidney injury (AKI) and MM who required dialysis recovered sufficient renal function to become independent from dialysis, with a median overall survival of less than 1 year. There are many other factors involved in determining patient survival. In this study we aimed to investigate the role of double filter-based extracorporeal treatment for removal of serum free light chains (sFLC) in acute myeloma kidney (AKI for MM) and to evaluate patient overall survival. All patients received Bortezomib-based chemotherapy and extracorporeal treatment for sFLC removal. For each session 2 dialyzers of the same kind were used. The dialytic dose was not related to the degree of renal function but to the removal of sFLC. The factors that have been found to be significantly associated with lower mortality were reduction of sFLC at day 12 and day 30, >50% reduction of sFLC at day 30, number of sessions and independence from dialysis. Among baseline characteristics, albumin level was statistically associated with the patients’ outcome. Our analysis highlights the importance of the early treatment for removal of sFLC in AKI for MM. These results indicate that the early removal of sFLC can improve patient’s outcome. Full article