Editor’s Choice Articles

Among the forms of idiopathic hyperhidrosis, those involving the forehead have the greatest impact on patients’ quality of life, as symptoms are not very controllable and are difficult to mask for patients. Although the local injection therapy with Incobotulinum toxin type A (IncoBTX-A therapy) can be considered a rational treatment, data from the literature describing both efficacy and safety of the treatment over the long term are poor. The aim of this report is to describe the single-center experience of five patients seeking treatment, for forehead hyperhidrosis with IncoBTX-A. To evaluate the benefits, safety profile and duration of anhidrosis, patients were treated following a standardized procedure and then followed until clinical relapse. The amount of sweating was measured by gravimetric testing, the extension of hyperhidrosis area was measured through Minor’s iodine starch test, and response to the treatment was evaluated using the Hyperhidrosis Disease Severity Scale (HDSS) and the Dermatology Life Quality Index (DLQI). In all treated patients, a significant anhidrotic effect was observed 4 weeks after the treatment and lasted for approximately 36 weeks. The reduction in sweat production was associated with significant amelioration of symptoms and quality of life for all treated patients. No serious side effects occurred; one patient complained of a mild transient bilateral ptosis. Although further wider studies are required, our preliminary results seem to encourage the use of IncoBTX-A in forehead hyperhidrosis. Full article

Clostridium botulinum and Clostridium tetani are Gram-positive, spore-forming, and anaerobic bacteria that produce the most potent neurotoxins, botulinum toxin (BoNT) and tetanus toxin (TeNT), responsible for flaccid and spastic paralysis, respectively. The main habitat of these toxigenic bacteria is the environment (soil, sediments, cadavers, decayed plants, intestinal content of healthy carrier animals). C. botulinum can grow and produce BoNT in food, leading to food-borne botulism, and in some circumstances, C. botulinum can colonize the intestinal tract and induce infant botulism or adult intestinal toxemia botulism. More rarely, C. botulinum colonizes wounds, whereas tetanus is always a result of wound contamination by C. tetani. The synthesis of neurotoxins is strictly regulated by complex regulatory networks. The highest levels of neurotoxins are produced at the end of the exponential growth and in the early stationary growth phase. Both microorganisms, except C. botulinum E, share an alternative sigma factor, BotR and TetR, respectively, the genes of which are located upstream of the neurotoxin genes. These factors are essential for neurotoxin gene expression. C. botulinum and C. tetani share also a two-component system (TCS) that negatively regulates neurotoxin synthesis, but each microorganism uses additional distinct sets of TCSs. Neurotoxin synthesis is interlocked with the general metabolism, and CodY, a master regulator of metabolism in Gram-positive bacteria, is involved in both clostridial species. The environmental and nutritional factors controlling neurotoxin synthesis are still poorly understood. The transition from amino acid to peptide metabolism seems to be an important factor. Moreover, a small non-coding RNA in C. tetani, and quorum-sensing systems in C. botulinum and possibly in C. tetani, also control toxin synthesis. However, both species use also distinct regulatory pathways; this reflects the adaptation of C. botulinum and C. tetani to different ecological niches. Full article

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs. Full article

Botulinum neurotoxins (BoNT) are a group of clostridial toxins that cause the potentially fatal neuroparalytic disease botulism. Although highly toxic, BoNTs are utilized as therapeutics to treat a range of neuromuscular conditions. Several serotypes (BoNT/A-/G, /X) have been identified with vastly differing toxicological profiles. Each serotype can be further sub-categorised into subtypes due to subtle variations in their protein sequence. These minor changes have been attributed to differences in both the duration of action and potency for BoNT/A subtypes. BoNTs are composed of three domains—a cell-binding domain, a translocation domain, and a catalytic domain. In this paper, we present the crystal structures of the botulinum neurotoxin A2 cell binding domain, both alone and in complex with its receptor ganglioside GD1a at 1.63 and 2.10 Å, respectively. The analysis of these structures reveals a potential redox-dependent Lys-O-Cys bridge close to the ganglioside binding site and a hinge motion between the H_CN and H_CC subdomains. Furthermore, we make a detailed comparison with the previously reported H_C/A2:SV2C structure for a comprehensive structural analysis of H_C/A2 receptor binding. Full article

The objective of this study was to evaluate whether low doses of zearalenone (ZEN) affect the carry-over of ZEN and its metabolites to intestinal tissues and the expression of CYP1A1 and GSTπ1 in the large intestine. Prepubertal gilts (with a BW of up to 14.5 kg) were exposed in group ZEN to daily ZEN5 doses of 5 μg/kg BW (n = 15); in group ZEN10, 10 μg/kg BW (n = 15); in group ZEN15, 15 μg/kg BW (n = 15); or were administered a placebo (group C, n = 15) throughout the experiment. After euthanasia, tissues were sampled on exposure days 7, 21, and 42 (D1, D2, and D3, respectively). The results confirmed that the administered ZEN doses (LOAEL, NOAEL, and MABEL) were appropriate to reliably assess the carry-over of ZEN. Based on the observations made during 42 days of exposure to pure ZEN, it can be hypothesized that all mycotoxins (ZEN, α-zearalenol, and β-zearalenol) contribute to a balance between intestinal cells and the expression of selected genes encoding enzymes that participate in biotransformation processes in the large intestine; modulate feminization processes in prepubertal gilts; and elicit flexible, adaptive responses of the macroorganism to mycotoxin exposure at the analyzed doses. Full article

Harmful algal blooms (HABs) have wide-ranging environmental impacts, including on aquatic species of social and commercial importance. In New Zealand (NZ), strategic growth of the aquaculture industry could be adversely affected by the occurrence of HABs. This review examines HAB species which are known to bloom both globally and in NZ and their effects on commercially important shellfish and fish species. Blooms of Karenia spp. have frequently been associated with mortalities of both fish and shellfish in NZ and the sub-lethal effects of other genera, notably Alexandrium spp., on shellfish (which includes paralysis, a lack of byssus production, and reduced growth) are also of concern. Climate change and anthropogenic impacts may alter HAB population structure and dynamics, as well as the physiological responses of fish and shellfish, potentially further compromising aquatic species. Those HAB species which have been detected in NZ and have the potential to bloom and harm marine life in the future are also discussed. The use of environmental DNA (eDNA) and relevant bioassays are practical tools which enable early detection of novel, problem HAB species and rapid toxin/HAB screening, and new data from HAB monitoring of aquaculture production sites using eDNA are presented. As aquaculture grows to supply a sizable proportion of the world’s protein, the effects of HABs in reducing productivity is of increasing significance. Research into the multiple stressor effects of climate change and HABs on cultured species and using local, recent, HAB strains is needed to accurately assess effects and inform stock management strategies. Full article

Harmful algal bloom (HAB) species Alexandrium catenella and Dinophysis acuminata are associated with paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) in humans, respectively. While PSP and DSP have been studied extensively, less is known about the effects of these HAB species or their associated toxins on shellfish. This study investigated A. catenella and D. acuminata toxicity in a larval oyster (Crassostrea virginica) bioassay. Larval activity and mortality were examined through 96-h laboratory exposures to live HAB cells (10–1000 cells/mL), cell lysates (1000 cells/mL equivalents), and purified toxins (10,000 cells/mL equivalents). Exposure to 1000 cells/mL live or lysed D. acuminata caused larval mortality (21.9 ± 7.0%, 10.2 ± 4.0%, respectively) while exposure to any tested cell concentration of live A. catenella, but not lysate, caused swimming arrest and/or mortality in >50% of larvae. Exposure to high concentrations of saxitoxin (STX) or okadaic acid (OA), toxins traditionally associated with PSP and DSP, respectively, had no effect on larval activity or mortality. In contrast, pectenotoxin-2 (PTX2) caused rapid larval mortality (49.6 ± 5.8% by 48 h) and completely immobilized larval oysters. The results indicate that the toxic effects of A. catenella and D. acuminata on shellfish are not linked to the primary toxins associated with PSP and DSP in humans, and that PTX2 is acutely toxic to larval oysters. Full article

Chromatin structure is a major regulator of DNA-associated processes, such as transcription, DNA repair, and replication. Histone post-translational modifications, or PTMs, play a key role on chromatin dynamics. PTMs are involved in a wide range of biological processes in eukaryotes, including fungal species. Their deposition/removal and their underlying functions have been extensively investigated in yeasts but much less in other fungi. Nonetheless, the major role of histone PTMs in regulating primary and secondary metabolisms of filamentous fungi, including human and plant pathogens, has been pinpointed. In this review, an overview of major identified PTMs and their respective functions in fungi is provided, with a focus on filamentous fungi when knowledge is available. To date, most of these studies investigated histone acetylations and methylations, but the development of new methodologies and technologies increasingly allows the wider exploration of other PTMs, such as phosphorylation, ubiquitylation, sumoylation, and acylation. Considering the increasing number of known PTMs and the full range of their possible interactions, investigations of the subsequent Histone Code, i.e., the biological consequence of the combinatorial language of all histone PTMs, from a functional point of view, are exponentially complex. Better knowledge about histone PTMs would make it possible to efficiently fight plant or human contamination, avoid the production of toxic secondary metabolites, or optimize the industrial biosynthesis of certain beneficial compounds. Full article

The Australasian region is home to the most diverse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared to elapids on other continents; however, their venom compositions are poorly known. The Australian elapid Hoplocephalus stephensii (Stephen’s banded snake) is an arboreal snake with a unique morphology. Human envenoming results in venom-induced consumption coagulopathy, without neurotoxicity. Using transcriptomics and a multi-step fractionation method involving reverse-phase high-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and bottom-up proteomics, we characterized the venom proteome of H. stephensii. 92% of the total protein component of the venom by weight was characterized, and included all dominant protein families and 4 secondary protein families. Eighteen toxins made up 76% of the venom, four previously characterized and 14 new toxins. The four dominant protein families made up 77% of the venom, including snake venom metalloprotease (SVMP; 36.7%; three identified toxins), phospholipase A₂ (PLA₂; 24.0%; five identified toxins), three-finger toxin (3FTx; 10.2%; two toxins) and snake venom serine protease (SVSP; 5.9%; one toxin; Hopsarin). Secondary protein families included L-amino acid oxidase (LAAO; 10.8%; one toxin), natriuretic peptide (NP; 0.8%; two toxins), cysteine-rich secretory protein (CRiSP; 1.7%; two toxins), c-type lectin (CTL; 1.1%; one toxin), and one minor protein family, nerve growth factor (NGF; 0.8%; one toxin). The venom composition of H. stephensii differs to other elapids, with a large proportion of SVMP and LAAO, and a relatively small amount of 3FTx. H. stephensii venom appeared to have less toxin diversity than other elapids, with only 18 toxins making up three-quarters of the venom. Full article

Over recent decades, the Norwegian cereal industry has had major practical and financial challenges associated with the occurrence of Fusarium head blight (FHB) pathogens and their associated mycotoxins in cereal grains. Deoxynivalenol (DON) is one of the most common Fusarium-mycotoxins in Norwegian oats, however T-2 toxin (T2) and HT-2 toxin (HT2) are also commonly detected. The aim of our study was to rank Nordic spring oat varieties and breeding lines by content of the most commonly occurring Fusarium mycotoxins (DON and HT2 + T2) as well as by the DNA content of their respective producers. We analyzed the content of mycotoxins and DNA of seven fungal species belonging to the FHB disease complex in grains of Nordic oat varieties and breeding lines harvested from oat field trials located in the main cereal cultivating district in South-East Norway in the years 2011–2020. Oat grains harvested from varieties with a high FHB resistance contained on average half the levels of mycotoxins compared with the most susceptible varieties, which implies that choice of variety may indeed impact on mycotoxin risk. The ranking of oat varieties according to HT2 + T2 levels corresponded with the ranking according to the DNA levels of Fusarium langsethiae, but differed from the ranking according to DON and Fusarium graminearum DNA. Separate tests are therefore necessary to determine the resistance towards HT2 + T2 and DON producers in oats. This creates practical challenges for the screening of FHB resistance in oats as today’s screening focuses on resistance to F. graminearum and DON. We identified oat varieties with generally low levels of both mycotoxins and FHB pathogens which should be preferred to mitigate mycotoxin risk in Norwegian oats. Full article

Crop diseases caused by Fusarium graminearum threaten crop production in both commercial and smallholder farming. F. graminearum produces deoxynivalenol mycotoxin, which is stable during food and feed processing. Therefore, the best way to prevent the sporulation of pathogens is to develop new prevention strategies. Plant-based pesticides, i.e., natural fungicides, have recently gained interest in crop protection as alternatives to synthetic fungicides. Herein we show that treatment with the methanolic extract of medicinal plant Zanthoxylum bungeanum (M20 extract), decreased F. graminearum growth and abrogated DON production. The F. graminearum DNA levels were monitored by a quantitative TaqMan real-time PCR, while DON accumulation was assessed by HPLC quantification. This M20 extract was mainly composed of four flavonoids: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The in vitro bioassay, which measured the percent inhibition of fungal growth, showed that co-inoculation of four F. graminearum strains with the M20 extract inhibited the fungal growth up to 48.5%. After biocontrol treatments, F. graminearum DNA level was reduced up to 85.5% compared to that of wheat heads, which received F. graminearum mixture only. Moreover, DON production was decreased in wheat heads by 73% after biocontrol treatment; meanwhile in wheat heads inoculated with F. graminearum conidia, an average of 2.263 ± 0.8 mg/kg DON was detected. Overall, this study is a successful case from in vitro research to in planta, giving useful information for wheat protection against F. graminearum responsible for Fusarium Head Blight and DON accumulation in grains. Further studies are needed to study the mechanism by which M20 extract inhibited the DON production and what changes happened to the DON biosynthetic pathway genes. Full article

Despite coagulotoxicity being a primary weapon for prey capture by Bothrops species (lancehead pit vipers) and coagulopathy being a major lethal clinical effect, a genus-wide comparison has not been undertaken. To fill this knowledge gap, we used thromboelastography to compare 37 venoms, from across the full range of geography, taxonomy, and ecology, for their action upon whole plasma and isolated fibrinogen. Potent procoagulant toxicity was shown to be the main venom effect of most of the species tested. However, the most basal species (B. pictus) was strongly anticoagulant; this is consistent with procoagulant toxicity being a novel trait that evolved within Bothrops subsequent to their split from anticoagulant American pit vipers. Intriguingly, two of the arboreal species studied (B. bilineatus and B. taeniatus) lacked procoagulant venom, suggesting differential evolutionary selection pressures. Notably, some terrestrial species have secondarily lost the procoagulant venom trait: the Mogi Mirim, Brazil locality of B. alternatus; San Andres, Mexico locality of B. asper; B. diporus; and the São Roque of B. jararaca. Direct action on fibrinogen was extremely variable; this is consistent with previous hypotheses regarding it being evolutionary decoupled due to procoagulant toxicity being the primary prey-capture weapon. However, human patients live long enough for fibrinogen depletion to be clinically significant. The extreme variability may be reflective of antivenom variability, with these results thereby providing a foundation for such future work of clinical relevance. Similarly, the venom diversification trends relative to ecological niche will also be useful for integration with natural history data, to reconstruct the evolutionary pressures shaping the venoms of these fascinating snakes. Full article

Patients with chronic kidney disease (CKD) have an elevated prevalence of atheromatous (ATH) and/or non-atheromatous (non-ATH) cardiovascular disease (CVD) due to an array of CKD-related risk factors, such as uremic toxins (UTs). Indeed, UTs have a major role in the emergence of a spectrum of CVDs, which constitute the leading cause of death in patients with end-stage renal disease. The European Uremic Toxin Work Group has identified over 100 UTs, more than 25 of which are dietary or gut-derived. Even though relationships between UTs and CVDs have been described in the literature, there are few reviews on the involvement of the most toxic compounds and the corresponding physiopathologic mechanisms. Here, we review the scientific literature on the dietary and gut-derived UTs with the greatest toxicity in vitro and in vivo. A better understanding of these toxins’ roles in the elevated prevalence of CVDs among CKD patients might facilitate the development of targeted treatments. Hence, we review (i) ATH and non-ATH CVDs and the respective levels of risk in patients with CKD and (ii) the mechanisms that underlie the influence of dietary and gut-derived UTs on CVDs. Full article

Despite antivenoms being the only established specific treatment for neuromuscular paralysis arising from snake envenoming, their ability to reverse the post-synaptic neurotoxicity in snake envenoming is poorly understood. We investigated the ability of five commercial antivenoms i.e., King cobra monovalent, Thai cobra monovalent, Thai neuro polyvalent, Indian polyvalent and Australian polyvalent antivenoms to reverse neurotoxicity induced by the venoms of King cobra (Ophiophagus hannah, 3 µg/mL), Indian cobra (Naja naja, 5 µg/mL) and Thai cobra (Naja kaouthia, 3 µg/mL) using the in vitro chick-biventer cervicis nerve–muscle preparation. All three venoms displayed post-synaptic neurotoxicity, which was prevented by all tested antivenoms (40 µL/mL) added to the bath prior to venom. All antivenoms partially reversed the established post-synaptic neuromuscular block after the addition of the three venoms during a 180 min observation period, but to varying degrees and at different rates. The neurotoxic effects of O. hannah venom recovered to a greater magnitude (based on twitch height restoration) and faster than the neurotoxicity of N. kaouthia venom, which recovered to a lower magnitude more slowly. The recovery of post-synaptic neurotoxicity by N. naja venom was hindered due to the likely presence of cytotoxins in the venom, which cause direct muscle damage. The observations made in this study provide further evidence that the commercial antivenoms are likely to actively reverse established α-neurotoxin-mediated neuromuscular paralysis in snake envenoming, and there is cross-neutralisation with different antivenoms. Full article

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

The corn earworm/bollworm, Helicoverpa zea (Boddie), is a pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1Ab and Vip3Aa20 are two common Bt toxins that are expressed in transgenic maize. The objective of this study was to determine the resistance allele frequency (RAF) to Cry1Ab and Vip3Aa20 in H. zea populations that were collected during 2018 and 2019 from four southeastern U.S. states: Louisiana, Mississippi, Georgia, and South Carolina. By using a group-mating approach, 104 F₂ iso-lines of H. zea were established from field collections with most iso-lines (85) from Louisiana. These F₂ iso-lines were screened for resistance alleles to Cry1Ab and Vip3Aa20, respectively. There was no correlation in larval survivorship between Cry1Ab and Vip3Aa20 when the iso-lines were exposed to these two toxins. RAF to Cry1Ab maize was high (0.256) and the RAFs were similar between Louisiana and the other three states and between the two sampling years. In contrast, no functional major resistance allele (RA) that allowed resistant insects to survive on Vip3Aa20 maize was detected and the expected RAF of major RAs with 95% probability was estimated to 0 to 0.0073. However, functional minor RAs to Vip3Aa20 maize were not uncommon; the estimated RAF for minor alleles was 0.028. The results provide further evidence that field resistance to Cry1Ab maize in H. zea has widely occurred, while major RAs to Vip3Aa20 maize are uncommon in the southeastern U.S. region. Information that was generated from this study should be useful in resistance monitoring and refinement of resistance management strategies to preserve Vip3A susceptibility in H. zea. Full article

Ten Amaryllidaceae alkaloids (AAs) were isolated for the first time from Pancratium maritimum collected in Calabria region, Italy. They belong to different subgroups of this family and were identified as lycorine, which is the main alkaloid, 9-O-demethyllycorine, haemanthidine, haemanthamine, 11-hydroxyvittatine, homolycorine, pancracine, obliquine, tazettine and vittatine. Haemanthidine was isolated as a scalar mixture of two 6-epimers, as already known also for other 6-hydroxycrinine alkaloids, but for the first time they were separated as 6,11-O,O′-di-p-bromobenzoyl esters. The evaluation of the cytotoxic and antiviral potentials of all isolated compounds was undertaken. Lycorine and haemanthidine showed cytotoxic activity on Hacat cells and A431 and AGS cancer cells while, pancracine exhibited selective cytotoxicity against A431 cells. We uncovered that in addition to lycorine and haemanthidine, haemanthamine and pancracine also possess antiretroviral abilities, inhibiting pseudotyped human immunodeficiency virus (HIV)−1 with EC50 of 25.3 µM and 18.5 µM respectively. Strikingly, all the AAs isolated from P. maritimum were able to impede dengue virus (DENV) replication (EC₅₀ ranged from 0.34–73.59 µM) at low to non-cytotoxic concentrations (CC₅₀ ranged from 6.25 µM to >100 µM). Haemanthamine (EC50 = 337 nM), pancracine (EC₅₀ = 357 nM) and haemanthidine (EC₅₀ = 476 nM) were the most potent anti-DENV inhibitors. Thus, this study uncovered new antiviral properties of P. maritimum isolated alkaloids, a significant finding that could lead to the development of new therapeutic strategies to fight viral infectious diseases. Full article

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

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

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

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

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

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

Sodium–glucose cotransporter (SGLT) inhibitors are a class of oral hypoglycemic agents, which, in recent years, have been shown to improve renal and cardiovascular outcomes in patients with diabetic and non-diabetic chronic kidney disease. There remains considerable debate regarding the potential glucose-independent mechanisms by which these benefits are conferred. SGLT inhibitors, to a variable extent, impair small intestinal glucose absorption, facilitating the delivery of glucose into the colon. This suppresses protein fermentation, and thus the generation of uremic toxins such as phenols and indoles. It is acknowledged that such a shift in gut microbial metabolism yields health benefits for the host. SGLT inhibition, in addition, may be hypothesized to foster the renal clearance of protein-bound uremic toxins. Altered generation and elimination of uremic toxins may be in the causal pathway between SGLT inhibition and improved cardiometabolic health. Present review calls for additional research. Full article

Pertussis, also known as whooping cough, is a respiratory disease caused by infection with Bordetella pertussis, which releases several virulence factors, including the AB-type pertussis toxin (PT). The characteristic symptom is severe, long-lasting paroxysmal coughing. Especially in newborns and infants, pertussis symptoms, such as leukocytosis, can become life-threatening. Despite an available vaccination, increasing case numbers have been reported worldwide, including Western countries such as Germany and the USA. Antibiotic treatment is available and important to prevent further transmission. However, antibiotics only reduce symptoms if administered in early stages, which rarely occurs due to a late diagnosis. Thus, no causative treatments against symptoms of whooping cough are currently available. The AB-type protein toxin PT is a main virulence factor and consists of a binding subunit that facilitates transport of an enzyme subunit into the cytosol of target cells. There, the enzyme subunit ADP-ribosylates inhibitory α-subunits of G-protein coupled receptors resulting in disturbed cAMP signaling. As an important virulence factor associated with severe symptoms, such as leukocytosis, and poor outcomes, PT represents an attractive drug target to develop novel therapeutic strategies. In this review, chaperone inhibitors, human peptides, small molecule inhibitors, and humanized antibodies are discussed as novel strategies to inhibit PT. Full article

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

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

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

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

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

Chronic kidney disease (CKD) is generally regarded as a final common pathway of several renal diseases, often leading to end-stage kidney disease (ESKD) and a need for renal replacement therapy. Estimated GFR (eGFR) has been used to predict this outcome recognizing its robust association with renal disease progression and the eventual need for dialysis in large, mainly cross-sectional epidemiological studies. However, GFR is implicitly limited as follows: (1) GFR reflects only one of the many physiological functions of the kidney; (2) it is dependent on several non-renal factors; (3) it has intrinsic variability that is a function of dietary intake, fluid and cardiovascular status, and blood pressure especially with impaired autoregulation or medication use; (4) it has been shown to change with age with a unique non-linear pattern; and (5) eGFR may not correlate with GFR in certain conditions and disease states. Yet, many clinicians, especially our non-nephrologist colleagues, tend to regard eGFR obtained from a simple laboratory test as both a valid reflection of renal function and a reliable diagnostic tool in establishing the diagnosis of CKD. What is the validity of these beliefs? This review will critically reassess the limitations of such single-focused attention, with a particular focus on inter-individual variability. What does science actually tell us about the usefulness of eGFR in diagnosing CKD? Full article

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

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

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

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

Fusarium is a species-rich group of mycotoxigenic plant pathogens that ranks as one of the most economically important fungal genera in the world. During growth and infection, they are able to produce a vast spectrum of low-molecular-weight compounds, so-called secondary metabolites (SMs). SMs often comprise toxic compounds (i.e., mycotoxins) that contaminate precious food and feed sources and cause adverse health effects in humans and livestock. In this context, understanding the regulation of their biosynthesis is crucial for the development of cropping strategies that aim at minimizing mycotoxin contamination in the field. Nevertheless, currently, only a fraction of SMs have been identified, and even fewer are considered for regular monitoring by regulatory authorities. Limitations to exploit their full chemical potential arise from the fact that the genes involved in their biosynthesis are often silent under standard laboratory conditions and only induced upon specific stimuli mimicking natural conditions in which biosynthesis of the respective SM becomes advantageous for the producer. This implies a complex regulatory network. Several components of these gene networks have been studied in the past, thereby greatly advancing the understanding of SM gene regulation and mycotoxin biosynthesis in general. This review aims at summarizing the latest advances in SM research in these notorious plant pathogens with a focus on chromatin structure. Full article

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

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

Fusarium spp. are ubiquitous fungi able to cause Fusarium head blight and Fusarium foot and root rot on wheat. Among relevant pathogenic species, Fusarium graminearum and Fusarium culmorum cause significant yield and quality loss and result in contamination of the grain with mycotoxins, mainly type B trichothecenes, which are a major health concern for humans and animals. Phenolic compounds of natural origin are being increasingly explored as fungicides on those pathogens. This review summarizes recent research activities related to the antifungal and anti-mycotoxigenic activity of natural phenolic compounds against Fusarium, including studies into the mechanisms of action of major exogenous phenolic inhibitors, their structure-activity interaction, and the combined effect of these compounds with other natural products or with conventional fungicides in mycotoxin modulation. The role of high-throughput analysis tools to decipher key signaling molecules able to modulate the production of mycotoxins and the development of sustainable formulations enhancing potential inhibitors’ efficacy are also discussed. Full article

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

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

The syndrome of uremic toxicity comprises a complex toxic milieu in-vivo, as numerous uremic substances accumulate and harm the organ systems. Among these substances, toxic and non-toxic players differently interfere with human cells. However, results from animal experiments are not always compatible with the expected reactions in human patients and studies on one organ system are limited in capturing the complexity of the uremic situation. In this narrative review, we present aspects relevant for cellular toxicity research based on our previous establishment of a human spermatozoa-based cell model, as follows: (i) applicability to compare the effects of more than 100 uremic substances, (ii) detection of the protective effects of uremic substances by the cellular responses towards the uremic milieu, (iii) inclusion of the drug milieu for cellular function, and (iv) transferability for clinical application, e.g., hemodialysis. Our technique allows the estimation of cell viability, vitality, and physiological state, not only restricted to acute or chronic kidney toxicity but also for other conditions, such as intoxications of unknown substances. The cellular models can clarify molecular mechanisms of action of toxins related to human physiology and therapy. Identification of uremic toxins retained during acute and chronic kidney injury enables further research on the removal or degradation of such products. Full article

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

We performed a systematic review and meta-analysis of randomised controlled trials (RCTs) conducted from January 2005 to June 2021 to update the evidence of Botulinum toxin A (BoNT-A) in neuropathic pain (NP) in addition to quality of life (QOL), mental health, and sleep outcomes. We conducted a Cochrane Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria analysis of RCTs from the following data sources: EMBASE, CINAHL, WHO International Clinical Trial Registry Platform, ClinicalTrials.gov, Cochrane database, Cochrane Clinical Trial Register, Australia New Zealand Clinical Trials Registry, and EU Clinical Trials Register. Meta-analysis of 17 studies showed a mean final VAS reduction in pain in the intervention group of 2.59 units (95% confidence interval: 1.79, 3.38) greater than the mean for the placebo group. The overall mean difference for sleep, Hospital Anxiety and Depression Scale (HADS) anxiety, HADS depression, and QOL mental and physical sub-scales were, respectively, 1.10 (95% CI: −1.71, 3.90), 1.41 (95% CI: −0.61, 3.43), −0.16 (95% CI: −1.95, 1.63), 0.85 (95% CI: −1.85, 3.56), and −0.71 (95% CI: −3.39, 1.97), indicating no significance. BoNT-A is effective for NP; however, small-scale RCTs to date have been limited in evidence. The reasons for this are discussed, and methods for future RCTs are developed to establish BoNT-A as the first-line agent. Full article

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

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

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

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

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

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