Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Characterization of a (p)ppApp Synthetase Belonging to a New Family of Polymorphic Toxin Associated with Temperate Phages.

Polymorphic toxins (PTs) are a broad family of toxins involved in interbacterial competition and pathogenesis. PTs are modular proteins that are comprised of a conserved N-terminal domain responsible for its transport, and a variable C-terminal domain bearing toxic activity. Although the mode of transport has yet to be elucidated, a new family of putative PTs containing an N-terminal MuF domain, resembling the Mu coliphage F protein, was identified in prophage genetic elements. The C-terminal toxin domains of these MuF PTs are predicted to bear nuclease, metallopeptidase, ADP-ribosyl transferase and RelA_SpoT activities. In this study, we characterized the MuF-RelA_SpoT toxin associated with the temperate phage of Streptococcus pneumoniae SPNA45. We show that the RelA_SpoT domain has (p)ppApp synthetase activity, which is bactericidal under our experimental conditions. We further determine that the two genes located downstream encode two immunity proteins, one binding to and inactivating the toxin and the other detoxifying the cell via a pppApp hydrolase activity. Finally, based on protein sequence alignments, we propose a signature for (p)ppApp synthetases that distinguishes them from (p)ppGpp synthetases.

In-situ synchrotron imaging, experimental, and computational investigations on the efficiency of Trametes versicolor laccase on detoxification of P-Cresyl Sulfate (PCS) Protein Bound Uremic Toxin (PBUT).

Protein bound uremic toxins (PBUTs) are small substances binding to larger proteins, mostly human serum albumin (HSA), and are challenging to remove by hemodialysis (HD). Among different classes of PBUTs, p-cresyl sulfate (PCS) is the most widely used marker molecule and major toxin, as 95 % is bound to HSA. PCS has a pro-inflammatory effect and increases both the uremia symptom score and multiple pathophysiological activities. High-flux HD to clear PCS leads to serious loss of HSA, which results in a high mortality rate. The goal of the present study is to investigate the efficacy of PCS detoxification in serum of HD patients using a biocompatible laccase enzyme from Trametes versicolor. Molecular docking was used to gain an in-depth understanding of the interactions between PCS and the laccase to identify the functional group(s) responsible for ligand-protein receptor interactions. UV-Vis spectroscopy and gas chromatography-mass spectrometry (GC-MS) were used to assess the detoxification of PCS. GC-MS was used to identify the detoxification byproducts and their toxicity was assessed using docking commutations. In situ synchrotron radiation micro-computed tomography (SR-µCT) imaging available at the Canadian Light Source (CLS) was conducted to assess HSA binding with PCS before and after detoxification with laccase and undertake the corresponding quantitative analysis. GC-MS analyses confirmed the detoxification of PCS with laccase at a concentration of 500 mg/L. The potential pathway of PCS detoxification in the presence of the laccase was identified. Increasing laccase concentration led to the formation of m-cresol, as indicated by the corresponding absorption in the UV-Vis spectra and a sharp peak on the GC-MS spectra. Our analysis provides insight into the general features of PCS binding on Sudlow site II, as well as insights into PCS detoxification product interactions. The average affinity energy for detoxification products was lower than that of PCS. Even though some byproducts showed potential toxicity, the level was lower than for PCS based on toxicity indexes (e.g., LD50/LC50, carcinogenicity, neurotoxicity, mutagenicity). In addition, these small compounds can also be more easily removed by HD compared to PCS. SR-µCT quantitative analysis showed adhesion of the HSA to a significant reduced extent in the presence of the laccase enzyme in bottom sections of the polyarylethersulfone (PAES) clinical HD membrane tested. Overall, this study opens new frontiers for PCS detoxification.

Toxic and antigenic characterization of Peruvian Micrurus surinamensis coral snake venom.

Micrurus surinamensis is a semi-aquatic coral snake found in primary forest region and can cause relevant human accidents. In this work we investigated the toxic and antigenic activities of the Peruvian Micrurus surinamensis venom (MsV). We found that MsV show hyaluronidase activity but lack LAAO and PLA2 enzymatic activities. Interestingly, MsV induce edematogenic responses but cannot cause nociceptive effects. Furthermore, MsV can reduce in vitro cell viability in MGSO-3 cell line derived from human breast cancer tissue. To evaluate its antigenic potential, rabbits were immunized with MsV, which proved to be immunogenic. ELISA, immunobloting and in vivo neutralization assays demonstrated that the specific rabbit anti-MsV antivenom is more efficient than the therapeutic Brazilian antivenom in recognizing and neutralizing the lethal activity of MsV. MsV differs in protein profile and biological activities from M. frontalis venom (MfV), used as control, which impairs its recognition and neutralization by Brazilian therapeutic anti-elapidic antivenom. We performed a SPOT immunoassay for the identification of B-cell linear epitopes in the main toxins described for MsV targeted by the elicited neutralizing antibodies previously produced. A membrane containing 15-mer peptides representing the sequences of five 3TFxs and five PLA2s was produced and probed with anti- MsV antibodies. Results revealed important regions in 3FTx toxins for venom neutralization. Identifying the main MsV components and its biological activities can be helpful in guiding the production of antivenoms and in the optimization of treatment for coral snake envenomation in Brazil.

PAT: a comprehensive database of prokaryotic antimicrobial toxins.

Antimicrobial toxins help prokaryotes win competitive advantages in intraspecific or interspecific conflicts and are also a critical factor affecting the pathogenicity of many pathogens that threaten human health. Although many studies have revealed that antagonism based on antimicrobial toxins plays a central role in prokaryotic life, a database on antimicrobial toxins remains lacking. Here, we present the prokaryotic antimicrobial toxin database (PAT, http://bioinfo.qd.sdu.edu.cn/PAT/), a comprehensive data resource collection on experimentally validated antimicrobial toxins. PAT has organized information, derived from the reported literature, on antimicrobial toxins, as well as the corresponding immunity proteins, delivery mechanisms, toxin activities, structural characteristics, sequences, etc. Moreover, we also predict potential antimicrobial toxins in prokaryotic reference genomes and show the taxonomic information and environmental distribution of typical antimicrobial toxins. These details have been fully incorporated into the PAT database, where users can browse, search, download, analyse and view informative statistics and detailed information. PAT resources have already been used in our prediction and identification of prokaryotic antimicrobial toxins and may contribute to promoting the efficient investigation of antimicrobial toxin functions, the discovery of novel antimicrobial toxins, and an improved understanding of the biological roles and significance of these toxins.

Pharmacological Screening of Venoms from Five Brazilian Micrurus Species on Different Ion Channels.

Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A2 and three-finger toxins, which are responsible for neuromuscular blockade-the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5' nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris, M. corallinus, M. frontalis, M. carvalhoi and M. decoratus. All five venoms induced full inhibition of the muscle-type α1ß1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1ß2 GABAAR. Interestingly, the screening on KV1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on NaV1.2 and NaV1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.

Neurotoxic and cytotoxic peptides underlie the painful stings of the tree nettle Urtica ferox.

The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.

A peptide toxin in ant venom mimics vertebrate EGF-like hormones to cause long-lasting hypersensitivity in mammals.

Venoms are excellent model systems for studying evolutionary processes associated with predator-prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.

Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease.

BACKGROUND: Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. METHODS: Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 â„ƒ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. RESULTS: DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. CONCLUSION: Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.

A Short Review of the Venoms and Toxins of Spider Wasps (Hymenoptera: Pompilidae).

Parasitoid wasps represent the plurality of venomous animals, but have received extremely little research in proportion to this taxonomic diversity. The lion's share of investigation into insect venoms has focused on eusocial hymenopterans, but even this small sampling shows great promise for the development of new active substances. The family Pompilidae is known as the spider wasps because of their reproductive habits which include hunting for spiders, delivering a paralyzing sting, and entombing them in burrows with one of the wasp's eggs to serve as food for the developing larva. The largest members of this family, especially the tarantula hawks of the genus Pepsis, have attained notoriety for their large size, dramatic coloration, long-term paralysis of their prey, and incredibly painful defensive stings. In this paper we review the existing research regarding the composition and function of pompilid venoms, discuss parallels from other venom literatures, identify possible avenues for the adaptation of pompilid toxins towards human purposes, and future directions of inquiry for the field.

Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens.

Bacterial human pathogens secrete initially inactive nucleotidyl cyclases that become potent enzymes by binding to actin inside eukaryotic host cells. The underlying molecular mechanism of this activation is, however, unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus bound to their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo-state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. Other nucleotidyl cyclase toxins that bind to calmodulin rather than actin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is conserved in these enzymes, albeit the different activator.

Characterization of Sialic Acid Affinity of the Binding Domain of Mistletoe Lectin Isoform One.

Sialic acid (Sia) is considered as one of the most important biomolecules of life since its derivatives and terminal orientations on cell membranes and macromolecules play a major role in many biological and pathological processes. To date, there is only a limited number of active molecules that can selectively bind to Sia and this limitation has made the study of this glycan challenging. The lectin superfamily is a well-known family of glycan binding proteins, which encompasses many strong glycan binding peptides with diverse glycan affinities. Mistletoe lectin (ML) is considered one of the most active members of lectin family which was initially classified in early studies as a galactose binding lectin; more recent studies have suggested that the peptide can also actively bind to Sia. However, the details with respect to Sia binding of ML and the domain responsible for this binding are left unanswered because no comprehensive studies have been instigated. In this study, we sought to identify the binding domain responsible for the sialic acid affinity of mistletoe lectin isoform I (MLI) in comparison to the binding activity of elderberry lectin isoform I (SNA), which has long been identified as a potent Sia binding lectin. In order to execute this, we performed computational carbohydrate-protein docking for MLB and SNA with Neu5Ac and ß-Galactose. We further analyzed the coding sequence of both lectins and identified their glycan binding domains, which were later cloned upstream and downstream to green fluorescent protein (GFP) and expressed in Escherichia coli (E. coli). Finally, the glycan affinity of the expressed fusion proteins was assessed by using different biochemical and cell-based assays and the Sia binding domains were identified.

Phosphorylation of the conserved C-terminal domain of ribosomal P-proteins impairs the mode of interaction with plant toxins.

The ribosome is subjected to post-translational modifications, including phosphorylation, that affect its biological activity. Among ribosomal elements, the P-proteins undergo phosphorylation within the C terminus, the element which interacts with trGTPases or ribosome-inactivating proteins (RIPs); however, the role of phosphorylation has never been elucidated. Here, we probed the function of phosphorylation on the interaction of P-proteins with RIPs using the ribosomal P1-P2 dimer. We determined the kinetic parameters of the interaction with the toxins using biolayer interferometry and microscale thermophoresis. The results present the first mechanistic insight into the function of P-protein phosphorylation, showing that introduction of a negative charge into the C terminus of P1-P2 proteins promotes α-helix formation and decreases the affinity of the P-proteins for the RIPs.

Chemical cues that attract cannibalistic cane toad (Rhinella marina) larvae to vulnerable embryos.

Chemical cues produced by late-stage embryos of the cane toad (Rhinella marina) attract older conspecific larvae, which are highly cannibalistic and can consume an entire clutch. To clarify the molecular basis of this attraction response, we presented captive tadpoles with components present in toad eggs. As previously reported, attractivity arises from the distinctive toxins (bufadienolides) produced by cane toads, with some toxins (e.g., bufagenins) much stronger attractants than others (e.g., bufotoxins). Extracts of frozen toad parotoid glands (rich in bufagenins) were more attractive than were fresh MeOH extracts of the parotoid secretion (rich in bufotoxins), and purified marinobufagin was more effective than marinobufotoxin. Cardenolide aglycones (e.g., digitoxigenin) were active attractors, whereas C-3 glycosides (e.g., digoxin, oubain) were far less effective. A structure-activity relationship study revealed that tadpole attractant potency strongly correlated with Na+/K+ ATPase inhibitory activity, suggesting that tadpoles monitor and rapidly react to perturbations to Na+/K+ ATPase activity.

Three-Finger Toxins from Brazilian Coral Snakes: From Molecular Framework to Insights in Biological Function.

Studies on 3FTxs around the world are showing the amazing diversity in these proteins both in structure and function. In Brazil, we have not realized the broad variety of their amino acid sequences and probable diversified structures and targets. In this context, this work aims to conduct an in silico systematic study on available 3FTxs found in Micrurus species from Brazil. We elaborated a specific guideline for this toxin family. First, we grouped them according to their structural homologue predicted by HHPred server and further curated manually. For each group, we selected one sequence and constructed a representative structural model. By looking at conserved features and comparing with the information available in the literature for this toxin family, we managed to point to potential biological functions. In parallel, the phylogenetic relationship was estimated for our database by maximum likelihood analyses and a phylogenetic tree was constructed including the homologous 3FTx previously characterized. Our results highlighted an astonishing diversity inside this family of toxins, showing some groups with expected functional similarities to known 3FTxs, and pointing out others with potential novel roles and perhaps structures. Moreover, this classification guideline may be useful to aid future studies on these abundant toxins.

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay.

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics.

MOTIVATION: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. RESULTS: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.

Targeting Toxins toward Tumors.

Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned.

Enhancing the power of liquid chromatography-Mass spectrometry for chemical fingerprinting of phytotoxins in the environment.

Phytotoxins are plant secondary metabolites. They have recently been considered as chemicals of emerging concern (CECs) and there is a growing interest in their environmental fate and potential threat to public health. Dedicated target and non-target screening (NTS) analysis of phytotoxins in environmental samples are sparse, meanwhile phytotoxins are rarely detected in NTS-based analysis due to lack of an efficient methodology. Development of new analytical measurement methods is therefore highly needed. In this study, we for the first time investigated key parameters of reversed phase liquid chromatography-high resolution mass spectrometry (RPLC-HRMS) for five major classes of phytotoxins (alkaloids, steroids, terpenoids, flavonoids and aromatic polyketides) in environmental matrices; the investigation included analytical conditions which have not yet been explored by others, e.g. ionization at alkaline pH above 9. As the outcome we established a new analytical method for target/suspect screening and NTS of phytotoxins in the environment, which significantly improved the detection sensitivity with up to 40 times compared to previous methods, and enabled the discovery of over 30 phytotoxins in a NTS-based environmental study. We also observed that the negative ionization of phenols could be facilitated by the number of hydroxyl groups on the ring rather than their position of substitution. This study is of interest for a better fundamental understanding of the behavior of phytotoxins in LC-MS. Dedicated target/suspect screening and NTS methods will facilitate a better risk characterization of phytotoxins in the environment and stimulate implementation of new public regulation on phytotoxins.