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Amanita poisoning is one of the most deadly types of mushroom poisoning. α-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for α-amanitin require expensive instruments. In this study, the α-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for α-amanitin. The detection limit was 0.369 μg/mL, while, in mushroom it was 0.372 μg/mL and in urine 0.337 μg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the α-amanitin aptamer and proposes a method of detecting α-amanitin.

The consumption of mushrooms has become increasingly popular, partly due to their nutritional and medicinal properties. This has increased the risk of confusion during picking, and thus of intoxication. In France, about 1300 cases of intoxication are observed each year, with deaths being mostly attributed to Amanita phalloides poisoning. Among amatoxins, α- and β-amanitins are the most widely studied toxins. Hepatotoxicity is the hallmark of these compounds, leading to hepatocellular failure within three days of ingestion. The toxic mechanisms of action mainly include RNA polymerase II inhibition and oxidative stress generation, leading to hepatic cell apoptosis or necrosis depending on the doses ingested. Currently, there is no international consensus concerning Amanita phalloides poisoning management. However, antidotes with antioxidant properties remain the most effective therapeutics to date suggesting the predominant role of oxidative stress in the pathophysiology. The partially elucidated mechanisms of action may reveal a suitable target for the development of an antidote. The aim of this review is to present an overview of the knowledge on amanitins, including the latest advances that could allow the proposal of new innovative and effective therapeutics.

Genomic deletion of the tumour suppressor TP53 frequently includes other neighbouring genes, such as the POLR2A housekeeping gene that encodes a crucial RNA polymerase II subunit; suppression of POLR2A with α-amanitin or by RNA interference selectively inhibits the tumorigenic potential of cancer cells, and in mouse models of cancer, tumours can be selectively targeted with α-amanitin coupled to antibodies, suggesting new therapeutic approaches for human cancers. Indirect targeting of TP53 The tumour suppressor gene TP53 is inactivated by mutation or deletion in a majority of human tumours. So far, attempts to restore the activity of its product, p53, have had little success owing to the complexity of p53 signalling. This paper suggests a new approach to targeting TP53 indirectly. Genomic deletion of TP53 frequently includes other neighbouring genes, such as the POLR2A housekeeping gene that encodes a crucial RNA polymerase II subunit. Xionbin Lu and colleagues show that loss of one copy of POLR2A renders cancer cells highly sensitive to inhibitors of RNA polymerase II, such as α-amanitin. In mouse models of cancer, tumours containing the POLR2A / TP53 co-deletion can be selectively targeted with α-amanitin conjugated to antibodies that target the cancer cells. Exploiting similar selective vulnerabilies for other genomic deletions that affect essential housekeeping in addition to tumour suppressor genes may pave a way towards selective therapies for a broad range of cancers. TP53 , a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours 1 , 2 . A tremendous effort has been made to restore p53 activity in cancer therapies 3 , 4 , 5 , 6 , 7 . However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin 8 , 9 . Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity 10 . However, we found that α-amanitin-based antibody–drug conjugates are highly effective therapeutic agents with reduced toxicity 11 . Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A . We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Among the toxic metabolites of the fungal world, those that, due to their strong biological effect, can seriously (even fatally) damage the life processes of humans (and certain groups of animals) stand out. Amatoxin-containing mushrooms and the poisonings caused by them stand out from the higher fungi, the mushrooms. There are already historical data and records about such poisonings, but scientific research on the responsible molecules began in the middle of the last century. The goals of this review work are as follows: presentation of the cosmopolitan mushroom species that produce amanitins (which are known from certain genera of four mushroom families), an overview of the chemical structure and specific properties of amanitins, a summary of the analytical methods applicable to them, a presentation of the “medical history” of poisonings, and a summary of the therapeutic methods used so far. The main responsible molecules (the amanitins) are bicyclic octapeptides, whose structure is characterized by an outer loop and an inner loop (bridge). It follows from the unusual properties of amanitins, especially their extreme stability (against heat, the acidic pH of the medium, and their resistance to human, and animal, digestive enzymes), that they are absorbed almost without hindrance and quickly transported to our vital organs. Adding to the problems is that accidental consumption causes no noticeable symptoms for a few hours (or even 24–36 h) after consumption, but the toxins already damage the metabolism of the target organs and the synthesis of nucleic acid and proteins. The biochemical catastrophe of the cells causes irreversible structural changes, which lead to necrotic damage (in the liver and kidneys) and death. The scientific topicality of the review is due to the recent publication of new data on the probable antidote molecule (ICR: indocyanine green) against amanitins. Further research can provide a new foundation for the therapeutic treatment of poisonings, and the toxicological situation, which currently still poses a deadly threat, could even be tamed into a controllable problem. We also draw attention to the review conclusions, as well as the mycological and social tasks related to amanitin poisonings (prevention of poisonings).

Amanita poisoning has a high mortality rate. The α-amanitin toxin in Amanita is the main lethal toxin. There is no specific detoxification drug for α-amanitin, and the clinical treatment mainly focuses on symptomatic and supportive therapy. The pathogenesis of α-amanitin mainly includes: α-amanitin can inhibit the activity of RNA polymeraseII in the nucleus, including the inhibition of the largest subunit of RNA polymeraseII, RNApb1, bridge helix, and trigger loop. In addition, α-amanitin acts in vivo through the enterohepatic circulation and transport system. α-Amanitin can cause the cell death. The existing mechanisms of cell damage mainly focus on apoptosis, oxidative stress, and autophagy. In addition to the pathogenic mechanism, α-amanitin also has a role in cancer treatment, which is the focus of current research. The mechanism of action of α-amanitin on the body is still being explored.

Deaths from the accidental ingestion of poisonous Amanita mushrooms occur every year due to the lack of a specific antidote against α-amanitin poisoning. Intervention and treatment can be promptly carried out to avoid serious consequences when the toxin can be effectively detected in whole blood before liver toxicity develops. Aptamers are molecular recognition units similar to antibodies, capable of specifically recognizing and detecting small molecules such as α-amanitin for which monoclonal antibodies are difficult to prepare. However, α-amanitin has a small molecular size and limited binding sites, which bring difficulties to aptamer selection. Moreover, achieving highly specific detection of α-amanitin in whole blood remains challenging due to the presence of potentially interfering components, such as human serum albumin (HSA). For these problems, we propose an aptamer selection method for small-molecule target α-amanitin, combining target-immobilized and library-immobilized SELEX to select high-affinity aptamers. To exclude HSA interference, counter-selection was introduced to remove HSA-bound sequences. Through these strategies, we successfully selected a highly specific α-amanitin aptamer with nanomolar affinity.

α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of isomers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P -ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryptorubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides. The occurrence of isomers of the bicyclic octapeptide α-amanitin, which presents a macrolactam with a tryptathionine cross-link forming a handle, has been reported under the term of atropoisomers. Here, the authors synthesize its analogs and analyse their isomers, proposing and describing for them the term ansamer.

Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable, and capable of modulating protein–protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the 25 amino-acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino-acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal eight residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology. Cyclic peptide macrocycles are promising anti-cancer and antimicrobial molecules. Here, the authors characterize the structure and catalytic mechanism of the prolyl oligopeptidase B from Basidiomycete fungi, showing that its dual macrocyclase-peptidase activity is crucial for amatoxin macrocyclization.

Introduction: The consumption of mushrooms (Basidiomycota) in Poland is one of the highest in Europe. It is particularly high in the 3rd quarter of each year, which is accompanied by an increase in the number of mushroom poisonings. This study aims to present difficulties with microscopic identification, the most popular method for diagnosing mushroom poisoning in hospital settings, when it comes to detecting Amanita phalloides spores in biological material.Materials and methods: Spore analysis was carried out using aqueous solutions containing reference spores of different mushrooms: death cap (Amanita phalloides), parasol mushroom (Macrolepiota procera), field mushroom (Agaricus campestris), yellow knight mushroom (Tricholoma equestre), and green cracking russula (Russula virescens). The spore analysis was also carried out for a meal (soup) containing selected spores. Spores were identified using a light microscope and staining with Sudan III and Meltzer’s reagent. A statistical analysis of mushroom poisoning cases was also performed at the Department of Clinical and Forensic Toxicology, Pomeranian Medical University in Szczecin using records for 2015–2019.Conlusions: Analysis of data from 2015–2019 from the Department of Clinical and Forensic Toxicology at the Pomeranian Medical University in Szczecin showed a marked increase in mushroom poisoning cases in the 3rd quarter of each year. Analysis of materials containing Amanita phalloides spores revealed their high similarity to oil drops and other cell structures present in biological material, resulting in the low reliability of microscopic identification. Therefore, as the absence of Amanita phalloides spores in the tested biological material does not rule out poisoning with this mushroom, a more advanced instrumental analysis (ELISA, LC/MS) is recommended.

Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode α-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. α-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable \"toxin\" region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.