Preservation of vision after CaMKII-mediated protection of retinal ganglion cells.

Retinal ganglion cells (RGCs) are the sole output neurons that transmit visual information from the retina to the brain. Diverse insults and pathological states cause degeneration of RGC somas and axons leading to irreversible vision loss. A fundamental question is whether manipulation of a key regulator of RGC survival can protect RGCs from diverse insults and pathological states, and ultimately preserve vision. Here, we report that CaMKII-CREB signaling is compromised after excitotoxic injury to RGC somas or optic nerve injury to RGC axons, and reactivation of this pathway robustly protects RGCs from both injuries. CaMKII activity also promotes RGC survival in the normal retina. Further, reactivation of CaMKII protects RGCs in two glaucoma models where RGCs degenerate from elevated intraocular pressure or genetic deficiency. Last, CaMKII reactivation protects long-distance RGC axon projections in vivo and preserves visual function, from the retina to the visual cortex, and visually guided behavior.

Brain Chimeroids reveal individual susceptibility to neurotoxic triggers.

Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.

Neurotoxic reactive astrocytes induce cell death via saturated lipids.

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease1-6. Here we report an approach to isolate one component of the long-sought astrocyte-derived toxic factor5,6. Notably, instead of a protein, saturated lipids contained in APOE and APOJ lipoparticles mediate astrocyte-induced toxicity. Eliminating the formation of long-chain saturated lipids by astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 mitigates astrocyte-mediated toxicity in vitro as well as in a model of acute axonal injury in vivo. These results suggest a mechanism by which astrocytes kill cells in the central nervous system.

Molecular forecasting of domoic acid during a pervasive toxic diatom bloom.

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis, this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis (dab) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters (sit1) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that coexpression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have colimited the diatom population along with low Si. Iron limitation represents an overlooked driver of both toxin production and ecological success of the low-iron-adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms.

Breaking muscle: neurotoxic and myotoxic effects of Central American snake venoms and the relative efficacies of antivenom and varespladib.

BACKGROUND: The snake genera Atropoides, Cerrophidion, and Metlapilcoatlus form a clade of neotropical pit vipers distributed across Mexico and Central America. This study evaluated the myotoxic and neurotoxic effects of nine species of Atropoides, Cerrophidion, and Metlapilcoatlus, and the neutralising efficacy of the ICP antivenom from Costa Rica against these effects, in the chick biventer cervicis nerve-muscle preparation. Given the prominence of PLA2s within the venom proteomes of these species, we also aimed to determine the neutralising potency of the PLA2 inhibitor, varespladib. RESULTS: All venoms showed myotoxic and potential neurotoxic effects, with differential intra-genera and inter-genera potency. This variation was also seen in the antivenom ability to neutralise the muscle damaging pathophysiological effects observed. Variation was also seen in the relative response to the PLA2 inhibitor varespladib. While the myotoxic effects of M. mexicanus and M. nummifer venoms were effectively neutralised by varespladib, indicating myotoxicity is PLA2 mediated, those of C. godmani and M. olmec venoms were not, revealing that the myotoxicity is driven by non-PLA2 toxin types. CONCLUSIONS: This study characterises the myotoxic and neurotoxic venom activity, as well as neutralisation of venom effects from the Atropoides, Cerrophidion, and Metlapilcoatlus clade of American crotalids. Our findings contribute significant clinical and evolutionary knowledge to a clade of poorly researched snakes. In addition, these results provide a platform for future research into the reciprocal interaction between ecological niche specialisation and venom evolution, as well as highlighting the need to test purified toxins to accurately evaluate the potential effects observed in these venoms.

Toxic to the touch: The makings of lethal mantles in pitohui birds and poison dart frogs.

How do chemically defended animals resist their own toxins? This intriguing question on the concept of autotoxicity is at the heart of how species interactions evolve. In this issue of Molecular Ecology (Molecular Ecology, 2024, 33), Bodawatta and colleagues report on how Papua New Guinean birds coopted deadly neurotoxins to create lethal mantles that protect against predators and parasites. Combining chemical screening of the plumage of a diverse collection of passerine birds with genome sequencing, the researchers unlocked a deeper understanding of how some birds sequester deadly batrachotoxin (BTX) from their food without poisoning themselves. They identified that birds impervious to BTX bear amino acid substitutions in the toxin-binding site of the voltage-gated sodium channel Nav1.4, whose function is essential for proper contraction and relaxation of vertebrate muscles. Comparative genetic and molecular docking analyses show that several of the substitutions associated with insensitivity to BTX may have become prevalent among toxic birds through positive selection. Intriguingly, poison dart frogs that also co-opted BTX in their lethal mantles were found to harbour similar toxin insensitivity substitutions in their Nav1.4 channels. Taken together, this sets up a powerful model system for studying the mechanisms behind convergent molecular evolution and how it may drive biological diversity.

Impacts on Atlantic Killifish from Neurotoxicants: Genes, Behavior, and Population-Relevant Outcomes.

Molecular, cellular, and organismal alterations are important descriptors of toxic effects, but our ability to extrapolate and predict ecological risks is limited by the availability of studies that link measurable end points to adverse population relevant outcomes such as cohort survival and growth. In this study, we used laboratory gene expression and behavior data from two populations of Atlantic killifish Fundulus heteroclitus [one reference site (SCOKF) and one PCB-contaminated site (NBHKF)] to inform individual-based models simulating cohort growth and survival from embryonic exposures to environmentally relevant concentrations of neurotoxicants. Methylmercury exposed SCOKF exhibited brain gene expression changes in the si:ch211-186j3.6, si:dkey-21c1.4, scamp1, and klhl6 genes, which coincided with changes in feeding and swimming behaviors, but our models simulated no growth or survival effects of exposures. PCB126-exposed SCOKF had lower physical activity levels coinciding with a general upregulation in nucleic and cellular brain gene sets (BGS) and downregulation in signaling, nucleic, and cellular BGS. The NBHKF, known to be tolerant to PCBs, had altered swimming behaviors that coincided with 98% fewer altered BGS. Our models simulated PCB126 decreased growth in SCOKF and survival in SCOKF and NBHKF. Overall, our study provides a unique demonstration linking molecular and behavioral data to develop quantitative, testable predictions of ecological risk.

Inhibition of mitochondrial transcription by the neurotoxin MPP.

The neurotoxin MPP+ triggers cell death of dopamine neurons and induces Parkinson's disease symptoms in mice and men, but the immediate transcriptional response to this neurotoxin has not been studied. We therefore treated human SH-SY5Y cells with a low dose (0.1 mM) of MPP+ and measured the effect on nascent transcription by precision run-on sequencing (PRO-seq). We found that transcription of the mitochondrial genome was significantly reduced already after 30 min, whereas nuclear gene transcription was unaffected. Inhibition of respiratory complex I by MPP+ led to reduced ATP production, that may explain the diminished activity of mitochondrial RNA polymerase. Our results show that MPP+ has a direct effect on mitochondrial function and transcription, and that other gene expression or epigenetic changes induced by this neurotoxin are secondary effects that reflect a cellular adaptation program.

The role of neutrophil extracellular traps in ß-methylamino L-alanine-induced liver injury in mice.

The non-protein amino acid ß-N-methylamino-L-alanine (BMAA), produced by cyanobacteria, has been recognized as a neurotoxin. L-serine as an antagonist of BMAA can effectively alleviate BMAA-induced neurotoxicity. Although BMAA has long been emphasized as a neurotoxin, with the emergence of BMAA detected in a variety of algae in freshwater around the world and its clear biological enrichment effect, it is particularly important to study the non-neurotoxic adverse effects of BMAA. However, there is only limited evidence to support the ability of BMAA to cause oxidative damage in the liver. The exact molecular mechanism of BMAA-induced liver injury is still unclear. The formation of neutrophil extracellular traps (NETs) is a 'double-edged sword' for the organism, excessive formation of NETs is associated with inflammatory diseases of the liver. Our results innovatively confirmed that BMAA was able to cause the formation of NETs in the liver during the liver injury. The possible mechanism may associated with the regulation of ERK/p38 and cGAS/STING signaling pathways. The massive formation of NETs was able to exacerbate the BMAA-induced oxidative stress and release of inflammatory factors in the mice liver. And the removal of NETs could alleviate this injury. This article will bring a new laboratory evidence for BMAA-induced non-neurotoxicity and immunotoxicity.

Insecticidal activity of the spider neurotoxin PPTX-04 through modulating insect voltage-gated sodium channel.

Ion channels on cell membrane are molecular targets of more than half peptide neurotoxins from spiders. From Pardosa pseudoannulata, a predatory spider on a range of insect pests, we characterized a peptide neurotoxin PPTX-04 with an insecticidal activity. PPTX-04 showed high toxicity to Nilaparvata lugens, a main prey of P. pseudoannulata, and the toxicity was not affected by the resistance to etofenprox (IUPAC chemical name:1-ethoxy-4-[2-methyl-1-[(3-phenoxyphenyl)methoxy]propan-2-yl]benzene, purity: 99%). On N. lugens voltage-gated sodium channel NlNav1 expressed in Xenopus oocytes, PPTX-04 prolonged the channel opening and induced tail currents, which is similar to pyrethroid insecticides. However, PPTX-04 potency on NlNav1 was not affected by mutations conferring pyrethroid resistance in insects, which revealed that PPTX-04 and pyrethroids should act on different receptors in NlNav1. In contrast, two mutations at the extracellular site 4 significantly reduced PPTX-04 potency, which indicated that PPTX-04 would act on a potential receptor containing the site 4 in NlNav1. The result from the molecular docking supported the conclusion that the binding pocket of PPTX-04 in NlNav1 should contain the site 4. In summary, PPTX-04 had high insecticidal activity through acting on a distinct receptor site in insect Nav, and was a potential resource to control insect pests and manage resistance to pyrethroids.

Neurotoxicology: a clinical systems-based review.

Neurological disease caused by toxins is widespread but under-recognised. Despite increasing public interest and a growing number of novel potential neurotoxins, diagnosis of neurotoxic disease is often delayed or missed, resulting in poorer patient outcomes. This article discusses neurotoxic syndromes using a systems-based approach, focusing on environmental and occupational agents. We do not discuss recreational drugs, pharmaceutical agents or developmental neurotoxins in detail. We aim to provide neurologists with a working understanding of the scenarios in which a clinical presentation may be due to a neurotoxin and how to approach confirmation of the diagnosis.

An Overview of the Pathophysiological Mechanisms of 3-Nitropropionic Acid (3-NPA) as a Neurotoxin in a Huntington's Disease Model and Its Relevance to Drug Discovery and Development.

Animal models are used to better understand the various mechanisms involved in the pathogenesis of diseases and explore potential pathways that will aid in discovering therapeutic targets. 3-Nitropropionic Acid (3-NPA) is a neurotoxin used to induce Huntington's disease (HD)-like symptoms in experimental animals. The 3-NPA is a fungus toxin that impairs the complex II (succinate dehydrogenase) activity of the mitochondria and reduces ATP synthesis, leading to excessive production of free radicals resulting in the degeneration of GABAergic medium spiny neurons (MSNs) in the striatum. This is characterized by motor impairments a key clinical manifestation of HD. 3-NPA has the potential to alter several cellular processes, including mitochondrial functions, oxidative stress, apoptosis, and neuroinflammation mimicking HD-like pathogenic conditions in animals. This review strives to provide a new insight towards the 3-NPA induced molecular dysfunctioning in developing an animal model of HD. Moreover, we summarise several preclinical studies that support the use of the 3-NPA-induced models for drug discovery and development in HD. This review is a collection of various articles that were published from 1977 to 2022 on Pubmed (1639), Web of Science (2139), and Scopus (2681), which are related to the 3-NPA induced animal model.

SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance.

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 µM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

Making the invisible visible: Using a qualitative system dynamics model to map disparities in cumulative environmental stressors and children's neurodevelopment.

BACKGROUND: The combined effects of multiple environmental toxicants and social stressor exposures are widely recognized as important public health problems, likely contributing to health inequities. However, US policy makers at state and federal levels typically focus on one stressor exposure at a time and have failed to develop comprehensive strategies to reduce multiple co-occurring exposures, mitigate cumulative risks and prevent harm. This research aimed to move from considering disparate environmental stressors in isolation to mapping the links between environmental, economic, social and health outcomes as a dynamic complex system using children's exposure to neurodevelopmental toxicants as an illustrative example. Such a model can be used to support a broad range of child developmental and environmental health policy stakeholders in improving their understanding of cumulative effects of multiple chemical, physical, biological and social environmental stressors as a complex system through a collaborative learning process. METHODS: We used system dynamics (SD) group model building to develop a qualitative causal theory linking multiple interacting streams of social stressors and environmental neurotoxicants impacting children's neurodevelopment. A 2 1/2-day interactive system dynamics workshop involving experts across multiple disciplines was convened to develop the model followed by qualitative survey on system insights. RESULTS: The SD causal map covered seven interconnected themes: environmental exposures, social environment, health status, education, employment, housing and advocacy. Potential high leverage intervention points for reducing disparities in children's cumulative neurotoxicant exposures and effects were identified. Workshop participants developed deeper level of understanding about the complexity of cumulative environmental health risks, increased their agreement about underlying causes, and enhanced their capabilities for integrating diverse forms of knowledge about the complex multi-level problem of cumulative chemical and non-chemical exposures. CONCLUSION: Group model building using SD can lead to important insights to into the sociological, policy, and institutional mechanisms through which disparities in cumulative impacts are transmitted, resisted, and understood.

Highly infectious prions are not directly neurotoxic.

Prions are infectious agents which cause rapidly lethal neurodegenerative diseases in humans and animals following long, clinically silent incubation periods. They are composed of multichain assemblies of misfolded cellular prion protein. While it has long been assumed that prions are themselves neurotoxic, recent development of methods to obtain exceptionally pure prions from mouse brain with maintained strain characteristics, and in which defined structures-paired rod-like double helical fibers-can be definitively correlated with infectivity, allowed a direct test of this assertion. Here we report that while brain homogenates from symptomatic prion-infected mice are highly toxic to cultured neurons, exceptionally pure intact high-titer infectious prions are not directly neurotoxic. We further show that treatment of brain homogenates from prion-infected mice with sodium lauroylsarcosine destroys toxicity without diminishing infectivity. This is consistent with models in which prion propagation and toxicity can be mechanistically uncoupled.

Biotin rescues mitochondrial dysfunction and neurotoxicity in a tauopathy model.

Mitochondrial and metabolic dysfunction are often implicated in neurological disease, but effective mechanism-based therapies remain elusive. We performed a genome-scale forward genetic screen in a Drosophila model of tauopathy, a class of neurodegenerative disorders characterized by the accumulation of the protein tau, and identified manipulation of the B-vitamin biotin as a potential therapeutic approach in tauopathy. We show that tau transgenic flies have an innate biotin deficiency due to tau-mediated relaxation of chromatin and consequent aberrant expression of multiple biotin-related genes, disrupting both carboxylase and mitochondrial function. Biotin depletion alone causes mitochondrial pathology and neurodegeneration in both flies and human neurons, implicating mitochondrial dysfunction as a mechanism in biotin deficiency. Finally, carboxylase biotin levels are reduced in mammalian tauopathies, including brains of human Alzheimer's disease patients. These results provide insight into pathogenic mechanisms of human biotin deficiency, the resulting effects on neuronal health, and a potential therapeutic pathway in the treatment of tau-mediated neurotoxicity.

Fifty Years of Animal Toxin Research at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS.

This review covers briefly the work carried out at our institute (IBCh), in many cases in collaboration with other Russian and foreign laboratories, for the last 50 years. It discusses the discoveries and studies of various animal toxins, including protein and peptide neurotoxins acting on the nicotinic acetylcholine receptors (nAChRs) and on other ion channels. Among the achievements are the determination of the primary structures of the α-bungarotoxin-like three-finger toxins (TFTs), covalently bound dimeric TFTs, glycosylated cytotoxin, inhibitory cystine knot toxins (ICK), modular ICKs, and such giant molecules as latrotoxins and peptide neurotoxins from the snake, as well as from other animal venoms. For a number of toxins, spatial structures were determined, mostly by 1H-NMR spectroscopy. Using this method in combination with molecular modeling, the molecular mechanisms of the interactions of several toxins with lipid membranes were established. In more detail are presented the results of recent years, among which are the discovery of α-bungarotoxin analogs distinguishing the two binding sites in the muscle-type nAChR, long-chain α-neurotoxins interacting with α9α10 nAChRs and with GABA-A receptors, and the strong antiviral effects of dimeric phospholipases A2. A summary of the toxins obtained from arthropod venoms includes only highly cited works describing the molecules' success story, which is associated with IBCh. In marine animals, versatile toxins in terms of structure and molecular targets were discovered, and careful work on α-conotoxins differing in specificity for individual nAChR subtypes gave information about their binding sites.

What Are the Neurotoxins in Hemotoxic Snake Venoms?

Snake venoms as tools for hunting are primarily aimed at the most vital systems of the prey, especially the nervous and circulatory systems. In general, snakes of the Elapidae family produce neurotoxic venoms comprising of toxins targeting the nervous system, while snakes of the Viperidae family and most rear-fanged snakes produce hemotoxic venoms directed mainly on blood coagulation. However, it is not all so clear. Some bites by viperids results in neurotoxic signs and it is now known that hemotoxic venoms do contain neurotoxic components. For example, viperid phospholipases A2 may manifest pre- or/and postsynaptic activity and be involved in pain and analgesia. There are other neurotoxins belonging to diverse families ranging from large multi-subunit proteins (e.g., C-type lectin-like proteins) to short peptide neurotoxins (e.g., waglerins and azemiopsin), which are found in hemotoxic venoms. Other neurotoxins from hemotoxic venoms include baptides, crotamine, cysteine-rich secretory proteins, Kunitz-type protease inhibitors, sarafotoxins and three-finger toxins. Some of these toxins exhibit postsynaptic activity, while others affect the functioning of voltage-dependent ion channels. This review represents the first attempt to systematize data on the neurotoxins from "non-neurotoxic" snake venom. The structural and functional characteristic of these neurotoxins affecting diverse targets in the nervous system are considered.

Resistance Is Not Futile: Widespread Convergent Evolution of Resistance to Alpha-Neurotoxic Snake Venoms in Caecilians (Amphibia: Gymnophiona).

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale.

Acetylcholine-Binding Protein Affinity Profiling of Neurotoxins in Snake Venoms with Parallel Toxin Identification.

Snakebite is considered a concerning issue and a neglected tropical disease. Three-finger toxins (3FTxs) in snake venoms primarily cause neurotoxic effects since they have high affinity for nicotinic acetylcholine receptors (nAChRs). Their small molecular size makes 3FTxs weakly immunogenic and therefore not appropriately targeted by current antivenoms. This study aims at presenting and applying an analytical method for investigating the therapeutic potential of the acetylcholine-binding protein (AChBP), an efficient nAChR mimic that can capture 3FTxs, for alternative treatment of elapid snakebites. In this analytical methodology, snake venom toxins were separated and characterised using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-throughput venomics. By subsequent nanofractionation analytics, binding profiling of toxins to the AChBP was achieved with a post-column plate reader-based fluorescence-enhancement ligand displacement bioassay. The integrated method was established and applied to profiling venoms of six elapid snakes (Naja mossambica, Ophiophagus hannah, Dendroaspis polylepis, Naja kaouthia, Naja haje and Bungarus multicinctus). The methodology demonstrated that the AChBP is able to effectively bind long-chain 3FTxs with relatively high affinity, but has low or no binding affinity towards short-chain 3FTxs, and as such provides an efficient analytical platform to investigate binding affinity of 3FTxs to the AChBP and mutants thereof and to rapidly identify bound toxins.