Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy.

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production.

Widespread and Differential Neurotoxicity in Venoms from the Bitis Genus of Viperid Snakes.

Research into the neurotoxic activity of venoms from species within the snake family Viperidae is relatively neglected compared with snakes in the Elapidae family. Previous studies into venoms from the Bitis genus of vipers have identified the presence of presynaptic phospholipase A2 neurotoxins in B. atropos and B. caudalis, as well as a postsynaptic phospholipase A2 in B. arietans. Yet, no studies have investigated how widespread neurotoxicity is across the Bitis genus or if they exhibit prey selectivity of their neurotoxins. Utilising a biolayer interferometry assay, we were able to assess the binding of crude venom from 14 species of Bitis to the neuromuscular α-1 nAChR orthosteric site across a wide range of vertebrate taxa mimotopes. Postsynaptic binding was seen for venoms from B. arietans, B. armata, B. atropos, B. caudalis, B. cornuta, B. peringueyi and B. rubida. To further explore the types of neurotoxins present, venoms from the representatives B. armata, B. caudalis, B. cornuta and B. rubida were additionally tested in the chick biventer cervicis nerve muscle preparation, which showed presynaptic and postsynaptic activity for B. caudalis and only presynaptic neurotoxicity for B. cornuta and B. rubida, with myotoxicity also evident for some species. These results, combined with the biolayer interferometry results, indicate complex neurotoxicity exerted by Bitis species, which varies dramatically by lineage tested upon. Our data also further support the importance of sampling across geographical localities, as significant intraspecific variation of postsynaptic neurotoxicity was reported across the different localities.

Analgesic effects of Phα1ß toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Analgesic effects of Phalfa1 beta toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

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.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

Isolation and pharmacological characterization of α-Elapitoxin-Na1a, a novel short-chain postsynaptic neurotoxin from the venom of the Chinese Cobra (Naja atra).

The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. Although previous studies have shown that postsynaptic neurotoxins account for 11-23% of N. atra venom, envenomed patients do not display marked signs of neurotoxicity. We have previously shown that the lack of clinical neurotoxicity following snake envenoming by some species with 'neurotoxic' venoms may be related to the high prevalence of short-chain postsynaptic neurotoxins in these venoms. In this study, we describe the isolation and characterization of α-Elapitoxin-Na1a (α-EPTX-Na1a; 6949 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 9% of N. atra crude venom. α-EPTX-Na1a (30-300 nM) produced concentration-dependent inhibition of indirect-twitches, with a t90 value of 17 ± 2 min at 300 nM, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior addition of either Chinese N. atra monovalent antivenom (0.3 U/ml) or Australian polyvalent snake antivenom (2.4 U/ml), prevented the in vitro neurotoxic effects of α-EPTX-Na1a (30 nM). Addition of each of these antivenoms at the t90 time point partially reversed the in vitro neurotoxicity caused by α-EPTX-Na1a (30 nM). The inhibition of indirect twitches by α-EPTX-Na1a (30 nM) was not reversed by repeatedly washing the tissue. α-EPTX-Na1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA2 value of 8.21. De novo protein sequencing of α-EPTX-Na1a revealed a typical short-chain postsynaptic neurotoxin profile of 62 amino acids which shared >98% amino acid sequence similarity with short-chain postsynaptic neurotoxins from other Naja species. When compared to short-chain neurotoxins isolated from cobras in China, α-EPTX-Na1a contained novel residues K47Q (i.e. lysine to glutamine), N48T (i.e. asparagine to threonine) and G49A (i.e. glycine to alanine). In conclusion, α-EPTX-Na1a is a potent, pseudo-irreversible, short-chain neurotoxin. The high prevalence of α-EPTX-Na1a in Chinese N. atra venom is likely to explain the mild neurotoxicity experienced by envenomed patients.

A novel Kunitz-type neurotoxin peptide identified from skin secretions of the frog Amolops loloensis.

A novel Kunitz-type neurotoxin peptide that inhibited voltage-gated sodium channel was purified and characterized from the skin secretions of rufous-spotted torrent frog, Amolops loloensis. It has a 240-bp cDNA encoding an 79-amino acid residue (aa) precursor protein containing 6 half-cysteines. The precursor was proven to release a 57-aa mature peptide with amino acid sequence, DRNPICNLPPKEGFCLWMMRRSFFNPSKGRCDTFGYRGCGGNKNNFETPRACKEACG. The mature was named amotoxin. Amotoxin shares sequence homology with other Kunitz-type toxins and also has three cysteine bridges. Amotoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.087 µM. To the best of our knowledge, this is the first gene-encoded neurotoxin found in Amolops loloensis. Recombinant amotoxin showed similar functional properties as the native amotoxin. The functional properties of amotoxin may provide insights into the ecological adaptation of amphibians and deepen our understanding about the biological function spectrum of amphibian skin peptides.

Preclinical and clinical research on the toxic and neurological effects of cassava (Manihot esculenta Crantz) consumption.

Cassava (Manihot esculenta Crantz) is a tropical plant that is used as fresh food, processed food, or raw material for the preparation of flours with high nutritional value. However, cassava contains cyanogenic glycosides, such as linamarin and lotaustralin, that can trigger severe toxic effects and some neurological disorders, including motor impairment, cognitive deterioration, and symptoms that characterize tropical ataxic neuropathy and spastic epidemic paraparesis (Konzo). These alterations that are associated with the consumption of cassava or its derivatives have been reported in both humans and experimental animals. The present review discusses and integrates preclinical and clinical evidence that indicates the toxic and neurological effects of cassava and its derivatives by affecting metabolic processes and the central nervous system. An exhaustive review of the literature was performed using specialized databases that focused on the toxic and neurological effects of the consumption of cassava and its derivatives. We sought to provide structured information that will contribute to understanding the undesirable effects of some foods and preventing health problems in vulnerable populations who consume these vegetables. Cassava contains cyanogenic glycosides that contribute to the development of neurological disorders when they are ingested inappropriately or for prolonged periods of time. Such high consumption can affect neurochemical and neurophysiological processes in particular brain structures and affect peripheral metabolic processes that impact wellness. Although some vegetables have high nutritional value and ameliorate food deficits in vulnerable populations, they can also predispose individuals to the development of neurological diseases.

Structure-Function Elucidation of a New α-Conotoxin, MilIA, from Conus milneedwardsi.

The a-Conotoxins are peptide toxins that are found in the venom of marine cone snails and they are potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies. We describe the isolation and characterization of α-conotoxin MilIA, the first conopeptide from the venom of Conus milneedwardsi. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs that were expressed in Xenopus laevis oocytes. MilIA, which is a member of the α3/5 family, is an antagonist of muscle type nAChRs with a high selectivity for muscle versus neuronal subtype nAChRs. Several analogues were designed and investigated for their activity in order to determine the key epitopes of MilIA. Native MilIA and analogues both showed activity at the fetal muscle type nAChR. Two single mutations (Met9 and Asn10) allowed for MilIA to strongly discriminate between the two types of muscle nAChRs. Moreover, one analogue, MilIA [∆1,M2R, M9G, N10K, H11K], displayed a remarkable enhanced potency when compared to native peptide. The key residues that are responsible for switching between muscle and neuronal nAChRs preference were elucidated. Interestingly, the same analogue showed a preference for α9α10 nAChRs among the neuronal types.

Three-finger toxins from the venom of Micrurus tschudii tschudii (desert coral snake): Isolation and characterization of tschuditoxin-I.

Venoms from Micrurus (New World coral snakes) display potent peripheral neurotoxicity which may cause death by respiratory paralysis, yet many are poorly or not characterized. The major venom components of coral snakes are three-finger toxins (3FTxs) and phospholipases A2, whose proportions vary among species. As a trend, venoms of Micrurus from South America contain high proportions of 3FTxs, in contrast to most North and Central American species. Micrurus tschudii tschudii, the 'Desert coral snake' from Perú, displays an extreme 3FTx-predominant venom phenotype, with ∼95% of its proteome belonging to this protein family. This study evaluated the toxicity of its major 3FTxs in mice. A lethal 3FTx, here named tschuditoxin-I, was purified and sequenced by MALDI-TOF-TOF and N-terminal degradation. Tschuditoxin-I showed highest similarity to MS-1, a short-chain α-neurotoxin from the aquatic, fish-eating coral snake M. surinamensis. The single amino acid substitution between these two toxins maps at the tip of the first ß-stranded 'finger' in the modeled structure of tschuditoxin-I, suggesting it may have a role in interaction with its target, which remains to be investigated. Owing to its lethal action, tschuditoxin-I is likely to be medically relevant in envenomings. In spite of its 74% sequence identity with an α-neurotoxin of M. nigrocinctus, an equine antivenom raised against venom of the latter did not immunorecognize tschuditoxin-I or M. t. tschudii venom by ELISA. This underscores the need of characterizing the biochemical and immunological properties of the main toxic components of Micrurus venoms, aiming to improve the limited para-specific coverage of current antivenoms.

Efecto de la acetona cianohidrina, un derivado de la yuca, sobre la actividad motora y la función renal y hepática en ratas Wistar / Effects of acetone cyanohydrin, a derivative of cassava, on motor activity and kidney and liver function in Wistar rats

Introducción: La acetona cianohidrina (ACH) es una sustancia tóxica resultante de la hidrólisis enzimática de linamarina, contenido en las raíces de yuca (Manihot esculenta Crantz); su consumo a largo plazo se asocia con 2 trastornos neurológicos: konzo y la neuropatía atáxica tropical. Estudios anteriores han evaluado las alteraciones conductuales después del consumo de esta sustancia, pero los efectos tóxicos sobre los procesos fisiológicos se desconocen. Método: Se asignaron 32 ratas Wistar macho a 4 grupos experimentales (n = 8): un grupo vehículo (solución salina 0,3 ml/rata, ip) y 3 grupos con ACH (PubChem CID: 6406) a concentraciones de 10, 15 y 20 mM, durante 28 días, cada 24 h. Se evaluó la actividad motora espontánea en campo abierto y la coordinación motora en pruebas de rotarod y nado a 0, 7, 14, 21 y 28 días de tratamiento. Al final de las pruebas conductuales (día 28) se tomaron muestras de sangre por punción transcardiaca para evaluar la función renal y hepática. Resultados: La ACH promovió alteraciones en la actividad locomotora y promovió tanto el nado lateral como la conducta de giro en la prueba de nado los días 21 y 28 del tratamiento. La ACH incrementó los parámetros de la función renal y hepática de una manera dependiente de la concentración, excepto la glucosa y la bilirrubina total. Conclusión: Estos datos indican que el contenido de este compuesto tóxico contenido en las raíces de yuca podría ser potencialmente peligroso bajo el consumo a largo plazo en sujetos vulnerables

Introduction: Acetone cyanohydrin (ACH) is a toxic substance present in cassava roots (Manihot esculenta Crantz) which results from enzymatic hydrolysis of linamarin. Long-term consumption is associated with 2 neurological disorders: konzo and tropical ataxic neuropathy. Previous studies have evaluated behavioural alterations linked to ACH consumption, but the toxic effects of this substance on physiological processes remain unknown. Method: 32 male Wistar rats were assigned to 4 experimental groups (n = 8 per group): a vehicle group (0.3 mL saline solution, IP) and 3 ACH groups (PubChem CID: 6406) dosed at 10, 15, and 20 mM/24h for 28 days. We evaluated spontaneous motor activity with the open field test and motor coordination with the rotarod and forced swimming tests at 0, 7, 14, 21, and 28 days of treatment. At the end of the assessment period (day 28), blood samples were collected by transcardiac puncture to evaluate kidney and liver function. Results: ACH caused alterations in locomotor activity and promoted both lateral swimming and spinning in the forced swimming test at 21 and 28 days of treatment. Furthermore, it led to an increase in the levels of the parameters of kidney and liver function in a concentration-dependent manner, except for glucose and total bilirubin. Conclusion: Our results suggest that long-term consumption of this toxic compound present in cassava roots may be potentially dangerous for vulnerable subjects

A Novel Phospholipase A2 Isolated from Palythoa caribaeorum Possesses Neurotoxic Activity.

Zoanthids of the genus Palythoa are distributed worldwide in shallow waters around coral reefs. Like all cnidarians, they possess nematocysts that contain a large diversity of toxins that paralyze their prey. This work was aimed at isolating and functionally characterizing a cnidarian neurotoxic phospholipase named A2-PLTX-Pcb1a for the first time. This phospholipase was isolated from the venomous extract of the zoanthid Palythoa caribaeorum. This enzyme, which is Ca2+-dependent, is a 149 amino acid residue protein. The analysis of the A2-PLTX-Pcb1a sequence showed neurotoxic domain similitude with other neurotoxic sPLA2´s, but a different catalytic histidine domain. This is remarkable, since A2-PLTX-Pcb1a displays properties like those of other known PLA2 enzymes.

Effective Extraction of Domoic Acid from Seafood Based on Postsynthetic-Modified Magnetic Zeolite Imidazolate Framework-8 Particles.

Domoic acid (DA) is a naturally occurring neurotoxin known to bioaccumulate in marine products. Despite its hypertoxicity, the enrichment and analysis of trace DA in complex marine organisms remains a challenge. We describe herein the fabrication of a postsynthetic-modified magnetic zeolite imidazolate framework-8 (Fe3O4 SPs@ZIF-8/Zn2+), based on Fe3O4 superparticles, for the adsorption of DA from complex biological matrices. The adsorption of DA is rapid (∼5 min) and occurs through strong electrostatic interactions and chelation with coordinatively unsaturated zinc sites on the surface of Fe3O4 SPs@ZIF-8/Zn2+. Employing our Fe3O4 SPs@ZIF-8/Zn2+ sorbent in a magnetic solid-phase extraction, followed by liquid chromatographic separation and tandem mass spectrometric detection, resulted in a facile, rapid, efficient, and sensitive method for the enrichment and detection of trace DA in marine products. After optimization, this method yielded satisfactory precision (relative standard deviation ≤3.4%; n = 5) with a high degree of linearity from 1.0 to 1000.0 pg mL-1 ( r2 = 0.9997) and a detection limit of 0.2 pg mL-1 (S/N = 3). Recoveries of 93.1-102.3% were obtained in spiked aquatic products. In addition, trace levels of DA (49.2 pg mL-1) were found in shellfish samples, confirming the applicability of our Fe3O4 SPs@ZIF-8/Zn2+ adsorbent for the detection of DA in seafood.

High-yield production of spider short-chain insecticidal neurotoxin Tx4(6-1) in Pichia pastoris and bioactivity assays in vivo.

Short-chain insecticidal neurotoxin Tx4(6-1) from the spider Phoneutria nigriventer can be prepared by reversed-phase high-performance liquid-chromatography (HPLC) fractionation of PhTx4, but this is difficult and represents an obstacle preventing analyses of its insecticidal activity against agricultural insect pests. Herein, we performed secretory expression of recombinant Tx4(6-1) using Pichia pastoris strain X33 as the host, and screened transformants using enzyme-linked immunosorbent assay (ELISA). In flasks, ∼5 mg/l rTx4(6-1) was expressed as a secreted protein following induction with methanol, and this was increased to 45 mg/l rTx4(6-1) in a fed-batch reactor. Approximately 4 mg of high-purity rTx4(6-1) was purified from a 400 ml fed-batch culture supernatant by Ni+-nitriloacetic acid affinity chromatography, followed by carboxymethyl (CM) sepharose ion-exchange chromatography. Purified rTx4(6-1) was determined by mass spectrometry (MS) analysis, revealing a molecular weight (MW) of 7660.5 Da, larger than the expected size due to O-linked glycosylation. Insect bioactivity tests of rTx4(6-1)-treated fifth-instar silkworm larvae (Bombyx mori Linnaeus) showed neurotoxin symptoms such as contraction paralysis, abdominal contraction, and mouth movement syndrome, with a half lethal dose at 12 h post-injection of ∼4.5-8.5 µg/g body weight. Dietary toxicity was not observed in silkworm larvae.

µ-TRTX-Ca1a: a novel neurotoxin from Cyriopagopus albostriatus with analgesic effects.

Human genetic and pharmacological studies have demonstrated that voltage-gated sodium channels (VGSCs) are promising therapeutic targets for the treatment of pain. Spider venom contains many toxins that modulate the activity of VGSCs. To date, only 0.01% of such spider toxins has been explored, and thus there is a great potential for discovery of novel VGSC modulators as useful pharmacological tools or potential therapeutics. In the current study, we identified a novel peptide, µ-TRTX-Ca1a (Ca1a), in the venom of the tarantula Cyriopagopus albostriatus. This peptide consisted of 38 residues, including 6 cysteines, i.e. IFECSISCEIEKEGNGKKCKPKKCKGGWKCKFNICVKV. In HEK293T or ND7/23 cells expressing mammalian VGSCs, this peptide exhibited the strongest inhibitory activity on Nav1.7 (IC50 378 nM), followed by Nav1.6 (IC50 547 nM), Nav1.2 (IC50 728 nM), Nav1.3 (IC50 2.2 µM) and Nav1.4 (IC50 3.2 µM), and produced negligible inhibitory effect on Nav1.5, Nav1.8, and Nav1.9, even at high concentrations of up to 10 µM. Furthermore, this peptide did not significantly affect the activation and inactivation of Nav1.7. Using site-directed mutagenesis of Nav1.7 and Nav1.4, we revealed that its binding site was localized to the DIIS3-S4 linker region involving the D816 and E818 residues. In three different mouse models of pain, pretreatment with Cala (100, 200, 500 µg/kg) dose-dependently suppressed the nociceptive responses induced by formalin, acetic acid or heat. These results suggest that Ca1a is a novel neurotoxin against VGSCs and has a potential to be developed as a novel analgesic.

A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1.

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic ß-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.

Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT-induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well-tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species-specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

Discovery Methodology of Novel Conotoxins from Conus Species.

Cone snail venoms provide an ideal resource for neuropharmacological tools and drug candidates discovery, which have become a research hotspot in neuroscience and new drug development. More than 1,000,000 natural peptides are produced by cone snails, but less than 0.1% of the estimated conotoxins has been characterized to date. Hence, the discovery of novel conotoxins from the huge conotoxin resources with high-throughput and sensitive methods becomes a crucial key for the conotoxin-based drug development. In this review, we introduce the discovery methodology of new conotoxins from various Conus species. It focuses on obtaining full N- to C-terminal sequences, regardless of disulfide bond connectivity through crude venom purification, conotoxin precusor gene cloning, venom duct transcriptomics, venom proteomics and multi-omic methods. The protocols, advantages, disadvantages, and developments of different approaches during the last decade are summarized and the promising prospects are discussed as well.

The Anemonia viridis Venom: Coupling Biochemical Purification and RNA-Seq for Translational Research.

Blue biotechnologies implement marine bio-resources for addressing practical concerns. The isolation of biologically active molecules from marine animals is one of the main ways this field develops. Strikingly, cnidaria are considered as sustainable resources for this purpose, as they possess unique cells for attack and protection, producing an articulated cocktail of bioactive substances. The Mediterranean sea anemone Anemonia viridis has been studied extensively for years. In this short review, we summarize advances in bioprospecting of the A. viridis toxin arsenal. A. viridis RNA datasets and toxin data mining approaches are briefly described. Analysis reveals the major pool of neurotoxins of A. viridis, which are particularly active on sodium and potassium channels. This review therefore integrates progress in both RNA-Seq based and biochemical-based bioprospecting of A. viridis toxins for biotechnological exploitation.