γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.

UNASSIGNED: Bungarus multicinctus is one of the top ten venomous snakes in China. Its venom is mainly neurotoxin-based. Novel antivenom drugs need to be further researched and developed.
UNASSIGNED: This study aimed to explore the molecular mechanism of Cynanchum paniculatum in treating Bungarus multicinctus bites based on network pharmacology. Material and methods. The potential active ingredients of Cynanchum paniculatum were screened and their SDF structures were obtained using the PubChem database and imported into the SwissTargetPrediction database, and targets were obtained for the antitoxin effects of Cynanchum paniculatum in the treatment of Bungarus multicinctus bites. The Cynanchum paniculatum-active compound-potential target network and protein-protein interaction network were constructed by using Cytoscape software, and then biological function analysis and KEGG pathway enrichment analysis were performed using the DAVID.
UNASSIGNED: Seven potential active components (cynapanoside C, cynatratoside B, tomentolide A, sitosterol, sarcostin, tomentogenin, and paeonol) and 286 drug targets were obtained, including 30 key targets for the treatment of bungarotoxin toxicity. The active components mainly acted on PIK3CA, MAPK1, MAP2K1, JAK2, FYN, ACHE, CHRNA7, CHRNA4, and CHRNB2, and they antagonized the inhibitory effect of bungarotoxin on the nervous system through cholinergic synapses and the neurotrophin signaling pathway.
UNASSIGNED: Cynanchum paniculatum exerts a therapeutic effect on Bungarus multicinctus bites through multiple active components, multiple targets, and multiple pathways. The findings provide a theoretical basis for the extraction of active components of Cynanchum paniculatum and for related antivenom experiments.

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (α-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and α-BTX, and in the second group, samples were stained with NF200, α-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

Bungarus multicinctus, the Chinese krait, is a highly venomous elapid snake which causes considerable morbidity and mortality in southern China. B. multicinctus venom contains pre-synaptic PLA2 neurotoxins (i.e., β-bungarotoxins) and post-synaptic neurotoxins (i.e., α-bungarotoxins). We examined the in vitro neurotoxicity of B. multicinctus venom, and the efficacy of specific monovalent Chinese B. multicinctus antivenom, and Australian polyvalent elapid snake antivenom, against venom-induced neurotoxicity. B. multicinctus venom (1-10 μg/mL) abolished indirect twitches in the chick biventer cervicis nerve-muscle preparation as well as attenuating contractile responses to exogenous ACh and CCh, but not KCl. This indicates a post-synaptic neurotoxic action but myotoxicity was not evident. Given that post-synaptic α-neurotoxins have a more rapid onset than pre-synaptic neurotoxins, the activity of the latter in the whole venom will be masked. The prior addition of Chinese B. multicinctus antivenom (12 U/mL) or Australian polyvalent snake antivenom (15 U/mL), markedly attenuated the neurotoxic actions of B. multicinctus venom (3 μg/mL) and prevented the inhibition of contractile responses to ACh and CCh. The addition of B. multicinctus antivenom (60 U/mL), or Australian polyvalent snake antivenom (50 U/mL), at the t90 time point after the addition of B. multicinctus venom (3 μg/mL), did not restore the twitch height over 180 min. The earlier addition of B. multicinctus antivenom (60 U/mL), at the t20 or t50 time points, also failed to prevent the neurotoxic effects of the venom but did delay the time to abolish twitches based on a comparison of t90 values. Repeated washing of the preparation with physiological salt solution, commencing at the t20 time point, failed to reverse the neurotoxic effects of venom or delay the time to abolish twitches. This study showed that B. multicinctus venom displays marked in vitro neurotoxicity in a skeletal muscle preparation which is not reversed by antivenom. This does not appear to be related to antivenom efficacy, but due to the irreversible/pseudo-irreversible nature of the neurotoxins.

Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of B. multicinctus envenomation is attributed to the lethal components of α-, β-, γ- and κ- bungarotoxins contained in the venom. Although anti-B. multicinctus sera were produced in Shanghai, Taiwan and Vietnam, the most widely clinic used product was term as B. multicinctus antivenin and manufactured by Shanghai Serum Bio-technology Co. Ltd. In the present investigation, high purity α-, β- and γ-bungarotoxins were separately isolated from B. multicinctus crude venom. Rabbit anti- α-, β- and γ-bungarotoxin antisera were prepared by common methods, respectively. LD50 values of α-, β- and γ-bungarotoxins were systematically determined via three administration pathways (intraperitoneal, intramuscular and intravenous injections) in Kunming mice. LD50 values of β-bungarotoxin were closely related with injection routines but those of both α- and γ-bungarotoxins were not dependent on the injection routines. Commercial B. multicinctus antivenin showed strong immunoreaction with high molecular weight fractions of the B. multicinctus but weakly recognized low molecular weight fractions like α- and γ-bungarotoxins. Although B. multicinctus antivenin showed immunoreaction with high molecular weight fractions of Bungarus fasciatus, Naja atra, Ophiophagus hannah venoms but the antivenin only demonstrated animal protection efficacy against O. hannah venom. These results indicated that the high molecular weight fractions of the O. hannah played an important role in venom lethality but those of B. fasciatus and N. atra did not have such a role.

Venom gland is a highly efficient venom production system to maintain their predatory arsenal. Venom toxins mRNA has been shown to increase abruptly in snake after venom expenditure, while the dynamics of venom accumulation during synthesis are poorly understood. Here, PacBio long-read sequencing, Illumina RNA sequencing (RNA-seq), and label-free proteome quantification were used to investigate the composition landscape and time- and temperature-dependent dynamics changes of the Bungarus multicinctus venom gland system. Transcriptome data (19.5223 Gb) from six adult B. multicinctus tissues were sequenced using PacBio RS II to generate a reference assembly, and average 7.28 Gb of clean RNA-seq data was obtained from venom glands by Illumina sequencing. Differentially expressed genes (DEGs) mainly were protein processing rather than venom toxins. Post-translational modifications provided the evidence of the significantly different proportions of toxins in the venom proteome with the changing of replenishment time and temperature, but constant of venom toxins mRNA in the venom gland transcriptome. Dynamic of toxins and genes involved in venom gland contraction suggesting the formation of the mature venom gland system would take at least 9 days. In addition, 59 toxin processing genes were identified, peptidylprolyl isomerase B of which underwent positive selection in Toxicofera. These results provide a reference for determining the extraction time of venom, production of polyclonal and monoclonal antibody for precise treatment plans of venomous snakebites, and construction of an in vitro synthesis system for snake venom protein.

Motor endplates (MEPs) are the important interfaces between peripheral nerves and muscle fibers. Investigation of the spatial distribution of MEPs could help us better understand neuromuscular functional activities and improve the diagnosis and therapy of related diseases. Methods: Fluorescent α-bungarotoxin was injected to label the motor endplates in whole-mount skeletal muscles, and tissue optical clearing combined with light-sheet microscopy was used to investigate the spatial distribution of MEPs and in-muscle nerve branches in different skeletal muscles in wild-type and transgenic fluorescent mice. Electrophysiology was used to determine the relationship between the spatial distribution of MEPs and muscle function. Results: The exact three-dimensional distribution of MEPs in whole skeletal muscles was first obtained. We found that the MEPs in the muscle were distributed in an organized pattern of lamella clusters, with no MEPs outside the lamella zone. Each MEP lamella was innervated by one independent in-muscle nerve branch and mediated an independent muscle subgroup contraction. Additionally, the MEPs changed along the lamella clusters after denervation and regained the initial pattern after reinnervation. The integrity and spatial distribution of MEPs could reflect the functional state of muscles. The signal absence of a certain MEP lamella could suggest a problem in certain part of the muscle. Conclusions: The MEP lamella clusters might be the basis of neuromuscular function, and the spatial distribution of MEPs could serve as a testbed for evaluating the functional status of muscle and the therapeutic targeting map related to MEPs.

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

The Kv1.3 channel plays potential roles in immune, inflammation and coagulation system. Many studies showed that Kv1.3 channel inhibitors have immunosuppressive and anti-inflammatory activities, but no Kv1.3 channel inhibitors have been found to have anticoagulation activities. Here, based on our previous work about Kv1.3 channel toxin peptide inhibitors, we first attempt to test anticoagulation activities of four known venom-derived Kv1.3 channel inhibitors with different structural folds: BmKTX with CSα/β structural fold, OmTx3 with CSα/α structural fold, BF9 with Kuntz-type structural fold, and SjAPI-2 with Ascaris-type structural fold. Our results showed that BmKTX and OmTx3 have no activities towards both intrinsic and extrinsic coagulation pathway, SjAPI-2 just has weak activity towards intrinsic coagulation pathway, and BF9 has potent activity towards intrinsic coagulation pathway with no apparent effect on extrinsic coagulation pathway. Enzyme and inhibitor reaction kinetics experiments further showed that BF9 inhibited intrinsic coagulation pathway-associated coagulation factor XIa, but have no apparent effects on common coagulation pathway coagulation factor IIa. Structure-activity relationship showed that Gly14, Asn17, Ala18 and Ile20 of BF9 are main residues involved in the inhibiting effect on factor XIa. To the best of our knowledge, BF9 is the first anticoagulant with Kv1.3 channel inhibitory activity. Together, our present studies found the first dual functional peptides with Kv1.3 channel and coagulation factor XIa inhibitory activities, and provided a new molecular template for the lead drug discovery towards immune and thrombosis-associated human diseases.

Sepsis-induced myocardial injury is a well-known cause of mortality. The cholinergic anti-inflammatory pathway (CHAIP) is a physiological mechanism by which the central nervous system regulates immune response through the vagus nerve and acetylcholine; the α7-nicotinic acetylcholine receptor (α7nAChR) is the main component of CHAIP; GTS-21, a synthetic α7nAChR selective agonist, has repeatedly shown its powerful anti-inflammatory effect. However, little is known about its effect on LPS-induced myocardial injury. We investigated the protective effects of GTS-21 on lipopolysaccharide (LPS)-induced cardiomyopathy via the cholinergic anti-inflammatory pathway in a mouse sepsis model. We constructed the model of myocardial injury in sepsis mice by C57BL/6 using LPS and determined the time of LPS treatment by hematoxylin-eosin (HE) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). C57BL/6 mice were randomized into five groups: blank control group, model group, α-bungarotoxin + LPS group, GTS-21 + LPS group, and α-bungarotoxin + GTS-21 + LPS group. The pathological results of myocardial tissue were detected by the HE method; the apoptosis rate was detected by the TUNEL method; the relative expressions of NF-κB p65, Caspase-3, Caspase-8, Bcl-2, Bax, p53, and a7nAChR were detected by real-time quantitative PCR (RT-PCR); and the protein expressions of IL-6, IL-1 β, TNF-α, and pSTAT3 were detected by western blot. The results showed that LPS-induced myocardial pathological and apoptosis changes were significant compared with the blank group, which was reversed by GTS-21; however, pretreatment with α-bungarotoxin obviously blocked the protective effect of GTS-21. NF-κB p65, Caspase-3, Caspase-8, Bax, p53, IL-6, IL-1β, TNF-α, and pSTAT3 were significantly increased in the model group, while a7nAChR and Bcl-2 were significantly decreased; GTS-21 treatment reversed that result, while pretreatment with α-bungarotoxin strengthened the result in the model. And pretreatment with α-bungarotoxin blocked the protective effect of GTS-21. GTS-21 can alleviate the LPS-induced damage in the heart via a7nAChR, and pretreatment with α-bungarotoxin obviously blocked the protective effect of GTS-21 on sepsis in mice.