Development of antibody-detection ELISA based on beta-bungarotoxin for evaluation of the neutralization potency of equine plasma against Bungarus multicinctus in Taiwan.

Animal testing has been the primary approach to assess the neutralization potency of antivenom for decades. However, the necessity to sacrifice large numbers of experimental animals during this process has recently raised substantial welfare concerns. Furthermore, the laborious and expensive nature of animal testing highlights the critical need to develop alternative in vitro assays. Here, we developed an antibody-detection enzyme-linked immunosorbent assay (ELISA) technique as an alternative approach to evaluate the neutralization potency of hyperimmunized equine plasma against B. multicinctus, a medically important venomous snake in Taiwan. Firstly, five major protein components of B. multicinctus venom, specifically, α-BTX, ß-BTX, γ-BTX, MTX, and NTL, were isolated. To rank their relative medical significance, a toxicity score system was utilized. Among the proteins tested, ß-BTX presenting the highest score was regarded as the major toxic component. Subsequently, antibody-detection ELISA was established based on the five major proteins and used to evaluate 55 hyperimmunized equine plasma samples with known neutralization potency. ELISA based on ß-BTX, the most lethal protein according to the toxicity score, exhibited the best sensitivity (75.6 %) and specificity (100 %) in discriminating between high-potency and low-potency plasma, supporting the hypothesis that highly toxic proteins offer better discriminatory power for potency evaluation. Additionally, a phospholipase A2 (PLA2) competition process was implemented to eliminate the antibodies targeting toxicologically irrelevant domains. This optimization greatly enhanced the performance of our assay, resulting in sensitivity of 97.6 % and specificity of 92.9 %. The newly developed antibody-detection ELISA presents a promising alternative to in vivo assays to determine the neutralization potency of antisera against B. multicinctus during the process of antivenom production.

Identification of poorly immunodepleted phospholipase A2 (PLA2) proteins of Bungarus fasciatus venom from Assam, India and evaluation of Indian polyvalent antivenom using third-generation antivenomics.

Bungarus fasciatus also referred to as the Banded krait is a snake which possesses venom and belongs to the Elapidae family. It is widely distributed across the Indian subcontinent and South East Asian countries and is responsible for numerous snakebites in the population. B. fasciatus possesses a neurotoxic venom and envenomation by the snake results in significant morbidity and occasional morbidity in the victim if not treated appropriately. In this study, the efficacy of Indian polyvalent antivenom (Premium Serums polyvalent antivenom) was evaluated against the venom of B. fasciatus from Guwahati, Assam (India) employing the Third-generation antivenomics technique followed by identification of venom proteins from three poorly immunodepleted peaks (P5, P6 and P7) using LC-MS/MS analysis. Seven proteins were identified from the three peaks and all these venom proteins belonged to the phospholipase A2 (PLA2) superfamily. The identified PLA2 proteins were corroborated by the in vitro enzymatic activities (PLA2 and Anticoagulant activity) exhibited by the three peaks and previous reports of pathological manifestation in the envenomated victims. Neutralization of enzymatic activities by Premium Serums polyvalent antivenom was also assessed in vitro for crude venom, P5, P6 and P7 which revealed moderate to poor inhibition. Inclusion of venom proteins/peptides, which are non-immunodepleted or poorly immunodepleted, into the immunization mixture of venom used for antivenom production may help in enhancing the efficacy of the polyvalent antivenom.

High-Throughput Venomics.

In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma, Echis ocellatus, Naja pallida, Bothrops asper, Bungarus multicinctus, Crotalus atrox, Daboia russelii, Naja naja, Naja nigricollis, Naja mossambica, and Ophiophagus hannah. Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.

Network Pharmacological Study on the Mechanism of Cynanchum paniculatum (Xuchangqing) in the Treatment of Bungarus multicinctus Bites.

Background: 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. Objective: 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. Results: 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. Conclusions: 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 structural and functional divergence of a neglected three-finger toxin subfamily in lethal elapids.

Bungarus multicinctus is a widely distributed and medically important elapid snake that produces lethal neurotoxic venom. To study and enhance existing antivenom, we explore the complete repertoire of its toxin genes based on de novo chromosome-level assembly and multi-tissue transcriptome data. Comparative genomic analyses suggest that the three-finger toxin family (3FTX) may evolve through the neofunctionalization of flanking LY6E. A long-neglected 3FTX subfamily (i.e., MKA-3FTX) is also investigated. Only one MKA-3FTX gene, which evolves a different protein conformation, is under positive selection and actively transcribed in the venom gland, functioning as a major toxin effector together with MKT-3FTX subfamily homologs. Furthermore, this lethal snake may acquire self-resistance to its ß-bungarotoxin via amino acid replacements on fast-evolving KCNA2. This study provides valuable resources for further evolutionary and structure-function studies of snake toxins, which are fundamental for the development of effective antivenoms and drug candidates.

On characterizing the Red-headed Krait (Bungarus flaviceps) venom: Decomplexation proteomics, immunoreactivity and toxicity cross-neutralization by hetero-specific antivenoms.

The Red-headed Krait (Bungarus flaviceps) is a medically important venomous snake species in Southeast Asia, while there is no specific antivenom available for its envenoming. This study investigated the venom composition through a decomplexation proteomic approach, and examined the immunoreactivity as well as cross-neutralization efficacy of two hetero-specific krait antivenoms, Bungarus candidus Monovalent Antivenom (BcMAV) and Bungarus fasciatus Monovalent Antivenom (BfMAV), against the venom of B. flaviceps from Peninsular Malaysia. A total of 43 non-redundant proteoforms belonging to 10 toxin families were identified in the venom proteome, which is dominated by phospholipases A2 including beta-bungarotoxin lethal subunit (56.20 % of total venom proteins), Kunitz-type serine protease inhibitors (19.40 %), metalloproteinases (12.85 %) and three-finger toxins (7.73 %). The proteome varied in quantitative aspect from the earlier reported Indonesian (Sumatran) sample, suggesting geographical venom variation. BcMAV and BfMAV were immunoreactive toward the B. flaviceps venom, with BcMAV being more efficacious in immunological binding. Both antivenoms cross-neutralized the venom lethality with varying efficacy, where BcMAV was more potent than BfMAV by ~13 times (normalized potency: 38.04 mg/g vs. 2.73 mg/g, defined as the venom amount completely neutralized by one-gram antivenom protein), supporting the potential utility of BcMAV for para-specific neutralization against B. flaviceps venom.

Efficacy of Silene arenosa extract on acetylcholinesterase in Bungarus sindanus (krait) venom.

OBJECTIVE: To examine the efficacy of Silene arenosa extract on acetylcholinesterase (AChE) of krait (Bungarus Sindanus) snake venom. METHODS: The present project designed to evaluate the inhibition of AChE by following standard procedures. RESULTS: Statistical analysis of the results showed that Silene arenosa exerted 73% inhibition against the krait venom acetylcholinesterase at fixed substrate acetylcholine (ACh) concentration (0.5 mM). Kinetic analysis using the Lineweaver Burk plot revealed that Silene arenosa caused a competitive type of inhibition i.e. Km values increased from 26.6 to 93.3 mM (26.6% to 93.3%) and Vmax remained constant in a concentration-dependent manner. Silene arenosa competes with the substrate to bind at the active site of the enzyme. The Kmapp of venom AChE for Silene arenosa increased from 60% to 81.6% and the Vmaxapp remains constant. Ki (inhibition constant was estimated to be 48 µg for snake venom; while the Km (Michaelis-Menten constant of AChE- substrate into AChE and product) was estimated to be 0.5 mM. The IC50 of AchE calculated for Silene arenosa was 67 µg. CONCLUSION: The present results suggest that Silene arenosa extract can be considered as an inhibitor of snake venom AChE.

Proteomics and neutralization of Bungarus multicinctus (Many-banded Krait) venom: Intra-specific comparisons between specimens from China and Taiwan.

The Many-banded Krait (Bungarus multicinctus) is a medically important venomous snake in East Asia. This study investigated the venom proteomes of B. multicinctus from Guangdong, southern China (BM-China) and insular Taiwan (BM-Taiwan), and the neutralization activities of two antivenom products (produced separately in China and Taiwan) against the lethal effect of the venoms. The venom proteomes of both specimens contained similar toxin families, notwithstanding small variations in the subtypes and abundances of minor components. More than 90% of the total venom proteins belong to three-finger toxins (3FTx, including alpha-neurotoxins) and phospholipases A2 (PLA2, including beta-bungarotoxins), supporting their key involvement in the pathophysiology of krait envenomation which manifests as pre- and post-synaptic neurotoxicity. The venoms exhibited potent neurotoxic and lethal effects with extremely low i.v. LD50 of 0.027 µg/g (Bm-China) and 0.087 µg/g (Bm-Taiwan), respectively, in mice. Bungarus multicinctus monovalent antivenom (BMMAV) produced in China and Neuro bivalent antivenom (NBAV) produced in Taiwan were immunoreactive toward both venoms and their toxin fractions. The antivenoms neutralized the venom lethality variably, with BMMAV being more efficacious than NBAV by approximately two-fold. Findings suggest that the monovalent antivenom has a higher potency presumably due to its species-specificity toward the krait venom.

A Wolf in Another Wolf's Clothing: Post-Genomic Regulation Dictates Venom Profiles of Medically-Important Cryptic Kraits in India.

The Common Krait (Bungarus caeruleus) shares a distribution range with many other 'phenotypically-similar' kraits across the Indian subcontinent. Despite several reports of fatal envenomings by other Bungarus species, commercial Indian antivenoms are only manufactured against B. caeruleus. It is, therefore, imperative to understand the distribution of genetically distinct lineages of kraits, the compositional differences in their venoms, and the consequent impact of venom variation on the (pre)clinical effectiveness of antivenom therapy. To address this knowledge gap, we conducted phylogenetic and comparative venomics investigations of kraits in Southern and Western India. Phylogenetic reconstructions using mitochondrial markers revealed a new species of krait, Romulus' krait (Bungarus romulusi sp. nov.), in Southern India. Additionally, we found that kraits with 17 mid-body dorsal scale rows in Western India do not represent a subspecies of the Sind Krait (B. sindanus walli) as previously believed, but are genetically very similar to B. sindanus in Pakistan. Furthermore, venom proteomics and comparative transcriptomics revealed completely contrasting venom profiles. While the venom gland transcriptomes of all three species were highly similar, venom proteomes and toxicity profiles differed significantly, suggesting the prominent role of post-genomic regulatory mechanisms in shaping the venoms of these cryptic kraits. In vitro venom recognition and in vivo neutralisation experiments revealed a strong negative impact of venom variability on the preclinical performance of commercial antivenoms. While the venom of B. caeruleus was neutralised as per the manufacturer's claim, performance against the venoms of B. sindanus and B. romulusi was poor, highlighting the need for regionally-effective antivenoms in India.

Kinetic analysis of effects of temperature and time on the regulation of venom expression in Bungarus multicinctus.

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.

Quantitative proteomic analysis of venom from Southern India common krait (Bungarus caeruleus) and identification of poorly immunogenic toxins by immune-profiling against commercial antivenom.

OBJECTIVES: To study the venom proteome composition of Southern India (SI) Common Krait (Bungarus caeruleus) and immunological cross-reactivity between venom against commercial antivenom. METHODS: Proteomic analysis was done by nano LC-MS/MS and toxins were quantitated by label-free analysis. The immunological cross-reactivity of venom towards polyvalent antivenom (PAV) was assessed by ELISA, Immunoblotting, and immuno-chromatographic methods. RESULTS: A total of 57 enzymatic and non-enzymatic proteins belonging to 12 snake venom protein families were identified. The three finger toxins (3FTx) (48.3%) and phospholipase A2 (PLA2) (37.6%) represented the most abundant non-enzymatic and enzymatic proteins, respectively. ß-bungarotoxin (12.9%), a presynaptic neurotoxin, was also identified. The venom proteome composition is well correlated with its enzymatic activities, reported pharmacological properties, and clinical manifestations of krait envenomation. Immuno-cross-reactivity studies demonstrated better recognition of high molecular weight proteins (>45 kDa) of this venom by PAVs compared to low molecular weight (<15 kDa) toxins such as PLA2 and 3FTxs. CONCLUSION: The poor recognition of <15 kDa mass SI B. caeruleus venom proteins is of grave concern for the successful treatment of krait envenomation. Therefore, emphasis should be given to improve the immunization protocols and/or supplement of antibodies raised specifically against the <15 kDa toxins of this venom.

Venom proteome of Bungarus sindanus (Sind krait) from Pakistan and in vivo cross-neutralization of toxicity using an Indian polyvalent antivenom.

The proteome of the Pakistani B. sindanus venom was investigated with reverse-phase HPLC and nano-ESI-LCMS/MS analysis. At least 36 distinct proteins belonging to 8 toxin protein families were identified. Three-finger toxin (3FTx), phospholipase A2 (including ß-bungarotoxin A-chains) and Kunitz-type serine protease inhibitor (KSPI) were the most abundant, constituting ~95% of total venom proteins. The other toxin proteins of low abundance are snake venom metalloproteinase (SVMP), L-amino acid oxidase (LAAO), acetylcholinesterase (AChE), vespryn and cysteine-rich secretory protein (CRiSP). The venom was highly lethal to mice with LD50 values of 0.04 µg/g (intravenous) and 0.15 µg/g (subcutaneous). The 3FTx proteins are diverse, comprising kappa-neurotoxins, neurotoxin-like protein, non-conventional toxins and muscarinic toxin-like proteins. Kappa-neurotoxins and ß-bungarotoxins represent the major toxins that mediate neurotoxicity in B. sindanus envenoming. Alpha-bungarotoxin, commonly present in the Southeast Asian krait venoms, was undetected. The Indian VINS Polyvalent Antivenom (VPAV) was immunoreactive toward the venom, and it moderately cross-neutralized the venom lethality (potency = 0.25 mg/ml). VPAV was able to reverse the neurotoxicity and prevent death in experimentally envenomed mice, but the recovery time was long. The unique toxin composition of B. sindanus venom may be considered in the formulation of a more effective pan-regional, polyspecific antivenom. BIOLOGICAL SIGNIFICANCE: Bungarus sindanus, an endemic krait species distributed mainly in the Sindh Province of Pakistan is a cause of snake envenomation. Its specific antivenom is, however, lacking. The proteomic study of its venom revealed a substantial presence of κ-bungarotoxins and ß-bungarotoxins. The toxin profile corroborates the potent neurotoxicity and lethality of the venom tested in vivo. The heterologous Indian VINS polyvalent antivenom (VPAV) cross-reacted with B. sindanus venom and cross-neutralized the venom neurotoxicity and lethality in mice, albeit the efficacy was moderate. The findings imply that B. sindanus and the phylogenetically related B. caeruleus of India share certain venom epitopes. Research should be advanced to improve the efficacy spectrum of a pan-regional polyspecific antivenom.

Expression and characterization of Flavikunin: A Kunitz-type serine protease inhibitor identified in the venom gland cDNA library of Bungarus flaviceps.

Trancriptomic analysis of the venom gland cDNA library of Bungarus flaviceps revealed Kunitz-type serine protease inhibitor as one of the major venom protein families with three groups A, B, C. One of the group B isoforms named Flavikunin, which lacked an extra cysteine residue involved in disulfide bond formation in ß-bungarotoxin, was synthesized, cloned, and overexpressed in Escherichia coli. To decipher the structure-function relationship, the P1 residue of Flavikunin, histidine, was mutated to alanine and arginine. Purified wild-type and mutant Flavikunins were screened against serine proteases-thrombin, factor Xa, trypsin, chymotrypsin, plasmin, and elastase. The wild-type and mutant Flavikunin (H∆R) inhibited plasmin with an IC 50 of 0.48 and 0.35 µM, respectively. The in-silico study showed that P1 residue of wild-type and mutant (H∆R) Flavikunin interacted with S1' and S1 site of plasmin, respectively. Thus, histidine at the P1 position was found to be involved in plasmin inhibition with mild anticoagulant activity.

Rational truncation of aptamer for cross-species application to detect krait envenomation.

In majority of snakebite cases, the snake responsible for the bite remains unidentified. The traditional snakebite diagnostics method relies upon clinical symptoms and blood coagulation assays that do not provide accurate diagnosis which is important for epidemiological as well as diagnostics point of view. On the other hand, high batch-to-batch variations in antibody performance limit its application for diagnostic assays. In recent years, nucleic acid aptamers have emerged as a strong chemical rival of antibodies due to several obvious advantages, including but not limited to in vitro generation, synthetic nature, ease of functionalization, high stability and adaptability to various diagnostic formats. In the current study, we have rationally truncated an aptamer developed for α-Toxin of Bungarus multicinctus and demonstrated its utility for the detection of venom of Bungarus caeruleus. The truncated aptamer α-Tox-T2 (26mer) is found to have greater affinity than its 40-mer parent counterpart α-Tox-FL. The truncated aptamers are characterized and compared with parent aptamer for their binding, selectivity, affinity, alteration in secondary structure and limit of detection. Altogether, our findings establish the cross-species application of a DNA aptamer generated for α-Toxin of Bungarus multicinctus (a snake found in Taiwan and China) for the reliable detection of venom of Bungarus caeruleus (a snake found in the Indian subcontinent).

An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms against Bungarus candidus and Naja naja.

In order to facilitate/expedite the production of effective and affordable snake antivenoms, a novel in vitro potency assay was previously developed. The assay is based on an antiserum's ability to bind to postsynaptic neurotoxin (PSNT) and thereby inhibit the PSNT binding to the nicotinic acetylcholine receptor (nAChR). The assay was shown to work well with antiserum against Thai Naja kaouthia which produces predominantly the lethal PSNTs. In this work, the assay is demonstrated to work well with antiserum/antivenom against Bungarus candidus (BC), which also produces lethal presynaptic neurotoxins, as well as antivenom against Sri Lankan Naja naja (NN), which produces an abundance of cytotoxins. The in vitro and in vivo median effective ratios (ER50s) for various batches of antisera against BC showed a correlation (R2) of 0.8922 (p < 0.001) while the corresponding value for the anti-NN antivenom was R2 = 0.7898 (p < 0.01). These results, together with the known toxin profiles of various genera of elapids, suggest that this in vitro assay could be used with antisera against other species of Bungarus and Naja and possibly other neurotoxic snake venoms worldwide. The assay should significantly save numerous lives of mice and accelerate production of life-saving antivenoms.

The antimicrobial potential of a new derivative of cathelicidin from Bungarus fasciatus against methicillin-resistant Staphylococcus aureus.

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32-128 µg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 µg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.

Proteomic characterization and comparison of venoms from two elapid snakes (Bungarus multicinctus and Naja atra) from China.

Bungarus multicinctus (many-banded krait) and Naja atra (Chinese cobra) are widely distributed and medically important venomous snakes in China; however, their venom proteomic profiles have not been fully compared. Here, we fractionated crude venoms and analyzed them using a combination of proteomic techniques. Three-finger toxins (3-FTx) and phospholipase A2 (PLA2) were most abundant in both species, respectively accounting for 32.6% and 66.4% of total B. multicinctus venom, and 84.3% and 12.2% of total N. atra venom. Venoms from these two species contained one common protein family and six less abundant species-specific protein families. The proteomic profiles of B. multicinctus and N. atra venoms and analysis of toxicological activity in mice suggested that 3-FTx and PLA2 are the major contributors to clinical symptoms caused by envenomation. The venoms differed in enzymatic activity, likely the result of inter-specific variation in the amount of related venom components. Antivenomics assessment revealed that a small number of venom components (3-FTxs and PLA2s in B. multicinctus, and 3-FTxs in N. atra) could not be immunocaptured completely, suggesting that we should pay attention to enhancing the immune response of these components in designing commercial antivenoms for B. multicinctus and N. atra. BIOLOGICAL SIGNIFICANCE: The proteomic profiles of venoms from two medically important snake species - B. multicinctus and N. atra - have been explored. Quantitative and qualitative differences are evident in both venoms when proteomic profiles and transcriptomic results are compared; this is a reminder that combined approaches are needed to explore the precise composition of snake venom. Two protein families (3-FTx and PLA2) of high abundance in these snake venoms are major players in the biochemical and pharmacological effects of envenomation. Elucidation of the proteomic profiles of these snake venoms is helpful in understanding composition-function relationships and will facilitate the clinical application of antivenoms.

Cathelicidin-BF, a Novel Antimicrobial Peptide from Bungarus fasciatus, Attenuates Disease in a Dextran Sulfate Sodium Model of Colitis.

Antimicrobial peptides are molecules of innate immunity. Cathelicidin-BF is the first cathelicidin peptide found in reptiles. However, the immunoregulatory and epithelial barrier protective properties of C-BF have not been reported. Inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, can lead to colon cancer, the third most common malignant tumor. The objective is to develop the new found cathelicidin-BF as a therapeutic to patients of ulcerative colitis. The morphology of the colon epithelium was observed by H&E staining; apoptosis index and infiltration of inflammatory cells in colonic epithelium were measured by TUNEL and immunohistochemistry; the expression level of endogenous mCRAMP was analyzed by immunofluorescence; and phosphorylation of the transcription factors c-jun and NF-κB in colon were analyzed by Western blot. Our results showed that the morphology of the colon epithelium in the C-BF+DSS group was improved compared with the DSS group. Apoptosis and infiltration of inflammatory cells in colonic epithelium were also significantly attenuated in the C-BF+DSS group compared with the DSS group, and the expression level of endogenous mCRAMP in the DSS group was significantly higher than other groups. DSS-induced phosphorylation level of c-jun and NF-κB while C-BF effectively inhibited phosphorylation of NF-κB (p65). The barrier protective effect of C-BF was still excellent. In conclusion, C-BF effectively attenuated inflammation and improved disrupted barrier function. Notably, this is the first report to demonstrate that C-BF attenuates DSS-induced UC both through the regulation of intestinal immune and retention of barrier function, and the exact pathway was through NF-κB.

Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity.

BACKGROUND: Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk. OBJECTIVES: Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants. METHODS AND RESULTS: We have isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor that prolonged activated partial thromboplastin time without significant effects on prothrombin time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial thromboplastin time at ~ 3 µmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A series of mutants were subsequently generated to improve the potency and selectivity of recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50 ~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type inhibitor of FXIa (Ki  = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice carotid artery in FeCl3 -induced thrombosis models. CONCLUSION: We have isolated an exogenous FXIa specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in vitro and in vivo efficacy. These proof-of-principle data supported the further development of Fasxiator as a novel anticoagulant candidate.

Recognition of Bungarus multicinctus venom by a DNA aptamer against ß-bungarotoxin.

Antibody-based technology is the main method for diagnosis and treatment of snake bite envenoming currently. However, the development of an antibody, polyclonal or monoclonal, is a complicated and costly procedure. Aptamers are single stranded oligonucleotides that recognize specific targets such as proteins and have shown great potential over the years as diagnostic and therapeutic agents. In contrast to antibodies, aptamers can be selected in vitro without immunization of animals, and synthesized chemically with extreme accuracy, low cost and high degree of purity. In this study we firstly report on the identification of DNA aptamers that bind to ß-bungarotoxin (ß-BuTx), a neurotoxin from the venom of Bungarus multicinctus. A plate-SELEX method was used for the selection of ß-BuTx specific aptamers. After 10 rounds of selection, four aptamer candidates were obtained, with the dissociation constant ranged from 65.9 nM to 995 nM measured by fluorescence spectroscopy. Competitive binding assays using both the fluorescently labeled and unlabeled aptamers revealed that the four aptamers bound to the same binding site of ß-BuTx. The best binder, ßB-1, bound specifically to ß-BuTx, but not to BSA, casein or α-Bungarotoxin. Moreover, electrophoretic mobility shift assay and enzyme-linked aptamer assay demonstrated that ßB-1 could discriminate B. multicinctus venom from other snake venoms tested. The results suggest that aptamer ßB-1 can serve as a useful tool for the design and development of drugs and diagnostic tests for ß-BuTx poisoning and B. multicinctus bites.