Analysis of the efficacy of Taiwanese freeze-dried neurotoxic antivenom against Naja kaouthia, Naja siamensis and Ophiophagus hannah through proteomics and animal model approaches

Liu, Chien-Chun; You, Chen-Hsien; Wang, Po-Jung; Yu, Jau‐Song; Huang, Guo-Jen; Liu, Chien-Hsin; Hsieh, Wen-Chin; Lin, Chun‐Yen

doi:10.1371/journal.pntd.0006138

Cited by 39 publications

(50 citation statements)

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“…The crude venom of N. atra was separated by RP-HPLC (Fig 1). Consistent with previous studies using similar methods [15,19], we purified and collected five major protein components. To confirm the identity of protein components in each fraction, we performed in-solution digestion together with LC-MS/MS.…”

Section: Description and Preparation Of Major Protein Components Frommentioning

confidence: 75%

“…CTXs, PLA 2 and NTX account for~90% of venom protein constituents [15,19]. The structure of CTXs, the most dominant proteins in N. atra venom, is characterized by a three-fingered fold comprising loops I-III [28].…”

Section: Discussionmentioning

confidence: 99%

“…The venom of N. atra was separated by RP-HPLC, as previously described [19]. Briefly, crude venom (100 μg protein) was dissolved in solvent A (water containing 0.1% trifluoroacetic acid (TFA), and separated by RP-HPLC using a Supelco Discovery 300 Å C 18 (4.6 × 150 mm, 3 μm particle size) column (Sigma-Aldrich, St. Louis, Missouri, USA).…”

Section: Reverse-phase High-performance Liquid Chromatography (Rmentioning

confidence: 99%

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Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models

et al. 2020

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Naja atra envenomation is one of the most significant clinical snakebite concerns in Taiwan. Taiwanese freeze-dried neurotoxic antivenom (FNAV) is currently used clinically for the treatment of cobra snakebite, and has been shown to limit the mortality of cobra envenomation to less than 1%. However, more than half of victims (60%) require surgery because of local tissue necrosis, a major problem in patients with cobra envenomation. Although the importance of evaluating the neutralizing effect of FNAV on this pathology is recognized, whether FNAV is able to prevent the local necrosis extension induced by N. atra venom has not been investigated in detail. Cytotoxins (CTXs) are considered as the major components of N. atra venom that cause necrosis. In the current study, we isolated CTXs from whole cobra venom and used both whole venom and purified CTXs to develop animal models for assessing the neutralization potential of FNAV against venom necrotizing activity. Local necrotic lesions were successfully produced in mice using CTXs in place of whole N. atra venom. FNAV was able to rescue mice from a subcutaneously injected lethal dose of cobra venom; however, it was unable to prevent CTX-induced dermo-necrosis. Furthermore, using the minimal necrosis dose (MND) of CTXs and venom proteome data, we found a dose of whole N. atra venom suitable for FNAV and developed a workable protocol for inducing local necrosis in rodent models that successfully imitated the clinical circumstance of cobra envenoming. This information provides a more comprehensive understanding of PLOS Neglected Tropical Diseases | https://doi.the pathophysiology of N. atra envenomation, and serves as a guide for improving current antivenom strategies and advancing clinical snakebite management in Taiwan. Author summaryNaja atra envenomation is an important public health issue in Taiwan. Although the mortality rate of cobra snakebite is controlled using antivenom, more than half of victims develop symptoms of local necrosis and require surgical intervention. Whether the Taiwanese freeze-dried neurotoxic antivenom (FNAV) currently in clinical use is able to prevent the local necrosis extension induced by N. atra venom is still unclear. In this study, we developed a dermo-necrosis animal model using purified cytotoxins (CTXs), the major necrosis-related proteins from N. atra venom. We found that FNAV was able to neutralize the lethality of whole cobra venom, but was unable to neutralize the necrosis induced by CTXs in vivo. This finding introduced an example that supplementary quality control assays may be necessary to determine the effectiveness of antivenoms in neutralizing specific pathology induced by the venom; only evaluating the rodent lethality prevention is insufficient. Our results provide insights that should help improve current antivenoms and advance cobra snakebite management in Taiwan.Pathogenesis of local necrosis induced by Naja atra venom PLOS Neglected Tropical Diseases | https://doi.org/10.The lyophilized fractions from RP-HPLC were d...

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Section: Description and Preparation Of Major Protein Components Frommentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Reverse-phase High-performance Liquid Chromatography (Rmentioning

confidence: 99%

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Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models

et al. 2020

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“…Recent proteomic studies validated that CTXs are indeed abundant in N. sumatrana and N. kaouthia venoms of various locales, constituting at least 20% of the total venom proteins (Yap et al, 2014;Tan et al, 2016a;Liu et al, 2017;Xu et al, 2017). The two major subgroups of CTX, i.e., P-type and S-type CTX, distribute variably among different Naja species.…”

Section: Discussionmentioning

confidence: 99%

Cytotoxicity of Snake Venoms and Cytotoxins From Two Southeast Asian Cobras (Naja sumatrana, Naja kaouthia): Exploration of Anticancer Potential, Selectivity, and Cell Death Mechanism

Chong

Tan

2020

Front. Mol. Biosci.

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Venoms of cobras ( Naja spp.) contain high abundances of cytotoxins, which contribute to tissue necrosis in cobra envenomation. The tissue-necrotizing activity of cobra cytotoxins, nevertheless, indicates anticancer potentials. This study set to explore the anticancer properties of the venoms and cytotoxins from Naja sumatrana (equatorial spitting cobra) and Naja kaouthia (monocled cobra), two highly venomous species in Southeast Asia. The cytotoxicity, selectivity, and cell death mechanisms of their venoms and cytotoxins (NS-CTX from N. sumatrana : NS-CTX; N. kaouthia : NK-CTX) were elucidated in human lung (A549), prostate (PC-3), and breast (MCF-7) cancer cell lines. Cytotoxins were purified through a sequential fractionation approach using cation-exchange chromatography, followed by C 18 reverse-phase high-performance liquid chromatography (HPLC) to homogeneity validated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by liquid chromatography-tandem mass spectrometry (LCMS/MS). The cobra venoms and their respective cytotoxins exhibited concentration-dependent growth inhibitory effects in all cell lines tested, with the cytotoxins being more potent compared to the corresponding whole venoms. NS-CTX and NK-CTX are, respectively, P-type and S-type isoforms of cytotoxin, based on the amino acid sequences as per LCMS/MS analysis. Both cytotoxins exhibited differential cytotoxic effects in the cell lines tested, with NS-CTX (P-type cytotoxin) being significantly more potent in inhibiting the growth of the cancer cells. Both cytotoxins demonstrated promising selectivity only for the A549 lung cancer cell line (selectivity index = 2.17 and 2.26, respectively) but not in prostate (PC-3) and breast (MCF-7) cancer cell lines (selectivity index < 1). Flow cytometry revealed that the A549 lung cancer cells treated with NS-CTX and NK-CTX underwent necrosis predominantly. Meanwhile, the cytotoxins induced mainly caspase-independent late apoptosis in the prostate (PC-3) and breast (MCF-7) cancer cells lines but lacked selectivity. The findings revealed the limitations and challenges that could be faced during the development of new cancer therapy from cobra cytotoxins, notwithstanding their potent anticancer effects. Further studies should aim to overcome these impediments to unleash the anticancer potentials of the cytotoxins.

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“…It would be a huge task or even impossible to develop a universal drug or antivenom for treatment of most venomous snake bites around the world. To solve this problem and collaborate with the WHO to find a better solution for responding to neglected tropical public health issues, many studies have investigated this issue and have discovered antivenoms with the potential capability for cross-neutralization of venoms from cross-regional or pan-species of snakes [21][22][23][24]. For example, the venom of N. kaouthia was the only Naja genus venom used to generate neuro-polyvalent snake antivenom (NPAV).…”

Section: Introductionmentioning

confidence: 99%

A Rapid and International Applicable Diagnostic Device for Cobra (Genus Naja) Snakebites

Lin

Sung

Liao

et al. 2020

Toxins

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Cobra snakes (genus Naja) are some of the most dangerous snake species in Asia and Africa, as their bites cause severe life-threatening respiratory failure and local tissue destruction, especially in the case of late diagnosis. The differential diagnosis of snakebite envenomation still mainly relies upon symptomatology, the patient’s description, and the experience of physicians. We have designed a rapid test, immunochromatographic test of cobra (ICT-Cobra), which obtained fair results in improving the diagnosis and treatment of Naja (N.) atra snakebites in Taiwan. In this study, we further investigated the feasibility of applying the kit for the detection of other cobra venoms based on the potential interspecies similarity. We firstly demonstrated the cross-reactivity between eight venoms of medically important cobra species and the rabbit anti-N. atra IgG that was used in ICT-Cobra by Western blotting and sandwich enzyme-linked immunosorbent assay. Then, ICT-Cobra was used to detect various concentrations of the eight venoms to elucidate its performance. Noticeable correlations between the cross-reactivity of venoms from genus Naja snakes and existing geographical characteristics were found. ICT-Cobra could detect venoms from other Asian cobras with variable detection limits comparable to those observed for N. atra, but the kit was less successful in the detection of venom from African cobras. The similar but slightly different venom components and the interaction between venom and rabbit anti-N. atra IgG led to variations in the detection limits. The transcontinental usage of ICT-Cobra might be possible due to the cross-reactivity of antibodies and similarities among the larger-sized proteins. This study showed that the close immunological relationships in the genus Naja could be used to develop a venom detection kit for the diagnosis of cobra envenomation in both Asian and African regions. Additional clinical studies and technical adjustments are still needed to improve the efficacy and broadening the application of ICT-Cobra in the future.

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Analysis of the efficacy of Taiwanese freeze-dried neurotoxic antivenom against Naja kaouthia, Naja siamensis and Ophiophagus hannah through proteomics and animal model approaches

Cited by 39 publications

References 46 publications

Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models

Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models

Cytotoxicity of Snake Venoms and Cytotoxins From Two Southeast Asian Cobras (Naja sumatrana, Naja kaouthia): Exploration of Anticancer Potential, Selectivity, and Cell Death Mechanism

A Rapid and International Applicable Diagnostic Device for Cobra (Genus Naja) Snakebites

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