Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Abstract Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.

Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.(AU)

Proteomic characterization of Naja mandalayensis venom

Background Naja mandalayensis is a spitting cobra from Myanmar. To the best of our knowledge, no studies on this venom composition have been conducted so far. On the other hand, few envenomation descriptions state that it elicits mainly local inflammation in the victims' eyes, the preferred target of this spiting cobra. Symptoms would typically include burning and painful sensation, conjunctivitis, edema and temporary loss of vision. Methods We have performed a liquid-chromatography (C18-RP-HPLC) mass spectrometry (ESI-IT-TOF/MS) based approach in order to biochemically characterize N. mandalayensis venom. Results A wide variety of three-finger toxins (cardiotoxins) and metallopeptidases were detected. Less abundant, but still representative, were cysteine-rich secretory proteins, L-amino-acid oxidases, phospholipases A2, venom 5'-nucleotidase and a serine peptidase inhibitor. Other proteins were present, but were detected in a relatively small concentration. Conclusion The present study set the basis for a better comprehension of the envenomation from a molecular perspective and, by increasing the interest and information available for this species, allows future venom comparisons among cobras and their diverse venom proteins.(AU)

Proteomic characterization of Naja mandalayensis venom

Naja mandalayensis is a spitting cobra from Myanmar. To the best of our knowledge, no studies on this venom composition have been conducted so far. On the other hand, few envenomation descriptions state that it elicits mainly local inflammation in the victims' eyes, the preferred target of this spiting cobra. Symptoms would typically include burning and painful sensation, conjunctivitis, edema and temporary loss of vision. Methods We have performed a liquid-chromatography (C18-RP-HPLC) mass spectrometry (ESI-IT-TOF/MS) based approach in order to biochemically characterize N. mandalayensis venom. Results A wide variety of three-finger toxins (cardiotoxins) and metallopeptidases were detected. Less abundant, but still representative, were cysteine-rich secretory proteins, L-amino-acid oxidases, phospholipases A2, venom 5'-nucleotidase and a serine peptidase inhibitor. Other proteins were present, but were detected in a relatively small concentration. Conclusion The present study set the basis for a better comprehension of the envenomation from a molecular perspective and, by increasing the interest and information available for this species, allows future venom comparisons among cobras and their diverse venom proteins.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Estudo clínico do envenenamento experimental por Micrurus surinamensis em cobaias (Cavia porcellus)

Micrurus surinamensis é uma espécie serpente peçonhenta encontrada na América do Sul, região da Amazônia, conhecida popularmente como coral verdadeira ou cobra-coral. Esta serpente, que difere das demais principalmente por seu hábito semi-aquático e alimentação baseada em peixes, possui veneno pouco estudado, especialmente em relação aos seus efeitos em mamíferos. Acidentes envolvendo corais verdadeiras são sempre graves pela composição do veneno que possuem, tais como toxinas de estrutura three finger toxin (3FTx) e fosfolipases (PLA2) em diferentes proporções. Não existem estudos que retratam o quadro clínico neurológico, os efeitos cardíacos, as alterações sanguíneas e as lesões microscópicas que podem ser provocadas pela peçonha de M. surinamensis. Desse modo, esta pesquisa teve como objetivo a avaliação de cobaias (Cavia porcellus) envenenadas experimentalmente com veneno da serpente M. surinamensis, enfatizando os sistemas nervoso e cardíaco (eletrocardiográfico), além da avaliação de exames laboratoriais sanguíneos e teciduais (microscopia óptica e eletrônica). Para tal, foram utilizados 12 animais machos distribuídos em dois grupos (n=6), denominados grupo veneno e grupo controle. Cada animal do grupo veneno recebeu 0,75 µg/g de peso vivo de veneno de M. surinamensis diluídos em 0,2 ml de PBS/BSA, injetado via intramuscular (IM). O grupo controle, que serviu como testemunha, recebeu 0,2 ml de PBS/BSA via IM. Os exames, físico geral e especificamente neurológico, foram realizados antes (Tempo zero T0) e após a inoculação do veneno ou PBS/BSA aos 30 minutos (Tempo 1 - T1), uma hora (Tempo 2 - T2) e duas horas (Tempo Final TF), aproximadamente. Eletrocardiogramas e exames hematológicos foram realizados nos tempos T0 e TF. Após 2h do envenenamento (TF), os animais que não foram a óbito foram eutanasiados, realizadas necropsia e coleta de órgãos para análise microscópica. Os animais que receberam veneno de M. surinamensis apresentaram alterações neurológicas com comprometimento do sistema nervoso central, periférico e autônomo, marcados por dificuldade respiratória progressiva, alterações motoras e no estado mental, quadriplegia flácida, paralisia muscular total e morte. O eletrocardiograma revelou graves alterações arrítmicas, como bloqueios atrioventriculares de 1º, 2º e 3º graus, extrassístoles supra e ventricular. No exame hematológico, o leucograma mostrou aumento na contagem de leucócitos totais causado especificamente por aumento de heterófilos e monócitos, além da diminuição de linfócitos. Os exames bioquímicos séricos revelaram aumento significativo das enzimas creatina quinase e sua fração MB, aspartato aminotransferase e lactato desidrogenase indicando lesão muscular esquelética e cardíaca. Na microscopia ótica e eletrônica foi visualizada necrose no músculo esquelético em dois animais que receberam veneno de M. surinamensis, todavia, ruptura das fibras musculares esqueléticas e cardíacas, somente foram visualizadas na microscopia eletrônica. Conclui-se que, o veneno de M. surinamensis em cobaias possui atividade neurotóxica e miotóxica super-aguda (até 2h) capaz de causar graves alterações neurológicas e elétricas cardíacas, culminando com o óbito dos animais.

Micrurus surinamensis is a species of poisonous snake found in South America, in the Amazon region and popularly known as true coral or coral snake. This serpent differs from the others mainly due to it is semi-aquatic nature and fish-based feeding and there is a lack of studies on its venom especially in relation to its effects on mammals. Accidents involving corals are always severe because of the composition of its venom, which has three finger toxins (3FTx) and phospholipases (PLA2) in different proportions. There are no studies involving the neurological clinical condition, the cardiac effects, the blood alterations and the microscopic lesions that may be caused by the venom of M. surinamensis. Thus, the aim of this study was to evaluate guinea pigs (Cavia porcellus) experimentally poisoned with M. surinamensis venom, emphasizing the nervous and the cardiac (electrocardiographic) systems, in addition to evaluating blood tests and tissue exams (optical and electron microscopy). In order to perform the trial, twelve male animals were randomly divided into two groups (n = 6): control group and venom group. Each animal in the venom group received 0.75 g/g of M. surinamensis venom diluted in 0.2 ml PBS / BSA, inoculated intramuscularly (IM). The control group received 0.2 ml of PSA / BSA intramuscularly (IM). All animals underwent general physical examination, more specifically neurological exam, which was performed prior to venom or PBS/BSA inoculation (Time zero T0), 30 minutes after inoculation (Time 1 - T1), one hour after inoculation (Time 2 - T2) and approximately two hours after inoculation (Final Time - TF). Electrocardiogram and hematological tests were performed at T0 and TF. The animals which did not die two hours after the poisoning (TF), were euthanized and their corpses underwent necropsy and organ collection for microscopic analysis. The animals that received M. surinamensis venom presented neurological alterations with central, peripheral and autonomic nervous system involvement, marked by progressive respiratory difficulty, motor and mental state alterations, flaccid quadriplegia, total muscle paralysis and death. The electrocardiogram revealed severe arrhythmic alterations, such as 1st, 2nd and 3rd degree atrioventricular block, supra and ventricular extrasystoles. The leukogram showed an increase in total leukocytes count, specifically due to an increase in heterophils and monocytes, in addition to a decrease in lymphocytes. Serum biochemistry revealed a significant increase in creatine kinase enzyme and its MB fraction, aspartate aminotransferase and lactate dehydrogenase, indicating skeletal and cardiac muscle injury. Optical microscopy and transmission electron microscopy showed skeletal muscle necrosis in two animals of the venom group, however, rupture of skeletal and cardiac muscle fibers were observed only in electron microscopy. In conclusion, the M. surinamensis venom has neurotoxic and super-acute myotoxic activity (2h) in guinea pigs and is capable of causing severe neurological and cardiac electrical changes culminating in the death of the animals.