snake identification
59339903
Rapid Identification of Phospholipase A2 Transcripts from Snake Venoms
Phospholipase A2 (PLA2) is a major component in snake venoms and it is found in many different isoforms. To identify transcripts encoding different PLA2 isoforms, we developed a simple, rapid procedure. Total RNA was extracted from the venoms of three cottonmouth snakes and two diamondback rattlesnakes, and further reverse transcribed into complementary DNA (cDNA) Using one pair of cottonmouth PLA2 specific primers and Reverse Transcription Polymerase Chain Reaction (RT PCR) technique, we identified 27 unique full length PLA2 transcripts, including nine sequences identical to the previously documented ones in the database and one novel GIII like PLA2. Two common transcripts respectively encoding Asp49 and Lys49 PLA2 isoforms were identified in all three cottonmouth venoms that contain more PLA2 transcripts than diamondback rattlesnake venoms. The placement of cloned PLA2 transcripts in snake venom PLA2s was further discussed by phylogenetic analysis. The procedure developed in this study paves the way for accelerated acquisition of transcriptome data on any other venom toxin families. The results obtained are crucial for insight into the structure and function of PLA2 isoforms for scientific and potential therapeutic purposes.
Toxins
Ying Jia Pablo Olvera F Rangel Bianca Mendez Samir Reddy
203892089
High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches
Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed phase liquid chromatography, and a post column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384 well plates before direct analysis using a plasma coagulation assay. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In depth identification analysis from seven medically important venoms, via mass spectrometry and nanoLC MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors. Author summary Snakebite is a neglected tropical disease that results in more than 100,000 deaths every year. Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to suffering life threating hemorrhage. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood coagulation cascade and based on the initial results and the medical relevance of the species, seven venoms were selected for in depth analysis of the responsible toxins using advanced identification techniques. Our findings reveal a number of anticoagulant toxins that have not yet been reported before as such. The methodology described herein not only enables the identification of both known and unknown toxins that cause impairment of the blood coagulation, but offers a throughput platform to effectively screen for inhibitory molecules relevant for the development of next generation snakebite treatments.
Julien Slagboom Marija Mladic Chunfang Xie Freek J Vonk Govert W Somsen Nicholas R Casewell Jeroen Kool
49342762
Proteomic characterization of six Taiwanese snake venoms: Identification of species specific proteins and development of a SISCAPA MRM assay for cobra venom factors.
Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Daboia russelii siamensis, Bungarus multicinctus and Naja atra are the six medically important venomous snake species in Taiwan. In this study, we characterized and compared their venom protein profiles using proteomic approaches. The major snake venom proteins were identified by GeLC MS/MS and the total venom proteome was characterized by in solution digestion coupled with LC MS/MS. A total of 27 52 proteins, categorized into 23 protein families, were identified in each snake's venom. The major venom components found in Viperidae species (D. acutus, T. stejnegeri, P. mucrosquamatus and D. russelii) were C type lectin, snake venom serine proteinase, venom metalloproteinase and phospholipase A2, whereas three finger toxin and phospholipase A2 were the major components detected in the venom of Elapidae snakes (B. multicinctus and N. atra) This study also provided the first demonstration of some low abundance proteins in these six snake venoms, including 5' nucleotidase, glutaminyl peptide cyclotransferase and phosphodiesterase, among others. Furthermore, we found that cobra venom factor (CVF) is a cobra specific protein. We produced anti peptide antibodies against CVF and used it to develop a highly sensitive SISCAPA MRM assay for quantifying CVF. The limit of detection and lower limit of quantification were 3.2 and 9.6 attomoles, respectively. This assay was used to precisely quantify CVF in 1 mg crude venom proteins from three Naja species and king cobra. The amount of CVF varied from 0.9 to 54.36 femtomoles (equivalent to 0.16 10.03 mg/g of venom protein) BIOLOGICAL SIGNIFICANCE There are six medically significant venomous snakes in Taiwan. The venoms of the four Viperidae species (Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus and Daboia russelii siamensis) cause local tissue swelling; this symptom is also seen in N. atra envenomation in humans, potentially complicating the differential diagnosis of envenomation by N. atra and Viperidae species. Thus, characterization of the venom proteomes of the six Taiwanese snakes, including the relative abundance of the major components and species specific protein(s) in each venom type, could be useful for future venom research, including the development of new assay(s) for detecting snake species specific venom protein(s) and new type(s) of antivenom.
Journal of proteomics
Chien-Chun Liu Chih-Chuan Lin Yung-Chin Hsiao Po-Jung Wang Jau-Song Yu
51612656
Rapid ligand fishing for identification of acetylcholinesterase binding peptides in snake venom reveals new properties of dendrotoxins
ABSTRACT Acetylcholinesterase (AChE) from Electrophorus electricus (eel) was immobilized on the surface of amino modified paramagnetic beads to serve as a model for the development, validation and application of a new affinity based ligand fishing assay for the discovery of bioactive peptides from complex protein mixtures such as venoms. Nano liquid chromatography mass spectrometry (nanoLC MS) was used for the analysis of trapped peptides. Using enzyme functionalized beads, the ligand fishing assay was evaluated and optimized using a peptide reference mixture composed of one acetylcholinesterase binder (fasciculin II) and five non binders (mambalgin 1, angiotensin II, bradykinin, cardiotoxin and &agr; bungarotoxin) As proof of concept, snake venom samples spiked with fasciculin II demonstrated assay selectivity and sensitivity, fishing the peptide binder from complex venom solutions at concentrations as low as 1.0 &mgr;g/mL. As negative controls for method validation, venoms of four different snake species, not known to harbor AChE binding peptides, were screened and no AChE binders were detected. The applicability of the ligand fishing assay was subsequently demonstrated with venom from the black mamba, Jameson's mamba and western green mamba (Dendroaspis spp. which have previously been reported to contain the AChE binding fasciculins. Unknown peptides (i.e. not fasciculins) with affinity to AChE were recovered from all mamba venoms tested. Tryptic digestion followed by nano LC MS analysis of the material recovered from black mamba venom identified the peptide with highest AChE binding affinity as dendrotoxin I, a pre synaptic neurotoxin previously not known to interact with AChE. Co incubation of AChE with various dendrotoxins in vitro revealed reduced inactivation of AChE activity over time, thus demonstrating that these toxins stabilize AChE. HighlightsAn affinity based assay was developed for the screening of AChE peptide binders in animal venoms.The assay was validated and applied to snake venoms.AChE binders were retrieved and analyzed by nanoLC MS.Dendrotoxin I from black mamba venom was revealed to bind to AChE allosterically and to stabilize AChE activity in vitro.
Toxicon official journal of the International Society on Toxinology
Kenia Lourenco Vanzolini Stuart Ainsworth Ben Bruyneel Volker Herzig Mitchell G L Seraus Govert W Somsen Nicholas R Casewell Quezia Bezerra Cass Jeroen Kool
53241705
Identification of a linear B cell epitope in the catalytic domain of bothropasin, a metalloproteinase from Bothrops jararaca snake venom
&NA; Bothropasin is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca venom, the snake responsible for most bites in Southeastern Brazil. SVMPs, such as bothropasin, are involved in the main bothropic envenoming symptoms, which include hemorrhage, inflammation, necrosis and blood coagulation deficiency. B cell epitope mapping of SVMPs can lead to the identification of peptides capable of inducing neutralizing antibodies without causing toxic effects, therefore improving anti venom production. Here, using the SPOT synthesis technique, we have identified an epitope located in the catalytic domain of bothropasin (202KARMYELANIVNEILRYLYMH222) which was synthesized and named BotEp1. The peptide was used to immunize Swiss mice and Anti BotEp1 serum cross reacted with bothropasin and crude venoms from B. jararaca and B. atrox venoms. Furthermore, Anti BotEp1 antibodies were able to completely neutralize the hemorrhagic activity of a chromatographic fraction from B. jararaca venom, which contains hemorrhagic SVMPs. In addition, the coagulation activity of the hemorrhagic fraction showed to be diminished when tested in serum from rabbit immunized with BotEp1 (compared to serum from non immunized animal) Our results show the identification of neutralizing epitopes in bothropasin and provide basis for the use of synthetic peptides to improve the production of immunotherapeutics.
Molecular immunology
Denis Alexis Molina Molina Clara Guerra-Duarte Dayane Lorena Naves de Souza Fernanda Costal-Oliveira Giovana Reis de Avila Vanete Thomaz Soccol Ricardo Andrez Machado-de-Avila Carlos Chavez-Olortegui
52145905
Detection and identification of antibacterial proteins in snake venoms using at line nanofractionation coupled to LC MS.
This study describes the application of at line nanofractionation to the screening of snake venoms for antibacterial activity against Gram positive and Gram negative bacteria, the detection of proteins of interest, and their partial or full identification. A method was developed to identify bioactive peptides in crude snake venoms based on reversed phase liquid chromatography (LC) with parallel nanofractionation onto 384 well plates and mass spectrometry (MS) Bioactivity assays were based on a resazurin reduction assay. Accurate masses of the bioactive peptides were determined, and peptides were then identified via nanoLC MS/MS analysis of tryptic digests, allowing full or partial identification of the bioactive proteins. Crude venoms from 41 species were screened for their antibacterial bioactivity. Venoms showing the highest activity were further screened using at line nanofractionation, which resulted in the elucidation of 28 bioactive proteins.
Toxicon official journal of the International Society on Toxinology
Marija Mladic Julien Slagboom Jeroen Kool Freek J Vonk Gilles P van Wezel Michael K Richardson
69617074
A Survey on Snake Species Identification using Image Processing Technique
Snakes play essential role in the eco system and are not indispensable. Deforestation are disturbing the habitat of snakes. Identification of the snake is important in planning treatment in certain areas of the world, but is not always possible. Snake bite is more common, local knowledge not may be sufficient to recognize the snake. Attempting to catch or kill the offending snake also puts one at risk and generally is not recommended. To prevent this proposed system is introduce to tackle this problem which can identify accurately the species of snake. General Terms Snake species identification, Object Detection, Moving Object Detection, Object Classification, YOLO algorithm, Image processing
Pranalini A Joshi Dhanesh Sarpale R G Sapkal Apeksha Rajput
89842928
Identification of sea snake meat adulteration in meat products using PCR RFLP of mitochondrial DNA
Abstract PCR RFLP based technique for identification of sea snakes in Thai waters was achieved by developing species specific markers. To distinguish between sea snake species, the PCR products of cytochrome b (Cyt b) 12S and 16S rRNA were sequenced and cut with different restriction endonuclease, Alu I and Hinf I. Each enzyme generated different sized fragments which specific to Cyt b of eight sea snake species. However, the identical pattern was found among Hydrophis group. This result could be resolved by using these enzymes 12S rRNA digestion. This technique was successfully applied to blood, shed skin, raw meat, cooked meat, sea snake fish binary admixture, and sea snake pork binary admixture. Hence, it could be applied for identification when sea snake meat adulteration in meat products and sold as meatballs to reduce production costs. Hopefully, this technique would improve sea snake species identification when morphological examination is no longer possible because the animals are already processed. This is very important to track when sea snake species are being hunted and also used to assess the conservation and management of the sea snakes in Thai waters, especially the Gulf of Thailand.
Sunutcha Suntrarachun Lawan Chanhome Montri Sumontha
8701305
A comparative study between standard Back Propagation and Resilient Propagation on snake identification accuracy
Identifying types of snakes is crucial for appropriate anti venom administration. We developed a Snake Bite Diagnosing System based on standard Back Propagation and Resilient Propagation Neural Networks. These systems were capable in differentiating between venomous and non venomous snakes. The accuracy of both systems were analyzed and compared. The post development comparative studies revealed that the Resilient Propagation technique yielded 83.33% 90.00% and 90.00% respectively for mean squared error, number of epoch and parameter setting. Whereas the Standard Back Propagation produced 85.00% 88.30% and 86.87% respectively. High accuracy in both systems enables early identification of type of snake and immediate specific anti venom can be administered. Hence, reduces the rate of morbidity and mortality.
2011 IEEE International Symposium on IT in Medicine and Education
Shamimi A Halim Azlin Ahmad Norzaidah Binti Md Noh Mohd Shazuan B Md Ali Safudin Rashidi Ahmad
13057602
Rapid screening and identification of ACE inhibitors in snake venoms using at line nanofractionation LC MS
This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at line nanofractionation approach, which combines liquid chromatography (LC) mass spectrometry (MS) and pharmacology in one platform. After initial LC separation of a crude venom, a post column flow split is introduced enabling parallel MS identification and high resolution fractionation onto 384 well plates. The plates are subsequently freeze dried and used in a fluorescence based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC50 1.1 mM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC50 3.5 mM. The identified peptides possess a high sequence similarity to other bradykinin potentiating peptides (BPPs) which are known ACE inhibitors found in snake venoms.
Analytical and Bioanalytical Chemistry
Marija Mladic Tessa de Waal Lindsey Burggraaff Julien Slagboom Govert W Somsen Wilfried M A Niessen R Manjunatha Kini Jeroen Kool