Standard Quality Characteristics and Efficacy of a New Third-Generation Antivenom Developed in Colombia Covering Micrurus spp. Venoms.

In Colombia, Micrurus snakebites are classified as severe according to the national clinical care guidelines and must be treated with specific antivenoms. Unfortunately, these types of antivenoms are scarce in certain areas of the country and are currently reported as an unavailable vital medicine. To address this issue, La Universidad de Antioquia, through its spin-off Tech Life Saving, is leading a project to develop third-generation polyvalent freeze-dried antivenom. The goal is to ensure access to this therapy, especially in rural and dispersed areas. This project aims to evaluate the physicochemical and preclinical parameters (standard quality characteristics) of a lab-scale anti-elapid antivenom batch. The antivenom is challenged against the venoms of several Micrurus species, including M. mipartitus, M. dumerilii, M. ancoralis, M. dissoleucus, M. lemniscatus, M. medemi, M. spixii, M. surinamensis, and M. isozonus, following the standard quality characteristics set by the World Health Organization (WHO). The antivenom demonstrates an appearance consistent with standards, 100% solubility within 4 min and 25 s, an extractable volume of 10.39 mL, a pH of 6.04, an albumin concentration of 0.377 mg/mL (equivalent to 1.22% of total protein), and a protein concentration of 30.97 mg/mL. Importantly, it maintains full integrity of its F(ab')2 fragments and exhibits purity over 98.5%. Furthermore, in mice toxicity evaluations, doses up to 15 mg/mouse show no toxic effects. The antivenom also demonstrates a significant recognition pattern against Micrurus venoms rich in phospholipase A2 (PLA2) content, as observed in M. dumerilii, M. dissoleucus, and M. isozonus. The effective dose 50 (ED50) indicates that a single vial (10 mL) can neutralize 2.33 mg of M. mipartitus venom and 3.99 mg of M. dumerilii venom. This new anti-elapid third-generation polyvalent and freeze-dried antivenom meets the physicochemical parameters set by the WHO and the regulators in Colombia. It demonstrates significant efficacy in neutralizing the venom of the most epidemiologically important Micrurus species in Colombia. Additionally, it recognizes seven other species of Micrurus venom with a higher affinity for venoms exhibiting PLA2 toxins. Fulfilling these parameters represents the first step toward proposing a new pharmacological alternative for treating snakebites in Colombia, particularly in dispersed rural areas, given that this antivenom is formulated as a freeze-dried product.

Epidemiology of Snake Bites Linked with the Antivenoms Production in Colombia 2008-2020: Produced Vials Do Not Meet the Needs.

Introduction: Snakebite envenomation is a public health event of mandatory reporting in Colombia. It is considered a medical emergency in which the government must guarantee antivenom availability. We describe snakebite epidemiological figures in Colombia between 2008 and 2020 and correlate them with antivenom manufacturing figures to determine rate coverage and the need for antivenom. Methods: We performed an ecological study based on secondary official figures from the National Health Institute, the National Institute for Surveillance of Medicines and Foods, the National Administrative Department of Statistics and the Ministry of Health and Social Protection. Absolute and relative frequencies were calculated with 95% confidence intervals, position measurements, dispersion and central tendency. Results: Through our research, we revealed that in the last 13 years (2008-2020), there were an average of 4467 annual snakebite envenomation cases affecting all the departments in Colombia. Antioquia reported the highest number of snakebites with 647 (95% CI 588-706) cases per year. The population incidence per 100,000 inhabitants was 9.5; the highest rates were found in Vaupés at 116.1 and Guaviare at 79.24. During the last seven years (2014-2020) Colombia produced an average of 21,104 antivenom vials per year, while the annual demand for antivenom is estimated at 54,440 units needed to guarantee access. Discussion: Colombia does not produce sufficient vials to cover their needs, and this is why only 74.4% of accidents (out of the 92% not classified as dry bites) were treated, and even 9.7% of the severe accidents did not receive the specific treatment (8% of the victims were classified as dry bites). Figures support the regular antivenom shortages declared by the Ministry of Health and Social Protection in the last 13 years (11 health emergency declarations). New efforts are needed to: 1) boost the production of GMP-based high-quality antivenom, that covers the national needs and is made availability, 2) a better estimation method to calculate the need for antivenom in Colombia, and 3) implementation of production-distribution chains guaranteeing access in remote communities.

Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia.

Scorpion stings are a public health event in Colombia lacking official epidemiological data, and are considered a medical emergency. Despite the two local producers of antivenoms, neither of them is currently manufacturing scorpion antivenoms. We present the characterization of a lab-scale process to produce the first specific scorpion antivenom for Colombia, formulated to cover scorpion stings produced by Tityus pachyurus, Tityus asthenes, Tityus fuhrmanii, Centruroides spp. To do so, rabbits were immunized by subcutaneous injection with each venom using an immunization program of 3 months. After each rabbit reached the required IgG concentration, rabbits were bled, and plasma was separated by decantation under refrigeration. Immunoglobulins were purified from each hyperimmune plasma using a methodology including precipitation with ammonium sulfate, thermocoagulation, and purification through an ultrafiltration process using a ready-to-use and reusable laboratory crossflow tangential cassette with a polyethersulfone membrane. Each hyperimmune plasma was processed by being separated and freeze-dried at the end of the process. Rabbits were able to produce specific IgG antibodies recognizing the respective immunization venom; even an in vitro interspecies cross-recognition was detected. The separation and purification processes allowed us to obtain IgG products without considerable contaminants (except for albumin). The process was characterized, and critical stages were identified.

Improving the Annotation of the Venom Gland Transcriptome of Pamphobeteus verdolaga, Prospecting Novel Bioactive Peptides.

Spider venoms constitute a trove of novel peptides with biotechnological interest. Paucity of next-generation-sequencing (NGS) data generation has led to a description of less than 1% of these peptides. Increasing evidence supports the underestimation of the assembled genes a single transcriptome assembler can predict. Here, the transcriptome of the venom gland of the spider Pamphobeteus verdolaga was re-assembled, using three free access algorithms, Trinity, SOAPdenovo-Trans, and SPAdes, to obtain a more complete annotation. Assembler's performance was evaluated by contig number, N50, read representation on the assembly, and BUSCO's terms retrieval against the arthropod dataset. Out of all the assembled sequences with all software, 39.26% were common between the three assemblers, and 27.88% were uniquely assembled by Trinity, while 27.65% were uniquely assembled by SPAdes. The non-redundant merging of all three assemblies' output permitted the annotation of 9232 sequences, which was 23% more when compared to each software and 28% more when compared to the previous P. verdolaga annotation; moreover, the description of 65 novel theraphotoxins was possible. In the generation of data for non-model organisms, as well as in the search for novel peptides with biotechnological interest, it is highly recommended to employ at least two different transcriptome assemblers.

Linking toxicity and predation in a venomous arthropod: the case of Tityus fuhrmanni (Scorpiones: Buthidae), a generalist predator scorpion.

BACKGROUND: Scorpions are arachnids that have a generalist diet, which use venom to subdue their prey. The study of their trophic ecology and capture behavior is still limited compared to other organisms, and aspects such as trophic specialization in this group have been little explored. METHODS: In order to determine the relationship between feeding behavior and venom toxicity in the scorpion species Tityus fuhrmanni, 33 specimens were offered prey with different morphologies and defense mechanisms: spiders, cockroaches and crickets. In each of the experiments we recorded the following aspects: acceptance rate, immobilization time and the number of capture attempts. The median lethal dose of T. fuhrmanni venom against the three different types of prey was also evaluated. RESULTS: We found that this species does not have a marked difference in acceptance for any of the evaluated prey, but the number of capture attempts of spiders is higher when compared to the other types of prey. The immobilization time is shorter in spiders compared to other prey and the LD50 was higher for cockroaches. CONCLUSIONS: These results indicate that T. fuhrmanni is a scorpion with a generalist diet, has a venom with a different potency among prey and is capable of discriminating between prey types and employing distinct strategies to subdue them.

Linking toxicity and predation in a venomous arthropod: the case of Tityus fuhrmanni (Scorpiones: Buthidae), a generalist predator scorpion

Background: Scorpions are arachnids that have a generalist diet, which use venom to subdue their prey. The study of their trophic ecology and capture behavior is still limited compared to other organisms, and aspects such as trophic specialization in this group have been little explored. Methods: In order to determine the relationship between feeding behavior and venom toxicity in the scorpion species Tityus fuhrmanni, 33 specimens were offered prey with different morphologies and defense mechanisms: spiders, cockroaches and crickets. In each of the experiments we recorded the following aspects: acceptance rate, immobilization time and the number of capture attempts. The median lethal dose of T. fuhrmanni venom against the three different types of prey was also evaluated. Results: We found that this species does not have a marked difference in acceptance for any of the evaluated prey, but the number of capture attempts of spiders is higher when compared to the other types of prey. The immobilization time is shorter in spiders compared to other prey and the LD50 was higher for cockroaches. Conclusions: These results indicate that T. fuhrmanni is a scorpion with a generalist diet, has a venom with a different potency among prey and is capable of discriminating between prey types and employing distinct strategies to subdue them.(AU)

Linking toxicity and predation in a venomous arthropod: the case of Tityus fuhrmanni (Scorpiones: Buthidae), a generalist predator scorpion

Abstract Background: Scorpions are arachnids that have a generalist diet, which use venom to subdue their prey. The study of their trophic ecology and capture behavior is still limited compared to other organisms, and aspects such as trophic specialization in this group have been little explored. Methods: In order to determine the relationship between feeding behavior and venom toxicity in the scorpion species Tityus fuhrmanni, 33 specimens were offered prey with different morphologies and defense mechanisms: spiders, cockroaches and crickets. In each of the experiments we recorded the following aspects: acceptance rate, immobilization time and the number of capture attempts. The median lethal dose of T. fuhrmanni venom against the three different types of prey was also evaluated. Results: We found that this species does not have a marked difference in acceptance for any of the evaluated prey, but the number of capture attempts of spiders is higher when compared to the other types of prey. The immobilization time is shorter in spiders compared to other prey and the LD50 was higher for cockroaches. Conclusions: These results indicate that T. fuhrmanni is a scorpion with a generalist diet, has a venom with a different potency among prey and is capable of discriminating between prey types and employing distinct strategies to subdue them.

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach.

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

MS/MS analysis of four scorpion venoms from Colombia: a descriptive approach.

BACKGROUND: Scorpions are widely known for the neurotoxic effects of their venoms, which contain peptides affecting ionic channels. Although Colombia is recognized for its scorpion diversity, only a few studies are available describing the venom content. METHODS: In this descriptive study, we analyzed the MS/MS sequence, electrophoretic and chromatographic profile linked to a bioinformatics analysis of the scorpions Chactas reticulatus (Chactidae), Opisthacanthus elatus (Hormuridae), Centruroides edwardsii (Buthidae) and Tityus asthenes (Buthidae) from Colombia. RESULTS: Each scorpion showed a specific electrophoretic and chromatographic profile. The electrophoretic profiles indicate the presence of high molecular mass compounds in all venoms, with a predominance of low molecular mass compounds in the Buthidae species. Chromatographic profiles showed a similar pattern as the electrophoretic profiles. From the MS/MS analysis of the chromatographic collected fractions, we obtained internal peptide sequences corresponding to proteins reported in scorpions from the respective family of the analyzed samples. Some of these proteins correspond to neurotoxins affecting ionic channels, antimicrobial peptides and metalloproteinase-like fragments. In the venom of Tityus asthenes, the MSn analysis allowed the detection of two toxins affecting sodium channels covering 50% and 84% of the sequence respectively, showing 100% sequence similarity. Two sequences from Tityus asthenes showed sequence similarity with a phospholipase from Opisthacanthus cayaporum indicating the presence of this type of toxin in this species for the first time. One sequence matching a hypothetical secreted protein from Hottentotta judaicus was found in three of the studied venoms. We found that this protein is common in the Buthidae family whereas it has been reported in other families - such as Scorpionidae - and may be part of the evolutionary puzzle of venoms in these arachnids. CONCLUSION: Buthidae venoms from Colombia can be considered an important source of peptides similar to toxins affecting ionic channels. An interesting predicted antimicrobial peptide was detected in three of the analyzed venoms.

MS/MS analysis of four scorpion venoms from Colombia: a descriptive approach

Background: Scorpions are widely known for the neurotoxic effects of their venoms, which contain peptides affecting ionic channels. Although Colombia is recognized for its scorpion diversity, only a few studies are available describing the venom content. Methods: In this descriptive study, we analyzed the MS/MS sequence, electrophoretic and chromatographic profile linked to a bioinformatics analysis of the scorpions Chactas reticulatus (Chactidae), Opisthacanthus elatus (Hormuridae), Centruroides edwardsii (Buthidae) and Tityus asthenes (Buthidae) from Colombia. Results: Each scorpion showed a specific electrophoretic and chromatographic profile. The electrophoretic profiles indicate the presence of high molecular mass compounds in all venoms, with a predominance of low molecular mass compounds in the Buthidae species. Chromatographic profiles showed a similar pattern as the electrophoretic profiles. From the MS/MS analysis of the chromatographic collected fractions, we obtained internal peptide sequences corresponding to proteins reported in scorpions from the respective family of the analyzed samples. Some of these proteins correspond to neurotoxins affecting ionic channels, antimicrobial peptides and metalloproteinase-like fragments. In the venom of Tityus asthenes, the MSn analysis allowed the detection of two toxins affecting sodium channels covering 50% and 84% of the sequence respectively, showing 100% sequence similarity. Two sequences from Tityus asthenes showed sequence similarity with a phospholipase from Opisthacanthus cayaporum indicating the presence of this type of toxin in this species for the first time. One sequence matching a hypothetical secreted protein from Hottentotta judaicus was found in three of the studied venoms. We found that this protein is common in the Buthidae family whereas it has been reported in other families - such as Scorpionidae - and may be part of the evolutionary puzzle of venoms in these arachnids. Conclusion: Buthidae venoms from Colombia can be considered an important source of peptides similar to toxins affecting ionic channels. An interesting predicted antimicrobial peptide was detected in three of the analyzed venoms.(AU)

Economic Evaluations of Interventions for Snakebites: A Systematic Review.

BACKGROUND: Snakebite is an often-neglected event with a high rate of mortality and is concentrated in poor areas. We aimed to assess the economic impact and health effects of the implementation of interventions for snakebites through a systematic review of the scientific literature. METHODS: Thirty search strategies were conducted in seven databases, applying PRISMA's identification, screening, selection, and inclusion phases. The reproducibility of the selection of studies and the extraction of information were guaranteed. The methodological quality was evaluated using the Consolidated Health Economic Evaluation Reporting Standards. Qualitative synthesis and meta-analysis were performed for determining the average cost-effectiveness (ACE) for each death and disability-adjusted life years (DALY) avoided. RESULTS: Six cost-effectiveness studies were included for the supply of antivenom (AV), taken as outcomes on days of hospitalization or in ICU, death and DALYs avoided. All studies only included institutional costs, and majority of them did not specify the analytical model or economic evaluation parameters and did not perform uncertainty analyses. The management protocol standardization with interdisciplinary attention improves ACE of AV. Cost-effectiveness ratio (CER) of treatment with AV was USD 1253 (constant value for the year 2017, adjusted by purchasing power parity) for each death avoided and USD 51 per DALY avoided. CONCLUSION: High cost-effectiveness of the AV treatment for snakebites was evidenced, which shows that the allocation of resources for this event should be a healthcare priority in addition to implementation of strategies that improve the access to, opportunity, and quality of hospital and pre-hospital care and reduce the cost of AV.

Venomic, Transcriptomic, and Bioactivity Analyses of Pamphobeteus verdolaga Venom Reveal Complex Disulfide-Rich Peptides That Modulate Calcium Channels.

Pamphobeteus verdolaga is a recently described Theraphosidae spider from the Andean region of Colombia. Previous reports partially characterized its venom profile. In this study, we conducted a detailed analysis that includes reversed-phase high-performance liquid chromatography (rp-HPLC), calcium influx assays, tandem mass spectrometry analysis (tMS/MS), and venom-gland transcriptome. rp-HPLC fractions of P. verdolaga venom showed activity on CaV2.2, CaV3.2, and NaV1.7 ion channels. Active fractions contained several peptides with molecular masses ranging from 3399.4 to 3839.6 Da. The tMS/MS analysis of active fraction displaying the strongest activity to inhibit calcium channels showed sequence fragments similar to one of the translated transcripts detected in the venom-gland transcriptome. The putative peptide of this translated transcript corresponded to a toxin, here named ω-theraphositoxin-Pv3a, a potential ion channel modulator toxin that is, in addition, very similar to other theraphositoxins affecting calcium channels (i.e., ω-theraphotoxin-Asp1a). Additionally, using this holistic approach, we found that P. verdolaga venom is an important source of disulfide-rich proteins expressing at least eight superfamilies.

Characterization of the Venom of C. d. cumanesis of Colombia: Proteomic Analysis and Antivenomic Study.

The Colombian rattlesnake Crotalus durissus cumanensis is distributed in three geographic zones of the country: the Atlantic Coast, the upper valley of the Magdalena River, and the eastern plains of the Colombian Orinoquía. Its venom induces neurological symptoms, such as eyelid ptosis, myasthenic facies, and paralysis of the respiratory muscles, which can lead to death. Identification and analysis of C. d. cumanensis showed nine groups of proteins responsible for the neurotoxic effect, of which the crotoxin complex was the most abundant (64.71%). Immunorecognition tests of C. d. cumanensis showed that the use of a commercial antivenom manufactured in Mexico resulted in immunoreactivity.

Target-Specificity in Scorpions; Comparing Lethality of Scorpion Venoms across Arthropods and Vertebrates.

Scorpions use their venom in defensive situations as well as for subduing prey. Since some species of scorpion use their venom more in defensive situations than others, this may have led to selection for differences in effectiveness in defensive situations. Here, we compared the LD50 of the venom of 10 species of scorpions on five different species of target organisms; two insects and three vertebrates. We found little correlation between the target species in the efficacy of the different scorpion venoms. Only the two insects showed a positive correlation, indicating that they responded similarly to the panel of scorpion venoms. We discuss the lack of positive correlation between the vertebrate target species in the light of their evolution and development. When comparing the responses of the target systems to individual scorpion venoms pairwise, we found that closely related scorpion species tend to elicit a similar response pattern across the target species. This was further reflected in a significant phylogenetic signal across the scorpion phylogeny for the LD50 in mice and in zebrafish. We also provide the first mouse LD50 value for Grosphusgrandidieri.

Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family.

The objective of this study was to characterize six different scorpion venoms using biological and biochemical methods, including a preliminary MS/MS and a post-translational modifications analysis. Despite the diversity of scorpion species of medical importance in Africa and Colombia, the venoms of these arachnids have been poorly studied in these two regions. We report the biochemical, electrophoretic, chromatographic profile, internal peptide sequences with a post-translational modification report, and a preliminary antitumor activity of five different scorpions of the Buthidae family, Androctonus amoreuxi, Babycurus jacksoni, Grosphus grandidieri, Hottentotta gentili and Tityus fuhrmanni, and one of the Scorpionidae family Pandinus imperator. No L-amino oxidase activity was detected in the evaluated venoms. Proteolytic activity using azocasein was detected only in G. grandidieri and P. imperator, indicating the possible presence of metalloproteinases in these two venoms. Proteolytic activity using NOBA was detected in all venoms indicating the possible presence of serine-proteinases. Phospholipase A2 activity was detected in the venoms of P. imperator, G. grandidieri, H. gentili and A. amoreuxi, with P. imperator venom being the most active. All venoms analyzed contained defensin-like proteins, alpha toxins, metalloproteinases, neuropeptides, DBP affecting ion channels, DBP with antimicrobial activity, among others. Venoms from P. imperator, G. grandidieri and T. fuhrmanni showed a dose-dependent cytotoxic activity over MCF-7 cells. Only two isolated RP-HPLC fractions from P. imperator and T. fuhrmanni showed cytotoxic activity over MCF-7. No cytotoxic activity was found in the venoms from A. amoreuxi, B. jacksoni, and H. gentili.

Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga.

Theraphosidae spider venoms are well known for possess a complex mixture of protein and non-protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenlty described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF-hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity.

Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga

Theraphosidae spider venoms are well known for possess a complex mixture of protein and non -protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenity described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF-hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity

Efecto citotóxico de fosfolipasas A2 del veneno de Crotalus durissus cumanensis de Colombia / Cytotoxic effect of A2 phospholipases of the venom of Crotalus durissus cumanensis from Colombia / Efeito citotóxico da fosfolipase A2 do veneno de Crotalus durissus cumanensis da Colômbia

Introducción. Los venenos de serpientes representan una fuente importante de proteínas y péptidos, los cuales exhiben diversas actividades biológicas, tales como antibacterianas, antiparasitarias, antivi-rales, antitumorales, antifúngicas y contra la agregación plaquetaria, entre otras.Las fosfolipasas A2 presentes en los venenos de serpientes son las proteínas más estudiadas en estos modelos. Se ha demostrado que las fosfolipasas A2, activas e inactivas, poseen actividad catalítica contra células tumorales. Objetivo. Aislar, purificar y caracterizar la fosfolipasa A2 del veneno de Crotalus durissus cumanensis para evaluar su actividad antitumoral in vitro. Materiales y métodos. El aislamiento, la purificación y la identificación de la crotoxina B se hizo mediante la cromatografía de exclusión molecular, la cromatografía líquida de alto rendimiento de fase inversa (Reversed Phase High-Performance Liquid Chromatography, RP-HPLC) y la espectrometría de masas. El efecto citotóxico sobre células tumorales (K562) y células normales (células mononucleares de sangre periférica) se determinó utilizando la técnica de MTT. Resultados. La separación y posterior identificación de la crotoxina B del veneno de C. d. cumanensis de Colombia, permitieron evidenciar que esta fosfolipasa A2 posee efecto citotóxico sobre las células mononucleares de sangre periférica con una dosis de 18,23 ± 0,57 µg/ml, mientras que, para las células K562, fue de 2,34 ± 0,199 µg/ml. Conclusiones. Los resultados sugieren la posibilidad de utilizar la crotoxina B aislada del veneno de C. d. cumanensis como un posible recurso terapéutico para su aplicación en humanos.

Introduction. Snake venoms are an important source of proteins and peptides, which display various biological activities such as antibacterial, antiparasitic, antiviral, antitumor, antifungal and against platelet aggregation, among others.Phospholipases A2 present in snake venoms are the most studied proteins in these models. Active and inactive A2 phospholipases have been shown to possess catalytic activity against tumor cells. Objective. To isolate, purify and characterize the phospholipase A2 of the venom of Crotalus durissus cumanensis to evaluate its in vitro antitumor activity. Materials and methods. Isolation, purification and identification of crotoxin B was done with Size Exclusion Chromatography, Reversed Phase High-Performance Liquid Chromatography, RP-HPLC, and Mass Spectrometry. The cytotoxic effect on tumor cells (K562) and normal cells (peripheral blood mononuclear cells) was determined using the MTT technique. Results. The separation and subsequent identification of crotoxin B, found in the venom of C. d. cumanensis from Colombia, showed that this phospholipase A2 has a cytotoxic effect on peripheral blood mononuclear cells at a dose of 18.23 ± 0.57 µg / ml, whereas for K562 cells, it was 2.34 ± 0.199 µg/ml Conclusions. The results suggest the use of crotoxin B, isolated from the venom of C. d. cumanensis, as a possible therapeutic resource for human application.

Introdução. Os venenos da serpentes constituem uma importante fonte de proteínas e péptidos, os quais exibem várias actividades biológicas, tais como agentes antibacterianos, antiparasitárias, antivi-rais, antitumorais, antifúngicas e contra a agregação de plaquetas, entre outros. As fosfolipases A2 presentes no veneno da serpentes são as proteínas mais estudadas nestes modelos. Tem sido demostrado que as fosfolipases A2, activas e inactivas, possuem actividade catalítica contra células tumorais. Objetivo. Isolar, purificar e caracterizar a fosfolipase A2 do veneno da Crotalus durissus cumanensis para avaliar a sua actividade anti-umoral in vitro. Materiais e métodos. O isolamento, a purificação e identificação da crotoxina B foi realizada por cromatografia de exclusão molecular, cromatografia líquida de alta eficiência de fase reversa (Reversed Phase High-Performance Liquid Chromatography, RP-HPLC) e espectrometria de massa. O efeito cito-tóxico sobre células tumorais (K562) e células normais (células mononucleares do sangue periférico) foi determinada usando a técnica de MTT. Resultados. A Separação e subsequente identificação da crotoxina B do veneno da C. d. cumanensis da Colômbia, permitiu constatar que esta fosfolipase A2 tem um efeito citotóxico em células mono-nucleares de sangue periférico, com uma dose de 18,23 ± 0,57 µg/ ml, enquanto que para as células K562, foi 2,34 ± 0,199 ug/ml. Conclusões. Os resultados sugerem a possibilidade de utilizar crotoxina B isolada a partir do veneno da C. d. cumanensis como recurso para o potencial uso terapêutico em humanos.

Venom from Opisthacanthus elatus scorpion of Colombia, could be more hemolytic and less neurotoxic than thought.

We report the first biochemical, biological, pharmacological and partial proteomic characterization studies of the Opisthancanthus elatus venom (Gervais, 1844) from Colombia. The Reverse Phase High-Performance Liquid Chromatography venom profile showed 28 main well-defined peaks, most eluting between 20 and 45min (18-30% of acetonitrile, respectively). High-resolution mass analysis indicates the presence of 106 components ranging from 806.59742Da to 16849.4139Da. O. elatus venom showed hemolytic activity and hydrolyzed the specific substrate BapNa suggesting the presence of proteins with serine-protease activity. Collected RP-HPLC fractions eluting at 52.6, 55.5, 55.8, 56.2, and 63.9min (PLA2 region between 33 and 40% of acetonitrile), showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of compounds with phospholipases A2 activity. These RP-HPLC fractions, showed molecular masses values up to 13978.19546Da, corroborating the possible presence of the mentioned enzymes. Tryptic digestion and MS/MS analysis showed the presence of a phospholipase like fragment, similar to on described in other Opisthacanthus genus studies. No coagulant activity was observed. No larvicidal or antimicrobial activity was observed at concentrations evaluated. Lethal and toxic activity is expected at doses above 100mg/kg, no neurotoxic effects were detected at lower doses. In conclusion, O. elatus exhibits a venom with a predominant phospholipase A2 activity than thought; mammal's neurotoxic activity is expected above the 100mg/kg, which is very high compared to the venom from other neurotoxic scorpions.

Aguapanela, a new tarantula genus from the Colombian Andes (Araneae, Theraphosidae).

A new monotypic genus of Theraphosidae is described from Colombia: Aguapanela Perafán & Cifuentes gen. nov. with only the type species Aguapanela arvi Perafán, Cifuentes & Estrada sp. nov., from Caldas and Medellin, Antioquia, Colombian Andes. The new genus differs from other theraphosid spiders mainly in the presence of stridulatory setae on the palps and legs I and II, together with the presence of type III and IV urticating setae. Males lack a tibial apophysis on leg I and have a simple palpal bulb with the subtegulum less extended than usual in Theraphosinae, elongated curved embolus, ventrally concave, and with two prolateral keels very flat and developed on the dorsal edge. The female spermathecae have two digitiform elongated and granulated seminal receptacles attached to a semicircular wide membranous base. We describe, diagnose and illustrate the new genus and give some biological remarks. Morphological, systematic and biogeographic aspects are discussed. Chromatographic and electrophoretic profiles of its venom are analyzed.