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Bites of "nonvenomous" snakes can sometimes be mistaken for the bites of venomous snakes. As an example of this confusion, this report describes confirmed bites by Philodryas olfersii and Bothrops jararaca. In the first case, a 55-y-old man with a history of controlled hypertension was bitten on his right forearm by P olfersii. Physical examination revealed extensive edema, erythema, and widespread ecchymoses throughout his right upper limb. Laboratory tests indicated leukocytosis and high D-dimer levels, but normal coagulation, suggestive of a resolved recent coagulopathy. He received only supportive treatment. In the second case, a healthy 35-y-old man was bitten by B jararaca. Although the anatomic region of the bite and the results of physical examination were similar to those in the first case, laboratory tests showed mild coagulopathy, leukocytosis, and high D-dimer levels. The patient was treated with antivenom. In both cases, the 20-min whole blood clotting test results were normal. Patients bitten by P olfersii may present with local symptoms resembling B jararaca envenomation. Without snake identification and the detection of venom-induced consumption coagulopathy, especially in places where the 20-min whole blood clotting test is the only clotting test available, it is almost impossible to establish an accurate and safe differential diagnosis. In this context, the best alternative is to take the risk of prescribing antivenom for a possible P olfersii bite rather than failing to do so for a real Bothrops bite. Late treatment for Bothrops bite can result in severe complications and sequelae.

Disintegrins are a group of cysteine-rich proteins found in a wide variety of snake venoms. These proteins selectively bind to integrins, which play a fundamental role in the regulation of many physiological and pathological processes. Here, we report the NMR chemical shift assignments for 1H, 15N, and 13C nuclei in the backbone and side chains of recombinant disintegrin Jarastatin (rJast), which was further validated by secondary structure prediction using the TALOS-N server. Taken together, these data are essential to perform NMR-based experiments, including structure determination, backbone dynamics, mapping ligand sites and enabling a deeper understanding of the effect of hydrophobic surface clusters, which are important elements to stabilize some 3D proteins structure/folding.

The majority (90%) of the snakebite envenomation in Brazil accounts for Bothrops from the Viperidae family. Some snake venom serine proteases provoke blood coagulation in ophidian accident victims because of their fibrinolytic activity, one of those proteases from Bothrops jararaca (B. jararaca) has been chosen for this study. Our objectives were to isolate and characterize the target serine protease; isolate, purify, and characterize the orange bagasse flavone (hesperetin, Hst), and investigate the interactions between the targets, enzyme, and hesperetin. The purified serine protease was named BjSP24 because of its molecular mass and proteolytic activity. BjSP24 was folded and characterized using circular dichroism and showed low alpha-helix contents (7.7%). BjSP24 exhibited sequence similarity to other known snake venom serine proteases as measured in the enzyme tryptic peptides' LC-MS/MS run. Hesperetin was obtained within the expected yield and with the predominance of 2S isomer (82%). It acted as a mixed inhibitor for the serine protease (SVSP) from Bothrops jararaca snake venom observed in three different in vitro experiments, fluorescence, kinetics, and SSTD-NMR. It is still to determine if hesperetin might aid-in reverting the on site blood clotting problems just after snakebite accidents.

Snake venoms, as complex mixtures of peptides and proteins, affect various vital systems of the organism. One of the main targets of the toxic components from snake venoms is the cardiovascular system. Venom proteins and peptides can act in different ways, exhibiting either cardiotoxic or cardioprotective effects. The principal classes of these compounds are cobra cardiotoxins, phospholipases A2, and natriuretic, as well as bradykinin-potentiating peptides. There is another group of proteins capable of enhancing angiogenesis, which include, e.g., vascular endothelial growth factors possessing hypotensive and cardioprotective activities. Venom proteins and peptides exhibiting cardiotropic and vasoactive effects are promising candidates for the design of new drugs capable of preventing or constricting the development of pathological processes in cardiovascular diseases, which are currently the leading cause of death worldwide. For example, a bradykinin-potentiating peptide from Bothrops jararaca snake venom was the first snake venom compound used to create the widely used antihypertensive drugs captopril and enalapril. In this paper, we review the current state of research on snake venom components affecting the cardiovascular system and analyse the mechanisms of physiological action of these toxins and the prospects for their medical application.

According to the World Health Organization, snakebite envenoming is a neglected disease that affects around 5.4 million people worldwide each year. In Brazil, in 2019 there were 29,000 cases of accidents, with 104 deaths. The genus Bothrops was responsible for 90% of reported envenomations, mainly the species B. jararaca. The current therapy is performed with an intravenous injection of antivenom, be it monovalent or polyvalent. However, this treatment has high manufacturing costs, may induce side effects, and it does not effectively neutralize tissue necrosis. The latter issue may lead to deformity or amputation of the affected limb. Therefore, new treatments are needed to aid or improve the efficacy of antivenoms. In this study, nine triazole compounds (AM11–AM19) were chemically synthesized, characterized using infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy analyses, and tested against some in vitro (hemolysis, coagulation, and proteolysis) and in vivo (hemorrhaging, lethal, and edema) activities of B. jararaca venom. Each compound was incubated with B. jararaca venom (incubation protocol) or injected after the venom (treatment protocol), and then, biological assays were performed. As a result, all the compounds inhibited the toxic activities of B. jararaca venom with different potencies in the incubation protocol, while the compound AM13 inhibited hemorrhaging in the treatment protocol. In addition, the compounds were devoid of toxicity, as shown through admetSAR analysis or in vitro cytotoxicity test. Thus, these compounds may be an important tool for the development of antivenom molecules to improve serum therapy for recovering patients envenomed by B. jararaca venom.

Snakebites are a serious occupational problem affecting rural populations of tropical and sub-tropical, developing countries. Envenomation caused by the snakes Bothrops jararaca and B. jararacussu is characterized by local pain, edema, hemorrhage, tissue necrosis, and death. Despite the fact that antivenom may prevent the death of bite victims, treatment does not prevent tissue necrosis, often leading to amputation or deformity of the victim’s affected limb.Therefore, more efficient therapies need to be investigated. In this work, we tested the ability of a carrageenan galactan polysaccharide, isolated from a clonal strain of the red alga, Chondrus crispus (commonly known as Irish moss) to inhibit toxic, in vitro (coagulation, hemolytic and proteolytic) or in vivo (hemorrhagic, edematogenic, myotoxic and lethal) activities of B. jararaca or B. jararacussu venom. When the polysaccharide was mixed together with the venoms, inhibition of their toxic activities was achieved, but with different potencies. Moreover, inhibition of hemorrhage, edema, lethality, or myotoxicity was observed, even if the polysaccharide was injected before or after the injection of venoms, regardless of the route of administration (i.e., intravenous, subcutaneous, and intraperitoneal). A gel formulation containing the polysaccharide of C. crispus also protected mice from hemorrhage after administration of the venoms. Thus, the lambda carrageenan polysaccharide, as produced by a strain of C. crispus, may aid antivenom to block the toxic activities of B. jararaca and B. jararacussu venom, as well as to aid in the development of a more efficient therapy for envenomation by these venomous snakes.

Enzymatic inhibition by natural compounds may represent a valuable adjuvant in snakebite serum therapy. The objective in this work was to evaluate possible in vitro interactions between vanillic acid and enzymes from Bothrops spp. and Crotalus durissus terrificus venoms, and also suggest a theory as how they interact based on molecular docking. Vanillic acid inhibited the phospholipase activity induced by Bothrops alternatus (∼25% inhibition); the caseinolytic activity induced by Bothrops atrox (∼30%), Bothrops jararacussu (∼44%), and C. d. terrificus (∼33%); the fibrinogenolysis induced by B. jararacussu, B. atrox, and C. d. terrificus (100%); the serine protease activity induced by Bothrops moojeni (∼45%) and Bothrops jararaca (∼66%); the hemolytic activity induced by B. moojeni (∼26%); the thrombolysis activity induced by B. atrox (∼30%) and B. jararacussu (∼20%); and the thrombotic activity induced by C. d. terrificus (∼8%). The compound was also capable of delaying the coagulation time in citrated plasma by 60, 35, and 75 Sec, when incubated with B. moojeni, B. atrox, and B. jararaca, respectively. The results obtained expand the possibilities for future pharmaceutical use of vanillic acid, considering the high homology degree among human and snake venom phospholipases A2 and proteases (involved in chronic inflammatory diseases). Also, this compound can be used as adjuvant to improve currently available treatments for ophidism victims.

Background: Snake venoms are natural sources of proteins and peptides with several biological activities. Therefore, this study aimed to evaluate the in vitro toxicity of crude Bothrops jararaca (B. jararaca) venom on different human tumor cells and investigate its action mechanism. Material and methods: The crude venom from B. jararaca was provided by State Foundation for Health Research and Production and Center of Toxicological Information of Rio Grande do Sul, Brazil. To investigate different sensitivities of normal and cancer cell lines, cell viability was measured by using the MTT and neutral red uptake assays.

This work describes the synthesis of new 1,3-benzoxathiol-2-one sulfonamides and evaluation of their ability to inhibit some in vitro (coagulant, proteolytic and hemolytic) and in vivo (hemorrhagic, edematogenic and lethality) toxic activities of Bothrops jararaca and Bothrops jararacussu venoms. Compounds have been synthesized from the coupling of intermediate 5-amino-6-methoxybenzo[d] [1,3]oxathiol-2-one 4 with benzenesulfonyl chlorides. Characterization of the products was achieved by nuclear magnetic resonance (NMR) and electrospray ionization mass spectra (ESI-MS) techniques. Biological assay results have shown that most of compounds inhibited the main toxic activities of the venom of the two snake species. Compound 5b (N-(6-methoxy-2-oxobenzo[d] [1,3]oxathiol-5-yl)-4-nitrobenzenesulfonamide) was the most efficient in inhibiting hemolysis of B. jararaca, and coagulation and proteolysis induced by both venoms. For in vivo activities, all compounds inhibited the edema, from 35 to 72%, and most of them exhibited antihemorrhagic and antilethality activities. Thus, the results pointed to the biological potential of these compounds, being promising molecules to treat envenomation by these snakes as well as to aid the current antivenom serum therapy.

Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (

Background: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. Objective: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. Methods: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. Results: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. Conclusion: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

The low molecular mass fraction from Bothrops jararaca snake venom contains proline-rich peptides (Bj-PROs), also known as bradykinin-potentiating peptides (BPPs), which were described as neuroprotective against H2O2-induced oxidative stress. Recently, we reported that the neuroprotective action of Bj-PRO-10c (

Cadmium (Cd) is a highly toxic contaminant causing diverse physiologic damage through the food chain, possibly influencing the demography of wild populations. Thus study aimed to evaluate the feasibility of using Bothrops jararaca as an indicator of Cd contamination. For this purpose, 20 B. jararaca individuals were divided into four groups: a control group and three treatment groups (n = 5 in each group), which received Cd-contaminated food (900 mg kg−1) once, twice, or thrice, with a 15-day interval between meals. Animal fitness was evaluated by righting response behavior. In addition, genotoxic effects (comet assay damage index – DI), Cd accumulation in the kidney, liver, heart, gallbladder, fat, venom gland, blood, and tail, and Cd contents in the feces were evaluated. Righting response was negatively related to the number of received meals. Comet assay DI was significantly increased in animals that were fed two or three times. After three contaminated meals, Cd distribution within the body was as follows (µg/g – median): liver (6.24) > kidney (1.88) > venom gland (0.93) > fat (0.85) > gallbladder (0.65) > tail (0.46) > blood (0.45) > heart (0.25). Cd contents in the liver, kidney, and feces were significantly greater in animals that consumed Cd-contaminated food for two or three times. However, Cd accumulation in the liver started to appear after the first meal comprising the highest Cd concentration, while accumulation in the kidney appeared after the third feeding. It can be concluded that B. jararaca is a feasible model for biomonitoring Cd using the liver as a prognostic organ for the bioaccumulation as well as righting response and the damage index as indicators of Cd effects at an experimental level. Field monitoring feces stood out as the most suitable indicator for Cd ingestion by B. jararaca.

Aqueous and KCl-soluble polysaccharides were extracted from Laurencia dendroidea (Rhodomelaceae, Ceramiales) and their chemical profile was accessed by anion-exchange chromatography, chemical and spectroscopic analyses. The homogeneous agaran DHS-4 (181.3 × 103 g. mol−1, 21.3% of NaSO3) presents A units mostly 2-sulfated (18.9 mol%), nonsubstituted (15.3 mol%) and 6-O-methylated (10.1 mol%), while B units are l -sugars composed predominantly by galactose 6-sulfate precursor units (19.2 mol%) and 3,6-anhydrogalactose (13.8 mol%), besides non-precursor galactose 6-sulfate units bearing d -xylose substituents on C-3 (8.1 mol%). The crude KCl-soluble DHS agaran (20.5% of NaSO3) inhibited proteolysis and hemolysis induced by Lachesis muta and Bothrops jararaca venoms. DHS was able to inhibit up to 75% the L. muta venom hemorrhagic effect and to reduce the lethality displayed by B. jararaca venom, increasing the mice survival time up to 3 times. Therefore, this agaran has high potential to be used as an additional tool to treat snakebite envenomation.

Made available in DSpace on 2022-04-29T08:32:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-01 The peak of activity of Bothrops jararaca is in the rainy season. This pitviper feeds mainly on small mammals and is preyed upon by several avian and mammal predators. However, the association between jararaca's seasonal activity with that of their prey and predators remains unknown. The present study tested the hypothesis that the peak activity of B. jararaca coincides with that of their prey and predators, mainly due to favorable climatic conditions during this period. Snake activity was inferred from standardized searches and animals deposited in herpetological collections. Seasonal activity of prey was inferred from monthly sampling carried out over one year using pitfall and Sherman traps. Plasticine models were used to infer predation risk. Analyses of predator and prey abundance were performed, taking into account climatic variables, and models explaining this relationship were proposed. Activity of both B. jararaca and its prey was higher in the rainy season. Activity of the latter was positively correlated with temperature and rainfall. Predator activity, however, was higher in the dry season and negatively correlated with temperature and rainfall but positively correlated with humidity. The peak activity of B. jararaca in the rainy season might favor foraging activities, since this period coincides with greater prey abundance and decreased predation risk. During the dry season when resource availability is low, predators feed opportunistically, encountering dangerous prey such as B. jararaca. In summary, the activity pattern showed by the snake clearly increases its survival. Laboratório de Ecologia e Evolução Instituto Butantan Cep 05593900 Programa de Pós-Graduação em Biologia Animal Instituto de Biociências Letras e Ciencias Exatas Universidade Estadual Paulista julio de Mesquita Filho Cep 15054000 São José Do Rio Preto Sp Programa de Pós-Graduação em Biologia Animal Instituto de Biociências Letras e Ciencias Exatas Universidade Estadual Paulista julio de Mesquita Filho Cep 15054000 São José Do Rio Preto Sp

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts., Author summary Envenomation by snakebites is a major burden to tropical and subtropical areas in the world. Many snake species produce venoms that, when injected into victims, cause bleedings and other associated symptoms and signs. This work aimed to understand the mechanisms that lead to a fall in blood platelet counts after bites by a snake that inhabits in southeastern Brazil, the lance-headed snake Bothrops jararaca (popularly known as jararaca). We used experimental approaches to understand the involvement of a protein from jararaca venom, called botrocetin, and a protein present in our blood (von Willebrand factor) in the fall of platelet counts. We observed that botrocetin alters von Willebrand factor, but this mechanism in not important for the decrease in platelet counts. We show that jararaca snake venom disturb blood platelets in a complex and intricate way, and that other venom compounds are involved in the decrease of platelet counts during snakebite envenomation.

Resultados de exámenes de heces han sido comparados a los obtenidos durante necropsias de serpientesinfectadas con helmintos. Muestras de heces de ejemplares de Bothrops jararaca e B. alternatus fueroninvestigadas y tres animales fueron positivos para huevos durante los exámenes: un caso representandoparásitos autóctonos de serpientes, otro relativo a especies introducidas accidentalmente junto con ratones naturalmente infectados que son ofrecidos como alimentación para las serpientes y otro con la asociación de ambos. Resultados de las necropsias de los mismos ejemplares indican la presencia de los nematodos Ophidascaris travassosi Vaz, 1938, Kalicephalus costatus (Rudolphi, 1819), K. inermis Molin, 1861, Kalicephalus sp. y Acanthorhabdias acanthorhabdias Pereira, 1927. Las especies Bothrops jararaca es un nuevo registro de huésped para Ophidascaris travassosi y Bothrops alternatus para A. acanthorhabdias; los resultados con relación a lesiones macroscópicas inducidas por los nematodos son presentados.

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome of Bothrops jararaca, a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A2 (PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option.

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

Background: In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom’s, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment This work was supported by grants from the São Paulo Research Foundation (FAPESP – 2017/04321-7; Cetics (2013/07467-1), Coordination for the Improvement of Higher Education Personnel (CAPES) and Fundação Butantan

Cancer is characterized by the development of abnormal cells that divide in an uncontrolled way and may spread into other tissues where they may infiltrate and destroy normal body tissue. Several previous reports have described biochemical anti-tumorigenic properties of crude snake venom or its components, including their capability of inhibiting cell proliferation and promoting cell death. However, to the best of our knowledge, there is no work describing cancer cell proteomic changes following treatment with snake venoms. In this work we describe the quantitative changes in proteomics of MCF7 and MDA-MB-231 breast tumor cell lines following treatment with Bothrops jararaca snake venom, as well as the functional implications of the proteomic changes. Cell lines were treated with sub-toxic doses at either 0.63 μg/mL (low) or 2.5 μg/mL (high) of B. jararaca venom for 24 h, conditions that cause no cell death per se. Proteomics analysis was conducted on a nano-scale liquid chromatography coupled on-line with mass spectrometry (nLC-MS/MS). More than 1000 proteins were identified and evaluated from each cell line treated with either the low or high dose of the snake venom. Protein profiling upon venom treatment showed differential expression of several proteins related to cancer cell metabolism, immune response, and inflammation. Among the identified proteins we highlight histone H3, SNX3, HEL-S-156an, MTCH2, RPS, MCC2, IGF2BP1, and GSTM3. These data suggest that sub-toxic doses of B. jararaca venom have potential to modulate cancer-development related protein targets in cancer cells. This work illustrates a novel biochemical strategy to identify therapeutic targets against cancer cell growth and survival.

Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A2 inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A2, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments., Graphical abstract This study investigated the small molecule inhibitors varespladib, marimastat and dimercaprol in neutralising coagulopathic Crotalinae (pit vipers) snake venom toxins by using nanofractionation analytics which combines bioassays, liquid chromatography and mass spectrometry in parallel. Results obtained highlight their potential as future snakebite treatments.Image 1

Cardiovascular diseases such as coronary and peripheral artery diseases, venous thrombosis, stroke, hypertension, and heart failure are enormous burden to health and economy globally. Snake venoms have been the sources of discovery of successful therapeutics targeting cardiovascular diseases. For example, the first-in-class angiotensin-converting enzyme inhibitor captopril was designed largely based on bradykinin-potentiating peptides from Bothrops jararaca venom. In the recent years, sensitive and high throughput approaches drive discovery and cataloging of new snake venom toxins. As one of the largest class of snake venom toxin, there are now>700 sequences of three-finger toxins (3FTxs) available, many of which are yet to be studied. While the function of 3FTxs are normally associated with neurotoxicity, increasingly more 3FTxs have been characterized to have pharmacological effects on cardiovascular systems. Here we focus on this family of snake venom toxins and their potential in developing therapeutics against cardiovascular diseases.

Accidents involving snakes from the genus Bothrops sp. constitute the most important cause of snake envenomation in Brazil. The Myrsine genus has been reported to be used in folk medicine against snakebites. In this work, the phytochemical profiles and ability of extracts from Myrsine parvifolia leaves to reduce the inflammatory process (edema, vascular permeability increase and leukocyte migration) induced by Bothrops jararaca venom were investigated in vivo. Chemical compounds were identified by chromatographic and spectroscopy techniques. Total polyphenol, tannin, and flavonoid contents were determined by spectrophotometric methods. Swiss male mice received an oral administration of extracts (100 mg/kg) in different protocols. Paw edema, intraperitoneal vascular permeability and pleurisy models in mice were used to evaluate the antiophidic potential of the extracts. Paw edema was induced by subplantar injection of B. jararaca venom and quantified as the increase in paw volume. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. Leukocyte migration was assessed by total and differential counts in the pleural cavity washes. Myricetin, myricetin-3-O-β-arabinopyranoside, quercetin and kaempferol were isolated from the ethyl acetate extract and identified as the primary compounds of the dichloromethane extract. Terpenes and fatty acids were identified in the hexane and dichloromethane extracts. The pretreated group with hydroethanolic and dichloromethane extract reduced total edema (40 and 52%, respectively), vascular permeability increase (32.4 and 32.2%, respectively) and leukocyte influx into the pleural cavity (42 and 39%, respectively), while the group treated with hexane extract showed only reduced edema (37%) induced by B. jararaca venom. The hydroethanolic extract showed better results in all of the tests performed and was also administered by the protocol of post-poisoning, showing maintenance of paw edema reduction and cell migration. These data indicate a potential anti-inflammatory activity of M. parvifolia in poisoning by B. jararaca, especially to reduce local poison effects.

We examined the ability of Bothrops jararaca venom (12.5 mg/kg) injected intraperitoneally (i.p.) to cause acute kidney injury (AKI) in rats. Blood urea and creatinine (AKI biomarkers, in g dL−1) were elevated after 2 h in venom-treated rats (urea: from 0.41 ± 0.1 to 0.7 ± 0.03; creatinine from 46.7 ± 3.1 to 85 ± 6.7; p n = 3 each), with no change in circulating reduced glutathione. Venom-treated rats survived for ∼6 h, at which point platelets were reduced (×103 µL−1; from 763.8 ± 30.2 to 52.5 ± 18.2) whereas leukocytes and erythrocytes were slightly increased (from 4.7 ± 0.3 to 6.6 ± 0.1 × 103 µL−1 and from 8.38 ± 0.1 to 9.2 ± 0.09 × 106 µL−1, respectively; p −1) and albumin (2.7 ± 0.1 g dL−1) were normal, whereas blood and urinary urea and creatinine were increased. All parameters returned to normal with antivenom given 2 h post-envenomation. The i.p. injection of venom caused AKI similar to that seen with other routes of administration.

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.

Thrombotic microangiopathy (TMA) is an uncommon and severe complication of snakebites, and is similar, in general, to hemolytic-uremic syndrome (HUS). We describe a case of TMA following envenomation by Bothrops jararaca.A 56-y-old-woman with controlled hypertension was transferred from a primary hospital to our ER ∼7 h after being bitten by B. jararaca in the distal left leg. She developed edema extending from the bite site to the proximal thigh, associated with intense radiating local pain, local paresthesia and ecchymosis at the bite site. Laboratory features upon admission revealed coagulopathy (20 min whole blood clotting time - WBCT20 20 min), thrombocytopenia (76,000 platelets/mmTMA following snakebite has been reported mainly in India, Sri Lanka and Australia, with several patients needing renal replacement therapy. Although controversial, plasmapheresis has also been used in some cases. Our patient developed microangiopathic hemolytic anemia, thrombocytopenia and acute kidney injury, a triad of features compatible with TMA similar to HUS. Despite the severity, the outcome following conservative treatment was good, with complete recovery.

Quantitative and qualitative alterations in the environment may have serious ecological consequences for populations of native species. Isolated forest fragments, such as those surrounded by highly urbanized areas, may function similarly to oceanic islands with differences in species diversity compared to mainland areas. These differences may include changes in prey availability and predator pressure, with consequent effects on growth and size of species that interact with those prey and predators. Here, we investigate body size, prey availability, and predation pressure in populations of Bothrops jararaca in two forest fragments within an urban environment in southeastern Brazil (a completely isolated, small forest fragment and a large, well-connected forest fragment). While we found no differences in mean body size or stoutness between the two populations, we did find larger specimens in the small isolated fragment. Prey availability and predator pressure also were significantly lower in the sm...

Snakes belonging to the genus Naja (Elapid family), also known as "spitting cobras", can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.

The number of snakes donated to the Brazilian Instituto Butantan has been decreasing in the past 10 years. This circumstance motivated us to compare the properties of five venom pools of Bothrops jararaca snake stored for up to 54 years. Results showed differences among venom pools regarding enzymatic and other biological activities, such as caseinolytic, phospholipase A2, hemorrhagic and coagulant activities, as well as antigenicity. Protein content, reverse-phase chromatographic profile, and immunorecognition by commercial Bothrops antivenom were comparable for all venom pools, although lethality of the most recent preparations was higher. Since the lowest functional activities did not always correspond to older venoms, differences among venom pools used for antivenom production during the period 1963–2008 may correlate with the different proportions of venoms from different localities used in their generation, rather than to long-term storage. We conclude that B. jararaca venoms properly stored for long periods of time retain their structural and pharmacological activities, thus representing useful materials for scientific research and antivenom production.

Snakebite envenoming affects millions of people worldwide, being officially considered a neglected tropical disease by the World Health Organization. The antivenom is effective in neutralizing the systemic effects of envenomation, but local effects are poorly neutralized, often leading to permanent disability. The natural resistance of the South American pit viper Bothrops jararaca to its venom is partly attributed to BJ46a, a natural snake venom metalloendopeptidase inhibitor. Upon complex formation, BJ46a binds non-covalently to the metalloendopeptidase, rendering it unable to exert its proteolytic activity. However, the structural features that govern this interaction are largely unknown. In this work, we applied structural mass spectrometry techniques (cross-linking-MS and hydrogen-deuterium exchange MS) and in silico analyses (molecular modeling, docking, and dynamics simulations) to understand the interaction between BJ46a and jararhagin, a metalloendopeptidase from B. jararaca venom. We explored the distance restraints generated from XL-MS experiments to guide the modeling of BJ46a and jararhagin, as well as the protein-protein docking simulations. HDX-MS data pinpointed regions of protection/deprotection at the interface of the BJ46a-jararhagin complex which, in addition to the molecular dynamics simulation data, reinforced our proposed interaction model. Ultimately, the structural understanding of snake venom metalloendopeptidases inhibition by BJ46a could lead to the rational design of drugs to improve anti-snake venom therapeutics, alleviating the high morbidity rates currently observed.

Data on the composition of local reptile assemblages in several Brazilian ecosystems can still be considered relatively restricted in scope in most cases. In this study, we conducted surveys in the Serra das Torres Natural Monument, located in the municipalities of Atílio Vivacqua, Muqui, and Mimoso do Sul, using the Rapid Assessments method (RAP) during 30 days in the rainy season of 2018. We sampled actively for approximately 1320 hours with a 6-10 person crew, supplemented by 720 hours of passive sampling (30 bucket-days) using pitfall traps with drift fences. We recorded 34 reptile species during our sampling method (2 amphisbaenid, 11 lizards, and 21 snakes) and an occasional encounter, after the end of sampling, that added a chelonian species to the list, Hydromedusa maximiliani, totaling 35 reptile species. The Dipsadidae was the family with the greatest snake species richness and, the Gymnophtalmidae had the greatest lizard species richness. The species richness recorded in the Serra das Torres Natural Monument (Ntotal = 35) represents ca. 27% of all reptile species found in the state of Espírito Santo (N = 130). The most abundant lizard species was Leposoma scincoides followed by Ecpleopus gaudichaudii and, the most abundant snake species was Bothrops jararaca being markedly higher than that recorded in similar studies. Twenty-seven percent of the reptile species recorded in our study are endemic to the Atlantic Forest and 30% (N = 10) have been recorded less than five times previously in the Brazilian state of Espírito Santo. Our study reinforces the need for the conservation of the Serra das Torres Natural Monument because of its importance as a reservoir of a considerable portion of the reptile biodiversity of Espírito Santo state, and of the Atlantic Forest biome as a whole. Resumo: O conhecimento das assembleias de répteis para muitos ecossistemas no Brasil pode ser considerado ainda relativamente restrito. Neste estudo, nós realizamos amostragens no Monumento Natural Serra das Torres, localizado nos municípios de Atílio Vivacqua, Muqui e Mimoso do Sul, no estado do Espírito Santo, utilizando o método de avaliação rápida (RAP) durante 30 dias na estação chuvosa de 2018. Amostramos cerca de 1320 horas de busca ativa durante os períodos diurno e noturno, com uma equipe de 6 a 10 pessoas, suplementada por 720 horas de amostragem com armadilhas de queda com cercas guia (30 dias-balde). Registramos 34 espécies de répteis squamatas durante as amostragens (2 anfisbenídeo, 11 lagartos e 21 serpentes) e um encontro ocasional posterior que acrescentou uma espécie de quelônio à lista, Hydromedusa maximiliani, totalizando 35 espécies de répteis. Dipsadidae foi a família com a maior riqueza de serpentes, e Gymnophtalmidae foi a família com maior riqueza de lagartos. A riqueza de espécies que registramos no Monumento Natural Serra das Torres (Ntotal = 35) representa ca. 27% de todas as espécies de répteis encontradas no estado do Espírito Santo (N = 130). A espécie de lagarto mais abundante foi Leposoma scincoides seguido por Ecpleopus gaudichaudii, enquanto a espécie mais abundante de serpente foi a Bothrops jararaca, sendo marcadamente maior do aquela registradas em estudos similares. Vinte e sete por cento das espécies de registradas em nosso estudo são endêmicas da Mata Atlântica e trinta por cento das espécies (N = 10) tinham menos de cinco indivíduos registrados anteriormente no estado do Espírito Santo. Nosso estudo reforça a necessidade de conservação do Monumento Natural Serra das Torres devido à sua importância como reservatório de uma considerável parcela da biodiversidade de répteis do estado do Espírito Santo, bem como do bioma Mata Atlântica.

Background: Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.

In this research, we have purified a serine protease from Bothrops jararaca venom (BjSP24). This enzyme showed molar mass of 24.4 kDa as determined using MALDI-TOF. BjSP24 inhibition by hesperitin (Hst) was studied using florescence, enzymatic kinetics and nuclear magnetic resonance techinique (Saturation Transferee Difference, STD-NMR).

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

Using approaches of transcriptomics and proteomics we have shown that the phenotype of Bothrops jararaca venom undergoes a significant rearrangement upon neonate to adult transition. Most regulatory processes in biology are intrinsically related to modifications of protein structure, function, and abundance. However, it is unclear to which extent intrinsic proteolysis affects toxins and snake venom phenotypes upon ontogenesis. Here we assessed the natural N-terminome of Bothrops jararaca newborn and adult venoms and explored the degree of N-terminal protein truncation in ontogenetic-based proteome variation. To this end we applied the Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to characterize venom collected in the presence of proteinase inhibitors. We identified natural N-terminal sequences in the newborn (71) and adult (84) venoms, from which only 37 were common to both. However, truncated toxins were found in higher number in the newborn (212) than in the adult (140) venom. Moreover, sequences N-terminally blocked by pyroglutamic acid were identified in the newborn (55) and adult (49) venoms. Most toxin classes identified by their natural N-terminal sequences showed a similar number of unique peptides in the newborn and adult venoms, however, those of serine proteinases and C-type lectins were more abundant in the adult venom. Truncated sequences from at least ten toxin classes were detected, however the catalytic and cysteine-rich domains of metalloproteinases were the most prone to proteolysis, mainly in the newborn venom. Our results underscore the pervasiveness of truncations in most toxin classes and highlight variable post-translational events in newborn and adult venoms.

- We report a mating of Bothrops jararaca observed in the wild, in the Serra do Japi, São Paulo, Brazil.

Proline-rich oligopeptides from Bothrops jararaca (Bj-PROs) produce potent and long-lasting antihypertensive effect through mechanisms that go beyond ACE inhibition. In this study we evaluated the renal function parameters of spontaneously hypertensive rats (SHR) injected with Bj-PRO-5a and -10c (0.47, 71 or 710 nmol/kg) found in the CNP-precursor of the snake. At 71 and 710 nmol/kg, Bj-PROs increased urinary flow rate (18.1–43.5%). At 71 nmol/kg, Bj-PRO 5a and 10c elevated sodium excretion (68.1 and 40.9%, respectively) and Bj-PRO-5a also increased urinary sodium/creatinine ratio (56.5%). At 0.47 nmol/kg, Bj-PROs did not change renal function. All doses of Bj-PROs reduced blood pressure (Δ = −13 to −24mmHg). We conclude that Bj-PROs reduce blood pressure and improve renal function of SHRs through diuretic and natriuretic mechanisms.

Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell-penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography-MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP - interestingly, a classical bradykinin-potentiating peptide - in the peptide-rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence-labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell-cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label-free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.

Components from animal venoms may vary according to the snake's age, gender and region of origin. Recently, we performed a proteomic analysis of Bothrops jararaca venom from southern ( BjSv ) and southeastern ( BjSEv ) Brazil, showing differences in the venom composition, as well as its biological activity. To continue the study, we report in this short communication the different effects induced by the BjSEv and BjSv on isolated kidney and MDCK renal cells. BjSEv decreased perfusion pressure (PP) and renal vascular resistance (RVR) and increased urinary flow (UF) and glomerular filtration rate (GFR), while BjSv did not alter PP and RVR and reduced UF and GFR. Both types of venom, more expressively BjSEv , reduced %TNa + , %TK + and %Cl − . In MDCK cells, the two types of venom showed cytotoxicity with IC 50 of 1.22 μg/mL for BjSv and 1.18 μg/mL for BjSEv and caused different profiles of cell death, with BjSv being more necrotic. In conclusion, we suggest that BjSv is more nephrotoxic than BjSEv .

We carried out an analysis of the venom gland proteome of Bothrops jararaca taking into account two distinct phases of its ontogenetic development (i.e., newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of B. jararaca venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated with transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon aging, the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186.

Background The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed. Objectives The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus). Methods Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes. Results There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells. Conclusions Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays.

Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q–I–P–P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. Biological significance Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout–vent length and weight, with females being larger than males. This sexual size dimorphism suggests the tendency for female specimens to feed on larger prey, and for male specimens to go on a diet similar to that of juveniles. Variation in the snake venom proteome is a ubiquitous phenomenon occurring at all taxonomic levels. At the intraspecific variation level, the individual contribution to the venom proteome is important but effects contributed by age and feeding habits may also affect the proteome phenotype. Whether sex-based factors play a role in venom variation of a species that shows sexual size dimorphism is poorly known. The use of proteomic strategies supported by transcriptomic data allows a more comprehensive assessment of venom proteomes uncovering components that are gender-specific.

We describe a new species of snail-eating snake from two localities within the Atlantic Forest of the states of Bahia and Minas Gerais, Brazil. The new species differs remarkably from all congeners by its dorsal pattern consisting of sharply bordered triangles resembling the pattern of sympatric pitvipers, more precisely Bothrops jararaca and B. pirajai. Parameters of external morphology of the new species resemble those of the Dipsas incerta species group. Its scalation, body/head shape, and color pattern are most similar to D. alternans, a species known to occur 360 km farther south, across the Rio Doce river. The new species differs from D. alternans by exhibiting triangular dorsal blotches and a higher number of pileus blotches, by the distribution of ventral spots and morphology of the supratemporal and premaxillae, as well as in hemipenial morphology. We also provide comparisons of the new species with all sympatric and/or parapatric congeners. The finding of a new snake species in the Atlantic rainforest of southern Bahia and adjacent Minas Gerais is particularly surprising as this region is easily accessible and represents a well-sampled area regarding herpetological surveys in the last decades.