Abstract Introduction: Sticholecitha serpentis is a poorly studied parasite of the snakes’ esophagus, with an unclear taxonomy. Currently, there are few records of S. serpentis in snakes in Brazil. Although it has been recorded in snakes of the family Viperidae, it has not yet been reported in Bothrops atrox. Objectives: To present new morphological characters, as well as ecological and parasitic relationships of S. serpentis with B. atrox. Methods: The samples were obtained in a dryland area in the Tapajós National Forest, Pará State, Brazil. Ten specimens of B. atrox were examined manually with the aid of tweezers, and three were positive for trematodes identified as S. serpentis in the oral cavity. Results: We provide scanning electron microscopy images, with detailed images of the S. serpentis tegument and its spines, as well as details of the dorsal keel using light microscopy methods. Moreover, we report the first record of S. serpentis parasitizing an Amazonian viperid, with infection frequency data of 30 %. Conclusion: We emphasize that the new morphological data presented in this manuscript provide additional information to facilitate the identification of this trematode in future studies.

Resumen Introducción: Sticholecitha serpentis es un parásito del esófago de serpientes poco estudiado y su sistemática no está clara. Actualmente, existen pocos registros del parásito en serpientes en Brasil. Aunque ha sido registrada en serpientes de la familia Viperidae, aún no ha sido reportada en Bothrops atrox. Objetivos: Presentar novedades en aspectos morfológicos, relaciones ecológicas y parasitarias de S. serpentis, parásito encontrado por primera vez en la cavidad bucal de B. atrox. Métodos: Las muestras provienen de un área seca en la Floresta Nacional de Tapajós, Estado de Pará, Brasil. Diez especímenes de B. atrox fueron examinados manualmente con la ayuda de pinzas. De estos individuos tres fueron positivos para trematodos compatibles con S. serpentis en la cavidad oral. Resultados: Se proporcionan imágenes de microscopía electrónica de barrido, con imágenes del tegumento de S. serpentis y sus espinas, y detalles de la quilla dorsal mediante métodos de microscopía óptica. Además, se reporta el primer registro de parasitismo por S. serpentis en un vipérido amazónico, con datos de frecuencia de infección del 30 %. Conclusión: Los nuevos datos morfológicos presentados en este manuscrito proporcionan información adicional para facilitar la identificación de este tremátodo en futuros estudios.

Abstract Variability in snake venom composition is well-documented and crucial for understanding snake ecology and predicting snakebites. In this study, we characterize the venom composition and biological activities of newborn female and male Bothrops moojeni and their mother. Our results reveal significant differences between the venom of newborn females and males, demonstrating a broad and diverse range of proteins. The venoms of newborn females showed higher serine protease effects, increased hemorrhagic activity, and greater lethality compared to the venom of newborn males. However, no differences were observed in phospholipase A2 and coagulant activity. The differences in protein composition and toxic activities between maternal and neonatal venom, as well as between the venoms of newborn females and males, contribute to understanding the diverse outcomes of snakebites. These results underscore the importance of considering sex and ontogeny in understanding venom composition in snakes. welldocumented documented snakebites study mother males proteins effects activity However A snakes

Resumo A variabilidade na composição dos venenos de serpentes é bem documentada e crucial para a compreensão da ecologia das serpentes ou do prognóstico dos envenenamentos ofídicos. Nesse estudo, caracterizamos a composição e a atividade biológica do veneno de fêmeas e machos recém-nascidos e sua mãe. Nossos resultados revelaram diferenças significativas entre o veneno de fêmeas e machos recém-nascidos, demonstrando uma ampla e diversificada gama de proteínas. Os venenos de fêmeas recém-nascidas apresentaram mais efeitos de serina protease, aumento da atividade hemorrágica e maior letalidade em comparação ao veneno dos machos recém-nascidos. No entanto, nenhuma diferença foi observada na fosfolipase A2 e na atividade coagulante. As diferenças na composição proteica e nas atividades tóxicas entre o veneno materno e neonatal, bem como entre os venenos de fêmeas e machos recém-nascidos contribuem para a compreensão dos diversos resultados dos acidentes ofídicos. Esses resultados ressaltam a importância de considerar o sexo e a ontogenia na compreensão da composição do veneno desses animais. ofídicos estudo recémnascidos recém nascidos mãe recémnascidos, nascidos, proteínas recémnascidas nascidas protease recémnascidos. nascidos. entanto coagulante neonatal animais

Abstract Background: The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a; <EDGPIPP) from Bothrops jararaca snake, on oxidative stress-induced toxicity in neuronal PC12 cells and astrocyte-like C6 cells. Methods: Both cells were pre-treated for four hours with different concentrations of PRO-7a, submitted to H2O2-induced damage for 20 h, and then the oxidative stress markers were analyzed. Also, two independent neuroprotective mechanisms were investigated: a) L-arginine metabolite generation via argininosuccinate synthetase (AsS) activity regulation to produce agmatine or polyamines with neuroprotective properties; b) M1 mAChR receptor subtype activation pathway to reduce oxidative stress and neuron injury. Results: PRO-7a was not cytoprotective in C6 cells, but potentiated the H2O2-induced damage to cell integrity at a concentration lower than 0.38 μM. However, PRO-7a at 1.56 µM, on the other hand, modified H2O2-induced toxicity in PC12 cells by restoring cell integrity, mitochondrial metabolism, ROS generation, and arginase indirect activity. The α-Methyl-DL-aspartic acid (MDLA) and L-NΩ-Nitroarginine methyl ester (L-Name), specific inhibitors of AsS and nitric oxide synthase (NOS), which catalyzes the synthesis of polyamines and NO from L-arginine, did not suppress PRO-7a-mediated cytoprotection against oxidative stress. It suggested that its mechanism is independent of the production of L-arginine metabolites with neuroprotective properties by increased AsS activity. On the other hand, the neuroprotective effect of PRO-7a was blocked in the presence of dicyclomine hydrochloride (DCH), an M1 mAChR antagonist. Conclusions: For the first time, this work provides evidence that PRO-7a-induced neuroprotection seems to be mediated through M1 mAChR activation in PC12 cells, which reduces oxidative stress independently of AsS activity and L-arginine bioavailability. Background loss injury death Therefore prolinerich proline rich PRO7a PROa PRO (PRO-7a <EDGPIPP EDGPIPP stressinduced induced PC PC1 astrocytelike astrocyte like C Methods pretreated pre treated PRO7a, 7a, H2O2induced HOinduced H2O2 H O 2 h analyzed Also investigated Larginine L arginine (AsS b M Results 038 0 38 0.3 μM However 156 1 56 1.5 µM hand metabolism αMethylDLaspartic α Methyl DL aspartic MDLA (MDLA LNΩNitroarginine NΩ Nitroarginine LName, LName Name , (L-Name) NOS, NOS (NOS) Larginine, arginine, PRO7amediated PROamediated DCH, DCH (DCH) antagonist Conclusions time PRO7ainduced PROainduced bioavailability HO H2O 03 3 0. 15 5 1. (L-Name (NOS amediated (DCH ainduced

Abstract Glioblastoma (GB) is the most common type of malignant tumor of the central nervous system, responsible for significant morbidity and with a 5-year overall relative survival of only 6.8%. Without advances in treatment in the last twenty years, the standard of care continues to be maximum safe resection, Temozolomide (TMZ), and radiotherapy. Many new trials are ongoing, and despite showing increased progression-free survival, these trials did not improve overall survival. They did not consider the adverse effects of these therapies. Therefore, an increasing number of bioprospecting studies have used snake venom molecules to search for new strategies to attack GB selectively without producing side effects. The present review aims to describe GB characteristics and current and new approaches for treatment considering their side effects. Besides, we focused on the antitumoral activity of snake venom proteins from the Viperidae family against GB, exploring the potential for drug design based on in vitro and in vivo studies. This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. In January 2024, a systematic search was performed in the PubMed, EMBASE, and Web of Science databases from January 2000 to December 2023. Search terms were selected based on the population/exposure/outcome (PEO) framework and combined using Boolean operators ("AND", "OR"). The search strategy used these terms: glioblastoma, glioma, high-grade glioma, WHO IV glioma, brain cancer, snake venom, Viperidae, and bioprospection. We identified 10 in vivo and in vitro studies with whole and isolated proteins from Viperidae venom that could have antitumor activity against glioblastoma. Studies in bioprospecting exploring the advantage of snake venom proteins against GB deserve to be investigated due to their high specificity, small size, inherent bioactivity, and few side effects to cross the blood-brain barrier (BBB) to reach the tumor microenvironment. (GB system 5year year 5 68 6 8 6.8% years resection TMZ, TMZ , (TMZ) radiotherapy ongoing progressionfree progression free therapies Therefore Besides MetaAnalysis Meta Analysis PRISMA (PRISMA guidelines 2024 PubMed EMBASE 200 2023 populationexposureoutcome population exposure outcome PEO (PEO AND, AND ("AND" OR. OR . "OR") glioblastoma glioma highgrade grade cancer bioprospection 1 specificity size bioactivity bloodbrain blood BBB (BBB microenvironment 6.8 (TMZ 202 20 ("AND "OR" 6. 2 "OR

Resumen Registramos la depredación de la serpiente de cascabel Crotalus helleri caliginis, endémica de la Isla Coronado Sur, Baja California, basados en el hallazgo de restos óseos de dos serpientes en las egagrópilas del Halcón peregrino Falco peregrinus. Este registro representa la primera depredación confirmada para la serpiente isleña y amplía el conocimiento de la dieta del Halcón peregrino en la isla.

Abstract We recorded the predation on the rattlesnake Crotalus helleri caliginis, endemic to Coronado Sur Island, Baja California, based on the discovery of bone remains of two snakes in the falcon pellets of the Peregrine Falcon Falco peregrinus. This record represents the first confirmed predation of the insular snake and expands our knowledge of the Peregrine Falcon diet on the island.

Abstract This study presents the first complete mitogenome of the Brazilian Atlantic bushmaster Lachesis with insights into snake evolution. The total length was 17,177 bp, consisting of 13 PCGs, 22 tRNAs, two rRNAs and a duplicate control region (CRs). Almost all genes were encoded by the heavy-strand, except for the ND6 gene and eight tRNAs (tRNA-Gln, Ala, Asn, Cys, Tyr, Ser[TGA anticodon], Glu, Pro). Only ATG, ATA, and ATC were starting codons for protein-coding sequences. Stop codons mainly were TAA, AGA, AGG, and TAG; whereas ND1, ND3, and CYTB terminated with incomplete stop codons. Phylogeny retrieved Lachesis within the Crotalinae as the sister group of Agkistrodon; and the Lachesis+Agkistrodon clade as the sister group of (Sistrurus+Crotalus)+Bothrops. The tree supports Crotalinae, Viperinae, and Azemiopinae in the Viperidae family, being sister taxa of Colubridae+(Elapidae+Psammophiidae). The mean genetic distance across 15 snake families and 57 nucleotide sequences was 0.37. The overall mean value of genetic distance across the Crotalinae was 0.23, with Lachesis muta exhibiting the shortest distance of 0.2 with Agkistrodon piscivorus, Protobothrops dabieshanensis and P. flavoviridis and the greatest 0.25 with Gloydius blomhoffii, Trimeresurus albolabris, S. miliarius, and Deinagkistrodon acutus. The complete Atlantic L. muta mitogenome presented herein is only the third annotated mitogenome from more than 430 described Brazilian snake species. evolution 17177 17 177 17,17 bp 1 PCGs 2 CRs. CRs . (CRs) heavystrand, heavystrand heavy strand, strand heavy-strand ND tRNAGln, tRNAGln tRNA Gln, Gln (tRNA-Gln Ala Asn Cys Tyr SerTGA Ser TGA anticodon, anticodon , anticodon] Glu Pro. Pro Pro) ATG ATA proteincoding protein coding TAA AGA AGG TAG ND1 ND3 LachesisAgkistrodon Sistrurus+Crotalus+Bothrops. SistrurusCrotalusBothrops Sistrurus+Crotalus +Bothrops. Sistrurus Crotalus Bothrops (Sistrurus+Crotalus)+Bothrops Viperinae family Colubridae+Elapidae+Psammophiidae. ColubridaeElapidaePsammophiidae Colubridae+ Elapidae+Psammophiidae Colubridae Elapidae Psammophiidae Colubridae+(Elapidae+Psammophiidae) 5 037 0 37 0.37 023 23 0.23 02 0. piscivorus P 025 25 blomhoffii albolabris S miliarius acutus L 43 species 1717 17,1 (CRs Sistrurus+Crotalus+Bothrops SistrurusCrotalus +Bothrops Colubridae+Elapidae+Psammophiidae ElapidaePsammophiidae Colubridae+(Elapidae+Psammophiidae 03 3 0.3 4 171 17,

Abstract Snakebite envenoming is a major global health problem that kills or disables half a million people in the world’s poorest countries. Identifying the biting snake and its habitat use is key to understanding snakebite eco-epidemiology and optimizing its clinical management. To prevent and combat the neglected snakebite disease, we characterize the morphology, geographic distribution, habitat use, and snakebites of medically important venomous snakes in the state of Rio de Janeiro (Brazil). Despite Philodryas spp. not being considered of medical importance by the Brazilian Ministry of Health, we also explore their data once the bites may require medical intervention, may cause death, and their consequences are underestimated. Methods: We assessed taxonomy and geographic data from specimens housed in scientific collections, the literature, and the Notifiable Diseases Information System. Our data revealed fragility in the morphological characters recommended to distinguish Bothrops jararaca from B. jararacussu, identify the subspecies of Crotalus durissus and distinguish the species of Philodryas. To help identify these species, we present an identification key to the venomous snake species from Rio de Janeiro based on the morphological data collected. We record the genera Bothrops and Micrurus in all mesoregions of the state. Here, we provide the first record of C. durissus in the Serrana region, supporting the hypothesis of geographic expansion of the species in the state. The crotalic antivenom must not be missing in Médio Paraíba, Centro-Sul Fluminense, and Serrana, where the rattlesnake C. durissus occurs. Bothrops bilineatus and Lachesis muta have historical records presented for the first time herein. However, these species are likely endangered or extinct in the state. There were 7,483 snakebites reported between 2001 and 2019, with an annual average of 393.8 cases. The Bothrops genus is responsible for the majority of accidents. The highest number of cases occurred in the Serrana region, the largest pole of family agriculture in Rio de Janeiro. We improve the identification of venomous snake species, better delimit their distribution, and update the number of cases of snakebites, thus providing greater precision in the attention to this problem in Rio de Janeiro. We emphasize the importance of clinical studies to test using bothropic-crotalic antivenom and heparin in all mesoregions to treat B. jararacussu envenomation; and mechanical ventilation, atropine, and anticholinesterases in the emergency health centers in the Metropolitana and Norte Fluminense regions due to the occurrence of the coral M. lemniscatus in these areas. worlds world s countries ecoepidemiology eco epidemiology management disease morphology distribution Brazil. Brazil . (Brazil) spp Health intervention death underestimated Methods collections literature System B collected Here C region Paraíba CentroSul Centro Sul occurs herein However 7483 7 483 7,48 200 2019 3938 393 8 393. accidents bothropiccrotalic bothropic envenomation ventilation atropine M areas (Brazil 748 48 7,4 20 201 39 74 4 7, 2 3

Resumo O envenenamento com serpentes de importância médica é um grande problema de saúde global que mata ou incapacita meio milhão de pessoas nos países mais pobres do mundo. A identificação de serpentes de importância médica e aspectos de sua história de vida é fundamental para compreender a eco-epidemiologia dos acidentes ofídicos e otimizar seu manejo clínico. Para prevenir e combater a doença negligenciada do ofidismo, caracterizamos os dados de morfologia, distribuição, uso de habitat e acidentes ofídicos das serpentes peçonhentas de importância médica no estado do Rio de Janeiro, Brasil. Apesar de Philodryas spp. não serem consideradas de importância médica pelo Ministério da Saúde do Brasil, também apresentamos seus dados porque suas picadas podem exigir intervenção médica, causar morte e suas consequências são subestimadas. Foram avaliados dados morfológicos e de habitat de espécimes pertencentes a coleções cientificas, literatura e dados do Sistema de Informação de Agravos de Notificação. Nossos dados revelaram caracteres morfológicos frágeis recomendados para distinguir Bothrops jararaca e B. jararacussu, para identificar as subespécies de cascavel Crotalus durissus e para distinguir as serpentes cipó Philodryas spp. Para auxiliar na identificação dessas espécies apresentamos uma chave de identificação para as mesmas com base nos dados morfológicos coletados. Registramos os gêneros Bothrops e Micrurus em todas as mesorregiões do estado. Já o soro anticrotálico não pode faltar no Médio Paraíba, Centro-Sul Fluminense e Serrana, onde encontramos a cascavel Crotalus durissus. Aqui estendemos o registro da cascavel pela primeira vez para a região Serrana, corroborando a hipótese da expansão da espécie no estado. Bothrops bilineatus e Lachesis muta possuem registros históricos aqui apresentados pela primeira vez. Essas espécies provavelmente estão ameaçadas ou extintas no estado. Foram 7,483 acidentes entre 2001-2019, com média anual de 393.8. O gênero Bothrops é responsável pela maioria dos acidentes. O maior número de casos ocorreu na região Serrana, maior polo da agricultura familiar do Rio de Janeiro. Aperfeiçoamos a identificação das espécies de serpentes de importância médica, delimitamos melhor sua distribuição e atualizamos os acidentes ofídicos, proporcionando maior precisão na atenção ao ofidismo no estado do Rio de Janeiro. Ressaltamos a importância de estudos clínicos para testar o uso de soro antibotrópico-crotálico e heparina em todas as mesorregiões para o tratamento do ofidismo B. jararacussu e ventilação mecânica, atropina e anticolinesterásicos nos prontos-socorros das regiões Metropolitana e Norte Fluminense pela ocorrência do coral M. lemniscatus. mundo ecoepidemiologia eco epidemiologia clínico morfologia Janeiro Brasil spp subestimadas cientificas Notificação B coletados Paraíba CentroSul Centro Sul Serrana 7483 7 483 7,48 20012019, 20012019 2001 2019, 2019 2001-2019 3938 393 8 393.8 antibotrópicocrotálico antibotrópico crotálico mecânica prontossocorros prontos socorros M lemniscatus 748 48 7,4 2001201 200 201 2001-201 39 393. 74 4 7, 200120 20 2001-20 3 20012 2 2001-2 2001-

ABSTRACT Snake venom disintegrins are low molecular weight, non-enzymatic proteins rich in cysteine, present in the venom of snakes from the families Viperidae, Crotalidae, Atractaspididae, Elapidae, and Colubridae. This family of proteins originated in venom through the proteolytic processing of metalloproteinases (SVMPs), which, in turn, evolved from a gene encoding an A Disintegrin And Metalloprotease (ADAM) molecule. Disintegrins have a recognition motif for integrins in their structure, allowing interaction with these transmembrane adhesion receptors and preventing their binding to proteins in the extracellular matrix and other cells. This interaction gives disintegrins their wide range of biological functions, including inhibition of platelet aggregation and antitumor activity. As a result, many studies have been conducted in an attempt to use these natural compounds as a basis for developing therapies for the treatment of various diseases. Furthermore, the FDA has approved Tirofiban and Eptifibatide as antiplatelet compounds, and they are synthesized from the structure of echistatin and barbourin, respectively. In this review, we discuss some of the main functional and structural characteristics of this class of proteins and their potential for therapeutic use. weight nonenzymatic non enzymatic cysteine Viperidae Crotalidae Atractaspididae Elapidae Colubridae SVMPs, SVMPs , (SVMPs) which turn ADAM (ADAM molecule cells functions activity result diseases Furthermore barbourin respectively review (SVMPs

Abstract Bothrops alternatus (yarará grande, víbora de la cruz) is a snake belonging to the Viperidae family and is widely distributed in Argentina. In the northeast, this snake group for the majority of the snakebite accidents. There are reports about envenomation in horses with clinical signs including swelling, hemorrhage, dermo and myonecrosis at the bite sight in addition to systemic effects such as impaired blood coagulation, hemorrhage and damage to vital organs. An additional consequence in the horse is laminitis which reduces the work capacity of the survivor on a long-term basis. This occurs independent of the site where the venom was injected. laminitis induces pain and claudication, and it can evolve to deform the hoof. This study uses explants from freshly slaughtered horses as an in-vitro assay from the limbs of normal horses in order to demonstrate the ability of Bothrops snake venom to cause laminitis. Explants were incubated with whole venom and phospholipases A2 (PLA2). The objective was to reproduce laminitis in vitro to corroborate the clinical data reported in snake venom accidents. Equine laminitis induced by whole venom was characterized by damage to the lamellar dermo-epidermal junction, loosening of the lamellar basement membrane and cytotoxic effects. Epidermal basal cells showed changes in the shape and size of their nuclei and other abnormalities.

Resumen Bothrops alternatus (yarará grande, víbora de la cruz) es una serpiente perteneciente a la familia Viperidae, y se encuentra ampliamente distribuida en Argentina. En el nordeste de nuestro país, esta serpiente ocasiona el mayor número de accidentes. Existen reportes de envenenamiento en equinos, siendo sus signos y síntomas más importantes: inflamación y hemorragia, dermo y mionecrosis en el área de inoculación del veneno. Por otro lado, la acción sistémica consiste en incoagulabilidad sanguínea, hemorragia y lesiones de órganos que pueden comprometer la salud del animal. Una secuela, debida a la intoxicación por esta serpiente, es la laminitis, que afecta la capacidad de trabajo del equino, independientemente del sitio de inoculación del veneno, debido a la acción sistémica del mismo. Esta patología produce dolor, claudicación, que luego puede progresar con deformación del casco. Se efectuó un estudio in vitro, tomando como modelo experimental muestras (explantes) de pie equino. Estas fueron incubadas con veneno entero y con Fosfolipasa A2 (PLA2) del mismo veneno con el objeto de reproducir laminitis a los efectos de corroborar datos clínicos reportados en accidentes por yarará como así también proponer una metodología de estudio de laminitis en explantes de pie equino. La laminitis inducida por el veneno entero se caracterizó por la separación de la unión dermo-epidérmica, desprendimiento de membrana basal y citotoxicidad y consecuentemente desprendimiento de unión dermo-epidérmica, permaneciendo las células basales epidérmicas con alteraciones en forma y tamaño de los núcleos, entre otras anormalidades. En contraste, las muestras incubadas con PLA2, las células basales epidérmicas mostraron anomalías citoplasmáticas y nucleares, pero con separación leve de la unión dermoepidérmica y presente solo en algunas áreas de las LES.

Resumen La diversidad de helmintos digenéticos de serpientes del Bioma Pampa es poco conocida, con pocos registros en Brasil, Argentina y Uruguay. El presente estudio documenta especies de Digenea que parasitan serpientes en el Bioma Pampa, sur de Brasil. Se examinaron 42 individuos pertenecientes a 11 especies de Dipsadidae y Viperidae. En 50% de las serpientes analizadas se encontraron un total de 178 ejemplares pertenecientes a 9 taxones de digéneos. Los hábitats y el comportamiento del hospedador pueden influir en la fauna parasitaria de las serpientes, ya que la diversidad y abundancia de helmintos fueron mayores en los hospedadores presentes en ambientes semiacuáticos y de pastizales. Además, 89% de los tremátodos digenéticos utilizan anuros como segundos hospedadores intermediarios; según la literatura, los anuros son el recurso alimenticio más común para estas serpientes. En este contexto, los estudios de dieta y helmintofauna de serpientes son complementarios y contribuyen a la comprensión de la biología de las especies y su papel en los ecosistemas.

Abstract The diversity of digenetic helminths of snakes from the Pampa Biome is poorly known, with few records in Brazil, Argentina, and Uruguay. The present study documents species of Digenea parasitizing snakes in the Pampa Biome, southern Brazil. Forty-two individuals belonging to 11 species of Dipsadidae and Viperidae were examined. A total of 178 specimens belonging to 9 taxa of digenetic trematodes were found in 50% of the snakes analyzed. Habitats and host behavior may influence the parasite fauna of snakes, since helminth diversity and abundance were greater in hosts present in semi-aquatic and grassland environments. Further, 89% of digenetic trematodes use anurans as second intermediate hosts; according to the literature, anurans are the most common food resource for these snakes. In this context, studies of diet and helminth fauna of snakes are complementary and contribute to the understanding of the biology of species and their role in ecosystems.

Abstract Background Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of β-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The β-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods β-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results Proteomic analysis revealed fragments on β-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. β-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion These findings indicate that β-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.

Abstract Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.

RESUMO: No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.

ABSTRACT: In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.

RESUMO: Embora raros, os micoplasmas estão incluídos entre as causas de doenças respiratórias em répteis e, na ordem Squamata, já foram realizados três relatos destes microrganismos causando doença em pítons. Este estudo teve como objetivo avaliar a ocorrência de espécies de Mycoplasma em serpentes em cativeiro. Foram avaliadas 26 serpentes das famílias Pythonidae (13), Boidae (7), Viperidae (5) e Colubridae (1) do RioZoo, Brasil. Os animais foram examinados para determinar sinais clínicos consistentes com qualquer doença infecciosa. Amostras de swab traqueal de cobras foram coletadas em meio Frey e analisadas por isolamento microbiológico e pela técnica da PCR para identificar Mycoplasma spp. As amostras positivas para o gênero Mycoplasma spp. foram submetidas ao sequenciamento genético para identificação das espécies. No isolamento, 19,23% (5/26) foram positivos, enquanto 65,38% (17/26) dos animais foram positivos por PCR. Com base nas análises das seis sequências obtidas, houve similaridade com o Mycoplasma spp. descrito anteriormente em um píton e M. agassizii e M. testudineum encontrados em quelônios. Este é o primeiro relato de Mycoplasma spp. em animais das famílias Boidae e Viperidae. Mycoplasma spp. foi detectado em serpentes com e sem sinais clínicos. Os micoplasmas encontrados apresentaram semelhança genética com outros já descritos em répteis. Não houve relação entre a presença de Mycoplasma spp. e sinais clínicos.

ABSTRACT: Although rare, mycoplasmas are included among the causes of respiratory diseases in reptiles and, in the order Squamata, three reports of these microorganisms causing diseases in pythons have already been reported. This study aimed to evaluate the occurrence of Mycoplasma species in captive snakes. A total of 26 snakes of the families Pythonidae (13), Boidae (7), Viperidae (5) and Colubridae (1) from RioZoo, Brazil, were evaluated. Animals were examined to determine clinical signs consistent with any infectious disease. Tracheal swab samples from snakes were collected in Frey medium and analyzed for the presence of Mycoplasma spp.by isolation and a genus-specific PCR. DNA sequencing analyses of six positive samples by PCR were carried out to identify the species. Using isolation 19.23% (5/26) was positive, while 65.38% (17/26) of the animals were positive by PCR. Based on the analyses of the six sequences obtained, there was similarity with a Mycoplasma spp. previously described in a phyton and, M. agassizii and M. testudineum reported in chelonians. This is the first report of Mycoplasma spp. in animals of the families Boidae and Viperidae. Mycoplasma spp. were detected in snakes with and without clinical signs. The mycoplasmas reported resented identity (range, 95% to 100%) to others already described in reptiles. There was no relationship between the presence of Mycoplasma spp. and clinical signs.

SUMMARY: The Viperidae venoms are composed of a mixture of constituents with enzymatic and non-enzymatic actions, which act on ultrastructural components of cells and tissues. Here, the number of mitochondria, mitochondrial area and the number of mitochondrial cristae from adrenal glands cortex treated with snake venoms were tested after 3, 6 and 24 hours of venom injections. The mitochondria quantitative changes showed a statistically significant decrease, in the number of mitochondria past 3, 6 and 24 h. There was an increase in the mitochondrial area after 6 h, where Crotalus vegrandis venom did not present significant differences with Crotalus pifanorum or Bothrops venezuelensis venoms. After 24 h, there was an escalation of mitochondrial area in all tested venoms. The number of mitochondrial cristae after 3 h did not present important differences with the control treatment. After 6 h, the number of mitochondrial cristae initiated to decrease under the activities of the 3 venoms action, until 24 h of observation. In the qualitative observations it was possible to witness an intense damage of the mitochondria, with loss and swelling of membranes, disappearance of cristae and the appearance of myelin figures, which started at 3 h after the Crotalus and Bothrops venoms injections. These damages probably were due to cytotoxic effects of phospholipases, metalloproteases and/or other proteolytic activities present in Viperidae snake venoms, being more evident in Crotalus venoms. As far as we know, these results define a novel finding that suggest that Viperidae snake venoms are extremely toxic to mammalian mitochondria.

RESUMEN: Los venenos de Viperidae tienen acciones enzimáticas y no enzimáticas, que actúan sobre la estructura celular. Aquí se probaron, a las 3, 6 y 24 horas de la inyección del veneno, el número de mitocondrias, el área mitocondrial y el número de crestas mitocondriales de la corteza de las glándulas adrenales. Los cambios cuantitativos de las mitocondrias mostraron una disminución en el número de mitocondrias a las 3, 6 y 24 h. Hubo un aumento en el área mitocondrial a las 6 h, donde el veneno de la serpiente Crotalus vegrandis no presentó diferencias significativas con los venenos de Crotalus pifanorum o Bothrops venezuelensis. Después de 24 h, hubo un aumento del área mitocondrial en todos los venenos. El número de crestas mitocondriales a las 3 h no presentó alteraciones o diferencias importantes con el tratamiento de control. Después de 6 h, el número de crestas mitocondriales comenzó a disminuir bajo la acción de los 3 venenos, hasta las 24 h de observación. En las observaciones cualitativas se observó un daño intenso de las mitocondrias, con pérdida y edema de las membranas, desaparición de las cristae y aparición de figuras mielínicas, que comenzó a las 3 h después de las inyecciones de veneno de Crotalus y Bothrops. Estos daños se debieron factiblemente a los efectos citotóxicos de componentes proteolíticos de los venenos. Creemos que estos resultados definen un nuevo y original hallazgo, que sugiere que los venenos de serpiente Viperidae son extremadamente tóxicos para las mitocondrias de mamíferos.