Avaliação do papel da crotoxina na modulação da carcinogênese pulmonar induzida em animais fenotipicamente selecionados para a resposta inflamatória aguda

Cancer is the main cause of death in the world, with lung cancer being responsible for most of the deaths, either because it is often diagnosed at a late stage or due to the lack of effective therapeutic methods. Crotoxin (CTX) is the main component of South American rattlesnake venom, corresponding to about 60% of its composition. In recent years, its immunomodulatory, anti-inflammatory, and antitumor properties have been described, making it a new possible candidate for therapeutic use. In order to study the regulatory role of CTX, lung carcinogenesis was induced by intraperitoneal injection of urethane (URT) in mice phenotypically selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory responses. From a genetic background, these animals have different sensitivity to urethane-induced lung tumorigenesis due to the fixation of alleles with opposite effects at the pas1 locus (Pulmonary adenoma susceptibility 1) during the selection process, with a susceptibility allele (pas1s ) in the AIRmin mice and a resistance allele (pas1r ) in the AIRmax mice. Considering these differences and the described effects of CTX, these animals were subjected to different treatments with CTX and evaluated regarding their tumorigenesis in terms of incidence and cellular dynamics in the lung. The results showed that the phenotypes of resistance and susceptibility in the mice were preserved, even after CTX administration. Additionally, possible modulatory effects of CTX were observed, mainly in histopathologic analysis on the groups where CTX was administered 10 days after tumor induction by urethane with a concentration of 8 μg/kg and 16 μg/kg, and also on the treatment where CTX was administered every 10 days with a concentration of 8 μg/kg.

O câncer é a principal causa de morte no mundo, sendo o câncer de pulmão o responsável pelo maior número de óbitos, seja por ser diagnosticado muitas vezes num estágio tardio ou pela falta de métodos terapêuticos eficazes. A crotoxina (CTX) é o principal componente do veneno da cascavel (Crotalus durissus terrificus), correspondendo a cerca de 60% da sua composição. Nos últimos anos, têm sido descritas as suas propriedades imunomoduladoras, anti-inflamatórias e antitumorais, sendo apresentada como uma possível candidata para utilização terapêutica. Com o intuito de estudar o papel regulador da CTX, foi induzida carcinogênese pulmonar por injeção intraperitoneal de uretana (URT), em animais fenotipicamente selecionados para resposta inflamatória aguda máxima (AIRmax) ou mínima (AIRmin). Do ponto de vista genético, estes animais apresentam uma sensibilidade diferenciada à tumorigênese pulmonar induzida por URT, relacionada à fixação de alelos de efeitos opostos no locus pas1 (Pulmonary adenoma susceptibillity 1) durante o processo de seleção, com um alelo de suscetibilidade (pas1s ) na linhagem AIRmin, e um alelo de resistência (pas1r ) na linhagem AIRmax. Considerando estas diferenças entre as linhagens e os efeitos descritos da CTX, esses animais foram submetidos a diferentes tratamentos com CTX e avaliados quanto à tumorigênese, no que concerne à incidência e dinâmica celular no pulmão. Os resultados demonstraram que os fenótipos de resistência e susceptibilidade estão preservados entre as linhagens, mesmo após a administração da CTX. Adicionalmente, foram observados possíveis efeitos moduladores da CTX, principalmente nas análises histopatológicas nos grupos de tratamento quando a CTX é aplicada 10 dias após a administração de URT na concentração de 8 μg/kg e 16 μg/kg, assim como quando aplicada continuamente de 10 em 10 dias numa concentração de 8 μg/kg.

Estudo da ação modulatória da crotoxina em animais selecionados fenotipicamente para resposta inflamatória aguda submetidos à carcinogênese cutânea por 7,12 dimetilbenzantraceno

The skin's immune system is made up of innate and adaptive immunity cells that act in defense against external agents, however, they can be affected when exposed to carcinogens such as polycyclic aromatic hydrocarbons (PAH), and suffer from their cytotoxic effects. An example of a PAH is 7,12 dimethylbenzanthracene (DMBA), which has toxic effects when interacting with the skin. Our analysis focused on the impacts of DMBA, alone or associated with TPA, on the skin of mice phenotypically selected for maximum (AIRmax) or minimum (AIRmin) acute inflammatory response, in the face of the appearance of papillomatous lesions. Above all, we studied the influence of Crotoxin (CTX) or crude crotalic venom on the evolution of these lesions. In a first experiment, animals from both lines received the following treatments: acetone (control), DMBA 5x and CTX, evaluated for 80 days. In a subsequent protocol, we associated DMBA and TPA, with the following groups: acetone (control), DMBA/TPA, DMBA/TPA/CTX EPI 2x and DMBA/TPA/Poison evaluated for 7, 20, 80 and 120 days. A final two-stage protocol was carried out with CTX EPI applications 1x per week and subcutaneous administration equally at a time, and the animals were evaluated for periods of 20, 40 and 80 days. Our findings revealed that in the first protocol, AIRmin were more sensitive to DMBA, especially when considering the multiplicity and incidence of injuries. In the second protocol (DMBA/TPA), AIRmax exhibited a high incidence of lesions that resembled carcinomas, especially in the groups that were exposed to CTX or poison. In the last approach, also with the two- stage protocol, we identified that the application of CTX EPI 1x per week inhibited the progression of lesions in both lineages, something that was not evidenced with the subcutaneous administration of CTX. However, histopathological data suggest that the subcutaneous route of CTX administration is important in inhibiting malignant evolution. Additionally, by evaluating cell populations, we distinguished two subpopulations of Tγδ lymphocytes that may play an important role in tumor progression. This highlights the importance of CTX as an immunomodulatory agent in studies on sensitivity to HPA-type carcinogens.

O sistema imunológico da pele é formado por células da imunidade inata e adaptativa que atuam na defesa contra agentes externos, porém, podem ser afetadas quando expostas a carcinógenos como os hidrocarbonetos policíclicos aromáticos (HPA), e sofrer com seus efeitos citotóxicos. Um exemplo de HPA é o 7,12 dimetilbenzantraceno (DMBA), que tem efeitos tóxicos ao interagir com a pele. Nossa análise tomou como foco os impactos do DMBA, isolado ou associado ao TPA, na pele de camundongos fenotipicamente selecionados para resposta inflamatória aguda máxima (AIRmax) ou mínima (AIRmin), frente ao surgimento de lesões papilomatosas. Sobretudo, estudamos a influência da Crotoxina (CTX) ou do veneno crotálico bruto na evolução dessas lesões. Em um primeiro experimento, animais de ambas as linhagens receberam os seguintes tratamentos: acetona (controle), DMBA 5x e CTX, avaliados por 80 dias. Em um protocolo subsequente, associamos DMBA e TPA, com os seguintes grupos: acetona (controle), DMBA/TPA, DMBA/TPA/CTX EPI 2x e DMBA/TPA/Veneno avaliados por 7, 20, 80 e 120 dias. Um último protocolo de dois estágios foi realizado com aplicações de CTX EPI 1x por semana e administração por via subcutânea igualmente por vez, e os animais foram avaliados por períodos de 20, 40 e 80 dias. Nossas descobertas revelaram que no primeiro protocolo, os AIRmin foram mais sensíveis ao DMBA, especialmente ao considerar a multiplicidade e incidência de lesões. Já no segundo protocolo (DMBA/TPA), os AIRmax exibiram alta incidência de lesões que se assemelhavam a carcinomas, principalmente nos grupos que foram expostos a CTX ou veneno. Na última abordagem, também com o protocolo de dois estágios, identificamos que a aplicação de CTX EPI 1x por semana, inibiu a progressão das lesões nas duas linhagens, algo que não foi evidenciado com a administração subcutânea de CTX. No entanto, dados histopatológicos sugerem que a via subcutânea para administração de CTX é importante na inibição da evolução maligna. Adicionalmente, ao avaliar as populações celulares, distinguimos duas subpopulações de linfócitos Tγδ que podem desempenhar uma função importante na progressão tumoral. Isso destaca a importância da CTX como agente imunomodulador em estudos sobre a sensibilidade a carcinógenos do tipo HPA.

Caracterização bioquímica de uma serinoprotease com efeito ativador do plasminogênio do veneno de Lachesis muta rhombeata

Snake venom serine proteases (SVSPs) are chymotrypsin-like proteins found in some venoms of the Viperidae family. These enzymes affect physiological processes of their prey and victims, acting mainly in the hemostatic, fibrinolytic and kinin systems. SVSPs belong to the PA clan, PA (S) subclan, S1 family and A subfamily of proteolytic enzymes. This work, describes a SVSP (Lmr-PA) isolated from the venom of Lachesis muta rhombeata which activates plasminogen. The proteinase was purified by combination of gel filtration and anionic exchange chromatographies. Its homogeneity was demonstrated by SDS-PAGE, reverse-phase HPLC, and two-dimensional electrophoresis. Lmr-PA is a 30-kDa single chain glycoprotein. Its amino acid sequence (61%) was determined by mass spectrometry on nLC-MS/MS. Lmr-PA activates plasminogen to release plasmin and degrades the plasmin substrate S-2251 as well as dimethylcasein. PMSF, the specific inhibitor of serine proteases completely blocked Lmr-PA activity. The proteinase cleaves the Aα chain and partially the Bβ and γ chains of fibrinogen. In addition the protease degrades laminin, nidogen and type IV collagen from Matrigel. The enzyme digests fibrin in presence of plasminogen in vitro. Deglycosylated Lmr-PA loses approximately 26% of its activity. In addition, Lmr-PA activity is inhibited by α2-macroglobulin at a ratio of 2:1 (α2-M:E) and α2-antiplasmin inhibits plasmin generated from plasminogen. Lmr-PA does not induce aggregation of washed human platelets but, aggregates platelets in presence of exogenous fibrinogen and binds to the platelet receptors glycoproteins (GP) GPIb and GPVI. Our data indicate that Lmr-PA is a plasminogen activating serine protease, like to previously reported LV-PA from Lachesis muta muta venom. These results suggested that Lmr- PA play a role in the pathology of snake envenomation and could be a useful model to study hemostatic disorders caused by snake bites.

As serinoproteases do veneno de serpentes (SVSPs) são proteínas semelhantes à quimotripsina presentes encontradas em alguns venenos da família Viperidae. Essas enzimas afetam os processos fisiológicos de suas presas e vítimas, atuando principalmente nos sistemas hemostático, fibrinolítico e cinina. As SVSPs pertencem ao clã PA, subclan PA (S), família S1 e subfamília A de enzimas proteolíticas. Este trabalho descreve uma SVSP (Lmr-PA) isolada do veneno de Lachesis muta rhombeata que ativa o plasminogênio. A proteinase foi purifica por combinação de cromatografias de filtração em gel e troca aniônica. Sua homogeneidade foi demonstrada por SDS-PAGE, HPLC de fase reversa e eletroforese bidimensional. Lmr-PA é uma glicoproteína de cadeia simples de 30 kDa. A sua sequência de aminoácidos (61%) foi determinada por espectrometria de massa em nLC-MS/MS. Lmr-PA ativa o plasminogênio para liberar plasmina e degrada o substrato de plasmina S-2251, bem como a dimetilcaseína. PMSF, um inibidor específico de serinoproteases bloqueou completamente a atividade da Lmr-PA. A proteinase cliva a cadeia Aα e parcialmente as cadeias Bβ e γ do fibrinogênio. Além disso, a protease degrada laminina, nidogênio e colágeno tipo IV de Matrigel. A enzima digere a fibrina na presença de plasminogênio in vitro. Lmr-PA desglicosilada perde aproximadamente 26% da sua atividade. Além disso, a atividade da Lmr-PA é inibida pela α2-macroglobulina em uma proporção de 2:1 (α2-M:E) e a α2-antiplasmina inibe a plasmina gerada a partir do plasminogênio. Lmr-PA não induz a agregação de plaquetas humanas lavadas, mas agrega plaquetas na presença de fibrinogênio exógeno e se liga às glicoproteínas dos receptores plaquetários (GP) GPIb e GPVI. Nossos dados indicam que a Lmr-PA é uma serinoprotease ativadora do plasminogênio semelhante a LV-PA relatada anteriormente do veneno de Lachesis muta muta. Esses resultados sugerem que a Lmr-PA desempenha um papel na patologia do envenenamento por serpentes e pode ser um modelo útil para estudar distúrbios hemostáticos causados por acidentes ofídicos.

Avaliação preditiva estrutural das serino peptidases de aranhas: as relações evolutivas e de estrutura/função

Serine peptidases (SP) are hydrolases represented in many living organisms, with chymotrypsins and trypsins being the main digestive SPs. Studies of genomes, transcriptomes and proteomes of spiders allow identifying these enzymes in different tissues and/or secretions of these animals and suggest that SPs are involved in poisoning and digestion, two very important processes for the evolutionary success of the group. In order to compare the SPs involved in these processes, in silico analyses of the databases of a total of 38 distinct species of spiders were performed, totaling 1,200 sequences of SPs. Proteomic analyses of Nephilingis cruentata, Stegodyphus mimosarum and Acanthoscurria geniculata show the presence of SPs in poisons (SPV) and digestive process (SPD). In general, the SPVs present a molecular mass in the range between 30 and 37 kDa and pI in the range of 4.65 and 9.79, presenting, in most sequences, only the catalytic domain. Fluid and digestive system SPDs mandatorily present the catalytic domain associated with CUB-LDLa, indicating that CUB-LDL SPcatalytic combination is a signature of the digestive SPs of spiders. These enzymes have a molecular mass of 36 to 140 kDa and pI between 4.4 and 5.6. Other differences found among SPV and SPD are: predicted patterns of glycosilation, cell addressing and structural prediction. Among the different digestive SPs there is a distinction between hydrophobicity, electrostatic potential, and substrate accessibility. Molecular docking also allowed predicting the interaction behavior of SPs with inhibitors that mimic substrates, as Kunitz type inhibitors. Analyses indicate that poison enzymes have greater accessibility of the catalytic triad to the substrate. Phylogenetic analyses show that the different groups of digestive SPs are represented in all studied spider species, keeping CUB-LDLa as a signature. The conservation of the CUB-LDLa domain was also observed in SPs of other Groups of Arachnida such as Scorpionidae, Acari and Opiliones, being even present in Limulus representative of the Chelicerata group. The association of these domains is possibly related to different functionalities of SPs in arachnids maintained throughout the evolutionary process.

As serino peptidases (SP) são hidrolases representadas em muitos organismos vivos, sendo as quimotripsinas e tripsinas as principais SPs digestivas. Estudos de genomas, transcriptomas e proteomas de aranhas permitem identificar essas enzimas em diferentes processos fisiológicos desses animais e sugerem que as SPs estão envolvidas no envenenamento e no processo digestivo, dois processos muito importantes para o sucesso evolutivo do grupo. Com o objetivo de comparar as SPs envolvidas nestes processos foram realizadas análises in silico dos bancos de dados de um total de 38 espécies distintas de aranhas, totalizando 1.200 sequências de SPs. Análises proteômicas de Nephilingis cruentata, Stegodyphus mimosarum e Acanthoscurria geniculata mostram a presença de SPs nos venenos (SPV) e no processo digestivo (SPD). Em geral, as SPVs apresentam uma massa molecular na faixa entre 30 e 37 kDa e pI na faixa de 4,65 e 9,79, apresentando, na maioria das sequências, apenas o domínio catalítico. As SPDs de fluido e sistema digestório apresentam obrigatoriamente o domínio catalítico associado a CUB-LDLa, indicando que a combinação CUB-LDLa-SP catalítico é uma assinatura das SPs digestivas de aranhas. Essas enzimas apresentam uma massa molecular de 36 a 140 kDa e o pI entre 4,4 e 5,6. Outras diferenças encontradas são: os padrões preditos de glicosilação, endereçamento celular e predição estrutural. Entre as diferentes SPs digestivas há distinção entre a hidrofobicidade, o potencial eletrostático e a acessibilidade ao substrato. Docking molecular permitiu ainda prever o comportamento de interação das SPs com inibidores que mimetizam substratos como inibidores do tipo Kunitz. As análises indicam que as enzimas de veneno têm maior acessibilidade da tríade catalítica ao substrato. Análises filogenéticas mostram que os diferentes grupos de SPs digestivas estão representados em todos as espécies de aranhas estudadas mantendo CUB-LDLa como assinatura. A conservação do domínio CUB-LDLa também foi observada em SPs de outros grupos de Arachnida como Scorpionidae, Acari e Opiliones, estando inclusive presente em Limulus polyphemus representante do grupo Chelicerata. A associação destes domínios possivelmente está relacionada a diferentes funcionalidades de SPs em aracnídeos mantidas ao longo do processo evolutivo.

Toxinas animais seletivas a canais iônicos: uma revisão / Ion channels selective animal toxins: a review

Animal poisons are glandular secretions that compromise biological systems. Their active biomolecules are called toxins. Many affect ion channels and ionotropic receptors, membrane proteins that control cellular ion flow. In this work a bibliographic survey was carried out about the main toxins of animal origin whose targets are ion channels. The groups with the highest toxin diversity were Conus ssp., Araneae, Scorpiones, Serpentes and Cnidaria. Toxins studies support basic and applied science. Despite their therapeutic potential, of all the studied poisons only ω-conotoxin MVIIA was approved for clinical use. So the field still has a lot to offer.

Venenos animais são secreções glandulares que comprometem sistemas biológicos. Suas biomoléculas ativas são denominadas toxinas. Muitas afetam canais iônicos e receptores ionotrópicos, proteínas de membrana que controlam o fluxo iônico celular. Neste trabalho foi realizado um levantamento bibliográfico sobre as principais toxinas de origem animal cujos alvos são canais iônicos. Os grupos com maior diversidade de toxinas foram Conus ssp., Araneae, Scorpiones, Serpentes e Cnidaria. Estudos sobre toxinas auxiliam a ciência de base e aplicada. Apesar do potencial terapêutico, dentre todos os venenos estudados apenas a ω-conotoxina MVIIA obteve aprovação para o uso clinico. Portanto, o campo ainda tem muito a oferecer.

Toxinology in Colombia: contributions of ophidism/scorpionism program and other research groups / Toxinología en Colombia: contribuciones del programa de ofidismo/escorpionismo y de otros grupos de investigación

Background: Toxinology is a sub-field of toxicology dedicated to studying toxins produced by animals, plants and, microorganisms. In Colombia, during the last thirty years, this area has been mainly investigated by Ophidism/Scorpionism Program of Universidad de Antioquia. However, some other research groups have also contributed to our knowledge of venoms and toxins, as well as their related effects and treatments. Objective: to highlight the most significant findings in toxinology made by the Ophidism/Scorpionism Program and other research groups in Colombia. Methods: 119 papers dealing with the history of ophidiology and toxinology in Colombia were collected and analyzed. Results: some useful terms are described to understand toxinology and its scope. Also, a brief history of ophidiology is presented, spanning from the discovery of America until present-day findings. Finally, an overall description of several results related to toxin isolation, characterization, antivenoms, clinical trials, description of new species, proteomic and transcriptomic, among others. The nineteens were characterized by the study of snakebites, their clinic manifestations, and the use of antivenoms. In addition, the ethnopharmacological studies of medicinal plants used in snakebite treatments began to be explored. The 2000s included the newly ethnopharmacology, toxin isolation, clinical trials, inhibitor studies, scorpion venom characterization, and scorpion stings features. Finally, from 2010 until today, proteomic and transcriptomic gave the most important findings. Conclusions: Toxinology works in Colombia have contributed to our knowledge about endemic species, clinical manifestations of snakebite and scorpion stings, and the development of new therapeutic agents. However, we invite Colciencias and other funding agencies to assign more resources to support a higher number of researchers in this field, since snakebite is considered a neglected tropical disease by the World Health Organization, which needs more attention from governments and scholars. Finally, the venoms of some species and their possible mode of action are still unknown to us. Besides, given the complexity of venoms, we are not yet aware of the potential use of toxins in current biomedicine. Thus, studies in toxinology must continue.

Antecedentes: La Toxinología es el campo de la Toxicología que estudia las toxinas producidas por animales, plantas y microorganismos. En Colombia, durante los últimos treinta años, los estudios realizados en esta área han sido desarrollados principalmente por el Programa de Ofidismo/Escorpionismo de la Universidad de Antioquia. Sin embargo, otros grupos de investigación también han contribuido en el conocimiento de venenos, toxinas, efectos y tratamientos. Objetivo: Destacar los hallazgos más relevantes en toxinología realizados por el Programa de Ofidismo Escorpionismo y otros grupos de investigación en Colombia. Métodos: Se recopilaron 119 artículos referentes a la historia de la ofidiología y la toxinología en Colombia. Resultados: Se describieron algunos términos útiles para el entendimiento de la toxinología y sus alcances. Se construyó una breve historia de la ofidiología que inicia con el descubrimiento de América y finaliza con hallazgos recientes. Se realizó una amplia descripción de varios resultados relacionados con el aislamiento y caracterización de toxinas, antivenenos, ensayos clínicos, descripciones de nuevas especies, proteómica y transcriptómica, entre otras. Así, la década de los noventa se caracterizó por el estudio de las mordeduras de serpientes, sus manifestaciones clínicas, el uso de antivenenos y la exploración de la etnofarmacología asociada a las mordeduras de serpiente. La década del 2000 incluyó nuevamente etnofarmacología, el aislamiento de toxinas, ensayos clínicos, estudios sobre inhibidores de toxinas, caracterización de venenos y picaduras de escorpión. Finalmente, desde 2010 hasta hoy, la proteómica y transcriptómica aportaron los hallazgos más importantes. Conclusiones: Los estudios de Toxinología en Colombia han contribuido al conocimiento de especies endémicas, manifestaciones clínicas de mordeduras de serpientes y picaduras escorpiones, y el desarrollo de nuevos agentes terapéuticos. No obstante, se invita a Colciencias y a otras agencias de financiamiento a apoyar la investigación en este campo, ya que es considerada una enfermedad tropical desatendida por la Organización Mundial de la Salud y necesita mayor atención por parte del gobierno e instituciones académicas. Además, dada la complejidad de los venenos, se desconoce el uso potencial de las toxinas en la biomedicina actual. Así, se deben continuar realizando estudios en toxinología.

Bothrops alternatus snake venom induces apoptosis of skeletal muscle cells in a rabbit model / O veneno da serpente Bothrops alternatus induz apoptose nas células musculares de coelhos

Este estudo objetiva reportar a ocorrência de apoptose in vivo induzida pelo veneno da serpente Bothrops alternatus em células musculares esqueléticas. Cinco coelhos machos, adultos, receberam 150µg/kg de veneno no músculo vasto lateral, enquanto outros cinco animais receberam 0,1% de BSA diluído em PBS no mesmo local. Após 12 horas, os animais foram eutanasiados, e amostras do local de inoculação foram coletadas para análise histopatológica. Foram evidenciadas necrose e hemorragia nas células musculares. Além disso, a análise imuno-histoquímica para identificação de caspase-3 ativada revelou marcações granulares e agregadas no citoplasma das células musculares, compatíveis com o processo de apoptose. Este é o primeiro relato que confirma o veneno de B. alternatus como causador de apoptose in vivo em células musculares esqueléticas.(AU)

Carbonylative negishi-type coupling of 2-Iodoglycals with alkyl and aryl halides

C-Glycosides are valuable organic compounds in the field of medicinal chemistry due to their ubiquity inside living systems and pronounced biological activity. Herein, we describe an approach to alkyl-ketones bearing glycal units via the Pd-catalyzed carbonylative coupling of 2-iodoglycals and alkyl and aryl halides. Examples bearing a variety of functional groups are presented as well as a mechanistic proposal for this transformation.

Variations in tetrodotoxin levels in populations of Taricha granulosa are expressed in the morphology of their cutaneous glands

Tetrodotoxin (TTX), one of the most toxic substances in nature, is present in bacteria, invertebrates, fishes, and amphibians. Marine organisms seem to bioaccumulate TTX from their food or acquire it from symbiotic bacteria, but its origin in amphibians is unclear. Taricha granulosa can exhibit high TTX levels, presumably concentrated in skin poison glands, acting as an agent of selection upon predatory garter snakes (Thamnophis). This co-evolutionary arms race induces variation in T. granulosa TTX levels, from very high to undetectable. Using morphology and biochemistry, we investigated differences in toxin localization and quality between two populations at the extremes of toxicity. TTX concentration within poison glands is related to the volume of a single cell type in which TTX occurs exclusively in distinctive secretory granules, suggesting a relationship between granule structure and chemical composition. TTX was detected in mucous glands in both populations, contradicting the general understanding that these glands do not secrete defensive chemicals and expanding currently held interpretations of amphibian skin gland functionality. Skin secretions of the two populations differed in low-mass molecules and proteins. Our results demonstrate that interpopulation variation in TTX levels is related to poison gland morphology.

Biochemical and biological characterization of the Hypanus americanus mucus: a perspective on stingray immunity and toxins

Stingrays skin secretions are largely studied due to the human envenoming medical relevance of the sting puncture that evolves to inflammatory events, including necrosis. Such toxic effects can be correlated to the biochemical composition of the sting mucus, according to the literature. Fish skin plays important biological roles, such as the control of the osmotic pressure gradient, protection against mechanical forces and microorganism infections. The mucus, on the other hand, is a rich and complex fluid, acting on swimming, nutrition and the innate immune system. The elasmobranch's epidermis is a tissue composed mainly by mucus secretory cells, and marine stingrays have already been described to present secretory glands spread throughout the body. Little is known about the biochemical composition of the stingray mucus, but recent studies have corroborated the importance of mucus in the envenomation process. Aiming to assess the mucus composition, a new non-invasive mucus collection method was developed that focused on peptides and proteins, and biological assays were performed to analyze the toxic and immune activities of the Hypanus americanus mucus. Pathophysiological characterization showed the presence of peptidases on the mucus, as well as the induction of edema and leukocyte recruitment in mice. The fractionated mucus improved phagocytosis on macrophages and showed antimicrobial activity against T. rubrumç. neoformans and C. albicans in vitro. The proteomic analyses showed the presence of immune-related proteins like actin, histones, hemoglobin, and ribosomal proteins. This protein pattern is similar to those reported for other fish mucus and stingray venoms. This is the first report depicting the Hypanus stingray mucus composition, highlighting its biochemical composition and importance for the stingray immune system and the possible role on the envenomation process.

Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation

Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganismsthat is stored in specialized glands located in the tegument. For some animals, such glands have accumulated inspecific regions of the body and formed prominent structures known as macroglands. The Bufonidae familydisplays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to beextremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though.Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a pro-teomic study on the parotoid macrogland secretion of the Asian bufonidDuttaphrynus melanostictus. By em-ploying the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions -bound and unbound–that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteinsrelated to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases,lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological ac-tivity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present inthe bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played bysuch molecules.Significance:The current approach allowed a detailed proteomic analysis of the several proteins synthesized intheD. melanostictusparotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within acomplex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological roleplayed by such proteins at the skin

Iron (III)-promoted synthesis of substituted 4H-Chalcogenochromenes and chemoselective functionalization

The iron(III)-promoted synthesis of densely-substituted 4H-chalcogenchromene from organochalcogen propargylamines in the presence of diaryl dichalcogenides is reported. Subsequent C2-functionalization with electrophiles and potassium trifluoroborate salts via Suzuki-Miyaura coupling reaction are also presented. A plausible mechanism based on HRMS experiments is proposed and discussed.

Reticulate evolution in nuclear Middle America causes discordance in the phylogeny of palm-pitvipers (Viperidae: Bothriechis)

Aim A number of processes can lead to weak or conflicting phylogenetic signals, especially in geographically dynamic regions where unstable landscapes and climates promote complex evolutionary histories. The Middle American pitviper genus Bothriechis has a complex biogeographic distribution and previous phylogenetic analyses have recovered conflicting topologies based on the data type used. Here, we tested whether historic conflicts in the phylogeny were the result of reticulate evolution and whether the inferred biogeographic history of the group would enable contact among reticulate lineages.Location Middle America. Taxon Palm-pitvipers (genus Bothriechis). Methods We generated a phylogenomic dataset using an anchored phylogenomics approach and inferred a genomics-based species tree and mitochondrial tree to assess incongruence among datasets. We then generated a dated phylogeny and conducted ancestral area reconstruction to examine the biogeographic history surrounding the diversification of these species. We additionally tested whether the discordance among trees is better explained by lineage sorting or reticulate evolution by testing models of reticulate evolution inferred through multiple methods. Results We found strong support for discordance in the phylogeny of Bothriechis and corresponding evidence for reticulate evolution among lineages with incongruent placement. Ancestralarea reconstruction placed these taxa in adjacent regions during the time period when reticulation was projected to take place and suggested a biogeographic history heavily influenced by vicariant processes. Main conclusions Reticulation among geographically proximate lineages has driven apparent genomic discordance in Bothriechis and is responsible for historical incongruence in the phylogeny. Inference of the order of events suggests that reticulation among nuclear Middle American taxa occurred during a time of geologic upheaval, promoting lineage divergence and secondary contact. Reticulate evolution and similar processes can have substantial impacts on the evolutionary trajectory of taxa and are important to explicitly test for in biogeographically complex regions.

Bottom-up proteomic analysis of polypeptide venom components of the giant ant Dinoponera quadriceps

nt species have specialized venom systems developed to sting and inoculate a biological cocktail of organic compounds, including peptide and polypeptide toxins, for the purpose of predation and defense. The genus Dinoponera comprises predatory giant ants that inoculate venom capable of causing long-lasting local pain, involuntary shaking, lymphadenopathy, and cardiac arrhythmias, among other symptoms. To deepen our knowledge about venom composition with regard to protein toxins and their roles in the chemical–ecological relationship and human health, we performed a bottom-up proteomics analysis of the crude venom of the giant ant D. quadriceps, popularly known as the "false" tocandiras. For this purpose, we used two different analytical approaches: (i) gel-based proteomics approach, wherein the crude venom was resolved by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and all protein bands were excised for analysis; (ii) solution-based proteomics approach, wherein the crude venom protein components were directly fragmented into tryptic peptides in solution for analysis. The proteomic data that resulted from these two methodologies were compared against a previously annotated transcriptomic database of D. quadriceps, and subsequently, a homology search was performed for all identified transcript products. The gel-based proteomics approach unequivocally identified nine toxins of high molecular mass in the venom, as for example, enzymes [hyaluronidase, phospholipase A1, dipeptidyl peptidase and glucose dehydrogenase/flavin adenine dinucleotide (FAD) quinone] and diverse venom allergens (homologous of the red fire ant Selenopsis invicta) and venom-related proteins (major royal jelly-like). Moreover, the solution-based proteomics revealed and confirmed the presence of several hydrolases, oxidoreductases, proteases, Kunitz-like polypeptides, and the less abundant inhibitor cysteine knot (ICK)-like (knottin) neurotoxins and insect defensin. Our results showed that the major components of the D. quadriceps venom are toxins that are highly likely to damage cell membranes and tissue, to cause neurotoxicity, and to induce allergic reactions, thus, expanding the knowledge about D. quadriceps venom composition and its potential biological effects on prey and victims.

Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus

Background: Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins. Methods: Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis. Results: Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomic-identified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes. Conclusions: Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.

Life history and reproduction of the neotropical caecilian Siphonops annulatus (Amphibia, Gymnophiona, Siphonopidae), with special emphasis on parental care

Due to their mainly fossorial way of life, caecilian amphibians are the least known order of terrestrial vertebrates. Here, we present new observations on the natural history and reproductive biology of the neotropical oviparous, siphonopid caecilian Siphonops annulatus from a long-term study of this species in the field and in captivity. In the studied population, mating occurs between the end of August and beginning of October, and oviposition between November and December, when rainfall peaks. Egg hatching occurs between the end of December and beginning of January. The complete cycle of maternal care, from oviposition to independent, self-sufficient offspring lasts about 3 months. After eclosion, the altricial young feed on the mother's specially modified skin (maternal dermatophagy) and are also supplied by a fluid released from coming from the maternal cloaca. Also presented are observations on the burrows, feeding and social behaviour of S. annulatus.

Evidence for snake venom plasticity in a long-term study with individual captive Bothrops atrox

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.

Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes)

Caenophidian snakes include the file snake genus Acrochordus and advanced colubroidean snakes that radiated mainly during the Neogene. Although caenophidian snakes are a well-supported clade, their inferred affinities, based either on molecular or morphological data, remain poorly known or controversial. Here, we provide an expanded molecular phylogenetic analysis of Caenophidia and use three non-parametric measures of support–Shimodaira-Hasegawa-Like test (SHL), Felsentein (FBP) and transfer (TBE) bootstrap measures–to evaluate the robustness of each clade in the molecular tree. That very different alternative support values are common suggests that results based on only one support value should be viewed with caution. Using a scheme to combine support values, we find 20.9% of the 1265 clades comprising the inferred caenophidian tree are unambiguously supported by both SHL and FBP values, while almost 37% are unsupported or ambiguously supported, revealing the substantial extent of phylogenetic problems within Caenophidia. Combined FBP/TBE support values show similar results, while SHL/TBE result in slightly higher combined values. We consider key morphological attributes of colubroidean cranial, vertebral and hemipenial anatomy and provide additional morphological evidence supporting the clades Colubroides, Colubriformes, and Endoglyptodonta. We review and revise the relevant caenophidian fossil record and provide a time-calibrated tree derived from our molecular data to discuss the main cladogenetic events that resulted in present-day patterns of caenophidian diversification. Our results suggest that all extant families of Colubroidea and Elapoidea composing the present-day endoglyptodont fauna originated rapidly within the early Oligocene–between approximately 33 and 28 Mya–following the major terrestrial faunal turnover known as the "Grande Coupure" and associated with the overall climate shift at the Eocene-Oligocene boundary. Our results further suggest that the caenophidian radiation originated within the Caenozoic, with the divergence between Colubroides and Acrochordidae occurring in the early Eocene, at ~ 56 Mya.

Synthesis of d-glyco-alkynone derivatives via carbonylative Sonogashira reaction

A carbonylative Sonogashira coupling approach to the synthesis of glyco-alkynones is described. Eighteen examples were obtained in moderate do nearly quantitative yields under mild conditions employing Mo(CO)6 as a safe carbon monoxide source. Functionalization of the alkynyl moiety via cycloaddition with organic azides provided six examples of glyco-triazoles.

Functional variability of Bothrops atrox venoms from three distinct areas across the Brazilian Amazon and consequences for human envenomings

Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.