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.

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.

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.

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.

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.

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.