Aline Lopes

Functional Variability of Snake Venom Metalloproteinases: Adaptive Advantages in Targeting Different Prey and Implications for Human Envenomation

Snake venom metalloproteinases (SVMPs) are major components in most viperid venoms that induce disturbances in the hemostatic system and tissues of animals envenomated by snakes. These disturbances are involved in human pathology of snake bites and appear to be essential for the capture and digestion of snakes prey and avoidance of predators. SVMPs are a versatile family of venom toxins acting on different hemostatic targets which are present in venoms in distinct structural forms. However, the reason why a large number of different SVMPs are expressed in some venoms is still unclear. In this study, we evaluated the interference of five isolated SVMPs in blood coagulation of humans, birds and small rodents. P-III class SVMPs (fractions Ic, IIb and IIc) possess gelatinolytic and hemorrhagic activities, and, of these, two also show fibrinolytic activity. P-I class SVMPs (fractions IVa and IVb) are only fibrinolytic. P-III class SVMPs reduced clotting time of human plasma. Fraction IIc was characterized as prothrombin activator and fraction Ic as factor X activator. In the absence of Ca2+, a firm clot was observed in chicken blood samples with fractions Ic, IIb and partially with fraction IIc. In contrast, without Ca2+, only fraction IIc was able to induce a firm clot in rat blood. In conclusion, functionally distinct forms of SVMPs were found in B. neuwiedi venom that affect distinct mechanisms in the coagulation system of humans, birds and small rodents. Distinct SVMPs appear to be more specialized to rat or chicken blood, strengthening the current hypothesis that toxin diversity enhances the possibilities of the snakes for hunting different prey or evading different predators. This functional diversity also impacts the complexity of human envenoming since different hemostatic mechanisms will be targeted by SVMPs accounting for the complexity of the response of humans to venoms.

P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

Background Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. Results Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. Conclusion We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.

Implementation of the Ramsar Convention on South American wetlands: an update

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Research and Reports in Biodiversity Studies 2015:4 47–58 Research and Reports in Biodiversity Studies Dovepress

Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.

Design, Immune Responses and Anti-Tumor Potential of an HPV16 E6E7 Multi-Epitope Vaccine

Cervical cancer is a common type of cancer among women worldwide and infection with high-risk human papillomavirus (HPVs) types represents the major risk factor for the etiopathogenesis of the disease. HPV-16 is the most frequently identified HPV type in cervical lesions and expression of E6 and E7 oncoproteins is required for the uncontrolled cellular proliferation. In the present study we report the design and experimental testing of a recombinant multi-epitope protein containing immunogenic epitopes of HPV-16 E6 and E7. Tumor preventive assays, based on the engraftment of TC-1 cells in mice, showed that the E6E7 multi-epitope protein induced a full preventive anti-tumor protection in wild-type mice, as well as in mice deficient in expression of CD4+ T cells and TLR4 receptor. Nonetheless, no anti-tumor protection was observed in mice deficient in CD8+ T cells. Also, the vaccine promoted high activation of E6/E7-specific T cells and in a therapeutic-approach, E6E7 protein conferred full anti-tumor protection in mice. These results show a potential use of this E6E7 multi-epitope antigen as a new and promising antigen for the development of a therapeutic vaccine against tumors induced by HPV.

Seed germination and seedling establishment of Amazonian floodplain trees

Many Amazonian floodplain trees fruit during the high-water period and thus benefit from long-distance dispersal by hydrographic corridors. Two different germination strategies can be observed: Species with buoyant seeds tend to germinate rapidly as soon seeds get in contact with river waters, while species with submerged seeds generally undergo longer dormancies and germinate when flood-waters recede. In Amazonian floodplains, tree seedlings get frequently fully submerged for various periods, inducing hypoxia, complete darkness, and elevated mechanical constraints brought up by water currents and sediment. However, observations from field inventories indicate that seedlings of several varzea tree species perform very well under submergence, whereas mortality rates increase during the terrestrial phases. Establishment strategies seems to change along the flooding gradient: Seedlings from highly flooded low-varzea tree species are well-adapted to tolerate the seasonal inundations, while seedlings from low and irregularly flooded high-varzea tree species are poorly-flood adapted thus reacting very sensitive to flood pulse variations.

O tempo de exposição e a dosagem de óleo afetando a produção de biomassa do capim semi-aquático amazônico Echinochloa polystachya

The effects of Urucu crude oil on growth and biomass production of Echinochloa polystachya were studied experimentally. Young plants contaminated with 5 oil doses (0 to 2.64 l.m-2) were monitored at 1, 5, 10, 15 and 20 days after exposure. A significant reduction was observed in leaf humidity and the ratio of live aerial/ total biomass related to the increase in oil dosage. Increased exposition time caused a reduction in the number of leaves, aerial biomass, proportion of live aerial/total biomass, total biomass, and an increase of dead aerial biomass. However, root biomass and leaf length were not affected by exposure to oil. The low tolerance of E. polystachya to Urucu crude oil has lead to the mortality of 90% of the plant population in 20 days at the 2.64 l treatment. The death of young individuals of E. polystachya by relatively low dosages of Urucu oil indicates that, in the case of an oil spill in the Amazon varzea, this species would be severely affected, endangering, consequently, the entire environment.

Effects of increasing temperature and, CO2 on quality of litter, shredders, and microorganisms in Amazonian aquatic systems

Climate change may affect the chemical composition of riparian leaf litter and, aquatic organisms and, consequently, leaf breakdown. We evaluated the effects of different scenarios combining increased temperature and carbon dioxide (CO2) on leaf detritus of Hevea spruceana (Benth) Müll. and decomposers (insect shredders and microorganisms). We hypothesized that simulated climate change (warming and elevated CO2) would: i) decrease leaf-litter quality, ii) decrease survival and leaf breakdown by shredders, and iii) increase microbial leaf breakdown and fungal biomass. We performed the experiment in four microcosm chambers that simulated air temperature and CO2 changes in relation to a real-time control tracking current conditions in Manaus, Amazonas, Brazil. The experiment lasted seven days. During the experiment mean air temperature and CO2 concentration ranged from 26.96 ± 0.98ºC and 537.86 ± 18.36 ppmv in the control to 31.75 ± 0.50ºC and 1636.96 ± 17.99 ppmv in the extreme chamber, respectively. However, phosphorus concentration in the leaf litter decreased with warming and elevated CO2. Leaf quality (percentage of carbon, nitrogen, phosphorus, cellulose and lignin) was not influenced by soil flooding. Fungal biomass and microbial leaf breakdown were positively influenced by temperature and CO2 increase and reached their highest values in the intermediate condition. Both total and shredder leaf breakdown, and shredder survival rate were similar among all climatic conditions. Thus, low leaf-litter quality due to climate change and higher leaf breakdown under intermediate conditions may indicate an increase of riparian metabolism due to temperature and CO2 increase, highlighting the risk (e.g., decreased productivity) of global warming for tropical streams.