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The worrisome emergence of pathogens resistant to conventional drugs has stimulated the search for new classes of antimicrobial and antiparasitic agents from natural sources. Antimicrobial peptides (AMPs), acting through mechanisms that do not rely on the interaction with a specific receptor, provide new possibilities for the development of drugs against resistant organisms. This study sought to purify and proteomically characterize the antimicrobial and antiparasitic peptidomes of B. atrox and B. jararacussu snake venoms against Gram-positive (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus—MRSA), Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, and the protozoan parasites Leishmania amazonensis and Plasmodium falciparum (clone W2, resistant to chloroquine). To this end, B. atrox and B. jararacussu venom peptides were purified by combination of 3 kDa cut-off Amicon® ultracentrifugal filters and reverse-phase high-performance liquid chromatography, and then identified by electrospray-ionization Ion-Trap/Time-of-Flight mass spectrometry. Fourteen distinct peptides, with masses ranging from 443.17 to 1383.73 Da and primary structure between 3 and 13 amino acid residues, were sequenced. Among them, 13 contained unique sequences, including 4 novel bradykinin-potentiating-like peptides (BPPs), and a snake venom metalloproteinase tripeptide inhibitor (SVMPi). Although commonly found in Viperidae venoms, except for Bax-12, the BPPs and SVMPi here reported had not been described in B. atrox and B. jararacussu venoms. Among the novel peptides, some exhibited bactericidal activity towards P. aeruginosa and S. aureus, had low hemolytic effect, and were devoid of antiparasitic activity. The identified novel antimicrobial peptides may be relevant in the development of new drugs for the management of multidrug-resistant Gram-negative and Gram-positive bacteria.

Purpose of review Systemic sclerosis (scleroderma, SSc) is a rare multisystem autoimmune disease characterized by autoantibodies, vasculopathy, and fibrosis of the skin and internal organs. This review aims to provide an overview and summary of the recent epidemiological studies in systemic sclerosis. Recent findings Global trends of scleroderma demonstrate greater prevalence of SSc in European, North, and South American patients compared with East Asian patients. However, the greatest prevalence (47 in 100 000), was found among the indigenous peoples in Canada. Phenotypical differences exist depending on the age of presentation with greater internal organ involvement and disease acceleration present in older patients. Sex differences include greater severity of disease expression, relative prevalence of diffuse cutaneous SSc, and organ involvement in males versus females. New studies conflict with previous data reporting greater proportion of pulmonary arterial hypertension in females. Furthermore, the effect of low median household income is demonstrated as a factor increasing risk of death in SSc patients. Summary Understanding the epidemiological factors in SSc enables patient care through patient classification, prognostication, and monitoring. Future research may emphasize enrichment of SSc patients in randomized trials who are more likely to progress or be treatment responsive, focused screening, and personalized patient care through the creation and validation of new SSc criteria and subsets.

We report a structural and functional proteomics characterization of the venoms of the two subspecies (bilineatus and smaragdinus) of the South American palm pitviper B. bilineatus from the Brazili...

The pathogenesis of connective tissue diseases (CTDs), such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), is characterized by derangements of the innate and adaptive immune system, and inflammatory pathways leading to autoimmunity, chronic cytokine production, and chronic inflammation. The diagnosis of these diseases is based on meeting established criteria with symptoms, signs and autoantibodies. However, there are pre-clinical states where criteria are not fulfilled but biochemical and autoimmune derangements are present. Understanding the underlying processes responsible for disease pathogenesis in pre-clinical states, which place patients at increased risk for the development of established connective tissue diseases, represents an opportunity for early identification and potentially enables timely treatment with the goal of limiting disease progression and improved prognosis. This scoping review describes the role of the innate and adaptive immune responses in the pre-clinical states of undifferentiated CTD at risk for SSc and prescleroderma, the evolution of antibodies from nonspecific to specific antinuclear antibodies prior to SLE development, and the signaling pathways and inflammatory markers of fibroblast, endothelial, and T cell activation underlying immune dysregulation in these pre-clinical states.

Digital ulcers (DUs) occur in half of the patients with systemic sclerosis (SSc) and require health care interventions for treatment and monitoring for complications. Our objective was to assess the impact of DUs on resource utilization, including hospitalizations, outpatient visits, and procedures within a large SSc Canadian registry in a matched cohort study.A total of 1,698 SSc patients who completed 1 or more 84-item Resource Utilization Questionnaire (RUQ) for a 12-month recall period between September 2005 and February 2020 were included (9,077 questionnaires). Organ involvement was assessed by the Disease Severity Scale (DSS) on the Medsger scale. Unadjusted and adjusted regression analyses compared the association between DUs and resource utilization.RUQs in 104 SSc patients with active DUs at 2 consecutive annual visits were compared with 104 patients without DUs matched 1:1 for age, sex, disease subtype, and duration. Over 1 year, DUs were associated with a higher number of tests (P ˂ 0.05) and visits to health professionals, especially to a rheumatologist (P ˂ 0.0001) and internist (P = 0.003), a greater need for an accompanying person (P ˂ 0.05) and aids purchased/received (P ˂ 0.05). Having DUs was associated with more severe disease, even after excluding the peripheral vascular domain from a total DSS score (9.7 ± 4.5 versus 5.6 ± 2.7, P ˂ 0.0001). After adjustment for disease severity in other organs, the presence of DUs remained a significant predictor of more frequent physician visits and more tests (P for all ˂ 0.05) by linear regression analysis.SSc patients with DUs used significantly more health care resources per year even after adjustment for disease severity in other organ systems.

Background:Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential.Methods:A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated.Results:Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 μg/mL against Leishmania infantum promastigotes and of 13.8 μg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 μg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 μg/mL.Conclusion:Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity.

A comparative venom proteomic analysis of the Brazilian southern coral snake, M. frontalis, the Amazon coral snake M. spixii spixii, and the aquatic coral snake M. surinamensis is reported. Venoms from M. frontalis and M. s. spixii were composed mainly (>90% of the total venom proteome) by 3FTxs and PLAs in different proportions, and minor proteins from 2 to 5 protein families. Conversely, the aquatic coral snake expressed a streamlined (95%) 3FTx venom with low abundance (4.2%) of PLA molecules. A compositional-lethal activity for natural prey correlation analysis suggests that M. surinamensis venom may has evolved under strong pressure to quickly immobilize aquatic prey. On the other hand, venoms from M. frontalis and M. s. spixii, whose diet consist mainly of amphisbaenians and colubrid snakes, may have been shaped through balancing selection. Our work provides strong evidence for the occurrence in M. frontalis venom, but not in those from M. s. spixi and M. surinamensis, of a KUN-PLA complex homologue to heterodimeric venom toxins from some long-tailed monadal coral snakes that target acid-sensing receptors ASIC1a/2 evoking pain. The M. frontalis protein would represent the first example of a KUN-PLA heterodimer in a South American short-tailed triadal coral snake venom., This study was partly supported by grant BFU 2017-89103-P from the Ministerio de Ciencia, Innovación y Universidades, Madrid (Spain)to JJC. The authors wish to gratefully acknowledge the collaboration ofDr. Kayena Delaix Zaqueo inM. surinamensisvenom extraction. Authorsalso express their gratitude to Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq/MCTIC), Coordenação deAperfeiçoamento de Pessoal de Nível Superior (CAPES/MEC) andFundação Rondônia de Amparo ao Desenvolvimento das AçõesCientíficas e Tecnológicas de Pesquisa do Estado de Rondônia(FAPERO) forfinancial support. The authors thank the Program forTechnological Development in Tools for Health-PDTIS-FIOCRUZ for theuse of its facilities.

Objective Systemic sclerosis (SSc) is characterized by vasculopathy, fibrosis of skin and internal organs, and autoimmunity with complications including interstitial lung disease, pulmonary hypertension, and digital ulcers with substantial morbidity and disability. Patients with SSc may require considerable healthcare resources with economic impact. The purpose of this systematic review was to provide a narrative synthesis of the economic impact and healthcare resource utilization associated with SSc. Methods MEDLINE and EMBASE were searched from inception to 20 January 2021. Studies were included if they provided information regarding the total, direct and indirect cost of SSc. The cost of SSc subtypes and associated complications was determined. Risk of bias assessments through the Joanna Briggs Institute cross-sectional and case series checklists, and the Newcastle–Ottawa Cohort and Case–Control study scales were performed. A narrative synthesis of included studies was planned. Results The number of publications retrieved was 1778, of which 34 were included representing 20 cross-sectional, 11 cohort, and three case–control studies. Studies used various methods of calculating cost including prevalence-based cost-of-illness approach and health resource units cost analysis. Overall SSc total annual cost ranged from USD $14 959 to $23 268 in USA, CAD $10 673 to $18 453 in Canada, €4607 to €30 797 in Europe, and AUD $7060 to $11 607 in Oceania. Annual cost for SSc-associated interstitial lung disease and pulmonary hypertension was USD $31 285–55 446 and $44 454–63 320, respectively. Conclusion Cost-calculation methodology varied greatly between included studies. SSc represents a significant patient and health resource economic burden. SSc-associated complications increase economic burden and are variable depending on geographical location and access.

Background Bleeding is the leading cause of maternal mortality in the world. Tranexamic acid reduces bleeding in trauma and surgery. Several systematic reviews of randomized trials have investigated tranexamic acid in the prevention of bleeding in cesarean delivery. However, the conclusions from systematic reviews are conflicting. This overview aims to summarize the evidence and explore the reasons for conflicting conclusions across the systematic reviews. Methods A comprehensive literature search of Medline, Embase, and Cochrane Database of Systematic Reviews was conducted from inception to April 2021. Screening, data extraction, and quality assessments were performed by two independent reviewers. A Measurement Tool to Assess Reviews 2 and the Risk of Bias Assessment Tool for Systematic Reviews were used for study appraisal. A qualitative synthesis of evidence is presented. Results In all, 14 systematic reviews were included in our analysis. Across these reviews, there were 32 relevant randomized trials. A modest reduction in blood transfusions and bleeding outcomes was found by most systematic reviews. Overall confidence in results varied from low to critically low. All of the included systematic reviews were at high risk of bias. Quality of evidence from randomized trials was uncertain. Conclusions Systematic reviews investigating prophylactic tranexamic acid in cesarean delivery are heterogeneous in terms of methodological and reporting quality. Tranexamic acid may reduce blood transfusion and bleeding outcomes, but rigorous well-designed research is needed due to the limitations of the included studies. Data on safety and adverse effects are insufficient to draw conclusions.

Systemic sclerosis (scleroderma, SSc) is a rare multisystem autoimmune disease characterized by autoantibodies, vasculopathy, and fibrosis of the skin and internal organs. This review aims to provide an overview and summary of the recent epidemiological studies in systemic sclerosis.Global trends of scleroderma demonstrate greater prevalence of SSc in European, North, and South American patients compared with East Asian patients. However, the greatest prevalence (47 in 100 000), was found among the indigenous peoples in Canada. Phenotypical differences exist depending on the age of presentation with greater internal organ involvement and disease acceleration present in older patients. Sex differences include greater severity of disease expression, relative prevalence of diffuse cutaneous SSc, and organ involvement in males versus females. New studies conflict with previous data reporting greater proportion of pulmonary arterial hypertension in females. Furthermore, the effect of low median household income is demonstrated as a factor increasing risk of death in SSc patients.Understanding the epidemiological factors in SSc enables patient care through patient classification, prognostication, and monitoring. Future research may emphasize enrichment of SSc patients in randomized trials who are more likely to progress or be treatment responsive, focused screening, and personalized patient care through the creation and validation of new SSc criteria and subsets.

Malaria is a parasitic infectious disease and was responsible for 400.000 deaths in 2018. Plasmodium falciparum represents the species that causes most human deaths due to severe malaria. In addition, studies prove the resistance of P. falciparum to drugs used to treat malaria, making the search for new drugs with antiplasmodial potential necessary. In this context, the literature describes snake venoms as a rich source of molecules with microbicidal potential, including phospholipases A2 (PLA2s). In this sense, the present study aimed to isolate basic PLA2s from Paraguayan Bothrops diporus venom and evaluate their antiplasmodial potential. Basic PLA2s were obtained using two chromatographic steps. Initially, B. diporus venom was subjected to ion exchange chromatography (IEC). The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the PLA2s. The toxins were tested for enzymatic activity using a chromogenic substrate and finally, the antiplasmodial, cytotoxic potential and hemolytic activity of the isolated toxins were evaluated. The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the two enzymatically active PLA2s, BdTX-I and BdTX-II and the PLA2 homologue BdTX-III. The antiplasmodial potential was evaluated and the toxins showed IC50 values of: 2.44, 0.0153 and 0.59 μg/mL respectively, presenting PLA2 selectivity according to the selectivity index results (SI) calculated against HepG2 cells. The results show that the 3 basic phospholipases isolated in this study have a potent antiparasitic effect against the W2 strain of P. falciparum. In view of the results obtained in this work, further research are necessary to determine the mechanism of action by which these toxins cause cell death in parasites., Highlights • Three basic PLA2s from the venom of Paraguayan Bothrops diporus have been isolated. • In vitro Antimalarial activity of PLA2s against intra-erythrocytic forms of Plasmodium falciparum were evaluated. • Cytotoxicity on HepG2 cells, hemolytic activity and Selectivity Index of PLA2s were analyzed.

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians’ skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions ofR. marinasecretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active againstStaphylococcus aureus,Pseudomonas aeruginosa,Escherichia coli,Plasmodium falciparum,Leishmania guyanensis, andLeishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents ofR.marinasecretion as well as the bioactive potential of these molecules.

Snake venom phospholipases A2 (PLA2 s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA2 homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA2 s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA2 from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.

Made available in DSpace on 2018-12-11T16:47:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107–116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment. Centro de Estudos de Biomoléculas Aplicadas a Saúde CEBio Fundação Oswaldo Cruz FIOCRUZ Fiocruz Rondônia and Departamento de Medicina Universidade Federal de Rondônia UNIR Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP Center for the Study of Biological Complexity Life Sciences Virginia Commonwealth University PVE-CNPq Centro Universitário São Lucas UNISL Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP

Made available in DSpace on 2018-12-11T16:47:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-01 Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100 μg/mL. The venom and BmajPLA2-II presented IC50 of 0.14 ± 0.08 and 6.41 ± 0.64 μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64 ± 7.94 and >150 μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated. Centro de Estudos de Biomoléculas Aplicadas à Saúde CEBio Fundação Oswaldo Cruz FIOCRUZ Fiocruz Rondônia Departamento de Medicina Universidade Federal de Rondônia UNIR Centro para el Desarrollo de la Investigación Científica CEDIC Laboratório da Plataforma de Bioensaios de Malária e Leishmaniose Fiocruz Rondônia Faculdade São Lucas FSL Departamento de Física e Biofísica Universidade Estadual Paulista UNESP Laboratório de Bioquímica e Biofísica Instituto Butantan Departamento de Física e Biofísica Universidade Estadual Paulista UNESP

Phospholipases A 2 (PLA 2 s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA 2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA 2 s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA 2 from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA 2 s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic phospholipase A 2 isoforms in snake venoms.

The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.

14 páginas, 3 figuras, 2 tablas. The datasets generated during the current study are available in Additional file 1 (Table S1) and Additional file 2 (Table S2). Raw mass spectrometric data are available from the corresponding authors on reasonable request., BACKGROUND: The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. METHODS: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. RESULTS: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. CONCLUSION: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead., This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia, Innovación y Universidades, Madrid (Spain) to JJC. The authors wish to express their gratitude to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCTIC), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/MEC) and Fundação Rondônia de Amparo ao Desenvolvimento das Ações Científicas e Tecnológicas de Pesquisa do Estado de Rondônia (FAPERO) for financial support.

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated. Elsevier

Malaria is a parasitic infectious disease and was responsible for 400.000 deaths in 2018. Plasmodium falciparum represents the species that causes most human deaths due to severe malaria. In addition, studies prove the resistance of P. falciparum to drugs used to treat malaria, making the search for new drugs with antiplasmodial potential necessary. In this context, the literature describes snake venoms as a rich source of molecules with microbicidal potential, including phospholipases A2 (PLA2s). In this sense, the present study aimed to isolate basic PLA2s from Paraguayan Bothrops diporus venom and evaluate their antiplasmodial potential. Basic PLA2s were obtained using two chromatographic steps. Initially, B. diporus venom was subjected to ion exchange chromatography (IEC). The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the PLA2s. The toxins were tested for enzymatic activity using a chromogenic substrate and finally, the antiplasmodial, cytotoxic potential and hemolytic activity of the isolated toxins were evaluated. The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the two enzymatically active PLA2s, BdTX-I and BdTX-II and the PLA2 homologue BdTX-III. The antiplasmodial potential was evaluated and the toxins showed IC50 values of: 2.44, 0.0153 and 0.59 μg/mL respectively, presenting PLA2 selectivity according to the selectivity index results (SI) calculated against HepG2 cells. The results show that the 3 basic phospholipases isolated in this study have a potent antiparasitic effect against the W2 strain of P. falciparum. In view of the results obtained in this work, further research are necessary to determine the mechanism of action by which these toxins cause cell death in parasites.

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of

Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones – a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs – and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms.

The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A2 called MsPLA2-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC50 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA2-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA2 from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA2-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA2-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential.

Background/Aims: To evaluate antileishmanial activity of crotamine, a toxin isolated from Crotalus durissus terrificus, in solution form and encapsulated in biodegradable microparticles in vitro. Methods: Particles were analyzed on-chip by surface plasmon resonance and characterized by testing their diameters, zeta potential and encapsulation rate. The viability of promastigotes as well as murine macrophages was assessed. Furthermore, the phagocytic index was determined for macrophages, and cell supernatants were collected for the determination of TNF-α levels. An infection assay using Leishmania amazonensis-infected macrophages was also conducted. Results: The diameters and zeta potential of control particles (1.35 μm; -12.3 mV) and of those containing crotamine (3.09 μm; -20.9 mV) were adequate for the assays conducted. Crotamine-loaded particles were better captured by macrophages than control particles (increase of 12% in the phagocytic index), leading to increased TNF-α levels (196 pg/ml), and they also induced a significant decrease in the numbers of amastigotes compared to infected macrophages only. Conclusion: The approach presented here opens the possibility of working with safe concentrations of encapsulated toxins to reach antileishmanial effects.

Snake venom phospholipases A

Crude venom ofBothrops jararacussuand isolated phospholipases A2(PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) byp-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2(PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

Bothrops mattogrossensissnake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2(PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s fromBothropsspecies. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms ofLeishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

L-amino acid oxidases are enzymes found in several organisms, including venoms of snakes, where they contribute to the toxicity of ophidian envenomation. Their toxicity is primarily due to enzymatic activity, but other mechanisms have been proposed recently which require further investigation. L-amino acid oxidases exert biological and pharmacological effects, including actions on platelet aggregation and the induction of apoptosis, hemorrhage, and cytotoxicity. These proteins present a high biotechnological potential for the development of antimicrobial, antitumor, and antiprotozoan agents. This review provides an overview of the biochemical properties and pharmacological effects of snake venom L-amino acid oxidases, their structure/activity relationship, and supposed mechanisms of action described so far.

The aim of the study was to evaluate the in vitro effects of Bothrops bilineata crude venom (BbV) on isolated human neutrophil function. We proved that BbV isn't toxic towards human neutrophils. During an incubation of human neutrophils with BbV hydrogen peroxide was produced. Moreover, BbV was able to stimulate neutrophil release of proinflammatory mediators such as IL-8 and IL-6 as well as PGE2 and NETs liberation. There is no data in the literature showing the effect of BbV on the production of IL-6 and IL-8 or NETs liberation by isolated human neutrophils. Taken together our results testify that BbV triggers relevant proinflammatory events in human neutrophils.

Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA2 (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin.

Phospholipases A

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A2 (PLA2), and BaltTX-II, an Asp49 catalytically active PLA2 isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA2 BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA2, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA2s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.

Snake venoms contain various proteins, especially phospholipases A

BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI⿿5.3, 15% sugar and a related molecular mass of 66,000 Da in its monomeric form, and 123,000 Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy.

Phospholipases A2 (PLA2s) from snake venoms comprise a group of 14-18 kDa proteins, responsible for several toxic effects induced by the whole venom. Considering this, studies aiming at the search for natural inhibitors of these proteins are very important. The present work had as objectives the isolation and functional/structural characterization of a γ-type phospholipase A2 inhibitor (PLI) from Bothrops jararacussu snake plasma, named γBjussuMIP. This acidic glycoprotein was isolated in a high purity level through affinity chromatography on CNBr-Sepharose 4B coupled with BthTXII, showing a pI ∼ 5.5 and molecular weight of 23,500 for the monomer (determined by SDS-PAGE), and 160,000 for the oligomer (determined by molecular exclusion chromatography on Sephacryl S-200). The interaction between γBjussuMIP (MIP) and Phospholipase A2 (PLA2) was confirmed using circular dichroism (CD) and emission fluorescence techniques. The helical content of the 1:1 molar mixture was higher than that calculated for the addition of the spectra of the unbound proteins indicating binding. The emission fluorescence experiments pointed that Trp residues in PLA2 participate in proteins interaction as blue shift of 4 nm was observed. The γBjussuMIP cDNA, obtained by PCR of the liver of B. jararacussu snake, revealed 543 bp codifying for a mature protein of 181 amino acid residues. Alignment of its amino acid sequence with those of other snake γPLIs showed 89-94% of similarity. γBjussuMIP mainly inhibited the pharmacological properties of Asp49 PLA2s, such as phospholipase, anticoagulant, myotoxic, edema inducing, cytotoxic, bactericidal and lethal activities. In addition, it showed to be able to supplement Bothrops antivenom, potentiating its antimyotoxic effect. The aspects broached in this work will be able to provide complementary information on possible mechanisms of action, relating structure and function, which could result in a better understanding of the inhibitory effects induced by γBjussuMIP.

Screening for new bioactive peptides in South American anurans has been pioneered in frogs of the genus Phyllomedusa. All frogs of this genus have venomous skin secretions, i.e., a complex mixture of bioactive peptides against potential predators and pathogens that presumably evolved in a scenario of predator-prey interaction and defense against microbial invasion. For every new anuran species studied new peptides are found, with homologies to hormones, neurotransmitters, antimicrobials, and several other peptides with unknown biological activity. From Vittorio Erspamer findings, this genus has been reported as a "treasure store" of bioactive peptides, and several groups focus their research on these species. From 1966 to 2009, more than 200 peptide sequences from different Phyllomedusa species were deposited in UniProt and other databases. During the last decade, the emergence of high-throughput molecular technologies involving de novo peptide sequencing via tandem mass spectrometry, cDNA cloning, pharmacological screening, and surface plasmon resonance applied to peptide discovery, led to fast structural data acquisition and the generation of peptide molecular libraries. Research groups on bioactive peptides in Brazil using these new technologies, accounted for the exponential increase of new molecules described in the last decade, much higher than in any previous decades. Recently, these secretions were also reported as a rich source of multiple antimicrobial peptides effective against multidrug resistant strains of bacteria, fungi, protozoa, and virus, providing instructive lessons for the development of new and more efficient nanotechnological-based therapies for infectious diseases treatment. Therefore, novel drugs arising from the identification and analysis of bioactive peptides from South American anuran biodiversity have a promising future role on nanobiotechnology.

Cancer, a disease that currently affects approximately 14 million people, is characterized by abnormal cell growth with altered replication capacity, which leads to the development of tumor masses without apoptotic control. Resistance to the drugs used in chemotherapy and their side effects stimulate scientific research seeking new therapies to combat this disease. Molecules from flora and fauna with cytotoxic activity against tumor cells have been studied for their potential to become a source of pharmaceutical agents. In this regard, snake venoms have a variety of proteins and peptides that have proven biotechnological potential. In several studies, antibacterial action and antitumor activity have been observed. One of the most widely studied venom components are phospholipases A2. Snake venom phospholipases A2 (svPLA2s) comprise a large class of molecules that catalyze the hydrolysis of the sn-2 position of phospholipids releasing fatty acids and lysophospholipids and are related to a broad spectrum of biotechnological activities. In addition to their specific cytotoxicity against some tumor cell lines, inhibitory activity of angiogenesis, adhesion and cell migration has been described. The antitumor activity of svPLA2s was observed both in vitro and in vivo, but little is known about the mechanism of action of these proteins in promoting this activity. In this review, the main structural and functional characteristics of svPLA2s are discussed, along with the mechanisms proposed, thus far, to explain their antitumor activity, targeting their potential use as a therapeutic alternative against cancer.

This study describes the biochemical and functional characterization of a new metalloproteinase named BbMP-1, isolated from Bothrops brazili venom. BbMP-1 was homogeneous on SDS-PAGE, presented molecular mass of 22,933Da and pI 6.4. The primary structure was partially elucidated with high identity with others metalloproteinases from Viperidae venoms. The enzymatic activity on azocasein was evaluated in different experimental conditions (pH, temperature). A significant reduction in enzyme activity after exposure to chelators of divalent cations (EDTA), reducing agents (DTT), pH less than 5.0 or temperatures higher than 45 °C was observed. BbMP-1 showed activity on fibrinogen degrading Aα chain quickly and to a lesser extent the Bβ chain. Also demostrated to be weakly hemorrhagic, presenting however, significant myotoxic and edematogenic activity. The in vitro activity of BbMP-1 against Plasmodium falciparum showed an IC50 of 3.2 ± 2.0 μg/mL. This study may help to understand the pathophysiological effects induced by this group of toxin and their participation in the symptoms observed in cases of snake envenomation. Moreover, this result is representative for this group of proteins and shows the biotechnological potential of BbMP-1 by the demonstration of its antiplasmodial activity.

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.

The thermal stability of a Schizolobium parahyba chymotrypsin inhibitor (SPCI) as a function of pH has been investigated using fluorescence, circular dichroism, and differential scanning calorimetry (DSC). The thermodynamic parameters derived from all methods are remarkably similar and strongly suggest the high stability of SPCI under a wide range of pH. The transition temperature (T(m)) values ranging from 57 to 85.3 degrees C at acidic, neutral, and alkaline pH are in good agreement with proteins from mesophilic and thermophilic organisms and corroborate previous data regarding the thermal stability of SPCI. All methods gave transitions curves adequately fitted to a two-state model of the unfolding process as judged by the cooperative ratio between the van't Hoff and the calorimetric enthalpy energies close to unity in all of the pH conditions analyzed, except at pH 3.0. Thermodynamic analysis using all these methods reveals that SPCI is thermally a highly stable protein, over the wide range of pH from 3.0 to 8.8, exhibiting high stability in the pH region of 5.0-7.0. The corresponding maximum stabilities, DeltaG(25), were obtained at pH 7.0 with values of 15.4 kcal mol(-1) (combined fluorescence and circular dichroism data), and 15.1 kcal mol(-1) (DSC), considering a DeltaC(p) of 1.72 +/- 0.24 kcal mol(-1) K(-1). The low histidine content ( approximately 1.7%) and the high acidic residue content ( approximately 22.5%) suggests a flat pH dependence of thermal stability in the region 2.0-8.8 and that the decrease in thermal stability at low pH can be due to the differences in pK values of the acidic groups.

Schizolobium parahyba chymotrypsin inhibitor (SPCI) was completely purified as a single polypeptide chain with two disulfide bonds, by TCA precipitation and ion exchange chromatography. This purification method is faster and more efficient than that previously reported: SPCI is stable from pH 2 to 12 at 25 degrees C, and is highly specific for chymotrypsin at pH 7-12. It weakly inhibits elastase and has no significant inhibitory effect against trypsin and alpha-amylase. SPCI is a thermostable protein and resists thermolysin digestion up to 70 degrees C.

Synthetic compounds derived from cinnamic acid were tested in cultures containing the promastigote form of Leishmania amazonensis and the dimethylsulphoxide solution of B2 compound (2.0 mg/mL) led to a 92% decrease of leishmania in 96 h of treatment. Then, different liposomal systems (diameters ∼200 nm) were prepared by the extrusion method in the presence and absence of compounds studied. DSC thermograms of the liposomes in the presence of these compounds caused changes in ΔH, Tm and ΔT1/2, compared to controls, indicating that there was an interaction of the compounds with the lipid bilayer. Assays with negatively charged liposomal systems containing these drugs in L. amazonensis cultures led to a 50–80% decrease in the number of leishmanias with a concentration to 100 times lower when compared to the B2 initial test. These liposomal systems are promoting more interaction and delivery of the compounds and proved to be an efficient, stable and promising system.

To evaluate antileishmanial activity of crotamine, a toxin isolated from Crotalus durissus terrificus, in solution form and encapsulated in biodegradable microparticles in vitro.Particles were analyzed on-chip by surface plasmon resonance and characterized by testing their diameters, zeta potential and encapsulation rate. The viability of promastigotes as well as murine macrophages was assessed. Furthermore, the phagocytic index was determined for macrophages, and cell supernatants were collected for the determination of TNF-α levels. An infection assay using Leishmania amazonensis-infected macrophages was also conducted.The diameters and zeta potential of control particles (1.35 μm; -12.3 mV) and of those containing crotamine (3.09 μm; -20.9 mV) were adequate for the assays conducted. Crotamine-loaded particles were better captured by macrophages than control particles (increase of 12% in the phagocytic index), leading to increased TNF-α levels (196 pg/ml), and they also induced a significant decrease in the numbers of amastigotes compared to infected macrophages only.The approach presented here opens the possibility of working with safe concentrations of encapsulated toxins to reach antileishmanial effects.

This paper presents a novel serine protease (SP) isolated fromBothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu2+significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated fromBothrops pirajaisnake venom.

For more than half a century, cytotoxic agents have been investigated as a possible treatment for cancer. Research on animal venoms has revealed their high toxicity on tissues and cell cultures, both normal and tumoral. Snake venoms show the highest cytotoxic potential, since ophidian accidents cause a large amount of tissue damage, suggesting a promising utilization of these venoms or their components as antitumoral agents. Over the last few years, we have studied the effects of snake venoms and their isolated enzymes on tumor cell cultures. Somein vivoassays showed antineoplastic activity against induced tumors in mice. In human beings, both the crude venom and isolated enzymes revealed antitumor activities in preliminary assays, with measurable clinical responses in the advanced treatment phase. These enzymes include metalloproteases (MP), disintegrins, L-amino acid oxidases (LAAOs), C-type lectins, and phospholipases A2(PLA2s). Their mechanisms of action include direct toxic action (PLA2s), free radical generation (LAAOs), apoptosis induction (PLA2s, MP, and LAAOs), and antiangiogenesis (disintegrins and lectins). Higher cytotoxic and cytostatic activities upon tumor cells than normal cells suggest the possibility for clinical applications. Further studies should be conducted to ensure the efficacy and safety of different snake venom compounds for cancer drug development.

Ophidian envenomation is an important health problem in Brazil and other South American countries. In folk medicine, especially in developing countries, several vegetal species are employed for the treatment of snakebites in communities that lack prompt access to serum therapy. However, the identification and characterization of the effects of several new plants or their isolated compounds, which are able to inhibit the activities of snake venom, are extremely important and such studies are imperative. Snake venom contains several organic and inorganic compounds; phospholipases A2(PLA2s) are one of the principal toxic components of venom. PLA2s display a wide variety of pharmacological activities, such as neurotoxicity, myotoxicity, cardiotoxicity, anticoagulant, hemorrhagic, and edema-inducing effects. PLA2inhibition is of pharmacological and therapeutic interests as these enzymes are involved in several inflammatory diseases. This review describes the results of several studies of plant extracts and their isolated active principles, when used against crude snake venoms or their toxic fractions. Isolated inhibitors, such as steroids, terpenoids, and phenolic compounds, are able to inhibit PLA2s from different snake venoms. The design of specific inhibitors of PLA2s might help in the development of new pharmaceutical drugs, more specific antivenom, or even as alternative approaches for treating snakebites.

The in vitro effects of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function were investigated. LAAO showed no toxicity on neutrophils. At non-cytotoxic concentrations, LAAO induced the superoxide anion production by isolated human neutrophil. This toxin, in its native form, is also able to stimulate the production of hydrogen peroxide in neutrophils, suggesting that its primary structure is essential for stimulation the cell. Moreover, the incubation of LAAO and phenol red medium did not induce the production of hydrogen peroxide. Furthermore, LAAO was able to stimulate neutrophils to release proinflammatory mediators such as IL-8 and TNF-a as well as NETs liberation. Together, the data showed that the LAAO triggers relevant proinflammatory events. Particular regions of the molecule distinct from the LAAO catalytic site may be involved in the onset of inflammatory events.