Resumo em Inglês:

Abstract Snakebite is a critical public health issue in tropical countries, particularly in Africa, where 20% of snakebites globally occur. In 2017, the WHO added snakebite envenoming to the category A of neglected tropical diseases. In 2019, thanks to broad institutional and international NGO support, including strong mobilization of African experts and governments, WHO launched a strategy for prevention and control of snakebite envenoming with more ambitious goals. In sub-Saharan Africa, accessibility of antivenoms and symptomatic, adjuvant or replacement therapy is a priority. Several antivenoms are available but their evaluation has not been properly carried out and they remain expensive. To date, there are no manufacturers of antivenom in sub-Saharan Africa (except in South Africa), which requires their importation from other continents. The lack of experience in antivenom choice and its use by health authorities, health personnel and population largely explains the shortage in sub-Saharan Africa. The deficiency of epidemiological data does not allow the implementation of appropriate and efficient care. It is crucial to strengthen the health system which does not have the necessary means for emergency management in general and envenoming in particular. Providing peripheral health centers with antivenoms would decrease complications and deaths. The motivation of communities at risk, identified through the epidemiological data, would be to reduce the delay in consultation that is detrimental to the efficiency of treatment. Partnerships need to be coordinated to optimize resources from international institutions, particularly African ones, and share the burden of treatment costs among all stakeholders. We propose here a project of progressive implementation of antivenom manufacturing in sub-Saharan Africa. The various steps, from the supply of appropriate venoms to the production of purified specific antibodies and vial filling, would be financed by international, regional and local funding promoting technology transfer from current manufacturers compensated by interest on the sale of antivenoms.

Resumo em Inglês:

Abstract Neglected Tropical Diseases (NTDs) comprise of a group of seventeen infectious conditions endemic in many developing countries. Among these diseases are three of protozoan origin, namely leishmaniasis, Chagas disease, and African trypanosomiasis, caused by the parasites Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei respectively. These diseases have their own unique challenges which are associated with the development of effective prevention and treatment methods. Collectively, these parasitic diseases cause more deaths worldwide than all other NTDs combined. Moreover, many current therapies for these diseases are limited in their efficacy, possessing harmful or potentially fatal side effects at therapeutic doses. It is therefore imperative that new treatment strategies for these parasitic diseases are developed. Nanoparticulate drug delivery systems have emerged as a promising area of research in the therapy and prevention of NTDs. These delivery systems provide novel mechanisms for targeted drug delivery within the host, maximizing therapeutic effects while minimizing systemic side effects. Currently approved drugs may also be repackaged using these delivery systems, allowing for their potential use in NTDs of protozoan origin. Current research on these novel delivery systems has provided insight into possible indications, with evidence demonstrating their improved ability to specifically target pathogens, penetrate barriers within the host, and reduce toxicity with lower dose regimens. In this review, we will examine current research on these delivery systems, focusing on applications in the treatment of leishmaniasis, Chagas disease, and African trypanosomiasis. Nanoparticulate systems present a unique therapeutic alternative through the repositioning of existing medications and directed drug delivery.

Resumo em Inglês:

Abstract Scorpion venoms are natural sources of molecules that have, in addition to their toxic function, potential therapeutic applications. In this source the neurotoxins can be found especially those that act on potassium channels. Potassium channels are responsible for maintaining the membrane potential in the excitable cells, especially the voltage-dependent potassium channels (Kv), including Kv1.3 channels. These channels (Kv1.3) are expressed by various types of tissues and cells, being part of several physiological processes. However, the major studies of Kv1.3 are performed on T cells due its importance on autoimmune diseases. Scorpion toxins capable of acting on potassium channels (KTx), mainly on Kv1.3 channels, have gained a prominent role for their possible ability to control inflammatory autoimmune diseases. Some of these toxins have already left bench trials and are being evaluated in clinical trials, presenting great therapeutic potential. Thus, scorpion toxins are important natural molecules that should not be overlooked in the treatment of autoimmune and other diseases.

Resumo em Inglês:

Abstract Spider venoms are known to contain proteins and polypeptides that perform various functions including antimicrobial, neurotoxic, analgesic, cytotoxic, necrotic, and hemagglutinic activities. Currently, several classes of natural molecules from spider venoms are potential sources of chemotherapeutics against tumor cells. Some of the spider peptide toxins produce lethal effects on tumor cells by regulating the cell cycle, activating caspase pathway or inactivating mitochondria. Some of them also target the various types of ion channels (including voltage-gated calcium channels, voltage-gated sodium channels, and acid-sensing ion channels) among other pain-related targets. Herein we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against the pathophysiological conditions including cancer and pain.

Resumo em Inglês:

Abstract In recent years feline leishmanial infections (FLI) have been studied more than ever before in various parts of the world. However, evidence-based knowledge on FLI has remained unavailable. The main objectives of this study were to investigate the status of felines infected by Leishmania spp. worldwide. Data were extracted from 10 available databases over the period of 1982 to 2017. Overall, 78 articles fulfilled the inclusion criteria and were used for data extraction in this systematic review. The overall FLI prevalence by both serological and molecular methods was estimated at 10% (95% CI: 8%-14%). In Italy, both the seroprevalence (24 %) and PCR prevalence (21 %) were found to be higher than in other countries. The most common diagnostic test used was the indirect fluorescent antibody test (38.5%). Studies on mixed-breed felines were more common than those on other breeds, while the most common parasite species was L. infantum (63%). Our findings suggest that cats act as primary and/or secondary reservoir hosts in the transmission of the Leishmania spp. to humans and also to dogs, by sandflies, at least in endemic foci. Moreover, available data confirm the enzootic stability situation of FLI in several countries including some in Europe.

Resumo em Inglês:

Abstract Traditional medicine plays an important role in the daily lives of people living in rural parts of Ethiopia. Despite the fact that Ethiopia has a long history of using traditional medicinal plants as an alternative medicine source, there is no checklist compiling these plants used for snakebite treatment. This review collected and compiled available knowledge on and practical usage of such plants in the country. A literature review on medicinal plants used to treat snakebites was conducted from 67 journal articles, PhD dissertation and MSc theses available online. Data that summarize scientific and folk names, administration methods, plant portion used for treatment and method of preparation of recipes were organized and analyzed based on citation frequency. The summarized results revealed the presence of 184 plant species distributed among 67 families that were cited for treating snakebite in Ethiopia. In this literature search, no single study was entirely dedicated to the study of traditional medicinal plants used for the treatment of snakebite in Ethiopia. Most of the species listed as a snakebite remedy were shrubs and climbers (44%) followed by herbs (33%) and trees (23%). Fabaceae was the most predominant family with the greatest number of species, followed by Solanaceae and Vitaceae. Remedies are mainly prepared from roots and leaves, through decoctions, infusions, powders and juices. Most remedies were administered orally (69%). The six most frequently mentioned therapeutically important plants were Nicotiana tabacum, Solanum incanum, Carissa spinanrum, Calpurnia aurea, Croton macrostachyus and Cynodon dactylon. Authors reviewed the vegetal substances involved in snakebite management and their action mode. In addition to screening the biologically active ingredients and pharmacological activities of these plant materials, future studies are needed to emphasize the conservation and cultivation of important medicinal plants of the country.

Resumo em Inglês:

Abstract In Brazil and in other tropical areas Zika virus infection was directly associated with clinical complications as microcephaly in newborn children whose mothers were infected during pregnancy and the Guillain-Barré syndrome in adults. Recently, research has been focused on developing new vaccines and drug candidates against Zika virus infection since none of those are available. In order to contribute to vaccine and drug development efforts, it becomes important the understanding of the molecular basis of the Zika virus recognition, infection and blockade. To this purpose, it is essential the structural determination of the Zika virus proteins. The genome sequencing of the Zika virus identified ten proteins, being three structural (protein E, protein C and protein prM) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Together, these proteins are the main targets for drugs and antibody recognition. Here we examine new discoveries on high-resolution structural biology of Zika virus, observing the interactions and functions of its proteins identified via state-of-art structural methodologies as X-ray crystallography, nuclear magnetic resonance spectroscopy and cryogenic electronic microscopy. The aim of the present study is to contribute to the understanding of the structural basis of Zika virus infection at an atomic level and to point out similarities and differences to others flaviviruses.

Resumo em Inglês:

Abstract Fibrin biopolymers, previously referred as “fibrin glue” or “fibrin sealants”, are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was “heterologous fibrin sealant”. The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.

Resumo em Inglês:

Abstract Background: Ant venoms express surface molecules that participate in antigen presentation involving pro- and anti-inflammatory cytokines. This work aims to investigate the expression of MHC-II, CD80 and CD86 on the polymorphonuclear cells (PMNs) in rats injected with samsum ant venom (SAV). Methods: Rats were divided into three groups - control, SAV-treated (intraperitoneal route, 600 μg/kg), and SAV-treated (subcutaneous route, 600 μg/kg). After five doses, animals were euthanized and samples collected for analysis. Results: The subcutaneous SAV-trated rats presented decreased levels of glutathione with increased cholesterol and triglyceride levels. Intraperitoneal SAV-treated animals displayed significantly reduced concentrations of both IFN-γ and IL-17 in comparison with the control group. However, intraperitoneal and subcutaneous SAV-treated rats were able to upregulate the expressions of MHC-II, CD80 and CD86 on PMNs in comparison with the control respectively. The histological examination showed severe lymphocyte depletion in the splenic white pulp of the intraperitoneal SAV-injected rats. Conclusion: Stimulation of PMNs by SAV leads to upregulation of MHC-II, CD 80, and CD 86, which plays critical roles in antigen presentation and consequently proliferation of T-cells. Subcutaneous route was more efficient than intraperitoneal by elevating MHC-II, CD80 and CD86 expression, disturbing oxidative stability and increasing lipogram concentration.

Resumo em Inglês:

Abstract Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 μg), goat (HU50 = 13.2 ± 0.3 μg), rabbit (HU50 = 34.7 ± 0.5 μg), and human (HU50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.

Resumo em Inglês:

Abstract Background: Background: Tuberculosis (TB) is an infectious lung disease with high worldwide incidence that severely compromises the quality of life in affected individuals. Clinical tests are currently employed to monitor pulmonary status and treatment progression. The present study aimed to apply a three-dimensional (3D) reconstruction method based on chest radiography to quantify lung-involvement volume of TB acute-phase patients before and after treatment. In addition, these results were compared with indices from conventional clinical exams to show the coincidence level. Methods: A 3D lung reconstruction method using patient chest radiography was applied to quantify lung-involvement volume using retrospective examinations of 50 patients who were diagnosed with pulmonary TB and treated with two different drugs schemes. Twenty-five patients were treated with Scheme I (rifampicin, isoniazid, and pyrazinamide), whereas twenty-five patients were treated with Scheme II (rifampicin, isoniazid, pyrazinamide, and ethambutol). Acute-phase reaction: Serum exams included C-reactive protein levels, erythrocyte sedimentation rate, and albumin levels. Pulmonary function was tested posttreatment. Results: We found strong agreement between lung involvement and serum indices pre- and posttreatment. Comparison of the functional severity degree with lung involvement based on 3D image quantification for both treatment schemes found a high correlation. Conclusions: The present 3D reconstruction method produced a satisfactory agreement with the acute-phase reaction, most notably a higher significance level with the C-reactive protein. We also found a quite reasonable coincidence between the 3D reconstruction method and the degree of functional lung impairment posttreatment. The performance of the quantification method was satisfactory when comparing the two treatment schemes. Thus, the 3D reconstruction quantification method may be useful tools for monitoring TB treatment. The association with serum indices are not only inexpensive and sensitive but also may be incorporated into the assessment of patients during TB treatment.

Resumo em Inglês:

Abstract Background: Three drugs - pentavalent antimonials, amphotericin B and pentamidine - are currently used for leishmaniasis treatment. They are administered for long periods, only parenterally, and have high cardiac, renal and hepatic toxicities. Therefore, the investigation of new compounds is required. Nitro-heterocyclic derivatives have been used as possible drug candidates to treat diseases caused by trypanosomatids. Methods: Leishmania (L.) amazonensis promastigotes (MHO/BR/73/M2269), maintained in the Laboratório de Soroepidemiologia - Instituto de Medicina Tropical- USP, were exposed to five nitroheterocyclic derivatives, with differences at phenyl-ring position 4: BSF-C4H9, BSF-H, BSF-NO2, BSF-CH3 and BSF-Cl, for 48 hours. After analyzing viability (MTT assay), we evaluated cellular-morphology activity of compounds by transmission electron microscopy (TEM) and measurement of apoptosis (phosphatidylserine expression) by flow cytometry. Results: EC50 of amphotericin B and BSF-CH3 were 0.50 (M and 0.39 (M respective. Other nitro-heterocyclic compounds presented EC50 higher than amphotericin B. All compounds showed greater AV- and PI-positive expression than amphotericin B at 100 (M, except BSF-NO2. TEM showed complete nuclear disfigurement with 100 (M of BSF-NO2, 25 and 6.25 (M of BSF-H, and 6.25 (M BSF-Cl; presence of vesicles within the flagellar pocket with 25 (M BSF-H; alteration of the kinetoplast with 25 (M BSF-C4H9, 25 (M of BSF-H, 6.25 (M BSF-CH3 and 6.25 (M of BSF-Cl. Conclusions: Nitro-heterocyclic compounds have shown activity against promastigotes of L. amazonensis, at lower concentrations. However, improvement of compound scaffolds are needed to assist the elucidation of the mechanism of action and to achieve greater activity.

Resumo em Inglês:

Abstract Background: Cutaneous leishmaniasis (CL) is a parasitic disease caused by the protozoan Leishmania spp. Pentavalent antimonial agents have been used as an effective therapy, despite their side effects and resistant cases. Their pharmacokinetics remain largely unexplored. This study aimed to investigate the pharmacokinetic profile of meglumine antimoniate in a murine model of cutaneous leishmaniasis using a radiotracer approach. Methods: Meglumine antimoniate was neutron-irradiated inside a nuclear reactor and was administered once intraperitoneally to uninfected and L. amazonensis-infected BALB/c mice. Different organs and tissues were collected and the total antimony was measured. Results: Higher antimony levels were found in infected than uninfected footpad (0.29% IA vs. 0.14% IA, p = 0.0057) and maintained the concentration. The animals accumulated and retained antimony in the liver, which cleared slowly. The kidney and intestinal uptake data support the hypothesis that antimony has two elimination pathways, first through renal excretion, followed by biliary excretion. Both processes demonstrated a biphasic elimination profile classified as fast and slow. In the blood, antimony followed a biexponential open model. Infected mice showed a lower maximum concentration (6.2% IA/mL vs. 11.8% IA/mL, p = 0.0001), a 2.5-fold smaller area under the curve, a 2.7-fold reduction in the mean residence time, and a 2.5-fold higher clearance rate when compared to the uninfected mice. Conclusions: neutron-irradiated meglumine antimoniate concentrates in infected footpad, while the infection affects antimony pharmacokinetics.

Resumo em Inglês:

Abstract Background: The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. Methods: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. Results: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.

Resumo em Inglês:

ABSTRACT Background: The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1. Methods: Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins. Results: When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion: These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.

Resumo em Inglês:

Abstract Background: Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom. Methods: LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes. Results: Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da. Conclusions: Novel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.

Resumo em Inglês:

Abstract Background: Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.

Resumo em Inglês:

Abstract Background: Studies on toad poison are relevant since they are considered a good source of toxins that act on different biological systems. Among the molecules found in the toad poison, it can be highlighted the cardiotonic heterosides, which have a known mechanism that inhibit Na+/K+-ATPase enzyme. However, these poisons have many other molecules that may have important biological actions. Therefore, this work evaluated the action of the low molecular weight components from Rhinella schneideri toad poison on Na+/K+-ATPase and their anticonvulsive and / or neurotoxic effects, in order to detect molecules with actions of biotechnological interest. Methods: Rhinella schneideri toad (male and female) poison was collected by pressuring their parotoid glands and immediately dried and stored at -20 °C. The poison was dialysed and the water containing the low molecular mass molecules (< 8 kDa) that permeate the dialysis membrane was collected, frozen and lyophilized, resulting in the sample used in the assays, named low molecular weight fraction (LMWF). Na+/K+ ATPase was isolated from rabbit kidneys and enzyme activity assays performed by the quantification of phosphate released due to enzyme activity in the presence of LMWF (1.0; 10; 50 and 100 µg/mL) from Rhinella schneideri poison. Evaluation of the L-Glutamate (L-Glu) excitatory amino acid uptake in brain-cortical synaptosomes of Wistar rats was performed using [3H]L-glutamate and different concentration of LMWF (10-5 to 10 µg/µL). Anticonvulsant assays were performed using pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA) to induce seizures in Wistar rats (n= 6), which were cannulated in the lateral ventricle and treated with different concentration of LMWF (0.25; 0.5; 1.0; 2.0; 3.0 and 4.0 µg/µL) 15 min prior to the injection of the seizure agent. Results: LMWF induced a concentration-dependent inhibition of Na+/K+-ATPase (IC50% = 107.5 μg/mL). The poison induces an increased uptake of the amino acid L-glutamate in brain-cortical synaptosomes of Wistar rats. This increase in the L-glutamate uptake was observed mainly at the lowest concentrations tested (10-5 to 10-2 µg/µL). In addition, this fraction showed a very relevant central neuroprotection on seizures induced by PTZ and NMDA. Conclusions: LMWF from Rhinella schneideri poison has low molecular weight compounds, which were able to inhibit Na+/K+-ATPase activity, increase the L-glutamate uptake and reduced seizures induced by PTZ and NMDA. These results showed that LMWF is a rich source of components with biological functions of high medical and scientific interest.

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ABSTRACT Background: Accidents caused by spiders of the genus Loxosceles constitute an important public health problem in Brazil. The venom of Loxosceles sp induces dermonecrosis at the bite site and systemic disease in severe cases. Traditional medicine based on plant-derived products has been proven to reduce the local effects of envenomation. The present study verified the healing effects of copaiba oil on lesions induced by the venom of L. intermedia. Methods: Cutaneous lesions were induced on the backs of rabbits by intradermal injection of L. intermedia venom. Copaiba oil was applied topically 6 hours after injection; the treatment was repeated for 30 days, after which animal skins were removed and processed for histopathological analysis. Blood samples were also collected before and 24 hours after venom inoculation to measure the hematological parameters. Results: Compared to the control group, the platelet count was reduced significantly in all groups inoculated with venom, accompanied by a decreased number of heterophils in the blood. The minimum necrotic dose (MND) was defined as 2.4 μg/kg. Topical treatment with copaiba oil demonstrated a differentiated healing profile: large skin lesions were observed 10 days after venom inoculation, whereas formation of a thick crust, without scarring was observed 30 days after venom inoculation. Histopathological analysis showed no significant difference after treatment. Nevertheless, the copaiba oil treatment induced a collagen distribution similar to control skin, in marked contrast to the group that received only the spider venom injection. Conclusions: We conclude that copaiba oil may interfere in the healing process and thus propose it as a possible topical treatment for cutaneous lesions induced by L. intermedia venom.

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Abstract Background: L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. Methods: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. Results: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in MAP2 fluorescence when compared to L-Glu insult. On the other hand, the riluzole treatment resulted in 17% increase of MAP2 fluorescence; therefore, the neuroprotection from RT10 was more efficacious. Conclusion: RT10 fraction exhibits neuroprotective effects against L-Glu excitotoxicity in neuron-glia cultured in vitro.

Resumo em Inglês:

Abstract Background: Ruminant feed containing animal byproduct proteins (ABPs) is prohibited in many countries due to its risk of transmitting prion diseases (PD). In most cases the entire herd is sacrificed, which causes great harm to the producer countries by preventing their exportation of ruminant derived-products. Methods: We used stable isotope ratio mass spectrometry (IRMS) of carbon (13C/12C) and nitrogen (15N/14N) to trace the animal protein in the blood of 15 buffaloes (Bubalus bubalis) divided into three experimental groups: 1 - received only vegetable protein (VP) during 117 days; 2 - received animal and vegetable protein (AVP); and 3 - received animal and vegetable protein with animal protein subsequently removed (AVPR). Groups 2 and 3 received diets containing 13.7% bovine meat and bone meal (MBM) added to a vegetable diet (from days 21-117 in the AVP group and until day 47 in the AVPR group, when MBM was removed). Results: On the 36th day, differences were detectable in the feeding profile (p <0.01) among the three experimental groups, which remained for a further 49 days (85th day). The AVPR group showed isotopic rate reversibility on the 110th day by presenting values similar to those in the control group (VP) (p> 0.05), indicating that it took 63 days to eliminate MBM in this group. Total atoms exchange (> 95%) of 13C and 15N was observed through incorporation of the diet into the AVP and AVPR groups. Conclusions: IRMS is an accurate and sensitive technique for tracing the feeding profile of ruminants through blood analysis, thus enabling investigation of ABP use.

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Abstract Background: This paper aims to highlight and analyze discrepancies in reporting of deaths due to venomous animals in Brazil, from 2001 to 2015, between two national information systems: The Notifiable Diseases Information System (Sistema de Informação de Agravos de Notificação - SINAN) and the Mortality Information System (Sistema de Informações sobre Mortalidade - SIM). Methods: Descriptive and comparative study of the SINAN and SIM information systems, was conducted via the following steps: collecting the death notices from SINAN and SIM; constructing tables and comparative graphics; and, only in scorpion sting fatalities, analyzing the distribution of deaths by age group as described in the specialized literature. Results: While SINAN identifies strong growth in the number of deaths from scorpion stings, SIM shows greater increase in the number of reported deaths from bee stings, especially in the South and Southeast regions. Notably, bees are the sole etiological agent that received more reports in SIM than in SINAN for every year in the period studied. The age-group distribution of the data on deaths from scorpion stings reinforced the indication of problems occurring in their registration in SINAN, especially since 2007, which may have an effect on analyses based on these data. Conclusion: Comparative analysis of these databases permits identification of important differences between profiles presented by these systems, which have equal relevance for Brazil as a whole and for its regions. These differences may influence the construction of various scenarios.

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Abstract Background: This work aimed to explore the action of natural prodigiosin on both bacterial organisms and Trypanosoma cruzi cells. Methods: Natural prodigiosin pigment was extracted and purified from cultures of Serratia marcescens. Two media, peanut broth and peptone glycerol broth, both recommended in the literature for prodigiosin production, were compared. The prodigiosin obtained was employed to explore its antimicrobial properties against both bacteria and Trypanosoma cruzi cells. Results: Peanut broth yielded four times more prodigiosin. The prodigiosin showed remarkable activity (minimal inhibitory concentrations in the range of 2-8 µM for bacteria and half maximal inhibitory concentration of 0.6 µM for Trypanosoma cruzi). In fact, the prodigiosin concentration required to inhibit parasite growth was as low as 0.25 mg/l versus 4.9 mg/l of benznidazole required. Furthermore, atomic force microscopy revealed marked morphological alterations in treated epimastigote forms, although no pore-formation activity was detected in protein-free environments. Conclusions: This work demonstrates the potential usefulness of prodigiosin against some gram-positive and gram-negative bacteria and Trypanosoma cruzi although further studies must be done in order to assess its value as a candidate molecule.

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ABSTRACT Background: Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. Methods: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. Results: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. Conclusions: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.

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ABSTRACT Background: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

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Abstract Background: Visceral leishmaniasis is a complex neglected tropical disease caused by Leishmania donovani complex. Its current treatment reveals strong limitations, especially high toxicity. In this context, natural products are important sources of new drug alternatives for VL therapy. Therefore, the antileishmanial and immunomodulatory activity of compounds isolated from Nectandra oppositifolia (Lauraceae) was investigated herein. Methods: The n-hexane extract from twigs of N. oppositifolia were subjected to HPLC/HRESIMS and bioactivity-guided fractionation to afford compounds 1 and 2 which were evaluated in vitro against Leishmania (L.) infantum chagasi and NCTC cells. Results: The n-hexane extract displayed activity against L. (L.) infantum chagasi and afforded isolinderanolide E (1) and secosubamolide A (2), which were effective against L. (L.) infantum chagasi promastigotes, with IC50 values of 57.9 and 24.9 µM, respectively. Compound 2 was effective against amastigotes (IC50 = 10.5 µM) and displayed moderate mammalian cytotoxicity (CC50 = 42 µM). The immunomodulatory studies of compound 2 suggested an anti-inflammatory activity, with suppression of IL-6, IL-10, TNF with lack of nitric oxide. Conclusion: This study showed the antileishmanial activity of compounds 1 and 2 isolated from N. oppositifolia. Furthermore, compound 2 demonstrated an antileishmanial activity towards amastigotes associated to an immunomodulatory effect.

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

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ABSTRACT Background: Several studies have pointed out that certain snake venoms contain compounds presenting cytotoxic activities that selectively interfere with cancer cell metabolism. In this study, Pseudocerastes persicus venom and its fractions were investigated for their anticancer potential on lung cancer cells. Methods: Lung cancer cells (A549) and normal fibroblast cells (Hu02) were treated with the P. persicus venom and its HPLC fractions and the cell cytotoxic effects were analyzed using MTT and lactate dehydrogenase release assays. Apoptosis was determined in venom-treated cell cultures using caspase-3 and caspase-9 assay kits. Results: The treatment of cells with HPLC fraction 21 (25-35 kDa) of P. persicus venom resulted in high LDH release in normal fibroblast cells and high caspase-3 and caspase-9 activities in lung cancer cells. These results indicate that fraction 21 induces apoptosis in cancer cells, whereas necrosis is predominantly caused by cell death in the normal cells. Fraction 21 at the final concentration of 10 μg/mL killed approximately 60% of lung cancer cells, while in normal fibroblast cells very low cell cytotoxic effect was observed. Conclusion: HPLC fraction 21 at low concentrations displayed promising anticancer properties with apoptosis induction in the lung cancer cells. This fraction may, therefore, be considered a promising candidate for further studies.

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ABSTRACT Background: Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. Methods: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. Results: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. Conclusions: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.

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Abstract Background: Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors. Methods: The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed. Results: V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4. Conclusion: The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.