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Disintegrins are a class of peptides found in snake venom that inhibit the activity of integrins, which are essential cell adhesion receptors in tumor progression and development. In this work, moojecin, a RGD disintegrin, was isolated from Bothrops moojeni snake venom, and its antitumor potential in acute myeloid leukemia (AML) HL-60 and THP-1 cells was characterized. The isolation was performed using a C 18 reverse-phase column in two chromatographic steps, and its molecular mass is 7417.84 Da. N-terminal and de novo sequencing was performed to identify moojecin. Moojecin did not show cytotoxic or antiproliferative activity in THP-1 and HL-60 at tested concentrations, but it exhibited significant antimigratory activity in both cell lines, as well as inhibition of angiogenesis in the tube formation assay on Matrigel in a dose-dependent manner. A stronger interaction with integrin αVβ3 was shown in integrin interaction assays compared to α5β1, and the platelet aggregation assay indicated an IC 50 of 5.039 μg/mL. Preliminary evaluation of disintegrin toxicity revealed no incidence of hemolysis or cytotoxic effects on peripheral blood mononuclear cells (PBMCs) across the tested concentrations. Thus, this is the first study to report the isolation, functional and structural characterization of a disintegrin from B. moojeni venom and bring a new perspective to assist in AML treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. There is no effective treatment for neurodegenerative diseases. Snake venoms are a cocktail of proteins and peptides with great therapeutic potential and might be useful in the treatment of neurodegenerative diseases. Crotapotin is the acid chain of crotoxin, the major component of Crotalus durissus collilineatus venom. PD is characterized by low levels of neurotrophins, and synaptic and axonal degeneration; therefore, neurotrophic compounds might delay the progression of PD. The neurotrophic potential of crotapotin has not been studied yet., Methods: We evaluated the neurotrophic potential of crotapotin in untreated PC12 cells, by assessing the induction of neurite outgrowth. The activation of the NGF signaling pathway was investigated through pharmacological inhibition of its main modulators. Additionally, its neuroprotective and neurorestorative effects were evaluated by assessing neurite outgrowth and cell viability in PC12 cells treated with the dopaminergic neurotoxin MPP + (1-methyl-4-phenylpyridinium), known to induce Parkinsonism in humans and animal models., Results: Crotapotin induced neuritogenesis in PC12 cells through the NGF-signaling pathway, more specifically, by activating the NGF-selective receptor trkA, and the PI3K/Akt and the MAPK/ERK cascades, which are involved in neuronal survival and differentiation. In addition, crotapotin had no cytotoxic effect and protected PC12 cells against the inhibitory effects of MPP + on cell viability and differentiation., Conclusion: These findings show, for the first time, that crotapotin has neurotrophic/neuroprotective/neurorestorative potential and might be beneficial in Parkinson's disease. Additional studies are necessary to evaluate the toxicity of crotapotin in other cell models., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Snake venom disintegrins are low molecular weight, non-enzymatic proteins rich in cysteine, present in the venom of snakes from the families Viperidae, Crotalidae, Atractaspididae, Elapidae, and Colubridae. This family of proteins originated in venom through the proteolytic processing of metalloproteinases (SVMPs), which, in turn, evolved from a gene encoding an A Disintegrin And Metalloprotease (ADAM) molecule. Disintegrins have a recognition motif for integrins in their structure, allowing interaction with these transmembrane adhesion receptors and preventing their binding to proteins in the extracellular matrix and other cells. This interaction gives disintegrins their wide range of biological functions, including inhibition of platelet aggregation and antitumor activity. As a result, many studies have been conducted in an attempt to use these natural compounds as a basis for developing therapies for the treatment of various diseases. Furthermore, the FDA has approved Tirofiban and Eptifibatide as antiplatelet compounds, and they are synthesized from the structure of echistatin and barbourin, respectively. In this review, we discuss some of the main functional and structural characteristics of this class of proteins and their potential for therapeutic use., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Bothrops snakebite envenomation (SBE) is consider an important health problem in Brazil, where Bothrops atrox is mainly responsible in the Brazilian Amazon. Local effects represent a relevant clinical issue, in which inflammatory signs and symptoms in the bite site represent a potential risk for short and long-term disabilities. Among local complications, secondary infections (SIs) are a common clinical finding during Bothrops atrox SBE and are described by the appearance of signs such as abscess, cellulitis or necrotizing fasciitis in the affected site. However, the influence of SI in the local events is still poorly understood. Therefore, the present study describes for the first time the impact of SBE wound infection on local manifestations and inflammatory response from patients of Bothrops atrox SBE in the Brazilian Amazon. This was an observational study carried out at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus (Brazil), involving victims of Bothrops SBE. Clinical and laboratorial data were collected along with blood samples for the quantification of circulating cytokines and chemokines before antivenom administrations (T0) and 24 h (T1), 48 h (T2), 72 h (T3) and 7 days after (T4). From the 94 patients included in this study, 42 presented SI (44.7%) and 52 were without SI (NSI, 55.3%). Patients classified as moderate envenoming presented an increased risk of developing SI (OR = 2.69; CI 95% = 1.08-6.66, p = 0.033), while patients with bites in hands showed a lower risk (OR = 0.20; CI 95% = 0.04-0.96, p = 0.045). During follow-up, SI patients presented a worsening of local temperature along with a sustained profile of edema and pain, while NSI patients showed a tendency to restore and were highlighted in patients where SI was diagnosed at T2. As for laboratorial parameters, leukocytes, erythrocyte sedimentation ratio, fibrinogen and C-reactive protein were found increased in patients with SI and more frequently in patients diagnosed with SI at T3. Higher levels of circulating IL-2, IL-10, IL-6, TNF, INF-γ and CXCL-10 were observed in SI patients along with marked correlations between these mediators and IL-4 and IL-17, showing a plurality in the profile with a mix of Th1/Th2/Th17 response. The present study reports for the first time the synergistic effects of local infection and envenoming on the inflammatory response represented by local manifestations, which reflected on laboratorial parameters and inflammatory mediators and thus help improve the clinical management of SI associated to Bothrops SBE.

Enterovirus A71 (EVA71) belongs to the Picornaviridae family and is the main etiological agent of hand, foot, and mouth disease (HFMD). There is no approved antiviral against EVA71, and therefore the search for novel anti-EVA71 therapeutics is essential. In this context, the antiviral activity of proteins isolated from snake venoms has been reported against a range of viruses. Here, the proteins CM10 and CM14 isolated from Bothrops moojeni, and Crotamin and PLA2 CB isolated from Crotalus durissus terrificus were investigated for their antiviral activity against EVA71 infection. CM14 and Crotamin possessed a selective index (SI) of 170.8 and 120.4, respectively, while CM10 and PLA2 CB had an SI of 67.4 and 12.5, respectively. CM14 inhibited all steps of viral replication (protective effect: 76 %; virucidal: 99 %; and post-entry: 99 %). Similarly, Crotamin inhibited up to 99 % of three steps. In contrast, CM10 and PLA2 CB impaired one or two steps of EVA71 replication, respectively. Further dose-response assays using increasing titres of EVA71 were performed and CM14 and Crotamin retained functionality with high concentrations of EVA71 (up to 1000 TCID 50 ). These data demonstrate that proteins isolated from snake venom are potent inhibitors of EVA71 and could be used as scaffolds for future development of novel antivirals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Phosphodiesterases are exonucleases that sequentially hydrolyse phosphodiester bonds of polynucleotides from the 3'-end and release 5-mononucleotides. After more than one decade without any advance in the study of Bothropic phosphodiesterases, we described here the isolation of the first phosphodiesterase from Bothrops jararacussu, which we named Bj-PDE. A five-step column chromatography procedure (size exclusion, hydrophobic interaction, cation exchange, lentil lectin affinity, and blue sepharose affinity) enabled isolation of Bj-PDE with preserved and stable enzymatic activity (bis(p-nitrophenyl) phosphate substrate), K m  = 6.9 mM (± 0.7 mM), k cat /K m  = 1.7 × 10 4 M -1  s -1 (± 0.2 × 10 4 M -1  s -1 ), MW = 116 kDa (SDS-PAGE), optimum activity around 45 °C at pH 8.0, and stability for 81 days at different storage temperatures (8, -20, and - 80 °C). Ca 2+ and Mg 2+ ions positively influenced Bj-PDE activity, while EDTA had the opposite action. Zn 2+ restored >50 % of enzyme activity after its inhibition by EDTA. The Bj-PDE partial sequence identified by mass spectrometry was very similar to the sequence of BATXPDE1 from Bothrops atrox, which was evolutionarily close to this new PDE. Therefore, our study represents an important progress on the isolation of this minor toxin and sheds new lights on the properties and bioprospection of bothropic phosphodiesterases., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Crotalus durissus snakebite represent 10 % of snakebite cases in Brazil, which cardiovascular disorders are associated with severe cases. Considering crotoxin (CTX) as the major venom component, the present study aimed to evaluate the hemodynamic alterations induced by CTX using in vivo and ex vivo approaches in a rat model. In vivo cardiac function parameters were analyzed from anesthetized rats treated with CTX or saline only (Sham), along with serum creatine kinase MB (CK-MB) and lung myeloperoxidase. From the same animals, hearts were isolated and functional parameters evaluated in Langendorff method ex vivo. CTX binding to myoblast cell line in vitro were evaluated using confocal microscopy and flow cytometry. CTX was capable of reducing arterial and diastolic blood pressure, heart rate, along with left ventricle pressure development or decay during systole (LVdP/dt max and LVdP/dt min ) in vivo, however no differences were found in the ex vivo approach, showing that intrinsic heart function was preserved. In vitro, CTX binding to myoblast cell line was mitigated by hexamethonium, a nicotinic acetylcholine receptor antagonist. The present study has shown that CTX induce hemodynamic failure in rats, which can help improve the clinical management of cardiovascular alterations during Crotalus durissus snakebite., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Suely Vilela Sampaio reports equipment, drugs, or supplies were provided by National Council for Scientific and Technological Development. Suely Vilela Sampaio reports equipment, drugs, or supplies was provided by State of Sao Paulo Research Foundation. Marco Aurelio Sartim reports equipment, drugs, or supplies was provided by Foundation for Research Support of Amazonas State. Wuelton Marcelo Monteiro reports writing assistance was provided by Foundation for Research Support of Amazonas State., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Zika virus is the etiologic agent of Zika fever, and has been previously associated with cases of microcephaly, drawing the attention of the health authorities worldwide. However, no vaccine or antiviral are currently available. Phospholipases A 2 (PLA 2 ) isolated from snake venoms have demonstrated antiviral activity against several viruses. Here we demonstrated the anti-ZIKV activity of bothropstoxins-I and II (BthTX-I and II) isolated from Bothrops jararacussu venom. Vero E6 cells were infected with ZIKVPE243 in the presence of compounds for 72 h, when virus titers were evaluated. BthTX-I and II presented strong dose-dependent inhibition of ZIKV, with a SI of 149.1 and 1.44 × 10 5 , respectively. These toxins mainly inhibited the early stages of the replicative cycle, such as during the entry of ZIKV into host cells, as shown by the potent virucidal effect, suggesting the action of these toxins on the virus particles. Moreover, BthTX-I and II presented significant activity towards post-entry stages of the ZIKV replicative cycle. Molecular docking analyses showed that BthTX-I and II potentially interact with DII and DIII domains from ZIKV Envelope protein. Our findings show that these PLA 2 s could be used as useful templates for the development of future antiviral candidate drugs against Zika fever., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

PEGylation was firstly described around 50 years ago and has been used for more than 30 years as a strategy to improve the drugability of biopharmaceuticals. However, it remains poorly employed in toxinology, even though it may be a promising strategy to empower these compounds in therapeutics. This work reports the PEGylation of rCollinein-1, a recombinant snake venom serine protease (SVSP), able to degrade fibrinogen and inhibit the hEAG1 potassium channel. We compared the functional, structural, and immunogenic properties of the non-PEGylated (rCollinein-1) and PEGylated (PEG-rCollinein-1) forms. PEG-rCollinein-1 shares similar kinetic parameters with rCollinein-1, maintaining its capability of degrading fibrinogen, but with reduced activity on hEAG1 channel. CD analysis revealed the maintenance of protein conformation after PEGylation, and thermal shift assays demonstrated similar thermostability. Both forms of the enzyme showed to be non-toxic to peripheral blood mononuclear cells (PBMC). In silico epitope prediction indicated three putative immunogenic peptides. However, immune response on mice showed PEG-rCollinein-1 was devoid of immunogenicity. PEGylation directed rCollinein-1 activity towards hemostasis control, broadening its possibilities to be employed as a defibrinogenant agent., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The abusive consumption of thermogenic supplements occurs worldwide and deserves special attention due to their use to stimulate weight loss and prevent obesity. Thermogenic formulations usually contain Synephrine (SN) and Caffeine (CAF), stimulating compounds extracted from natural sources, but no genetic toxicology studies have predicted this hazardous combination potential. This study examined the toxicogenomic responses induced by SN and CAF, either alone or in combination, in the human hepatic cell line HepG2 in vitro. SN (0.03-30 μM) and CAF (0.6-600 μM) alone did neither decrease cell viability nor induce DNA damage, as assessed using the MTT and comet assays, respectively. SN (3 μM) and CAF (30-600 μM) were combined at concentrations similar to those found in commercial dietary supplements. SN/CAF at 3:90 and 3:600 μM ratios significantly decreased cell viability and increased DNA damage levels in HepG2 cells. CAF (600 μM) and the SN/CAF association at 3:60, 3:90, and 3:600 μM ratios promoted cell death by apoptosis, as demonstrated by flow cytometry. Similar results were observed in gene expression (RT-qPCR): SN/CAF up-regulated the expression of apoptosis- (BCL-2 and CASP9) and DNA repair-related (XPC) genes. SN/CAF at 3:90 μM also downregulated the expression of cell cycle control (CDKN1A) genes. In conclusion, the SN/CAF combination reduces cell viability by inducing apoptosis, damages DNA, and modulates the transcriptional expression of apoptosis-, cell cycle-, and DNA repair-related genes in human hepatic (HepG2) cells in vitro. These effects can be worrisome to consumers of thermogenic supplements., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever, a globally spreading mosquito-borne disease. There is no approved antiviral or vaccine against CHIKV, highlighting an urgent need for novel therapies. In this context, snake venom proteins have demonstrated antiviral activity against several viruses, including arboviruses which are relevant to public health. In particular, the phospholipase A2 CB (PLA2 CB ), a protein isolated from the venom of Crotalus durissus terrificus was previously shown to possess anti-inflammatory, antiparasitic, antibacterial and antiviral activities. In this study, we investigated the multiple effects of PLA2 CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2 CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2 CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2 CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2 CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2 CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2 CB on the CHIKV replicative cycle, and suggest that PLA2 CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells.

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl + leukemic cells and improve the disease treatment., Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl + cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro ., Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl + cells., Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H 2 O 2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy., Competing Interests: Competing interests: The authors report no conflicts of interest.

Background: Snake venom phospholipases A 2 (svPLA 2 ) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA 2 s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated., Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB., Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA 2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB., Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Adipose tissue secretes proinflammatory mediators which promote systemic and adipose tissue inflammation seen in obesity. Group IIA (GIIA)-secreted phospholipase A 2 (sPLA 2 ) enzymes are found to be elevated in plasma and adipose tissue from obese patients and are active during inflammation, generating proinflammatory mediators, including prostaglandin E 2 (PGE 2 ). PGE 2 exerts anti-lipolytic actions and increases triacylglycerol levels in adipose tissue. However, the inflammatory actions of GIIA sPLA 2 s in adipose tissue cells and mechanisms leading to increased PGE 2 levels in these cells are unclear. This study investigates the ability of a representative GIIA sPLA 2 , MT-III, to activate proinflammatory responses in preadipocytes, focusing on the biosynthesis of prostaglandins, adipocytokines and mechanisms involved in these effects. Our results showed that MT-III induced biosynthesis of PGE 2 , PGI 2 , MCP-1, IL-6 and gene expression of leptin and adiponectin in preadipocytes. The MT-III-induced PGE 2 biosynthesis was dependent on cytosolic PLA 2 (cPLA 2 )-α, cyclooxygenases (COX)-1 and COX-2 pathways and regulated by a positive loop via the EP 4 receptor. Moreover, MT-III upregulated COX-2 and microsomal prostaglandin synthase (mPGES)-1 protein expression. MCP-1 biosynthesis induced by MT-III was dependent on the EP4 receptor, while IL-6 biosynthesis was dependent on EP3 receptor engagement by PGE 2 . These data highlight preadipocytes as targets for GIIA sPLA 2 s and provide insight into the roles played by this group of sPLA 2 s in obesity.

Respiratory compromise in Crotalus durissus terrificus (C.d.t.) snakebite is an important pathological condition. Considering that crotoxin (CTX), a phospholipase A 2 from C.d.t. venom, is the main component of the venom, the present work investigated the toxin effects on respiratory failure. Lung mechanics, morphology and soluble markers were evaluated from Swiss male mice, and mechanism determined using drugs/inhibitors of eicosanoids biosynthesis pathway and autonomic nervous system. Acute respiratory failure was observed, with an early phase (within 2 h) characterized by enhanced presence of eicosanoids, including prostaglandin E2, that accounted for the increased vascular permeability in the lung. The alterations of early phase were inhibited by indomethacin. The late phase (peaked 12 h) was marked by neutrophil infiltration, presence of pro-inflammatory cytokines/chemokines, and morphological alterations characterized by alveolar septal thickening and bronchoconstriction. In addition, lung mechanical function was impaired, with decreased lung compliance and inspiratory capacity. Hexamethonium, a nicotinic acetylcholine receptor antagonist, hampered late phase damages indicating that CTX-induced lung impairment could be associated with cholinergic transmission. The findings reported herein highlight the impact of CTX on respiratory compromise, and introduce the use of nicotinic blockers and prostanoids biosynthesis inhibitors as possible symptomatic therapy to Crotalus durissus terrificus snakebite.

Snake venom serine proteases (SVSPs) are complex and multifunctional enzymes, acting primarily on hemostasis. In this work, we report the hitherto unknown inhibitory effect of a SVSP, named collinein-1, isolated from the venom of Crotalus durissus collilineatus, on a cancer-relevant voltage-gated potassium channel (hEAG1). Among 12 voltage-gated ion channels tested, collinein-1 selectively inhibited hEAG1 currents, with a mechanism independent of its enzymatic activity. Corroboratively, we demonstrated that collinein-1 reduced the viability of human breast cancer cell line MCF7 (high expression of hEAG1), but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines (HepG2 and MCF10A, respectively), which present low expression of hEAG1. In order to obtain both functional and structural validation of this unexpected discovery, where an unusually large ligand acts as an inhibitor of an ion channel, a recombinant and catalytically inactive mutant of collinein-1 (His43Arg) was produced and found to preserve its capability to inhibit hEAG1. A molecular docking model was proposed in which Arg79 of the SVSP 99-loop interacts directly with the potassium selectivity filter of the hEAG1 channel.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Inflammation and coagulopathies are important systemic events following snakebite. Snake venom galactoside-binding lectins (SVgalLs) are known modulators of the immune response with no direct effect on hemostasis. Considering the crosstalk between inflammation and coagulation, the present study investigated how BJcuL, a proinflammatory SVgalL isolated from Bothrops jararacussu venom, mediated the inflammation-induced procoagulant activity. We examined the proinflammatory cytokine production and procoagulant tissue factor (TF) activity in human whole blood and monocyte-rich cell suspension (MR-PBMC) treated with BJcuL. This lectin increased production of the cytokines TNF-α, IL-1β and IL-6, upregulated TF expression on the cell surface, and induced procoagulant activity. The proinflammatory behavior was mediated by the direct interaction between the lectin and toll-like receptor 4, via binding to β-galactoside-containing glycoconjugates on the cell surface, and activation of NFκ-B signaling. Interestingly, the BJcuL-induced inflammation was directly associated with the procoagulant activity of MR-PBMC cells. In whole blood culture, the lectin exhibited similar behavior, i.e. it induced cytokine production and MR-PBMC TF-mediated procoagulant activity. Therefore, the present study is the first report on the inflammation-induced procoagulant activity of SVgalLs, and it indicates that BJcuL is an important factor associated with coagulopathy in patients with snake envenomation., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The synthetic peptide p-BTX-I is based on the native peptide (formed by glutamic acid, valine and tryptophan) isolated from Bothrops atrox venom. We have previously demonstrated its neuroprotective and neurotrophic properties in PC12 cells treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Now, we have investigated the neuroprotective effects and mechanisms of p-BTX-I against the toxicity of acrolein in PC12 cells. Studies have demonstrated that acrolein might play an important role in the etiology of Alzheimer's disease (AD), which is characterized by neuronal and synaptic loss. Our results showed that not only acrolein reduced cell differentiation and cell viability, but also altered the expression of markers of synaptic communication (synapsin I), energy metabolism (AMPK-α, Sirt I and glucose uptake), and cytoskeleton (β-III-tubulin). Treatment with p-BTX-I increased the percentage of differentiation in cells treated with acrolein and significantly attenuated cell viability loss, besides counteracting the negative effects of acrolein on synapsin I, AMPK-α, Sirt I, glucose uptake, and β-III-tubulin. Additionally, p-BTX-I alone increased the expression of apolipoprotein E (apoE) gene, associated with the proteolytic degradation of β-amyloid peptide aggregates, a hallmark of AD. Taken together, these findings demonstrate that p-BTX-I protects against acrolein-induced neurotoxicity and might be a tool for the development of novel drugs for the treatment of neurodegenerative diseases.

The aim of this study was to evaluate the immunomodulatory effects of two toxins from Bothrops snake venoms (the P-I metalloprotease Batroxase and the thrombin-like serine protease Moojase) on human peripheral blood mononuclear cells (PBMC), also investigating changes in the expression of genes related to epigenetic alterations and their immunotherapeutic potential. After 24 h of PBMC stimulation, Batroxase (2 μg/mL) and Moojase (4 μg/mL) increased some cytokine levels (including IL-6 and IL-10), but did not promote cell death processes (apoptosis/necrosis) or alterations in the global DNA methylation levels. Gene expression experiments (RT-qPCR) showed that most of the genes with altered transcript levels encode enzymes that act on histones, such as acetyltransferases (HAT1), deacetylases (HDACs), methyltransferases (DOT1L) or demethylases (KDM5B), indicating that these toxins may alter gene regulation through epigenetic changes mainly related to histones and to methyl-CpG binding proteins (MECP2). Subsequently, the immunotherapeutic potential of these toxins was evaluated using in vitro cytotoxicity assays with NK cells and K562 leukemic cells. Both toxins were able to potentiate the NK cell cytotoxic effects against K562 tumor cells, and the effect of Batroxase was dependent on the concomitant stimulus with IL-2, whereas Moojase increased the NK cytotoxicity independently of IL-2. Thus, Batroxase and Moojase presented interesting immunomodulatory effects that could be explored for the development of new strategies in anticancer immunotherapies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

CRISPs represent a family of cysteine-rich secretory proteins with molecular mass between 20 and 30 kDa and a highly conserved specific pattern of 16 cysteine residues. In this work, we isolated and characterized a novel CRISP from Bothrops alternatus venom, named BaltCRP, also evaluating its effects on different isoforms of potassium channels (Kv1.1; Kv1.2; Kv1.3; Kv1.4; Kv1.5; Kv2.1; Kv10.1 and Shaker) and on inflammatory processes in vivo. This toxin has a molecular mass of 24.4 kDa and pI around 7.8. Electrophysiological experiments using voltage clamp techniques showed that BaltCRP can affect the currents of Kv1.1; Kv1.3; Kv2.1 and Shaker channels. In addition, BaltCRP induced inflammatory responses characterized by an increase of leukocytes in the peritoneal cavity of mice, also stimulating the production of mediators such IL-6, IL-1β, IL-10, PGE 2 , PGD 2 , LTB 4 and CysLTs. Altogether, these results demonstrated that BaltCRP can help understand the biological effects evoked by snake venom CRISPs, which could eventually lead to the development of new molecules with therapeutic potential., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Endothelium plays an important modulatory role due to the synthesis and secretion of molecules that act on hemostasis and fibrinolysis. As there are no literature data about the effect of crotoxin (CTX) - the main component of Crotalus durissus terrificus snake venom - on human endothelial cells, the present study examined how CTX (25-200 μg/mL) affects the endothelial production of the hemostatic factors antithrombin III, protein C, protein S, plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (t-PA), and vWF in human umbilical vein endothelial cells (HUVEC) in vitro. Production of hemostatic factors was assessed in HUVEC cells treated with CTX (25-200 μg/mL) in the presence or absence of lypopolysaccharide (LPS; 1 μg/mL; 24 h). We found that CTX alone did not exert pro- or anticoagulant effect on hemostasis, and pro- or antifibrinolytic effect on fibrinolysis. LPS alone had procoagulant (increased vWF and reduced protein C and S levels) and antifibrinolytic action (reduced t-PA and increased PAI-1 levels). However, CTX exerted anticoagulant and profibrinolytic action in the presence of LPS by lowering the levels of vWF and t-PA and elevating the levels of protein C and PAI-1. Therefore can be used as a potential tool against thrombosis development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Vascular endothelium plays an important modulatory role due to the production of molecules that mediate vasomotricity, inflammation, and leukocyte adhesion and rolling. Here we addressed whether crotoxin (25-200 μg/mL) - the main component of Crotalus durissus terrificus snake venom - interferes with cell viability, apotosis/necrosis, and cell response to oxidative stress in human umbilical vein endothelial cells (HUVEC) in vitro. We also examined whether crotoxin alters the levels of interleukins, adhesion molecules, and endothelial vasoactive factors in HUVEC cells treated or not with lipopolysaccharide (LPS; 1 μg/mL; 24 h). Crotoxin was not cytotoxic towards HUVEC cells, and downregulated the LPS-induced production of adhesion molecules (VCAM-1, ICAM-1, and E-selectin), vasoactive factors (endothelin-1 and prostaglandin I2), and interleukins (IL-6, IL-8, and IL1β), as well as protected cells against H 2 O 2 -induced oxidative stress. Hence, crotoxin played anti-inflammatory, antioxidant, immunomodulating, and vasoactive actions on HUVEC cells, in vitro. Considering that the initial stages of atherosclerosis is characterized by vasoconstriction, increased levels of adhesion molecules, inflammatory cytokines, and oxidative stress in the vascular endothelium; and crotoxin downmodulated all these events, our findings indicate that the actions of crotoxin here demonstrated suggest that it may have an anti-atherogenic action in vivo, which deserves to be tested in future studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Hepatocellular carcinoma incidence rates have increased worldwide, which encouraged the development of new chemotherapeutic drugs. l-Amino acid oxidases from snake venoms are cytotoxic towards human tumor cells in in vitro monoculture systems, which do not simulate the tumor microenvironment. We examined the antitumor potential of BjussuLAAO-II, an l-amino acid oxidase from Bothrops jararacussu venom, in hepatocarcinoma cells (HepG2) in monoculture and co-culture with human umbilical vein endothelial cells (HUVEC) in vitro. All the concentrations tested (0.25-5.00 μg/mL) were cytotoxic (MTT and clonogenic survival assays) towards HepG2 and HUVEC cells in monoculture, and increased oxidative stress by 2',7'-dichlorofluorescin diacetate fluorescence assay. Only 1.00 and 5.00 μg/mL exerted these effects in HepG2 cells co-cultured with HUVEC cells, and were genotoxic (comet assay) to HUVEC cells in monoculture. BjussuLAAO-II at 5.00 μg/mL induced DNA, but not chromosomal damage (micronucleus assay) in HepG2 cells in mono- and co-culture. The cytotoxicity and genotoxicity was more pronounced in monoculture, indicating that the tumor microenvironment influences the cellular response. BjussuLAAO-II caused cell death and DNA damage in HepG2 cells in vitro by inducing oxidative stress. Therefore, BjussuLAAO-II is a promising molecule for the development of new antitumor drugs., (Published by Elsevier B.V.)

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A 2 (PLA 2 -CB). We showed previously the antiviral effect of PLA 2 -CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA 2 -CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA 2 -CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

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., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Individual variations studies are important to understand the snakebite envenoming and to improve the antivenom production and its effectiveness. In this way, the objective of this study was a comparative analysis of intraspecific variation in the venom composition of 22 Crotalus durissus collilineatus specimens through proteomic techniques. Venoms were fractionated by RP-FPLC, and analyzed by SDS-PAGE and mass spectrometry. Although similar, chromatographic and electrophoretic profiles showed significant qualitative and quantitative differences. Some venom components were identified for the very first time in C. d. collilineatus, such as glutathione peroxidase, nerve growth factor, 5'-nucleotidase, angiotensin-converting enzyme, carboxypeptidase, phosphodiesterase, glutaminyl cyclase and phospholipase B. Regarding hyaluronidase activity, 2 venoms did not present detectable enzyme activity in the tested amounts. Additionally, in vivo crotalic envenoming in mice showed that venoms from different specimens resulted in diversified changes of biochemical and immunological parameters, such as creatine kinase and interleukin 6. This study demonstrated significant intraspecific variations in the venom of C. d. collilineatus, which may impact the production and effectiveness of the antivenom therapy. BIOLOGICAL SIGNIFICANCE: This study performed the proteomic and functional analyzes of 22 C. d. collilineatus individual venoms and verified the occurrence of quali and quantitative variations among them. The venoms evaluated caused envenomings with different changes in biochemical and immunological parameters. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness. In addition, this study was able to identify for the first time 8 different proteins in this subspecies venom, increasing knowledge about its composition and showing that it is a source of these proteins with possible biotechnological applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Chronic myeloid leukemia (CML) is a BCR-ABL1 + myeloproliferative neoplasm marked by increased myeloproliferation and presence of leukemic cells resistant to apoptosis. The current first-line therapy for CML is administration of the tyrosine kinase inhibitors imatinib mesylate, dasatinib or nilotinib. Although effective to treat CML, some patients have become resistant to this therapy, leading to disease progression and death. Thus, the discovery of new compounds to improve CML therapy is still challenging. Here we addressed whether MjTX-I, a phospholipase A 2 isolated from Bothrops moojeni snake venom, affects the viability of imatinib mesylate-resistant Bcr-Abl + cell lines., Methods: We examined the cytotoxic and pro-apoptotic effect of MjTX-I in K562-S and K562-R Bcr-Abl + cells and in the non-tumor HEK-293 cell line and peripheral blood mononuclear cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the hypotonic fluorescent solution methods, associated with detection of caspases 3, 8, and 9 activation and poly (ADP-ribose) polymerase (PARP) cleavage. We also analyzed the MjTX-I potential to modulate the expression of apoptosis-related genes in K562-S and K562-R cells., Results: MjTX-I decreased the viability of K562-S and K562-R cells by 60 to 65%, without affecting the viability of the non-tumor cells, i.e. it exerted selective cytotoxicity towards Bcr-Abl + cell lines. In leukemic cell lines, the toxin induced apoptosis, activated caspases 3, 8, and 9, cleaved PARP, downregulated expression of the anti-apoptotic gene BCL-2 , and upregulated expression of the pro-apoptotic gene BAD ., Conclusion: The antitumor effect of MjTX-I is associated with its potential to induce apoptosis and cytotoxicity in Bcr-Abl positive cell lines sensitive and resistant to imatinib mesylate, indicating that MjTX-I is a promising candidate drug to upgrade the CML therapy., Competing Interests: We declare that The Human Research Ethics Committee of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil, approved this study protocol, which was registered under the code CEP/FCFRP 032/2018. We declare that the study was conducted with the consent of the human participants.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

For the past 80 years, Crotoxin has become one of the most investigated isolated toxins from snake venoms, partially due to its major role as the main toxic component in the venom of the South American rattlesnake Crotalus durissus terrificus . However, in the past decades, progressive studies have led researchers to shift their focus on Crotoxin, opening novel perspectives and applications as a therapeutic approach. Although this toxin acts on a wide variety of biological events, the modulation of immune responses is considered as one of its most relevant behaviors. Therefore, the present review describes the scientific investigations on the capacity of Crotoxin to modulate anti-inflammatory and immunosuppressive responses, and its application as a medicinal immunopharmacological approach. In addition, this review will also discuss its mechanisms, involving cellular and molecular pathways, capable of improving pathological alterations related to immune-associated disorders., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms., Methods and Results: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn 2+ , Al 3+ , Cu 2+ and Ni 2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4 °C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU., Conclusions: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80⁻6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

Background: Our group has previously performed a proteomic study verifying that individual variations can occur among Crotalus durissus collilineatus venoms. These variations may lead to differences in venom toxicity and may result in lack of neutralization of some components by antivenom. In this way, this study aimed to evaluate the Brazilian anticrotalic serum capacity in recognizing twenty-two Crotalus durissus collilineatus venoms, as well as their fractions., Methods: The indirect enzyme-linked immunosorbent assay (ELISA) was chosen to evaluate the efficacy of heterologous anticrotalic serum produced by Instituto Butantan (Brazil) in recognizing the twenty-two Crotalus durissus collilineatus venoms and the pool of them. Moreover, the venom pool was fractionated using reversed-phase fast protein liquid chromatography (RP-FPLC) and the obtained fractions were analyzed concerning antivenom recognition., Results: Evaluation of venom variability by ELISA showed that all venom samples were recognized by the Brazilian anticrotalic antivenom. However, some particular venom fractions were poorly recognized., Conclusion: This study demonstrated that the Brazilian anticrotalic serum recognizes all the different twenty-two venoms of C. d. collilineatus and their fractions, although in a quantitatively different way, which may impact the effectiveness of the antivenom therapy. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: Snake venom phospholipases A 2 (PLA 2 s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA 2 isolated from Bothrops jararaca venom, named BJ-PLA 2 -I., Methods and Results: For its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA 2 -I showed acidic characteristics, with pI~ 4.4 and molecular mass of 14.2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA 2 s, including 100% identity with BJ-PLA 2 , an Asp49 PLA 2 previously isolated from B. jararaca venom. Being an Asp49 PLA 2 , BJ-PLA 2 -I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA 2 -I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 μg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1β and PGE 2 were observed in the inflammatory exudate induced by BJ-PLA 2 -I, while nitric oxide, TNF-α, IL-10 and LTB 4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA 2 -I (2.5-160 μg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated., Conclusions: BJ-PLA 2 -I was characterized as an acidic Asp49 PLA 2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA 2 s., Competing Interests: Animal care procedures were performed according to the Brazilian College of Animal Experimentation (COBEA) guidelines and the experimental protocols were approved by the Committee for Ethics on Animal Use (CEUA) from FCFRP-USP (Proc. n° 2012.1.414.53.4). All experiments involving human blood were in accordance with the authorization of the Research Ethics Committee of FCFRP-USP (CEP/FCFRP protocol n° 353).Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Snake venom serine proteases (SVSPs) are commonly described as capable of affecting hemostasis by interacting with several coagulation system components. In this study, we describe the isolation and characterization of BjSP from Bothrops jararaca snake venom, a serine protease with distinctive properties. This enzyme was isolated by three consecutive chromatographic steps and showed acidic character (pI 4.4), molecular mass of 28 kDa and N-carbohydrate content around 10%. Its partial amino acid sequence presented 100% identity to a serine protease cDNA clone previously identified from B. jararaca venom gland, but not yet isolated or characterized. BjSP was significantly inhibited by specific serine protease inhibitors and showed high stability at different pH values and temperatures. The enzyme displayed no effects on washed platelets, but was able to degrade fibrin clots in vitro and also the Aα and Bβ chains of fibrinogen differently from thrombin, forming additional fibrinopeptides derived from the Bβ chain, which should be related to its inability to coagulate fibrinogen solutions or platelet-poor plasma. In the mapping of catalytic subsites, the protease showed high hydrolytic specificity for tyrosine, especially in subsite S1. Additionally, its amidolytic activity on different chromogenic substrates suggests possible effects on other factors of the coagulation cascade. In conclusion, BjSP is a serine protease that acts nonspecifically on fibrinogen, generating different Bβ fibrinopeptides and thus not forming fibrin clots. Its distinguished properties in comparison to most SVSPs stimulate further studies in an attempt to validate its potential as a defibrinogenating agent., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Due to the strong bitter taste, sacha inchi seeds are usually consumed after roasting, which also contributes to the elimination of antinutrients. Sacha inchi plants fully adapted to cultivation under sub-tropical climate conditions were produced in southeastern Brazil. Our main goal was to evaluate the effect of dry heating (roasting) on the antinutrient content of these seeds. We also investigated the effects of the applied roasting treatments on the antioxidant activity, proximate composition and oxidative stability of the seeds. To the best of our knowledge, this is the first report on antinutrients of sacha inchi seeds cultivated under sub-tropical conditions, outside their native tropical environment. Except for saponins, which are not heat-labile compounds, the contents of all assessed antinutrients continually reduced with the increase in roasting temperature. Roasting improved antioxidant activity and phenolic content in the seeds at the highest temperature. Oxidation changes occurred in the seed oil, and they increased with temperature. However, maximum peroxide value was within the acceptable consumption limits. As a conclusion, roasting treatments can be applied to minimize the antinutrient potential in sacha inchi seeds. Knowledge on the composition and proper processing of sacha inchi cultivated under sub-tropical conditions may support future efforts focused on the development of new production areas.

Envenomation by Bothrops snakes promotes the release of inflammatory mediators, whose effects during this context are not well understood. These mediators include chemokines, cytokines and eicosanoids. Indeed, Bothrops snake envenomation results in local and systemic perturbations, including leukocyte recruitment, edema, pain and extensive tissue damage. Recently, our group demonstrated that leukotriene B 4 (LTB 4 ) and prostaglandin E 2 (PGE 2 ) regulate macrophage production of interleukin-1β (IL-1β) induced by scorpion venom. Whether these molecular mechanisms also affect host cell responses to snake venoms, such as those from B. jararaca or B. jararacussu, is unknown. In this study, we demonstrate that B. jararaca or B. jararacussu venoms induce macrophage inflammatory protein 1-alpha (MIP-1α) and IL-1β production by THP-1 (cell line of human peripheral blood monocytes) and AMJ2-C11 (cell line of mouse alveolar macrophage). We also show that venoms from both Bothrops species induce NF-κB activation in THP-1 cells. Furthermore, we observed that treatment of THP-1 and AMJ2-C11 cell lines with exogenous PGE 2 reduces MIP-1α production, while increasing IL-1β production in cells stimulated by B. jararaca or B. jararacussu venoms. Interestingly, exogenous LTB 4 had the opposite effect by reducing IL-1β and increasing MIP-1α release. Our results suggest that, differential eicosanoid metabolism in myeloid cells is tightly associated with the production of cytokines and chemokines after stimulation with B. jararaca or B. jararacussu venoms., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT 2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H 2 O 2 , and is also capable of modulating genes related to the DNA repair system and antioxidant pathways., (Copyright © 2018. Published by Elsevier B.V.)

Venom small peptides that target neurotrophin receptors might be beneficial in neurodegeneration, including Parkinsońs disease (PD). Their small size, ease of synthesis, structural stability and target selectivity make them important tools to overcome the limitations of endogenous neurotrophins as therapeutic agents. Additionally, they might be optimized to improve resistance to enzymatic degradation, bioavailability, potency and, mainly, lipophilicity, important to cross the blood brain barrier (BBB). Here, we evaluated the neuroprotective effects and mechanisms of the synthetic snake-venom-based peptide p-BTX-I (Glu-Val-Trp) in PC12 cells treated with MPP + (1-methyl-4-phenylpyridinium), a dopaminergic neurotoxin that induces Parkinsonism in vivo. The peptide p-BTX-I induced neuritogenesis, which was reduced by (i) k252a, antagonist of the NGF-selective receptor, trkA (tropomyosin receptor kinase A); (ii) LY294002, inhibitor of the PI3 K/AKT pathway and (iii) U0126, inhibitor of the MAPK-ERK pathway. Besides that, p-BTX-I also increased the expression of GAP-43 and synapsin, which are molecular markers of axonal growth and synaptic communication. In addition, the peptide increased the viability and differentiation of cells exposed to MPP + , known to inhibit neuritogenesis. Altogether, our findings suggest that the synthetic peptide p-BTX-I protects PC12 cells from MPP + toxicity by a mechanism that mimics the neurotrophic action of NGF. Therefore, the molecular structure of p-BTX-I might be relevant in the development of drugs aimed at restoring the axonal connectivity in neurodegenerative processes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Venom composition varies across snakes from all taxonomic levels and is influenced by the snakes&rsquo; age, habitat, diet, and sexual dimorphism. The present study reports the first in-depth investigation of venom composition in male and female Bothrops moojeni ( B. moojeni ) snakes (BmooM and BmooF, respectively) through three proteomics approaches associated with functional, cytotoxic, and immunoreactivity characterization. Compared with BmooM venom, BmooF venom exhibited weaker hyaluronidase, metalloproteinase, and phospholipase activity; stronger recognition by anti-bothropic serum; 1.4-fold stronger cytotoxicity; and greater number of peptides. The increased L-amino acid oxidase expression probably accounted for the stronger immunoreactivity and cytotoxicity of BmooF venom. BmooF and BmooM venom shared only 19% peptides. Some venom components were gender-specific, such as phospholipases B, phospholipase inhibitor, and hyaluronidases in BmooM, and cysteine-rich secretory proteins in BmooF. In conclusion, we describe herein the first proteomics study of B. moojeni snake venom and an in-depth characterization of gender-specific differences in venom composition. Altogether, our findings not only stress the importance of considering the snake&rsquo;s gender during antivenom production, but also help to identify new potential drugs and biotechnological tools.

Colorectal carcinoma is one of the most common cancers in adults. As chemotherapy, the first-choice treatment for colorectal carcinoma, is often infeasible due to acquired tumor resistance and several adverse effects, it is important to discover and explore new molecules with better therapeutic action. Snake venom toxins have shown promising results with high cytotoxicity against tumor cells, but their mechanisms of action remain unclear. Here we examined how BjussuLAAO-II, an L-amino acid oxidase isolated from Bothrops jararacussu snake venom, exerts cytotoxicity towards colorectal adenocarcinoma human cells (Caco-2) and human umbilical vein endothelial cell line (HUVEC). A 24-h treatment with BjussuLAAO-II at 0.25 - 5.00 μg/mL diminished cell viability by decreasing (i) mitochondrial activity, assessed by reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and resazurin; (ii) the activity of acid phosphatases; and (iii) lysosomal function, assessed by neutral red uptake. BjussuLAAO-II also increased intracellular levels of reactive oxygen species and DNA damage, as assessed by fluorescence and the comet assay, respectively. BjussuLAAO-II altered the expression of cell proliferation-related genes, as determined by RT-qPCR: it elevated the expression of the inflammatory cytokine genes TNF and IL6, and lowered the expression of the apoptotic-related genes BAX, BCL2, and RELA. Therefore, BjussuLAAO-II induces Caco-2 cells death by acting on multiple intracellular targets, providing important data for further studies to assess whether these effects are seen in both tumor and normal cells, with the aim of selecting this drug for possible therapeutic purposes in the future., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Snake venoms are complex mixtures of organic and inorganic compounds, including proteins belonging to the protease (serine and metalloproteinases), oxidase (L-amino acid oxidases), and phospholipase (especially phospholipases A 2 ) enzyme classes. These toxins account for the serious deleterious effects of snake envenomations, such as tissue necrosis, neurotoxicity, and hemorrhage. In addition to their toxic effects, snake venom toxins have served as important tools for investigating the mechanisms underlying envenomation and discovering new pharmacologically active compounds with immunotherapeutic potential. In this sense, the present review discusses the new findings and therapeutic perspectives in the immune modulating potential of enzymatic toxins from snake venoms belonging to the classes metalloproteinase, serine protease, L-amino acid oxidase, and phospholipase A 2 ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Snakebites are a serious health problem in tropical countries. In Brazil, the genus Bothrops (Viperidae family) causes most of the ophidic accidents, characterized by proteolysis and haemorrhage. Snake venoms are rich sources of toxins with great therapeutic and biotechnological potential and omics approaches is a valuable tool for identification of new bioactive components in the venom. In this study, we described the first transcriptome of the venom gland of Bothrops moojeni snake, using the next-generation sequencing with the Illumina platform. We identified: (i) 20 venom components classes, among which metalloproteases were the most expressed ones, followed by serine proteases and phospholipases; and (ii) the 33 full-length amino acid sequences of toxins that have never been reported before in B. moojeni venom, such as one cysteine-rich secretory protein (Moojin), two hyaluronidases (BmooHyal-1 and BmooHyal-2), and one three-finger toxin (Bmoo-3FTx). Altogether, the transcripts identified herein represent a starting point for the analysis of structure-function relationships of toxins, which shall help develop novel biological tools and therapeutic drugs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle.

A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC 50 =4.56μg/mL) and Trypanosoma cruzi (IC 50 =4.85μg/mL). This toxin also induced cytotoxicity (IC 50 =1.80μg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Snake venom toxins that activate coagulation factors are key players in the process of venom-induced coagulopathy, and account for severe clinical manifestations. The present study applies a variety of biochemical, hematological, and histopathological approaches to broadly investigate the intravascular and systemic effects of moojenactivase (MooA), the first described PIIId subclass metalloprotease isolated from Bothrops sp. venom that activates coagulation factors. MooA induced consumption coagulopathy with high toxic potency, characterized by prolongation of prothrombin and activated partial thromboplastin time, consumption of fibrinogen and the plasma coagulation factors X and II, and thrombocytopenia. MooA promoted leukocytosis and expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, accompanied by tissue factor-dependent procoagulant activity in peripheral blood mononuclear cells. This metalloprotease also caused intravascular hemolysis, elevated plasma levels of creatine kinase-MB, aspartate transaminase, and urea/creatinine, and induced morphopathological alterations in erythrocytes, heart, kidney, and lungs associated with thrombosis and hemorrhage. Diagnosis of MooA-induced disseminated intravascular coagulation represents an important approach to better understand the pathophysiology of Bothrops envenomation and develop novel therapeutic strategies targeting hemostatic disturbances., (Copyright © 2017. Published by Elsevier B.V.)