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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.

Purpose: To evaluate the effects of topical insulin in patients with persistent corneal epithelial defects that are refractory to the standard treatment., Methods: A retrospective, hospital-based, clinical study was performed on 17 eyes of 16 patients with different types of refractory persistent epithelial defects who were treated with topical insulin. The treatment was continued until the defect either was resolved or persisted after three months. Patients' demographic information, etiology, comorbidities, and clinical data were reviewed. The rate of epithelial healing was considered as the primary outcome measure., Results: Neurotrophic keratitis was the most common cause of persistent epithelial defects (58.8%), and within this category, herpetic eye disease was the main comorbidity (44.4%). The mean follow-up time was 17.91 months. Eleven out of fifteen eyes (77.3%) had complete improvement and only one patient did not respond to the treatment. The mean time of reepithelization for the eyes with full recovery was 31.27 days (ranging from 6 to 61 days). The best-corrected visual acuity improved significantly after treatment ( P < 0.005), and there were no reports of complications or side effects during the study period., Conclusion: Our results suggest that topical insulin, due to its good safety profile, availability, and affordability, could be a good therapeutic alternative for persistent epithelial defects., Competing Interests: None., (Copyright © 2024 Almeida et al.)

Cat-transmitted sporotrichosis is caused by the emerging fungal pathogen Sporothrix brasiliensis and constitutes a significant public health issue that affects people living in resource-poor urban centers in Brazil. The lack of knowledge about transmission dynamics makes it difficult to propose public health policies to contain the advance of sporotrichosis. We describe the recent emergence of 1,176 cases of sporotrichosis in cats (2016 to 2021) in the metropolitan region of Recife, Brazil, leading to significant zoonotic transmission and an overwhelming occurrence of S. brasiliensis as the etiological agent. Most cases were from cats in the cities of Olinda (408/1,176; 34.70%), Jaboatão dos Guararapes (332/1,176; 28.23%), and Recife (237/1,176; 20.15%). Molecular typing using amplified fragment length polymorphism (EcoRI-GA/MseI-AG) revealed low polymorphic information content (PIC = 0.2499) and heterozygosity (H = 0.2928), typical of an outbreak scenario. Dendrogram and multivariate cluster analysis revealed that isolates from Pernambuco are closely related to Rio de Janeiro isolates. We report a substantial occurrence of MAT1-2 idiomorphs in the metropolitan region of Recife (0:60 ratio; χ 2  = 60.000, P < 0.0001). The limited population differentiation and genetic diversity of the isolates from Pernambuco suggest a recent introduction, possibly via a founder effect, from the parental population in Rio de Janeiro. Our findings emphasize the critical importance of molecular surveillance of S. brasiliensis for outbreak response. A comprehensive one-health strategy is mandatory to control the spread of cat-transmitted sporotrichosis driven by S. brasiliensis, encompassing sanitary barriers, quick diagnosis, and treatment., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Forest Islands and their adjacent natural grasslands are vulnerable and sensitive ecosystems to the actions of severe fires, which result in losses of their resilience, which makes the potential of passive restoration of these environments unfeasible after such events. This study aims to verify, through an autochthonous species exclusive to these Forest Islands, whether it can develop in Histosols around a Forest Island that has been degraded by fire for years. The place of study and collection of the material tested was in the Sempre-Vivas National Park. Histosols samples were collected for analysis of chemical and physical attributes and experimental conduction in a seedling nursery. The performance of Richeria grandis was evaluated in these Histosols from seed vigor tests, initial plant growth in a greenhouse. R. grandis manages to develop in Histosols around the degraded Forest Island, disregarding possible interspecific field competitions. The physical and chemical characteristics of the Histosols around the island do not prevent the effective restoration of this phytocenosis. R. grandis showed the same seed vigor for all Histosols tested and all seedlings survived until the end of the experiment. It was observed that the seedlings grown in the Histosols of the island of the forest, showed a behavior of greater height, number of leaves and moisture content, and the place with exposed Histosols, with the highest fire severity, provided the lowest development in height, diameter and number of leaves. According to ecophysiological analyses, the species is under some environmental stress regardless of the treatment., Competing Interests: Declaration of competing interest There is no conflict of interest between authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Single-haptic iris-claw intraocular lens (IOL) dislocation is not an uncommon complication. A few different surgical techniques are available for its refixation but usually involve a more invasive approach. We aim to demonstrate an original and simple approach for refixation of single haptic retropupillary iris-claw IOL subluxations., Methods: We present a case of an 80-year-old male with a single haptic retropupillary iris-claw IOL subluxation in a previously vitrectomized eye. We used a new surgical approach to refix this type of subluxation., Results: This new technique uses only two 30-gauge needles (one of them connected to a viscosurgical device) to re-enclavate the subluxated haptic of the retropupillary iris-claw IOL. By minimising surgical manipulation, the patient's postoperative period was uneventful., Conclusions: We highlight a new, quick, safe, and unusual surgical approach to single-haptic retropupillary iris-claw IOL dislocation in vitrectomized eyes. Because of its characteristics, we named it the "fencing" IOL repositioning technique., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Optic neuropathies, such as glaucoma, are some of the leading causes of irreversible blindness worldwide. There has been a lot of research for potential therapies that could attenuate and even reduce the impact of the pathological pathways that lead to the loss of retinal ganglion cells (RGCs). In recent years, vitamin B 3 (nicotinamide) has gained some interest as a viable option for these neurodegenerative diseases due to its fundamental role in enhancing the mitochondria metabolism of the RGCs. This review focuses on elucidating the impact of vitamin B 3 on retinal cells, especially when in a dysfunctional state like what happens in optic neuropathies, especially glaucoma. This review also summarizes the existing and future research on the clinical effects of vitamin B 3 in these optic neuropathies, and determines appropriate recommendations regarding its dosing, efficacy, and eventual side effects.

Background: Leishmaniasis, a neglected disease caused by the parasite Leishmania, is treated with drugs associated with high toxicity and limited efficacy, in addition to constant reports of the emergence of resistant parasites. In this context, snake serums emerge as good candidates since they are natural sources with the potential to yield novel drugs., Objectives: We aimed to show the antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, against Leishmania (Leishmania) amazonensis., Methods: Promastigotes forms were exposed to γCdcPLI, and we assessed the parasite viability and cell cycle, as well as invasion and proliferation assays., Findings: Despite the low cytotoxicity effect on macrophages, our data indicate that γCdcPLI has a direct effect on parasites promoting an arrest in the G1 phase and reduction in the G2/M phase at the highest dose tested. Moreover, this PLA2 inhibitor reduced the parasite infectivity when promastigotes were pre-treated. Also, we demonstrated that the γCdcPLI treatment modulated the host cell environment impairing early and late steps of the parasitism., Main Conclusions: γCdcPLI is an interesting tool for the discovery of new essential targets on the parasite, as well as an alternative compound to improve the effectiveness of the leishmaniasis treatment.

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies., 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 Inc. All rights reserved.)

This study reports the isolation of CollinLAAO-I, a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom, its biochemical characterization and leishmanicidal potential in Leishmania spp. CollinLAAO-I (63.1 kDa) was successfully isolated with high purity using two chromatographic steps and represents 2.5% of total venom proteins. CollinLAAO-I displayed high enzymatic activity (4262.83 U/mg/min), significantly reducing after 28 days. The enzymatic activity of CollinLAAO-I revealed higher affinity for hydrophobic amino acids such as L-leucine, high enzymatic activity in a wide pH range (6.0-10.0), at temperatures from 0 to 25 °C, and showed complete inhibition in the presence of Na + and K + . Cytotoxicity assays revealed IC 50 of 18.49 and 11.66 μg/mL for Leishmania (L.) amazonensis and Leishmania (L.) infantum, respectively, and the cytotoxicity was completely suppressed by catalase. CollinLAAO-I significantly increased the intracellular concentration of reactive oxygen species (ROS) and reduced the mitochondrial potential of both Leishmania species. Furthermore, CollinLAAO-I decreased the parasite capacity to infect macrophages by around 70%, indicating that even subtoxic concentrations of CollinLAAO-I can interfere with Leishmania vital processes. Thus, the results obtained for CollinLAAO-I provide important support for developing therapeutic strategies against leishmaniasis., 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 Ltd. All rights reserved.)

One-third of the world's population is estimated to be affected by toxoplasmosis. Pregnancy-related Toxoplasma gondii infection can cause vertical transmission, infect the fetus, and cause miscarriage, stillbirth, and fetal death. The current study showed that both human trophoblast cells (BeWo lineage) and human explant villous were resistant to T. gondii infection after incubation with BjussuLAAO-II, an l-amino acid oxidase isolated from Bothrops jararacussu. Almost 90% of the parasite's ability to proliferate in BeWo cells was decreased by the toxin at 1.56 μg/mL and showed an irreversible anti-T. gondii effect. Also, BjussuLAAO-II impaired the key events of adhesion and invasion of T. gondii tachyzoites in BeWo cells. BjussuLAAO-II antiparasitic properties were associated with the intracellular production of reactive oxygen species and hydrogen peroxide, since the presence of catalase restored the parasite's growth and invasion. In addition, T. gondii growth in human villous explants was decreased to approximately 51% by the toxin treatment at 12.5 μg/mL. Furthermore, BjussuLAAO-II treatment altered IL-6, IL-8, IL-10 and MIF cytokines levels, assuming a pro-inflammatory profile in the control of T. gondii infection. This study contributes to the potential use of a snake venom l-amino acid oxidase for the development of agents against congenital toxoplasmosis and the discovery of new targets in parasites and host cells., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Objectives: Up to one quarter of the patients with colorectal cancer (CRC) develop peritoneal carcinomatosis (PM). The aims of this retrospective study were to characterize the histological response of the PM of CRC to preoperative chemotherapy and evaluate the potential prognostic value, in terms of survival., Methods: This retrospective unicentric study evaluated a group of 30 patients treated between 2010 and 2020 at the São João University Hospital Center with preoperative chemotherapy, followed by cytoreduction surgery plus hyperthermic intraperitoneal chemotherapy. The evaluation of the histological response was done using two scores: the tumor regression grading (TRG) and the peritoneal regression grading score (PRGS)., Results: Mean post-procedure survival is higher in the PRGS 1-2 group (74.19 months) vs. the PRGS 3-4 group (25.27 months) (p=0.045), as well as in the TRG 1-2 group (74.58 months) vs. TRG 4-5 (25.27 months) (p=0.032). As for progression-free survival (PFS), the PRGS 1-2 group had a mean value of 58.03 months vs. PRGS 3-4 which had 11.67 months (p=0.002). Similar was observed with the TRG 1-2 group, which had a mean PFS of 61.68 months vs. TRG 4-5 with 11.67 months (p=0.003)., Conclusions: A better histological response to preoperative chemotherapy, represented as a lower PRGS and TRG value, is associated with longer post-procedure survival and progression-free survival in this group of patients. That is, these two scores have prognostic value., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

The genus Odontomachus is widely distributed in neotropical areas throughout Central and South America. It is a stinging ant that subdues its prey (insects) by injecting them a cocktail of toxic molecules (venom). Ant venoms are generally composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Odontomachus chelifer is an ant that inhabits neotropical regions from Mexico to Argentina. Unlike the venom of other animals such as scorpions, spiders and snakes, this ant venom has seldom been analyzed comprehensively, and their compositions are not yet completely known. In the present study, we performed a partial investigation of enzymatic and functional activities of O. chelifer ant venom, and we provide a global insight on the transcripts expressed in the venom gland to better understand their properties. The crude venom showed phospholipase A 2 and antiparasitic activities. RNA sequencing (Illumina platform) of the venom gland of O. chelifer generated 61, 422, 898 reads and de novo assembly Trinity generated 50,220 contigs. BUSCO analysis against Arthropoda&#95;db10 showed that 92.89% of the BUSCO groups have complete gene representation (single-copy or duplicated), while 4.05% are only partially recovered, and 3.06% are missing. The 30 most expressed genes in O. chelifer venom gland transcriptome included important transcripts involved in venom function such as U-poneritoxin (01)-Om1a-like (pilosulin), chitinase 2, venom allergen 3, chymotrypsin 1 and 2 and glutathione S-transferase. Analysis of the molecular function revealed that the largest number of transcripts were related to catalytic activity, including phospholipases. These data emphasize the potential of O. chelifer venom for prospection of molecules with biotechnological application., 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 Ltd. All rights reserved.)

Objective: To investigate vascular and perivascular abnormalities in the carotid arteries using ultrasound, as well as to evaluate their association with mortality and clinical variables in hospitalized patients with coronavirus disease 2019 (COVID-19)., Materials and Methods: This was a prospective study in which 53 hospitalized patients with severe COVID-19 were evaluated and underwent carotid ultrasound. We documented the carotid ultrasound findings in these patients. Clinical, demographic, laboratory, and imaging features were analyzed and compared by statistical analysis to detect correlations between them., Results: Carotid ultrasound demonstrated luminal surface irregularity in 29 patients (55%), carotid plaques in 30 (57%), perivascular infiltration in four (8%), and increased intima-media thickness (IMT) in 31 (58%). Of the 31 patients with increased IMT, 19 (61%) died, and the association between increased IMT and COVID-19-related mortality was significant ( p = 0.03). Logistic regression showed that the risk of death was 85% in patients who had increased IMT in combination with acute kidney injury at admission or a history of chronic kidney disease ( p < 0.05)., Conclusion: In hospitalized patients with severe COVID-19, carotid ultrasound can show increased IMT, luminal surface irregularity, carotid plaques, and perivascular infiltrates. The combination of increased IMT and kidney damage appears to increase the risk of death in such patients.

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.)

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a K D of 9.146 × 10 -7  M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA 2 , thus proposing a new mechanism of PLA 2 inhibition, and presenting more evidence of GA's potential as an antivenom compound., (Copyright © 2021. Published by Elsevier B.V.)

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.)

Antiangiogenic strategies are promising tools for cancer treatment and several other disorders. In this sense, phospholipases A 2 (PLA 2 s) from snake venom have been described to possess antiangiogenic properties. In this study, we evaluated both in vitro and ex vivo antiangiogenic effects induced by BnSP-7, a Lys49 PLA 2 isolated from Bothrops pauloensis snake venom. BnSP-7 was able to inhibit endothelial cell (HUVEC) proliferation, which was indeed confirmed by a modulation of cell cycle progression. Interestingly, BnSP-7 also inhibited the adhesion and migration of HUVECs and blocked in vitro angiogenesis in a VEGF-dependent manner, an important proangiogenic factor. Finally, BnSP-7 was capable of inhibiting sprouting angiogenic process through an ex vivo aortic ring assay. Taken together, these results indicate that BnSP-7 has potent in vitro and ex vivo antiangiogenic effect., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.)

Ruthenium complexes have been extensively explored as potential molecules for cancer treatment. Considering our previous findings on the remarkable cytotoxic activity exhibited by the ruthenium (II) complex 3-hydroxy-4-methoxybenzoate (hmxbato)-cis-[Ru II (ŋ 2 -O 2 CC 7 H 7 O 2 )(dppm) 2 ]PF 6 against Leishmania promastigotes and also the similar metabolic characteristics between trypanosomatids and tumor cells, the present study aimed to analyze the anticancer potential of hmxbato against lung tumor cells, as well as the partial death mechanisms involved. Hmxbato demonstrated selective cytotoxicity against A549 lung tumor cells. In addition, this complex at a concentration of 3.8 µM was able to expressively increase the generation of reactive oxygen species (ROS) in tumor cells, causing an oxidative stress that may culminate in: (1) reduction in cellular proliferation; (2) changes in cell morphology and organization patterns of the actin cytoskeleton; (3) cell arrest in the G2/M phase of the cell cycle; (4) apoptosis; (5) changes in the mitochondrial membrane potential and (6) initial DNA damage. Furthermore, we demonstrated that the induction of programmed cell death can occur by the intrinsic apoptotic pathway through the activation of caspases. It is also worth highlighting that hmxbato exhibited predominant actions on A549 tumor cells in comparison to BEAS-2B normal bronchial epithelium cells, which makes this complex an interesting candidate for the design of new drugs against lung cancer.

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.

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.)

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.)

Drug delivery systems can overcome cancer drug resistance, improving the efficacy of chemotherapy agents. Poly (lactic acid) (PLA) microparticles are an interesting alternative because their hydrophobic surface and small particle size could facilitate interactions with cells. In this study, two poloxamers (PLX 407 and 188) were applied to modulate the structural features, the drug release behavior and the cell viability from spray-dried microparticles. Five formulations with different PLA: PLX blend ratio (100:0, 75:25, 50:50, 25:50, and 0:100) were well-characterized by SEM, particle size analysis, FTIR spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction analysis (XRD). The spray-dried microparticles showed higher drug loading, spherical-shape, and smaller particle size. The type of poloxamer and blend ratio affected their structural and functional properties such as morphology, crystallinity, blend miscibility, drug release rate, and cell viability. The methotrexate (MTX), a model drug, was loaded in amorphous spray-dried microparticles. Moreover, the drug release studies demonstrated that PLX induced a leaching-effect of MTX from PLA: PLX blends, suggesting the formation of MTX/PLX micelles in aqueous medium. This finding was better established by cell viability assays. Therefore, biocompatible PLA: PLX blends showed promising in vitro results, and further in vivo studies will be performed to evaluate the performance of this chemotherapeutic agent.

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.

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.

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 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 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.

Background: Although it is well known that allergic diseases involve a strong Th2 immune response, with production of high levels of specific IgE allergen, knowledge on the association between filarial infection and allergies, among paediatric patients is scarce., Objective: To evaluate the allergic response patterns in cases of filarial infection by comparing peripheral eosinophils, total IgE levels, immediate hypersensitivity and cytokine levels in children and adolescents in Brazil., Methods: This was an exploratory study with three groups: (I) with filarial infection and without allergic diseases; (II) without filarial infection and with allergic diseases; and (III) without filarial infection and without allergic diseases. The prick test and specific IgE tests for aeroallergens were performed using five antigens. Peripheral eosinophils and total IgE were also evaluated. IL-4 and IL-5 were determined using whole-blood culture stimulated by three antigens., Results: Eosinophilia and elevated levels of total IgE (≥400IU/dl) were observed in all groups. The prick test was positive in 56.6% of the cases. Group I presented hypersensitive responses similar to the allergic disease groups. In the whole-blood culture stimulated by Dermatophagoides pteronyssinus, average IL-4 production did not differ significantly among the groups, but IL5 production resulting from stimulation was greater in the allergic disease groups (p<0.05)., Conclusions: The allergic response pattern in group with filarial infection was similar to that of the groups with and without allergic diseases, but the response to IL-5 in the culture stimulated by D. pteronyssinus was an exclusive characteristic of the allergic group., (Copyright © 2018 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

The isolation and identification of a series of new pseudoceratidine (1) derivatives from the sponge Tedania brasiliensis enabled the evaluation of their antiparasitic activity against Plasmodium falciparum, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) infantum, and Trypanosoma cruzi, the causative agents of malaria, cutaneous leishmaniasis, visceral leishmaniasis, and Chagas disease, respectively. The new 3-debromopseudoceratidine (4), 20-debromopseudoceratidine (5), 4-bromopseudoceratidine (6), 19-bromopseudoceratidine (7), and 4,19-dibromopseudoceratidine (8) are reported. New tedamides A-D (9-12), with an unprecedented 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-2-carboxamide moiety, are also described. Compounds 4 and 5, 6 and 7, 9 and 10, and 11 and 12 have been isolated as pairs of inseparable structural isomers differing in their sites of bromination or oxidation. Tedamides 9+10 and 11+12 were obtained as optically active pairs, indicating an enzymatic formation rather than an artifactual origin. N 12 -Acetylpseudoceratidine (2) and N 12 -formylpseudoceratidine (3) were obtained by derivatization of pseudoceratidine (1). The antiparasitic activity of pseudoceratidine (1) led us to synthesize 23 derivatives (16, 17, 20, 21, 23, 25, 27-29, 31, 33, 35, 38, 39, 42, 43, 46, 47, 50, and 51) with variations in the polyamine chain and aromatic moiety in sufficient amounts for biological evaluation in antiparasitic assays. The measured antimalarial activity of pseudoceratidine (1) and derivatives 4, 5, 16, 23, 25, 31, and 50 provided an initial SAR evaluation of these compounds as potential leads for antiparasitics against Leishmania amastigotes and against P. falciparum. The results obtained indicate that pseudoceratidine represents a promising scaffold for the development of new antimalarial drugs.

Purpose: The study aims to assess the influence of the stage of chronic kidney disease and glomerular filtration rate on prevalence and degree of erectile dysfunction., Materials and Methods: This transversal study, conducted from May 2013 to December 2015, included patients with chronic kidney disease in conservative treatment, stages III/IV/V. Erectile dysfunction was evaluated by the International Index of Erectile Function. Data classically associated with erectile dysfunction were obtained by medical record review. Erectile dysfunction, degree of erectile dysfunction, and other main variables associated with erectile dysfunction were compared between patients with chronic kidney disease on conservative treatment stages III versus IV/V using the Chi-square test. The relationship between score of the International Index of Erectile Dysfunction and glomerular filtration rate was established by Pearson correlation coefficient., Results: Two hundred and forty five patients with chronic kidney disease in con-servative treatment participated of the study. The prevalence of erectile dysfunction in patients with chronic kidney disease in stages IV/V was greater than in stage III. Glomerular filtration rate positively correlated with score of the International Index of Erectile Dysfunction., Conclusions: The study suggests that chronic kidney disease progression (glomerular filtration rate decrease and advance in chronic kidney disease stages) worsen erectile function. Hypothetically, diagnosis and treatment of erectile dysfunction may be anticipated with the analysis of chronic kidney disease progression., Competing Interests: Conflict of interest: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Population with chronic kidney disease (CKD) has had many problems, and some of these have arisen from sexual disorders. The present study intends to determine the prevalence and the associated factors with erectile dysfunction (ED) among patients with CKD on conservative treatment (CKDCT). This transversal study was conducted from May 2013 to December 2015. The tools used were: medical records and the International Index of Erectile Function. Data were analyzed by univariate and multivariate logistic regression analysis. Among 245 patients that have participated of this study, ED was present in 71.02% and it was severe in 36.73%. Age greater than 50 years, body mass index lower than 25, diabetes mellitus, stages IV/V of CKD, cardiac conduction disturbances, benign prostatic hyperplasia, smoking, alcohol use, albumin <3.5 g per 100 ml and creatinine clearance between 15 and 29 ml min -1 per  1.73 m 2 were associated with ED. Time of CKD was the only variable associated with ED independent of the presence of other factors. ED prevalence in patients with CKDCT is high and it is severe in more than half of them. Several factors are associated with ED in this population but the principal one is the time of CKD.

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.)

Bothrops envenomations can promote severe inflammatory responses by inducing edema, pain, leukocyte recruitment and release of chemical mediators by local cells. In the present study, two toxins from Bothrops atrox venom (the P-I metalloprotease Batroxase and the acidic phospholipase A 2 BatroxPLA 2 ) were evaluated in relation to their inflammatory effects induced in vivo and in vitro, mainly focusing on the participation of different immune cells and inflammatory mediators. Both toxins mainly promoted acute inflammatory responses with significant recruitment of neutrophils in the early hours (1-4h) after administration into the peritoneal cavity of C57BL/6 mice, and increased infiltration of mononuclear cells especially after 24h. Among the mediators induced by both toxins are IL-6, IL-10 and PGE 2 , with Batroxase also inducing the release of L-1β, and BatroxPLA 2 of LTB 4 and CysLTs. These responses pointed to possible involvement of immune cells such as macrophages and mast cells, which were then evaluated in vitro. Mice peritoneal macrophages stimulated with Batroxase produced significant levels of IL-6, IL-1β, PGE 2 and LTB 4 , whereas stimulus with BatroxPLA 2 induced increases of IL-6, PGE 2 and LTB 4 . Furthermore, both toxins were able to stimulate degranulation of RBL-2H3 mast cells, but with distinct concentration-dependent effects. Altogether, these results indicated that Batroxase and BatroxPLA 2 promoted local and acute inflammatory responses related to macrophages and mast cells and to the production of several mediators. Our findings should contribute for better understanding the different mechanisms of toxicity induced by P-I metalloproteases and phospholipases A 2 after snakebite envenomations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1β, LTB 4 and PGE 2 . In vitro, IL-6 and IL-1β production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1β that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them.

Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections-unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.

Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN W279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

Objective: To describe an unusual clinical presentation of intra-abdominal extralobar pulmonary sequestration in a 2-year, 9 month-old patient and assess diagnostic and treatment aspects of this pathology., Case Description: An undefined intra-abdominal mass was identified in the right adrenal region in a male fetus. Postnatal evaluation with ultrasound images, computed tomography, magnetic resonance imaging and laboratory testing was insufficient to determine the nature of the lesion. After two years, laparoscopic resection of the mass and histopathological examination of the surgical specimen allowed to establish the diagnosis of intra-abdominal extralobar pulmonary sequestration., Comments: This malformation can be monitored clinically; however, surgical excision is often performed, probably due to the impossibility of attaining diagnosis with non-invasive methods, such as in the present case, in which the lesion appeared in an unusual position for intra-abdominal extralobar pulmonary sequestration. Therefore, the surgical approach seems to be the key to attain the diagnosis and establish the conduct for this type of congenital malformation., (Copyright © 2015 Sociedade de Pediatria de São Paulo. Publicado por Elsevier Editora Ltda. All rights reserved.)

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Bcr-Abl tyrosine kinase protein, which confers resistance to apoptosis in leukemic cells. Tyrosine kinase inhibitors (TKIs) are effectively used to treat CML; however, CML patients in the advanced (CML-AP) and chronic (CML-CP) phases of the disease are usually resistant to TKI therapy. Thus, it is necessary to seek for novel agents to treat CML, such as the enzyme l-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) snake venom. We examined the antitumor effect of CR-LAAO in Bcr-Abl(+) cell lines and peripheral blood mononuclear cells (PBMC) from healthy subjects and CML patients. CR-LAAO was more cytotoxic towards Bcr-Abl(+) cell lines than towards healthy subjects' PBMC. The H2O2 produced during the enzymatic action of CR-LAAO mediated its cytotoxic effect. The CR-LAAO induced apoptosis in Bcr-Abl(+) cells, as detected by caspases 3, 8, and 9 activation, loss of mitochondrial membrane potential, and DNA damage. CR-LAAO elicited apoptosis in PBMC from CML-CP patients without TKI treatment more strongly than in PBMC from healthy subjects and TKI-treated CML-CP and CML-AP patients. The antitumor effect of CR-LAAO against Bcr-Abl(+) cells makes this toxin a promising candidate to CML therapy., (Copyright © 2016. Published by Elsevier B.V.)

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

Background: Phospholipases A2 (PLA2s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA2 isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines., Methods: The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I., Results: It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %)., Conclusions: These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.

CR-LAAO is an L-amino acid oxidase from Calloselasma rhodostoma snake venom that has been broadly studied regarding its structural and biochemical characteristics, however, few studies have investigated its pharmacological effects. The present study aimed at the evaluation of the biotechnological potential of CR-LAAO by determining its bactericidal, antifungal, leishmanicidal and trypanocidal activity, as well as its cytotoxicity on human tumor and non-tumor cell lines. After 24 h of preincubation, CR-LAAO showed bactericidal effects against both Staphylococcus aureus (MIC 0.78 μg/mL) and Escherichia coli (MIC 31.25 μg/mL) strains, inducing dismantle of bacterial cell walls. After 6 h of preincubation with Candida albicans, CR-LAAO was able to inhibit 80% of the yeast growth, and it also showed cytotoxic activity on Leishmania species and Trypanosoma cruzi. Additionally, CR-LAAO showed high cytotoxicity on HepG2 and HL-60 tumor cells (IC50 10.78 and 1.7 μg/mL), with lower effects on human mononuclear cells (PBMC). The cytotoxic effects of CR-LAAO were significantly inhibited in the presence of catalase, which suggests the involvement of hydrogen peroxide in its mechanisms of toxicity. Therefore, CR-LAAO showed promising pharmacological effects, and these results provide important information for the development of therapeutic strategies with directed action, such as more effective antimicrobial agents., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bacteria have evolved a remarkable array of sophisticated nanomachines to export various virulence factors across the bacterial cell envelope. In recent years, considerable progress has been made towards elucidating the structural and molecular mechanisms of the six secretion systems (types I-VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone-usher pathway and the curli secretion machinery. These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In this Review, we explore the structural and mechanistic relationships between these single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies.