Transcriptomic and biochemical analysis from the venom gland of the neotropical ant Odontomachus chelifer.

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 A2 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_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.

Antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, on Leishmania (Leishmania) amazonensis

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.

Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action

BACKGROUND Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

Antiangiogenic effects of phospholipase A2 Lys49 BnSP-7 from Bothrops pauloensis snake venom on endothelial cells: An in vitro and ex vivo approach.

Antiangiogenic strategies are promising tools for cancer treatment and several other disorders. In this sense, phospholipases A2 (PLA2s) 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 PLA2 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.

Ruthenium (II) complex cis-[RuII(ŋ2-O2CC7H7O2)(dppm)2]PF6-hmxbato induces ROS-mediated apoptosis in lung tumor cells producing selective cytotoxicity.

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-[RuII(ŋ2-O2CC7H7O2)(dppm)2]PF6 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.

Genotoxic effects of BnSP-6, a Lys-49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on MDA-MB-231 breast cancer cells.

Herein we evaluated the genotoxic effects of BnSP-6, a Lys-49 phospholipase A2 (PLA2) from Bothrops pauloensis, on breast cancer cells. BnSP-6 was able to induce a higher cytotoxic and genotoxic activity in MDA-MB-231 cells, when compared to MCF10A (a non-tumorigenic breast cell line), suggesting that this protein presented a possible preference for cancer cells. BnSP-6 inhibited MDA-MB-231 proliferation at 24, 48 and 72 h. In addition, BnSP-6 induced significant increase in the percentage of TUNEL-positive cells, a marker of DNA damage. To obtain novel insight into the direct DNA damage interference in MDA-MB-231 survival and proliferation, we evaluated cell cycle progression. BnSP-6 produced a significant decrease in 2N (G1) and an increase in the G2/M phase and this capacity is likely related to the modulation of expression of progression cell cycle-associated genes (CCND1, CCNE1, CDC25A, CHEK2, E2F1, CDH-1 and NF-kB). Taken together, these results indicate that BnSP-6 induces DNA damage in breast cancer cells and is an attractive model for developing innovative therapeutic agents against breast cancer.

Angiogenenic effects of BpLec, a C-type lectin isolated from Bothrops pauloensis snake venom.

The present work reports the effects of a C-type lectin (BpLec) isolated from Bothrops pauloensis snake venom upon in vitro and in vivo angiogenesis models. Initially, we noted that BpLec was not cytotoxic to endothelial cells (tEnd) in doses up to 40µg/mL, but lower doses (2.5µg/mL, 5µg/mL, 10µg/mL and 20µg/mL) reduced tEnd cells adhesion to some extracellular matrix proteins and inhibited the in vitro vessel formation in Matrigel assay stimulated by bFGF. ß-galactosides (d-lactose, N-acetyl-d-galactosamine and d-galactose) at 400mM reversed the effect of BpLec on tEnd cells adhesion, whereas d-galactose (400mM) partially reversed BpLec property of inhibiting vessel formation by tEnd cells in Matrigel. In vivo assays showed that BpLec increased hemoglobin content and capillary vessels number in polyether-polyurethane sponge discs subcutaneously implanted into dorsal skin mice. Additionally, BpLec also reduced collagen deposition and did not induce a pro-inflammatory response, as demonstrated by the decreased the secretion of some inflammatory cytokines, whereas myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities were not altered by BpLec. Taken together, our results indicate that BpLec might represent an interesting angiogenesis and inflammatory modulator that could also be used for searching possible therapeutic targets involved in these processes.

In vitro antitumor and antiangiogenic effects of Bothropoidin, a metalloproteinase from Bothrops pauloensis snake venom.

Breast cancer is a highly malignant carcinoma and remains the second leading cause of mortality among women. The antitumor effects of metalloproteinases and disintegrins from snake venom on various types of cancer cells have been investigated. In this study, we evaluated the antitumor and antiangiogenic effects on MDA-MB-231 human breast cancer cells and endothelial cells induced by Bothropoidin, a disintegrin-like metalloproteinase isolated from Bothrops pauloensis snake venom. At 24h after treatment at 100µg/mL, Bothropoidin exerted a moderate cytotoxic effect of 30% on MDA-MB-231 versus 10% cytotoxicity against MCF10A (a non-tumorigenic breast cell line), a significant difference that suggests a possible preference by this protein for targets in cancer cells. Early and late apoptosis of MDA-MB-231 was observed after Bothropoidin treatment (10µg/mL and 40µg/mL). Furthermore, this toxin inhibited not only the adhesion of MDA-MB-231 cells in a dose-dependent manner but also cell migration by approximately 45%. In addition, Bothropoidin decreased endothelial cells viability and adhesion in Matrigel and inhibited in vitro angiogenesis in Matrigel stimulated by bFGF, showing significantly fewer formed vessels. The results demonstrated that Bothropoidin has potent in vitro antitumor and antiangiogenic effect and represents a biotechnological tool for elucidating the antitumor effect of disintegrins-like metalloproteinases in cancer cells.

Human breast cancer cell death induced by BnSP-6, a Lys-49 PLA2 homologue from Bothrops pauloensis venom.

This work shows the antitumoral effects of BnSP-6, a Lys 49 PLA2 isolated from Bothrops pauloensis venom, on human breast cancer MDA-MB-231 cells. BnSP-6 caused a dose-dependent cytotoxicity and inhibited cell adhesion. Interestingly, cytotoxic activity of BnSP-6 was significantly lower against MCF10A, a non-tumorigenic breast cell line, suggesting that this PLA2 presented a possible preference for targets in cancer cells. Analysis of cell death on MDA-MB-231 cells showed that BnSP-6 stimulated the autophagy process, as evidenced by labeling of autophagic vacuoles. Moreover, apoptosis assays showed that BnSP-6 induced both early and late apoptosis. Apoptosis of MDA-MB-231 cells was also confirmed by up-regulation of different genes related to the apoptosis pathway, such as TNF, TNFRSF10B, TNFRSF1A and CASP8 and decreased expression of anti-apoptotic genes (BCL2 and BCL2L). In addition, BnSP-6 caused a remarkable increase in gene expression of BRCA2 and TP53 tumor suppressors. Finally, BnSP-6 induced down-regulation of Angiopoetin 1 gene (potent pro-angiogenic factor) and inhibited adhesion and migration of MDA-MB-231 cells suggesting pharmaceutical applications of this PLA2 as an antiangiogenic and anti-metastatic agent. Taken together, our results show that the PLA2 BnSP-6 presents anticancer potential that can be exploited as prototype for the design of new therapies.

Molecular cloning and pharmacological properties of an acidic PLA2 from Bothrops pauloensis snake venom.

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A(2) (PLA(2)) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA(2)-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA(2)-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA(2)-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA(2)-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA(2)s. The phylogenetic analyses showed that BpPLA(2)-TXI forms a group with other acidic D49 PLA(2)s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.

Perfil de proteínas e açúcares do plasma seminal e sua relação com os parâmetros andrológicos de touros da raça nelore / Perfil de proteínas e açúcares do plasma seminal e sua relação com os parâmetros andrológicos de touros da raça nelore = Proteins and sugars profile from the seminal plasma and its relationship with andrological parameters of nelore bulls

A avaliação da fertilidade de um touro é realizada considerando diferentes aspectos do animal e de seu sêmen. O objetivo deste estudo foi conhecer o perfil bioquímico de proteínas e açúcares redutores do plasma seminal em touros da raça Nelore com 24 meses de idade e sua relação com as características do sêmen. Foram utilizados 20 animais e as amostras de sêmen foram coletadas pelo método de eletroejaculação. As dosagens de proteínas e de açúcares redutores foram determinadas pelos métodos de Bradford com modificações e de Miller (ácido 3-5 dinitrosalicílico), respectivamente. A concentração de proteínas no plasma seminal variou de 5,90 a 166,33 mg/mL, com média de 61,41±44,60 mg/mL e a concentração de açúcares redutores variou de 0,78 a 19,88 mg/mL, com média de 9,31±4,54 mg/mL. A correlação entre a concentração de proteínas e açúcares redutores foi significativa com r = 0,88 (p<0,05), sendo também significativa entre elas e a concentração e patologia espermáticas. O perfil de proteínas e açúcares redutores do plasma seminal de touros da raça Nelore com 24 meses de idade apresentou variação entre os animais, o que demonstra que a produção destes elementos tem característica individual e são importantes para a qualidade do sêmen.

The fertility evaluation of a bull is accomplished considering different aspects of the animal and its semen. The objective of this study was to know the biochemical profile of proteins and reducing sugars in the seminal plasma of within 24-month Nelore bulls and its relation to semen characteristics. Twenty animals were used and their semen samples were collected using electrical ejaculation. The dosages of proteins and reducing sugars were determined by the Bradford method with modifications and Miller (3-5 dinitrosalycilic acid), respectively. It was obtained in the concentration of proteins in the seminal plasma range from 5.90 to 166.33 mg/mL, average of 61,41±44,60 mg/mL and in the concentration of reducing sugars ranging from 0.78 to 19.88 mg/mL, average 9,31±4,54 mg/mL. The correlation between protein and sugar concentrations was significant with r= 0.88 (p <0.05). This correlation was also significant with concentration and spermatic pathology. The biochemical profile of proteins and reducing sugars in seminal plasma of with 24-month Nelore bulls showed a variation among the animals, demonstrating that the production of these elements is an individual characteristic and has great importance in the semen quality

Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay in patients from Northern Paraná State, Brazil.

American cutaneous leishmaniasis (ACL) is an endemic disease caused by Leishmania parasites. The ACL diagnosis is commonly accomplished by parasitological and immunological methods. The objective of this work was to evaluate the enzyme immunoassay (EIA) for ACL laboratory diagnosis. IgG antibodies against Leishmania (Viannia) braziliensis promastigotes were researched. For the method standardization 240 sera were used: 72 from patients with positive parasitological diagnosis, 38 from normal individuals and 113 from individuals with other pathologies. The sensibility was 93% and the specificity was 70%. Regarding diagnosis, EIA showed 97% positivity in patients with ACL and 97% negativity in patients with other cutaneous lesions. The EIA presented a good performance when used for diagnosis, thus, it may become an important tool for it and for further ACL epidemiological studies in endemic areas.

Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay in patients from Northern Paraná State, Brazil

American cutaneous leishmaniasis (ACL) is an endemic disease caused by Leishmania parasites. The ACL diagnosis is commonly accomplished by parasitological and immunological methods. The objective of this work was to evaluate the enzyme immunoassay (EIA) for ACL laboratorial diagnosis. IgG antibodies against Leishmania (Viannia) braziliensis promastigotes were researched. For the method standardization 240 sera were used: 72 from patients with positive parasitological diagnosis, 38 from normal individuals and 113 from individuals with other pathologies. The sensibility was 93 percent and the specificity was 70 percent. Regarding diagnosis, EIA showed 97 percent positivity in patients with ACL and 97 percent negativity in patients with other cutaneous lesions. The EIA presented a good performance when used for diagnosis, thus, it may become an important tool for it and for further ACL epidemiological studies in endemic areas.

Caracterização de microdomínios da membrana resistentes a detergentes não-iônico em promastigotas de Leishmania (Viannia) braziliensis: papel dos microdomínios na infectividade / Characterization of non-ionic detergent resistant membrane microdomains in Leishmania (Viannia)braziliensis promastigote: role of microdmains in the infectivity

A elucidação estrutural de glicoconjugados de superfície de Leishmania é um tema de grande relevância uma vez que estes componentes mostram-se envolvidos em diversos processos na interação do parasita com a célula hospedeira. Desse modo, seis frações glicolipídicas, denominadas de B1 a B6, foram purificadas de promastigotas de L. (v. ) braziliensis por cromatografia líquida de alta resolução (HPLC) . Todas as frações foram lábeis à hidrólise alcalina suave e as análises por cromatografia gasosa acoplada a espectrometria de massa (GC/MS) demonstraram a presença de manose, galactose, glucose, glucosamina e myo-inositol, sugerindo que os glicolipídeos sejam glicoinositolfosfolipídeos (GIPLs) . A análise dos alditóis acetato parcialmente metilados (PMAAs) mostrou a presença de 2,4 -di -O-metil-mani tol tetracetato, sugerindo uma estrutura glicana ramificada contendo resíduo de galactose terminal, decorrente da presença de 2,3,4,6-tetra-O-metil galactitol diacetato entre os PMAAs . A reatividade com o anticorpo monoclonal (mAb) 8T-3 também sugere a presença de resíduos de Galb1-+3Galcx na porção glicana dos GIPLs. Estes resultados indicam que promastigotas de L. (v. ) braziliensis expressam GIPLs, os quais possivelmente possam ser incluídos nos GIPLs tipo híbrido apresentando, entretanto, novas estruturas glicanas...