Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Abstract Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.

Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.(AU)

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Toxic systemic reaction after bee stings in a bitch

Background: Bee sting poisonings are common in dogs, and toxic systemic presentation may represent a life-threatening condition. Apis mellifera venom is a complex mixture of melitin, apamine, phospholipase, hyaluronidase and degranulating peptides, that causes local injury at the site of inoculation and multiple organ complications, including hemolysis, kidney injury, muscular damage, cardiovascular and respiratory complications. The present work reports a complete and detailed description of a dog's systemic toxic reaction to bee stings, including history, clinical signs, laboratory findings, emergency care and development, as well as possible association with later immunomediated arthritis. Case: A 6-year-old female German Shepperd suffered multiple bee stings. First care was conducted by a veterinary at the site, where he only received promethazine, meloxicam and dexamethasone. After 24 h and significant progression of symptoms, the animal was forwarded to a specialized veterinary hospital. The patient was evaluated throughout 9 days, and presented intense edema, respiratory distress, tongue necrosis and grade II of acute kidney injury. Extensive laboratory exams were conducted throughout the hospitalization. Main laboratory findings included polycythemia, leukocytosis by neutrophilia and monocytosis, thrombocytopenia and azotemia. Urinalysis evidenced turbid aspect, dark yellow color and intense proteinuria, reinforcing kidney damage. Abdominal ultrasound examination identified blood clots in the bladder, and liver with reduced echogenicity and echotexture, suggesting acute inflammation. Therapy aimed to stabilize the patient, control kidney damage and avoid anaphylaxis. Treatment included intensive care support, promethazine, hydrocortisone, dexamethasone, dipyrone, methadone, metronidazole, ampicillin, clindamycin and tramadol. Following successful treatment, the animal presented immunomediated polyarthritis, possibly associated to both the poisoning and later diagnosed hemoparasitosis (both Erlichia and Babesia). Discussion: Massive bee attacks can cause severe complications, however, data regarding emergency care records are scarce. Based on clinical signs and laboratory findings, the patient presented toxic systemic reaction, including grade II of acute kidney injury and significant cardiorespiratory distress. Another important complication was tongue necrosis, that demanded attention and special supportive care, including feeding tube and specific feed. Treatment also focused in reducing edema and control possible anaphylaxis, providing analgesia and antibiotic therapy. Laboratory findings have been previously described, with evidence of immune-mediated reaction. Follow-up consultations revealed normal parameters, and an unusual presentation of claudication. Investigation concluded that polyarthritis could be responsible for such finding and may be a result of the deposition of immunomediated complexes in the joints, due in this case to the bee poisoning and later positive diagnosis for both Erlichia and Babesia. Systemic reactions to bee stings are complex, and full clinical and laboratory profile aid in both the prognosis and treatment options. Special attention must be given to tongue damage and supportive care is essential for maintaining feeding conditions. Arthritis should be considered as possible complication, reinforcing the importance of follow-up consultations.

Venom composition of Trimeresurus albolabris, T. insularis, T. puniceus and T. purpureomaculatus from Indonesia

Background: Several studies have been published on the characterization of Trimeresurus venoms. However, there is still limited information concerning the venom composition of Trimeresurus species distributed throughout Indonesia, which contributes to significant snakebite envenomation cases. The present study describes a comparative on the composition of T. albolabris, T. insularis, T. puniceus, and T. purpureomaculatus venoms originated from Indonesia. Methods: Protein content in the venom of four Trimeresurus species was determined using Bradford assay, and the venom proteome was elucidated using one-dimension SDS PAGE nano-ESI- LCMS/MS shotgun proteomics. Results: The venom of T. albolabris contained the highest protein content of 11.1 mg/mL, followed by T. puniceus, T. insularis and T. purpureomaculatus venom with 10.7 mg/mL, 8.9 mg/mL and 5.54 mg/mL protein, respectively. In total, our venomic analysis identified 65 proteins belonging to 16 protein families in T. purpureomaculatus; 64 proteins belonging to 18 protein families in T. albolabris; 58 different proteins belonging to 14 protein families in T. puniceus; and 48 different proteins belonging to 14 protein familiesin T. insularis. Four major proteins identified in all venoms belonged to snake venom metalloproteinase, C-type lectin, snake venom serine protease, and phospholipase A2. There were 11 common proteins in all venoms, and T. puniceus venom has the highest number of unique proteins compared to the other three venoms. Cluster analysis of the proteins and venoms showed that T. puniceus venom has the most distinct venom composition. Conclusions: Overall, the results highlighted venom compositional variation of four Trimeresurus spp. from Indonesia. The venoms appear to be highly similar, comprising at least four protein families that correlate with venom's toxin properties and function. This study adds more information on venom variability among Trimeresurus species within the close geographic origin and may contribute to the development of optimum heterologous antivenom.(AU)

Biochemical and proteomic analyses of venom from a new pit viper, Protobothrops kelomohy

Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.(AU)

Comparative compositional and functional venomic profiles among venom specimens from juvenile, subadult and adult Russell's viper ( Daboia siamensis ): correlation with renal pathophysiology in experimental rabbits

Abstract Background: Eastern Russell's viper (Daboia siamensis) is one of the most medically significant snakes responsible for the development of acute renal failure. However, variation of the clinical picture and renal pathophysiology following bites by young and adult D. siamensis have not been elucidated. Methods: In this study, we analyzed the venomic profiles of D. siamensis at different maturation stages of juvenile, subadult and adult groups. The same pooled venom from each group was subjected to enzymatic, electrophoretic and proteomic analysis, including sublethal toxicity (0.1 mg/kg iv.) examined on bodily functions by comparing the venom compositional and functional profiles among venom specimens from juvenile, subadult and adult D. siamensis by correlating them with the renal pathophysiology in experimental rabbits. Results: The comparative studies revealed that juvenile venom possessed higher phospholipase A2 , metalloproteinase and serine proteinase levels, while subadult and adult venoms contained more L-amino acid oxidase, phosphodiesterase, the Kunitz-type serine protease inhibitor, disintegrin families and endothelial growth factor. An in vivo study revealed that the adult and subadult venoms caused persistent hypotension and bradycardia, while thrombocytopenia was a more characteristic effect of juvenile venom. All venom age groups showed significant reductions in renal hemodynamics and electrolyte excretions. The juvenile venom caused a higher tubulonephrosis lesion score than adult and subadult venoms. Conclusions: The D. siamensis venom shows an ontogenetic shift in its compositions and activities. Renal function alterations after envenomation depend on either the synergistic actions of different venom components or the disproportionate expression between the concentrations of enzymatic and non-enzymatic proteins in each age venom group. The high proportion of enzymatic toxin proteins in the juvenile venom results in greater nephrotoxicity.(AU)

Bothrops envenomation in dogs: local and systemic manifestations

Background: Snakebite envenoming is a condition that affects humans and domestic animals worldwide. Identification of the snake species involved in the envenomation is infrequent. Bothrops envenomation presents typical clinicopathological features. This report describes epidemiological, clinical, and pathological data of 2 cases of Bothrops envenomation in dogs, including the first case of Bothrops moojeni snake striking a domestic animal in Brazil. Cases: Case 1. A dog was witnessed to have a Bothrops moojeni snakebite on a farm. In the first 24 h, acute lameness, pain, diffuse swelling, focal bleeding at the left forelimb, and increased whole-blood clotting time were observed in the envenomed dog. Polyvalent antivenom was administered in addition to fluid therapy, analgesics, corticosteroids, and antibiotics. On the 5th day, the animal presented spontaneous bleeding at the wound site, thrombocytopenia, and increased whole-blood clotting time. An additional dose of polyvalent antivenom was administered, and local treatment at the snakebite site was initiated. After 13 days, the dog showed no clinical or laboratory changes and recovered entirely. Case 2. A mongrel dog was taken for a necropsy to determine the cause of death. Grossly, major findings included swelling in the nasal plane that extended to the neck and dissecting hemorrhage in the subcutaneous tissue and adjacent musculature. Hemorrhages were observed in the heart, parietal pleura, left forelimb, lumbar region, and perirenal tissue. Marked necrosis and disruption of small blood vessels and lymphatics within the deep dermis and subcutaneous tissue were the main microscopic findings close to the snakebite site. Additionally, degeneration and necrosis of muscle fibers and dissecting hemorrhage were observed in the head and neck tissues surrounding the snakebite site. Kidneys showed marked interstitial hemorrhage and acute tubular nephrosis. Discussion: Bothrops envenoming is characterized by local (hemorrhage, dermonecrosis, and myonecrosis) and systemic (coagulative disorders, systemic hemorrhage, and acute kidney injury) changes due to the effect of the main venom components such as phospholipase A2 and metalloproteinases. These changes are hallmarks for the bothropic envenomation, supporting the diagnosis in cases 1 and 2. In case 1, the dog developed a Bothrops moojeni snakebite envenomation, but the immediate treatment with antivenom allowed a favorable outcome. In case 2, gross and microscopic findings supported the presumptive diagnosis of fatal bothropic envenomation. A marked local reaction such as swelling, pain, bleeding, bruising, and tissue necrosis was observed in case 1. In case 2, the most significant local changes were swelling and edema at the head and neck, hemorrhage in the subcutaneous tissue, and adjacent musculature. Systemic effects were observed clinically as spontaneous bleeding, thrombocytopenia, increased whole-blood clotting time (Case 1), systemic hemorrhages, and acute tubular nephrosis (Case 2). A proper treatment probably prevented the development of acute renal failure in Case 1. Herein, we show the first case of accidental snakebite envenomation by B. moojeni in a dog in Brazil. Information is scarce on the identification of venomous snake species striking domestic animals. Fast detection of well-determined clinical and pathological findings of Bothrops envenomation is essential for a correct diagnosis, therapeutics, and a good prognosis, even in cases with an unknown history.

Comparison of biological activities of Tityus pachyurus venom from two Colombian regions

Abstract Background: In the present study, we have tested whether specimens of the medically relevant scorpion Tityus pachyurus, collected from two climatically and ecologically different regions, differ in the biological activities of the venom. Methods: Scorpions were collected in Tolima and Huila, Colombia. Chemical profiles of the crude venom were obtained from 80 scorpions for each region, using SDS-PAGE and RP-HPLC. Assays for phospholipase A2, direct and indirect hemolytic, proteolytic, neuromuscular, antibacterial, and insecticidal activities were carried out. Results: The electrophoretic profiles of venom from the two regions showed similar bands of 6-14 kDa, 36-45 kDa, 65 kDa and 97 kDa. However, bands between 36 kDa and 65 kDa were observed with more intensity in venoms from Tolima, and a 95 kDa band occurred only in venoms from Huila. The chromatographic profile of the venoms showed differences in the intensity of some peaks, which could be associated with changes in the abundance of some components between both populations. Phospholipase A2 and hemolytic activities were not observable, whereas both venoms showed proteolytic activity towards casein. Insecticidal activity of the venoms from both regions showed significant variation in potency, the bactericidal activity was variable and low for both venoms. Moreover, no differences were observed in the neuromuscular activity assay. Conclusion: Our results reveal some variation in the activity of the venom between both populations, which could be explained by the ecological adaptations like differences in feeding, altitude and/or diverse predator exposure. However more in-depth studies are necessary to determine the drivers behind the differences in venom composition and activities.

Use of protein kinase C and phospholipase A2 inhibitors in bovine endometrial cells treated with estradiol and calcium ionophore / Uso de inibidores de proteína quinase C e fosfolipase A2 em células endometriais bovinas tratadas com estradiol e ionóforo de cálcio

The release of endometrial prostaglandin-F2α (PGF2α) in bovine females can be induced in vivo by estradiol (E2). However, its role in this mechanism has not been clarified. We hypothesized that E2 stimulates the activity and abundance of protein kinase C (PKC) and phospholipase A2 (PLA2). Our objective in this study was to analyze the effects of PKC and PLA2 inhibitors on PGF2α synthesis induced by E2 and calcium ionophore (CI) in bovine endometrial cells (BEND cells; Experiment 1). Additionally, we evaluated the abundance of PKC and PLA2 in endometrial explants of cows treated or not with E2 17 days after estrus (D17, D0 = estrus; Experiment 2). In Experiment 1, BEND cells were submitted to a PKC inhibitor (10 µM of C25H24N4O2; bisindolylmaleimide I, or BIS I), a PLA2 inhibitor (20 µM of arachydoniltrifluoromethane or AACOCF3), or none. The BEND cells were subsequently treated with E2 and CI, and PGF2α concentrations were measured in the culture medium through radioimmunoassay. For DIF-12 (PGF2α concentration 12 h after treatment subtracted from PGF2α concentration at hour 0), no PKC inhibitor effect was observed (P= 0.2709). However, DIF-12 was lower (P < 0.05) for groups treated with the PLA2 inhibitor and PLA2 inhibitor + CI + E2 groups than the control and CI + E2 groups. Thus, AACOCF3 was an efficient PLA2 inhibitor in the BEND cells culture system, and E2 did not stimulate the synthesis of PKC and PLA2. In Experiment 2, cyclic Nellore heifers received none (n = 5) or 3 mg (n = 6) of 17ß-E2 on D17 and were slaughtered 2 h after administration. The abundance of PKC and PLA2 in the endometrial tissue was evaluated using Western blotting analysis. No E2 effect was observed on PKC (P = 0.08) and PLA2 (P = 0.56). We concluded that E2 did not stimulate the activity and abundance of PKC and PLA2.(AU)

A liberação endometrial de prostaglandina-F2α (PGF2α) em fêmeas bovinas pode ser induzida in vivo pelo estradiol (E2). Entretanto o seu mecanismo de ação ainda não foi bem esclarecido. Nossa hipótese é que o E2 estimula a atividade e a abundância da proteína quinase C (PKC) e da fosfolipase A2 (PLA2). Nosso objetivo com este estudo foi analizar os efeitos de inibidores de PKC e PLA2 na síntese de PGF2α induzida por E2 e ionóforo de cálcio (CI) em células endometriais bovinas (células BEND; Experimento 1). Adicionalmente, nós avaliamos a abundância de PKC e PLA2 em explantes endometriais de vacas tratadas com ou sem E2 17 dias após o estro (D17, D0 = estro; Experimento 2). No Experimento 1, células BEND foram submetidas ao inibidor de PKC (10 µM de C25H24N4O2; bisindolylmaleimide I, ou BIS I), e ao inibidor de PLA2 (20 µM de arachydoniltrifluoromethane ou AACOCF3) ou a nenhum inibidor. As células BEND foram subsequentemente tratadas com E2 e CI e concentrações de PGF2α foram mensuradas no meio de cultura por radioimunoenssaio. Para DIF-12 (concentração de PGF2α 12 horas depois do tratamento, subtraída da concentração de PGF2α na hora 0), não foi observado efeito do inibidor de PKC (P = 0.2709). Entretanto DIF-12 foi menor (P < 0.05) nos grupos tratados com inibidor de PLA2 e inibidor de PLA2 + CI + E2 quando comparados com o grupo controle e o grupo CI + E2. O AACOCF3 foi um eficiente inibidor de PLA2 em sistema de cultura de células BEND e o E2 não estimulou a síntese de PKC e PLA2. No Experimento 2, novilhas Nelore cíclicas receberam 3 mg de 17ß-E2 (n = 6) ou nenhum tratamento (n = 5) no D17 e foram abatidas duas horas depois da administração dos tratamentos. A quantidade de PKC and PLA2 no tecido endometrial foi avaliada pela técnica de Western Blotting. Não foi observado efeito do E2 sobre a PKC (P= 0.08) e nem sobre a PLA2 (P= 0.56). Conclui-se que o E2 não estimulou a atividade e abundância de PKC e PLA2.(AU)

Use of protein kinase C and phospholipase A2 inhibitors in bovine endometrial cells treated with estradiol and calcium ionophore / Uso de inibidores de proteína quinase C e fosfolipase A2 em células endometriais bovinas tratadas com estradiol e ionóforo de cálcio

The release of endometrial prostaglandin-F2α (PGF2α) in bovine females can be induced in vivo by estradiol (E2). However, its role in this mechanism has not been clarified. We hypothesized that E2 stimulates the activity and abundance of protein kinase C (PKC) and phospholipase A2 (PLA2). Our objective in this study was to analyze the effects of PKC and PLA2 inhibitors on PGF2α synthesis induced by E2 and calcium ionophore (CI) in bovine endometrial cells (BEND cells; Experiment 1). Additionally, we evaluated the abundance of PKC and PLA2 in endometrial explants of cows treated or not with E2 17 days after estrus (D17, D0 = estrus; Experiment 2). In Experiment 1, BEND cells were submitted to a PKC inhibitor (10 µM of C25H24N4O2; bisindolylmaleimide I, or BIS I), a PLA2 inhibitor (20 µM of arachydoniltrifluoromethane or AACOCF3), or none. The BEND cells were subsequently treated with E2 and CI, and PGF2α concentrations were measured in the culture medium through radioimmunoassay. For DIF-12 (PGF2α concentration 12 h after treatment subtracted from PGF2α concentration at hour 0), no PKC inhibitor effect was observed (P= 0.2709). However, DIF-12 was lower (P < 0.05) for groups treated with the PLA2 inhibitor and PLA2 inhibitor + CI + E2 groups than the control and CI + E2 groups. Thus, AACOCF3 was an efficient PLA2 inhibitor in the BEND cells culture system, and E2 did not stimulate the synthesis of PKC and PLA2. In Experiment 2, cyclic Nellore heifers received none (n = 5) or 3 mg (n = 6) of 17ß-E2 on D17 and were slaughtered 2 h after administration. The abundance of PKC and PLA2 in the endometrial tissue was evaluated using Western blotting analysis. No E2 effect was observed on PKC (P = 0.08) and PLA2 (P = 0.56). We concluded that E2 did not stimulate the activity and abundance of PKC and PLA2.(AU)

A liberação endometrial de prostaglandina-F2α (PGF2α) em fêmeas bovinas pode ser induzida in vivo pelo estradiol (E2). Entretanto o seu mecanismo de ação ainda não foi bem esclarecido. Nossa hipótese é que o E2 estimula a atividade e a abundância da proteína quinase C (PKC) e da fosfolipase A2 (PLA2). Nosso objetivo com este estudo foi analizar os efeitos de inibidores de PKC e PLA2 na síntese de PGF2α induzida por E2 e ionóforo de cálcio (CI) em células endometriais bovinas (células BEND; Experimento 1). Adicionalmente, nós avaliamos a abundância de PKC e PLA2 em explantes endometriais de vacas tratadas com ou sem E2 17 dias após o estro (D17, D0 = estro; Experimento 2). No Experimento 1, células BEND foram submetidas ao inibidor de PKC (10 µM de C25H24N4O2; bisindolylmaleimide I, ou BIS I), e ao inibidor de PLA2 (20 µM de arachydoniltrifluoromethane ou AACOCF3) ou a nenhum inibidor. As células BEND foram subsequentemente tratadas com E2 e CI e concentrações de PGF2α foram mensuradas no meio de cultura por radioimunoenssaio. Para DIF-12 (concentração de PGF2α 12 horas depois do tratamento, subtraída da concentração de PGF2α na hora 0), não foi observado efeito do inibidor de PKC (P = 0.2709). Entretanto DIF-12 foi menor (P < 0.05) nos grupos tratados com inibidor de PLA2 e inibidor de PLA2 + CI + E2 quando comparados com o grupo controle e o grupo CI + E2. O AACOCF3 foi um eficiente inibidor de PLA2 em sistema de cultura de células BEND e o E2 não estimulou a síntese de PKC e PLA2. No Experimento 2, novilhas Nelore cíclicas receberam 3 mg de 17ß-E2 (n = 6) ou nenhum tratamento (n = 5) no D17 e foram abatidas duas horas depois da administração dos tratamentos. A quantidade de PKC and PLA2 no tecido endometrial foi avaliada pela técnica de Western Blotting. Não foi observado efeito do E2 sobre a PKC (P= 0.08) e nem sobre a PLA2 (P= 0.56). Conclui-se que o E2 não estimulou a atividade e abundância de PKC e PLA2.(AU)

Comparison of biological activities of Tityus pachyurus venom from two Colombian regions

Background: In the present study, we have tested whether specimens of the medically relevant scorpion Tityus pachyurus, collected from two climatically and ecologically different regions, differ in the biological activities of the venom. Methods: Scorpions were collected in Tolima and Huila, Colombia. Chemical profiles of the crude venom were obtained from 80 scorpions for each region, using SDS-PAGE and RP-HPLC. Assays for phospholipase A2, direct and indirect hemolytic, proteolytic, neuromuscular, antibacterial, and insecticidal activities were carried out. Results: The electrophoretic profiles of venom from the two regions showed similar bands of 6-14 kDa, 36-45 kDa, 65 kDa and 97 kDa. However, bands between 36 kDa and 65 kDa were observed with more intensity in venoms from Tolima, and a 95 kDa band occurred only in venoms from Huila. The chromatographic profile of the venoms showed differences in the intensity of some peaks, which could be associated with changes in the abundance of some components between both populations. Phospholipase A2 and hemolytic activities were not observable, whereas both venoms showed proteolytic activity towards casein. Insecticidal activity of the venoms from both regions showed significant variation in potency, the bactericidal activity was variable and low for both venoms. Moreover, no differences were observed in the neuromuscular activity assay. Conclusion: Our results reveal some variation in the activity of the venom between both populations, which could be explained by the ecological adaptations like differences in feeding, altitude and/or diverse predator exposure. However more in-depth studies are necessary to determine the drivers behind the differences in venom composition and activities.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

Antimicrobial effect of bothropstoxin-i in broilers / Atividade antimicrobiana da bothropstoxin-i em frangos de corte

Bacterial resistance is a sanitary issue explained by indiscriminate use of nonprescription drugs, and antimicrobial use in food production for growth promotion. Bothropstoxin-I (BthTx-I) is a phospholipase A2 (PLA2) from Bothrops jararacussu venom, which has a known antimicrobial effect. The goal of this study was the unprecedented evaluation of in vivo antimicrobial activity of BthTx-I in broilers. Microbiological, biochemical, and histological parameters were determined using 84 21-day old broilers that were kept in cages with four birds each at a density of 625 cm2/broiler. The experiment was randomized by three treatments with seven repetitions of four broilers each that lasted seven days. The treatments were: 1) bacitracin zinc diet; 2) PLA2-BthTx-I; 3) without additives. The data obtained from the studied variables was subjected to analysis of variance and an F-test at the 5% significance level. Averages of each variable in each treatment were compared by Tukey’s test. Broiler bacterial cloacal counts showed that BthTx-I decreased the microbial population without reducing body weight, intestinal morphology, or liver or kidney histopathological damage. The toxin showed in vivo activity, being an alternative for better performance in the production of broiler chickens, because it acted by decreasing the microbial load of potentially pathogenic bacteria in the intestinal(AU)

A resistência bacteriana é uma questão sanitária, explicada pelo uso indiscriminado de medicamentos sem receita médica e pelo uso de antimicrobianos na produção de alimentos para promover o crescimento. Bothropstoxin-I (BthTx-I) é uma fosfolipase A2 (PLA2) obtida do veneno da Bothrops jararacussu. A PLA2 do veneno de cobra tem efeito antimicrobiano conhecido. Objetivou-se com este estudo avaliar sem precedentes a atividade antimicrobiana in vivo de BthTx-I em frangos de corte. Os parâmetros microbiológicos, bioquímicos e histológicos foram realizados em 84 frangos de corte com 21 dias de idade mantidos em gaiolas com quatro animais cada e densidade de 625 cm2/frango. O experimento foi dividido em três tratamentos com sete repetições de quatro frangos cada um, com duração de sete dias. Os tratamentos foram: 1) dieta com bacitracina de zinco; 2) PLA2-BthTx-I; 3) sem aditivos. Os dados obtidos das variáveis estudadas foram submetidos à análise de variância e teste F ao nível de significância de 5%. As médias dos tratamentos em cada variável foram comparadas pelo teste de Tukey. A contagem cloacal bacteriana de frangos de corte mostrou que o BthTx-I diminuiu a população microbiana sem comprometer o peso corporal, a morfologia intestinal ou causar danos histopatológico no fígado e rins. Concluiu-se que a toxina apresentou atividade in vivo, sendo uma alternativa para um melhor desempenho na produção de frangos de corte, pois agiu diminuindo a carga microbiana de bactérias potencialmente patogênicas na microbiota intestinal das aves e não causou danos musculares, hepáticos ou renais na dosagem avaliada.(AU)

Antimicrobial effect of bothropstoxin-i in broilers / Atividade antimicrobiana da bothropstoxin-i em frangos de corte

Bacterial resistance is a sanitary issue explained by indiscriminate use of nonprescription drugs, and antimicrobial use in food production for growth promotion. Bothropstoxin-I (BthTx-I) is a phospholipase A2 (PLA2) from Bothrops jararacussu venom, which has a known antimicrobial effect. The goal of this study was the unprecedented evaluation of in vivo antimicrobial activity of BthTx-I in broilers. Microbiological, biochemical, and histological parameters were determined using 84 21-day old broilers that were kept in cages with four birds each at a density of 625 cm2/broiler. The experiment was randomized by three treatments with seven repetitions of four broilers each that lasted seven days. The treatments were: 1) bacitracin zinc diet; 2) PLA2-BthTx-I; 3) without additives. The data obtained from the studied variables was subjected to analysis of variance and an F-test at the 5% significance level. Averages of each variable in each treatment were compared by Tukey’s test. Broiler bacterial cloacal counts showed that BthTx-I decreased the microbial population without reducing body weight, intestinal morphology, or liver or kidney histopathological damage. The toxin showed in vivo activity, being an alternative for better performance in the production of broiler chickens, because it acted by decreasing the microbial load of potentially pathogenic bacteria in the intestinal

A resistência bacteriana é uma questão sanitária, explicada pelo uso indiscriminado de medicamentos sem receita médica e pelo uso de antimicrobianos na produção de alimentos para promover o crescimento. Bothropstoxin-I (BthTx-I) é uma fosfolipase A2 (PLA2) obtida do veneno da Bothrops jararacussu. A PLA2 do veneno de cobra tem efeito antimicrobiano conhecido. Objetivou-se com este estudo avaliar sem precedentes a atividade antimicrobiana in vivo de BthTx-I em frangos de corte. Os parâmetros microbiológicos, bioquímicos e histológicos foram realizados em 84 frangos de corte com 21 dias de idade mantidos em gaiolas com quatro animais cada e densidade de 625 cm2/frango. O experimento foi dividido em três tratamentos com sete repetições de quatro frangos cada um, com duração de sete dias. Os tratamentos foram: 1) dieta com bacitracina de zinco; 2) PLA2-BthTx-I; 3) sem aditivos. Os dados obtidos das variáveis estudadas foram submetidos à análise de variância e teste F ao nível de significância de 5%. As médias dos tratamentos em cada variável foram comparadas pelo teste de Tukey. A contagem cloacal bacteriana de frangos de corte mostrou que o BthTx-I diminuiu a população microbiana sem comprometer o peso corporal, a morfologia intestinal ou causar danos histopatológico no fígado e rins. Concluiu-se que a toxina apresentou atividade in vivo, sendo uma alternativa para um melhor desempenho na produção de frangos de corte, pois agiu diminuindo a carga microbiana de bactérias potencialmente patogênicas na microbiota intestinal das aves e não causou danos musculares, hepáticos ou renais na dosagem avaliada.

In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victim’s local effects and time to heal.

MS/MS analysis of four scorpion venoms from Colombia: a descriptive approach

Background: Scorpions are widely known for the neurotoxic effects of their venoms, which contain peptides affecting ionic channels. Although Colombia is recognized for its scorpion diversity, only a few studies are available describing the venom content. Methods: In this descriptive study, we analyzed the MS/MS sequence, electrophoretic and chromatographic profile linked to a bioinformatics analysis of the scorpions Chactas reticulatus (Chactidae), Opisthacanthus elatus (Hormuridae), Centruroides edwardsii (Buthidae) and Tityus asthenes (Buthidae) from Colombia. Results: Each scorpion showed a specific electrophoretic and chromatographic profile. The electrophoretic profiles indicate the presence of high molecular mass compounds in all venoms, with a predominance of low molecular mass compounds in the Buthidae species. Chromatographic profiles showed a similar pattern as the electrophoretic profiles. From the MS/MS analysis of the chromatographic collected fractions, we obtained internal peptide sequences corresponding to proteins reported in scorpions from the respective family of the analyzed samples. Some of these proteins correspond to neurotoxins affecting ionic channels, antimicrobial peptides and metalloproteinase-like fragments. In the venom of Tityus asthenes, the MSn analysis allowed the detection of two toxins affecting sodium channels covering 50% and 84% of the sequence respectively, showing 100% sequence similarity. Two sequences from Tityus asthenes showed sequence similarity with a phospholipase from Opisthacanthus cayaporum indicating the presence of this type of toxin in this species for the first time. One sequence matching a hypothetical secreted protein from Hottentotta judaicus was found in three of the studied venoms. We found that this protein is common in the Buthidae family whereas it has been reported in other families - such as Scorpionidae - and may be part of the evolutionary puzzle of venoms in these arachnids. Conclusion: Buthidae venoms from Colombia can be considered an important source of peptides similar to toxins affecting ionic channels. An interesting predicted antimicrobial peptide was detected in three of the analyzed venoms.(AU)

Effects of venoms on neutrophil respiratory burst: a major inflammatory function

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.(AU)

In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victims local effects and time to heal.(AU)