Tetanus in a canine: clinical and therapeutic aspects / Tétano em um canino: aspectos clínicos e terapêuticos

Tetanus is a serious and rare disease in small animals, of an acute nature and mediated by the action of the neurotoxin tetanospasmin, from the bacillus Clostridium tetani. This report describes a case of tetanus in a canine, with emphasis on its clinical and therapeutic aspects, in addition to its clinical recovery. A canine, female, Pitbull breed, five months old, in status epilepticus, was attended at the Veterinary Hospital of the University of Passo Fundo. After stabilizing the patient, the animal was found in a trestle position, with generalized spastic paralysis, risus sardonicus, trismus, erect tail and ears, and difficulty in expanding the thorax. The presumptive diagnosis of tetanus was established based on the anamnesis, clinical signs, and laboratory findings. The established supportive treatment included hydro electrolytic replacement, antitetanus serum, antibiotic therapy, analgesia, myorelaxant and anticonvulsant drugs. The patient's intensive management was carried out with urethral and gastroesophageal probing, changes of position every two hours, and reduction of environmental stimuli. Physiotherapy and acupuncture were also used to complement the treatment and accelerate recovery. The patient was discharged one month after the beginning of the treatment, presenting satisfactory evolution. In the present case, the clinical examination in association with the characteristic clinical signs of the disease, added to the detailed anamnesis, was essential for the presumptive diagnosis of tetanus. In addition, intensive management, drug treatment, physiotherapy, and acupuncture enabled the evolution of the clinical condition to cure.

O tétano é uma doença grave e rara em pequenos animais, de caráter agudo e mediada pela ação da neurotoxina tetanospasmina, proveniente do bacilo Clostridium tetani. Neste relato descreve-se um caso de tétano em um canino, com ênfase em seus aspectos clínicos e terapêuticos, além da sua recuperação clínica. Foi atendido no Hospital Veterinário da Universidade de Passo Fundo, um canino, fêmea, da raça Pitbull, com cinco meses, em status epilepticus. Após a estabilização da paciente, constataram-se animal em posição de cavalete, com paralisia espástica generalizada, risus sardonicus, trismo, cauda e orelhas eretas e dificuldade em expandir o tórax. O diagnóstico presuntivo de tétano foi firmado a partir da anamnese, sinais clínicos e achados laboratoriais. O tratamento suporte estabelecido incluiu reposição hidroeletrolítica, soro antitetânico, antibioticoterapia, analgesia, fármacos miorrelaxantes e anticonvulsivantes. O manejo intensivo da paciente foi realizado com sondagem uretral e nasoesofágica, trocas de decúbito a cada duas horas e diminuição de estímulos ambientais. A fisioterapia e a acupuntura também foram utilizadas para complementar o tratamento e acelerar a recuperação. A paciente recebeu alta médica um mês após o início do tratamento, apresentando evolução satisfatória. No presente caso, o exame clínico em associação aos sinais clínicos característicos da doença, acrescidos da anamnese minuciosa foram fundamentais ao diagnóstico presuntivo de tétano. Além disso, o manejo intensivo, o tratamento medicamentoso, assim como a realização de fisioterapia e acupuntura, possibilitaram a evolução do quadro clínico à cura.

ß-micrustoxin (Mlx-9), a PLA2 from Micrurus lemniscatus snake venom: biochemical characterization and anti-proliferative effect mediated by p53

Background: Endogenous phospholipases A2 (PLA2 ) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM médium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.(AU)

Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp

Background: Scorpion neurotoxins such as those that modify the mammalian voltagegated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes ­ one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 ­ and another non-native toxin ­ named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions ­ were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.(AU)

Surto de botulismo tipo C em aves de subsistência em Santa Catarina, Brasil / Outbreak of type C botulism in backyard poultry in Santa Catarina, Brazil

Background: Botulism is a disease caused by the ingestion of neurotoxin produced by Clostridium botulinum, characterizedby flaccid paralysis, which can lead to high mortality. They have seven types of neurotoxins (A, B, C, D, E, F, and G) and,in birds, most cases are attributed to type C. They are considered sources of botulinum toxins where the decomposition oforganic matter occurs, like stagnant water and rotting food. The main feature of the disease in birds is ascending symmetricflaccid paralysis. The present study aims to describe an outbreak of type C botulism in backyard poultry in the state ofSanta Catarina, Southern Brazil.Case: A visit was made to the property with 160 backyard poultry with a history of high mortality in the municipality ofAgrolândia, Santa Catarina. Clinical signs were characterized by paralysis of the pelvic limbs, neck and pendular wings,which progressed to death within 48 h. There was a mortality rate of 37.5% (60/160) between March and May 2019. Thesebirds were kept in an overcrowded environment, with different species (chickens, ducks, teals, and turkeys) fed irregularly.The water supplied was provided from kitchen exhaust, accumulating in puddles on the floor that contained organic matterresidues such as animal feces, food waste and bone fragments. The disposal of the carcasses of birds that died was in thesame enclosure, buried superficially, facilitating the access of other birds to dig them up and consume them. Necropsywas performed on 2 chickens and one duck, no macroscopic or histopathological lesions were observed. Blood, liver, andgastrointestinal content samples were sent for research and identification of botulinum toxin through the serum neutralization test in mice. The presence of type C botulinum toxin was confirmed in the liver chicken of one sampled animals.Discussion: The identification of type C botulism toxin enabled the characterization of the outbreak, which is...

Surto de botulismo tipo C em aves de subsistência em Santa Catarina, Brasil / Outbreak of type C botulism in backyard poultry in Santa Catarina, Brazil

Background: Botulism is a disease caused by the ingestion of neurotoxin produced by Clostridium botulinum, characterizedby flaccid paralysis, which can lead to high mortality. They have seven types of neurotoxins (A, B, C, D, E, F, and G) and,in birds, most cases are attributed to type C. They are considered sources of botulinum toxins where the decomposition oforganic matter occurs, like stagnant water and rotting food. The main feature of the disease in birds is ascending symmetricflaccid paralysis. The present study aims to describe an outbreak of type C botulism in backyard poultry in the state ofSanta Catarina, Southern Brazil.Case: A visit was made to the property with 160 backyard poultry with a history of high mortality in the municipality ofAgrolândia, Santa Catarina. Clinical signs were characterized by paralysis of the pelvic limbs, neck and pendular wings,which progressed to death within 48 h. There was a mortality rate of 37.5% (60/160) between March and May 2019. Thesebirds were kept in an overcrowded environment, with different species (chickens, ducks, teals, and turkeys) fed irregularly.The water supplied was provided from kitchen exhaust, accumulating in puddles on the floor that contained organic matterresidues such as animal feces, food waste and bone fragments. The disposal of the carcasses of birds that died was in thesame enclosure, buried superficially, facilitating the access of other birds to dig them up and consume them. Necropsywas performed on 2 chickens and one duck, no macroscopic or histopathological lesions were observed. Blood, liver, andgastrointestinal content samples were sent for research and identification of botulinum toxin through the serum neutralization test in mice. The presence of type C botulinum toxin was confirmed in the liver chicken of one sampled animals.Discussion: The identification of type C botulism toxin enabled the characterization of the outbreak, which is...(AU)

Doramectin intoxication in malnourished 15-month-old cattle / Intoxicação por doramectina em bovinos malnutridos de 15 meses de idade

Macrocyclic lactones are widely used as endectocides in farm animals. Intoxications occur in situations of overdose and/or malnutrition, in young animals, and in genetically sensitive breeds. We describe the intoxication by doramectin in malnourished 15-month-old cattle that received 1.6 times the recommended dose. The animals presented salivation, ataxia, motor incoordination, reluctance to move, and sternal recumbency. Two animals recovered spontaneously; one died and was necropsied. No gross or microscopic changes were observed. This study suggests that doramectin may cause intoxication when administered to malnourished cattle in doses higher than those recommended and that knowing the history is essential to establish a diagnosis.

As lactonas macrocíciclas são amplamente utilizadas como endectocidas em animais de produção. Casos de intoxicação ocorrem em situações de sobredosagem e/ou desnutrição, em animais jovens ou em raças geneticamente suscetíveis. Descreve-se a intoxicação por doramectina em bovinos desnutridos, com 15 meses de idade, que receberam uma dose 1,6 vezes maior que a dose recomendada. Os animais apresentaram salivação, ataxia, incoordenação motora, relutância em se movimentar e decúbito esternal. Dois animais se recuperaram espontaneamente; um morreu e foi necropsiado. Não foram observadas alterações macro e microscópicas. Esse relato sugere que a doramectina pode causar intoxicação em bovinos desnutridos quando administrada em doses maiores que as recomendadas e que o histórico é fundamental para estabelecer o diagnóstico.

Type C botulism in dogs from rural properties located in Goiânia, Brazil

Botulism is a disease usually fatal, caused by the ingestion of neurotoxins produced by Clostridium botulinum. In dogs, intoxication is caused by the ingestion of botulinum toxin type C, and animals often recover spontaneously. The present study describes the occurrence of type C botulism in two dogs domiciled on neighboring rural properties in the municipality of Goiânia, state of Goiás, Brazil, probably associated with ingestion of decomposing bovine carcass. Upon clinical evaluation, the dogs were alert in the lateral decubitus position with ascending flaccid paralysis, absence of eyelid reflexes, and reduced muscle tone. Due to their worsening clinical symptoms, the animals died within 12 h and 3 days after supportive treatment. Botulinum toxin type C was identified, in the serum and feces of both dogs, by seroneutralization in mice with homologous monovalent antitoxin. The results of the high-throughput gene sequencing showed that the abundance of C. botulinum in the fecal microbiota of one of the affected dogs was low (0.53%). In this way, the present study highlights the need of sanitary practices related to the appropriate collection and disposal of bovine carcasses in rural areas since they represent a risk factor for the occurrence of botulism in dogs domiciled on rural properties.

Outbreak of organophosphorus compound-induced delayed neurotoxicity in water buffaloes / Surto de neuropatia tardia induzida por organofosforados em búfalos

Forty 1-2-y-old water buffaloes were simultaneously treated with trichlorfon and chlorpyrifos products in the recommended dose for cattle. After a week, 19 animals started presenting clinical signs characterized by apathy, diarrhea, aggressiveness, dehydration, and motor incoordination, followed by flaccid paralysis and permanent lateral recumbency. All affected buffaloes died after a clinical course of 1-4 days. Reduction of serum cholinesterase activity in three cases was indicative of significant exposure to organophosphorus compounds (OPs). Pathological examination of three buffaloes revealed no gross and histological lesions. By thin layer chromatography, chlorpyrifos residues and trace of trichlorfon residues were detected in fresh tissue samples. The epidemiological, clinical, pathological, and toxicological findings were highly compatible with OPs-induced delayed neurotoxicity, a neurological manifestation rarely described in domestic animals.

Quarenta búfalos foram simultaneamente tratados com clorpirifós e triclorfom na dose recomendada para bovinos. Após uma semana, 19 animais apresentaram sinais clínicos caracterizados por apatia, diarreia, agressividade, desidratação e incoordenação motora, seguidos por paralisia flácida e decúbito lateral permanente. Todos os búfalos afetados morreram após um curso clínico de 1-4 dias. Redução da atividade da colinesterase sérica em três casos foi indicativa de exposição significativa a organofosforados (OPs). O exame patológico de três búfalos não revelou lesões macroscópicas e histológicas. Por cromatografia em camada delgada, resíduos de clorpirifós e traços de resíduos de triclorfon foram detectados em amostras de tecidos frescos. Os achados epidemiológicos, clínicos, patológicos e toxicológicos foram compatíveis com neuropatia tardia induzida por OPs, uma manifestação neurológica raramente descrita em animais domésticos.

Outbreak of organophosphorus compound-induced delayed neurotoxicity in water buffaloes / Surto de neuropatia tardia induzida por organofosforados em búfalos

Forty 1-2-y-old water buffaloes were simultaneously treated with trichlorfon and chlorpyrifos products in the recommended dose for cattle. After a week, 19 animals started presenting clinical signs characterized by apathy, diarrhea, aggressiveness, dehydration, and motor incoordination, followed by flaccid paralysis and permanent lateral recumbency. All affected buffaloes died after a clinical course of 1-4 days. Reduction of serum cholinesterase activity in three cases was indicative of significant exposure to organophosphorus compounds (OPs). Pathological examination of three buffaloes revealed no gross and histological lesions. By thin layer chromatography, chlorpyrifos residues and trace of trichlorfon residues were detected in fresh tissue samples. The epidemiological, clinical, pathological, and toxicological findings were highly compatible with OPs-induced delayed neurotoxicity, a neurological manifestation rarely described in domestic animals.(AU)

Quarenta búfalos foram simultaneamente tratados com clorpirifós e triclorfom na dose recomendada para bovinos. Após uma semana, 19 animais apresentaram sinais clínicos caracterizados por apatia, diarreia, agressividade, desidratação e incoordenação motora, seguidos por paralisia flácida e decúbito lateral permanente. Todos os búfalos afetados morreram após um curso clínico de 1-4 dias. Redução da atividade da colinesterase sérica em três casos foi indicativa de exposição significativa a organofosforados (OPs). O exame patológico de três búfalos não revelou lesões macroscópicas e histológicas. Por cromatografia em camada delgada, resíduos de clorpirifós e traços de resíduos de triclorfon foram detectados em amostras de tecidos frescos. Os achados epidemiológicos, clínicos, patológicos e toxicológicos foram compatíveis com neuropatia tardia induzida por OPs, uma manifestação neurológica raramente descrita em animais domésticos.(AU)

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species. Methods: In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail. Results: The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned. Conclusion: Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.(AU)

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species. Methods: In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail. Results: The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned. Conclusion: Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.(AU)

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)

Analgesic effects of Phα1ß toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Analgesic effects of Phalfa1 beta toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

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)

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)

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. Methods Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. Conclusion Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.(AU)

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. Methods Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. Conclusion Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.(AU)

Analgesic effects of Ph1 toxin: a review of mechanisms of action involving pain pathways

Abstract Ph1 is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Ph1 to treat chronic pain reverted opioid tolerance with a safer profile than -conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Ph1 (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Ph1 antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.