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Background: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

Temporary passive immunity such as serotherapy against venoms requires the full knowledge of all venom's components. Here, four venoms from Moroccan common yellow scorpions belonging to Buthus occitanus, subspecies tunetanus , paris, malhommei, and mardochei , all collected in four different restricted areas, were analysed in deep. They were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and their molecular masse profile determined by off-line MALDI-TOF mass spectrometry. Characterisation of their main components was achieved by enzyme-linked immunosorbent assay (ELISA) using specific antisera against the major lethal scorpion toxins identified so far, i.e. voltage-gated sodium channels (Na v ) modulators α- and β-toxins, as well as diverse potassium channel pore blocker toxins. For fractions with identical RP-HPLC retention times, we observe that their relative quantities show large differences. Moreover, identical masses present simultaneously in the four venoms are infrequent. ELISAs show that the majority of the RP-HPLC compounds cross-react with the antiserum against the “α-like” toxin Bot I, which has been previously identified in the Algerian Buthus occitanus tunetanus venom. Moreover, minor fractions were recognised by the antiserum against the highly lethal “classical” α-toxin of reference AaH II from the Androctonus australis venom. As such, our results bring new sights for further improving scorpion venom serotherapy in Morocco.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A peçonha da aranha "armadeira" Phoneutria nigriventer possui diversos componentes biologicamente ativos, dentre eles a toxina PnTx4(6-1), um peptídeo composto por 48 resíduos de aminoácidos e massa molecular de 5,2 kDa. PnTx4(6-1), também denominada δ-ctenitoxin-Pn1a, foi inicialmente descrita como uma neurotoxina inseticida que se liga ao sítio 3 de canais para sódio em sinaptossomas de corda nervosa de barata. Quando injetada por via intracerebroventricular em camundongos (30 μg), PnTx4(6-1) não provocou toxicidade aparente, além disso, esta toxina não apresentou efeito em canais para sódio de músculo esquelético (rSKM1) e de cérebro (rBIIA) de ratos. Posteriormente demonstramos que PnTx4(6-1) possui efeito antinociceptivo frente a três modelos de dor: i) inflamatório, evocado por carragenina; ii) nociceptivo, evocado por prostaglandina E2 e iii) neuropático, evocado por constrição do nervo ciático. Utilizando-se antagonistas verificamos que o sistema canabinoide, via receptor CB1, e o sistema opioide, via receptores μ e δ, estão envolvidos no efeito antinociceptivo evocado por PnTx4(6-1). No presente trabalho baseado na sequencia de aminoácidos de PnTx4(6-1), propusemos e sintetizamos um peptídeo linear, de 13 resíduos de aminoácidos denominado Pep13, na tentativa de reproduzir ou aumentar os efeitos antinociceptivos da toxina nativa. Verificamos que, assim como a toxina nativa, Pep13 apresenta ação antinociceptiva quando administrado por via intratecal e este efeito, também, relaciona-se aos sistemas canabinoide e opioide. Além disto, quando o efeito periférico foi avaliado, por administração intraplantar, Pep13 foi capaz de reverter o limiar hiperalgésico evocado por prostaglandina E2. Relacionada, ainda, ao efeito antinociceptivo periférico foi observada a participação do sistema opioide, uma vez que o antagonista não específico (naloxona) foi capaz de prevenir parcialmente o efeito de Pep13. Com relação ao sistema canabinoide, com a utilização de antagonistas específicos, observou-se a participação dos receptores CB1. Diferentemente da inseto-toxina nativa, Pep13 não causou nenhum efeito aparente quando administrado em moscas. Em conclusão, o peptídeo sintético Pep13, cuja sequência possui 35 resíduos de aminoácidos a menos que a toxina nativa PnTx4(6-1), reproduz os efeitos antinociceptivos apresentados pela mesma, sem entretanto, apresentar toxicidade para insetos e podendo assim, se tornar um bom modelo para novos analgésicos. The venom of the “armed” spider Phoneutria nigriventer has several biologically active peptides, one of them the toxin PnTx4(6-1) is a peptide composed of 48 amino acid residues and molecular mass of 5.2 kDa. PnTx4(6-1), also named δ-ctenitoxin-Pn1a, was initially described as a insecticidal neurotoxin, that binds to site 3 of sodium channels, in nerve cord synaptosomes of cockroach. When intracerebral injected in (30 μg) in mice, PnTx4(6-1) caused no apparent toxicity, moreover this toxin did not affect sodium channels of skeletal muscle (rSKM1) and brain (rBIIa), both of rats. Subsequently, we demonstrated that PnTx4(6-1) has antinociceptive effect in three pain models: i) inflammatory, evoked by carrageenan; ii) nociceptive, evoked by prostaglandin E2 and iii) neuropathic, evoked by constriction of the sciatic nerve. Using diverse receptors antagonists, we verified that the cannabinoid system, via the CB1 receptor, and the opioid system, through the μ and δ receptors, are both involved in the antinociceptive effect of PnTx4(6-1). In the present work, based on PnTx4(6-1) amino acid sequence we proposed and synthesized a linear peptide, with 13 amino acid residues named Pep13, based on the amino acid sequence of PnTx4(6-1) in order to try to reproduce or increase the analgesic effect of the toxin, with a simpler molecule. We verified, similarly to the toxin, the peptide has antinociceptive activity, when intrathecally administered, and this effect is also related to the cannabinoid and opioid systems. In addition, when the peripheral effect was evaluated, by intraplantar admistration, Pep13 was able to reverse the hyperalgesic threshold evoked by prostaglandin E2. Still related to the peripheral antinociceptive effect, the participation of the opioid system was observed, since the nonspecific antagonist (naloxone) was able to partially prevent the effect of Pep13. Regarding the cannabinoid system, by using specific antagonists, the participation of CB1 receptors was observed. Differently from the native toxin, which has a high toxicity to insects, the peptide caused no apparent effect when administered in flies. In conclusion, the synthetic peptide Pep13, which has 35 amino acid residues less than the native toxin PnTx4(6-1), reproduces the antinociceptive effects presented therein, without presenting however insect toxicity and may become a good model for new analgesics.

PnTx4(6-1), henceforth renamed δ-Ctenitoxin-Pn1a (δ-CNTX-Pn1a), a peptide from Phoneutria nigriventer spider venom, initially described as an insect toxin, binds to site 3 of sodium channels in nerve cord synaptosomes and slows down sodium current inactivation in isolated axons in cockroaches (Periplaneta americana). δ-CNTX-Pn1a does not cause any apparent toxicity to mice, when intracerebroventricularly injected (30 μg). In this study, we evaluated the antinociceptive effect of δ-CNTX-Pn1a in three animal pain models and investigated its mechanism of action in acute pain. In the inflammatory pain model, induced by carrageenan, δ-CNTX-Pn1a restored the nociceptive threshold of rats, when intraplantarly injected, 2 h and 30 min after carrageenan administration. Concerning the neuropathic pain model, δ-CNTX-Pn1a, when intrathecally administered, reversed the hyperalgesia evoked by sciatic nerve constriction. In the acute pain model, induced by prostaglandin E2, intrathecal administration of δ-CNTX-Pn1a caused a dose-dependent antinociceptive effect. Using antagonists of the receptors, we showed that the antinociceptive effect of δ-CNTX-Pn1a involves both the cannabinoid system, through CB1 receptors, and the opioid system, through μ and δ receptors. Our data show, for the first time, that δ-Ctenitoxin-Pn1a is able to induce antinociception in inflammatory, neuropathic and acute pain models.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo A peçonha da aranha Phoneutria nigriventer, conhecida como “aranha armadeira”, possui diversos componentes biologicamente ativos. Entre os sintomas decorrentes da picada desse animal destacam-se dor local, excitação, salivação, lacrimejamento, priapismo, convulsão e paralisia flácida e espástica dos membros anteriores e posteriores. A toxina PnTx4(6-1) foi isolada da fração PhTx4 deste veneno, sendo um polipeptídeo de cadeia única composto por 48 resíduos de aminoácidos, cuja massa é de 5244,6 Da. PnTx4(6-1) foi inicialmente descrita como uma neurotoxina inseticida, que liga-se ao sítio 3 de canais para sódio em sinaptossomas de barata (Periplaneta americana). Entretanto, canais para sódio de músculo esquelético (rSKM1) e cérebro (rBIIA), ambos de ratos, não foram alvos da toxina PnTx4(6-1). Estes resultados corroboraram resultados anteriores em que a injeção intracerebral da toxina purificada (15 e 30 μg), em camundongos, não provocou toxicidade aparente. O presente estudo avaliou o efeito antinociceptivo da toxina PnTx4(6-1) frente a hiperalgesia evocada por carragenina, prostaglandina E2 e por constrição do nervo ciático. A atividade antihiperalgésica foi avaliada pelo teste de retirada da pata do rato submetida à compressão, descrito originalmente por Randall & Selitto, em 1957. A hiperalgesia no modelo de dor inflamatória foi causada pela injeção intraplantar de 250 μg de carragenina. Quando administrada no mesmo local, após intervalo de duas horas e trinta minutos, a toxina PnTx4(6-1) (5 μg) foi capaz de restaurar o limiar nociceptivo da pata dos animais. A hiperalgesia, no modelo de dor nociceptiva, foi evocada pela injeção intraplantar de 2 μg de prostaglandina E2. A administração central da toxina, por injeção intratecal, causou um efeito antinociceptivo dose-dependente. As doses de 200 e 400 pmoles reverteram totalmente o efeito hiperalgésico evocado por prostaglandina E2, tendo duração de 20 minutos. Medidas na pata contralateral revelaram que PnTx4(6-1), quando administrada pela via intratecal, não altera o limiar nociceptivo da pata não tratada. Com relação ao modelo de dor neuropática a toxina na dose de 100 pmoles reverteu, com duração de dez minutos, a hiperalgesia evocada por constrição do nervo ciático. A investigação das propriedades biológicas das toxinas animais e a perspectiva de sua utilização para o alívio da dor demonstram forte potencial terapêutico. The venom of the “armed” spider Phoneutria nigriventer has several biologically active components including peptides, proteins, amino acids, salts etc. The symptoms resulting from this animal’s bite comprise local pain, arousal, salivation, lacrimation, priapism, seizures and spastic and flaccid paralysis of the anterior and posterior limbs. The toxin PnTx4(6-1), isolated from the PhTx4 fraction of these venom, is a single chain polypeptide composed of 48 amino acid residues, with a mass of 5244.6 Da. This polypeptide was initially described as a neurotoxin insecticide. PnTx4(6-1) appears to bind to site 3 of sodium channels in nerve cord synaptosomes of cockroach (Periplaneta americana) and slow down inactivation of sodium current in isolated axon of insect, without affecting sodium channels of skeletal muscle (rSKM1) and brain (rBIIA), both of rats. This result corroborated previous assays in which the intracerebral injection of the toxin (up to 30μg) in mice caused no apparent toxicity. The present study evaluated the antinociceptive effect of the PnTx4(6-1) in three experimental pain models, using the test paw withdrawal, subjected to compression (Randall & Selitto, 1957). The hyperalgesia in inflammatory pain model was caused by intraplantar injection of 250 μg of carrageenan. When administered in the same place, after an interval of two hours and thirty minutes, PnTx4(6-1) (5 μg) was able to restore the nociceptive threshold of the animals. The hyperalgesia in nociceptive pain model, was evoked by intraplantar injection of 2 μg of prostaglandin E2 (PGE2). The central administration of PnTx4(6-1) by intrathecal injection caused a dose-dependent antinociceptive effect against the hyperalgesia induced by PGE2. Briefly, a dose of 100 pmoles was able to raise the nociceptive threshold of the paw of rats, and the doses of 200 and 400 pmoles totally reversed the hyperalgesic effect evoked by PGE2, with duration of 30 minutes. The measurements in the contralateral paw revealed that PnTx4(6-1), when centrally administered, does not alter the nociceptive threshold of the paw untreated. Concerning to the neuropathic pain model the toxin (100 pmoles) reversed, lasting ten minutes, the hyperalgesia evoked by constriction of the sciatic nerve. This research with PnTx4(6-1) indicates a strong therapeutic potential to this molecule as a possible model to new analgesic.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A resistência antimicrobiana é um dos grandes problemas mundiais de saúde pública onde se faz necessário um trabalho de conscientização do uso adequado dos antibióticos. Neste contexto, é pertinente a abordagem deste tema no ambiente escolar, sendo a escola o local de aproximação dos estudantes com o conhecimento científico e de formação de indivíduos responsáveis por suas ações. Essa temática foi abordada com os estudantes do 3º ano do Ensino Médio de uma escola pública de Minas Gerais por meio de uma sequência de ensino que utiliza a abordagem investigativa. O trabalho teve como objetivo avaliar se o uso do ensino por investigação possibilita que os alunos explorem a temática e se esse contribui para a construção do conhecimento científico sobre o tema. As aulas estão suspensas em todas as escolas de Minas Gerais, devido à pandemia da COVID-19. Portanto, a sequência de ensino deste trabalho foi aplicada em um ambiente virtual de aprendizagem, por meio da ferramenta Google Sala de Aula. Durante a aplicação da sequência foi possível observar que o acesso a internet é um fator limitador para a participação dos estudantes nas atividades desenvolvidas no ambiente virtual. Em contrapartida, os resultados obtidos demonstram que é possível desenvolver uma sequência de ensino investigativa, como por exemplo, sobre resistência bacteriana, nesse ambiente. Sendo assim, é fundamental o papel do professor em criar atividades com a abordagem investigativa, a fim de que permita a construção crítica do conhecimento pelo estudante. Antimicrobial resistance is one of the major global public health problems where awareness work on the appropriate use of antibiotics is necessary. In this context, it is pertinent to approach this theme in the school environment, with the school being the place where students get closer to scientific knowledge and to the training of individuals responsible for their actions. This theme was approached with the students of the 3rd year of high school in a public school in Minas Gerais through a teaching sequence that uses the investigative approach. This work aimed to assess whether the use of teaching by investigation allows students to explore the theme and whether it contributes to the construction of scientific knowledge on the subject. Classes are suspended in all schools in Minas Gerais, due to the COVID-19 pandemic. Therefore, the teaching sequence was applied in a virtual learning environment, using the Google Classroom tool. During the application of the sequence, it was possible to observe that internet access is a limiting factor for the participation of students in activities developed in the virtual environment. On the other hand, the results obtained demonstrate that it is possible to develop an investigative teaching sequence, as for example, on bacterial resistance, in this environment. Therefore, the role of the teacher in creating activities with an investigative approach is essential, in order to allow the critical construction of knowledge by the student.