Abstract Background: Scorpion neurotoxins such as those that modify the mammalian voltage-gated 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.

Resumen Este trabajo recoge la experiencia pedagógica de monumentación y medición de puntos a partir de la utilización del Sistema de Posicionamiento Global (GPS), aplicando el método estático con un equipo diferencial (DGPS), fue realizado en la Universidad Pedagógica Experimental Libertador - Instituto Pedagógico de Maturín, Venezuela. Esta actividad estuvo enmarcada en los contenidos programáticos de las asignaturas Cartografía y Fotogrametría, Ordenamiento Territorial e Introducción a los Sistemas de Información Geográfica. El propósito fue involucrar a los estudiantes en el uso de nuevas tecnologías de información geográfica, entre ellas el Sistema de Posicionamiento Global. Para efectuar la monumentación se contó con la colaboración de la empresa MARFRAN C.A. quien facilitó los equipos DGPS y el talento humano especializado como apoyo para la ejecución de la práctica en todos sus momentos, es decir, planificación, monumentación de puntos y medición, y fase de post-procesamiento de datos. La base utilizada para las mediciones la constituyó un punto CARSON de la Red Geodésica Venezolana (REGVEN), ubicado en el Aeropuerto de Maturín. Como resultados se obtuvieron coordenadas de tres puntos monumentados, medidos en el Datum SIRGAS-REGVEN, de orden "B", a través de la aplicación del software de post-proceso Astech Solution; estos puntos GPS servirán de apoyo para la ejecución de futuras prácticas, pero además para efectuar chequeo y/o verificación de equipos de medición empleados en Geodesía y Topografía. Igualmente, la experiencia de involucrar a los estudiantes con el uso de modernas tecnologías de información geográfica resultó ser significativa desde el punto de vista pedagógico.

Abstract This paper gathers the pedagogical experience of monumentation and measurement of points through the use of the Global Positioning System (GPS), applying the static method with differential equipment (DGPS), conducted at Universidad Pedagógica Experimental Libertador - Instituto Pedagógico de Maturín, Venezuela. This activity was included in the syllabus of the courses Cartography and Photogrammetry, Territorial Planning, and Introduction to Geographic Information Systems. The purpose was to involve students in the use of new geographic information technologies, including the Global Positioning System. For monumentation, we had the support of MARFRAN C.A., which provided the DGPS equipment and specialized human talent as support to carry out all stages of the activity, namely planning, monumentation of points and measurements, and post-data processing. The basis used for the measurements was a CARSON point from the Venezuelan Geodetic Network (REGVEN) located in the Maturín Airport. As a result, we obtained the monumented points, measured in the SIRGAS-REGVEN datum, "B" order, by applying the Astech Solution post-process software; these GPS points will serve as support for future practice, but also to perform check and/ or verification of the measurement equipment used in Geodesy and Topography. The experience of involving the students with the use of modern geographic information technologies was also significant from a pedagogical point of view.

Resumo Este trabalho, realizado na Universidade Pedagógica Experimental Libertador - Instituto Pedagógico de Maturin, Venezuela, coleta a experiência pedagógica de monumentação e medição de pontos a partir do uso de Sistema de Posicionamento Global (GPS), aplicando [MCAF2] o método estático com um dispositivo diferencial (DGPS). Esta atividade seguiu os conteúdos programáticos das disciplinas Cartografia e Fotogrametria, Ordenamento Territorial e Introdução aos Sistemas de Informação Geográfica. O propósito foi envolver os estudantes no uso de novas tecnologias de informação geográficas, entre elas o Sistema de Posicionamento Global. Para realizar a monumentação, tivemos a ajuda da empresa MARFRAN C.A, que contribuiu com os dispositivos DGPS e o talento humano especializado para a execução da prática em todos seus estágios, ou seja, planejamento, monumentação de pontos e mediação, assim como a fase de pós-processamento de dados. A base utilizada para as mediações foi construída por um ponto CARSON da Rede Geodésica Venezuelana (REGEVEN), localizado no Aeroporto de Maturin. Como resultados, obtivemos coordenadas de três pontos monumentados, medidos no Datum SIRGAS-REGEVEN, de ordem B, através da aplicação do software de pós-processo Astech Solution; esses pontos GPS servirão de apoio para a execução de futuras práticas, assim como para efetuar revisão ou verificação de dispositivos de medição utilizados em Geodésia e Topografia. Igualmente, a experiência de envolver os estudantes no uso de modernas tecnologias de informação geográfica foi significativa desde o ponto de vista pedagógico.

Abstract Background The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.

<title>Abstract</title><sec><title>Background:</title><p>The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.</p></sec><sec><title>Methods:</title><p>The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider <italic>Brachypelma albiceps.</italic>It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a⁺. Each vector was transfected into E. <italic>coli</italic> M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.</p></sec><sec><title>Results:</title><p>Both expression systems pQE40 and pET28a⁺ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under <italic>in vitro</italic>conditions. The <italic>in vitro</italic> folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.</p></sec><sec><title>Conclusions:</title><p>The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.</p></sec>