Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Targeted innovative design of Bt Cry toxin insecticidal mimics / 生物工程学报

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Therapeutic effect of Panax notoginseng saponins combined with cyclophosphamide in mice bearing hepatocellular carcinoma H22 cell xenograft / 南方医科大学学报

OBJECTIVE@#To investigate the therapeutic effects of total saponins from Panax notognseng (PNS) combined with cyclophosphamide (CTX) in mice bearing hepatocellular carcinoma H22 cell xenograft.@*METHODS@#We examined the effects of treatment with different concentrations of PNS on H22 cell proliferation for 24 to 72 h in vitro using CCK8 colorimetric assay. Annexin V/PI double fluorescence staining was used to detect the effect of PNS on apoptosis of H22 cells. Mouse models bearing H22 cell xenograft were established and treated with CTX (25 mg/kg), PNS (120, 240 or 480 mg/kg), alone or in combinations. After treatments for consecutive 10 days, the mice were euthanized for examinations of carbon clearance ability of the monocytes and macrophages, splenic lymphocyte proliferation, tumor necrosis factor (TNF-α), interleukin-2 (IL-2), serum hemolysin antibody level, blood indicators, and the tumor inhibition rate.@*RESULTS@#Treatment with PNS concentration-dependently inhibited the proliferation and significantly promoted apoptosis of cultured H22 cells (P < 0.01). In the tumor-bearing mouse models, PNS alone and its combination with CTX both resulted in obvious enhancement of phagocytosis of the monocyte-macrophages, stimulated the proliferation of splenic lymphocytes, promoted the release of TNF-α and IL-2 and the production of serum hemolysin antibody, and increased the number of white blood cells, red blood cells and lymphocytes in the peripheral blood. Treatment with 480 mg/kg PNS combined with CTX resulted in a tumor inhibition rate of 83.28% (P < 0.01) and a life prolonging rate of 131.25% in the mouse models (P < 0.05).@*CONCLUSION@#PNS alone or in combination with CTX can improve the immunity and tumor inhibition rate and prolong the survival time of H22 tumor-bearing mice.

Advances in receptor-mediated resistance mechanisms of Lepidopteran insects to Bacillus thuringiensis toxin / 生物工程学报

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.

Detección de genes que codifican hemolisinas en cepas de Staphylococcus aureus aisladas en pantallas de teléfonos móviles de estudiantes de último año de odontología en Cuenca-Ecuador, 2020-2021 / Detection of hemolysin-encoding genes in Staphylococcus aureus strains isolated from cell phone screens of senior dental students in Cuenca-Ecuador, 2020-2021

Staphylococcus aureus es la especie más común implicada en las enfermedades infecciosas que causan morbilidad y mortalidad a nivel mundial. Posee los genes hla, hlb, hld, hlg, hlg-v que codifican para hemolisinas. Las hemolisinas son reconocidas como un factor de virulencia potencial que ataca a la membrana y produce destrucción de las plaquetas y necrosis. Tienen la capacidad de sobrevivir por largos periodos en superficies inertes como en pantallas de teléfonos móviles. Estudio observacional de tipo transversal descriptivo. Se aislaron en un estudio previo 16 cepas de Staphylococcus aureus a partir de 92 muestras de pantallas de teléfonos móviles de estudiantes de odontología. Se utilizó la técnica de reacción en cadena de la polimerasa para detectar los genes que codifican para hemolisinas. El gen hla se detectó en 75% (12/16) de cepas de Staphylococcus aureus, hlb en 25% (4/16), hld 75% (12/16), hlg 75% (12/16), hlg-v 13% (2/16). Este estudio evidencia el alto porcentaje de cepas virulentas que poseen genes que codifican para hemolisinas en pantallas de teléfonos móviles, lo que puede contribuir a la diseminación de este patógeno. Es imperioso implementar medidas para la desinfección de teléfonos móviles (AU)

Staphylococcus aureus is the most common species implicated in infectious diseases causing morbidity and mortality worldwide. It has the hla, hlb, hld, hlg, hlg-v genes encoding for hemolysins. Hemolysins are recognized as a potential virulence factor that attacks the membrane and causes platelet destruction and necrosis. They have the ability to survive for long periods on inert surfaces such as cell phone screens. Observational descriptive cross-sectional study. Sixteen strains of Staphylococcus aureus were isolated in a previous study from 92 samples of cell phone screens of dental students; the polymerase chain reaction technique was used to detect genes coding for hemolysins. The hla gene was detected in 75% (12/16) of Staphylococcus aureus strains, hlb in 25% (4/16), hld 75% (12/16), hlg 75% (12/16), hlg-v 13 % (2/16). This study evidences the high percentage of virulent strains that possess genes encoding for hemolysins in cell phone screens, which may contribute to the dissemination of this pathogen. It is imperative to implement measures for the disinfection of cell phones (AU)

Efficiency of biological control for fall armyworm resistant to the protein Cry1F / Eficiência do controle biológico da lagarta-do-cartucho resistente à proteína Cry1F

Abstract Understanding the ecological and toxicological relationship between genetically modified cultivars (GM) and biological control agents is of great importance for discussions related to the compatability of GM cultivars and integrated management strategies for pest resistance. The present study evaluated the search behavior and predatory capacity of Orius insidiosus (Say) (Hemiptera: Anthocoridae) and Doru luteipes (Scudder) (Dermaptera: Forficulidae) on eggs and caterpillars of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistant or not to the protein Cry1F expressed in Bt corn. To determine the search time, a stopwatch was run until the capture of the first prey, predation capacity was evaluated by counting the prey remaining after 24 hours of infestation. The injuries of S. frugiperda in genetically modified and conventional corn in the presence and absence of predators was also evaluated. The predators were not able to distinguish between resistant and susceptible prey (eggs or caterpillars), given the predatory behaviour observed. There was no difference in searching time or predatory capacity between the predators for eggs and caterpillars of either resistant or susceptible S. frugiperda. In the presence of predators, the injury scores for resistant S. frugiperda on the Bt corn plants were lower. It was concluded that O. insidiosus and D. luteipes did not notice the presence of the protein Cry1F in the prey S. frugiperda, which may facilitate the combined use of GM corn and biological control in integrated management programs and for management of pest resistance.

Resumo O entendimento de relações ecológicas e toxicológicas envolvendo culturas geneticamente modificadas (GM) e agentes de controle biológico é de grande importância para discussões relativas à compatibilidade de culturas GM com estratégias de manejo integrado e manejo de resistência de pragas. Este trabalho avaliou o comportamento de busca e a capacidade predatória de Orius insidiosus (Say) (Hemiptera: Anthocoridae) e Doru luteipes (Scudder) (Dermaptera: Forficulidae) sobre ovos e lagartas de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistente ou não à proteína Cry1F expressa em milho Bt. Para determinar o tempo de busca foi utilizado um cronômetro que foi disparado até a captura da primeira presa; a capacidade de predação foi avaliada através da contagem das presas remanescentes 24 h após infestação. Também foram avaliadas as injúrias de S. frugiperda em milho transgênico e milho convencional na presença ou ausência dos predadores. Os predadores não foram capazes de distinguir entre presas (ovos ou lagartas) resistentes e suscetíveis, considerando os comportamentos predatórios avaliados. Não houve diferença no tempo de busca e capacidade predatória sobre ovos e lagartas de S. frugiperda resistente ou suscetível entre os predadores. Na presença dos predadores, as notas de injúria de S. frugiperda resistente nas plantas de milho Bt foram menores. Conclui-se que O. insidiosus e D. luteipes não percebem a presença da proteína Cry1F na presa S. frugiperda, o que pode contribuir para o uso integrado de milho GM e controle biológico em programas de manejo integrado e manejo de resistência de pragas.

Behavior and development of Tetranychus ludeni Zacher, 1913 (Acari: Tetranychidae) and physiological stress in genetically modified cotton expressing Cry1F and Cry1Ac proteins / Comportamento e desenvolvimento de Tetranychus ludeni Zacher, 1913 (Acari: Tetranychidae) e estresse fisiológico em algodoeiro geneticamente modificado expressando as proteínas Cry1F e Cry1Ac

Genetically modified plants are one of the tactics used in integrated pest management - IPM. There is great concern about the impact of these plants on non-target organisms. On the other hand, there is little information in the literature on the effects of transgenics (Bacillus thuringiensis) Bt on populations of phytophagous mites, and the physiological responses that this attack promotes on plants. The objective of this work was to evaluate the biology of the T. ludeni mite in Bt cotton, expressing the Cry1F and Cry1Ac proteins. To evaluate the behavior of food and oviposition preference of the T. ludeni with Bt cotton and isohybrid. Verify if the physiological stress caused by T. ludeni's attack is differentiated in Bt cotton. The mites were reared in Bt cotton and isohybrid, in a total of 40 replicates in the completely randomized design and the biological cycle was evaluated. The food preference and oviposition analysis were done with 10 replicates, with choice. The physiological stress was evaluated through chlorophyll fluorescence, under greenhouse conditions. The data of the T. ludeni biology were analyzed by Student's t-test, for food and oviposition preference the chi-square test was performed. Regression models were fitted for the fluorescence parameters. The model identity test was used to evaluate the differences between Bt and isohybrid treatments. Cry1F and Cry1Ac proteins have not affected the biology of T. ludeni. The photosynthetic parameters in Bt cotton plants were less influenced by T. ludeni infestation.

O uso de plantas geneticamente modificadas é uma das táticas utilizadas no manejo integrado de pragas - MIP. Observa-se grande preocupação com o impacto dessas plantas sobre organismos não alvos. Por outro lado, existe pouca informação na literatura sobre efeitos dos transgênicos (Bacillus thuringiensis) Bt em populações de ácaros fitófagos, e as respostas fisiológicas que esse ataque promove nas plantas. Objetivou-se com esse trabalho avaliar a biologia do ácaro T. ludeni em algodoeiro Bt, expressando as proteínas Cry1F e Cry1Ac. Avaliar se há comportamento de preferência alimentar e postura de T. ludeni em relação ao algodoeiro Bt e seu iso-híbrido. E verificar se o estresse fisiológico causado pelo ataque de T. ludeni é diferenciado em algodoeiro Bt. Os ácaros foram criados em algodoeiro Bt e iso-híbrido, em um total de 40 repetições no delineamento inteiramente casualizado, onde foi avaliado o ciclo biológico. A análise de preferência alimentar e de posturas foi feita com 10 repetições, com escolha. O estresse fisiológico foi avaliando através da fluorescência da clorofila, em casa de vegetação. Os dados da biologia de T. ludeni foram analisados pelo teste t Student, para preferência alimentar e postura foi realizado o teste qui-quadrado. Para os parâmetros da fluorescência, foram ajustados modelos de regressão. Para testar as diferenças entre Bt e iso-híbrido foi utilizado o teste de identidade de modelos. As proteínas Cry1F e Cry1Ac não afetaram a biologia de T. ludeni. Os parâmetros fotossintéticos em plantas de algodoeiro Bt foram menos influenciados pela infestação de T. ludeni.

Titulación de hemolisinas en concentrados plaquetarios obtenidos a partir de buffy coat de donantes voluntarios de sangre de grupo O Rh D positivo / Hemolysin titration in platelet concentrates obtained from buffy coat of voluntary blood donors of group O Rh D positive

Los posibles efectos adversos que se producen en transfusiones incompatibles ABO son un riesgo latente en el uso de concentrados de plaquetas grupo O, por lo que la titulación de hemolisinas anti-A/B constituye una de las estrategias para su prevención. El objetivo de este estudio fue determinar la frecuencia de títulos de hemolisinas de isotipos IgG e IgM anti-A/B en donantes de sangre. Se trató de un estudio descriptivo, transversal y aleatorio simple con un tamaño muestral de 308 muestras. Se aplicó la metodología en tubo, gel salino y anti-inmunoglobulina IgG y, mediante soluciones seriadas, se evidenció el título. Adicionalmente, se realizó una encuesta sobre los posibles factores de riesgo para el aumento de estos títulos. Se aplicó estadística descriptiva mediante el uso del software informático SPSS versión 22.0 y la relación entre variables independientes a través del análisis estadístico de Chi-cuadrado y, para establecer la concordancia de las lecturas visuales de las tarjetas de gel, se aplicó el índice kappa. Se determinó la existencia de hemolisinas de isotipo IgG e IgM anti-A/B de títulos superiores a 1/64. Existió una relación estadísticamente significativa entre embarazos y títulos de IgG anti-A/B >1/128 y el aumento de hemolisinas de isotipo IgM y la ingesta de probióticos. Los resultados demostraron la necesidad de implementar la titulación de hemolisinas previo a la transfusión de concentrados plaquetarios no isogrupo, por lo que se recomienda una investigación de riesgo-beneficio y el seguimiento de pacientes con transfusiones de concentrados plaquetarios incompatibles ABO.

The possible adverse effects that occur in incompatible ABO transfusions are a latent risk in the use of group O platelet concentrates, so the titration of anti-A/B hemolysins is one of the strategies for its prevention. The objective of this study was to determine the frequency of hemolysins titers IgG and IgM anti-A/B isotypes in blood donours. It was a simple randomized descriptive cross-sectional study with a sample size of 308 samples. The methodology was applied in tube, saline gel and anti-IgG anti-immunoglobulin and by means of serial solutions the title was verified. Additionally, a survey was conducted on the possible risk factors for the increase in securities. Descriptive statistics were used through the application of the SPSS version 22.0 software and the relationship between independent variables through the Chi-square statistical analysis and the kappa index was applied to match the visual readings of the gel cards. The existence of IgG and IgM anti-A/B isotype hemolysins of titers greater than 1/64 was determined. There was a statistically significant relationship between pregnancies and anti-A/B IgG titres>1/128; and the increase in IgM isotype hemolysins and probiotic intake. The results demonstrate the need to implement hemolysin titration prior to transfusion of non-isogroup platelet concentrates, so a risk-benefit investigation and follow-up of patients with transfusions of ABO incompatible platelet concentrates is recommended.

Os possíveis efeitos adversos que ocorrem em transfusões incompatíveis ABO são um risco latente no uso de concentrados de plaquetas do grupo O, portanto a titulação de hemolisinas anti-A/B é uma das estratégias para sua prevenção. O objetivo deste estudo foi determinar a frequência de títulos de hemolisinas de isotipos IgG e IgM anti-A/B em doadores de sangue. Trata-se de um estudo descritivo transversal aleatório simples, com tamanho de amostra de 308 amostras. A metodologia foi aplicada em tubo, gel salino e anti-imunoglobulina IgG e utilizando soluções em série, o título foi verificado. Além disso, foi realizada uma pesquisa sobre os possíveis fatores de risco para o aumento destes títulos. A estatística descritiva foi utilizada através da aplicação do software informático SPSS versão 22.0 e a relação entre variáveis independentes por meio da análise estatística do qui-quadrado e, para estabelecer a concordância com as leituras visuais dos cartões de gel, o índice kappa foi aplicado. Foi determinada a existência de hemolisinas de isotipo IgG e IgM anti-A/B de títulos maiores que 1/64. Existiu uma relação estatisticamente significante entre gestações e títulos de IgG anti-A/B>1/128; e o aumento de hemolisinas do isotipo IgM e a ingestão de probióticos. Os resultados demonstram a necessidade de implementar a titulação da hemolisina antes da transfusão de concentrados de plaquetas não isogrupo, por isso, recomenda-se uma investigação de risco-benefício e acompanhamento de pacientes com transfusões de concentrados de plaquetas incompatíveis com ABO.

ISA 61 VG adjuvant enhances protective immune response of Listeria monocytogenes inactivated vaccine / 生物工程学报

Listeria monocytogenes (Lm) is zoonotic pathogen that can cause listeriosis, and vaccine is one of the effective methods to prevent this pathogen infection. In this study, we developed a novel vaccine that is a mixture of inactivated bacteria and Montanide™ ISA 61 VG, a mineral oil adjuvant, and evaluated the safety and immune response characteristics of this vaccine. The mice immunized with the ISA 61 VG adjuvant had high safety, and it could induce significantly higher titer of anti-listeriolysin O (LLO) antibody and higher value of IgG2a/IgG1 ratio compared with the group without the adjuvant. In particular, it could provide 100% immune protection against lethal doses of Lm challenge in mice. In summary, ISA 61VG adjuvant significantly enhanced the ability of inactivated listeria vaccine to induce humoral and cellular immune responses, thereby enhanced the protective immune response in the host, and it is a potential vaccine candidate for the prevention of Lm infection in humans and animals.

Cepas chilenas de origen clínico de Vibrio cholerae no-O1, no-O139 portan los genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF del sistema de secreción T3SS2 presentes en una isla de patogenicidad / Chilean strains of clinical origin of non-O1, non-O139 Vibrio cholerae carry the genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF from secretion system T3SS2 present in an island of pathogenicity

Resumen Introducción. Los factores de virulencia de las cepas de Vibrio cholerae no-O1, no-O139 no son claramente conocidos. La cepa de origen septicémico NN1 Vibrio cholerae no-O1, no-O139 fue secuenciada previamente mediante la plataforma Illumina, detectándose en su genoma un fragmento de la isla de patogenicidad VPaI-7 de V. parahaemolyticus. Objetivo: detectar los genes de virulencia vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF en cepas chilenas clínicas de V. cholerae no-O1, no-O139. Material y Métodos: Un total de 9 cepas chilenas de origen clínico de Vibrio cholerae no-O1, no-O139 aisladas entre 2006-2012 fueron analizadas mediante ensayos de reacción de polimerasa en cadena (RPC, en inglés PCR) convencional para los genes de secreción tipo III codificados en dicha isla: vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF. Adicionalmente se determinó la presencia de los genes de virulencia hylA y rtxA. Además, se realizaron ensayos de repetitive element palindromic PCR (REP-PCR) y Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Resultados: la mayoría (6/9) de las cepas chilenas de V. cholerae no-O1, no-O139 contiene todos los genes de secreción tipo III vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF, codificados en una isla de patogenicidad. Además, el total de las cepas (9/9) contiene los genes de virulencia hylA y rtxA. Conclusión: Estos resultados sugieren fuertemente la posibilidad que dichas cepas posean un potencial de virulencia importante en seres humanos.

Backgound: The virulence factors of the Vibrio cholerae non-O1, non-O139 strains are not clearly known. The strain of septicemic origin NN1 Vibrio cholerae non-O1, non-O139 was sequenced previously by the Illumina platform. A fragment of the pathogenicity island VPaI-7 of V. parahaemolyticus was detected in its genome. Aim: To detect the virulence genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF in Chilean strains of V. cholerae non-O1, non-O139. Methods: A total of 9 Chilean strains of clinical origin of Vibrio cholerae non-O1, non-O139 isolated between 2006-2012 were analyzed by conventional PCR assays for type III secretion genes encoded on that island: vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF. Additionally, the presence of the virulence genes hylA and rtxA was determined. In addition, REP-PCR and ERIC-PCR assays were performed. Results: most (6/9) Chilean V. cholerae non-O1, non-O139 strains contain the type III secretion genes vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF, encoded in an island of pathogenicity. In addition, all (9/9) the strains contain the virulence genes hylA and rtxA. Conclusion: These results strongly suggest the possibility that those strains possess an important virulence potential in humans.

Owlet moths (Lepidoptera: Noctuoidea) associated with Bt and non- Bt soybean in the brazilian savanna / Noctuóides (Lepidoptera: Noctuoidea) associados a soja Bt e não-Bt no Cerrado brasileiro

Abstract The use of GMO expressing Bt toxin in soybean production has increased significantly in the last years in Brazil in order to manage the damage caused by lepidopteran pests. In this study, we compared the richness and abundance of owlet moths (Noctuoidea) associated with Bt and non-Bt soybean. We determined the temporal variations as a function of phenology, and correlated the population variations of the most common species with meteorological variables. The research was conducted at the experimental area of Embrapa Cerrados. The collection method used was differentiated being suppressive and absolute. A total of 13 species were collected, of which eight occurred on Bt soybeans. The most representative taxa were Chrysodeixis includens (72.87%), Anticarsia gemmatalis (18.17%) and Spodoptera spp (5.22%). The number of larvae belonging to species targeted by the Bt technology was 10 times lower on Bt than on non-Bt soybeans. Utetheisa ornatrix and Elaphria deltoides were recorded on soybean for the first time, observing larvae of both species in non-Bt soybean and those of U. ornatrix also in Bt soybean. Only A. gemmatalis larvae correlated (p <0.05) negatively with precipitation. This study provided field information on the abundance and species richness of owlet moths on non-Bt soybeans, associated with the effects of Bt soybean. When considering the different levels of infestation between cultivars as a criterion, larvae monitoring is of substantial importance in order to develop the lost control program.

Resumo O uso de OGM que expressam toxina Bt na produção de soja tem aumentado significativamente nos últimos anos no Brasil e são utilizados para conter os danos causados ​​pelos lepidópteros pragas. Neste estudo comparamos a riqueza e a abundância de Noctuoides (Noctuoidea) associados à soja Bt e não-Bt. Determinamos as variações temporais em função da fenologia e correlacionamos às variações populacionais das espécies mais comuns com variáveis ​​meteorológicas. A pesquisa foi conduzida na área experimental da Embrapa Cerrados. O método de coleta utilizado foi diferenciado sendo supressivo e absoluto. Um total de 13 espécies foram coletadas, das quais oito ocorreram em soja Bt. Os taxa mais representativos foram Chrysodeixis includens, Anticarsia gemmatalis e Spodoptera spp. O número de larvas pertencentes às espécies alvo da tecnologia Bt foram 10 vezes menores na soja Bt do que em soja não-Bt . Utetheisa ornatrix e Elaphria deltoides foram registradas na soja pela primeira vez, observando-se larvas de ambas espécies na soja não-Bt e as de U. ornatrix também na soja Bt. Somente as larvas de A. gemmatalis se correlacionaram (p <0,05) negativamente com a precipitação. Este estudo forneceu informações em campo sobre a abundância e riqueza de espécies na soja não- Bt, associada aos efeitos da soja Bt. A importância do monitoramento das lagartas é substancial, a fim de tomar a melhor decisão de controle, considerando-se os diferentes níveis de infestação entre cultivares como critério.

Preparation and purification of Cry1Ah protein candidate reference material / 生物工程学报

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.

Quercetina atenúa la virulencia de Staphylococcus aureus al disminuir la secreción de alfa toxina / Quercetin attenuates Staphylococcus aureus virulence by reducing alpha-toxin secretion

Alfa toxina, una proteína formadora de poros con actividad citotóxica, es uno de los principales factores de virulencia secretados por la mayoría de las cepas de Staphylococcus aureus. Se ha establecido la relevancia de esta proteína en la patogenia de la neumonía asociada a infecciones por S. aureus. Por lo tanto, la inhibición de la secreción de alfa toxina puede ser una alternativa en el control de las infecciones causadas por este microorganismo. En este trabajo mostramos que quercetina, un flavonoide de origen natural, inhibe de manera dosis dependiente la actividad hemolítica y disminuye la secreción de alfa toxina en sobrenadantes de cultivos de S. aureus sensible y resistente a meticilina. Además, quercetina previene de manera significativa el daño de células alveolares humanas cuando se co-cultivan con S. aureus. Nuestros datos sugieren que quercetina puede disminuir la virulencia de S. aureus al afectar la secreción de alfa toxina.

Alpha toxin, a pore-forming protein with cytotoxic activity, is one of the major virulence factors secreted by most strains of Staphylococcus aureus. The relevance of this protein in the pathogenesis of pneumonia associated with S. aureus infections has already been esta blished. Therefore, inhibiting alpha toxin secretion can be an alternative for controlling these infections. This study shows that quercetin, a naturally occurring flavonoid, inhibits hemolytic activity in a dose-dependent manner and reduces alpha toxin secretion in culture supernatants of methicillin-sensitive and methicillin-resistant S. aureus. Furthermore, quercetin significantly prevents damage to human alveolar cells when co-cultured with S. aureus. Our results suggest that quercetin can reduce S. aureus virulence by affecting alpha-toxin secretion.

Serogroup identification, phenotypic detection of hemolysis and extended spectrum beta-lactamases of Escherichia coli isolated from psittacine of illegal wildlife trade in Fortaleza, Brazil / Identificação de sorogrupo e detecção fenotípica de betalactamase de espectro estendido e produção de hemolisina em Escherichia coli isoladas de psitacídeos do tráfico de animais silvestres em Fortaleza-CE

This study aimed to identify serogroups of Escherichia coli important for human health in isolates from psittacine of illegal wildlife trade in Ceará State. In addition, hemolysis and production of Extended Spectrum Beta-Lactamases (ESBL) was assessed in the isolates. A total of 78 E. coli strains isolated from different Psittaciformes species from a wildlife rehabilitation center in Fortaleza, Brazil. The isolates used in this study were previously identified and stored. Serogroup identification was performed using polyvalent sera for EPEC (O55, O111, O119, O114, O125, O86, O126, O127, O128), EIEC (O136, O124) and EHEC (O157). ESBL detection was performed with double disk synergy method. For hemolysis detection, isolates were inoculated in blood agar base enriched with ovine blood. Only 31 (39.7%) isolates were seropositive and the most frequent were O127, O114, O128 and O111. There was no agglutination for serogroups O55, O124, O136 or O157. Considering both seropositive and seronegative isolates, 9 (11.5%) and 35 (44.9%) presented hemolysis and ESBL production, respectively. In conclusion, the investigated psittacine from illegal wildlife trade hosted ESBL-producing E. coli strains and some belong to important serogroups often linked to severe human infections.(AU)

Este trabalho teve como objetivo identificar sorogrupos de E. coli importantes para a saúde humana, oriundos de psitacídeos provenientes do tráfico no estado do Ceará, assim como detectar atividade hemolítica e produção de betalactamase de espectro estendido (ESBL). Foram testadas 78 cepas de Escherichia coli provenientes de psitaciformes do Centro de Triagem de Animais Silvestres, Fortaleza, CE. Para a identificação dos sorogrupos, utilizaram-se soros polivalentes EPEC (O55, O111, O119, O114, O125, O86, O126, O127, O128), EIEC (O136, O124) e EHEC (O157). Para detecção de ESBL, as cepas foram submetidas ao método de aproximação de disco e, para a detecção de hemolisina, foram plaqueadas em ágar sangue base enriquecido com sangue de carneiro. No geral, 31 (39,7%) das amostras foram soropositivas. Os sorogrupos mais frequentemente detectados foram O127, O114, O128 e O111. Não houve positividade para os sorogrupos O55, O124, O136 e O157. Considerando-se as amostras sororreagentes e não sororreagentes, observou-se que nove (11,5%) e 35 (44,9%) cepas de E. coli apresentaram produção de hemolisinas e de ESBL, respectivamente. Em conclusão, constatou-se que psitacídeos provenientes do tráfico de animais silvestres albergam cepas de E. coli produtoras de ESBL e providas de importantes sorogrupos implicados em graves infecções humanas.(AU)

Interaction of Bombyx mori aminopeptidase N and cadherin-like protein with Bacillus thuringiensis Cry1Ac toxin / 生物工程学报

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.

Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina / Diversidad y distribución de genes de toxinas insecticidas Lepidoptera-específicos en cepas de Bacillus thuringiensis de Argentina

A total of 268 Bacillus thuringiensis strains obtained from different sources of Argentina were analyzed to determine the diversity and distribution of the cryl, cry2, cry8, cry9 and vip3A genes encoding for lepidopteran-specific insecticidal proteins. Twin strains were excluded. Ten different profiles were detected among the 80 selected B. thuringiensis strains. Two of these profiles (cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (35/80), and cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (25/80)) pooled 75% of the strains. The existence of this low diversity is rare, since in most of the studied collections a great diversity of insecticidal toxin gene profiles has been described. In addition, the most frequently detected profile was also most frequently derived from soil (70%), stored product dust (59%) and spider webs (50%). In contrast, the cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa profiles were mainly detected in strains isolated from leaves (40%) and dead insect larvae (50%). Six of the identified insecticidal toxin gene profiles were discovered in strains isolated from stored product dust and leaves indicating higher diversity of profiles in these kinds of sources than in others. Some strains with high insecticidal activity against Epinotia aporema (Lepidoptera) larvae were identified, which is important to explore future microbial strategies for the control of this crop pest in the region.

Se analizaron 268 cepas de Bacillus thuringiensis obtenidas de diferentes fuentes de Argentina con el objeto de determinar la diversidad y distribución de genes cryl, cry2, cry8, cry9 y vip3A, que codifican proteínas insecticidas lepidóptero-específicas. Se excluyeron las cepas gemelas. Se detectaron solo diez perfiles diferentes entre los 80 B. thuringiensis seleccionados. Dos de estos perfiles, el cry1Aa, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (35/80) y el cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (25/80), comprendieron el 75% de las cepas seleccionadas. La existencia de esta baja diversidad es una rareza, ya que en la mayor parte de las colecciones estudiadas se ha descrito una gran diversidad de perfiles de genes de toxinas insecticidas. El perfil detectado con mayor frecuencia se obtuvo principalmente de cepas procedentes de suelo (el 70% de los de esa fuente lo tenían), también fue mayoritario entre los procedentes de polvo de producto almacenado (59%) y en los que procedían de telas de arana (50%). En cambio, el perfil cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa se detectó principalmente en las cepas aisladas de hojas (40%) y de larvas de insectos muertos (50%). Seis de los perfiles identificados fueron encontrados en cepas aisladas de polvo de producto almacenado y de hojas, lo que indica una mayor diversidad de perfiles en estas fuentes que en otras. Se identificaron algunas cepas con alta actividad insecticida contra larvas de Epinotia aporema (Lepidoptera), hallazgo importante para explorar en el futuro estrategias microbianas para el control de esta plaga en la región.

Characterization of Cry toxins from autochthonous Bacillus thuringiensis isolates from Mexico / Caracterización de toxinas Cry de aislados de Bacillus thuringiensis autóctonos de México

Abstract: Background: Chemical pesticides, widely used in agriculture and vector-borne disease control, have shown toxic effects on the environment and the people in contact with them. Bacillus thuringiensis is a widely used bacterium for alternative and safer control of insect pests. Its toxins are specific for insects but innocuous for mammals and may be used as powerful adjuvants when applied with vaccines. The objective of this work was to characterize some autochthonous B. thuringiensis strains, which could be used for the control of a local pest (Diatraea considerata Heinrich) that affects sugar cane crops in Sinaloa, Mexico. Also, to evaluate these strains as a source of Cry toxins, which may be used in the future as adjuvants for some vaccines. Methods: Eight strains from field-collected dead insects were isolated. These were microbiologically identified as B. thuringiensis and confirmed by amplification and sequencing of 16S rDNA. Bioassays were performed to evaluate their pathogenicity against D. considerata, and Cry toxins were identified by proteomic analyses. Results: An increased mortality among larvae infected with strain Bt-D was observed, and its toxin was identified as Cry1Ac. Conclusions: The observed data showed that the selected strain was pathogenic to D. considerata and seemed to produce Cry1Ac protein, which has been reported as an adjuvant in different types of immunization.

Resumen: Introducción: Los pesticidas químicos, ampliamente usados en agricultura y en el control de vectores transmisores de enfermedades, han mostrado efectos tóxicos sobre el medio ambiente y las personas expuestas a ellos. Bacillus thuringiensis es una bacteria ampliamente utilizada como una alternativa segura y eficaz en el control biológico de plagas agrícolas. Sus toxinas son específicas de insectos, pero inocuas para mamíferos, e incluso poseen gran potencial para ser usadas como adyuvantes en vacunas. El objetivo de este trabajo fue caracterizar cepas autóctonas de B. thuringiensis con efectividad contra el gusano barrenador (Diatraea considerata Heinrich) de la caña de azúcar en cultivos del estado de Sinaloa, México, y como fuente de proteínas Cry, con potencial de utilizarse como adyuvantes en vacunas. Métodos: Se lograron aislar ocho cepas a partir de insectos muertos en campos agrícolas, las cuales fueron identificadas microbiológicamente como B. thuringiensis, lo que se confirmó por amplificación y secuenciación del 16S rDNA. La efectividad de los aislados para el control del gusano barrenador fue evaluada mediante bioensayos y las toxinas Cry fueron identificadas por análisis proteómico. Resultados: Se observó una mortalidad elevada en las larvas infectadas con las cepas de estudio. Particularmente, la cepa Bt-D, de la cual el análisis molecular mostró que posee una toxina tipo Cry1Ac. Conclusiones: Los resultados mostraron que la cepa Bt-D posee un elevado potencial patogénico hacia D. considerata y produce la proteína Cry1Ac, de la cual existen reportes de su aplicación como adyuvante en diferentes formas de inmunización.

Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects

ABSTRACT In this study, the cry1Ab gene of previously characterized and Lepidoptera-, Diptera-, and Coleoptera-active Bacillus thuringiensis SY49-1 strain was cloned, expressed and individually tested on Ephestia kuehniella (Lepidoptera: Pyralidae) and Plodia interpunctella (Lepidoptera: Pyralidae) larvae. pET-cry1Ab plasmids were constructed by ligating the cry1Ab into pET28a (+) expression vector. Constructed plasmids were transferred to an Escherichia coli BL21 (DE3) strain rendered competent with CaCl2. Isopropyl β-D-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, ∼130 kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000 µg g-1 caused 40% and 64% mortality on P. interpunctella and E. kuehniella larvae, respectively. However, the mortality rates of Bt SY49-1 strains' spore-crystal mixture at the same dose were observed to be 70% on P. interpunctella and 90% on E. kuehniella larvae. The results indicated that cry1Ab may be considered as a good candidate in transgenic crop production and as an alternative biocontrol agent in controlling stored product moths.

Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.