In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer.

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods.

MAIN CONCLUSION: The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10 weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2-ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 µg g-1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 µg g-1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis).

BACKGROUND: The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis. RESULTS: Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability. CONCLUSIONS: The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

Molecular approaches to improve the insecticidal activity of Bacillus thuringiensis Cry toxins.

Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme.

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 108 α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control.

Variant Cry1Ia toxins generated by DNA shuffling are active against sugarcane giant borer.

Sugarcane giant borer (Telchin licus licus) is a serious sugarcane pest in Americas whose endophytic lifestyle hampers effective chemical and biological controls. Therefore, development of alternative control methods is extremely important. Envisaging development of transgenic plants resistant to this pest, we investigated the effect of the Bacillus thuringiensis Cry protein Cry1Ia12synth (truncated protein lacking C-terminus with plant codon usage) and variants against T. l. licus. cry1Ia12synth gene was used to generate mutated variants, which were screened for toxicity toward T. l. licus. For that purpose, an innovative technique combining cry gene shuffling with phage-display was used to build a combinatorial library comprising 1.97x10(5) Cry1Ia12synth variants. Screening of this library for variants binding to T. l. licus Brush Border Midgut Vesicles led to the identification of hundreds of clones, out of which 30 were randomly chosen for toxicity testing. Bioassays revealed four variants exhibiting activity against T. l. licus as compared to the non-toxic Cry1Ia12synth. Eight single substitutions sites were found in these active variants. Based on theoretical molecular modelling, the probable implications of these mutations are discussed. Therefore, we have four genes encoding Cry1Ia12synth variants active against T. l. licus promising for future development of resistant transgenic sugarcane lines.

O f�rmaco 17 α -etlnilestradiol: seus poss�veis efeitos � sa�de humana e animal por exposi��es ambientais / The drug alpha etlnilestradiol 17: its possible effects to human and animal health by environmental exposures

Este trabalho contextualiza o f�rmaco 17 α-etinilestradiol como um interferente end�crino de grande potencial estrog�nico, ambientalmente persistente, biodisponivel por via oral ao ser humano, e de cont�nua entrada nos compartimentos ambientais (principalmente nos corpos h�dricos).Efeitos na sa�de humana e animal por exposi��es ambientais, t�cnicas de identifica��o e caracteriza��o laboratorial, e as concentra��es encontradas dessa subst�ncia em diversas matrizes aqu�ticas do mundo s�o explicitados,utilizando como ferramenta metodol�gica a revis�o da literatura cient�fica nacional e internacional sobre o tema .Este trabalho mostra estudos cient�ficos que relatam a presen�a do 17 α-etinilestradiol em concentra��es ambientais baixas (em torno de nanogramas por litro), mas que poderiam promover, na biota exposta, efeitos como indu��o de feminiza��o de esp�cies machos, altera��o da propor��o entre os sexos da prole, infertilidade e redu��o da fecundidade.Estas concentra��es relatadas n�o seriam capazes, isoladamente, de gerar efeitos na esp�cie humana, segundo pesquisas de avalia��o de risco a sa�de. Por�m, discute-se a contribui��o desse f�rmaco em uma exposi��o cr�nica e m�ltipla a diversos interferentes end�crinos estrog�nicos presentes na �gua e alimentos.

Determina��o de estrog�nios em afluentes da Lagoa de Araruama / Determination of estrogens in tributaries of Araruama Lake

Ao longo deste trabalho, foram promovidas, atrav�s de 2 campanhas em meses distintos (novembro e dezembro de 2012), determina��es de caracter�sticas f�sico-quimicas, de eutrofiza��o, e tamb�m das concentra��es de estrog�nios (por imunoensaio enzim�tico ou por GC-MS) em afluentes que desembocam na lagoa de Araruama-RJ. (...) Os resultados dos par�metros f�sico-qu�micos e de eutrofiza��o mostram que a qualidade das �guas dos afluentes na primeira campanha estava pior que na segunda campanha, com piores resultados para os pontos de coleta C (rio regam�, fortemente influenciado por efluente tratado da ETE de Araruama) e D (efluente tratado da ETE de Cabo Frio). Os resultados cromatogr�ficos para o analito 17-α etinilestradiol foram comparados com os obtidos por metodologia por imunoensaio ELISA monoclonal, cujo LQ � de (...) Para as amostras analisadas, n�o se obteve concentra��es acima do limite de quantifica��o para este analito em nenhum dos 2 m�todos. Entretanto, no m�todo cromatogr�fico, � relatado que, na primeira campanha, os pontos de coleta C e D tiveram concentra��es pouco acima do limite de identifica��o para estrona e (...) estradiol, no ponto C, e para os 3 estrog�nios no ponto D. Baseado nos resultados encontrados, existe a suspeita de que os estrog�nios possam estar produzindo efeitos reprodutivos � biota residente e que um poss�vel aumento populacional na regi�o de estudo (em virtude de um feriado ocorrido pouco antes da primeira campanha) possa estar envolvida na explica��o dos resultados distintos entre as campanhas, embora outros fatores desconhecidos ou ignorados que justifiquem esses achados n�o possam ser descartados. (AU)

Determinação de estrogênios em afluentes da Lagoa de Araruama / Determination of estrogens in tributaries of Araruama Lake

Ao longo deste trabalho, foram promovidas, através de 2 campanhas em meses distintos (novembro e dezembro de 2012), determinações de características físico-quimicas, de eutrofização, e também das concentrações de estrogênios (por imunoensaio enzimático ou por GC-MS) em afluentes que desembocam na lagoa de Araruama-RJ. (...) Os resultados dos parâmetros físico-químicos e de eutrofização mostram que a qualidade das águas dos afluentes na primeira campanha estava pior que na segunda campanha, com piores resultados para os pontos de coleta C (rio regamé, fortemente influenciado por efluente tratado da ETE de Araruama) e D (efluente tratado da ETE de Cabo Frio). Os resultados cromatográficos para o analito 17-α etinilestradiol foram comparados com os obtidos por metodologia por imunoensaio ELISA monoclonal, cujo LQ é de (...) Para as amostras analisadas, não se obteve concentrações acima do limite de quantificação para este analito em nenhum dos 2 métodos. Entretanto, no método cromatográfico, é relatado que, na primeira campanha, os pontos de coleta C e D tiveram concentrações pouco acima do limite de identificação para estrona e (...) estradiol, no ponto C, e para os 3 estrogênios no ponto D. Baseado nos resultados encontrados, existe a suspeita de que os estrogênios possam estar produzindo efeitos reprodutivos à biota residente e que um possível aumento populacional na região de estudo (em virtude de um feriado ocorrido pouco antes da primeira campanha) possa estar envolvida na explicação dos resultados distintos entre as campanhas, embora outros fatores desconhecidos ou ignorados que justifiquem esses achados não possam ser descartados.

O fármaco 17 α -etlnilestradiol: seus possíveis efeitos à saúde humana e animal por exposições ambientais / The drug alpha etlnilestradiol 17: its possible effects to human and animal health by environmental exposures

Este trabalho contextualiza o fármaco 17 α-etinilestradiol como um interferente endócrino de grande potencial estrogênico, ambientalmente persistente, biodisponivel por via oral ao ser humano, e de contínua entrada nos compartimentos ambientais (principalmente nos corpos hídricos).Efeitos na saúde humana e animal por exposições ambientais, técnicas de identificação e caracterização laboratorial, e as concentrações encontradas dessa substância em diversas matrizes aquáticas do mundo são explicitados,utilizando como ferramenta metodológica a revisão da literatura científica nacional e internacional sobre o tema .Este trabalho mostra estudos científicos que relatam a presença do 17 α-etinilestradiol em concentrações ambientais baixas (em torno de nanogramas por litro), mas que poderiam promover, na biota exposta, efeitos como indução de feminização de espécies machos, alteração da proporção entre os sexos da prole, infertilidade e redução da fecundidade.Estas concentrações relatadas não seriam capazes, isoladamente, de gerar efeitos na espécie humana, segundo pesquisas de avaliação de risco a saúde. Porém, discute-se a contribuição desse fármaco em uma exposição crônica e múltipla a diversos interferentes endócrinos estrogênicos presentes na água e alimentos.

Aterosclerose: fatores de risco e fatores de risco / Atherosclerosis: risk factors and risk factors

Desde o estudo de Framingham (1948) sabe-se que inúmeros fatores de risco precipitam o aparecimento da doença aterosclerótica e suas terríveis conseqüências. Os fatores de risco bem definidos estão presentes em somente 50 por cento dos casos de aterosclerose. Observou-se que em pessoas que sofrem isquemia ou infarto do miocárdio há um aumento significativo dos marcadores de inflamação, como, por exemplo, a velocidade de sedimentação das hemácias e a proteína C-reativa. Esse fato levantou a hipótese da existência de um agente exógeno, uma infecção crônica capaz de promover o desenvolvimento e(ou) a ruptura da placa ateromatosa. Muitos e variados agentes infecciosos têm sido indiciados, porém os mais estudados são a Chlamydia pneumoniae, o citomegalivírus, o Helicobacter pylori e diversas bactérias periodontais