Horizontal gene transfer in silkworm, Bombyx mori

<p>Abstract</p> <p>Background</p> <p>The domesticated silkworm, <it>Bombyx mori</it>, is the model insect for the order Lepidoptera, has economically important values, and has gained some representative behavioral characteristics compared to its wild ancestor. The genome of <it>B. mori </it>has been fully sequenced while function analysis of <it>BmChi-h </it>and <it>BmSuc1 </it>genes revealed that horizontal gene transfer (HGT) maybe bestow a clear selective advantage to <it>B. mori</it>. However, the role of HGT in the evolutionary history of <it>B. mori </it>is largely unexplored. In this study, we compare the whole genome of <it>B. mori </it>with those of 382 prokaryotic and eukaryotic species to investigate the potential HGTs.</p> <p>Results</p> <p>Ten candidate HGT events were defined in <it>B. mori </it>by comprehensive sequence analysis using Maximum Likelihood and Bayesian method combining with EST checking. Phylogenetic analysis of the candidate HGT genes suggested that one HGT was plant-to- <it>B. mori </it>transfer while nine were bacteria-to- <it>B. mori </it>transfer. Furthermore, functional analysis based on expression, coexpression and related literature searching revealed that several HGT candidate genes have added important characters, such as resistance to pathogen, to <it>B. mori</it>.</p> <p>Conclusions</p> <p>Results from this study clearly demonstrated that HGTs play an important role in the evolution of <it>B. mori </it>although the number of HGT events in <it>B. mori </it>is in general smaller than those of microbes and other insects. In particular, interdomain HGTs in <it>B. mori </it>may give rise to functional, persistent, and possibly evolutionarily significant new genes.</p

Sphingomyelinase D Activity in Sicarius tropicus Venom : Toxic Potential and Clues to the Evolution of SMases D in the Sicariidae Family

The spider family Sicariidae includes three genera, Hexophthalma, Sicarius and Loxosceles. The three genera share a common characteristic in their venoms: the presence of Sphingomyelinases D (SMase D). SMases D are considered the toxins that cause the main pathological effects of the Loxosceles venom, that is, those responsible for the development of loxoscelism. Some studies have shown that Sicarius spiders have less or undetectable SMase D activity in their venoms, when compared to Hexophthalma. In contrast, our group has shown that Sicarius ornatus, a Brazilian species, has active SMase D and toxic potential to envenomation. However, few species of Sicarius have been characterized for their toxic potential. In order to contribute to a better understanding about the toxicity of Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from Sicarius tropicus and compare them with that from Loxosceles laeta, one of the most toxic Loxosceles venoms. We show here that S. tropicus venom presents active SMases D. However, regarding hemolysis development, it seems that these toxins in this species present different molecular mechanisms of action than that described for Loxosceles venoms, whereas it is similar to those present in bacteria containing SMase D. Besides, our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms' composition may play a role in the toxic potential of venoms from Sicarius species.Peer reviewe

Development of an Assay for Sphingomyelinase D Products in Venom from Loxosceles reclusa and Other Spiders

The venoms of Loxosceles reclusa, the brown recluse spider, and some other Loxosceles species cause dermonecrotic lesions—a dying of the flesh—and in severe cases, systemic injury and death. Since this type of injury results from a bite, diagnosis can be cryptic unless the culprit spider is available to be identified by an expert. The distribution of Loxosceles reclusa is also not well known, which further complicates diagnosis. These spiders are of additional interest because one of the agents in the venom is a sphingomyelinase D (SMase D) that cleaves the head group of sphingomyelin (SM) and causes intramolecular transphosphatidylation to produce ceramide 1,3-cyclic phosphate (Cer(1,3)P), a lipid that is not known to be present in humans otherwise. Therefore, a simple assay to analyze the presence of SMase D, by either activity assays or detection of its product Cer(1,3)P, could aid in identification of spider bites and possibly facilitate development of a treatment. A key reagent for such studies is Cer(1,3)P which is currently not commercially available, so we have developed a method for its synthesis and identified conditions for distinguishing Cer(1,3)P from the substrate SM and alternative hydrolysis products (ceramide-1-phosphate and ceramide) by thin-layer chromatography. The availability of this chemical and its synthesis procedure enables an assay of SMase D for better characterization of this important component of brown recluse spider venom (and possibly other organisms). Moreover, the assay could be used to search for enzymes that hydrolyze Cer(1,3)P, and such enzymes might prove useful in the development of a treatment for these wounds.Undergraduat

Role of \u3ci\u3eIxodes scapularis\u3c/i\u3e Sphingomyelinase-Like Protein (IsSMase) in Tick Pathogen Interactions

Arthropod-borne diseases are one of the major concerns throughout the world. Ixodes scapularis (hard tick) is one of the major vectors that is involved in arthropod-borne disease transmission. Langat virus (LGTV) is a model pathogen that is very similar to other medically important flaviviruses such as Tick-Borne Encephalitis virus (TBEV) and Powassan virus (POWV). Sphingomyelinase-like protein (IsSMase, a Sphingomyelinase D or SMase D, a venomous protein ortholog of spiders) is an enzyme present in ticks that helps to catalyze the hydrolysis of the sphingomyelin (cell membrane lipid) into phosphocholine and ceramide. The objective of our study is to delineate the role of IsSMase in exosome biogenesis upon LGTV infection in ticks. Our previous study showed that LGTV-infection enhanced the production and release of exosomes to mediate the transmission of flavi-viral proteins and infectious RNA genomes from the arthropod to the vertebrate host. Understanding the mechanism(s) of arthropod-borne flavivirus transmission via exosome biogenesis is very important. My MS thesis project explored the detailed role of IsSMase in tickborne viral replication and pathogenesis and provided molecular insights of viral modulated survival strategies in ticks. Our data, in specific, suggests an important role for IsSMase in regulating viral replication in ticks, and in general a mechanism for anti-viral pathways in medically important vectors

Obtención y caracterización de las proteínas recombinantes PLD y CP40, factores de virulencia de Corynebacterium pseudotuberculosis ovis con potencial inmunogénico

El objetivo del presente trabajo fue obtener y caracterizar las proteínas recombinantes PLD y CP40 de C. pseudotuberculosis de un aislado mexicano (2J-L), antígenos con potencial inmunogénico.La Linfadenitis caseosa es una enfermedad causada por Corynebacterium pseudotuberculosis biovar ovis, bacteria intracelular que provoca la formación de abscesos cutáneos y/o viscerales, y ocasiona importantes pérdidas económica en la producción de pequeños rumiantes. Existen vacunas comerciales; sin embargo, la protección conferida no ha sido eficaz para el control de la enfermedad. Actualmente el empleo de la tecnología del ADN recombinante ha permitido obtener proteínas con potencialidades inmunogénicas que podrían ser una alternativa para el desarrollo de nuevas vacunas, debido a esto el objetivo del presente trabajo fue obtener y caracterizar las proteínas recombinantes PLD y CP40 de C. pseudotuberculosis de un aislado mexicano (2J-L), antígenos con potencial inmunogénico. Los genes pld y cp40 se amplificaron mediante PCR, se clonaron en el plásmido pGEM-T Easy y se secuenciaron, para el análisis bioinformático. Las proteínas PLDr 2J-L y CP40r 2J-L se expresaron en cepas de E. coli BL21 (DE3) transformadas con los plásmidos pET28a-pld y pET28a-cp40. El análisis filogenético en base a la secuencia de los genes pld y cp40 permitió agrupar el aislado 2J-L junto a cepas del biovar ovis, presentando una mayor relación con la cepa de origen mexicano MEX29. La proteína PLDr 2J-L presentó un 100% de identidad con respecto a la secuencia de las proteínas de las cepas de origen mexicano incluidas en el estudio. La proteína CP40r 2J-L presentó un 100% de identidad con respecto a MEX1, MEX2, MEX9 y MEX29, mientras que un 99% con respecto a CP40 de MEX25 debido a una variación de (V/L) en la posición 247. La caracterización de ambas proteínas se realizó in silico con la determinación de las propiedades físico-químicas, composición y predicción de las estructuras secundarias y modelado en 3D. El potencial inmunogénico de las proteínas se determinó mediante la predicción de epítopos lineales y discontinuos de células B y epítopos de células T presentados en el contexto del MHC I. Por primera vez se presenta un mapeo in silico de epítopos de células B y T para PLD y CP40 a partir de un aislado mexicano. Todos los epítopos identificados fueron conservados en un 100%, por las proteínas PLD y CP40 de las cepas de origen mexicano MEX1, MEX2, MEX9, MEX25 y MEX29. El programa Vaxign identificó ambas proteínas como antigénicas con función de adhesión y VaxiJen clasificó ambas proteínas como antígenos inmunogénicos probables para un valor de corte de 0.4. Las proteínas PLD y CP40 de C. pseudotuberculosis aislado mexicano 2J-L, obtenidas por vía recombinante, constituyen una fuente de antígenos potencialmente inmunogénicos para el desarrollo de una vacuna o medios diagnósticos

Mapeamento estrutural do sítio ativo de fosfolipases D presente no veneno de aranha-marrom (Loxosceles intermedia) : características bioquímicas e biológicas

Orientador : Prof. Dr. Silvio Sanches VeigaCoorientador : Prof. Dr. Olga Meiri ChaimTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 25/02/2016Inclui referências : f. 33-34;53-61Área de concentração :Biologia Celular e MolecularResumo: Loxoscelismo é a denominação do quadro clínico provocado em pacientes picados por aranhas do gênero Loxosceles, caracterizado principalmente por causar lesão dermonecrótica com intensa resposta inflamatória no local da picada, e raramente ocorrem manifestações sistêmicas, como distúrbios renais e hematológicos. O veneno loxoscélico é um líquido cristalino e incolor composto essencialmente por proteínas e peptídeos entre 5 a 40kDa, contendo cerca de 30?g de proteínas. Sabe-se que as toxinas da família das fosfolipases-D são capazes de desencadear a maioria dos efeitos biológicos causados pelo envenenamento, tornando-se necessário o aprofundamento da ação completa desta toxina. Algumas isoformas de fosfolipases-D de veneno de aranha-marrom já foram identificadas e biologicamente caracterizadas. Após análise do transcriptoma da glândula produtora de veneno de Loxosceles intermedia, algumas prováveis novas isoformas desta toxina foram identificadas e, entre elas, uma se destacou por sua presença em proporção relevante. O objetivo deste trabalho foi a clonagem, expressão heteróloga e purificação desta nova isoforma de toxina dermonecrótica, de modo a permitir sua avaliação biológica por testes in vitro e in vivo. A partir de técnicas de biologia molecular, foi possível obter a sequência nucleotídica e, por sua vez, aminoacídica completas da LiRecDT7, relatando que esta possui uma substituição conservativa no resíduo de aminoácido Asp233'Glu233. Houve pouco rendimento na expressão da toxina recombinante na forma solúvel e o processo de purificação por cromatografia mostrou-se pouco eficiente. Os testes de atividade esfingomielinásica sugerem que esta toxina como enzima está ativa. Os resultados obtidos até o presente demonstram que esta toxina é uma nova isoforma de fosfolipase-D presente no veneno de L. intermedia. Ainda assim, o processo de expressão e purificação devem ser otimizados, para avaliá-la biologicamente, contribuindo para a compreensão da composição do veneno dessa espécie, além de ser mais uma alternativa de ferramenta com potencial de aplicação biotecnológica. Palavras-chave: aranha, Loxosceles intermedia, veneno, fosfolipase-DAbstract: Loxoscelism is the name of the clinical picture provoked in patients bitten by Loxosceles spiders. It is mainly characterized by dermonecrotic lesions with an intense inflammatory response at the site of the bite, and rarely systemic manifestations such as renal and hematological disorders. The venom produced by Loxosceles spiders is a crystalline, colorless liquid consisting mainly of proteins and peptides between 5 and 40kDa. It is known that the phospholipase-D family of toxins present in this venom is capable of causing most of the biological effects observed in accidents with the spiders. Some isoforms of phospholipases-D have been identified in the Loxosceles venom and biologically characterized. Transcriptome analysis of the venom gland of Loxosceles intermedia revealed some potential novel isoforms of this toxin and among them, one stood out for its presence in significant proportion. The aim of this work was the cloning, heterologous expression and purification of this new isoform of dermonecrotic toxin, named LiRecDT7, to allow their biological evaluation by in vitro and in vivo analysis. Using molecular biology techniques, it was possible to obtain the nucleotide sequence and, in turn, the complete amino acid sequence of LiRecDT7. Further analysis showed a conservative substitution at amino acid residue Glu233'Asp233. The expression of recombinant toxin in soluble form resulted in a low yield and the process of purification by chromatography proved to be inefficient. Nevertheless, preliminary assays that tested the sphingomyelinasic activity of LiRecDT7 suggest that this toxin is active as an enzyme. The results show that LiRecDT7 is a new isoform of phospholipase-D present in the venom of L. intermedia. Still, the expression and purification process must be optimized to allow further assessments. Thus, the results will help to expand the knowledge concerning the composition of the venom of this species, besides being an alternative tool with potential biotechnological applications. Key-words: spider, Loxosceles intermedia, venom, phospholipase-

Estudo dos peptídeos pertencentes à família ICK presentes no veneno das aranhas do gênero Loxosceles

Orientador : Prof. Dr. Silvio Sanches VeigaCoorientador : Profª. Drª. Olga Meiri ChaimTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 26/02/2015Inclui referências : f. 102-115Área de concentraçãoResumo: O conteúdo total do veneno das aranhas do gênero Loxosceles permanece ainda desconhecido, entretanto, muitos estudos têm mostrado que se constitui uma mistura complexa de compostos biologicamente ativos. Por meio de análises eletroforéticas, observa-se a predominância de moléculas de baixa massa molecular (3-45 kDa), enquanto moléculas de alta massa molecular são menos abundantes. Os venenos de aranhas estão funcionalmente relacionados à defesa contra predadores e também à paralisia e captura de presas, especialmente insetos. As aranhas desenvolveram um arsenal de moléculas inseticidas, resultando em uma biblioteca combinatória de peptídeos que tem sido aprimorada durante sua evolução. Comumente, tais peptídeos consistem em moléculas de cadeia única com massa molecular variando de 3 a 10 kDa, ricos em resíduos de cisteína, os quais estabelecem pontes dissulfeto intramoleculares características. Essas pontes se organizam em um motivo estrutural característico denominado "nó de cistina inibidor" ou ICK (Inhibitor Cystine Knot) e, por isso, os peptídeos que o contém são denominados peptídeo ICK ou notinas ("knottins"). Recentemente, análises do transcriptoma da glândula de veneno de L. intermedia (GREMSKI et al., 2010) revelaram ESTs com similaridade a peptídeos ICK previamente descritas como LiTx (De CASTRO et al., 2004). Sequências relacionadas à LiTx3, por exemplo, foram as mais abundantes no transcriptoma de L. intermedia, representando aproximadamente 13,9% de todas as ESTs obtidas e compreendendo 32% dos mRNAs codificantes de toxinas; as sequências relativas aos demais grupos de peptídeos ICK, LiTx1, LiTx2 e LiTx4 representaram 6,2%, 11,4% e 3,8% de todos os transcritos codificantes de toxinas, respectivamente. Devido a alta proporção de sequências codificantes para peptídeos ICK os objetivos deste trabalho foram o rastreamento de sequências codificantes de peptídeos ICK em outras duas aranhas do gênero (Loxosceles gaucho e Loxosceles laeta), bem como a obtenção de um peptídeo ICK semelhante à LiTx3 de forma recombinante em Pichia pastoris e sua caracterização biológica. A partir do RNA total extraído das glândulas de veneno de L. laeta e L. gaucho, procedeu-se o rastreamento de sequências relacionadas a peptídeos ICK; sequências codificantes para todos os grupos de peptídeos ICK já descritos (LiTx1-4) foram encontradas, algumas revelando sutis diferenças na sua estrutura primária, outras revelando divergências importantes como a não presença de sequências consenso para modificações pós-traducionais importantes para a atividade biológica dos mesmos. Quanto à produção do peptídeo recombinante, várias formas foram obtidas; entretanto, análises de SDS-PAGE e western blotting mostraram que os peptídeos obtidos ou não apresentavam a conformação nativa ou apresentavam glicosilação indesejada. A atividade biológica desses peptídeos foi testada in vivo em insetos e in vitro em cultura de células, contudo nenhum efeito tóxico pode ser comprovado. As sequências encontradas em L. laeta e L. gaucho representam potenciais moléculas a serem exploradas do ponto de vista biotecnológico, ao passo que o peptídeo recombinante estudado deve ser expresso em outros modelos com intuito de obtê-lo em conformação adequada e, assim, comprovar sua atividade biológica. Palavras-chave: Peptídeo ICK, notinas, Loxosceles, veneno, Pichia pastoris.Abstract: The whole content of Loxosceles spider venom still remains unknown, but several studies have shown that is a complex mixture of biologically active and inactive components. By eletrophoretic analysis, the predominance of low molecular mass molecules (3-45kDa) can be observed, while high molecular mass ones are less abundant. Spider venoms are functionally related to defense against predators as well as to paralyze and capture a natural prey, especially insects. Spiders had developed an arsenal of insecticidal molecules, resulting in a combinatorial peptide library of peptides that has been improved during evolution. Commonly, such peptides consist in single chain molecules ranging between 3- 10 kDa and are rich in cystein residues, which form intramolecular disulfide bridges. These bridges establish a structural motif "Inhibitor Cystine Knot" (ICK), then, these peptides are named ICK peptides or "knottins". Recently, a transcriptome analysis of L. intermedia venomous gland (GREMSKI et al., 2010) has revealed ESTs with similarity to ICK peptides previously described as LiTx (De CASTRO et al., 2004). LiTx3-related sequences were the most abundant in the L. intermedia transcriptome representing about 13.9% of all ESTs obtained and comprise 32% of toxin-encoding messengers; the sequences related to the other groups of ICK peptides, LiTx1, LiTx2 e LiTx4, represented 6,2%, 11,4% and 3,8% of all EST coding for toxins, respectively. Due to the high proportion of sequences encoding ICK peptides verified by the transcriptome analyses, the present study aimed screening ESTs related to these peptides in other two Loxosceles species (L. gaucho and L. laeta), as well as the obtainment of an ICK recombinant peptide with high similarity to LiTx3 in Pichia pastoris and its biological activity characterization. From total RNA purified from the venom glands of L. gaucho and L. laeta, it was performed the screening of ICK peptides sequences. ESTs coding for all groups of ICK peptides already described were found (LiTx1-4), some of them revealed subtle differences in their primary structures while others showed important divergences, for example, the absence of consensus sequences for posttranslational modifications that are essential for biological activities. The recombinant peptide was produced in different forms, however, SDS-PAGE and western blotting analyses indicated that they were not properly folded or presented unwanted glycosylation. The biological activity of the recombinant peptide was tested in insects microinjection's assays and in vitro cultivated cells, nevertheless no toxic effects were proven. The sequences identified form L. laeta and L. gaucho RNA represent potential molecules to be biotechnologically explored, whereas the studied recombinant peptide must be expressed in other heterologous expressions models in order to obtain it in the native conformation and, thus, verify its biological activity. Keywords: ICK peptides, knottins, Loxosceles, venom, toxins, Pichia pastoris. recombinant peptide was tested in insects microinjection's assays and in vitro cultivated cells, nevertheless no toxic effects were proven. The sequences identified form L. laeta and L. gaucho RNA represent potential molecules to be biotechnologically explored, whereas the studied recombinant peptide must be expressed in other heterologous expressions models in order to obtain it in the native conformation and, thus, verify its biological activity. Keywords: ICK peptides, knottins, Loxosceles, venom, toxins, Pichia pastoris

Actualidad y prospectiva de la investigación científica en el Centro Universitario Amecameca de la Universidad Autónoma del Estado de México

Con responsabilidad, se organizó un programa cuya finalidad fuera publicitar con transparencia dichos avances, a través de un esfuerzo de rendición de cuentas a la comunidad inmediata, la universitaria, y a la comunidad abierta, la sociedad que la principal referencia para tal efecto. El programa se concretiza a través del presente libro, conformado con una inspiración de investigación multidisciplinaria; sin embargo, para llegar a tal fin, el reto es realizar el proceso de búsqueda y generación de conocimiento transitando hacia la colaboración de los cuerpos académicos, que puedan construir nuevos conocimientos fortalecidos por la convergencia de diferentes campos del saber. En consecuencia, la primera etapa de esta estrategia es la publicidad de los trabajos investigativos ejercidos, para hacer un balance al día, pero también proyectar el futuro de cada campo y área del conocimiento. La organización explicativa está organizada por tres bloques representativos del quehacer en la generación de conocimiento del Centro Universitario, un primer bloque centra el interés en las humanidades, educación y sustentabilidad; el segundo bloque lo integra la reflexión científica sobre la construcción democrática, derechos humanos y equidad de género; en el tercer segmento se destina a la seguridad alimentaria, salud pública y sistemas agropecuarios. La actualidad de la investigación eleva la producción lograda y lo que en el momento se encuentra en construcción y los alcances que produce para la docencia, la investigación misma, y para la sociedad en general. La prospectiva es un área que todos los capítulos desarrollan con el propósito de delinear los alcances innovadores por andar en teoría, metodología e incluso en los saberes mismo