Una vacuna de vesículas de membrana externa de Vibrio cholerae es aún una promesa: Caracterización proteómica / Outer membrane vesicle vaccine from Vibrio cholerae is still a promise: Proteomic characterization

Las epidemias de cólera afectan a un gran número de países africanos, asiáticos y del Caribe. Los cambios climatológicos y las constantes migraciones hacen que esta enfermedad se extienda, por lo que resulta necesario disponer de vacunas protectoras. En el presente trabajo se caracterizó una nueva vacuna de vesículas de membrana externa (VMEs) obtenidas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa cepa C7258, en el Instituto Finlay de vacunas (Cuba), a través de métodos proteómicos. Se identificaron 53 proteínas presentes en las VME (4 proteínas por banda electroforética) separadas por electroforesis unidimensional (1D) y digeridas con tripsina. Los fragmentos obtenidos fueron separados por cromatografía líquida de alta resolución (HPLC) acoplada a espectrometría de masa, secuenciados e identificados mediante bases de datos de proteínas Swiss-Prot y TrEMBL. El patrón proteico obtenido presentó algunas de las proteínas (12 proteínas citoplasmáticas y 5 proteínas de membrana externa) sugeridas dentro del proteoma de buena calidad para candidatos vacunales. Se estudiaron las mejores condiciones para la separación de las proteínas a través de electroforesis bidimensional. Las VME evaluadas cuentan con una composición fundamentada en proteínas necesarias para garantizar una respuesta inmune que proteja contra Vibrio cholerae O1 biotipo El Tor serotipo Ogawa.

Cholera epidemics affect a large number of African, Asian and Caribbean countries. The climate changes and the constant migrations cause this disease to spread, making it is necessary to obtain protective vaccines. In the present work, a new vaccine of outer membrane vesicles (OMV) from V. cholerae O1 El Tor biotype Ogawa serotype strain C7258 at Finlay Institute of vaccines (Cuba) was characterized by proteomic methods. A total of 53 proteins present in the OMV (approximate ratio of 4 proteins by electrophoresis band) were identified, separated by one dimension electrophoresis and digested by tripsin method. The fragments were separated by high performance liquid chromatography (HPLC) coupled to mass spectrometry, sequenced and identified, using Swiss-Prot and TrEMBL protein databases. The pattern showed some proteins (12 cytoplasmic proteins and 5 outer membrane proteins) suggested within the highest quality proteome for vaccine candidate. The best conditions for proteins separation by two dimension electrophoresis were studied. The OMV composition was based on proteins described to the immunity response and protection against V. cholerae O1 El Tor biotype Ogawa serotype.

As epidemias de cólera afetam um grande número de países africanos, asiáticos e caribenhos. As mudanças climáticas e as constantes migrações fazem com que esta doença se espalhe, portanto é necessário ter vacinas protectoras. No presente trabalho, uma nova vacina de vesículas de membrana externa (VMEs) obtidas de Vibrio cholerae 01 biotipo El Tor sorotipo Ogawa cepa C7258, no Instituto de Vacinas Finlay (Cuba), através de métodos proteômicos. Foram identificadas 53 proteínas presentes nas VME (4 proteínas por banda eletroforética) separadas por eletroforese unidimensional (1D) e digeridas com tripsina. Os fragmentos obtidos foram separados por cromatografia de alta resolução (HPLC) acoplada a espectrometria de massa, sequenciados e identificados usando bancos de dados de proteínas Swiss-Prot e TrEMBL. O padrão proteico obtido apresentou algumas das proteínas (12 proteínas citoplasmáticas e 5 proteínas de membrana externa) sugeridas dentro do proteoma de boa qualidade para candidatos vacinais. As melhores condições para a separação de proteínas através de eletroforese bidimensional foram estudadas. As VME avaliados possuem uma composição baseada em proteínas necessárias para garantir uma resposta imune que proteja contra Vibrio cholerae O1 biotipo El Tor sorotipo Ogawa.

Caracterización proteómica de vesículas de membrana externa extraídas de Shigella sonnei / Proteomic characterisation of outer membrane vesicle extracted from Shigella sonnei

Las vacunas compuestas por vesículas de membrana externa (VMEs) previenen de manera exitosa la enfermedad meningocócica del serogrupo B. Esta plataforma tecnológica de obtención puede ser aplicada para otros patógenos bacterianos Gram negativos. Una vacuna de VMEs desarrollada contra Shigella sonnei fue obtenida a través de una extracción de componentes celulares y su caracterización por electroforesis en geles de poliacrilamida unidimensional (1D). Se estudiaron las mejores condiciones de solubilización de las muestras, separación electroforética e identificación a través de espectrometría de masas acoplada a cromatografía de alta presión después del corte de las bandas y su tratamiento enzimático con tripsina. En esta etapa se identificaron un total de 57 proteínas en 23 bandas (2,5 proteínas por banda escindida), 47 de las proteínas no repetidas. Las proteínas inmunogénicas presentes en VMEs de S. sonnei fueron cuantificadas en cuanto a masa molecular por 1D-Western blotting en membranas de nitrocelulosa con anticuerpos obtenidos a partir de ratones inmunizados con las VMEs. Como que las bandas electroforéticas 1D contenían más de una proteína, se estudiaron las mejores condiciones de separación por el método de electroforesis bidimensional (2D) para el establecimiento del mapa proteico; tal que el incremento del tamaño de las tiras, el tiempo de focalización y la aplicación catódica garantizaron la mayor resolución(AU)

Outer membrane vesicles (OMVs) vaccines successfully prevent Group B meningococcal disease. This platform technology may be applied against other Gram negative bacterial pathogens. An OMV vaccine against Shigella sonnei was prepared by detergent extraction of cells and characterised by one-dimensional polyacrylamide gel electrophoresis (1D). The protein components were quantified by staining and scanning and the composition of bands were defined by coupled high-performance low chromatography/electrospray ionization tandem mass spectrometry after band excision and in-gel trypsin digestion. 57 proteins contained in 23 bands (2.5 proteins/split band) were detected, 47 of them were not repeated. The S. sonnei OMVs immunogenic proteins were identified by 1D-immunoblotting, after transfer of proteins to a nitrocellulose membrane and treatment with antibodies generated by immunisation of mice with the OMVs. The bands detected by 1D had more than a single proteins, that is why we studied the best conditions for molecular separation by two-dimension electrophoresis (2D) for establishing the protein map; such that the increase of strips size, time of isoelectric focusing (IEF) and cup-cathodic loading guaranteed the highest resolution(AU)

Using fish assemblages in different habitats to develop a management plan for the Upper Essequibo Conservation Concession, Guyana

The Upper Essequibo Conservation Concession is a reserve in central-eastern Guyana managed by Conservation International. The site is uninhabited by people and poorly studied. The first scientific fish survey was in 2007 in conjunction with the filming of the BBC nature documentary Lost Land of the Jaguar. Aquatic habitats were primarily flowing water, ranging from the main channel of the Essequibo River to small forest creeks. Ponds and seasonally flooded forests were uncommon. Large predatory fishes were abundant in the Essequibo River. Fishes tolerant of low oxygen levels were common in flooded forests and small forest creeks. There was zero similarity between the fish assemblages of the Essequibo River and flooded forests / small forest creeks. The rest of the habitats and fish assemblages formed a continuum between these extremes. Imminent threats to the Upper Essequibo Conservation Concession include logging, mining, and over-fishing. Because of the heterogeneous distribution of fish assemblages, and because each threat will differentially affect different habitats, a two-pronged approach focusing on the ends of the habitat / fish assemblage continuum should be implemented in order to conserve the entire fish biodiversity of the Upper Essequibo Conservation Concession.

La Upper Essequibo Conservation Concession es una reserva en Guyana centro-oriental, administrada por Conservation International. El sitio está deshabitada por personas y mal estudiado. El primer estudio científico de peces fue en 2007 junto con la filmación del BBC documental naturaleza Lost Land of the Jaguar. Hábitats acuáticos fueron el canal principal del Río Essequibo a quebradas pequeñas del bosque. Lagos pequeños y bosques inundados estacionalmente eran infrecuentes. Grandes peces depredadores abundaban en el Río Essequibo. Peces tolerantes con bajos niveles de oxígeno eran comunes en quebradas pequeñas del bosque y bosques inundados. Hubo cero similitud entre las comunidades de peces del Río Essequibo y bosques inundados / quebradas pequeñas del bosque. El resto de los hábitats y comunidades de peces forma un continuum entre estos extremos. Las amenazas inminentes a la Upper Essequibo Conservation Concession incluyen deforestación, la minería y sobrepesca. Debido a la distribución heterogénea de comunidades de peces y porque cada amenaza diferencialmente afectarán a diferentes hábitats, un enfoque doble centrándose en los extremos del hábitat / comunidades de peces continuum debe aplicarse con el fin de conservar la biodiversidad de peces entero de la Upper Essequibo Conservation Concession.

á-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an á-N-acetylglucosamine (á-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Vaccines based on the cell surface carbohydrates of pathogenic bacteria

Vacinas glicoconjugadas, cujo carboidrato da superfície de um microrganismo está covalentemente ligado a uma proteína carreadora, vêm sendo consideradas como efetivas para gerar respostas imunes que previnem um grande número de doenças. A tecnologia é genérica e aplicável a vários patógenos, se os anticorpos contra os carboidratos de superfície forem capazes de proteger contra a infecção. Três vacinas contra Haemophilus influenzae tipo b, Neissseria meningitidis Grupo C e sete sorotipos de Streptococcus pneumoniae já foram licenciadas e muitas outras estão em desenvolvimento. Este artigo discute o racional para o desenvolvimento e uso de vacinas glicoconjugadas; os mecanismos pelos quais elas induzem respostas imune dependentes de célula T e suas implicações para o seu desenvolvimento; o papel dos métodos físico-químicos na caracterização e no controle de qualidade dessas vacinas; e os produtos novos que estão em desenvolvimento.

Post-clipping cerebrovascular compliance in an infant with bilateral anterior circulation aneurysms

We present an uncommon case of an infant 12 months, presenting with a seizure due to subarachnoid hemorrhage secondary to an anterior circulation aneurysm. Following angiography, a right A1/A2 junction aneurysm was clipped by a right pterional approach. Angiography 5 months after clipping revealed loss of flow in the right anterior cerebral artery and a de novo L A1/A2 aneurysm, which was electively clipped by a left pterional approach. Follow angiography 5 monthslater showed loss left anterior cerebral artery flow and the anterior cerebral artery territory perfused by posterior cerebral arteries. Cerebral angiography 8 months later did not show further aneurysms and demostrated the posterior circulation vessels perfusing the anterior circulation vascular territory The child remains neurologically intact

Strategies for the structure determination of parasite glycoconjugates using fast atom bombardment mass spectrometry

Fast atom bombardment mass spectrometry (FAB MS) is a sensitive and powerful technique for the analysis of thermally labile and involatile materials. In this paper we describe the application of the method to the characterization of oligosaccharides isolated from the cell surface glycoconjugates of parasitic protozoa. The spectra typically contain both molecular ions, which define the monosaccharide composition, and fragment ions which are related to the structure of the intact molecules. The use of additional techniques such as chemical derivatization and colisional activation enhances fragmentation and simplifies interpretation of the data, enabling the determination of residue sequence, the positions of branch points, and the location of noncarbohydrate substituents. We have applied these techniques to the characterization of phosphoinositol oligosaccharides from members of the Trypanosoma family, including Leptomonas smueli, Endotrypanum schaudinni and Leishmania adleri. Although it is not usually possible to determine the complete structure of oligosaccharide by mass spectrometric methods alone, the information gained greatly simplifies the interpretation of the results from other techiniques such as nuclear magnetic resonance and methylation analysis.