Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Endovascular treatment of cerebral aneurysms: a retrospective study of 163 embolized aneurysms Tratamento endovascular de aneurismas cerebrais: estudo de 163 aneurismas embolizados

OBJECTIVE: To present the results of cerebral aneurysms treated by endovascular technique. METHOD: Retrospective analysis of patient files of Hospital Geral de Fortaleza, Brazil. RESULTS: We report the results of 163 cerebral aneurysms treated by endovascular techniques from January 2002 to October 2005. Patients with ruptured aneurysms (87.2%), according to Hunt-Hess scale were: 33.7% HH I, 28.4% HH II, 24.1% HH III, 13.8% HH IV. The Fisher scale grade IV was the most common (39.7%). Remodeling, coil embolization, arterial occlusion and histoacryl embolization were the techniques employed. Effective occlusion was achieved in 87.7%, partial occlusion in 5.3% and non-effective occlusion in 7.0% of the patients. Glasgow outcome scale results were: 76.3% GOS 5, 5.0% GOS 4, 5.8% GOS 3, 1.4% GOS 2 and 11.5% GOS 1. CONCLUSION: Endovascular treatment seems to be feasible within Brazilian public health system, with results as good as those obtained in larger international centers.<br>OBJETIVO: Apresentar os resultados de aneurismas tratados pela técnica endovascular. MÉTODO: Análise retrospectiva de prontuários do Hospital Geral de Fortaleza, Brasil. RESULTADOS: Apresentamos os resultados de 163 aneurismas cerebrais tratados por técnicas endovasculares de Janeiro de 2002 a Outubro de 2005. Os pacientes com aneurismas rotos (87,2%) eram, segundo a escala de Hunt-Hess: 33,7% HH1; 28.4% HH II, 24.1% HH III, 13.8% HH IV. O Grau IV da escala de Fisher foi o mais comum (39,7%). Empregaram-se as técnicas de remodeling, espiras metálicas, oclusão arterial e embolização com histoacryl. Foi obtida oclusão efetiva em 87,7%, oclusão parcial em 5,3% e oclusão não-efetiva em 7,0% dos casos. De acordo com a Glasgow outcome scale, os resultados foram: 76,3% GOS 5, 5,0% GOS 4, 5,8% GOS 3, 1,4% GOS 2 e 11,5% GOS 1. CONCLUSÃO: A aplicação de técnica endovasculares mostra-se viável na rede pública brasileira, com resultados comparáveis aos de grandes centros mundiais