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

Numerical analysis of two-layered elastic beams considering inter-layer slip and delamination

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-kappaB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-kappaB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs

Reactivation of HIV latency by a newly modified Ingenol derivative via protein kinase C&dgr;–NF-&kgr;B signaling

ObjectiveAlthough HAART effectively suppresses viral replication, it fails to eradicate latent viral reservoirs. The 'shock and kill' strategy involves the activation of HIV from latent reservoirs and targeting them for eradication. Our goal was to develop new approaches for activating HIV from latent reservoirs.DesignWe investigated capacity of Ingenol B (IngB), a newly modified derivative of Ingenol ester that was originally isolated from a Brazilian plant in Amazon, for its capacity and mechanisms of HIV reactivation.MethodsReactivation of HIV-1 by IngB was evaluated in J-Lat A1 cell culture model of HIV latency as well as in purified primary CD4 T cells from long-term HAART-treated virologically-suppressed HIV-infected individuals. The underlining molecular mechanisms of viral reactivation were investigated using flow cytometry, RT-qPCR and chromatin immunoprecipitation.ResultsIngB is highly effective in reactivating HIV in J-Lat A1 cells with relatively low cellular toxicity. It is also able to reactivate latent HIV in purified CD4 T cells from HAART-treated HIV-positive individuals ex vivo. Our data show that IngB may reactivate HIV expression by both activating protein kinase C (PKC)δ-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and directly inducing NF-κB protein expression. Importantly, IngB has a synergistic effect with JQ1, a BET bromodomain inhibitor, in latent HIV reactivation.ConclusionsIngB is a new promising compound to activate latent HIV reservoirs. Our data suggest that formulating novel derivatives from Ingenol esters may be an innovative approach to develop new lead compounds to reactivate latent HIV

Estudo brasileiro de validação para localização e lista de qualidade formal do Rorschach-SC: não-pacientes psiquiátricos Estudio brasileño de validación para localización y lista de calidad formal del Rorschach-SC: no-pacientes psiquiátricos Brazilian validation study to locate and list of formal quality of the Rorschach-CS: non-psychiatric patients

Este estudo se refere à validação do Atlas de Localização de respostas e da Lista de Qualidade Formal de respostas e conteúdos, FQ, do Rorschach-SC de uma amostra brasileira. Foram selecionados, 46 adultos não-pacientes psiquiátricos, de ambos os sexos, níveis sociais e escolaridade diversos. Os instrumentos utilizados foram o SRQ-20 e o Método de Rorschach. As respostas ao Rorschach foram codificadas segundo as áreas e a lista de qualidade formal norte-americanas e brasileiras. As análises comparativas realizadas por meio da ANOVA não encontraram diferenças entre os dois grupos, no que diz respeito às áreas de localização, W, D, Dd nas áreas brasileiras e norte-americanas, comprovando-se a validade das áreas brasileiras. Não foram também encontradas diferenças quanto à qualidade formal FQo e X+%, nas listas brasileiras e norte-americanas, comprovando-se a validade da lista brasileira para essas duas variáveis. Os resultados indicam validade para o atlas brasileiro.<br>Este estudio se refiere a la validación del Atlas de Localización de respuestas y de la Lista de Calidad Formal de respuestas y contenidos, FQ, del Rorschach-SC de una muestra brasileña. Fueron seleccionados, 46 adultos no-pacientes psiquiátricos, de ambos sexos, niveles sociales y de escolaridad diversos. Los instrumentos utilizados fueron el SRQ-20 y el Método de Rorschach. Las respuestas al Rorschach fueron codificadas según las áreas y la lista de calidad formal norteamericanas y brasileñas. Los análisis comparativos realizados, por medio de la ANOVA, no encontraron diferencias entre los dos grupos en lo que respecta a las áreas de localización, W, D, Dd en las áreas brasileñas y norteamericanas, comprobando la validez de las áreas brasileñas. No fueron también encontradas diferencias cuanto a la calidad formal FQo y X+%, en las listas brasileñas y norteamericanas, comprobando la validez de la lista brasileña para esas dos variables. Los resultados indicaron validez para el atlas brasileño.<br>This study aimed at validating the Atlas Rorschach-CS Location Areas and Formal Quality List from a Brazilian sample. 46 adults, non-patients psychiatric cases, both genders and different educational levels and social status were selected. The instruments used were the SRQ-20 and the Rorschach method. The Rorschach responses were scoredaccording to the areas and the North-American and Brazilian Lists of formal quality . The comparative analyzes performed by ANOVA did not established differences between the two groups regarding the Location Areas W, D, Dd in both North-American and Brazilian areas, proving the validity of the Brazilian areas. Differences were not found as regarding the Formal Quality FQo and X+% both using the North-American and Brazilian lists, proving the validity of the Brazilian list for these two variables.. The results indicate validity to the Brazilian Atlas