Dengue-2 Structural Proteins Associate with Human Proteins to Produce a Coagulation and Innate Immune Response Biased Interactome

<p>Abstract</p> <p>Background</p> <p>Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem.</p> <p>Methods</p> <p>A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology.</p> <p>Results</p> <p>Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.</p> <p>Conclusions</p> <p>Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.</p

In vitro investigation of Dimethylsulfoxide upon human lymphocyte proliferation

Dimetilsulfóxido (DMSO) é um solvente amplamente utilizado para diluir ou incorporar componentes ou misturas em meios aquosos para ensaios biológicos in vitro. Neste trabalho, seus efeitos sobre a proliferação de linfócitos humanos foram avaliados observando-se a multiplicação celular por citometria de fluxo, análise morfológica em citocentrifugados corados e quantificação de AgNOR. Células mononucleares de sangue periférico humano foram induzidas a proliferar por fitohemaglutinina na presença de DMSO (150-300mM), por 5 dias a 37°C e 5% de CO2. Observou-se que o DMSO inibiu significativamente, proporcional à concentração, a proliferação de linfócitos. A morfologia celular mostrou predominância de linfócitos nas culturas tratadas com DMSO, enquanto baixos números de AgNORs, que são segmentos de DNA que transcrevem RNA ribossomal, foram encontrados. Estes resultados demonstram que o DMSO é um solvente inapropriado para estudos biológicos que envolvam imunomodulação de linfócitos humanos.Dimethylsulfoxide (DMSO) is a solvent widely used for diluting or including compounds or mixtures for in vitro biological assays. In this work, the effects of DMSO on human lymphocytes proliferation were evaluated through cell multiplication in a flow cytometer, morphological analyses on stained smears, and AgNOR quantification. Human peripheral blood mononuclear cells induced to proliferate by phytohemagglutinin were treated with DMSO (150- 300mM) for 5 days at 37 °C, and 5% CO2. DMSO inhibited significantly the lymphocyte proliferation in a dosedependent manner. Lymphocyte morphology predominated in DMSO-treated cultures while low numbers of Ag- NOR, which are DNA segments that transcribe ribosomal RNA, were found. The results showed that DMSO is not appropriate for using as a solvent for biological assays involving human lymphocytes immunomodulation.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Alkaloid-rich fraction of Himatanthus lancifolius contains anti-tumor agents against leukemic cells

The effects of the alkaloid-rich fraction of Himatanthus lancifolius (Müll. Arg) Woodson on normal marrow cells and leukemic cell lines were investigated. After 48 h exposure, the proliferation assay showed significant cell growth inhibition for Daudi (0.1-10 µg/mL), K-562 (1-10 µg/mL), and REH cells (10-100 µg/mL), yet was inert for normal marrow cells. A similar inhibition profile was observed in clonogenic assays. This alkaloid-rich fraction, in which uleine is the main compound, showed no signs of toxicity to any cells up to 10 µg/mL. Cell feature analyses after induction of differentiation showed maintenance of the initial phenotype. Flow cytometric expression of Annexin-V and 7-AAD in K-562 and Daudi cells has indicated that the cells were not undergoing apoptosis or necrosis, suggesting cytostatic activity for tumor cells<br>Os efeitos da fração rica em alcalóides indólicos de Himatanthus lancifolius (Müll. Arg) Woodson sobre células normais de medula óssea e linhagens celulares leucêmicas foram investigados. Após 48 horas de exposição, os ensaios de proliferação demonstraram efeitos inibitórios significativos para as linhagens Daudi (0,1-10 µg/mL), K-562 (1-10 µg/mL) e REH (10-100 µg/mL), enquanto mostrou-se inerte sobre células normais de medula óssea. Os perfis de inibição se repetiram nos ensaios clonogênicos. A fração rica em alcalóides, na qual a uleína é a substância majoritária, não demonstrou toxicidade até a dose de 10 µg/mL para nenhuma das células incluídas no estudo. Da mesma forma, não se observou influência dessa fração sobre a diferenciação celular dessas linhagens, mas manutenção de seu estado maturacional inicial. O conjunto de dados descritos associado à baixa co-expressão de anexina-V e 7-AAD sugerem que esta fração exerce atividade citostática para células tumorais

Influence of rosmarinic acid and Salvia officinalis extracts on melanogenesis of B16F10 cells

Melanin is a photoprotective skin pigment, and pathologies characterized by hypo or hyperpigmentation are common. New compounds that regulate melanogenesis are, therefore, opportune, and many natural products with this property, as polyphenols, have been described. Salvia officinalis L., Lamiaceae, is a widely used food spice that contains high amounts of phenol derivates, including rosmarinic acid. The aim of this work was to evaluate the contribution of rosmarinic acid in the melanogenic activity of sage extracts. Fluid and aqueous extracts of sage and purified rosmarinic acid were assayed for B16F10 cytotoxicity and, then, evaluated on melanin production and tyrosinase activity. While sage extracts showed a concentration-dependent ability to significantly increase melanin production without necessarily changing the enzymatic activity, rosmarinic acid showed a dual behavior on melanogenesis, increasing melanin biosynthesis and tyrosinase activity at low concentrations and decreasing it at higher levels. Rosmarinic acid may collaborate with sage extracts activity on melanogenesis, although other compounds may be involved. This is the first time that a dual action of rosmarinic acid on melanogenesis is reported, which may be useful in further studies for therapeutic formulations to treat skin pigmentation disorders