Dengue Virus Ensures Its Fusion in Late Endosomes Using Compartment-Specific Lipids

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design

Monitoring the sardine (Sardinella brasiliensis) fermentation process to obtain anchovies Monitoramento do processo de fermentação da sardinha, Sardinella brasiliensis, para obtenção de anchovas

Anchovies are traditional fish preserves, prepared from fermented fish of the engraulidae family, mainly in European countries. In Brazil, sardines (Sardinella brasiliensis) are an alternative fish for preparing these types of preserves, provided that the preservation process results in a high quality product. In this research, sardines were prepared for preservation and physicochemical, microbiological and sensory analyses were carried out during the preservation process. Whole or eviscerated sardines, with or without condiments/preservatives and with 20% of salt (w/w) were used. Sardines were analyzed fresh, and at 1, 15, 30, 45 and 60 days along the preservation process. The use of whole sardines, with or without condiments/preservatives, presented best results, with increased non-proteic nitrogen in the dry matter, higher levels of total volatile bases and higher contents of lactic acid and sodium chloride. The higher acidity observed in the whole sardine treatments resulted in better control of halophylic mesophilic microorganisms, which were kept under 1.4 x 10³ CFU g-1 in both treatments. Total coliforms and Staphylococcus aureus reached 21 and 3.0 x 10² CFU g-1, respectively. Escherichia coli and Salmonella spp were not present in the fresh sardines or in any of the four treatments, indicating that the concentration of salt used was appropriate to maintain the product under adequate microbiological control. Both whole or eviscerated sardines under the conditions of this experiment were appropriate in terms of the microbiological safety of the preserves. Treatments using whole fish, either with or without condiments/preservatives, also presented better sensorial properties such as color, flavor, taste and texture, as compared to the eviscerated fish treatments. Whole sardines produced good quality, anchovy-type preserves, which can be used for consumption and marketing purposes.<br>As sardinhas brasileiras podem ser utilizadas para o preparo de pescado fermentado, à semelhança do que é feito com as anchovas na Europa, desde que o processamento permita a obtenção de um produto com qualidade. O objetivo desta pesquisa foi monitorar o processamento de fermentação de sardinhas, Sardinella brasiliensis, utilizando 4 tratamentos, a saber: peixes inteiros e eviscerados, ambos com ou sem condimentos, e 20% de sal. As sardinhas foram analisadas in natura e nos períodos de 1; 15; 30; 45 e 60 dias de fermentação. O pescado mantido com vísceras apresentou maior facilidade para fermentação e revelou maiores teores de bases voláteis, nitrogênio não protéico e acidez em ácido lático (19,82 mg 100 g-1). A contagem total de mesófilos se manteve na faixa de 10³ UFC g-1. Coliformes totais e Staphilococcus aureus apresentaram baixas contagens. Escherichia coli e Salmonella não foram detectadas. O processamento do pescado com vísceras, não interferiu na segurança microbiológica e propiciou os melhores resultados para cor, aroma, sabor e textura do produto final