SachaInchi (Plukenetiavolubilis) is a nut that has been grown in the Amazon Rainforest and the high Andes Mountains of Peru for countless centuries. The oil of this nut, natural source of omega 3, 6 ...and 9, has been recognized by its high antioxidant capacity in humans. In this work, oil from Sacha Inchi was fortified with two commercial antioxidants (Ecoprol 2020 and tocopherol) in order to prepare a fresh cheese from cow's milk. The antioxidant capacities of Sacha Inchi and commercial antioxidants were used as preservatives with the purpose to increase the shelf-life of fresh cheese besides nutritional content.The factorial method was necessary to prepare seven formulations in order to find the optimal concentration of the antioxidants added to Sacha Inchi oil and the addition of this oil to the fresh cheese.A sensory analysis was performed to choose the best formulation.The results showed that an oil formulation (F4) with tocopherol (150 mg/kg of oil) and Ecoprol2020 (1000 mg/kg of oil) displayed the lowest peroxide values (PI: 2.6 ± 0.1 meq O2/kg of oil, p < 0.001) and it was able to reduce approximately 50% of fatty acid oxidation in SachaInchi oil in relation to the PI control. Then, F4 was used to elaborate further nine formulations (F’1 –F’9), enriched with Sacha Inchi oil (1 to 4%) to prepare the fresh cheese. Microbiological analysis for all formulations were performed (limits of mold, yeasts, coliforms, salmonella, and bacteria) in order to meet the legal requirements of health and safety in Peru. The cheese taste acceptability was determined through the sensorial evaluation, which reached 7.2according to the 9-hedonic scale for F'5. Thus, an optimum fresh cheese was obtained from the formulation (F’5) with 22.5g/L of salt and 2.5% of Sacha Inchi oil enriched with 150 mg/kg of tocopherol and 1000 mg/kg of Ecoprol 2020. The cheese shelf-life was also evaluated, increasing it up from 7 days to 16 days in refrigeration.
We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in
Spodoptera frugiperda (
Sf21) insect cells correlates well with the efficiency of ...transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small (∼25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in
Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells.
Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.
To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies.
Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors ...were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP, were cloned into plasmids driven by RNA polymerase III promoter H1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry.
The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%.
By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.
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