In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes.This system is useful to study regulation of Drosha activity in physiological and pathological conditions.     

Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth

The demand for recombinant proteins both for biopharmaceutical and technical applications is rapidly growing, and therefore the need to establish highly productive expression systems is steadily increasing. Yeasts, such as Pichia pastoris, are among the widely used production platforms with a strong emphasis on secreted proteins. Protein secretion is a limiting factor of productivity. There is strong evidence that secretion is coupled to specific growth rate (µ) in yeast, being higher at higher µ. For maximum productivity and product titer, high specific secretion rates at low µ would be desired. At high secretion rates cultures contain a large fraction of cells in the G2 and M phases of cell cycle. Consequently, the cell design target of a high fraction of cells in G2 + M phase was achieved by constitutive overexpression of the cyclin gene CLB2. Together with predictive process modeling this reverse engineered production strain improved the space time yield (STY) of an antibody Fab fragment by 18% and the product titer by 53%. This concept was verified with another secreted protein, human trypsinogen. Biotechnol. Bioeng. 2011;108: 2403–2412. © 2011 Wiley Periodicals, Inc.     

Spoilage-related activity of Carnobacterium maltaromaticum strains in air-stored and vacuum-packed meat

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.     

Cooling of pacu (Piaractus mesopotamicus) embryos at various stages of development for 6 or 10 hours

The objective of this research was to verify the effects of cooling embryos of pacu, Piaractus mesopotamicus, in four stages of development during two stocking periods. The stages of embryo development were at: blastoderm, ∼64 cells—1.4 h after fertilization (haf); 25% of the epiboly movement—5.2 haf; blastoporous closing—8.0 haf; and optical vesicle appearing—13.3 haf. Embryos were exposed to a cryoprotectant solution containing methanol (10%) and sucrose (0.5 M). Thereafter, embryos were submitted to a cooling curve until they reached −8 °C, and then kept cooled for 6 or 10 h. In addition, for each stage of embryonic development, a control group with uncooled embryos was used to compare hatching rates. The total number of larvae from the first two stages of ontogenetic development (1.4 and 5.2 haf) was lower compared to the other stages (0.0 and 8.0 haf). There was no significant difference between stages 8.0 and 13.3 haf for the total number of larvae (49.9 ± 6.7% and 55.2 ± 6.7%, respectively). Embryo diameter varied according to embryonic stage, providing evidence of differences in membrane permeability. There was a negative correlation between embryo diameter and the total number of larvae (r = −0.372). In conclusion, use of embryonic stages 8.0 and 13.3 haf were recommended for maintaining cooled pacu embryos at −8 °C for 6 or 10 h.     

Production of recombinant proteins and metabolites in yeasts

Recombinant DNA (rDNA) technologies allow the production of a wide range of peptides, proteins and metabolites from naturally non-producing cells. Since human insulin was the first heterologous compound produced in a laboratory in 1977, rDNA technology has become one of the most important technologies developed in the 20th century. Recombinant protein and metabolites production is a multi-billion dollar market. The development of a new product begins with the choice of the cell factory. The final application of the compound dictates the main criteria that should be taken into consideration: (1) quality, (2) quantity, (3) yield and (4) space time yield of the desired product. Quantity and quality are the most predominant requirements that must be considered for the commercial production of a protein. Quantity and yield are the requirements for the production of a metabolite. Finally, space time yield is crucial for any production process. It therefore becomes clear why the perfect host does not exist yet, and why—despite important advances in rDNA applications in higher eukaryotic cells—microbial biodiversity continues to represent a potential source of attractive cell factories. In this review, we compare the advantages and limitations of the principal yeast and bacterial workhorse systems.     

Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.     

Rapid and reliable identification of Staphylococcus equorum by a species-specific PCR assay targeting the sodA gene

Rapid and reliable identification of Staphylococcus (S.) equorum was achieved by species-specific PCR assays. A set of primers targeting the manganese-dependent superoxide dismutase (sodA) gene of S.equorum was designed. Species-specificity of the primer set was evaluated by using a total of 112 strains (including 27 reference strains of the DSM collection), representing 26 different species of the genus Staphylococcus, 3 species of the genus Kocuria, and different strains of Macrococcus caseolyticus. By using primers SdAEqF and SdAEqR the expected PCR fragment was obtained only when DNA from S. equorum strains was used as template. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. equorum strains.     

The mouse liver content of carbonic anhydrase III and glutathione S-tranferases A3 and P1 depend on dietary supply of methionine and cysteine

The contents of glutathione S-transferase (GST) subunits, carbonic anhydrase III (CAIII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a 230 kDa protein are affected by protein deprivation in mouse liver. In order to know if particular amino acids control these contents, the effects of feeding for 5 days with diets containing different amino acids were examined. After an exploration using SDS-PAGE analysis, the action of selected diets was further examined by distinct techniques. The 230 kDa protein was identified as fatty acid synthase (FAS) by both mass spectrometry and amino acid sequence analyses. Dietary tests showed that: (1) a protein-free diet (PFD) increased the content of glutathione S-transferases P1 and M1, and glyceraldehyde-3-phosphate dehydrogenase, while the content of glutathione S-transferase A3, fatty acid synthase and carbonic anhydrase III decreased; (2) a protein-free diet having either methionine or cysteine preserved the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anydrase III; (3) a protein-free diet having threonine preserved partially the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anhydrase III; (4) a protein-free diet having methionine, threonine and cysteine prevented in part the loss of fatty acid synthase; and (5) the glyceraldehyde-3-phosphate dehydrogenase content was controlled by increased carbohydrate level and/or by lower amino acid content of diets, but not by any specific amino acid. These data indicate that methionine and cysteine exert a main role on the control of liver glutathione S-transferases A3 and P1, and carbonic anhydrase III. Thus, they emerge necessary to prevent unsafe alterations of liver metabolism caused by protein deprivation.