Handheld virus concentration method using a hollow fiber filter module.

A virus concentration method is required for viral vaccine manufacturing and virus-related research. However, concentration methods, such as ultracentrifugation, often require capital investment. We report a simple and easy-to-use handheld syringe method for virus concentration using a hollow fiber (HF) filter module, which can be applicable to viruses of different sizes, without incorporating any special machines or reagents. This virus concentration method does not use pumps, which might cause shear stress for virus particles; therefore, it is useful for stress-sensitive virus particles, and virus-like particles, as well as other proteins. The clarified harvest of flavivirus (Zika virus) was concentrated using an HF filter module and compared with a centrifugal ultrafiltration device (CUD) for demonstration of the HF filter method. The HF filter method achieved concentration of the virus solution in less time than the CUD. The yield comparison of the recovered virus solution indicated that recovery from the developed method was comparable to using the CUD, and infectivity was maintained throughout.•The Zika virus was concentrated from 200 mL to 5 mL within 45 min using the HF filter and handheld syringe module method.•The handheld HF filter method may be applicable to stress-sensitive viruses and proteins of different sizes.•The virus concentration process should be conducted in a safety cabinet, which is preferred for virus containment.

Landscape- and Local-Scale Actions Are Essential to Conserve Regional Macrophyte Biodiversity.

Regional-scale pond diversity is supported by high variation in community composition. To effectively and efficiently conserve pond regional diversity, it is essential to recognize the community types in a focal region and the scales of the factors influencing the occurrence of respective community types. Based on a flora survey and GIS analysis of 367 ponds in western Japan, we developed a multinomial regression model that describes the relationship between aquatic macrophyte community type (based on cluster analysis) and five environmental factors that differ in the spatial scale at which they operate (i.e., landscape or local scale) and origin (i.e., natural or anthropogenic). A change in topographic configuration resulted in a transition of the community types with high species richness. Increasing urban and agricultural area around ponds resulted in a decrease in species-rich community occurrence; an increase in urban area increased the probability of a pond having no macrophytes, whereas that of paddy field increased the probability of a pond having only a few macrophytes. Pond surface area and proportion of artificial embankment significantly defined the pond community: greater embankment proportions increased the probability of ponds having few or no macrophytes. Our results suggest that conserving regional pond biodiversity will require actions not only at a local scale but also at a sufficiently large spatial scale to cover the full gradient of topographic configurations that influence the macrophyte species composition in ponds.

Synthesis of functional dipeptide carnosine from nonprotected amino acids using carnosinase-displaying yeast cells.

Carnosine (beta-alanyl-L-histidine) is one of the bioactive dipeptides and has antioxidant, antiglycation, and cytoplasmic buffering properties. In this study, to synthesize carnosine from nonprotected amino acids as substrates, we cloned the carnosinase (CN1) gene and constructed a whole-cell biocatalyst displaying CN1 on the yeast cell surface with alpha-agglutinin as the anchor protein. The display of CN1 was confirmed by immunofluorescent labeling, and CN1-displaying yeast cells showed hydrolytic activity for carnosine. When carnosine was synthesized by the reverse reaction of CN1, organic solvents were added to the reaction mixture to reduce the water content. The CN1-displaying yeast cells were lyophilized and examined for organic solvent tolerance. Results showed that the CN1-displaying yeast cells retained their original hydrolytic activity in hydrophobic organic solvents. In the hydrophobic organic solvents and hydrophobic ionic liquids, the CN1-displaying yeast cells catalyzed carnosine synthesis, and carnosine was synthesized from nonprotected amino acids in only one step. The results of this research suggest that the whole-cell biocatalyst displaying CN1 on the yeast cell surface can be used to synthesize carnosine with ease and convenience.

Enhancement of display efficiency in yeast display system by vector engineering and gene disruption.

Vector engineering and gene disruption in host cells were attempted for the enhancement of alpha-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.

Effective NADH-dependent oxidation of 7beta-hydroxy-delta8-tetrahydrocannabinol to the corresponding ketone by Japanese monkey hepatic microsomes.

The NADH-dependent activity by hepatic microsomes of Japanese monkeys for 7-oxo-Delta(8)-tetrahydrocannabinol (7-oxo-Delta(8)-THC) formation from 7beta-hydroxy-Delta(8)-THC exhibited about 70% of the NADPH-dependent activity (100%) at the substrate concentration of 72.7 microM, although NADPH was an obligatory cofactor for maximal activity. Both NADH- and NADPH-dependent activities were significantly inhibited by the typical P450 inhibitors, such as SKF525-A and metyrapone. Both activities were almost completely inhibited by the NADPH-P450 reductase inhibitor diphenyliodonium chloride. The ratio of NADH- and NADPH-dependent activities varied significantly according to the substrate concentration. Interestingly, the NADH-dependent activity was higher than that of NADPH at low substrate concentrations of 13-50 microM. The ratio was also affected by the cofactor concentration. In the reconstituted system of CYP3A8 purified from hepatic microsomes of Japanese monkeys as a major enzyme responsible for the NADPH-dependent oxidation, NADH as well as NADPH could sustain the oxidation of 7beta-hydroxy-Delta(8)-THC to the corresponding ketone. The NADH-dependent oxidation of 7beta-hydroxy-Delta(8)-THC by monkey livers is mainly catalyzed by CYP3A8 as well as the NADPH-dependent oxidation. These results indicate that NADH as a cofactor may be also useful for the oxidation of 7beta-hydroxy-Delta(8)-THC, and that the cofactor requirement for the reaction is varied by the concentrations of substrate and/or cofactor.