Membrane fusion and infection abilities of baculovirus virions are preserved during freezing and thawing in the presence of trehalose

Budded viruses (BVs) of baculovirus such as Autographa californica nucleopolyhedrovirus (AcNPV) have recently been studied as biological nanomaterials, and methods for their longer-term storage without deterioration would be desirable. The cryopreservation of virions with a naturally occurring saccharide like trehalose as a cryoprotectant is known to be useful for maintaining the viral structure and function. In this study, we examined how useful trehalose is as protectant for BV cryopreservation during repeated freeze-thaw cycles: 1) membrane fusion between liposomes (multilamellar vesicles, MLVs) and BVs, 2) infection of insect culture cells (Sf9 cells) by RFP-expressing BVs, and 3) morphologies of these BVs were investigated by fluorescent dequenching assay, fluorescence microscopy, and transmission electron microscopy (TEM), respectively. The results suggest that the BVs deteriorate in quality with each freeze-thaw cycle, and this deterioration can be diminished with the use of trehalose to an extent similar to that seen with storage on ice. Trehalose serves as a cryoprotectant on baculovirus budded virus particles during repeated freeze-thaw cycles.</p

pH Switching That Crosses over the Isoelectric Point (pI) Can Improve the Entrapment of Proteins within Giant Liposomes by Enhancing Protein–Membrane Interaction

The ability to encapsulate various molecules including proteins within giant liposomes is needed for studies on model cell membranes and artificial cells. In this report, we demonstrate how to improve the efficiency of protein entrapment with the gentle hydration (natural swelling) method, which is a well-known protocol for the preparation of giant liposomes. We found that when the initial pH of a solution was kept below the pI of a target protein during hydration and then changed to above the pI, the protein could be entrapped more efficiently compared to the sample that was kept at above the pI during the hydration. An examination of the ratio of the mass of entrapped protein to the moles of phospholipid in liposomes (dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylglycerol (DOPG)) indicated that entrapment of target proteins like bovine serum albumin, myoglobin, and lysozyme could be improved using this procedure, and this trend was consistent with microscopic observations at the level of a single giant liposome. The conditions that resulted in good efficiencies were affected by the NaCl concentration and the temperature of the hydration solution, implying that protein entrapment in giant liposomes may be enhanced by associative interaction between lipid lamellar membranes and target proteins

Crowding by Anionic Nanoparticles Causes DNA Double-Strand Instability and Compaction

Up to the present, DNA structural transitions caused by cationic polymers as well as in concentrated solutions of neutral polymers are well documented, while a little is known about DNA interaction with like-charge species. Herein, changes in the structure of DNA induced by anionic nanoparticles of different sizes (20–130 nm) were investigated by combining single-molecule DNA fluorescent microscopy, to monitor the conformational dynamics of long-chain DNA, with spectroscopic methods, to gain insight into changes in the secondary structure of DNA. The results showed that several percent of negatively charged silica nanoparticles induced DNA compaction from a coil to a globule, and this change was accompanied by a decrease in the melting temperature of the DNA double helix. DNA was compacted into toroidal condensates with reduced diameters of about 20–30 nm. Smaller 20 nm nanoparticles triggered a DNA coil–globule transition at lower concentrations, but the exclusion volume for each type of nanoparticle at the point of complete DNA collapse, as estimated by taking into account the depth of the ionic atmosphere, was found to be almost the same

The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins

<div><p>Budded virus (BV) particles of baculovirus (<i>Autographa californica</i> nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.</p></div

Opposite effect of polyamines on <i>In vitro</i> gene expression: Enhancement at low concentrations but inhibition at high concentrations - Fig 1

Chemical formulas of (A) spermidine, [SPD(3+)] and (B) spermine, [SP(4+)].</p

Gene expression efficiency depending on the DNA concentration at a fixed concentration of SPD(3+), 1.5 mM.

<p>Gene expression efficiency depending on the DNA concentration at a fixed concentration of SPD(3+), 1.5 mM.</p

AFM images of DNA conformations at different concentrations of SP(4+).

<p>SP(4+) concentration is (A) 0.004 mM (control), (B) 0.007 mM, (C) 0.06 mM, and (D) 0.6 mM.</p

Gene expression efficiency depending on the concentrations of polyamines, SPD(3+) and SP(4+).

The longitudinal axis shows the relative emission intensity of luciferin-luciferase reaction, which corresponds to the efficiency of gene expression. DNA concentration was fixed at 0.3 μM.</p

Schematic representation of the nature of the interaction of polyamine with DNA and also with RNA polymerase depending on the polyamine concentration.

(A): elongated DNA without polyamine, (B): parallel alignment of DNA segments with the inset of the AFM image of Fig 5(C), (C): compact DNA with the inset of AFM of Fig 5(D).</p

Data_Sheet_1_Aqueous/Aqueous Micro Phase Separation: Construction of an Artificial Model of Cellular Assembly.PDF

To artificially construct a three-dimensional cell assembly, we investigated the availability of long-duration microdroplets that emerged near a critical point in an aqueous two-phase system (ATPS) with the hydrophilic binary polymers, polyethylene glycol (PEG), and dextran (DEX), as host containers. We found that erythrocytes (horse red blood cells; RBCs) and NAMRU mouse mammary gland epithelial cells (NMuMG cells) were completely and spontaneously entrapped inside DEX-rich microdroplets. RBCs and NMuMG cells were located in the interior and at the periphery of the droplets at PEG/DEX = 5%:5%. In contrast, the cells exhibited opposite localizations at PEG/DEX = 10%:5%, where, interestingly, NMuMG cells apparently assembled to achieve cell adhesion. We simply interpreted such specific localizations by considering the alternative responses of these cells to the properties of the PEG/DEX interfaces with different gradients in polymer concentrations.</p